Cosmetic composition for shitening, moisture removing, and wrink removal containing low molecule
By isolating low molecular peptides from the alcoholic meal, a cosmetic composition with whitening, moisturizing and wrinkle improvement effects was prepared, which solved the problem of insufficient research on physiological active substances in the utilization of alcoholic meals in the prior art, and achieved significant skin Aging relief effect.
Patent Information
- Application Number
- CN202311064622.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2023-06-30
- Filing Date
- 2023-08-22
- Publication Date
- 2024-12-31
AI Technical Summary
The prior art has failed to effectively separate physiological active substances with whitening, moisturizing and wrinkle improvement effects from wine meals, and the research on the utilization of functional cosmetic materials mainly focuses on the preparation methods and effects of simple extracts or fermented substances. There is a lack of research on the physiologically active substances of its components.
Low molecular peptides with less than 1000 Da, including anionic, cationic and nonionic low molecular peptides, were isolated by treating proteolytic enzymes to the wine meal, and inducing enzyme reaction, precipitation and chromatography through specific steps, whitening, Cosmetic composition for moisturizing and wrinkle improvement.
Isolation of low molecular peptides from wine meals was achieved, and cosmetic compositions with significant whitening, moisturizing and wrinkle improvement effects were prepared. By reducing the expression of tyrosinase-related protein 1 and increasing the expression of type I collagen and laminin-5 , improve the expression of silk polyprotein and relieve skin aging.
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Abstract
Description
Technical Field
[0001] The present invention relates to a cosmetic composition containing a low-molecular peptide isolated from sake lees. More specifically, the present invention relates to a cosmetic composition for skin whitening, skin wrinkle improvement or skin moisturizing, wherein a low-molecular peptide having whitening, moisturizing and wrinkle improvement activities is isolated from sake lees, and the low-molecular peptide is contained as an active ingredient to exhibit effects of reducing the expression of tyrosinase-related protein 1 (TRP-1), increasing the expression of type I collagen (Collagen Type 1) and laminin-5 (Laminin-5), and increasing the expression of filaggrin (Filaggrin). Background Art
[0002] With the development of industry in modern society, the increase in demand for various resources also brings about the increase in various wastes, which causes serious problems such as environmental pollution and natural disasters caused by the global greenhouse effect. It is known that the countless pollutants that cause environmental pollution damage the skin barrier, bringing about the adverse effect of accelerating skin aging. The skin aging phenomenon caused by the external environment is mainly due to ultraviolet rays penetrating the epidermis into the dermis, damaging the collagen and elastin in the dermis to induce skin wrinkles and excessively generate harmful reactive oxygen species, thereby gradually accelerating skin aging. In addition, pollutants such as external dust bring various chemical stimuli to keratinocytes and lipid membranes, thereby causing inflammation and inducing problems such as pigmentation, dry skin, damaged skin barrier, and skin wrinkles.
[0003] On the other hand, as industrial development leads to an increase in waste and environmental pollution, there is a growing interest in sustainable materials, and increasing the value of waste through "upcycling," which is defined as recycling waste materials to create products with new value, is also attracting attention. In particular, the amount of food processing byproducts discarded after food processing is as high as 5.48 million tons per year in Korea alone, and many researchers are actively studying the recycling of raw materials that do not burden the environment, not just the containers or packaging materials used to package products.
[0004] Sake lees (dregs), a representative byproduct of food processing, are byproducts of brewing or ethanol production. As oil prices rise, the production of ethanol as a substitute for oil has increased, and the production of sake lees has also increased. Sake lees have been proven to have high feed value and have been used as feed for a variety of livestock. They contain crude fat, crude protein, fiber, fermented yeast bodies, and various minerals as main components, so their usable value is gradually increasing. Such sake lees have been studied in the fields of food additives, functional food materials, bioenergy production, feed and fertilizer utilization, and there are also many studies on the use of functional sachets and cosmetic materials. However, existing research only includes the preparation methods and effects of simple extracts or fermented products, and there is still a blank report on which components of sake lees exert these functions.
[0005] Therefore, the present invention intends to separate and purify physiologically active substances with whitening, moisturizing and wrinkle-improving activities from sake lees, which is a typical byproduct of food processing, to prepare high-quality sake lees extract. As a result, low-molecular peptides were separated from sake lees and the whitening, moisturizing and wrinkle-improving effects of a cosmetic composition containing the low-molecular peptides were confirmed, thereby developing a material that can alleviate skin aging.
[0006] Prior art literature
[0007] Patent Literature
[0008] Patent Document 1: Korean Patent No. 10-1643696 (granted on July 22, 2016) discloses a cosmetic composition for removing heavy metals and dust containing sake lees.
[0009] Patent Document 2: Korean Patent No. 10-0736327 (granted on June 29, 2007) discloses functional natural cosmetics utilizing sake lees for moisturizing, whitening, and wrinkle improvement, and a method for producing the same. Summary of the invention
[0010] Technical issues
[0011] The present invention aims to separate physiologically active substances having whitening, moisturizing and wrinkle-improving effects from sake lees and prepare sake lees low-molecular peptides containing the same. Furthermore, by preparing a dosage form containing the low-molecular peptide and confirming its efficacy, a cosmetic composition containing sake lees extract showing excellent whitening, moisturizing and wrinkle-improving effects is developed and provided.
[0012] Technical Solution
[0013] The present invention provides a cosmetic composition containing, as an active ingredient, a low-molecular-weight peptide of 1000 Da or less separated by treating sake lees with a proteolytic enzyme.
[0014] The present invention provides a cosmetic composition containing anionic low-molecular peptides of 1000 Da or less separated by treating sake lees with a proteolytic enzyme, cationic low-molecular peptides of 1000 Da or less separated by treating sake lees with a proteolytic enzyme, and nonionic low-molecular peptides of 1000 Da or less separated by treating sake lees with a proteolytic enzyme.
[0015] In the cosmetic composition of the present invention, the above-mentioned low-molecular peptide is characterized in that it can be obtained by a process comprising the following steps: step (a), soaking sake lees in pure water; step (b), after the soaking in the above step (a), adding subtilisin derived from Bacillus to induce a primary enzyme reaction; step (c), after the primary enzyme reaction in the above step (b), adding pepsin to induce a secondary enzyme reaction; step (d), after the secondary enzyme reaction in the above step (c), recovering the supernatant by centrifugation; and step (e), adding ethanol to the supernatant recovered in the above step (d) to induce a precipitation reaction, and recovering the supernatant by centrifugation.
[0016] In the cosmetic composition of the present invention, the above-mentioned anionic low-molecular peptide is characterized in that it can be obtained by a process comprising the following steps: step (a), soaking sake lees in pure water; step (b), after the soaking in the above step (a), adding subtilisin derived from Bacillus to induce a primary enzyme reaction; step (c), after the primary enzyme reaction in the above step (b), adding pepsin to induce a secondary enzyme reaction; step (d), after the secondary enzyme reaction in the above step (c), recovering the supernatant by centrifugation; step (e), adding ethanol to the supernatant recovered in the above step (d) to induce a precipitation reaction, and recovering the supernatant by centrifugation; and step (f), obtaining the anionic peptide by passing the supernatant recovered in the above step (e) through an anion exchange resin.
[0017] In the cosmetic composition of the present invention, the cationic low-molecular peptide is characterized in that it can be obtained by a process comprising the following steps: step (a), soaking sake lees in pure water; step (b), after the soaking in the above step (a), adding subtilisin derived from Bacillus to induce a primary enzyme reaction; step (c), after the primary enzyme reaction in the above step (b), adding pepsin to induce a secondary enzyme reaction; step (d), after the secondary enzyme reaction in the above step (c), recovering the supernatant by centrifugation; step (e), adding ethanol to the supernatant recovered in the above step (d) to induce a precipitation reaction, and recovering the supernatant by centrifugation; and step (f'), obtaining the cationic peptide by passing the supernatant recovered in the above step (e) through a cation exchange resin.
[0018] In the cosmetic composition of the present invention, the nonionic low-molecular peptide is characterized in that it can be obtained by a process comprising the following steps: step (a), soaking sake lees in pure water; step (b), after the soaking in the above step (a), adding subtilisin derived from Bacillus to induce a primary enzyme reaction; step (c), after the primary enzyme reaction in the above step (b), adding pepsin to induce a secondary enzyme reaction; step (d), after the secondary enzyme reaction in the above step (c), recovering the supernatant by centrifugation; step (e), adding ethanol to the supernatant recovered in the above step (d) to induce a precipitation reaction, and recovering the supernatant by centrifugation; step (f1), passing the supernatant recovered in the above step (e) through an anion exchange resin to obtain a primary eluate; step (f2), passing the eluate obtained in the above step (f1) through a cation exchange resin to obtain a secondary eluate; and step (g), filtering the secondary eluate obtained in the above step (f2) to obtain a filtrate.
[0019] In the present invention, the above cosmetic composition is characterized in that it can be used for skin whitening.
[0020] In the present invention, the cosmetic composition is characterized in that it can be used to improve skin wrinkles.
[0021] In the present invention, the cosmetic composition is characterized in that it can be used for moisturizing the skin.
[0022] Effects of the Invention
[0023] The present invention provides a method for separating low-molecular peptides from sake lees and a cosmetic composition containing the same.
[0024] Furthermore, the cosmetic composition of the present invention has the effects of inhibiting pigmentation by reducing the amount of melanin produced, inhibiting pigmentation by reducing the expression of tyrosinase related protein 1 (TRP-1), improving wrinkles by increasing the expression of type I collagen and laminin-5, and moisturizing by increasing the expression of filaggrin, thereby alleviating skin aging. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 These are the results of molecular weight analysis of low molecular weight peptides isolated from sake lees.
[0026] Figure 2 The results are analysis results of the surface potentials of anionic low-molecular peptides, cationic low-molecular peptides, and nonionic low-molecular peptides separated from sake lees.
[0027] Figure 3 These are the results of measuring the melanin production-reducing effect of low-molecular-weight peptides isolated from sake lees.
[0028] Figure 4 These are the results of measuring the effect of low-molecular-weight peptides isolated from sake lees on reducing the expression of tyrosinase-related protein 1.
[0029] Figure 5 These are the results of measuring the effect of low-molecular-weight peptides isolated from sake lees on increasing the expression of type I collagen.
[0030] Figure 6 These are the results of measuring the effect of low-molecular-weight peptides isolated from sake lees on increasing the expression of laminin-5.
[0031] Figure 7 These are the results of measuring the effect of low-molecular-weight peptides isolated from sake lees on increasing filaggrin expression. DETAILED DESCRIPTION
[0032] Sake lees (lees) are byproducts of brewing or ethanol production, and their value is gradually increasing as they contain crude fat, crude protein, fiber, fermented yeast, various minerals, etc. Such sake lees are being studied in the fields of food additives and functional food materials, as well as functional sachets and cosmetic materials. However, existing research only includes the preparation methods and effects of simple extracts or fermented products, and there is little research on the physiologically active substances contained in sake lees that have these effects.
[0033] Therefore, the present inventors isolated low-molecular peptides as physiologically active substances that exhibit whitening, moisturizing and wrinkle-improving effects from sake lees and utilized them as cosmetic compositions, judging that they could be developed into new functional materials with excellent physiological activity, safety and stability.
[0034] The present invention provides a cosmetic composition containing, as an active ingredient, a low-molecular-weight peptide of 1000 Da or less separated by treating sake lees with a proteolytic enzyme.
[0035] Furthermore, the present invention provides a cosmetic composition comprising an anionic low-molecular peptide of 1000 Da or less separated by treating sake lees with a proteolytic enzyme, a cationic low-molecular peptide of 1000 Da or less separated by treating sake lees with a proteolytic enzyme, and a nonionic low-molecular peptide of 1000 Da or less separated by treating sake lees with a proteolytic enzyme. In this case, preferably, the cosmetic composition is prepared by mixing the anionic low-molecular peptide, the cationic low-molecular peptide, and the nonionic low-molecular peptide in a weight ratio of 1:0.5 to 1.5:0.5 to 1.5.
[0036] 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 prepared by treating proteolytic enzymes. The preparation method thereof will be described in detail according to each step in the following content.
[0037] Step (a): Soaking the Sake Lees
[0038] This step is a process of soaking sake lees in pure water. Sake lees, also known as sake lees, can be used without restriction as long as it is a byproduct obtained during brewing or ethanol production. Preferably, the dried sake lees are powdered for use, and more preferably, the dried and powdered sake lees are soaked in pure water and the pH is adjusted to 6.5 to 7.5.
[0039] Step (b): Inducing an enzyme reaction
[0040] This step is a process of inducing a primary enzyme reaction by adding subtilisin derived from Bacillus after the soaking in the above step (a).
[0041] Subtilisin is a proteolytic enzyme used to hydrolyze proteins in sake lees. In this step, by using subtilisin to hydrolyze proteins in sake lees into larger units, such hydrolysis can further accelerate protein hydrolysis in the following secondary enzyme reaction to obtain low molecular peptides.
[0042] In this step, the primary enzyme reaction can be carried out using an ultra-high pressure enzyme reactor equipped with a polymer coated with subtilisin, a protease isolated from Bacillus. In this case, preferably, the reaction is carried out at a temperature of 50°C to 60°C under a pressure of 80 bar to 120 bar, and preferably, the reaction time is 12 hours to 36 hours.
[0043] Step (c): Inducing secondary enzyme reaction
[0044] This step is a process of adding pepsin to induce a secondary enzyme reaction after the primary enzyme reaction in the above step (b).
[0045] Pepsin is a proteolytic enzyme secreted by the stomach. In this step, the protein in the sake lees that has been hydrolyzed once by adding subtilisin in the above step (b) is hydrolyzed into lower molecules by adding pepsin.
[0046] In this step, preferably, the secondary enzyme reaction can be carried out using an ultra-high pressure enzyme reactor equipped with a polymer coated with pepsin. In this case, preferably, the reaction is carried out at a pressure of 80 bar to 120 bar and a temperature of 35° C. to 45° C., and preferably, the reaction time is 3 hours to 9 hours.
[0047] After the secondary enzyme reaction in this step is completed, it is preferred to boil the solution after the secondary reaction to inactivate the excess enzyme.
[0048] Step (d): Centrifugation
[0049] This step is a process of recovering the supernatant by centrifugation after the secondary enzyme reaction in step (c). That is, the solution after the secondary enzyme reaction in step (c) is centrifuged to recover the supernatant.
[0050] Step (e): Recovering the supernatant
[0051] This step is a process of adding ethanol to the supernatant recovered in the above step (d) to induce a precipitation reaction, and then recovering the supernatant by centrifugation.
[0052] In this step, ethanol precipitation is induced by adding ethanol to the supernatant recovered in the above step (d). In this case, 0.8 to 1.2 weight ratios of ethanol may be added relative to 1 weight of the supernatant, and more preferably, 1 weight ratio of ethanol is added relative to 1 weight of the supernatant. When ethanol is added in this way, precipitation is induced and the precipitate sinks to the bottom. Preferably, the ethanol precipitation is performed at a refrigerated temperature.
[0053] On the other hand, by carrying out this step, low-molecular peptides can be prepared from sake lees. That is, after inducing ethanol precipitation reaction through the above process, the supernatant obtained by centrifugation can be filtered and the filtrate can be freeze-dried as needed to prepare sake lees low-molecular peptides.
[0054] Step (f), step (f'), step (f1), step (f2): chromatography
[0055] In order to obtain anionic peptides, the supernatant recovered in the above step (e) is passed through an anion exchange resin.
[0056] In order to obtain a cationic peptide, the supernatant recovered in the above step (e) is passed through a cation exchange resin.
[0057] In order to obtain nonionic peptides, the following steps are performed: step (f1), passing the supernatant recovered in the above step (e) through an anion exchange resin to obtain a primary eluate; step (f2), passing the eluate obtained in the above step (f1) through a cation exchange resin to obtain a secondary eluate. Then, filtering the secondary eluate obtained in the above step (f2) to obtain a filtrate.
[0058] More specifically, the anionic peptide, cationic peptide and nonionic peptide in step (f), step (f'), step (f1) and step (f2) of the present invention can be obtained by separation as follows.
[0059] First, in order to obtain anionic peptides, an anion exchange resin is activated in 8 to 12 times of a 0.5M sodium hydroxide (NaOH) aqueous solution and then loaded into a hollow column tube. Then, 8 to 12 times of the resin capacity of distilled water is flowed in for washing. After the supernatant recovered in the above step (e) or the low-molecular peptide obtained from the supernatant is dissolved in distilled water and loaded into a hollow column equipped with an anion exchange resin, 8 to 12 times of the resin capacity of distilled water is continuously flowed in at an appropriate rate to adsorb the low-molecular peptide with anions. Then, 8 to 12 times of a 2M sodium chloride (NaCl) aqueous solution is used to flow in at an appropriate rate for elution. On the other hand, the eluted eluate is passed through a 0.1μm to 0.3μm microfiltration membrane to obtain a filtrate, and the obtained filtrate can be freeze-dried as needed to prepare sake lees anionic low-molecular peptides.
[0060] In order to obtain cationic peptides, a cation exchange resin is activated in an 8- to 12-fold 0.5M sodium hydroxide (NaOH) aqueous solution and then loaded into a hollow column tube. Then, 8- to 12-fold distilled water of the resin capacity is flowed in for washing. After the supernatant recovered in the above step (e) or the low-molecular peptide obtained from the supernatant is dissolved in distilled water and loaded into a hollow column containing a cation exchange resin, 8- to 12-fold distilled water of the resin capacity is continuously flowed in at an appropriate rate to adsorb the low-molecular peptide with cations. Then, 8- to 12-fold 2M sodium chloride (NaCl) aqueous solution is used to flow in at an appropriate rate for elution. On the other hand, the eluted eluate is passed through a 0.1μm to 0.3μm microfiltration membrane to obtain a filtrate, and the obtained filtrate can be freeze-dried as needed to prepare a sake lees cationic low-molecular peptide.
[0061] For nonionic peptides, anion exchange resin and cation exchange resin are activated in 8 to 12 times of 0.5M sodium hydroxide (NaOH) aqueous solution, respectively, and then loaded into a hollow column tube. Then, distilled water 8 to 12 times the volume of the resin is flowed in for washing. The supernatant recovered in the above step (e) or the low-molecular peptide obtained from the supernatant is dissolved in distilled water and loaded into a hollow column equipped with anion exchange resin, and distilled water 8 to 12 times the volume of the resin is continuously flowed at an appropriate speed for primary elution. Then, after the above primary eluate is raised to a hollow column equipped with a cation exchange resin, distilled water 8 to 12 times the volume of the resin is continuously flowed at an appropriate speed for secondary elution. The eluate of the secondary elution is passed through a 0.1μm to 0.3μm microfiltration membrane to obtain a filtrate, and the obtained filtrate can be freeze-dried as needed to prepare sake lees nonionic low-molecular peptides.
[0062] On the other hand, in the cosmetic composition of the present invention, the cosmetic composition is characterized in that it can be used for skin whitening. On the other hand, the skin whitening effect can be exerted in a manner of suppressing pigmentation by reducing the expression of tyrosinase-related protein 1.
[0063] In the cosmetic composition of the present invention, the cosmetic composition is characterized in that it can be used to improve skin wrinkles. On the other hand, the skin wrinkle improvement effect can be exerted by increasing the expression of type I collagen and laminin-5.
[0064] In the cosmetic composition of the present invention, the cosmetic composition is characterized in that it can be used for skin moisturizing. On the other hand, the skin moisturizing effect can be exerted by increasing the expression of filaggrin.
[0065] On the other hand, the cosmetic composition of the present invention preferably contains 0.01% by weight to 30.0% by weight of the low-molecular peptide of the present invention relative to the total weight of the cosmetic composition.
[0066] In the cosmetic composition of the present invention, the above-mentioned cosmetic composition can be prepared into any dosage form commonly prepared in the technical field to which the present invention belongs. As an example, it can be prepared into: a basic cosmetic dosage form selected from the group consisting of a solution, a suspension, an emulsion, a patch, a lotion, a gel, a water-soluble liquid, a cream, an essence, a surfactant-containing cleanser, an oil, an oil-in-water (O / W) type, and an oil-in-water (W / O) type; a facial mask dosage form; a body cleansing dosage form; an exfoliating gel; an oil-in-water type or an oil-in-water type base cream. But it is not limited to this.
[0067] In the cosmetic composition of the present invention, the above-mentioned cosmetic composition can be repeatedly used with other cosmetic compositions other than the present invention. In addition, the cosmetic composition of the present invention can be used according to the usual usage method, and the number of uses can vary depending on the skin condition or preference of the user.
[0068] The present invention is described in more detail below through the following embodiments and experimental examples. However, the scope of the present invention is not limited to the following embodiments and experimental examples, but should also include variations of equivalent technical concepts.
[0069] Example 1: Preparation of low molecular weight peptides from sake lees
[0070] In the examples, a low-molecular peptide derived from sake lees was prepared according to the following method.
[0071] After the dried sake lees were powdered by a pulverizer, they were soaked in pure water and the pH was adjusted to 7, and then placed in an ultra-high pressure enzyme reactor equipped with a polymer coated with subtilisin derived from Bacillus, and the temperature was maintained at 55°C under a pressure of 100 bar for 24 hours of enzymatic decomposition reaction. Then, after the solution was recovered from the reactor, the pH of the solution was adjusted to 3, and the solution was placed in an ultra-high pressure enzyme reactor equipped with a polymer coated with pepsin, and the temperature was maintained at 40°C under a pressure of 100 bar for 6 hours of enzymatic decomposition reaction, and then the reaction was terminated by heating for 30 minutes to inactivate the excess enzyme. Then, the solution was recovered from the reactor and centrifuged to obtain a supernatant, and ethanol was added to the supernatant obtained above at a weight ratio of 1:1 and placed at low temperature, and the supernatant of the precipitate was removed by centrifugation. Then, the filtrate was obtained by passing it through a 0.2μm microfiltration membrane, and the obtained filtrate was freeze-dried to prepare sake lees low molecular peptide powder.
[0072] Experimental Example 1: Determination of the molecular weight of low molecular weight peptides derived from sake lees (Example 1)
[0073] In this experiment, an experiment was conducted to measure the molecular weight of the above-prepared Example 1. In order to measure the molecular weight, a matrix-assisted laser desorption-ionization time-of-flight mass spectrometer (MALDI-TOF MS) was used.
[0074] After Example 1 was dissolved in a 50% (v / v) methanol / water mixture, 1 μl was taken and mixed with 1 μl of sinapinic acid (SA) matrix (50% (v / v) acetonitrile containing 0.1% trifluoroacetic acid (TFA) / 50% (v / v) water). Then, the above mixture was spotted on a stainless steel MALDI plate and dried at room temperature. The analysis was performed using Microflex LRF MALDI-TOF mass analysis and Bruker UltrafleXtreme (BrukerDaltonics, Bremen, Germany), and the intensity of each ion was calculated by summing the peaks from the first isotope peak to the third isotope peak. Mass spectrum acquisition and processing were performed using FlexAnalysis software (ver.3.3, BrukerDaltonics, Bremen, Germany), and the results are shown as follows. Figure 1 shown.
[0075] Figure 1The results of confirming the molecular weight of the low molecular weight peptide (Example 1) derived from sake lees show that the molecular weight distribution of Example 1 is below 1000 Da, and more specifically, most of the molecular weight distribution is below 500 Da. This indicates that the prepared Example 1 is well hydrolyzed into low molecular weight.
[0076] Example 2: Preparation of anionic low molecular weight peptides from sake lees
[0077] (1) Preparation of anion exchange resin (affinity resin) for peptide separation
[0078] First, in order to separate anionic low-molecular peptides derived from sake lees, an affinity resin for separating anionic peptides was prepared as follows.
[0079] Agarose beads, which are biopolymers, were swollen in 10-fold 1N sodium chloride aqueous solution and filtered, and then the filtered agarose was stirred in 10-fold 3N sodium hydroxide aqueous solution for 30 minutes for activation. 1,3-propane sultone was added to the activated agarose and stirred at 60°C for 3 hours for reaction. Then, 10-fold distilled water was added and allowed to stand, and the washing step of removing the supernatant was performed 4 times, and the pH was adjusted to 7, and the above washing step was performed 2 more times before filtering.
[0080] (2) Isolation of anionic low-molecular peptides from sake lees
[0081] The anion exchange resin for peptide separation prepared in the above Example 2-(1) is activated in 10 times of 0.5M sodium hydroxide aqueous solution and loaded into each hollow column tube. Then, distilled water 10 times the volume of the above resin is flowed in for washing. The sake lees low molecular peptide powder prepared in the above Example 1 is dissolved in distilled water and a specified amount is loaded into the hollow column containing the anion exchange resin. Then, distilled water 10 times the volume of the resin is continuously flowed in at an appropriate rate to adsorb the low molecular peptides with anions. Then, 10 times of 2M sodium chloride aqueous solution is flowed in at an appropriate rate for elution and passed through a 0.2μm microfiltration membrane to obtain a filtrate. The filtrate obtained is freeze-dried to prepare sake lees anion low molecular peptide powder.
[0082] Example 3: Preparation of cationic low molecular weight peptides from sake lees
[0083] (1) Preparation of cation exchange resin (affinity resin) for peptide separation
[0084] First, in order to separate the cationic low-molecular peptides derived from sake lees, an affinity resin for separating cationic peptides was prepared as follows.
[0085] Agarose beads, which are biopolymers, were swelled in a 10-fold 1N sodium chloride aqueous solution and filtered, and then the filtered agarose was stirred in a 10-fold 5N sodium hydroxide aqueous solution for 30 minutes for activation. Glycidyltrimethylammonium chloride was added to the activated agarose and stirred at 55°C for 3 hours for reaction. Then, 10-fold distilled water was added and allowed to stand, and a washing step of removing the supernatant was performed 4 times, and after adjusting the pH to 7, the above washing step was performed 2 more times and then filtered.
[0086] (2) Isolation of cationic low-molecular peptides from sake lees
[0087] The peptide separation cation exchange resin prepared in the above Example 3-(1) is activated in 10 times of 0.5M sodium hydroxide aqueous solution and loaded into a hollow column tube. Then, distilled water 10 times the volume of the above resin is flowed in for washing. The sake lees low molecular peptide powder prepared in the above Example 1 is dissolved in distilled water and a specified amount is loaded into a hollow column filled with cation exchange resin. Then, distilled water 10 times the volume of the resin is continuously flowed in at an appropriate rate to adsorb the low molecular peptides with cations. Then, 10 times of 2M sodium chloride aqueous solution is flowed in at an appropriate rate for elution and passed through a 0.2μm microfiltration membrane to obtain a filtrate. The filtrate obtained is freeze-dried to prepare sake lees cationic low molecular peptide powder.
[0088] Example 4: Preparation of nonionic low-molecular peptides from sake lees
[0089] (1) Isolation of nonionic low-molecular peptides from sake lees
[0090] The anion exchange resin and cation exchange resin for peptide separation prepared in the above-mentioned Example 2-(1) and Example 3-(1) are activated in 10 times of 0.5M sodium hydroxide aqueous solution, respectively, and loaded into each hollow column tube. Then, distilled water 10 times the volume of the above-mentioned resin is flowed in for washing. The sake lees low molecular peptide powder prepared in the above-mentioned Example 1 is dissolved in distilled water and loaded into the hollow column equipped with anion exchange resin in a specified amount, and distilled water 10 times the volume of the resin is continuously flowed at an appropriate rate for elution. After the above-mentioned eluate is loaded into the hollow column equipped with cation exchange resin, distilled water 10 times the volume of the resin is continuously flowed at an appropriate rate for elution. The above-mentioned eluate is passed through a 0.2μm microfiltration membrane to obtain a filtrate, and the obtained filtrate is freeze-dried to prepare sake lees non-ionic low molecular peptide powder.
[0091] Experimental Example 2: Measurement of surface potential
[0092] In this experiment, the surface potential of Examples 2 to 4 was measured. To analyze the surface charge, the surface charge of each peptide was confirmed using a zeta potential analyzer, which is a measurement device for dynamic light scattering and electrophoretic light scattering, and the measurement was repeated three times to obtain the average value of the measured values.
[0093] Figure 2 The results of analyzing the surface potential of anionic low-molecular peptides (Example 2), cationic low-molecular peptides (Example 3), and nonionic low-molecular peptides (Example 4) separated from sake lees are shown. Figure 2 As shown, the zeta potential of Example 2 (anionic low-molecular peptide derived from sake lees) was measured to be -67.64 mV, the zeta potential of Example 3 (cationic low-molecular peptide derived from sake lees) was 47.89 mV, and the zeta potential of Example 4 (nonionic low-molecular peptide derived from sake lees) was 4.73 mV. This indicates that the sake lees low-molecular peptides from Example 1 were well separated according to different charges.
[0094] Examples 5 to 8: Preparation of a mixture using the low molecular weight peptides of Examples 2 to 4 obtained from sake lees
[0095] In this example, the anionic low-molecular peptides derived from sake lees (Example 2), the cationic low-molecular peptides derived from sake lees (Example 3), and the non-ionic low-molecular peptides derived from sake lees (Example 4) prepared in Examples 2 to 4 are mixed in different mixing ratios to prepare mixtures containing low-molecular peptides in Examples 5 to 8.
[0096] In order to confirm the whitening, moisturizing and wrinkle improvement effects of Examples 2 to 4, the anionic low-molecular peptides, cationic low-molecular peptides and nonionic low-molecular peptides separated from sake lees were mixed in different mixing ratios shown in Table 1 below to prepare mixtures of Examples 5 to 8. Table 1 below shows the mixing ratios (weight ratios) of the anionic low-molecular peptides derived from sake lees (Example 2), the cationic low-molecular peptides derived from sake lees (Example 3), and the nonionic low-molecular peptides derived from sake lees (Example 4).
[0097] Table 1
[0098] Weight Ratio Example 2 Example 3 Example 4 Example 5 1 1 0 Example 6 1 0 1 Example 7 0 1 1 Example 8 1 1 1
[0099] Comparative Example 1: Preparation of sake lees extract according to existing methods
[0100] In Comparative Example 1, a sake lees extract was prepared according to a downstream method. The existing sake lees extract refers to an extract prepared by a common method disclosed in existing literature or patents. The sake lees extract of Comparative Example 1 was prepared by the following method.
[0101] The dried sake lees were powdered using a grinder, extracted with a 95% (V / V) ethanol aqueous solution under reflux for 3 hours three times, and then cold-soaked. The powder was filtered and concentrated using Whatman #4 filter paper and freeze-dried to prepare sake lees extract powder.
[0102] Comparative Example 2: Preparation of fermented sake lees according to existing methods
[0103] In Comparative Example 2, a fermented sake lees product was prepared according to an existing method. The existing sake lees extract refers to an extract prepared by a common method disclosed in existing literature or patents, and the method authorized in Korean Patent No. 10-0736327 was used.
[0104] 5 g of sodium bicarbonate was added to 1 kg of sake lees and mixed, and then 2 L of distilled water was added to the mixture to make a slurry, and 1 g of yeast was added. After culturing in a 30°C incubator for 24 hours, the mixture was filtered using Whatman #4 filter paper, concentrated, and freeze-dried to prepare sake lees fermentation powder.
[0105] Experimental Example 3: Determination of melanin production inhibitory effect
[0106] In this experiment, experiments were conducted to confirm the melanin production inhibitory effects of the peptides derived from sake lees prepared in Examples 1 to 4, the mixtures containing the peptides derived from sake lees prepared in Examples 5 to 8 (Examples 2 to 4), and Comparison Example 1 (existing sake lees extract) and Comparison Example 2 (existing sake lees fermentation product).
[0107] 2.5×10 3 After the mouse melanin-forming (B16F10) cells were dispensed into a 96-well plate, the cells were stabilized for one day in a cell culture incubator at 37°C and 5% CO2. The samples were diluted to different concentrations and treated, and arbutin, which is known to be a whitening agent, was used as a positive control group. After the α-melanocyte stimulating hormone (MSH) as a stimulant was treated and reacted for 72 hours, the absorbance was measured at a wavelength of 405nm using a spectrophotometer (microplate reader).
[0108] The results of the experiments are summarized as shown in Table 2 below. When the sake lees-derived peptides prepared in Examples 1 to 4 of the present invention or the mixture containing sake lees-derived peptides (Examples 2 to 4) prepared in Examples 5 to 8 were treated, it was confirmed that the melanin production inhibitory effect was higher than that of Comparative Example 1 (existing sake lees extract) and Comparative Example 2 (existing sake lees fermentation product). In addition, it was confirmed that the melanin production inhibitory effect was higher when Example 2 (anionic low-molecular peptides derived from sake lees) and Example 3 (cationic low-molecular peptides derived from sake lees) separated by different charges were treated compared to the case of Example 1 (low-molecular peptides derived from sake lees), and it was confirmed that the highest pigment production inhibitory effect was exerted in the mixture of Example 8 containing Example 2 (anionic low-molecular peptides derived from sake lees), Example 3 (cationic low-molecular peptides derived from sake lees) and Example 4 (non-ionic low-molecular peptides derived from sake lees).
[0109] Table 2
[0110]
[0111] Experimental Example 4: Determination of the inhibitory effect of MMP-1 production
[0112] In this experiment, experiments were conducted to confirm the MMP-1 production inhibitory effects of the peptides derived from sake lees prepared in Examples 1 to 4, the mixtures containing the peptides derived from sake lees prepared in Examples 5 to 8 (Examples 2 to 4), and Comparison Example 1 (existing sake lees extract) and Comparison Example 2 (existing sake lees fermentation product).
[0113] 5×10 4 Human dermal fibroblasts (HDFn) were dispensed into 24-well plates and incubated in a cell culture incubator at 37°C and 5% CO2 for one day. 2 Treated with long-wave ultraviolet rays (UVA). Each sample was diluted to different concentrations while replacing the culture medium supplemented with 2% fetal bovine serum (FBS). 100 μM adenosine (Adenosine, which has been reported as a raw material with wrinkle functionality) was treated as a positive control group. After 48 hours of reaction, the MMP-1 expression level in the supernatant was confirmed using the R&D systems kit (DY901), and the particles were treated with MTT solution (Solution) to confirm cytotoxicity.
[0114] After MMP-1 ELISA analysis, the absorbance at a wavelength of 450 nm was measured to calculate the amount of MMP-1 produced, and then the MMP-1 expression inhibition rate (%) was calculated by comparison with the control group. The calculation formula is shown in the following mathematical formula 1.
[0115] Mathematical formula 1
[0116]
[0117] The results of the experiments are summarized as shown in Table 3 below. When the sake lees-derived peptides prepared in Examples 1 to 4 of the present invention or the mixture containing sake lees-derived peptides (Examples 2 to 4) prepared in Examples 5 to 8 were treated, it was confirmed that the MMP-1 production inhibitory activity was higher than that of Comparative Example 1 (existing sake lees extract) and Comparative Example 2 (existing sake lees fermentation product). In addition, it was confirmed that the MMP-1 production inhibitory activity was higher when Example 2 (anionic low-molecular peptides derived from sake lees) and Example 3 (cationic low-molecular peptides derived from sake lees) separated by different charges were treated than when Example 1 (low-molecular peptides derived from sake lees) was treated. The highest MMP-1 production inhibitory activity was confirmed in the mixture of Example 8 containing Example 2 (anionic low-molecular peptides derived from sake lees), Example 3 (cationic low-molecular peptides derived from sake lees) and Example 4 (non-ionic low-molecular peptides derived from sake lees).
[0118] Table 3
[0119] Sample name MMP-1 inhibition rate (%) Adenosine 42.31 Comparative Example 1 24.52 Comparative Example 2 27.38 Example 1 32.47 Example 2 35.14 Example 3 37.91 Example 4 33.46 Example 5 38.52 Example 6 41.88 Example 7 43.29 Example 8 47.83
[0120] Formulation Example 1: Preparation of a cosmetic composition formulation using a mixture containing a peptide derived from sake lees (Example 8)
[0121] In this formulation example, a cosmetic composition was prepared using Example 8 (a mixture containing a peptide derived from sake lees) with the composition shown in Table 4 below. On the other hand, Comparative Formulation Example 1 without Example 8 was prepared as a control group. Table 4 below shows the compositions of the cosmetic compositions of Formulation Example 1 and Comparative Formulation Example 1.
[0122] Table 4
[0123]
[0124] According to Table 4 above, cetyl ethylhexanoate, hydrogenated polydecene, shea butter, C14-22 alcohols and polyglyceryl-2 stearate were heated to 80°C to dissolve to prepare a transparent emulsified system. After adding the emulsified system to the aqueous phase in which trisodium ethylenediamine disuccinate, glycerin, propylene glycol and sodium polyacryloyldimethyl taurate were dispersed in pure water and heated to 70°C, the mixture was emulsified at 5000 rpm for 5 minutes using a homomixer. 1,2-Hexanediol and ethylhexylglycerol were added at a temperature of 45°C and stirred for 3 minutes and then cooled to 30°C. Then, Example 8 was added and stirred for 3 minutes and then degassed to prepare a cosmetic composition of Formulation Example 1 of a cream formulation. On the other hand, Comparative Formulation Example 1 (control group) was prepared in the same manner as Formulation Example 1 except that Example 8 was not added.
[0125] Experimental Example 5: Determination of the pigmentation inhibition effect using a reconstructed skin model
[0126] In the experiment, in order to confirm the pigmentation inhibition effect of the cosmetic composition prepared in the above-mentioned Formulation Example 1, an experiment to confirm the melanin production amount and the expression amount of tyrosinase-related protein 1 was carried out using a reconstructed skin model.
[0127] (1) Preparation of reconstructed skin tissue
[0128] After fibroblasts, type I collagen aqueous solution, 10×DMEM:F12 (3:1) medium, 10× buffer and hyaluronic acid were mixed well and placed inside the culture insert of a 12-well culture plate, they were reacted in a cell culture incubator at 5% CO2 and 37°C for 2 hours to solidify, and then fibroblast medium was placed inside and outside and cultured overnight. Then, the culture medium was changed for 5 days to prepare a dermis layer with fibroblasts. Keratinocyte-SFM medium containing bovine pituitary extract (BPE, Bovine Pituitary Extract), insulin, hydrocortisone, vitamin C, and epidermal growth factor (EGF, epidermal growth factor) was used to seed (seeding) pre-cultured keratinocytes (primary keratinocytes) and melanocytes on the upper part of the prepared dermis and cultured overnight. Then, after culturing for 5 days to induce the proliferation of epidermal cells, 2 mM Ca 2+ Then, all the medium was removed and a plate containing epidermal growth factor (EGF), 2 mM Ca 2+ Primary keratinocytes were cultured in air-exposed culture medium for 12 days to induce the formation of the epidermis, thereby preparing reconstructed skin tissue.
[0129] (2) Sample processing and culture
[0130] After adding α-melanocyte stimulating hormone (α-MSH) (100 nM) to the prepared reconstructed skin tissue (RST) to induce excessive melanin formation, 20 μl of Formulation Example 1 and Comparative Formulation Example 1 (control group) were treated once every 2 days and cultured for 5 days. Then, the tissue was fixed in a 10% neutral formalin solution for 24 hours, embedded in an OCT frozen section embedding medium (OCT Compound), cut at a thickness of 12 μm, attached to a gelatin-coated slide, and stored after drying for staining.
[0131] (3) Melanin staining and immunofluorescence staining
[0132] After staining using the Fontana-Masson kit (ab160669, abcam), the image results were analyzed and the black content was quantified as follows: Figure 3 As shown. The tissue sections prepared on the slide were placed in distilled water and treated with ammoniacal silver solution, and then left to stand in a slide warmer at 60°C for 30 minutes. After washing with distilled water again, they were treated with 0.1% gold chloride for 1 minute and washed with distilled water, and then treated in 5% sodium thiosulfate (Hypo) for 2 minutes. Then, they were washed with distilled water and treated with Nuclear-fast red for 5 minutes, and finally, they were gently washed with distilled water, dehydrated and cleared, sealed in Canada balsam, and observed under a microscope.
[0133] The following immunofluorescence staining method was used to analyze tyrosinase-related protein 1, a protein associated with melanin formation. Figure 4As shown. After the tissue sections prepared on the slides were hydrated in distilled water, they were placed in a 0.01M citric acid (Citrate) solution (pH 6.0) and boiled in a microwave for 15 minutes, and then allowed to stand at room temperature for 20 minutes. Blocked with anti-goat serum and reacted with primary antibody tyrosinase-related protein 1 at 4°C overnight. Then, after washing with phosphate buffer solution (PBS), reacted with goat anti-mouse IgG Alexa Fluor 488 as a secondary antibody at room temperature for 1 hour, washed with phosphate buffer solution, sealed with mounting medium containing DaPI and observed under a fluorescence microscope. The positive area of staining in the photos taken at a magnification of 200 times using a fluorescence microscope was digitized using Image J program to calculate the rate of change, and the calculation formula is shown in the following mathematical formula 2.
[0134] Mathematical formula 2
[0135]
[0136]
[0137] (4) Analysis of melanin content
[0138] Melanin staining is black, and the bar graph of the analyzed content is as follows Figure 3 As shown. Figure 3 As shown in the bar graph, it can be confirmed that in the case of the formulation example 1 of the treatment example 8, the melanin content is significantly reduced compared with the comparative formulation example 1 which is the untreated group.
[0139] (5) Analysis of tyrosinase-related protein 1 content
[0140] Tyrosinase-related protein 1 is an enzyme protein related to melanin formation, which is stained dark brown in the epidermis. The experimental results are as follows Figure 4 As shown. Figure 4 As shown in the bar graph, it can be confirmed that when the formulation example 1 of Example 8 was treated, the expression level of tyrosinase-related protein 1 was significantly reduced compared with the comparative formulation example 1 which was the untreated group.
[0141] Experimental Example 6: Determination of wrinkle improvement and moisturizing effects using a reconstructed skin model
[0142] In this experiment, in order to confirm the wrinkle improvement and moisturizing effects of the cosmetic composition prepared in Formulation Example 1, an experiment to confirm the expression levels of type I collagen, laminin-5, and filaggrin was conducted using a reconstructed skin model.
[0143] (1) Preparation of reconstructed skin tissue
[0144] The reconstructed skin tissue was prepared in the same manner as in Experimental Example 5-(1) above.
[0145] (2) Sample processing and staining
[0146] The prepared reconstructed skin tissue was treated with 20 μl of Formulation Example 1 and Comparative Formulation Example 1 (control group) at a frequency of once every two days and cultured for 5 days. Then, the tissue was fixed in a 10% neutral formalin solution for 24 hours, embedded in an OCT frozen section embedding medium, cut at a thickness of 12 μm, attached to a gelatin-coated slide, and stored after drying for staining.
[0147] For morphological observation, Masson's trichrome kit (MST-100T, BIOGNOST) was used for staining. The tissue sections prepared on the slide were placed in distilled water for hydration treatment, and then treated with bouin's solution at 56°C for 1 hour, cooled at room temperature for 20 minutes, and washed with distilled water. The cell nuclei were stained with Weigert's iron hematoxylin for 20 minutes and then washed, and Biebroch scarlet-acid fuchsin solution was used for staining for 10 minutes and then washed with distilled water. Phosphomolybdic acid-orange G solution was used for mordanting for 5 minutes and decolorization, and aniline blue staining solution was used to stain collagen fibers. After 0.1% acetic acid was used to fade the unbound light blue, the cells were dehydrated and cleared, and sealed in Canada gum for observation under a microscope.
[0148] Type I collagen, laminin-5, and fillagrin, proteins associated with wrinkle improvement and moisturizing effects, were analyzed by the following fluorescent staining method. The tissue sections prepared on the slide were hydrated in distilled water, then placed in a 0.01 M citrate solution (pH 6.0) and boiled in a microwave for 15 minutes, and then left to stand at room temperature for 20 minutes. Anti-goat serum was used for blocking and reacted with fillagrin (1:100, Santa Cruz), laminin-5 (1:500, abcam), and type I collagen (1:500, invitrogen) at 4°C overnight. Then, the sections were washed with phosphate buffered saline (PBS), reacted with goat anti-mouse IgG Alexa Fluor 488 as a secondary antibody for 1 hour at room temperature, washed with phosphate buffered saline, sealed with a DAPI-containing sealing solution, and observed under a fluorescence microscope. The positive staining area in the photograph taken at a magnification of 200 times using a fluorescence microscope was quantified using the Image J program to calculate the change rate. The calculation formula is shown in the above-mentioned Mathematical Formula 2.
[0149] (3) Analysis of type I collagen
[0150] Type I collagen is a protein that occupies 70% of the dermis and plays an important role in preventing skin wrinkles. Increased expression of type I collagen indicates that it is possible to prevent skin wrinkle formation. Figure 5 The results of measuring the effect of increasing the expression of type I collagen are shown. The blue light indicates the stained cell nucleus, and type I collagen is stained as grass green fluorescence in the dermis. Figure 5 As shown in the bar graph, it can be confirmed that in the case of the treatment of Formulation Example 1 of Example 8, the expression level of type I collagen was significantly increased compared with the comparative formulation example 1 which is the untreated group.
[0151] (4) Analysis of laminin-5
[0152] Laminin-5 is a protein that constitutes the dermal-epidermal junction (DEJ) responsible for the connection between the epidermis and the dermis. Its expression decreases with the progress of aging, causing skin damage and hindering nutrient supply. Therefore, increased expression of laminin-5 indicates that skin aging can be prevented. Figure 6 The results of measuring the effect of increasing the expression of laminin-5 are shown. The blue light indicates the stained nucleus, and laminin-5 is stained as a linear morphology with grass green fluorescence at the epidermal-dermal junction. Figure 6As shown in the bar graph, it was confirmed that when the formulation example 1 of Example 8 was treated, the expression level of laminin-5 was significantly increased compared with the comparative formulation example 1 which was an untreated group.
[0153] (5) Analysis of filaggrin
[0154] Filaggrin is a protein that plays an important role in the formation of the stratum corneum in the epidermis. Increased expression of filaggrin indicates the formation of a normal skin barrier to prevent the evaporation of skin water. Figure 7 The results of measuring the effect of increasing filaggrin expression are shown. Blue light indicates stained cell nuclei, and filaggrin is stained as grass green fluorescence in the stratum corneum. Figure 7 As shown in the bar graph, it can be confirmed that in the case of the treatment of Formulation Example 1 of Example 8, the expression level of filaggrin was significantly increased compared with the comparative formulation example 1 which was the untreated group.
[0155] As described above, the present invention has confirmed that the low-molecular peptide isolated from sake lees and the cosmetic composition containing the same are effective in whitening, moisturizing and wrinkle improvement. That is, compared with the existing sake lees extract or sake lees fermentation product, the low-molecular peptide derived from sake lees of the present invention has effective whitening, moisturizing and wrinkle improvement effects. The present invention contains the low-molecular peptide isolated from sake lees, which not only has the pigmentation inhibition effect caused by the reduction of the expression of tyrosinase-related protein 1 and the wrinkle improvement effect caused by the increase of the expression of type I collagen and laminin-5, but also has the moisturizing effect caused by the increase of the expression of filaggrin, so that a cosmetic composition effective in inhibiting skin aging can be prepared.
Claims
1. A cosmetic composition, characterized in that It contains, as an active ingredient, a low-molecular-weight peptide of 1,000 Da or less separated by treating sake lees with a proteolytic enzyme.
2. A cosmetic composition, characterized in that The invention comprises anionic low-molecular peptides of 1000 Da or less separated by treating sake lees with a proteolytic enzyme, cationic low-molecular peptides of 1000 Da or less separated by treating sake lees with a proteolytic enzyme, and nonionic low-molecular peptides of 1000 Da or less separated by treating sake lees with a proteolytic enzyme.
3. The cosmetic composition according to claim 1, characterized in that The above-mentioned low molecular weight peptide is obtained by a process comprising the following steps: Step (a), soaking the wine lees in pure water; Step (b), after the soaking in the above step (a), adding subtilisin derived from Bacillus to induce a primary enzyme reaction; Step (c), adding pepsin to induce a secondary enzyme reaction after the primary enzyme reaction in the above step (b); Step (d), after the secondary enzyme reaction in step (c), recovering the supernatant by centrifugation; and Step (e), adding ethanol to the supernatant recovered in the above step (d) to induce a precipitation reaction, and then recovering the supernatant by centrifugation.
4. The cosmetic composition according to claim 2, characterized in that The above-mentioned anionic low-molecular peptide is obtained by a process comprising the following steps: Step (a), soaking the wine lees in pure water; Step (b), after the soaking in the above step (a), adding subtilisin derived from Bacillus to induce a primary enzyme reaction; Step (c), adding pepsin to induce a secondary enzyme reaction after the primary enzyme reaction in the above step (b); Step (d), after the secondary enzyme reaction in step (c), recovering the supernatant by centrifugation; Step (e), adding ethanol to the supernatant recovered in the above step (d) to induce a precipitation reaction, and recovering the supernatant by centrifugation; as well as Step (f) is to pass the supernatant recovered in the above step (e) through an anion exchange resin to obtain anionic peptides.
5. The cosmetic composition according to claim 2, characterized in that: The above cationic low molecular weight peptide is obtained by a process comprising the following steps: Step (a), soaking the wine lees in pure water; Step (b), after the soaking in the above step (a), adding subtilisin derived from Bacillus to induce a primary enzyme reaction; Step (c), adding pepsin to induce a secondary enzyme reaction after the primary enzyme reaction in the above step (b); Step (d), after the secondary enzyme reaction in step (c), recovering the supernatant by centrifugation; Step (e), adding ethanol to the supernatant recovered in the above step (d) to induce a precipitation reaction, and recovering the supernatant by centrifugation; as well as In step (f'), the supernatant recovered in the above step (e) is passed through a cation exchange resin to obtain a cationic peptide.
6. The cosmetic composition according to claim 2, characterized in that The above nonionic low molecular weight peptide is obtained by a process comprising the following steps: Step (a), soaking the wine lees in pure water; Step (b), after the soaking in the above step (a), adding subtilisin derived from Bacillus to induce a primary enzyme reaction; Step (c), adding pepsin to induce a secondary enzyme reaction after the primary enzyme reaction in the above step (b); Step (d), after the secondary enzyme reaction in step (c), recovering the supernatant by centrifugation; Step (e), adding ethanol to the supernatant recovered in the above step (d) to induce a precipitation reaction, and recovering the supernatant by centrifugation; Step (f1), passing the supernatant recovered in the above step (e) through an anion exchange resin to obtain a primary eluate; Step (f2), passing the eluate obtained in the above step (f1) through a cation exchange resin to obtain a secondary eluate; as well as Step (g), filtering the secondary eluate obtained in the above step (f2) to obtain a filtrate.
7. The cosmetic composition according to claim 1 or 2, characterized in that: The above cosmetic composition is used for skin whitening.
8. The cosmetic composition according to claim 1 or 2, characterized in that: The above cosmetic composition is used for improving skin wrinkles.
9. The cosmetic composition according to claim 1 or 2, characterized in that: The above cosmetic composition is used for moisturizing the skin.