A cosmetic composition for soothing irritation and improving moisturizing properties, containing an enzyme-treated and low-temperature aged low-molecular-weight hyaluronic acid hydrolysate as an active ingredient, and a method for producing the hydrolysate thereof.

Abstract

To provide a cosmetic composition for alleviating irritation and improving moisturization, containing enzymatically processed and low-temperature aged low-molecular-weight hyaluronic acid hydrolysate as an active ingredient, and a method for manufacturing hydrolysate thereof.SOLUTION: The present invention includes: a step (Step 1), which comprises mixing hyaluronic acid and mixed enzymes comprising proteinase and pectinase in distilled water, and conducting an enzymatic reaction to obtain mixed-enzyme-treated hyaluronic acid; and a step (Step 2), which comprises subjecting the mixed-enzyme-treated hyaluronic acid obtained in Step 1 to steam treatment, and subsequently to low-temperature aging treatment at 5°C to 20°C for 1 day to 5 days, wherein the low-molecular-weight hyaluronic acid prepared by the method of the present invention achieves a weight-average molecular weight (Mw) of 500 Da or less, resulting in significantly improved skin absorption rate and providing significantly superior effects in alleviating skin irritation and providing moisturization.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a cosmetic composition for alleviating irritation and improving moisture retention, which contains an enzymatically treated and low-temperature aged low molecular weight hyaluronic acid hydrolyzate as an active ingredient, and a method for producing the hydrolyzate. More specifically, when producing hyaluronic acid as an enzymatically treated and low-temperature aged hydrolyzate, a low molecular weight hyaluronic acid hydrolyzate with a weight average molecular weight (Mw) of 500 Da or less is obtained, and its histamine inhibitory ability, NO production inhibitory ability, and the effect of improving moisture retention are confirmed.

Background Art

[0002] Hyaluronic acid is one of the glycosaminoglycans, which is known to exist in the extracellular matrix, participate in maintaining the moisture of tissues, storing and diffusing cell growth factors and nutritional components, and being synthesized by keratinocytes and fibroblasts. A decrease in hyaluronic acid in the skin is known to cause a lack of firmness and an increase in wrinkles in the skin.

[0003] Most hyaluronic acid produced by conventional methods is a high molecular polysaccharide with a high degree of polymerization of 500,000 daltons (500,000 Da = 500 kDa) or more, so it cannot pass through the skin. That is, if hyaluronic acid of 500,000 daltons or more is applied to the skin, due to its property of trying to absorb moisture in the air, it can prevent the evaporation of moisture from the skin, but it cannot penetrate the skin, so it stays on the surface of the skin and is easily washed away from the surface of the skin, making it difficult to sustain the moisturizing effect on the skin. For this reason, various attempts to reduce the molecular weight of hyaluronic acid have been reported.

[0004] As a prior art document for reducing the molecular weight of hyaluronic acid, Patent Document 1 relates to a method for producing a hyaluronic acid oligomer containing ultra-low molecular weight hyaluronic acid, and the production method discloses a method for producing low molecular weight hyaluronic acid by causing a deacetylation reaction. The composition is reported to have an effect of alleviating and improving wrinkles by promoting the activity of fibroblasts and the synthesis of collagen.

[0005] Furthermore, Patent Document 2 relates to a method for producing low-molecular-weight hyaluronic acid by gamma irradiation and treatment with a degrading enzyme, providing a method for producing low-molecular-weight hyaluronic acid hydrolysate by treating recombinant protein enzyme with hyaluronic acid and then irradiating it with gamma rays. However, this method utilizes chemicals or recombinant proteins that are difficult to purify, making it somewhat difficult to apply industrially.

[0006] In addition, the inventors diligently researched how to maximize the performance of hyaluronic acid and, as a result, discovered that when hyaluronic acid is produced as a hydrolysate by simultaneously enzymatic treatment and low-temperature maturation, it is possible to reduce the molecular weight of the hyaluronic acid and provide excellent irritation-relieving and moisturizing effects, thus completing the present invention. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Korean Registered Patent Publication No. 10-2548168 [Patent Document 2] Korean Registered Patent Publication No. 10-2013603 [Overview of the project] [Problems that the invention aims to solve]

[0008] The first object of the present invention is to provide a method for producing low-molecular-weight hyaluronic acid.

[0009] A second object of the present invention is to provide low-molecular-weight hyaluronic acid with a weight-average molecular weight (Mw) of 500 Da or less, produced by the above-described manufacturing method.

[0010] A third object of the present invention is to provide a cosmetic composition for soothing and moisturizing skin irritation, which contains low-molecular-weight hyaluronic acid produced by the above-described manufacturing method.

[0011] A fourth object of the present invention is to provide a cosmetic composition for mitigating skin irritation and moisturizing, which contains low-molecular-weight hyaluronic acid with a weight-average molecular weight (Mw) of 500 Da or less. [Means for solving the problem]

[0012] To achieve the above objective, the present invention comprises the steps of: (Step 1) mixing hyaluronic acid with distilled water and proteinase and pectinase as mixed enzymes to perform an enzymatic reaction to obtain hyaluronic acid treated with mixed enzymes; The present invention provides a method for producing low-molecular-weight hyaluronic acid, comprising the steps of: steaming the hyaluronic acid treated with mixed enzymes in step 1, followed by low-temperature aging at 5 to 20°C for 1 to 5 days (step 2).

[0013] In the manufacturing method according to the present invention, step 1 is a step in which a hyaluronic acid raw material with a high weight-average molecular weight (Mw) is hydrolyzed by mixed enzyme treatment. The weight-average molecular weight (Mw) of commercially available hyaluronic acid raw materials is approximately at the level of 1 million to 2 million Da.

[0014] The proteinase in step 1 can be one or more of the following: Flavorzyme, Protamex, α-chymotrypsin, subtilisin Carlsberg, thermolysin, papain, fungal protease, pepsin, etc.

[0015] The pectinase used in step 1 can be Pectinex Ultra SP-L, Viscozyme L, Ultrazyme AFP, Pectinex Ultra AFP, Pectinex Ultra clear, α-herbzyme, etc., either individually or in combination of two or more.

[0016] Preferably, the mixed enzyme can use 1 to 5 parts by weight of proteinase based on 1 part by weight of pectinase, preferably 2 to 4 parts by weight of proteinase based on 1 part by weight of pectinase, and more preferably 3 parts by weight of proteinase based on 1 part by weight of pectinase.

[0017] If the weight percentage of the mixed enzyme deviates from the stated percentage, a problem may arise in which the hydrolytic efficacy against hyaluronic acid decreases. Specifically, it may not be possible to achieve a weight-average molecular weight (Mw) of hyaluronic acid of 500 Da or less.

[0018] Furthermore, in step 1, 2 to 6 parts by weight of the mixed enzyme can be used based on 100 parts by weight of hyaluronic acid, preferably 3 to 5 parts by weight, and more preferably 3.5 to 4.5 parts by weight.

[0019] In the manufacturing method according to the present invention, step 2 is a step in which the hydrolyzed hyaluronic acid is subjected to low-temperature aging treatment to further reduce its molecular weight.

[0020] Preferably, the steaming process in step 2 can be carried out at 70-90°C for 2-4 hours, and the low-temperature aging can be carried out at 14-18°C for 2-4 days.

[0021] The weight average molecular weight (Mw) of the low-molecular-weight hyaluronic acid produced by the production method according to the present invention is characterized by being 500 Da or less.

[0022] The present invention also provides a low-molecular-weight hyaluronic acid having a weight average molecular weight (Mw) of 500 Da or less produced by the above production method.

[0023] The low-molecular-weight hyaluronic acid having a weight average molecular weight (Mw) of 500 Da or less has a significantly improved skin absorption rate and remarkable effects on alleviating skin irritation and moisturizing efficacy.

[0024] Furthermore, the present invention provides a cosmetic composition for alleviating skin irritation and moisturizing containing the low-molecular-weight hyaluronic acid produced by the above production method.

[0025] The present invention also provides a cosmetic composition for alleviating skin irritation and moisturizing containing a low-molecular-weight hyaluronic acid having a weight average molecular weight (Mw) of 5 yet another.

Effects of the Invention

[0026] The low-molecular-weight hyaluronic acid produced by the production method according to the present invention achieves a weight average molecular weight (Mw) of 500 Da or less, thereby significantly improving the skin absorption rate and having remarkable effects on alleviating skin irritation and moisturizing efficacy.

Brief Description of the Drawings

[0027] [Figure 1] Figure 1 is a graph showing the results of cytotoxicity experiments of hyaluronic acid samples produced in Example 1 and Comparative Examples 1 to 4. [Figure 2] Figure 2 is a graph showing the results of histamine inhibitory activity of hyaluronic acid samples produced in Example 1 and Comparative Examples 1 to 4. [Figure 3] Figure 3 is a graph showing the results of the ability to inhibit NO (Nitric oxide) production of hyaluronic acid samples produced in Example 1 and Comparative Examples 1 to 4. [Figure 4]Figure 4 is a graph showing the percentage increase in skin moisture content after treatment with the hyaluronic acid sample prepared in Example 1 and the control sample (Comparative Example 1). [Figure 5] Figure 5 is a spectral image showing the molecular weight of the hyaluronic acid hydrolysate from Example 1. [Figure 6] Figure 6 is a table summarizing the peak information in the spectrum in which the molecular weight of the hyaluronic acid hydrolysate of Example 1 was measured. Gray cells indicate matrix peak molecular weights. [Modes for carrying out the invention]

[0028] The present invention will be described in detail below.

[0029] [Cosmetic composition]

[0030] The present invention provides a cosmetic composition containing an active ingredient.

[0031] The cosmetic composition may be in the form of, for example, a solution, gel, solid or kneaded anhydrous product, emulsion obtained by dispersing an oil phase in an aqueous phase, suspension, microemulsion, microcapsule, microgranulocyte or ionic (liposome) or nonionic vesicle dispersants. More specifically, it can be provided in the form of a lotion, emulsion, cream, skin, serum, essence, emulsion, powder, cosmetic ointment, spray, gel, face pack, cleanser, soap, shampoo, conditioner, bath additive, cleanser or concealer stick. It may also be manufactured in the form of a foam or an aerosol composition further comprising a compressed propellant.

[0032] Furthermore, the cosmetic composition may further contain, in addition to the active substance of the present invention, adjuvants commonly used in the cosmetic field, such as fatty substances, organic solvents, solvents, concentrates and gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion sequestering agents and chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic surfactants, lipid vesicles, or any other ingredients commonly used in cosmetics.

[0033] In a cosmetic composition containing the active substance of the present invention, the active substance of the present invention can be added to the cosmetic composition in an amount of 0.1 to 50% by weight, preferably 1 to 10% by weight.

[0034] When the active substance of the present invention is used as a topical skin preparation, it may further contain adjuvants commonly used in dermatology, such as fatty substances, organic solvents, solvents, concentrates and gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion sequestering agents and chelating agents, preservatives, vitamins, blocking agents, humectants, essential oils, dyes, pigments, hydrophilic or lipophilic surfactants, lipid vesicles, or any other components commonly used in topical skin preparations. Furthermore, these components may be introduced in amounts commonly used in dermatology.

[0035] The present invention will be described in more detail below with reference to the following examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0036] <Example 1> Production of mixed enzymes and hyaluronic acid hydrolysate treated with low-temperature aging (mixed enzymes: proteinase and pectinase 3:1% by weight)

[0037] Stage 1: Mixed enzyme treatment stage

[0038] 100g of hyaluronic acid powder (200 mesh) was mixed with 3g of Flavorzyme (Novozyme) as a proteinase and 1g of Pectinex Ultra SP-L (Novozyme) as a pectinase, and 300mL of water was added and the mixture was reacted at 50°C for 120 minutes.

[0039] Stage 2: Low-temperature aging process

[0040] The mixed enzyme-treated hyaluronic acid prepared in step 1 was subjected to primary steaming at 80°C for 3 hours, followed by low-temperature aging at 16°C for 3 days.

[0041] The hyaluronic acid hydrolysate, which had been treated with the mixed enzyme and low-temperature aging in parallel, was extracted by stirring with 3 L of purified water at 80°C for 120 minutes, then concentrated under reduced pressure and freeze-dried to obtain the hyaluronic acid hydrolysate treated with the mixed enzyme and low-temperature aging.

[0042] <Example 2> Production of mixed enzymes and hyaluronic acid hydrolysate treated with low-temperature aging (mixed enzymes: proteinase and pectinase 2:1% by weight)

[0043] Hyaluronic acid hydrolysate was obtained in the same manner as in Example 1, except that in step 1 of Example 1, proteinase and pectinase were used in a ratio of 2:1% by weight in a total weight of 4 g of mixed enzymes.

[0044] <Example 3> Production of mixed enzymes and hyaluronic acid hydrolysate treated with low-temperature aging (mixed enzymes: proteinase and pectinase 4:1% by weight)

[0045] Hyaluronic acid hydrolysate was obtained in the same manner as in Example 1, except that in step 1 of Example 1, proteinase and pectinase were used in a ratio of 4:1% by weight in a total weight of 4 g of mixed enzymes.

[0046] <Example 4> Production of mixed enzymes and hyaluronic acid hydrolysate treated with low-temperature aging (mixed enzymes: proteinase and pectinase 1:1 by weight)

[0047] Hyaluronic acid hydrolysate was obtained in the same manner as in Example 1, except that in step 1 of Example 1, proteinase and pectinase were used in a 1:1% by weight ratio in a total weight of 4 g of mixed enzymes.

[0048] <Example 5> Production of mixed enzymes and hyaluronic acid hydrolysate treated with low-temperature aging (mixed enzymes: proteinase and pectinase 5:1% by weight)

[0049] Hyaluronic acid hydrolysate was obtained in the same manner as in Example 1, except that in step 1 of Example 1, proteinase and pectinase were used in a ratio of 5:1% by weight in a total weight of 4 g of mixed enzymes.

[0050] <Comparative Example 1> Preparation of hyaluronic acid (groups without mixed enzyme treatment and without low-temperature maturation treatment)

[0051] Without performing steps 1 and 2 of Example 1, 100 g of hyaluronic acid powder (200 mesh) was mixed with 3 L of purified water, stirred and extracted at 80°C for 120 minutes, and then concentrated under reduced pressure and freeze-dried to obtain hyaluronic acid.

[0052] Specifically, 100g of hyaluronic acid powder (200 mesh) was mixed with 3L of purified water and extracted at 80°C for 120 minutes. The mixture was then concentrated under reduced pressure and freeze-dried to obtain hyaluronic acid.

[0053] <Comparative Example 2> Production of hyaluronic acid hydrolysate treated with mixed enzymes (untreated, low-temperature aging group)

[0054] Except for not performing step 2 in Example 1, the procedure was carried out in the same manner as in Example 1 to produce a mixed enzyme-treated hyaluronic acid hydrolysate.

[0055] Specifically, 100 g of hyaluronic acid powder (200 mesh) was mixed with 3 g of proteinase and 1 g of pectinase, and 300 mL of water was added and the mixture was reacted at 50°C for 120 minutes. The hyaluronic acid treated with the mixed enzymes was then extracted by stirring with 3 L of purified water at 80°C for 120 minutes, followed by vacuum concentration and freeze-drying to obtain a hydrolyzed mixed enzyme-treated hyaluronic acid product.

[0056] <Comparative Example 3> Production of Hyaluronic Acid Hydrolysate Treated by Single Enzyme (Single Enzyme Treatment and Untreated Group with Low Temperature Aging)

[0057] Hyaluronic acid hydrolysate treated with a single enzyme was produced by following the same procedure as in Example 1, except that 1 g of proteinase was used instead of the mixed enzyme in step 1 of Example 1, and step 2 was not performed.

[0058] Specifically, 100 g of hyaluronic acid powder (200 mesh) was treated with 1 g of proteinase, and 300 mL of water was added and the mixture was reacted at 50°C for 120 minutes. 3 L of purified water was added to the single-enzyme-treated hyaluronic acid, and the mixture was stirred and extracted at 80°C for 120 minutes. The mixture was then concentrated under reduced pressure and freeze-dried to obtain a single-enzyme-treated hyaluronic acid hydrolysate.

[0059] <Comparative Example 4> Production of low-temperature aged hyaluronic acid hydrolysate (untreated with mixed enzymes)

[0060] Except for not performing step 1 of Example 1, the procedure was carried out in the same manner as in Example 1 to produce a hyaluronic acid hydrolysate that had undergone low-temperature aging treatment.

[0061] Specifically, 100g of hyaluronic acid powder (200 mesh) was subjected to primary steaming at 80°C for 3 hours and then aged at 16°C for 3 days. The hyaluronic acid hydrolysate that had undergone low-temperature aging treatment was then extracted by stirring with 3L of purified water at 80°C for 120 minutes, followed by vacuum concentration and freeze-drying to obtain the hyaluronic acid hydrolysate that had undergone low-temperature aging treatment.

[0062] <Comparative Example 5> Production of hyaluronic acid hydrolysate treated with proteinase alone (single enzyme treatment and low-temperature aging treatment group)

[0063] Hyaluronic acid hydrolysate was obtained in the same manner as in Example 1, except that 4 g of proteinase was used alone instead of the mixed enzyme used in Example 1.

[0064] <Comparative Example 6> Production of hyaluronic acid hydrolysate treated with pectinase alone (single enzyme treatment and low-temperature aging treatment group)

[0065] Hyaluronic acid hydrolysate was obtained in the same manner as in Example 1, except that 4 g of pectinase was used alone instead of the mixed enzyme used in Example 1.

[0066] <Comparative Example 7> Production of mixed enzymes and hyaluronic acid hydrolysate treated with low-temperature aging (mixed enzymes: proteinase and pectinase 0.8:1% by weight)

[0067] Hyaluronic acid hydrolysate was obtained in the same manner as in Example 1, except that in step 1 of Example 1, proteinase and pectinase were used in a ratio of 0.8:1% by weight in a total weight of 4 g of mixed enzymes.

[0068] <Comparative Example 8> Production of mixed enzymes and hyaluronic acid hydrolysate treated with low-temperature aging (mixed enzymes: proteinase and pectinase 5.2:1% by weight)

[0069] Hyaluronic acid hydrolysate was obtained in the same manner as in Example 1, except that in step 1 of Example 1, proteinase and pectinase were used in a ratio of 5.2:1% by weight in a total weight of 4 g of mixed enzymes.

[0070] <Experimental Example 1> Cytotoxicity Test: WST Assay

[0071] To confirm whether or not the hyaluronic acid samples prepared in Example 1 and Comparative Examples 1-4 were irritating to cells, cytotoxicity experiments were conducted on macrophages (RAW264.7).

[0072] RAW264.7 cells, a type of macrophage, were placed in 24-well plates at a rate of 1.0 × 10⁶ cells using DMEM medium containing 1% penicillin / streptomycin and 10% FBS (fetal bovine serum). 4 After counting and dispensing cells / wells uniformly, the cells were cultured for 24 hours under conditions of 37°C and 5% CO2. After 24 hours of culture, the hyaluronic acid samples prepared in Example 1 and Comparative Examples 1-4 were mixed with culture medium at their respective concentrations (0%, 0.5%, 1%, 2%, and 4%), and 1 mL of each mixture was added to each well. The mixture was then incubated in an incubator at 37°C and 5% CO2 for 24 hours. Subsequently, only the supernatant from each well was collected separately, and WST-1 assay solution (ez-cytox) was added to each well. The mixture was incubated in an incubator for 2 hours, and the absorbance was measured at 450 nm using an ELISA reader.

[0073] Cell viability was calculated using the following formula 1, compared to a treatment group (untreated group) whose sample was not treated.

[0074] [Formula 1] Cell viability (%) = (Absorbance of the treated sample group / Absorbance of the untreated sample group) × 100

[0075] Figure 1 is a graph showing the results of cytotoxicity experiments on hyaluronic acid samples produced in Example 1 and Comparative Examples 1-4.

[0076] As shown in Figure 1, none of the hyaluronic acid samples produced in Example 1 and Comparative Examples 1-4 showed cytotoxicity, confirming that they can be used as safe materials in cosmetics.

[0077] <Experimental Example 2> Stimulus Relaxation Test: Histamine Inhibitory Activity

[0078] β-hexosaminidase is a substance that constitutes the granules of mast cells, and histamine secretion due to mast cell degranulation is proportional to the amount of β-hexosaminidase released. Therefore, the degranulation and histamine secretion inhibitory effect of the ginseng saponin fraction was confirmed by measuring the amount of β-hexosaminidase released in RBL-2H3 rat mast cell lines. After suspending RBL-2H3 cells in DMEM containing 10% FBS, 2 × 10⁶ cells were placed in each well of a 24-well plate. 5 After dispensing individual cells, each well was sensitized with 0.5 μg / m³ of LDNP-IgE and cultured overnight in a 5% CO2 incubator. Subsequently, the cells in each well were washed twice with Silaganian buffer (119 mM NaCl, 5 mM KCl, 5.6 mM glucose, 0.4 mM MgCl2, 25 mM HEPES, 40 mM NaOH, 1 mM CaCl2, 0.1% BSA, pH 7.2), followed by a total reaction with Silaganian buffer at 37°C for 10 minutes. After adding the test substance, the cells were reacted again for 10 minutes. Subsequently, the cells were treated with the antigen (DNP-BSA, 10 μg / ml) at 37°C for 30 minutes to degranulate them, then left on ice for 10 minutes to terminate the reaction. 20 μL of the supernatant was taken and transferred to a 96-well plate, and 1 mM p-nitrophenyl-N-acetyl-β-D-glucosaminid was added and the cells were cultured for 1 hour. After adding a stop solution (0.1 M Na2CO3 / NaHCO3), the absorbance was measured at 405 nm by ELISA and quantified.

[0079] Figure 2 is a graph showing the results of the histamine inhibitory activity of the hyaluronic acid samples prepared in Example 1 and Comparative Examples 1-4.

[0080] As shown in Figure 2, the hyaluronic acid sample prepared in Comparative Example 1 showed significantly superior histamine inhibitory activity compared to Comparative Examples 1-4, and it was found that the histamine inhibitory activity improved in a concentration-dependent manner up to a treatment concentration of 2.0%.

[0081] <Experimental Example 3> Flame-retardant efficacy test: NO (Nitric oxide) production inhibitory activity

[0082] To measure the anti-inflammatory activity of the hyaluronic acid samples prepared in Example 1 and Comparative Examples 1-4, experiments were conducted to measure the concentration of NO produced by the inflammation-inducing response.

[0083] RAW264.7 cells, a type of macrophage, were placed in 24-well plates at a rate of 1.0 × 10⁶ cells using DMEM medium containing 1% penicillin / streptomycin and 10% FBS (fetal bovine serum). 4 After counting and dispensing cells / wells uniformly, the cells were cultured for 24 hours under conditions of 37°C and 5% CO2. After 24 hours of culture, the hyaluronic acid samples prepared in Example 1 and Comparative Examples 1-4 were mixed with the culture medium at a concentration of 50 μg / mL, and 1 mL was added to each well. The cells were then incubated in an incubator at 37°C and 5% CO2 for 24 hours. At this time, LPS (Lipo polysaccharide), an inflammatory pro-inflammatory factor that expresses NO, was added at a concentration of 1 μg / mL and the cells were incubated at 37°C and 5% CO2 for 24 hours. Subsequently, the supernatant from each well was collected separately. Then, 100 mL of the culture medium was added to each well using an NO detection kit and placed in a 96-well plate. 50 μL of Griess reagent A (N-1-naphthylethylenediamine (NEDHC)) and 50 μL of Griess reagent B (sulfanilamide) were added to each well, and after reacting for 10 minutes, the absorbance was measured at 540 nm using an ELISA plate reader.

[0084] Figure 3 is a graph showing the results of the NO (Nitric oxide) production inhibitory activity of the hyaluronic acid samples prepared in Example 1 and Comparative Examples 1-4.

[0085] As shown in Figure 3, it was found that the hyaluronic acid sample prepared in Comparative Example 1 exhibited significantly superior NO production inhibitory ability compared to Comparative Examples 1-4.

[0086] <Experiment Example 4> Moisture Improvement Test: Clinical Evaluation of the Moisture Improvement Efficacy of 20 Layers of Skin

[0087] Clinical evaluations were conducted to measure the moisturizing effect of the hyaluronic acid samples prepared in Example 1 and Comparative Examples 1-4.

[0088] The hyaluronic acid samples prepared in Examples and Comparative Examples 1-4 were diluted to a concentration of 50 μg / mL, and then applied to the forearms of the subjects, 10 cm away from both wrists. The moisture content of the skin was measured using an Epsilon E100 to measure the moisture content of the skin, and the amount of moisture inside the skin was measured by repeatedly applying the same pressure for 2 seconds and stripping using Scotch Magic Invisible Tape (3M).

[0089] Figure 4 is a graph showing the percentage increase in skin moisture content after treatment with the hyaluronic acid sample prepared in Example 1 and the control sample (Comparative Example 1).

[0090] As shown in Figure 4, it was found that the hyaluronic acid sample treatment group prepared in Comparative Example 1 showed superior moisturizing effect up to 20 layers of skin compared to the control sample (Comparative Example 1).

[0091] <Experimental Example 5> Evaluation of Hyaluronic Acid Molecular Weight

[0092] The molecular weights of the hyaluronic acid hydrolysates obtained in Comparative Examples 1-8 and Examples 1-5 were measured and compared.

[0093] The molecular weights of the mixed enzyme and the hyaluronic acid hydrolysate treated with low-temperature aging in Example 1 were determined by molecular weight confirmation analysis conducted by the Gyeonggi Provincial Economic and Chemical Promotion Agency (GBSA), and the results are shown in Figures 5 and 6.

[0094] For molecular weight measurement, a matrix solution was prepared by adding 10 mg / mL of DHB (2,5-dihydroxybenzoic acid) to a 0.1% TFA / ACN (1:1, v / v) solvent. 2 μL of the sample from Example 1 was directly mixed with 2 μL of the matrix solution onto a MALDI target and vacuum-dried. An autoflex maX (Bruker Daltonics) was used as the molecular weight measurement device. The measurement device settings were as follows:

[0095] 1. Equipment control: Flex Control 3.4 (manufactured by Bruker Daltonics) 2. Analysis Mode: Linear Mode 3. Polarity: Positive 4. Detection: m / z 100~100,000 5. Laser repetition rate: 2,000Hz 6. Number of shots: 500 shots 7.Deflection:On, 100Da 8. Voltage

[0096] [Table 1]

[0097] Figure 5 is a spectral image showing the molecular weight of the hyaluronic acid hydrolysate from Example 1.

[0098] Figure 6 is a table summarizing the peak information in the spectrum in which the molecular weight of the hyaluronic acid hydrolysate of Example 1 was measured. Gray cells indicate matrix peak molecular weights.

[0099] As shown in Figures 5 and 6, the weighted average molecular weight excluding the matrix peak was confirmed to be 368.3 Da.

[0100] The molecular weight measurements of aluronic acid obtained in Examples 2-5 and Comparative Examples 1-8, performed in the same manner as the molecular weight measurement method in Example 1, are shown in Tables 1 and 2 below.

[0101] [Table 2]

[0102] [Table 3]

[0103] As shown in Table 1 above, it was found that using a weight percentage of proteinase and pectinase mixed enzyme in the range of 1 to 5:1 resulted in a significantly higher hyaluronic acid hydrolysis rate.

[0104] As shown in Table 2 above, Comparative Examples 1 and 4 were samples that differed only in whether or not they underwent low-temperature aging treatment. A comparison of these samples revealed that low-temperature aging treatment had a hydrolytic effect. This result can also be seen by comparing Comparative Example 2 with Example 1. Furthermore, Comparative Examples 5 and 6 were samples treated with single enzymes and were compared with Examples 1 to 5. A comparison of these revealed that the hydrolysis rate was significantly improved in the mixed enzyme-treated samples compared to those treated with single enzymes.

[0105] Examples of cosmetic manufacturing

[0106] The active substance according to the present invention can be manufactured in many forms of cosmetics depending on the purpose. The following are examples of methods for manufacturing several cosmetics containing the active substance according to the present invention as an active ingredient, but the present invention is not limited to these.

[0107] <Example of cosmetic manufacturing 1> Manufacturing of softening lotion

[0108] [Table 4]

[0109] <Example of cosmetic manufacturing> Manufacturing of nourishing cream

[0110] [Table 5]

[0111] The present invention has been described in detail, focusing on preferred embodiments. Those with ordinary skill in the art to which the present invention pertains will understand that the present invention can be embodied in modified forms that do not depart from its essence. Therefore, the disclosed embodiments should be considered in an explanatory rather than restrictive manner. The scope of the present invention is defined not by the foregoing description, but particularly by the claims, and all differences within the equivalent scope should be construed as belonging to the present invention.

Claims

1. Step 1 involves mixing hyaluronic acid with distilled water and proteinase and pectinase as mixed enzymes to carry out an enzymatic reaction to obtain hyaluronic acid treated with mixed enzymes, The process includes a step (step 2) in which the hyaluronic acid treated with mixed enzymes in step 1 is subjected to steam aging, followed by low-temperature aging at 5 to 20°C for 1 to 5 days, A method for producing hyaluronic acid hydrolysate.

2. The manufacturing method according to claim 1, characterized in that the proteinase in step 1 is Flavorzyme, Protamex, α-chymotrypsin, subtilisin Carlsberg, thermolysin, papain, fungal protease, or pepsin.

3. The pectinase in step 1 is Pectinex Ultra SP-L, Viscozyme L The manufacturing method according to claim 1, characterized in that it is L), Ultrazyme AFP, Pectinex Ultra AFP, Pectinex Ultra Clear, and α-herbzyme.

4. The manufacturing method according to claim 1, characterized in that the mixed enzyme contains 1 to 5 parts by weight of proteinase based on 1 part by weight of pectinase.

5. The manufacturing method according to claim 4, characterized in that the mixed enzyme contains 2 to 4 parts by weight of proteinase based on 1 part by weight of pectinase.

6. The manufacturing method according to claim 1, characterized in that the steaming treatment in step 2 is carried out at 70 to 90°C for 2 to 4 hours, and the low-temperature aging is carried out at 14 to 18°C ​​for 2 to 4 days.

7. The manufacturing method according to claim 1, characterized in that the weight-average molecular weight (Mw) of the hyaluronic acid produced by the above manufacturing method is 500 Da or less.

8. A hyaluronic acid hydrolysate with a weight-average molecular weight (Mw) of 500 Da or less, produced by the manufacturing method described in claim 1.

9. A cosmetic composition for soothing skin irritation and moisturizing, comprising a hyaluronic acid hydrolysate produced by the manufacturing method described in claim 1.

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