Method for preparing low-molecular-weight oat peptide mixture, composition comprising same, and use thereof

Abstract

One aspect relates to a method for preparing a low-molecular-weight oat peptide mixture, comprising the steps of: adding an acid to an oat extract so as to obtain an oat protein precipitate; and adding an enzyme complex to the oat protein precipitate so as to obtain a low-molecular-weight oat peptide mixture. It has been identified that the low-molecular-weight oat peptide mixture prepared using the method has excellent effects of strengthening the skin barrier, alleviating skin inflammation, alleviating itchiness, and alleviating skin redness, and has a superior synergistic effect when used in combination with panthenol, and thus can be used as a cosmetic composition, a composition for external application to the skin, a pharmaceutical composition, and the like.

Description

Method for preparing a low molecular weight oat peptide mixture, a composition comprising the same, and uses thereof

[0001] The present invention relates to a method for preparing a low molecular weight oat peptide mixture, a composition containing the same, and the use thereof.

[0002] Biopeptides extracted from plants have higher skin penetration than high molecular weight proteins, allowing them to reach deeper skin layers. Due to their diverse biological activities, they are attracting attention as functional food and cosmetic ingredients for skin and hair care.

[0003] Currently, peptides are mainly obtained through chemical synthesis or partial digestion of animal proteins, but due to the harmfulness of chemical solvents, interest in eco-friendly plant-based raw materials is increasing. However, effective extraction methods for industrially useful low-molecular-weight proteins derived from plants have not yet been identified.

[0004] Under this technical background, the inventors made diligent efforts to produce a plant extract with an increased content of low molecular weight proteins and to improve the physiological activity of said extract, and as a result, developed a method for producing a low molecular weight oat peptide mixture. Furthermore, the present invention was completed by confirming that the mixture produced according to this method has an excellent effect in improving skin condition.

[0005] One aspect provides a method for preparing a low-molecular-weight oat peptide mixture, comprising the steps of: adding an acid to an oat extract to obtain an oat protein precipitate; and adding an enzyme complex to the oat protein precipitate to obtain a low-molecular-weight oat peptide mixture.

[0006] Another aspect is to provide a cosmetic composition for improving skin condition comprising a low molecular weight oat peptide mixture prepared by the above method.

[0007] Another aspect is to provide a topical skin composition for improving skin condition comprising a low molecular weight oat peptide mixture prepared by the above method.

[0008] Another aspect is to provide a pharmaceutical composition for the prevention or treatment of skin inflammatory diseases comprising a mixture of low molecular weight oat peptides prepared by the above method.

[0009] Another aspect is to provide a use of a low-molecular-weight oat peptide mixture prepared by the above method for improving skin condition.

[0010] Another aspect is to provide a use of a low molecular weight oat peptide mixture prepared by the above method for the prevention or treatment of skin inflammatory diseases.

[0011] Another aspect is to provide a method for improving skin condition, comprising the step of administering an effective amount of a low-molecular-weight oat peptide mixture prepared by the above method to an individual.

[0012] Another aspect is to provide a method for preventing or treating skin inflammatory diseases, comprising the step of administering an effective amount of a low-molecular-weight oat peptide mixture prepared by the above method to an individual.

[0013] One aspect provides a method for preparing a low-molecular-weight oat peptide mixture, comprising the steps of: adding an acid to an oat extract to obtain an oat protein precipitate; and adding an enzyme complex to the oat protein precipitate to obtain a low-molecular-weight oat peptide mixture.

[0014] The term "oats (Avena sativa)" refers to cultivated oats, a biennial grass belonging to the Poaceae family, also known as *yeonmaek* or *jakmaek*. Originating from the Armenian region of Central Asia, it is a major crop with the sixth-highest global production volume, following wheat, corn, rice, barley, and sorghum. Oats reach a height of 1 meter; their stems stand upright and are nearly hairless, except for downward-pointing hairs at the nodes. The leaves are 15–30 cm long and 6–12 mm wide, slightly wider than those of wheat, and are dark green. Oats have long been recognized as a health food due to their richness in protein, dietary fiber, vitamins, and minerals; they are particularly attracting attention as a functional food ingredient due to their high beta-glucan content.

[0015] The above oats may be varieties such as Joyang, Daeyang, Seonyang, Suyang, Samhan, Donghan, Johan, Punghan, Dark Horse, High Speed, etc., and specifically may be the Daeyang variety.

[0016] The term "extract" refers to an active ingredient isolated from a natural product. The extract may be obtained by an extraction process using water, an organic solvent, or a mixture thereof, and includes the extract liquid, the dried powder thereof, or any form formulated using the extract liquid. It includes the extract itself and all formulations of extracts that can be formed using the extract liquid, such as the extract liquid obtained by the extraction treatment of oats, the diluted or concentrated extract liquid, the dried product obtained by drying the extract liquid, the modified or purified product, fraction, or mixture thereof of the extract liquid.

[0017] The above extract may be prepared using general manufacturing methods known in the art and is not particularly limited. For example, it may be prepared by sonication extraction, supercritical extraction, vacuum extraction, reflux extraction, hot-water extraction, autoclave extraction, high-pressure extraction, enzyme extraction, or by a known method using an extraction solvent, but is not limited thereto.

[0018] The above oat extract may be an extract obtained by extracting oats with a C1-C6 alcohol, water, or a mixture thereof as a solvent, and specifically, may be an oat water extract.

[0019] The above oat extract may be an extract obtained using water or hot water as a solvent. The water may be, for example, distilled water, purified water, sterile water, or hot water.

[0020] The above oat extract may be obtained by mixing 1 kg of ground oats with 10 kg of purified water with a pH of 8.0 to 14.0, for example, pH 8.0 to 12.0, pH 9.0 to 13.0, pH 10.0 to 12.0, or pH 11.0 and stirring the mixture.

[0021] The above oat extract may be obtained by mixing 1 kg of ground oats with 10 kg of purified water and stirring the mixture at 30 to 70°C, for example, 40 to 60°C, 45 to 55°C, or 50°C.

[0022] The above oat extract may be obtained by mixing 1 kg of ground oats with 10 kg of purified water and stirring for 3 to 60 hours, for example, 6 to 60 hours, 12 to 60 hours, 24 to 60 hours, 36 to 60 hours, 42 to 54 hours, 46 to 50 hours, or 48 hours.

[0023] In one embodiment, 1 kg of ground oats may be mixed with 10 kg of purified water with a pH of 11.0 and stirred for 48 hours at 50°C.

[0024] The step of adding acid to the oat extract to obtain an oat protein precipitate may be for obtaining oat protein by removing the supernatant after protein precipitation of the oat extract using a protein precipitation method.

[0025] The term "acid" refers to hydrogen ions (H₂) in an aqueous solution. + It refers to any substance capable of dissociating ). The acid may be a weak acid or a strong acid. The acid may be one used to separate proteins from oat extract.

[0026] The term "protein precipitation" refers to the phenomenon in which a protein in solution separates from the solution as its solubility decreases and it is converted into an insoluble solid form. This may include phase changes of proteins caused by changes in temperature, pH, or ionic strength, the addition of organic solvents or salts, or changes in other chemical or physical conditions.

[0027] The term "oat protein precipitate" refers to a protein component extracted from oats. The oat protein precipitate may be obtained by protein precipitating an oat extract, and specifically, may be obtained by protein precipitating an oat extract through pH and temperature control.

[0028] The above oat protein precipitate may be adjusted to a pH of 1.0 to 5.0, for example, pH 2.0 to 4.0, pH 2.5 to 3.5, or pH 3.0 by adding acid to an oat water extract.

[0029] The above oat protein precipitate may be produced by adding acid to an oat water extract and allowing protein precipitation to proceed for 3 to 60 hours, for example, 6 to 60 hours, 12 to 60 hours, 24 to 60 hours, 36 to 60 hours, 42 to 54 hours, 46 to 50 hours, or 48 hours.

[0030] The above oat protein precipitate may be produced by adding acid to an oat water extract and then proceeding with protein precipitation at 1°C to 10°C, for example, 1°C to 5°C, 2°C to 5°C, 3°C to 5°C, or 4°C.

[0031] In one embodiment, the protein precipitate may be produced by adding acid to an oat water extract to adjust the pH to 3.0 and proceeding with protein precipitation under low temperature conditions of 4°C for 48 hours.

[0032] The above method may additionally include a step of removing the supernatant after protein precipitation.

[0033] The step of adding an enzyme complex to the oat protein precipitate to obtain a low-molecular-weight oat peptide mixture may be for low-molecular-weight oat protein peptides to be low-molecular-weight oat peptides by using a complex enzyme reaction on the oat protein precipitate to obtain a low-molecular-weight oat peptide mixture.

[0034] The term "enzyme complex" refers to an enzyme mixture comprising two or more enzymes combined for a synergistic effect in a protein enzyme reaction. By treating with the enzyme complex, proteins can be hydrolyzed into amino acids or peptides to obtain low-molecular-weight proteins.

[0035] The enzyme complex may be added in an amount of 0.001 to 50 wt% relative to the weight of the oat protein precipitate, for example, 0.01 to 50 wt%, 0.1 to 50 wt%, 1 to 50 wt%, 5 to 50 wt%, 10 to 50 wt%, 0.001 to 40 wt%, 0.01 to 40 wt%, 0.1 to 40 wt%, 1 to 40 wt%, 5 to 40 wt%, 10 to 50 wt%, 0.001 to 30 wt%, 0.01 to 30 wt%, 0.1 to 30 wt%, 1 to 30 wt%, 5 to 30 wt%, or 10 to 30 wt%. If the above range is exceeded, the enzyme reaction may not proceed sufficiently, making it impossible to obtain low-molecular-weight proteins, or there may be problems such as increased manufacturing costs due to the excessive use of enzymes.

[0036] The above enzyme complex may include one or more proteolytic enzymes, and may include, for example, one or more proteolytic enzymes derived from plants, microorganisms, animals, etc.

[0037] The above enzyme complex may comprise one or more selected from the group consisting of papain, bromelain, alkalase, and Protamax, specifically may comprise one or more selected from the group consisting of papain and bromelain, and more specifically may comprise papain and bromelain.

[0038] The term "Papain" refers to the cysteine ​​protease enzyme extracted from papaya (Carica papaya) and wild papaya (Vasconcellea cundinamarcensis). It is characterized by maintaining activity over a wide pH and temperature range.

[0039] The term "Bromelain" refers to a mixture of proteolytic enzymes extracted from the pineapple plant (Ananas comosus). It is heat-resistant and has the characteristic of maintaining its activity even after being boiled for 60 minutes.

[0040] The term "Alcalase" refers to a food-grade endopeptidase derived from the Bacillus licheniformis strain. It is characterized by broad substrate specificity, enabling it to hydrolyze a wide range of proteins, as well as high thermal and pH stability.

[0041] The term "Protamax" refers to a protease derived from a Bacillus strain. The above-mentioned Protamax is a mixture of endo-proteases, and its enzyme activity may be 1.4 to 1.6 AU / g.

[0042] The above enzyme complex may contain papain and bromelain in a weight ratio of 1:0.1 to 20, for example, 1:0.1 to 10, 1:0.1 to 2, 1:0.5 to 1.5, or 1:1. If the ratio is outside the above range, there may be a problem in that the enzyme reaction is not sufficiently carried out and a low-molecular-weight protein cannot be obtained.

[0043] The above enzyme reaction may proceed at pH 5.0 to 9.0, for example, pH 6.0 to 8.0, pH 6.5 to 7.5, or pH 7.

[0044] The above enzyme reaction may be carried out at 30 to 70°C, for example, 40 to 60°C, 45 to 55°C, or 50°C.

[0045] The above enzyme reaction may be carried out for 12 to 36 hours, for example, 18 to 30 hours, 21 to 27 hours, 23 to 25 hours, or 24 hours.

[0046] In one embodiment, the enzyme reaction may be carried out for 24 hours under conditions of pH 7.0 and 50°C.

[0047] The term "low molecular weight oat peptide mixture" refers to a mixture of oat peptides that have been reduced in molecular weight by enzyme treatment. The "low molecular weight oat peptide mixture" can be used interchangeably with "oat peptide hydrolysate."

[0048] The above low molecular weight oat peptide mixture may have an average molecular weight (MW) of less than 5000 Da, 4000 Da, 3500 Da, 3000 Da, 2900 Da, 2800 Da, 2700 Da, 2600 Da, 2500 Da, 2400 Da, 2300 Da, 2200 Da, 2100 Da, or 2000 Da. If it falls outside the above range, there may be problems such as reduced skin penetration ability or reduced stability of the active ingredient.

[0049] In one embodiment, it was confirmed that the average molecular weight of the low molecular weight oat peptide mixture prepared by the above method was less than 2500 Da, confirming that the low molecular weight effect on oat protein through protein precipitation and complex enzyme reaction is excellent.

[0050] The above method may additionally include an enzyme inactivation step after the complex enzyme reaction.

[0051] The above method may additionally include a step of preservative treatment after enzyme inactivation.

[0052] The above enzyme inactivation may be carried out at 80 to 110°C, for example, 85 to 105°C, 90 to 100°C, or 95°C.

[0053] The above enzyme inactivation may be carried out for 5 minutes to 5 hours, for example, 10 minutes to 3 hours, 20 minutes to 2 hours, 30 minutes to 1 hour 30 minutes, or 1 hour.

[0054] The above method may additionally include a filtration step after preservation treatment.

[0055] Another aspect provides a cosmetic composition for improving skin condition comprising a mixture of low molecular weight oat peptides prepared by the above method. In the cosmetic composition, any terms or elements mentioned that are identical to those already mentioned are as described above.

[0056] The term "improvement of skin condition" refers to all actions that enhance the health and appearance of the skin, and may include improving skin elasticity, improving skin texture, skin whitening, strengthening the skin barrier, moisturizing the skin, improving skin inflammation, improving skin redness, soothing the skin, regulating sebum secretion, antioxidant effects, promoting skin regeneration, healing skin wounds, improving skin damage, and preventing skin aging.

[0057] The above skin condition improvement may be strengthening the skin barrier, improving skin inflammation, soothing the skin, improving itching, or improving skin redness.

[0058] The term "skin barrier strengthening" refers to improving or maintaining the structure and function of the stratum corneum, the outermost layer of the epidermis.

[0059] The term "improvement of skin inflammation" refers to any action that suppresses or improves inflammation.

[0060] In one embodiment, it was confirmed that the low molecular weight oat peptide mixture showed a significantly superior effect in increasing the expression levels of ELOVL 4, ABCA12, Filaggrin, and Claudin-1, or decreasing the expression levels of TNF-α, IL-6, and TSLP, thereby confirming that it has an excellent skin barrier strengthening effect and skin inflammation improvement effect.

[0061] The term "skin soothing" refers to promoting the recovery of irritated or sensitized skin to its normal physiological state. This may include actions that alleviate inflammatory responses or damage to the skin barrier, or relieve symptoms of skin irritation such as redness, swelling, itching, and burning sensations.

[0062] The term "improvement of skin redness" refers to any action that soothes a condition in which the skin surface has turned red due to external stimuli, internal factors, or environmental changes, and restores the skin to its natural tone. The above-mentioned improvement of skin redness may include actions that alleviate erythema or facial flushing caused by ultraviolet rays, heat, or physical / chemical stimuli, regulate abnormal dilation of microvessels, or suppress an increase in blood flow caused by an inflammatory response.

[0063] In one embodiment, it was confirmed that the Hemoglobin average value, which indicates the degree of skin redness of the low molecular weight oat peptide mixture, was significantly reduced, thus confirming that there is an excellent skin redness improvement effect.

[0064] The above composition may further include panthenol.

[0065] The term "Panthenol" refers to provitamin B5, which is a precursor of vitamin B3. It is also called dexpanthenol or pantothenol, and its trade name is Bepanthen. When panthenol is applied to a living organism, it is rapidly oxidized and converted into pantothenic acid, and exists as a viscous, transparent liquid at room temperature. The above panthenol may be D-panthenol or DL-panthenol.

[0066] The above panthenol is 30 to 70 wt%, for example, 5 to 80 wt%, 10 to 70 wt%, 20 to 70 wt%, 25 to 70 wt%, 30 to 70 wt%, 35 to 70 wt%, 40 to 70 wt%, 45 to 70 wt%, 5 to 60 wt%, 10 to 60 wt%, 20 to 60 wt%, 25 to 60 wt%, 30 to 60 wt%, 35 to 60 wt%, 40 to 60 wt%, 45 to 60 wt%, 5 to 55 wt%, 10 to 55 wt%, 20 to 55 wt%, 25 to 55 wt%, 30 to 55 wt%, 35 to 55 wt%, 40 to 55 wt%, based on the total weight of the composition. It may be included in weight%, 45 to 55 weight%, or 50 weight%. If it falls outside the above range, there may be a problem in that the synergistic effect between the low molecular weight oat peptide mixture and pentanol is not excellent.

[0067] In one embodiment, it was confirmed that the composition containing the low molecular weight oat peptide mixture and the pentanol had a significantly superior effect in increasing Claudin-1 expression and reducing IL-1β and TNF-α expression, thereby confirming that it has an excellent skin barrier strengthening effect and skin soothing effect.

[0068] In one embodiment, it was confirmed that the Hemoglobin average value, which indicates the degree of skin redness of the composition containing the low molecular weight oat peptide mixture and the pantanol, was significantly reduced, thus confirming that there is an excellent skin redness improvement effect.

[0069] The above cosmetic composition may be prepared in a formulation comprising a lotion (skin lotion), skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, eye cream, hand cream, foundation, essence, nourishing essence, eye essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, cleansing water, cleansing oil, cleansing tissue, hand sanitizer, body lotion, body cream, body cleanser, suspension, gel, powder, paste, shampoo, hair essence, treatment, rinse, scalp pack, mask pack or sheet, or aerosol composition. Compositions of such formulations may be prepared according to methods conventional in the field.

[0070] The ingredients included in the above cosmetic composition may include ingredients commonly used in cosmetic compositions in addition to the low molecular weight oat peptide mixture, and may include, for example, conventional auxiliary agents and carriers such as stabilizers, solubilizers, vitamins, pigments, and fragrances.

[0071] Another aspect provides a topical skin composition for improving skin condition comprising a low molecular weight oat peptide mixture prepared by the above method. In the topical skin composition, any terms or elements mentioned that are identical to those already mentioned are as described above.

[0072] The term "external skin preparation" is a concept that generally encompasses all compositions used externally, and may refer to a wide range of products that can be applied to cosmetic compositions containing various cosmetics such as basic cosmetics, makeup cosmetics, hair cosmetics, and shaving cosmetics, or various pharmaceuticals or quasi-pharmaceuticals such as ointments. For example, the above-mentioned quasi-pharmaceuticals may be disinfectant cleansers, shower foams, mouthwash, wet wipes, detergent soaps, hand washes, humidifier fillers, masks, ointments, coating agents, or filter fillers.

[0073] The above-mentioned external skin composition may additionally contain ingredients that are typically incorporated into external preparations according to the use and properties of the external preparation composition, in addition to the low molecular weight oat peptide mixture, specifically moisturizers, UV absorbers, vitamins, animal and plant extracts, digestive agents, whitening agents, vasodilators, astringents, cooling agents, hormones, etc.

[0074] The formulation of the above-mentioned external skin composition may take an appropriate form depending on the intended use and the properties of the external composition, and specifically may be an aqueous solution type, a solubilization type, an emulsion type, a liquid type, a gel type, a paste type, an ointment type, an aerosol type, a water-oil two-layer type, or a water-oil-powder three-layer type, but the formulation and form of the external agent of the present invention are not limited by the above-mentioned formulation.

[0075] In addition, the above-mentioned topical skin preparation may further include a mechanism necessary to penetrate or transfer the active ingredient into the skin tissue.

[0076] Another aspect provides a pharmaceutical composition for the prevention or treatment of skin inflammatory diseases comprising a mixture of low molecular weight oat peptides prepared by the above method. In the pharmaceutical composition, any terms or elements mentioned that are identical to those already mentioned are as described above.

[0077] The term "skin inflammatory disease" refers to any disease characterized by an inflammatory reaction occurring in skin tissue due to various factors. The skin inflammatory disease may include any disease accompanied by an inflammatory reaction that may occur on the skin, such as redness (erythema), swelling, fever, pain, and itching, regardless of the cause. The skin inflammatory disease may include, for example, skin wounds, dermatitis, pruritus, eczematous skin disease, dry eczema, erythema, urticaria, psoriasis, drug rash, acne, scalp inflammatory disease, seborrheic dermatitis or scalp dermatitis, atopic dermatitis, contact dermatitis, or stress-induced inflammation.

[0078] The term "prevention" refers to any act in which the possibility of developing a skin inflammatory disease is suppressed or delayed by the administration of the composition of the present invention.

[0079] The term "treatment" refers to any act of improving or beneficially altering the symptoms of a skin inflammatory disease through the administration of the composition of the present invention.

[0080] The above pharmaceutical composition may be formulated and used in the form of tablets, pills, powders, granules, capsules, suspensions, oral liquids, emulsions, or syrups, as well as external preparations, suppositories, or injectable solutions, according to conventional methods. Specifically, when formulating, it may be prepared using diluents or excipients such as commonly used fillers, weighting agents, binders, wetting agents, disintegrants, and surfactants.

[0081] The above pharmaceutical composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage may be appropriately selected by a person skilled in the art, although it depends on the patient's condition and body weight, the severity of the disease, the form of the drug, the route of administration, and the time.

[0082] Another aspect is to provide a use of a low-molecular-weight oat peptide mixture prepared by the above method for improving skin condition.

[0083] Another aspect is to provide a use of a low molecular weight oat peptide mixture prepared by the above method for the prevention or treatment of skin inflammatory diseases.

[0084] Another aspect is to provide a method for improving skin condition, comprising the step of administering an effective amount of a low-molecular-weight oat peptide mixture prepared by the above method to an individual.

[0085] Another aspect is to provide a method for preventing or treating skin inflammatory diseases, comprising the step of administering an effective amount of a low-molecular-weight oat peptide mixture prepared by the above method to an individual.

[0086] In the above aspects, any terms or elements mentioned that are identical to those already mentioned are as stated above.

[0087] In the above aspects, "effective amount" refers to the content of a composition in which the active ingredient in the mixture is contained in an amount sufficient to provide a therapeutic, corrective, or preventive effect to an individual to whom the mixture is to be administered. The effective amount may include the meaning of a "preventive effective amount," a "corrective effective amount," or a "therapeutic effective amount."

[0088] In the above aspects, "individual" may mean any individual requiring improvement of skin condition or prevention or treatment of skin inflammatory disease. The individual may include or exclude humans, and may include mammals, and the mammals may include, for example, humans, dogs, cats, rabbits, rats, mice, rats, hamsters, guinea pigs, horses, cattle, pigs, sheep, monkeys, chimpanzees, etc. The individual may include companion animals, and the companion animals may include, for example, dogs, cats, rabbits, hamsters, guinea pigs, etc.

[0089] In the above aspects, "administration" means administering an effective amount of the mixture directly to an individual to form it within the individual's body. The administration may be oral or parenteral, and may include transdermal administration, subcutaneous administration, or application to the skin. Suitable dosages of the mixture or composition may be prescribed differently depending on factors such as the method of formulation, mode of administration, age, body weight, sex, pathological condition, food, time of administration, route of administration, rate of excretion, and response sensitivity.

[0090] One aspect relates to a method for preparing a low-molecular-weight oat peptide mixture, comprising the steps of: adding an acid to an oat extract to obtain an oat protein precipitate; and adding an enzyme complex to the oat protein precipitate to obtain a low-molecular-weight oat peptide mixture. It has been confirmed that the low-molecular-weight oat peptide mixture prepared by the above method has excellent effects on strengthening the skin barrier, improving skin inflammation, improving itching, and improving skin redness, and exhibits even better synergistic effects when used in combination with panthenol, and can be used as a cosmetic composition, a topical skin preparation composition, a pharmaceutical composition, etc.

[0091] Figure 1 is a figure showing the protein molecular weight distribution of Example 1.

[0092] Figure 2 is a figure showing the protein molecular weight distribution of Comparative Examples 1 to 4.

[0093] Figure 3 shows the results of analyzing the gene expression levels of the barrier strengthening factor ELOVL4 in the examples and comparative examples.*p<0.05 vs. None,**p<0.01 vs. None.

[0094] Figure 4 shows the results of analyzing the gene expression levels of the barrier strengthening factor ABCA12 in the examples and comparative examples.*p<0.05 vs. None,**p<0.01 vs. None.

[0095] Figure 5 shows the results of analyzing the expression levels of the barrier strengthening factor Filaggrin gene in the examples and comparative examples.*p<0.05 vs. None,**p<0.01 vs. None.

[0096] Figure 6 shows the results of analyzing the expression levels of the barrier strengthening factor Claudin-1 gene in the examples and comparative examples.*p<0.05 vs. None,**p<0.01 vs. None.

[0097] Figure 7 shows the results of analyzing the gene expression levels of the inflammatory factors TNF-α, IL-6, and the pruritus factor TSLP in Example 1.###p<0.005 vs. None,***p<0.005 vs. Veh.

[0098] Figure 8 shows the results of analyzing the gene expression levels of the barrier strengthening factor Claudin-1 of Example 2 to confirm the synergistic effect of the low-molecular-weight oat peptide mixture and panthenol.*p<0.05 vs. (+),**p<0.01 vs. (+),***p<0.001 vs. (+),###p<0.001 vs. None.

[0099] Figure 9 shows the results of analyzing the gene expression levels of the sedative factors IL-1β and TNF-α in Examples 1 and 2 to confirm the synergistic effect of the low-molecular-weight oat peptide mixture and panthenol.*p<0.05 vs. (+),**p<0.01 vs. (+),***p<0.001 vs. (+),###p<0.001 vs. None.

[0100] Figure 10 shows the results of evaluating the degree of improvement in redness before and after skin damage and after application of the test product and control product.

[0101] Figure 11 shows the results of measuring the average hemoglobin value according to the application of samples and skin damage before and after the test product and control product.

[0102] Figure 12 shows the results of evaluating the skin moisture improvement effect (left) and itching improvement effect (right) of the test product.

[0103] The following examples will be explained in more detail. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

[0104] [Example]

[0105] Example 1. Preparation of a low molecular weight oat peptide mixture.

[0106] 1 kg of ground oats (Avena sativa) was mixed with 10 kg of purified water with a pH of 10.0–12.0 and stirred for 48 hours at 50°C. Afterward, the oat extract was filtered using filter paper. Acid was added to adjust the pH of the filtered oat extract to 2.0–4.0, and protein precipitation was carried out for 48 hours under low temperature conditions at 4°C.

[0107] After protein precipitation, the supernatant was removed, and a complex enzyme reaction for protein demolecule was carried out. Papain and bromelain were mixed in a weight ratio of 1:1, and the enzyme complex was added at 10 to 30% by weight relative to the weight of the oat protein precipitate. The enzyme reaction was carried out for 24 hours under conditions of pH 6.0–8.0 and 50°C. Subsequently, to inactivate the enzyme, the reaction was carried out at 95°C for 1 hour, followed by preservation treatment. Finally, the mixture was filtered through a membrane filter to prepare a demolecule oat peptide mixture derived from the oat water extract (Example 1).

[0108] Example 2. Preparation of a composition comprising a low molecular weight oat peptide mixture and panthenol.

[0109] Panthenol was mixed with the low molecular weight oat peptide mixture obtained by the method of Example 1 as shown in Table 1 below, and stirred continuously for 10 seconds to prepare a composition (Example 2) containing the low molecular weight oat peptide mixture and panthenol.

[0110] (Unit: weight%) Example 2 Low molecular weight oat peptide mixture 50 Panthenol 50

[0111] [Comparative Example]

[0112] Comparative Example 1. Preparation of oat water extract.

[0113] Comparative Example 1 was prepared in the same manner as Example 1, except that protein precipitation and enzyme reaction were not performed in Example 1.

[0114] Specifically, 1 kg of ground oats (Avena sativa) was mixed with 10 kg of purified water with a pH of 10.0 to 12.0 and stirred for 48 hours at 50°C. Afterward, the oat extract was filtered using filter paper and treated with preservatives. Finally, the oat water extract (Comparative Example 1) was prepared by filtering with a membrane filter.

[0115] Comparative Example 2. Preparation of oat protein precipitate.

[0116] Comparative Example 2 was prepared in the same manner as Example 1, except that the enzyme reaction was not performed in Example 1.

[0117] Specifically, 1 kg of ground oats (Avena sativa) was mixed with 10 kg of purified water with a pH of 10.0–12.0 and stirred for 48 hours at 50°C. Afterward, the oat extract was filtered using filter paper. The pH of the filtered oat extract was adjusted to 2.0–4.0, and protein precipitation was carried out for 48 hours under low temperature conditions at 4°C.

[0118] After protein precipitation, the supernatant was removed, and the mixture was neutralized to a pH of 6.0–8.0 and subjected to preservative treatment. Finally, the mixture was filtered through a membrane filter to produce an oat protein precipitate (Comparative Example 2).

[0119] Comparative Example 3. Preparation of an oat peptide mixture by bromelain single enzyme reaction.

[0120] Comparative Example 3 was prepared in the same manner as Example 1, except that a bromelain single enzyme reaction was performed instead of a complex enzyme reaction in Example 1.

[0121] Specifically, 1 kg of ground oats (Avena sativa) was mixed with 10 kg of purified water with a pH of 10.0–12.0 and stirred for 48 hours at 50°C. Afterward, the oat extract was filtered using filter paper. The pH of the filtered oat extract was adjusted to 2.0–4.0, and protein precipitation was carried out for 48 hours under low temperature conditions at 4°C.

[0122] After protein precipitation, the supernatant was removed, and a bromelain single-enzyme reaction was carried out to reduce the protein molecular weight. Bromelain enzyme was added at 10 to 30% by weight relative to the weight of the oat protein precipitate. The enzyme reaction was carried out for 12 to 24 hours under conditions of pH 6.0 to 8.0 and 40 to 50°C. Subsequently, to inactivate the enzyme, the reaction was carried out for 1 hour at 90 to 95°C, followed by preservation treatment. Finally, the mixture was filtered through a membrane filter to prepare an oat peptide mixture (Comparative Example 3) by the bromelain single-enzyme reaction.

[0123] Comparative Example 4. Preparation of an oat peptide mixture by papain single-enzyme reaction.

[0124] Comparative Example 4 was prepared in the same manner as Example 1, except that a papain single enzyme reaction was performed instead of a complex enzyme reaction in Example 1.

[0125] Specifically, 1 kg of ground oats (Avena sativa) was mixed with 10 kg of purified water with a pH of 10.0–12.0 and stirred for 48 hours at 50°C. Afterward, the oat extract was filtered using filter paper. The pH of the filtered oat extract was adjusted to 2.0–4.0, and protein precipitation was carried out for 48 hours under low temperature conditions at 4°C.

[0126] After protein precipitation, the supernatant was removed, and a papain single-enzyme reaction was carried out to reduce the protein molecular weight. Papain enzyme was added at 10 to 30% by weight relative to the weight of the oat protein precipitate. The enzyme reaction was carried out for 12 to 24 hours under conditions of pH 6.0 to 8.0 and 40 to 50°C. Subsequently, to inactivate the enzyme, the reaction was carried out for 1 hour at 90 to 95°C, followed by preservation treatment. Finally, the mixture was filtered through a membrane filter to prepare an oat peptide mixture (Comparative Example 4) by the papain single-enzyme reaction.

[0127] Comparative Example 5. Preparation of Panthenol.

[0128] 100% DL-Panthenol (Manufacturer: JIANXI TONGDE CHEMICAL) was used.

[0129] [Experimental Example]

[0130] Experimental Example 1. Molecular weight analysis.

[0131] To determine the average molecular weight of the oat compositions prepared in the above examples and comparative examples, an experiment was conducted in the following manner.

[0132] Specifically, GPC analysis was performed at 40°C under isocratic conditions using a Shimadzu SIL-40 HPLC-GPC System, a Shodex OHpak column (8.0 x 300, 6 μm), and a 0.1 M NaNO3 aqueous solution.

[0133] As a result, as shown in FIGS. 1 and 2 and Table 2 below, the average molecular weight of Comparative Example 1, which is an oat water extract, is approximately 2228 Da, and Comparative Examples 2 to 4, in which protein precipitation and enzymatic reactions were performed using the same amount of single enzyme, have an average molecular weight of 2500 to 2700 Da. In contrast, in the case of Example 1, which was reduced in molecular weight using protein precipitation and a complex enzymatic reaction, it was confirmed that it has an average molecular weight of less than 1950 Da, thus confirming the excellent synergistic effect of protein precipitation and complex enzymatic reactions using an enzyme complex according to one aspect.

[0134] Average Molecular Weight (MW) of Treatment Groups Example 1: 1946.3 Comparative Example 1: 2228.4 Comparative Example 2: 2589.5 Comparative Example 3: 2563.0 Comparative Example 4: 2705.0

[0135] Experimental Example 2. Confirmation of skin barrier strengthening effect.

[0136] To confirm the skin barrier effect of the low molecular weight oat peptide mixture of Example 1 and the oat water extract prepared in Comparative Example 1, an experiment was conducted in the following manner.

[0137] Specifically, to confirm the expression levels of skin barrier strengthening factors (ELOVL 4, ABCA12, Filaggrin, Claudin-1), HaCaT human keratinocyte cells were cultured in DMEM medium (Dulbecco's modified Eagle's Medium, Gibco 1210-0038) containing 10% fetal bovine serum and 1% antibiotic-antimycotic solution, and all cultures were performed in a 37°C, 5% CO2 incubator. The cultured cell lines were treated with 1 or 10 ppm of the oat water extract (Comparative Example 1) or 1 or 10 ppm of the low molecular weight oat peptide mixture (Example 1) and cultured for an additional 24 hours. 1 μM of retinoic acid (RA) was used as a positive control. Real-time polymerase chain reaction was performed on a real-time PCR machine using primers for ELOVL 4, ABCA12, Filaggrin, and Claudin-1, which are factors associated with skin barrier strengthening. The expression levels of ELOVL 4, ABCA12, Filaggrin, and Claudin-1 were finally analyzed after correction for the β-actin gene.

[0138] As a result, as shown in FIGS. 3 to 6, it was confirmed that the low molecular weight oat peptide mixture (Example 1) showed a significantly superior effect of increasing the expression levels of ELOVL 4, ABCA12, Filaggrin, and Claudin-1 compared to the same concentration of oat water extract (Comparative Example 1), indicating that the skin barrier strengthening effect is excellent.

[0139] Experimental Example 3. Confirmation of effects for improving skin inflammation and skin itching.

[0140] In order to confirm the skin inflammation improvement effect and skin itching improvement effect of the low molecular weight oat peptide mixture of Example 1 and the oat water extract prepared in Comparative Example 1, an experiment was conducted in the following manner.

[0141] Specifically, to determine the expression levels of skin inflammatory factors (TNF-α, IL-6) and pruritus factor (TSLP), HaCaT human keratinocyte cells were cultured in DMEM medium (Dulbecco's modified Eagle's Medium, Gibco 1210-0038) containing 10% fetal bovine serum and 1% antibiotic-antimycotic solution, and all cultures were performed in a 37°C, 5% CO2 incubator. Cells were seeded into each well containing DMEM medium free of FBS, and inflammation was induced in the keratinocyte cell line by adding Poly I:C at 10 µg / ml and IL-4 at 10 ng / ml. Next, the low molecular weight oat peptide mixture (Example 1) was treated at concentrations of 1 and 10 ppm, followed by additional culture for 4 hours. Dexamethasone 1 μM was used as a positive control. The expression levels of TNF-α, IL-6, and TSLP were finally analyzed after correction for the β-actin gene.

[0142] As a result, as shown in Figure 7, it was confirmed that the low molecular weight oat peptide mixture (Example 1) had a significantly superior effect in reducing the expression levels of TNF-α, IL-6, and TSLP, indicating that it has excellent effects in improving skin inflammation and skin itching.

[0143] Experimental Example 4. Confirmation of skin itching improvement effect.

[0144] To confirm the skin itching improvement effect of the low molecular weight oat peptide mixture of Example 1 above, an experiment was conducted in the following manner.

[0145] Specifically, a clinical evaluation regarding the efficacy of relieving skin itching was conducted on five adults with an average age of 29.0 years. A test product in the form of a cream containing 0.02 wt% of Example 1 was used as a sample, and the subjects continuously applied the sample (test product) to the skin areas experiencing itching twice a day for a total of 4 weeks. First, to measure the moisturizing improvement efficacy, the skin area of ​​the test subjects was cleaned with alcohol and then stabilized by waiting for 10 minutes under constant temperature and humidity conditions (22±2℃, relative humidity 40–60%). Subsequently, skin moisture levels were measured using a Corneometer (CK electronic, Germany) before sample application (week 0), and after 2 and 4 weeks of use, respectively. Moisture levels were measured a total of three times to calculate the average value, and the change compared to before use was analyzed.

[0146] At the same time, a Subjective Self-Assessment of pruritus was conducted by the subjects. The degree of itching was subdivided into a 6-point scale ranging from 0 points (not felt at all) to 5 points (discomfort in daily life), and surveys were conducted before sample use (0 weeks), 2 weeks after use, and 4 weeks after use.

[0147] As a result, as shown in Figure 12, it was confirmed that the application of the test product containing a low molecular weight oat peptide mixture (Example 1) improved skin moisture levels compared to before use and reduced itching caused by dryness, thereby effectively alleviating itching symptoms caused by dryness.

[0148] Experimental Example 5. Confirmation of skin redness improvement effect.

[0149] To confirm the skin redness improvement effect of the low molecular weight oat peptide mixture of Example 1 above, an experiment was conducted in the following manner.

[0150] Specifically, the average hemoglobin value was analyzed using the skin analysis device Antera 3D (Miravex, UK). First, one adult (31 years old) was selected as a subject. The inner forearm, the test site, was washed with IVORY soap, and the skin condition was stabilized by waiting for 10 minutes in a constant temperature and humidity room (22±2℃, relative humidity 40–60%) to minimize the influence of the external environment. Subsequently, the initial redness of the area (before damage) was photographed using Antera 3D. Then, to induce skin damage caused by physical stimulation, D-squame tape was attached to the test site, and the redness (after damage) was photographed and measured using the same device after stripping 10 times.

[0151] Subsequently, a test product in the form of a cream containing 0.02 wt% of Example 1 and a control product in the form of a cream not containing Example 1 were applied to the damaged area, respectively. The control product was prepared in the same manner as the test product, except that it did not contain Example 1, and an untreated control area was established for comparison. The redness recovery rate (%) was calculated according to Formula 1 below based on the hemoglobin average value obtained from a total of three repeated measurements. The hemoglobin value obtained through Antera 3D analysis is a value representing the degree of skin erythema, and it was determined that the skin soothing and redness improvement efficacy of the composition of the present invention is superior as the value decreases.

[0152] [Formula 1]

[0153]

[0154] As a result, as shown in Figures 10 and 11, it was confirmed that the redness of the damaged skin was significantly reduced upon application of the test product containing the low molecular weight oat peptide mixture (Example 1) compared to the unapplied control group or control product, indicating that the skin redness improvement effect is excellent.

[0155] Experimental Example 6. Confirmation of synergistic effect of low molecular weight oat peptide mixture and panthenol.

[0156] In order to confirm the synergistic effect of the composition containing the low molecular weight oat peptide mixture and panthenol prepared in Example 2 above, an experiment was conducted to confirm the skin barrier strengthening effect and skin soothing effect in the following manner.

[0157] Specifically, to determine the expression levels of skin soothing factors (IL-1β, TNF-α) and barrier strengthening factors (CLDN1), HaCaT human keratinocyte cells were cultured in DMEM medium (Dulbecco's modified Eagle's Medium, Gibco 1210-0038) containing 10% fetal bovine serum and 1% antibiotic-antimycotic solution, and all cultures were performed in a 37°C, 5% CO2 incubator. Cells were seeded into each well containing DMEM medium without FBS, and inflammation was induced in the keratinocyte cell line by adding Poly I:C at 10 µg / ml and IL-4 at 10 ng / ml. Next, the low molecular weight oat peptide mixture (Example 1), panthenol (Comparative Example 5), or a composition containing the low molecular weight oat peptide mixture and panthenol (Example 2) was treated at a concentration of 10 ppm, followed by additional incubation for 4 hours and 24 hours for sedation and barrier evaluation, respectively. Dexamethasone 1 μM and retinoic acid (RA) 1 μM were used as positive controls. Real-time polymerase chain reaction was performed on a real-time PCR machine using primers for IL-1β, TNF-α, and Claudin-1. The expression levels of IL-1β, TNF-α, and Claudin-1 were finally analyzed after correction for the β-actin gene.

[0158] As a result, as shown in Figures 8 and 9, compared to the use of the low molecular weight oat peptide mixture (Example 1) or panthenol (Comparative Example 5) alone under the same concentration conditions (10 ppm), it was confirmed that the composition containing the low molecular weight oat peptide mixture and panthenol (Example 2) showed an excellent effect of increasing Claudin-1 expression and decreasing IL-1β and TNF-α expression, indicating that it has an excellent effect in improving skin inflammation and skin itching.

[0159] In summary, it was confirmed that the low molecular weight oat peptide mixture prepared by the method according to one aspect exhibits significantly superior effects in increasing the expression levels of ELOVL 4, ABCA12, Filaggrin, and Claudin-1, or decreasing the expression levels of TNF-α, IL-6, and TSLP, and has excellent effects in strengthening the skin barrier, improving skin inflammation, alleviating itching, and improving skin redness. Furthermore, it was confirmed that when used in combination with panthenol, it produces a synergistic effect with even superior skin barrier strengthening and skin soothing effects, and thus can be used in cosmetic compositions, topical skin compositions, pharmaceutical compositions, etc.

Claims

1. A step of adding acid to oat extract to obtain an oat protein precipitate; and A method for preparing a low-molecular-weight oat peptide mixture, comprising the step of adding an enzyme complex to the oat protein precipitate to obtain a low-molecular-weight oat peptide mixture.

2. A method for preparing a low molecular weight oat peptide mixture according to claim 1, wherein the oat extract is an extract obtained by extracting oats with a C1-C6 alcohol, water, or a mixture thereof as a solvent.

3. A method for preparing a low-molecular-weight oat peptide mixture according to claim 1, wherein the enzyme complex is added in an amount of 0.1 to 10 weight% relative to the weight of the oat protein precipitate.

4. A method for preparing a low molecular weight oat peptide mixture according to claim 1, wherein the enzyme complex comprises one or more selected from the group consisting of papain, bromelain, alkalase, and protamax.

5. A method for preparing a low molecular weight oat peptide mixture according to claim 4, wherein the enzyme complex comprises papain and bromelain in a weight ratio of 1:0.1 to 10.

6. A method for preparing a low molecular weight oat peptide mixture according to claim 1, wherein the low molecular weight oat peptide mixture has an average molecular weight (MW) of less than 2500 Da.

7. A cosmetic composition for improving skin condition comprising a low molecular weight oat peptide mixture prepared by the method of any one of claims 1 to 6.

8. A cosmetic composition according to claim 7, wherein the improvement of the skin condition is strengthening the skin barrier, improving skin inflammation, soothing the skin, improving itching, or improving skin redness.

9. A cosmetic composition according to claim 7, wherein the composition further comprises panthenol.

10. A cosmetic composition according to claim 9, wherein the panthenol is included in an amount of 30 to 70 weight% based on the total weight of the composition.

11. A topical skin composition for improving skin condition comprising a low molecular weight oat peptide mixture prepared by the method of any one of claims 1 to 6.

12. A pharmaceutical composition for the prevention or treatment of skin inflammatory diseases comprising a low molecular weight oat peptide mixture prepared by the method of any one of claims 1 to 6.

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