Method for extracting low-molecular weight collagen using pollack skin, low-molecular weight collagen, and cosmetic composition comprising same with skin regeneration efficacy

Abstract

The present invention relates to a method for extracting low-molecular weight collagen using pollack skin, low-molecular weight collagen, and a cosmetic composition comprising same with skin regeneration efficacy.

Description

Title of the invention

[0001] Method for extracting low molecular weight collagen using pollack skin, low molecular weight collagen, and a cosmetic composition having skin regeneration efficacy containing the same

[0002] The present invention relates to a method for extracting low molecular weight collagen using pollack skin, low molecular weight collagen, and a cosmetic composition having skin regeneration efficacy containing the same.

[0003] As environmental pollution resulting from modern industrialization, as well as fine dust and ultraviolet rays in the atmosphere, have undesirable effects on the skin of modern people, leading to skin roughness and accelerated skin aging, there is a growing demand for preventing dryness and skin roughness by blocking moisture evaporation and maintaining skin moisture for a long time, as well as for improving wrinkles caused by photoaging.

[0004] The skin is the outermost organ of the human body and serves as a barrier protecting our body from harmful external substances such as microorganisms, irritants, and pollutants. The skin is composed of the epidermis, dermis, and subcutaneous fat layer. The epidermis is divided into the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale according to the keratinization process. Most of the cells composing the epidermis are keratinocytes, which form the skin barrier by synthesizing proteins such as transglutaminase-1 and filaggrin, and also participate in skin moisturization by synthesizing hyaluronic acid synthase (HAS), which produces hyaluronic acid, and aquaporin-3, which is involved in the movement of water and glycerol (Jiwon H, et al., 2021). Therefore, strengthening the skin barrier can be considered an essential requirement for skin health.

[0005] The skin is broadly divided into three layers in order from the outside: the epidermis, dermis, and subcutaneous adipose tissue. It functions to protect the human body from physical and chemical stimuli from the external environment. In particular, the skin regulates the evaporation of approximately 65–70% of the body's water. Among these, the epidermis is divided from the outside in the order of the stratum corneum, stratum granulosum, stratum spinosum, and stratum basale. The stratum corneum of the epidermis contains about 10–20% water and is located at the outermost layer of the body, inhibiting water evaporation from the body while blocking the excessive penetration of substances from the outside (J. Invest. Dermatol. 80(Suppl.), 44-49. 1983). The cells constituting this stratum corneum contain high concentrations of Natural Moisturizing Factor (NMF), a water-soluble component, which not only helps the skin exhibit flexibility but also helps maintain adequate moisture (J. Invest. Dermatol. 54, 24-31, 1970). However, due to causes such as environmental changes, changes in lifestyle patterns, various stresses and environmental pollution arising from social life, frequent washing due to makeup habits, and natural skin aging due to increasing age, the moisture in the stratum corneum decreases, causing the skin to become dry, rough on the surface, and lose its luster, appearing dull. Consequently, the importance of skin moisturization is growing.

[0006] Recently, among the factors for skin health, natural moisturization, anti-aging, skin regeneration, and elasticity improvement are important, along with strengthening the skin barrier.

[0007] In particular, collagen, which is essential for skin regeneration and elasticity improvement, is an abundant fibrous structural protein of animal origin that accounts for 30% of the total protein in vertebrates and invertebrates. It forms a triple helix structure and plays a role in forming most of the organic matter in the skin, bones, and teeth in various forms, with particularly high content in bones and skin (dermis). The basic unit of collagen is troprocollagen, which possesses physical or biological stability through intramolecular or intermolecular covalent cross-linking. While the amino acid composition of collagen varies slightly depending on the type, glycine typically accounts for about one-third of the total, proline for one-fourth, and hydroxyproline for one-seventh. Hydroxyproline, an amino acid constituting collagen, forms a specific ratio (12.5–14%) along with hydroxylysine, serving as an indicator component for quantifying collagen; it is known that the composition ratio of collagen varies depending on the species and age of the organism.

[0008] Regarding research on collagen derived from marine products, many studies have been reported on collagen extracted from the connective tissues and skin of vertebrates and invertebrates such as starfish, squid, jellyfish, and skates. Various studies on these fish collagens, including extraction conditions and physicochemical properties, have been reported using species such as the red snapper (Lutjanus lutjanus) (Kittiphattanabawon et al. 2005), sea bass (Lates niloticus) (Muyonga et al. 2004), yellowfin tuna (Thunnus albacares) (Woo et al. 2008), skate (Sebastes mentella) (Wang et al. 2008), minke whale (Balaenoptera acutorostrata) (Nagai et al. 2008), black snapper (Pogonias cromis), and Japanese snapper (Archosargus probatocephalsu) (Ogawa et al. 2003).

[0009] Most collagen research regarding the aforementioned seafood utilizes skins, bones, and scales obtained as by-products of fishery processing. Since skins and bones obtained as fishery processing by-products account for approximately 30% or more of the total volume and contain large amounts of collagen, the production of fish collagen using these by-products to replace collagen derived from land animals is of great significance.

[0010] Collagen has been widely utilized in the pharmaceutical, cosmetic, and food industries, and recently, its applications have expanded to include anti-aging, skin elasticity improvement, and arthritis prevention. Currently, most collagen products on the market are derived from land animals such as cattle and pigs; however, recent outbreaks of Mad Cow Disease and Foot-and-Mouth Disease have caused difficulties in collagen production. Consequently, there is a growing need for research to replace land-based collagen, and interest is increasing in the extraction of collagen using aquatic resources that are safe from Mad Cow Disease and Foot-and-Mouth Disease.

[0011] However, in Korea, which consumes a large amount of seafood amidst a shortage of collagen sources, fish processing residues are increasing during the processing of fish and shellfish. Among these, pollock consumption is approximately 400,000 tons, with skin accounting for 8 to 10% of the total, resulting in a significant amount of byproduct discharge. However, most of this is discarded due to low utilization rates, leading not only to resource waste but also to environmental pollution. Recently, there has been an increasing trend in the utilization of refined collagen from pollock skin, and it is believed that utilizing this byproduct as an industrial material would be of great significance.

[0012] However, hydrolyzing proteins has the disadvantage of causing the loss of essential amino acids such as tryptophan and cysteine. Furthermore, while acid hydrolysis requires controlling the salt generated during the neutralization process, enzymatic hydrolysis does not require such a process, making it easier to adjust salt levels during the product manufacturing stage. Additionally, acid-based protein degradation typically breaks down proteins to the amino acid ends, whereas enzymatic hydrolysis limits the breakdown of proteins, making it difficult to extract collagen from natural products.

[0013] Therefore, there is a need to research methods for easily and safely extracting low-molecular-weight collagen from natural extracts and to develop cosmetics with excellent wrinkle improvement, antioxidant activity, and skin regeneration efficacy by incorporating this into skin regeneration cosmetic compositions.

[0014] Patent Document 1: Korean Registered Patent Document No. 10-1971291

[0015] One embodiment of the present specification aims to provide a method for extracting low molecular weight collagen using pollack skin, low molecular weight collagen, and a cosmetic composition having skin regeneration efficacy containing the same.

[0016] One embodiment of the present specification provides a method for extracting low-molecular-weight collagen comprising: a cleaning step of cleaning a pollack to separate the skin, washing it once with water, and then washing it twice under running water; a step of pre-treating the pollack skin by washing it with distilled water containing a natural extract; a step of extracting collagen by boiling the pre-treated pollack skin in purified water; and a step of demolding the collagen, wherein the step of demolding the collagen comprises: a) an acid treatment step of adjusting the pH of the collagen to 2 to 4; b) a step of demolding the collagen once by adding a primary acidic active enzyme to the acid-treated collagen; c) a step of neutralizing the pH of the collagen once demolded to 7 to 8; and d) a step of demolding the collagen once by adding a secondary neutral active enzyme to the neutralized collagen once demolded.

[0017] One embodiment of the present specification provides a method for extracting low molecular weight collagen, comprising: b) a step of adding a primary acidic active enzyme to acid-treated collagen to perform primary low molecular weight reduction, and then passing it through an ultrafiltration membrane to increase the content of low molecular weight collagen of 3,000 Da or less; and c) a step of neutralizing the pH of the primary low molecular weight collagen to 7 to 8; and then passing it through an ultrafiltration membrane to increase the content of low molecular weight collagen of 1,000 Da or less.

[0018] According to one embodiment of the present specification, a method for extracting low molecular weight collagen is provided, wherein the primary acidic active enzyme comprises pepsin, the secondary neutral active enzyme comprises pancreatin and nutrasase, and the pancreatin comprises 0.01 parts by weight or more and 10 parts by weight or less based on 100 parts by weight of the secondary neutral active enzyme; and the nutrasase comprises 90 parts by weight or more and 99.99 parts by weight or less.

[0019] According to one embodiment of the present specification, a method for extracting low molecular weight collagen is provided, further comprising: d) a step of adding a secondary neutral active enzyme to the neutralized primary low molecular weight collagen to perform secondary low molecular weight reduction, and then adding a tertiary neutral active enzyme to the secondary low molecular weight collagen to perform tertiary low molecular weight reduction; and a step of passing through an ultrafiltration membrane to increase the content of low molecular weight collagen of 3,000 Da or less, wherein the tertiary neutral active enzyme is collagenase.

[0020] According to one embodiment of the present specification, a method for extracting low molecular weight collagen is provided, wherein the active temperature in the first low molecular weight step; the second low molecular weight step and the third low molecular weight step is 30°C or higher and 40°C or lower, and the active time is within 12 hours.

[0021] In one embodiment of the present application, the natural extract comprises green tea extract and lemon extract, wherein the lemon extract comprises 20 parts by weight or more and 40 parts by weight or less based on 100 parts by weight of the natural extract; and the green tea extract comprises 60 parts by weight or more and 80 parts by weight or less. A method for extracting low molecular weight collagen is provided.

[0022] One embodiment of the present application provides low molecular weight collagen extracted through the above-described method for extracting low molecular weight collagen.

[0023] In the present application, the low molecular weight collagen provides a low molecular weight collagen containing 60 wt% or more of collagen with a molecular weight of 1,000 Da or less.

[0024] One embodiment of the present application provides a cosmetic composition having wound healing and skin regeneration efficacy comprising the aforementioned low molecular weight collagen; and an amino acid including proline.

[0025] Finally, the present application provides a cosmetic composition having wound healing and skin regeneration efficacy, wherein, based on 100 parts by weight of the total cosmetic composition, the low molecular weight collagen is included in an amount of 1 part by weight or more and 5 parts by weight or less, and the amino acid is included in an amount of 10 parts by weight or more and 15 parts by weight or less.

[0026] The present invention is characterized by preparing pollack in a unique manner to separate the skin, extracting collagen from the skin, and, in particular, forming low-molecular-weight collagen using enzymes under specific pH conditions.

[0027] When a cosmetic composition is formed using amino acids containing proline along with the low molecular weight collagen, the effects of wrinkle improvement and skin elasticity enhancement can be enhanced. Furthermore, by using natural materials, it is possible to reduce allergic reactions and skin irritation, which were previously problematic, while simultaneously strengthening the skin barrier and providing effects of natural moisturization, anti-aging, skin regeneration, and elasticity improvement.

[0028] Accordingly, the present invention aims to provide a cosmetic composition comprising low molecular weight collagen extracted from pollock, which has a skin irritation reduction effect, a skin elasticity enhancement effect, and an excellent regenerative function.

[0029] Figure 1 is data analyzing the weight average molecular weight and composition content of low molecular weight collagen according to Example 1.

[0030] Figures 1 to 4 are diagrams showing an antioxidant evaluation according to the present application.

[0031] Figure 5 is a figure showing a cell viability experiment according to the present application.

[0032] Figure 6 is a figure for confirming the intracellular regenerative effect of Example 1 according to the present application.

[0033] The inventors have completed the present invention by researching and developing a cosmetic composition capable of achieving all of the aforementioned effects, namely, significantly improved skin irritation, improved skin elasticity enhancement, and improved skin regeneration and long-lasting moisturization. As a result, they confirmed that all of these effects can be achieved by preparing pollock in a unique manner to separate the skin, extracting collagen from the skin, and, in particular, using collagen that has been broken down into low molecular weight collagen using enzymes under specific pH conditions, while simultaneously using an amino acid containing proline. Therefore, the objective of the invention is to provide a cosmetic composition that exhibits improved effects. Other objectives and advantages of the present invention will become more apparent from the following detailed description of the invention and the claims.

[0034]

[0035] Method for Extracting Low Molecular Weight Collagen

[0036] The present application provides a method for extracting low-molecular-weight collagen comprising: a cleaning step of cleaning a pollack to separate the skin, washing it once with water, and then washing it twice under running water; a step of pre-treating the pollack skin by washing it with distilled water containing a natural extract; a step of extracting collagen by boiling the pre-treated pollack skin in purified water; and a step of demolding the collagen, wherein the step of demolding the collagen comprises: a) an acid treatment step of adjusting the pH of the collagen to 2 to 4; b) a step of demolding the collagen once by adding a primary acidic active enzyme to the acid-treated collagen; c) a step of neutralizing the pH of the demolded collagen once by adding a secondary neutral active enzyme to the collagen once by demolding it

[0037] One embodiment of the present application relates to a cleaning step in which a pollack is cleaned to separate the skin, washed once with water, and then washed twice under running water.

[0038] The above cleaning step corresponds to a step of removing physical impurities or foreign substances by washing the dried or semi-dried pollack with water once and then washing it under running water twice, and corresponds to a step of separating the head, tail, and body parts of the pollack and, in particular, separating and securing only the skin of the pollack.

[0039] Next, the present application provides a step of pre-treating the pollack skin by washing the pollack skin with distilled water containing a natural extract.

[0040] The above step is intended to increase the efficiency of the enzyme reaction described later by removing fatty components and minerals from the pollack skin, and for this purpose, it includes a step of washing the surface several times with distilled water to remove surface impurities and inorganic salts.

[0041] At this time, the present application is characterized by including a specific natural extract in the pretreatment step.

[0042] The term "extract" in this specification follows the dictionary definition and refers to a product obtained by concentrating a specific component in an extract, wherein a specific active component is separated using a suitable solvent, and the solvent is completely or mostly evaporated to obtain a remaining mass or powder which is then adjusted according to standards. In this specification, the term "extract" may be used to include all three forms: semi-fluid or syrup-like, pill-like or solid, dried powder, etc.

[0043] In particular, the present application provides a method for extracting low molecular weight collagen, wherein the natural extract comprises green tea extract and lemon extract, and based on 100 parts by weight of the natural extract, the lemon extract comprises 20 parts by weight or more and 40 parts by weight or less; and the green tea extract comprises 60 parts by weight or more and 80 parts by weight or less.

[0044] In another embodiment, based on 100 parts by weight of the natural extract, the lemon extract comprises 30 parts by weight; and the green tea extract comprises 70 parts by weight.

[0045] As described above, by pre-treating pollack skin with natural extracts containing the above amounts, the acidic components of lemon and catechins of green tea can aid in fat breakdown, and when used within the above range, the removal of impurities and inorganic salts along with fat breakdown can be particularly effective.

[0046] The present application comprises the steps of: boiling the pretreated pollack skin in purified water to extract collagen; and breaking down the collagen into low molecular weights.

[0047] If the existing step was to separate the pollock skin and facilitate the extraction of collagen from the pollock skin, the above step corresponds to the step of extracting collagen from the pollock skin and reducing its molecular weight, which can be considered a characteristic part of the present application.

[0048] In the present application, the pre-treated pollack skin is placed in purified water and boiled to first extract the collagen component.

[0049] Subsequently, the method includes a step of reducing the collagen to a low molecular weight. Collagen is composed of various molecular weights and is known to be particularly excellent for wrinkle improvement and skin regeneration. However, since the aforementioned effects are negligible in the case of high molecular weight collagen, the cosmetic composition according to the present application uses low molecular weight collagen.

[0050] In the present application, the step of reducing the collagen may include a) an acid treatment step of reducing the pH of the collagen to 2 to 4, b) a step of reducing the collagen by adding a primary acidic active enzyme to the acid-treated collagen, c) a step of neutralizing the pH of the primary reduced collagen to 7 to 8; and d) a step of reducing the collagen by adding a secondary neutral active enzyme to the neutralized primary reduced collagen.

[0051] The step of reducing the molecular weight of collagen according to the present application is characterized by being carried out in a first and second step as described above, and is further characterized by including the third step of reducing molecular weight described later to obtain collagen with a further reduced molecular weight compared to existing low molecular weight collagen.

[0052] First, the first low-molecular-weighting step includes a) an acid treatment step of adjusting the pH of the collagen to 2 to 4, and b) a step of adding a primary acidic active enzyme to the acid-treated collagen to perform the first low-molecular-weighting.

[0053] In other words, the first step of demoleculing the collagen corresponds to the step of demoleculing the collagen by first introducing an enzyme that is active under acidic conditions.

[0054] In the present application, the primary acid-active enzyme may be pepsin. Pepsin is an enzyme effective in breaking down collagen into small molecules by cleaving the N-terminus of the collagen in pollock skin, and since the enzyme exhibits good activity in acidic conditions, the present application is characterized by first lowering the pH of the extracted collagen to control it to an acidic state.

[0055] The above b) further includes a step of adding a primary acidic active enzyme to acid-treated collagen to perform primary low molecular weight reduction, and then passing it through an ultrafiltration membrane to increase the content of low molecular weight collagen of 3,000 Da or less.

[0056] In the case of the ultrafiltration membrane according to the present application, the pore size within the filtration membrane may be 50 nm or less, specifically 40 nm or less, 1 nm or more, or 10 nm or more.

[0057] In the present application, the step of passing through the ultrafiltration membrane may include the step of pressurizing the collagen with nitrogen or compressed air to pass it through the ultrafiltration membrane.

[0058] As described above, collagen with a low molecular weight can be obtained by passing the primary low molecular weight collagen through an ultrafiltration membrane.

[0059] In the present application, the pH of the collagen can be adjusted to a range of 2 to 4 by adding 5N-HCl, and the first low-molecular-weighting step can be carried out for 6 to 12 hours.

[0060] The present application is characterized by secondarily low-molecular-weighting the primary low-molecular-weight collagen.

[0061] That is, it includes c) a step of neutralizing the pH of the primary low-molecular-weight collagen to 7 to 8; and d) a step of secondary low-molecular-weighting by adding a secondary neutralizing enzyme to the neutralized primary low-molecular-weight collagen.

[0062] The present application is characterized by steps of first and second demoleculing, and in particular, focuses on the demoleculing of collagen by adjusting the pH of each step so that the enzyme used can be optimized and activated.

[0063] In the present application, the secondary neutral active enzyme comprises pancreatin and nutrasase, wherein, based on 100 parts by weight of the secondary neutral active enzyme, the pancreatin may comprise 0.01 parts by weight or more and 10 parts by weight or less; and the nutrasase may comprise 90 parts by weight or more and 99.99 parts by weight or less.

[0064] More specifically, based on 100 parts by weight of the secondary neutral active enzyme, the pancreatin may comprise 0.01 parts by weight or more and 10 parts by weight or less, specifically 5 parts by weight, and the nutrase may comprise 95 parts by weight.

[0065] When the above-mentioned pancreatin and nutrasase are used together, they can degrade collagen more effectively. They contain various proteolytic enzymes, enabling extensive degradation, and exhibit good activity, particularly under neutral conditions as described above. However, since non-specific degradation may occur with pancreatin, it is necessary to adjust the amount used within the aforementioned range; when used within this range, effective degradation of collagen can be achieved. Additionally, nutrasase is characterized by high activity at neutral pH, allowing for additional degradation at neutral pH after pretreatment with pepsin.

[0066] The above includes c) a step of neutralizing the pH of the primary low-molecular-weight collagen to 7 to 8; and then a step of passing it through an ultrafiltration membrane to increase the content of low-molecular-weight collagen of 1,000 Da or less.

[0067] The above ultrafiltration membrane is the same as described above.

[0068] The above secondary low-molecular-weighting step may be carried out for 3 to 4 hours, and through the secondary low-molecular-weighting step as described above, decomposition can be carried out more finely.

[0069] It may optionally include a step of tertiary low molecular weight reduction.

[0070] That is, d) a step of adding a secondary neutralizing enzyme to the neutralized primary low-molecular-weight collagen to perform secondary low-molecular-weight reduction, followed by a step of adding a tertiary neutralizing enzyme to the secondary low-molecular-weight collagen to perform tertiary low-molecular-weight reduction; and a step of passing through an ultrafiltration membrane to increase the content of low-molecular-weight collagen of 300 Da or less; wherein the tertiary neutralizing enzyme may be collagenase.

[0071] In the present application, the collagenase is a collagen-specific degrading enzyme, but since it is expensive and the reaction time may be long, it corresponds to a step of including it if the goal is to achieve very low molecular weight of 300 Da or less.

[0072] A very small molecular weight can be obtained through the above third low-molecular-weighting step, and the above third low-molecular-weighting step can be carried out for 12 hours.

[0073] In the present application, the active temperature in the first low-molecular-weighting step; the second low-molecular-weighting step and the third low-molecular-weighting step is 30°C or higher and 40°C or lower, and the active time may be within 12 hours.

[0074] In the case of the primary, secondary, and tertiary enzymes according to the present application, the reaction is most active at the above temperature and can be efficient, and if the above temperature range is exceeded, the enzyme is inactivated and the objective of low molecular weight cannot be achieved.

[0075] In the present application, the final low-molecular-weight collagen can be obtained by further including: d) a step of removing substrates, by-products, and enzymes after a step of adding a secondary neutralizing active enzyme to the neutralized primary low-molecular-weight collagen to perform secondary low-molecular-weight reduction; and a step of separating insoluble substances and solutions by rotating at high speed through centrifugation.

[0076] In other words, high-purity collagen is obtained by removing unreacted substrates, byproducts, and enzymes after the enzymatic reaction, and the final product can be secured by separating insoluble substances and the solution through high-speed centrifugation.

[0077]

[0078] Low molecular weight collagen

[0079] The present application provides low molecular weight collagen extracted through the low molecular weight collagen extraction method described above.

[0080] In the present application, the low molecular weight collagen may contain 60 wt% or more of collagen with a molecular weight of 1,000 Da or less.

[0081] In the present application, the low molecular weight collagen may be 1,000 Da or less, specifically 200 Da or less, more specifically 180 Da or less, and may contain 60 wt% or more and 95 wt% or less of collagen of 50 Da or more.

[0082] In the present application, the average molecular weight of the low molecular weight collagen may be 100 Da to 1,050 Da.

[0083] More specifically in the present application, the average molecular weight of the low molecular weight collagen may be 100 Da to 1,050 Da, 150 Da to 950 Da, or 150 Da to 300 Da.

[0084] That is, by undergoing the manufacturing method described above to produce low molecular weight collagen having the molecular weight and weight described above, when included in the cosmetic composition described later, it possesses the characteristic of excellent skin regeneration effect.

[0085] In the present application, the average molecular weight of the low molecular weight collagen may be 100 Da to 1,050 Da.

[0086] <Cosmetic composition having wound healing and skin regeneration efficacy>

[0087] The present application provides a cosmetic composition having wound healing and skin regeneration efficacy comprising the above-mentioned low molecular weight collagen; and an amino acid including proline.

[0088] As described above, the present application comprises low molecular weight collagen produced by a specific manufacturing method, and the cosmetic composition containing it is characterized by improved skin regeneration efficacy along with wrinkle improvement, skin antioxidant and wound healing effects.

[0089] In the present application, a cosmetic composition having wound healing and skin regeneration efficacy is provided, wherein, based on 100 parts by weight of the total cosmetic composition, the low molecular weight collagen is included in an amount of 1 part by weight or more and 5 parts by weight or less, and the amino acid is included in an amount of 10 parts by weight or more and 15 parts by weight or less.

[0090] The above amino acid has an amino acid sequence of glycine-hydroxyproline, and the proline may have 15.0 to 20.0 mol% of the total molar amount of the above amino acid.

[0091] The cosmetic composition of the present invention may be prepared in any formulation conventionally manufactured in the industry, for example, as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, surfactant-containing cleansing, powder foundation, emulsion foundation, wax foundation, and spray, but is not limited thereto. More specifically, it may be prepared in the form of a softening lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, or a wash-off pack.

[0092] In the case where the formulation of the present invention is a paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, cellulose derivative, polyethylene glycol, silicone, bentonite, kaolin, silica, talc, or zinc oxide may be used as a carrier component.

[0093] When the formulation of the present invention is a solution or an emulsion, a solvent, a solubilizing agent, or an emulsifying agent is used as a carrier component, such as water, ethanol, glycerin, methylpropanol, pentylene glycol, isoprene glycol, propylene glycol, 1,3-butylene glycol, glycerol aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan. When the formulation of the present invention is a suspension, a liquid diluent such as water, ethanol, or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracanthera may be used as a carrier component. In the case where the formulation of the present invention is a cleansing agent containing a surfactant, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester, etc. may be used as a carrier component.

[0094] The cosmetic composition according to the present invention can be prepared by mixing the above components in a physical manner. In the present invention, the method of mixing raw materials or homogenizing uses a known microdispersion method, and the method is not particularly limited, but, for example, methods such as sonication, microfluidization, or homogenization may be used.

[0095] The above method may be carried out using known equipment such as an ultrahomogenizer, a microfluidizer, or a homomixer, but is not limited thereto.

[0096] According to one embodiment of the present specification, the cosmetic composition may include an antioxidant substance. The antioxidant substance may include, but is not limited to, an antioxidant active ingredient having the ability to significantly reduce or prevent the destruction and depletion of skin function and structure damaged by oxidation, such as extracts derived from natural products like animals, plants, and minerals, fermented extracts, amino acids, peptides, and proteins. For example, acetamidocaproic acid, acetyl benzoyloxy prasterone, acetyl cysteine, 2-acetylhydroquinone, adamantanylcarboxamido hydroxybenzamide, aminoethanesulfinic acid, aminopropyl ascorbyl phosphate, angoroside C, anserine, apigenin, arbutin, alpha-albutin, ascorbic acid, asiaticoside, benzoquanamine, BHA (butylhydroxyanisol), BHT (butylated Hydroxy Toluene), bis-hydroxyethyl tocopherylsuccinoylamido hydroxypropane, bis-(tetramethyl hydroxypiperidinyl) sebacate, butylated xylenol, t-butylbenzamido hydroxylbenzamide,4-butylresorcinol, caffeic acid, calcium ascorbate, carnosic acid, carotenoids, chitosan ascorbate, chlorogenic acid, cobalt DNA, copper adenosine triphosphate, copper pyridoxal 5-phosphate, curcumin, cysteine, cysteine ​​hydrochloride, decapeptide-6, decapeptide-7, decursinol, decylmercaptomethylimidazole, Diamylhydroquinone, di-t-butylhydroquinone, dicetyl thiodipropionate, dicyclopentadiene / t-butylcresol copolymer, digalloyl trioleate, diisooctyl sebacate, dilauryl thiodipropionate, dimethoxybenzamido phenylhydroxyacetamide, dimethoxy di-p-cresol, dimethylmethoxy chromanol, dimyristyl thiodipropionate, dioleyl Dioleyl tocopheryl methylsilanolDiosmine, diretinyl ether, disodium ascorbyl sulfate, distearyl thiodipropionate, ditridecyl thiodipropionate, dodecyl gallate, Dunaliella bardawil powder, ellagic acid, epigallocatechin gallate, ergothioneine, Eriobotrya japonica leaf protoplasts, erythorbic acid, ethylbisiminomethylguaiacol manganese chloride, ethyl ferulate, Ethylhexyl ferulate, ferulicacid, feruloyl soy glycerides, furfuryl palmitate, genistein glucoside, ginkgo leaf terpenoids, glucosylrutin, glyceryl chromonyl ether, glyceryl diferulate, hesperetin, hexyloxy trimethylphenol, honokiol, hydrolyzed proanthocyanidin, hydroquinone, p-hydroxyanisole,Hydroxydecyl ubiquinone, hydroxylamine hydrochloride (hydroxylamine HCl), hydroxylamine sulfate, hydroxyphenyl dihydroxybenzamide, inosinol hexaniacinate hexaascorbate, isooctyl caprylate / caprate, isooctyl thioglycolate, isoquercitrin, kaempferol, kojic acid, kojyl glucoside, kojyl methylenedioxycinnamate, black tea extract (kou-cha ekisu), lycopene, Madecassoside, magnesium ascorbate, magnolol, manganese adenosine triphosphate, matrine, melatonin, methoxy PEG-7 ascorbic acid, methoxytrimethylphenyl dihydroxyphenyl propanol, methyl di-t-butyl hydroxyhydrocinnamate, methylene di-t-butylcresol, methyl methacrylate / trimethoxysilylpropyl methacrylate crosspolymer,Methyl myristic acid, niacinamide hydroxybenzoate, nictoflorin, nordihydroguaiaretic acid, octanicotinoyl epigallocatechin gallate, octapeptide-4, oligopeptide-28, paeonol, palmatine, PEG / PPG-2 / 5 tocopheryl ether, perillyl alcohol, phenethyl caffeate, phenylethyl resorcinol, phenylthioglycolic acid, phloretin, Phloroglucinol, piceatannol, piperlonguminine, porphyridium polysaccharide, potassium ascorbyl tocopheryl phosphate, potassium sulfite, PPG-2 tocophereth-5, propyl gallate, protocatechuicaldehyde, puerarin, pyridoxine hydroxybenzoate, phridyloxide t-butylnitrone, quercetin, resacetophenone, resorcinol, Resveratrol,Retinyl formyl aspartamate, rosmarinic acid, rutin, rutinyl succinate, ryoku-cha ekisu, salnacedin, sodium ascorbate, sodium sulfite, sodium tocopheryl phosphate, sodium erythorbate, sodium phosphono-pyridoxylidenerhodanine, sodium thioglycolate, sodium zinc histidine dithiooctanamide, sorbityl furfural, Stearyl gallate, succinoyl ascorbate pentapeptide-6, tangeritin, TBHQ (tert-butylhydroquinone), tetrabutyl ethylidinebisphenol, tetrahydrodemethoxydiferuloylmethane, tetrahydrocurcumin diacetate, tetrahydrodiferuloylmethane, tetramethylbutyl dihydroxybenzamide, tetramethylchromanol glucoside, thioctic acid, thiodiglycol, thiolactic acid,Thiosalicylic acid, thioaurine, thymol trimethoxycinnamate, tococysteamide, tocophereth-5, tocopherol, tocophersolan, tocopheryl acetate, tocoquinone, toluene, o-tolyl biguanide, totarol, tripropylene glycol, tris-BHT mesitylene, tris(nonylphenyl)phosphate, trisodium ascorbyl isopalmitate phosphate, tyrosyl hisdidine It may be one or more selected from the group consisting of hydrochloride (tyrosyl histidine HCl), ubiquinol, ubiquinone, Ulva lactuca powder, oolong tea extract, xylyl dibutylbenzofuranone, zinc ATP (zincadenosine triphosphate), zinc dibutyldithiocarbamate, zinc fructose diphosphate, zinc pyridoxal 5-phosphate, and derivatives thereof.

[0097] More specifically, the antioxidant substance may be selected from the group consisting of acetamidocaproic acid, acetylbenzoyloxy prasterone, acetyl cysteine, 2-acetylhydroquinone, adamantanylcarboxamido hydroxybenzamide, aminoethanesulfinic acid, and aminopropyl ascorbyl phosphate.

[0098] A cosmetic composition according to one embodiment of the present specification may include other ingredients commonly used in cosmetics as needed.

[0099] Other ingredients that may be added include oils, moisturizers, emollients, UV absorbers, pH adjusters, blood circulation promoters, cooling agents, and restrictive agents.

[0100] Examples of the above-mentioned oil components include ester-based oils, hydrocarbon-based oils, silicone-based oils, fluorine-based oils, animal oils, and plant oils.

[0101] The above ester-based oils include tri-2-ethylhexanoate glyceryl, 2-ethylhexanoate cetyl, isopropyl myristate, butyl myristate, isopropyl palmitate, ethyl stearate, octyl palmitate, isocetyl isocetyl isostearate, butyl stearate, ethyl linoleate, isopropyl linoleate, ethyl oleate, isocetyl myristate, isostearyl myristate, isostearyl palmitate, octyldodecyl myristate, isocetyl isostearate, diethyl sebacate, diisopropyl adipate, isoalkyl neopentanoate, tri(capryl, capric acid)glyceryl, tri-2-ethylhexanoate trimethylolpropane, triisostearate trimethylolpropane, tetra-2-ethylhexanoate pentaerlislitol, cetyl caprylate, decyl lauricate, Hexyl laurate, Decyl myristate, Myristyl myristate, Cetyl myristate, Stearyl stearate, Decyl oleate, Cetyl lisinooleate, Isostearyl laurate, Isotridecyl myristate, Isocetyl palmitate, Octyl stearate, Isocetyl stearate, Isodecyl oleate, Octyldodecyl oleate, Octyldodecyl linoleate, Isopropyl isostearate, Cetostearyl 2-ethylhexanoate, Stearyl 2-ethylhexanoate, Hexyl isostearate, Ethylene glycol dioctanoate, Ethylene glycol dioleate, Propylene glycol dicaprine, Propylene glycol dicaprylate, Neopentyl glycol dicaprine, Neopentyl glycol dioctanoate, Glyceryl tricaprylate, Glyceryl triundecylate, Glyceryl triisopalmitate, Glyceryl triisostearate, Octyldodecyl neopentanoate, Isostearyl octanoate, Octyl isononanoate, Hexyldecyl neodecanoate, Octyldodecyl neodecanoate, Isocetyl isostearate, Isostearyl isostearate, Octyldecyl isostearate, Polyglycerin oleic acid ester, Polyglycerin isostearic acid ester, Triisocetyl citrate, Triisoalkyl citrate, Triisooctyl citrate, Lauryl lactate, Myristyl lactate, Cetyl lactate, Octyldecyl lactate, Triethyl citrate, Acetyltriethyl citrate, Acetyltributyl citrate, Trioctyl citrate, Diisostearyl malate, 2-ethylhexyl hydroxystearate, Di2-ethylhexyl succinate, Diisobutyl adipate, diisopropyl sebacate, dioctyl sebacate, cholesteryl stearate,Examples include esters such as cholesteryl isostearate, cholesteryl hydroxystearate, cholesteryl oleate, dihydrocholesteryl oleate, pitsteryl isostearate, pitsteryl oleate, isocetyl 12-stealloylhydroxystearate, stearyl 12-stealloylhydroxystearate, and isostearyl 12-stealloylhydroxystearate.

[0102] Examples of the above hydrocarbon oils include squalene, liquid paraffin, alpha-olefin oligomer, isoparaffin, ceresin, paraffin, liquid isoparaffin, polybutene, microcrystalline wax, Vaseline, etc.

[0103] Examples of the above silicone-based oils include polymethylsilicon, methylphenylsilicon, methylcyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecamethylcyclosiloxane, dimethylsiloxane methylcetyloxysiloxane copolymer, dimethylsiloxane methylstealoxysiloxane copolymer, alkyl-modified silicone oil, amino-modified silicone oil, etc.

[0104] Examples of the above-mentioned fluorine-based oils include perfluoropolyethers.

[0105] The above animal or plant oils include avocado oil, almond oil, olive oil, sesame oil, rice bran oil, soybean oil, corn oil, rapeseed oil, apricot kernel oil, palm kernel oil, palm oil, castor oil, sunflower oil, grapeseed oil, cottonseed oil, coconut oil, cucurbit nut oil, wheat germ oil, rice germ oil, shea butter, laurel oil, magma damia nut oil, egg yolk oil, beef tallow, horse oil, mink oil, orange raffia oil, jojoba oil, candelilla wax, carnaba wax, liquid lanole, hydrogenated castor oil, etc.

[0106] Examples of the above moisturizers include water-soluble low-molecular-weight moisturizers, oil-soluble molecular moisturizers, water-soluble polymers, oil-soluble polymers, etc.

[0107] Examples of the above-mentioned water-soluble low molecular weight moisturizers include serine, glutamine, sorbitol, mannitol, pyrrolidone-sodium carboxylate, glycerin, propylene glycol, 1,3-butylene glycol, ethylene glycol, polyethylene glycol B (degree of polymerization n = 2 or more), polypropylene glycol (degree of polymerization n = 2 or more), polyglycerin B (degree of polymerization n = 2 or more), lactic acid, lactate, etc.

[0108] Examples of the above-mentioned fat-soluble low-molecular-weight moisturizers include cholesterol and cholesterol esters.

[0109] Examples of the above water-soluble polymers include carboxyvinyl polymer, polyaspartate, tragacanth, xanthan gum, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, water-soluble chitin, chitosan, dextrin, etc.

[0110] Examples of the above-mentioned lipid-soluble polymers include polyvinylpyrrolidone eicocene copolymer, polyvinylpyrrolidone hexadecene copolymer, nitrocellulose, dextrin fatty acid ester, and polymeric silicone.

[0111] Examples of the above emollients include long-chain acylglutamic acid cholesteryl ester, hydroxystearic acid cholesteryl, 12-hydroxystearic acid, stearic acid, rosin acid, lanolin fatty acid cholesteryl ester, etc.

[0112] The above-mentioned ultraviolet absorbers include para-aminobenzoic acid, ethyl para-aminobenzoate, amyl para-aminobenzoate, octyl para-aminobenzoate, ethylene glycol salicylate, phenyl salicylate, octyl salicylate, benzyl salicylate, butylphenyl salicylate, homomentyl salicylate, benzyl cinnamonate, paramethoxycinnamic acid-2-ethoxyethyl paramethoxycinnamic acid, octyl paramethoxycinnamic acid, diparamethoxycinnamic acid mono-2-ethylhexaneglyceryl, isopropyl paramethoxycinnamic acid, a mixture of diisopropyl and diisopropyl cinnamonic acid esters, urocanic acid, ethyl urocanic acid, hydroxymethoxybenzophenone, hydroxymethoxybenzophenonesulfonic acid and its salts, dihydroxymethoxybenzophenone, sodium dihydroxymethoxybenzophenonedisulfonate, dihydroxybenzophenone, tetrahydroxybenzophenone, Examples include 4-tert-butyl-4'-methoxydibenzoylmethane, 2,4,6-trianilino-p-(carbo-2'-ethylhexyl-1'-oxy)-1,3,5-triazine, 2-(2-hydroxy-5-methylphenyl)benzotriazole, etc.

[0113] Examples of the above pH adjusting agents include citric acid, sodium citrate, malic acid, sodium malate, fumaric acid, sodium fumarate, succinic acid, sodium succinate, sodium hydroxide, sodium monohydrogen phosphate, etc.

[0114] In addition, the ingredients that may be added in addition are not limited thereto, and any of the above ingredients may be incorporated within a range that does not impair the purpose and effect of the present invention, but may preferably be incorporated in an amount of 0.01 to 5 parts by weight, more preferably 0.01 to 3 parts by weight, based on the total weight.

[0115] According to the most preferred embodiment of the present invention, the cosmetic composition of the present invention has an emulsion formulation. Such an emulsion formulation allows the cosmetic composition of the present invention to best exhibit the effects, namely (a) the reduction of skin irritation, (b) the improvement of skin elasticity enhancement, and (c) the improvement of the duration of moisturizing effects. An emulsion applied by being absorbed into the skin surface (especially dry areas) is a cosmetic formulation that has been used for a long time and is used not only on the face but also for parts of the body and the whole body. The functions of the emulsion formulation of the present invention are as follows. First, the stratum corneum of the skin is repaired and softened by moisture derived from the emulsion, moisturizers, and emollients, as well as by moisture coming from the subcutaneous tissue due to the occlusive effect of the applied emulsion.

[0116] The components used in the emulsion include purified water, humectants, film-forming agents and thickeners, oils (emollients), powdered colorants, pharmaceuticals, preservatives, surfactants, buffers, etc. The cosmetic composition of the present invention, exemplified in the following examples, is prepared according to a conventional method for preparing emulsion formulations by adding a plant-derived protein surfactant, a hydrolyzed protein component, and human stem cell culture medium to these components of the emulsion.

[0117] The present invention will be explained in more detail below through examples. These examples are intended to explain the invention more specifically, and the scope of the invention is not limited by these examples according to the gist of the invention.

[0118]

[0119] <Preparation Example>

[0120] <Manufacture of Low Molecular Weight Collagen>

[0121] <Example 1>

[0122] Semi-dried pollack (Bada Nuri Co., Ltd.) was washed once with water at 5℃ and then washed a second time under running water, after which the pollack skin and pollack flesh were separated. Subsequently, a natural extract consisting of lemon extract and green tea extract mixed in a ratio of 3:7 was added to distilled water at a concentration of about 3 wt% to decompose impurities, inorganic salts, and fats contained in the pollack skin, thereby pre-treating the product. Then, the pre-treated pollack skin was boiled at 60℃ to extract collagen.

[0123] Subsequently, collagen extracted from pollack skin was reduced in molecular weight using the pH, temperature, time, and enzymes as shown in Table 1 below. Then, unreacted substrates, by-products, and enzymes were removed to obtain high-purity collagen. The insoluble substances and solution were separated by high-speed centrifugation, and the results are listed in Table 2 below.

[0124] [Table 1] 1st Low Molecular Weighting 2nd Low Molecular Weighting 3rd Low Molecular Weighting Example 1: Enzyme Pepsin Pancreatin:Nutriase = 5:95 Collagenase Conditions pH 2~3, 37℃, 6~12 hours, Ultrafiltration 3,000 MW CO pH 7, 37℃, 3~4 hours, Ultrafiltration 1,000 MW CO pH 7, 37℃, 12 hours, Ultrafiltration 500 MW CO Example 2: Enzyme Pepsin Pancreatin:Nutriase = 5:95 Conditions pH 2~3, 37℃, 6~12 hours, Ultrafiltration 3,000 MW CO pH 7, 37℃, 3~4 hours, Ultrafiltration 1,000 MW CO Comparative Example 1 Enzyme Pepsin:Pancreatin:Nutriase = 5:5:90 -- Conditions pH 2–3, 37℃, 6–12 hours, ultrafiltration 3,000 MWCO -- Comparative Example 2 Enzyme Pepsin:Pancreatin:Nutriase = 5:5:90 -- Conditions pH 7, 37℃, 6–12 hours, ultrafiltration 3,000 MWCO -- Comparative Example 3 Enzyme Pepsin Pancreatin:Nutriase = 5:95 Collagenase Conditions pH 7, 37℃, 6–12 hours, ultrafiltration 3,000 MWCO pH 2–3, 37℃, 3–4 hours, ultrafiltration 1,000 MWCO pH 7, 37℃, 12 hours, ultrafiltration 500 MWCO Comparative Example 4 Enzyme Pepsin -- Conditions pH 7, 37℃, 6~12 hours, ultrafiltration 3,000MWCO--

[0125] In Table 1 above, MWCO stands for Molecular Weight Cut-off, which means that molecules with a molecular weight below a certain level are allowed to pass through, while molecules with a molecular weight above a certain level are not allowed to pass through (Cut off).

[0126] [Table 2] Content of 1000 Mw or less Weight Average Molecular Weight Example 162.4 wt% 1,000 Dalton Example 261.7 wt% 1,050 Dalton Comparative Example 141.5 wt% 2,100 Dalton Comparative Example 242.3 wt% 2,050 Dalton Comparative Example 348.2 wt% 1,900 Dalton Comparative Example 447.7 wt% 1,950 Dalton

[0127] Specifically, FIG. 1 is a data analysis of the weight average molecular weight and composition content of low molecular weight collagen according to Example 1, and the remaining examples and comparative examples were analyzed in the same way and listed in Table 2 above.

[0128] As can be seen in Table 2 above, it was confirmed that Examples 1 and 2, in which collagen was extracted using the manufacturing method according to the present application, were well reduced in molecular weight compared to the comparative example. This corresponds to the result of appropriately using specific enzymes according to pH in 2 to 3 stages.

[0129]

[0130] <Preparation of cosmetic compositions>

[0131] A cosmetic composition was prepared by mixing with purified water, comprising 1 to 5 parts by weight and 10 to 15 parts by weight of amino acids including low molecular weight collagen and proline according to the above-described examples and comparative examples, and 5 parts by weight of other moisturizers (butylene glycol), emulsifiers (cetearyl oliveate), conditioning agents (squalane), surfactants (tromethamine), pH adjusters (tromethamine), and fragrances.

[0132]

[0133] <Experimental Example>

[0134] <Experimental Example 1: Antioxidant Evaluation>

[0135] For the antioxidant evaluation, experiments were conducted by increasing the content of low molecular weight collagen in Example 1 described above (prepared at 100 ppm, 500 ppm, and 1000 ppm), and the antioxidant evaluation was performed through DPPH evaluation, ABTS evaluation, and SOD evaluation, and the results are described in Section 4.

[0136] Specifically, Figure 2 shows the DPPH evaluation, Figure 3 shows the ABTS evaluation, and Figure 4 shows the SOD evaluation results. It can be seen that the antioxidant effect increases as the content of low molecular weight collagen increases, and accordingly, it was confirmed that the content of low molecular weight collagen and each effect are concentration-dependent.

[0137]

[0138] <Experimental Example 2: Cell Viability Experiment>

[0139] Cells (HS68) were inoculated into DMEM medium containing 4.5 g / l glucose, 10% serum, and 1% antibiotics and cultured at 37°C for 24 hours under 5% CO2 conditions. The number of inoculated cells was 1 × 10⁴.

[0140] After incubation, the experimental samples were applied to each culture well. The experimental samples used were those from the Examples and Comparative Examples. After applying each experimental sample, the wells were incubated again for 24 hours. 10 μL of a solution in which MTT was dissolved at a concentration of 5 mg / mL in salin-phosphate buffer (PBS) was added to each well, and the reaction was carried out at 37°C for 4 hours. After completely discarding the contents of the wells, 100 μL of acid-treated isopropanol (0.1N HCl) containing 10% Triton X-10, an MTT solution, was added to each well and stirred for 20 minutes. The absorbance was then measured at 570 nm using an ELISA meter, and the results are shown in Figure 5.

[0141] Specifically, in the case of the cosmetic compositions of the embodiments and comparative examples of the present invention, it was confirmed that they have stability by having a value of approximately 100% in cell viability experiments.

[0142]

[0143] <Experimental Example 3: Skin Irritation Test>

[0144] Using the closed patch test method, the experimental products of the example and comparative example were attached to the forearms of 20 healthy male and female subjects for 24 hours each, then removed to check for skin irritation. The experimental results are shown in Table 3.

[0145] At this time, the skin irritation judgment criteria were as follows.

[0146] 0: No stimulation

[0147] 1: Weak stimulation

[0148] 2: Visually detectable minor irritation

[0149] 3: Visually observable irritation

[0150] 4: Strong stimulation

[0151]

[0152] <Experimental Example 4: Skin Elasticity Effect>

[0153] Forty healthy women were randomly divided into two groups of 20 each in a constant temperature and humidity room maintained at 24–26°C and 40% humidity. The experimental products of the examples and comparative examples were applied to the left and right faces of each group, and after 3 months of use, skin elasticity was measured using a Cutometer SEM 575, a skin elasticity measuring device, and the average values ​​were compared. The results are shown in Table 3.

[0154] At this time, the elasticity effect was indicated as 0 being the most improved on a scale of 5, with poor performance.

[0155]

[0156] <Experimental Example 5: Skin Moisturizing Effect>

[0157] In a constant temperature and humidity room maintained at 24–26°C and 40% humidity, the experimental products of the examples and comparative examples were applied to the faces of 40 healthy women, and after 4 hours, skin moisture was measured using a Corneometer CM 820. The experimental results are shown in Table 3.

[0158] At this time, the moisturizing effect was indicated on a scale of 0, where poor results were rated as 5, with 5 being the most improved.

[0159]

[0160] <Experimental Example 6: Skin Wrinkle Improvement Effect>

[0161] Forty healthy women were tested, and two sets of the experimental products from the example and comparative example were applied to the left and right sides of their faces. After three months of use, skin replicas were created, and skin wrinkles were analyzed using a Skin Visiometer SV 400. The wrinkle improvement effect was compared using Ra (average wrinkle length) and Rz (average distance between wrinkle grooves) values. The experimental results are shown in Table 2.

[0162] At this time, the wrinkle improvement effect was indicated on a scale of 0, where poor was the standard, and 5, where the improvement was the greatest.

[0163] [Table 3] Experimental Example 3 Experimental Example 4 Experimental Example 5 Experimental Example 6 Example 187.30.14.84.6 Example 281.20.44.34.7 Comparative Example 180.22.812.12.2 Comparative Example 281.32.661.92.1 Comparative Example 379.33.11.71.4 Comparative Example 480.32.92.31.9

[0164] As shown in Table 3, the experimental results of the sensory evaluation confirmed that when using the cosmetic compositions of Examples 1 and 2 of the present invention, skin irritation, moisturizing effect, wrinkle improvement, and elasticity were enhanced. This is predicted to be a result of the excellent purity of low molecular weight collagen.

[0165]

[0166] <Experimental Example 7: Skin Regeneration Effect>

[0167] 1) Intracellular collagen production test

[0168] Experiments related to intracellular collagen production were conducted using the cosmetic compositions prepared in the above examples and comparative examples.

[0169] Collagen is synthesized in the cell in the form of procollagen, and during secretion outside the cell, the propeptide is cleaved to form extracellular collagen fibrils. The measured amount of free propeptide reflected the amount of collagen synthesized in the cell. This experiment was conducted by evaluating the degree of intracellular collagen production by measuring procollagen type 1 carboxyl-terminal peptide, a free propeptide secreted into the culture medium, using the EIA method, and determining the degree of increase in collagen production by comparing this with a control group. The results are shown in Table 4.

[0170] [Table 4] Intracellular Collagen Production Test Example 1126 Example 2128 Comparative Example 1101 Comparative Example 2102 Comparative Example 399 Comparative Example 4102

[0171] As can be seen in Table 4, it was confirmed that the intracellular collagen production efficacy of the cosmetic composition according to the present invention is significantly high. This is because, in the case of Examples 1 and 2 according to the present application, low molecular weight collagen produced by a specific manufacturing method was used, resulting in high purity low molecular weight collagen.

[0172] 2) In vitro skin cell regenerative activity test

[0173] Experiments related to skin cell regeneration activity were conducted using the topical skin preparation composition prepared in Example 1 above.

[0174] The experiment was conducted to confirm skin regenerative ability by measuring the time and extent of recovery after inducing a wound on skin cells, specifically dermal cells (Human dermal fibroblasts) and epidermal cells (HaCaT). Figure 6 shows the data comparing the case where the composition prepared in Example 1 was used with the case where a substance generally known to be effective for skin regeneration (Madecassol, Centella Asiatica leaf extract 10%) was used.

[0175] The central part of each Data in Fig. 6 is the wounded area, and as can be seen from the photograph, it was found that the effect of the external skin composition of the present invention is excellent even when compared to when a substance known to be effective for skin regeneration is used.

[0176] As described above, although the present invention has been explained with reference to preferred embodiments, those skilled in the art will understand that various modifications and changes can be made to the present invention without departing from the spirit and scope of the invention as set forth in the following claims.

Claims

A method for extracting low molecular weight collagen comprising: a step of cleaning a pollack to separate the skin, washing it once with water, and then washing it twice under running water; a step of pre-treating the pollack skin by washing it with distilled water containing a natural extract; a step of extracting collagen by boiling the pre-treated pollack skin in purified water; and a step of demolding the collagen, wherein the step of demolding the collagen comprises: a) a step of acid treatment to lower the pH of the collagen to 2 to 4; b) a step of demolding the collagen once by adding a primary acidic active enzyme to the acid-treated collagen; c) a step of neutralizing the pH of the collagen to lower the molecular weight to 7 to 8; and d) a step of demolding the collagen once by adding a secondary neutral active enzyme to the neutralized collagen. A method for extracting low molecular weight collagen according to claim 1, further comprising: b) a step of adding a primary acidic active enzyme to acid-treated collagen to perform primary low molecular weight reduction, and subsequently passing it through an ultrafiltration membrane to increase the content of low molecular weight collagen of 3,000 Da or less; and c) a step of neutralizing the pH of the primary low molecular weight collagen to 7 to 8; and subsequently passing it through an ultrafiltration membrane to increase the content of low molecular weight collagen of 1,000 Da or less. A method for extracting low molecular weight collagen according to claim 1, wherein the primary acidic active enzyme comprises pepsin, the secondary neutral active enzyme comprises pancreatin and nutrasase, and the pancreatin comprises 0.01 parts by weight or more and 10 parts by weight or less based on 100 parts by weight of the secondary neutral active enzyme; and the nutrasase comprises 90 parts by weight or more and 99.99 parts by weight or less. A method for extracting low molecular weight collagen according to claim 1, further comprising: d) a step of adding a secondary neutral active enzyme to the neutralized primary low molecular weight collagen to perform secondary low molecular weight reduction, and then adding a tertiary neutral active enzyme to the secondary low molecular weight collagen to perform tertiary low molecular weight reduction; and a step of passing through an ultrafiltration membrane to increase the content of low molecular weight collagen of 3,000 Da or less; wherein the tertiary neutral active enzyme is collagenase. A method for extracting low molecular weight collagen according to claim 4, wherein the active temperature in the first low molecular weight step; the second low molecular weight step and the third low molecular weight step is 30℃ or higher and 40℃ or lower, and the active time is within 12 hours. A method for extracting low molecular weight collagen according to claim 1, wherein the natural extract comprises green tea extract and lemon extract, and wherein, based on 100 parts by weight of the natural extract, the lemon extract comprises 20 parts by weight or more and 40 parts by weight or less; and the green tea extract comprises 60 parts by weight or more and 80 parts by weight or less. Low molecular weight collagen extracted through the method for extracting low molecular weight collagen according to any one of claims 1 to 6. In claim 7, the low molecular weight collagen is a low molecular weight collagen containing 60 wt% or more of collagen with a molecular weight of 1,000 Da or less. A cosmetic composition having wound healing and skin regeneration efficacy comprising low molecular weight collagen according to claim 7; and an amino acid including proline. A cosmetic composition having wound healing and skin regeneration efficacy according to claim 9, wherein, based on 100 parts by weight of the total cosmetic composition, the low molecular weight collagen is included in an amount of 1 part by weight or more and 5 parts by weight or less, and the amino acid is included in an amount of 10 parts by weight or more and 15 parts by weight or less.

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