Method for producing vegetable collagen

Abstract

The present invention relates to a method for producing plant-based collagen and plant-based collagen produced therefrom. Specifically, the present invention can provide a method for producing plant-based collagen with a higher yield from plant raw materials, and the hibiscus collagen produced by the method of the present invention has excellent skin improvement efficacy.

Description

Technology Field

[0001] The present invention relates to a method for producing plant-based collagen and plant-based collagen produced therefrom. Background Technology

[0002] Collagen is a major component of connective tissues in the human body, such as the dermis, cartilage, and bone. It is widely used in the cosmetics and health functional food industries because it is closely related to maintaining skin moisture, enhancing elasticity, and improving wrinkles. Most collagen raw materials currently on the market are extracted from animal sources such as fish scales or pigs and cows, and are generally supplied in the form of peptides with reduced molecular weight.

[0003] However, limitations have been pointed out regarding collagen based on animal-derived ingredients due to consumers' religious and ethical aversion, the characteristic off-flavor and reluctance to consume animal-derived ingredients, concerns regarding allergic reactions and safety, risks related to livestock diseases such as BSE, and sustainability issues.

[0004] Accordingly, the development of vegan collagen or plant-derived collagen substitute materials capable of replacing animal-derived ingredients has recently been actively underway. Generally, plants do not contain collagen protein itself, but they do contain protein and peptide precursors with collagen-specific amino acid compositions such as glycine (Gly), proline (Pro), and hydroxyproline (Hyp), or various plant-based components (polyphenols, vitamin C, etc.) that promote collagen synthesis in the body. Therefore, process technology is required to enzymatically hydrolyze plant proteins to convert them into low-molecular-weight peptides or to remove impurities to maximize functionality.

[0005] In conventional technology, hydrolyzed peptides using proteins from some cereals and legumes, such as soybeans, peas, rice, and oats, have been reported; however, these methods alone have limitations in securing the Hyp content characteristic of collagen, and were insufficient in terms of off-flavor and functional efficacy (skin moisturization, elasticity improvement, etc.). In addition, existing processes had problems with low sensory palatability and reduced bioavailability due to the persistence of impurities (lipids, pigments, etc.). Prior art literature

[0006] Republic of Korea Published Patent No. 10-2024-0177776 The problem to be solved

[0007] In one aspect, the present invention aims to provide a method for producing plant-based collagen, comprising: (a) a step of preparing a plant extract from a plant raw material; (b) an enzymatic hydrolysis step of treating the plant extract with a proteolytic enzyme; (c) an enzymatic inactivation step of heating the hydrolysate obtained in step (b); (d) a step of fractionating the enzymatic inactivation solution obtained in step (c) through a filtration process to obtain low molecular weight peptides and freeze-drying the recovered solution; and (e) a step of treating the freeze-dried product obtained in step (d) with supercritical carbon dioxide.

[0008] In another aspect, the present invention aims to provide a skin-improving composition comprising hibiscus collagen as an active ingredient.

[0009] In another aspect, the present invention aims to provide a food composition comprising hibiscus collagen as an active ingredient. means of solving the problem

[0010] In one aspect, the present invention provides a method for producing plant-based collagen, comprising: (a) a step of preparing a plant extract from a plant raw material; (b) an enzymatic hydrolysis step of treating the plant extract with a proteolytic enzyme; (c) an enzymatic inactivation step of heating the hydrolysate obtained in step (b); (d) a step of fractionating the enzymatic inactivation solution obtained in step (c) through a filtration process to obtain low molecular weight peptides and freeze-drying the recovered solution; and (e) a step of treating the freeze-dried product obtained in step (d) with supercritical carbon dioxide.

[0011] In an exemplary embodiment, step (a) may involve mixing dried plant raw materials with water in a ratio of 1:10 to 1:30 (w / w) and extracting at 90 to 110°C for 20 to 60 minutes to obtain an extract.

[0012] In an exemplary embodiment, step (b) may involve adjusting the pH of the extract to 6.4 to 7.2, adding 0.1 to 0.3 wt% of a proteolytic enzyme relative to the total weight of the solution, and hydrolyzing by stirring at 50 to 60°C for 2 to 5 hours.

[0013] In an exemplary embodiment, step (c) may involve heating the hydrolysate at 80 to 110°C for 5 to 15 minutes to inactivate the enzyme.

[0014] In an exemplary embodiment, step (d) may involve fractionating the enzyme inactivation solution with an ultrafiltration membrane to obtain low molecular weight peptides and freeze-drying the recovered solution. For example, the ultrafiltration membrane may be performed by removing high molecular weights through an MWCO of 10 kDa and then concentrating low molecular weight peptides through a 3 kDa cutoff. Additionally, following the ultrafiltration process, a nanofiltration (NF) process may be applied to more precisely fractionate and purify components within a specific molecular weight range (e.g., 300–500 Da) among the low molecular weight peptides.

[0015] In an exemplary embodiment, step (e) may be supercritical carbon dioxide treatment comprising static treatment for 10 to 30 minutes and dynamic treatment for 30 to 90 minutes under conditions of 200 to 300 bar and 40 to 50 ℃.

[0016] In an exemplary embodiment, the protein-degrading enzyme may be a protease.

[0017] In an exemplary embodiment, the plant raw material may be one or more selected from the group consisting of hibiscus, apple, carrot, spinach, kale, and broccoli.

[0018] In another aspect, the present invention provides a skin improvement composition comprising hibiscus collagen as an active ingredient.

[0019] In an exemplary embodiment, the hibiscus collagen may comprise one or more of the following features: (a) a glycine (Gly) content of 26.8 mol% to 28.8 mol%, (b) a proline (Pro) content of 11.4 mol% to 13.4 mol%, and (c) a hydroxyproline (Hyp) content of 1.5 mol% to 2.2 mol%.

[0020] In an exemplary embodiment, the skin improvement may be for skin moisturization, skin elasticity improvement, skin tone improvement, or skin roughness relief.

[0021] In an exemplary embodiment, the composition may be for cosmetic use.

[0022] In another aspect, the present invention provides a food composition comprising hibiscus collagen as an active ingredient.

[0023] In an exemplary embodiment, the composition may be for use in a health functional food.

[0024] In an exemplary embodiment, the composition may be for general food use.

[0025] In an exemplary embodiment, the composition may be formulated into tablets, granules, pills, powders, drinks, tea bags, candies, confectionery, beverages, jellies, etc. Effects of the invention

[0026] The present invention can produce plant-based collagen with a higher content from plant raw materials.

[0027] Plant-based collagen produced by the method of the present invention has superior skin improvement efficacy compared to conventional plant-based collagen.

[0028] The plant-based collagen produced by the method of the present invention has excellent efficacy in increasing the secretion of Pro-collagen I C-peptide in human fibroblasts, increasing COL1A1 mRNA expression, and inhibiting MMP-1 protein expression.

[0029] Plant-based collagen produced by the method of the present invention has excellent effects in moisturizing the skin, improving skin elasticity, improving skin tone, or alleviating skin roughness. Brief explanation of the drawing

[0030] Figure 1 shows a manufacturing process diagram of hibiscus collagen according to one embodiment of the present invention. Specific details for implementing the invention

[0031] The present invention will be described in detail below.

[0033] In one aspect, the present invention provides a method for producing plant-based collagen, comprising: (a) a step of preparing a plant extract from a plant raw material; (b) an enzymatic hydrolysis step of treating the plant extract with a proteolytic enzyme; (c) an enzymatic inactivation step of heating the hydrolysate obtained in step (b); (d) a step of fractionating the enzymatic inactivation solution obtained in step (c) through a filtration process to obtain low molecular weight peptides and freeze-drying the recovered solution; and (e) a step of treating the freeze-dried product obtained in step (d) with supercritical carbon dioxide.

[0034] The above plant raw materials may include not only flower parts (dried flowers, petals) but also leaves, stems, seeds, and fruits, as well as any parts containing edible or functional ingredients. Additionally, the above plant raw materials may include any plant containing plant-based collagen without limitation. For example, the above plant raw materials may include plants containing a collagen-like amino acid composition or ingredients that promote collagen synthesis, but are not limited thereto.

[0035] For example, the above step (a) may be performed using a plant extraction method conventional in the art, but is not limited thereto. For example, the above step (a) may be obtained by extracting, separating, and fractionating from nature using extraction, separation, and fractionating methods known in the art, and may be extracted according to various extraction or fractionation solvents and extraction methods. Furthermore, for example, the extraction method of the above extract may include hot water extraction, cold maceration extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, elution, pressing, etc., but is not limited thereto.

[0036] The inventors have confirmed that when the supercritical carbon dioxide treatment process of step (e) is added to the method for manufacturing plant-based collagen, the purity of the plant-based collagen is improved, the stability of the collagen indicator is increased, and particle dispersibility is improved, which can further increase the absorption rate in the body.

[0037] In an exemplary embodiment, step (a) may involve mixing a dried plant raw material with water in a ratio of 1:10 to 1:30 (w / w) and extracting at 90 to 110°C for 20 to 60 minutes to obtain an extract. For example, the mixing ratio of the dried plant raw material and the water may be 1:10 (w / w) or more, 1:12 (w / w) or more, 1:14 (w / w) or more, 1:16 (w / w) or more, 1:18 (w / w) or more, or 1:20 (w / w) or more, and may be 1:30 (w / w) or less, 1:28 (w / w) or less, 1:26 (w / w) or less, 1:24 (w / w) or less, 1:22 (w / w) or less, or 1:20 (w / w) or less, but is not limited thereto. In addition, for example, the extraction temperature may be 90°C or higher, 92°C or higher, 94°C or higher, 96°C or higher, 98°C or higher, or 100°C or higher, and may be 110°C or lower, 108°C or lower, 106°C or lower, 104°C or lower, 102°C or lower, or 100°C or lower, but is not limited thereto. In addition, for example, the extraction time may be 20 minutes or more, 24 minutes or more, 28 minutes or more, 32 minutes or more, 36 minutes or more, or 40 minutes or more, and may be 60 minutes or less, 56 minutes or less, 52 minutes or less, 48 ​​minutes or less, 44 minutes or less, or 40 minutes or less, but is not limited thereto.

[0038] In an exemplary embodiment, step (b) may involve adjusting the pH of the extract to 6.4 to 7.2, adding a proteolytic enzyme in an amount of 0.1 to 0.3 wt% relative to the total weight of the solution, and hydrolyzing by stirring at 50 to 60°C for 2 to 5 hours. For example, the adjusted pH of the extract may be 6.4 or higher, 6.5 or higher, 6.6 or higher, 6.7 or higher, or 6.8 or higher, and may be 7.2 or lower, 7.1 or lower, 7.0 or lower, 6.9 or lower, or 6.8 or lower, but is not limited thereto. In addition, for example, the protein-degrading enzyme may be added in an amount of 0.1 wt% or more, 0.12 wt% or more, 0.14 wt% or more, 0.16 wt% or more, 0.18 wt% or more, or 0.20 wt% or more relative to the total weight of the solution, or 0.3 wt% or less, 0.28 wt% or less, 0.26 wt% or less, 0.24 wt% or less, 0.22 wt% or less, or 0.20 wt% or less, but is not limited thereto. In addition, for example, the hydrolysis temperature may be 50°C or higher, 52°C or higher, 54°C or higher, or 55°C or higher, and may be 60°C or lower, 58°C or lower, 56°C or lower, or 55°C or lower, but is not limited thereto. In addition, for example, the hydrolysis time may be 2 hours or more, 2.2 hours or more, 2.4 hours or more, 2.6 hours or more, 2.8 hours or more, or 3 hours or more, and may be 5 hours or less, 4.5 hours or less, 4 hours or less, 3.8 hours or less, 3.6 hours or less, 3.4 hours or less, 3.2 hours or less, or 3 hours or less, but is not limited thereto.

[0039] In an exemplary embodiment, the hydrolysate obtained after step (b) has a diverse molecular weight distribution due to the action of proteolytic enzymes. At this time, the hydrolysate may contain not only low molecular weight peptides but also high molecular weight impurities, residual proteins, enzyme proteins, etc. Therefore, in the subsequent step (c), the hydrolysate is heated at 80 to 110°C for a certain period of time to inactivate the proteolytic enzymes and terminate the reaction. This enzyme inactivation step functions as a pretreatment process to prevent excessive hydrolysis and to stably perform the subsequent membrane separation process.

[0040] In an exemplary embodiment, step (c) may involve heating the hydrolysate at 80 to 110°C for 5 to 15 minutes to inactivate the enzyme. For example, the temperature during heating may be 80°C or higher, 85°C or higher, 90°C or higher, 92.5°C or higher, or 95°C or higher, and may be 110°C or lower, 105°C or lower, 100°C or lower, 97.5°C or lower, or 95°C or lower, but is not limited thereto. Additionally, for example, the time during heating may be 5 minutes or more, 6 minutes or more, 7 minutes or more, 8 minutes or more, 9 minutes or more, or 10 minutes or more, and may be 15 minutes or less, 14 minutes or less, 13 minutes or less, 12 minutes or less, 11 minutes or less, or 10 minutes or less, but is not limited thereto.

[0041] In an exemplary embodiment, step (d) may involve fractionating the enzyme-inactivated solution using a membrane separation process of ultrafiltration (including nanofiltration if necessary) to obtain low molecular weight peptides, and freeze-drying the recovered solution.

[0042] In an exemplary embodiment, step (d) may apply an ultrafiltration membrane with an MWCO of 10 kDa or less to remove high molecular weight components and selectively concentrate low molecular weight peptides.

[0043] Additionally, high molecular weight components can be removed by applying the above ultrafiltration membrane, and low molecular weight peptides can be selectively concentrated by applying a nanofiltration membrane with an MWCO of 3 kDa or less.

[0044] For example, in the ultrafiltration process, the MWCO may be 10 kDa or less, 9.5 kDa or less, 9.0 kDa or less, 8.5 kDa or less, 8.0 kDa or less, or 7.5 kDa or less, and in the nanofiltration process, the MWCO may be 3.0 kDa or less, 2.8 kDa or less, 2.6 kDa or less, 2.4 kDa or less, 2.2 kDa or less, or 2.0 kDa or less, but is not limited thereto.

[0045] Unlike when using a single membrane, this filtration process continuously performs high molecular weight removal and low molecular weight peptide concentration, thereby optimizing the amino acid composition and, in particular, significantly improving the hydroxyproline (Hyp) content.

[0046] In addition, the above filtration process can further purify the low molecular weight peptide fraction by performing repeated washing (diafiltration) on the permeate or residue at each stage. Through this process, residual impurities are ultimately minimized, and the concentration of low molecular weight peptides increases, resulting in an improved absorption rate in the body.

[0047] Meanwhile, in another exemplary embodiment, after step (b), that is, before enzyme inactivation, some filtration processes may be performed to remove impurities in advance, and then step (c) may be passed to finally proceed with the filtration of step (d).

[0048] In an exemplary embodiment, step (e) may involve supercritical carbon dioxide treatment of the freeze-dried material, including static treatment for 10 to 30 minutes and dynamic treatment for 30 to 90 minutes at 200 to 300 bar and 40 to 50 ℃. For example, the pressure during the supercritical carbon dioxide treatment may be 200 bar or more, 210 bar or more, 220 bar or more, 230 bar or more, 240 bar or more, or 250 bar or more, and may be 300 bar or less, 290 bar or less, 280 bar or less, 270 bar or less, or 260 bar or less, but is not limited thereto. In addition, for example, the temperature during the supercritical carbon dioxide treatment may be 40°C or higher, 42°C or higher, 44°C or higher, or 45°C or higher, and may be 50°C or lower, 48°C or lower, or 46°C or lower, but is not limited thereto. In addition, for example, the time during the static treatment may be 10 minutes or more, 12 minutes or more, 14 minutes or more, or 15 minutes or more, and may be 30 minutes or less, 25 minutes or less, or 20 minutes or less, but is not limited thereto. In addition, for example, the time during the dynamic treatment may be 30 minutes or more, 35 minutes or more, 40 minutes or more, 45 minutes or more, 50 minutes or more, or 55 minutes or more, and may be 90 minutes or less, 85 minutes or less, 80 minutes or less, 75 minutes or less, or 70 minutes or less, but is not limited thereto.

[0049] In an exemplary embodiment, the protein-degrading enzyme may be a protease. The inventors have confirmed that when the protein-degrading enzyme is a protease, plant-based collagen can be produced with higher efficiency compared to pectinase.

[0050] In an exemplary embodiment, the plant raw material may be one or more selected from the group consisting of hibiscus, apple, carrot, spinach, kale, and broccoli.

[0052] In another aspect, the present invention provides a skin improvement composition comprising hibiscus collagen as an active ingredient.

[0053] The above hibiscus refers to a plant belonging to the genus Hibiscus of the family Malvaceae, order Malvales, of dicotyledonous plants. The above hibiscus contains various beneficial components, including anthocyanin and hydroxycitric acid (HCA), which are antioxidant substances that produce a red color, and helps with metabolism and the elimination of waste products.

[0054] The above hibiscus collagen refers to plant-based collagen derived from raw hibiscus material. The above hibiscus collagen may be manufactured by the above manufacturing method, but is not limited thereto.

[0055] In an exemplary embodiment, the hibiscus collagen may include one or more of the following features: (a) a glycine (Gly) content of 26.8 mol% to 28.8 mol%, (b) a proline (Pro) content of 11.4 mol% to 13.4 mol%, and (c) a hydroxyproline (Hyp) content of 1.5 mol% to 2.2 mol%. For example, the glycine content in the hibiscus collagen may be 26.8 mol% or more, 27 mol% or more, 27.2 mol% or more, 27.4 mol% or more, 27.6 mol% or more, or 27.8 mol% or more, and may be 28.8 mol% or less, 28.6 mol% or less, 28.4 mol% or less, 28.2 mol% or less, 28 mol% or less, or 27.8 mol% or less, but is not limited thereto. In addition, for example, the proline content in the hibiscus collagen may be 11.4 mol% or more, 11.6 mol% or more, 11.8 mol% or more, 12 mol% or more, 12.2 mol% or more, or 12.4 mol% or more, and may be 13.4 mol% or less, 13.2 mol% or less, 13 mol% or less, 12.8 mol% or less, 12.6 mol% or less, 12.4 mol% or less, or 12.2 mol% or less, but is not limited thereto. In addition, for example, the hydroxyproline content in the hibiscus collagen may be 1.5 mol% or more, 1.55 mol% or more, 1.6 mol% or more, 1.65 mol% or more, or 1.7 mol% or more, and may be 2.2 mol% or less, 2.15 mol% or less, 2.1 mol% or less, 2.05 mol% or less, or 2 mol% or less, but is not limited thereto.

[0056] In an exemplary embodiment, the skin improvement may be for skin moisturization, skin elasticity improvement, skin tone improvement, or skin roughness relief.

[0057] In an exemplary embodiment, the composition may be for use in a health functional food.

[0058] The formulation of the above-mentioned composition for health functional foods is not particularly limited, but may be formulated, for example, into tablets, granules, pills, powders, liquids such as drinks, caramels, gels, bars, tea bags, etc. For each formulation of the food composition, in addition to the active ingredient, ingredients commonly used in the relevant field can be appropriately selected and combined by a person skilled in the art without difficulty according to the formulation or purpose of use, and a synergistic effect may occur when applied simultaneously with other raw materials.

[0059] The above composition for health functional foods may be administered by various methods, such as simple ingestion, drinking, injection, spray, or squeeze. The above composition for health functional foods may be, for example, various food products such as chewing gum, caramel products, candies, frozen desserts, and confectionery, or beverage products such as soft drinks, mineral water, and alcoholic beverages.

[0060] In an exemplary embodiment, the composition may be for general food use. The composition for general food use may be applied to various processed foods that are consumed daily, regardless of health functional claims, and may be applied, for example, to bakery products (bread, cakes, cookies, etc.), cereals and processed grain products, soy milk and grain beverages, dairy products (yogurt, cheese, ice cream, etc.), sauces, dressings, jellies, powdered soups, instant foods, Home Meal Replacements (HMR), and various beverages. Such general food compositions may also include the plant-based collagen of the present invention as an active ingredient to provide effects such as improved skin health, elasticity, and enhanced absorption rate in the body.

[0061] The above food compositions (including those for health functional foods and general foods) may contain food additives in addition to their active ingredients. Food additives can generally be understood as substances added to, mixed with, or permeated into food during the manufacturing, processing, or preservation of food; since they are consumed daily and over a long period along with food, their safety must be guaranteed. Food additive codes in accordance with national laws governing the manufacturing and distribution of food (the "Food Sanitation Act" in Korea) restrictively define food additives with guaranteed safety in terms of composition or function. The Korean Food Additive Code (Notification of the Ministry of Food and Drug Safety, "Standards and Specifications for Food Additives") classifies food additives into chemically synthesized products, natural additives, and mixed preparations in terms of composition, while these food additives are classified into sweeteners, flavoring agents, preservatives, emulsifiers, acidulants, and thickeners in terms of function.

[0062] Sweeteners are used to impart a suitable sweetness to food, and both natural and synthetic sweeteners may be used in the food composition according to one aspect of the present invention. Preferably, natural sweeteners are used, and examples of natural sweeteners include corn syrup solids, honey, sucrose, fructose, lactose, maltose, etc.

[0063] Flavoring agents are used to improve taste or aroma, and both natural and synthetic types may be used. Preferably, natural ones are used. When natural ones are used, nutritional enhancement can be achieved in addition to flavor. Natural flavoring agents may be obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, etc., or from green tea leaves, Solomon's seal, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, etc. Additionally, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo, etc., may be used. Natural flavoring agents may be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents may be used, and synthetic flavoring agents may include esters, alcohols, aldehydes, terpenes, etc.

[0064] Calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. may be used as preservatives. In addition, acacia gum, carboxymethylcellulose, xanthan gum, pectin, etc. may be used as emulsifiers, and citric acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, etc. may be used as acidifiers. Acidifiers may be added to the food composition to achieve an appropriate acidity, in addition to for the purpose of enhancing flavor, for the purpose of inhibiting the growth of microorganisms.

[0065] As thickening agents, suspending agents, precipitating agents, gel-forming agents, puffing agents, etc. may be used.

[0066] In addition to the food additives mentioned above, the above-mentioned health functional food may include physiologically active substances or minerals known in the industry and whose safety as food additives is guaranteed, for the purpose of supplementing and reinforcing functionality and nutritional value.

[0067] In an exemplary embodiment, the composition may be for cosmetic use.

[0068] The above cosmetic composition may have, for example, formulations such as softening lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, eye cream, eye essence, essence, cleansing cream, cleansing lotion, cleansing foam, cleansing water, pack, powder, body lotion, body cream, body essence, body cleanser, hair dye, shampoo, rinse, hair styling agent, hair conditioner, ointment, gel, cream, patch, spray, powder, and skin adhesive type, but is not limited thereto. In addition, for each formulation, other ingredients other than the essential ingredients mentioned above may be appropriately selected and combined by a person skilled in the art without difficulty according to the type of other external preparation or purpose of use. The cosmetic composition according to one embodiment may be provided in any formulation suitable for local application. For example, it may be provided in the form of a solution, an emulsion obtained by dispersing an oil phase in an aqueous phase, an emulsion obtained by dispersing an aqueous phase in an oil phase, a suspension, a solid, a gel, a powder, a paste, a microneedle, a foam, or an aerosol composition. Compositions of such formulations may be prepared according to conventional methods in the relevant field. A cosmetic composition according to one embodiment may additionally include functional additives and ingredients included in general cosmetic compositions in addition to the compounds of this specification. The functional additives may include ingredients selected from the group consisting of water-soluble vitamins, fat-soluble vitamins, high molecular weight peptides, high molecular weight polysaccharides, sphingolipids, and seaweed extracts. A cosmetic composition according to this specification may include other ingredients that can provide a synergistic effect to the main effect, preferably within a range that does not impair the main effect. In addition, the cosmetic composition according to the present specification may further include a moisturizer, an emollient, a surfactant, a UV absorber, a preservative, a disinfectant, an antioxidant, a pH adjuster, organic and inorganic pigments, a fragrance, a cooling agent, or a limiting agent. The amount of the above ingredients can be easily selected by a person skilled in the art within a range that does not impair the purpose and effect of the present specification, and the amount is 0. based on the total weight of the composition.It may be 0.01 to 10 weight%, specifically 0.01 to 3 weight%.

[0070] Embodiments of the present invention will be described in detail below with reference to the drawings. The description below is intended only to illustrate the embodiments and is not intended to limit or restrict the scope of the rights according to the present invention. Anything that can be easily inferred by a person skilled in the art from the detailed description and embodiments of the invention should be interpreted as falling within the scope of the rights according to the present invention. Detailed descriptions of matters widely known to those skilled in the art regarding the present invention are omitted.

[0071] The terms used in this invention are described as general terms widely used in the technical field relating to this invention; however, the meaning of the terms used in this invention may vary depending on the intent of those skilled in the field, the emergence of new technologies, examination standards, or case law. Some terms may be selected at the discretion of the applicant, and in such cases, the meaning of the arbitrarily selected terms will be explained in detail. The terms used in this invention should be interpreted not merely in their dictionary meanings, but in a sense that reflects the overall context of the specification.

[0073] Preparation Example

[0075] 1. Preparation of Hibiscus Collagen (Example 1)

[0076] (1) Extraction step

[0077] Dried hibiscus flower powder was mixed with distilled water at a ratio of 1:20 (w / w). The mixture was extracted once at 100°C for 40 minutes to obtain an extract containing hibiscus protein. After cooling to room temperature, the solids were filtered out.

[0078] (2) Enzyme hydrolysis step

[0079] After adjusting the pH of the extract to 6.8 by titrating with a 1 N NaOH aqueous solution, 0.20 wt% of protease was added relative to the total weight of the solution, and hydrolysis was carried out by stirring at 55 ℃ for 3 hours.

[0080] (3) Enzyme inactivation stage

[0081] The reaction solution in which hydrolysis was completed was heated at 95°C for 8 minutes to inactivate the protease, and then immediately cooled.

[0082] (4) Filtration step

[0083] The above enzyme inactivation solution was first fractionated using an MWCO 10 kDa ultrafiltration membrane to remove high molecular weight impurities, and then the permeate was fractionated again using a 3 kDa cutoff ultrafiltration membrane to obtain a low molecular weight peptide concentrate. At each step, the permeate was washed (diafiltration) with three times the volume of distilled water to remove residual impurities.

[0084] (5) Fractionation, concentration, and drying steps

[0085] To obtain low molecular weight peptides, the permeate obtained from an ultrafiltration membrane process of approximately 3 kDa was recovered (diafiltration of more than 3 times the volume was performed at each stage), and then concentrated under reduced pressure and freeze-dried to obtain a prepared powder.

[0086] (6) Supercritical CO2 post-treatment step

[0087] The freeze-dried powder was loaded into a supercritical extractor and post-treated with SC-CO2 (conditions: 250 bar, 45 ℃, static 15 min + dynamic 60 min, CO2 usage = 20 kg / kg-powder, separator: 100 bar, 35 ℃ (recovery of degreasing / aromatic (aromatic component) removal material)). After treatment, the powder was nitrogen-exchanged and packaged to obtain hibiscus collagen (Example 1).

[0089] 2. Preparation of Hibiscus Collagen (Example 2)

[0090] In the filtration step (4) of the manufacturing process of Example 1 above, the concentrate was fractionated using a 3 kDa cutoff ultrafiltration membrane, and then passed through a nanofiltration membrane (NF) to precisely fractionate and purify ultra-low molecular weight components of 300 to 500 Da or less. Other processes are the same as in Example 1.

[0092] 3. Other plant-based collagen manufacturing

[0093] Apple collagen and carrot collagen were also prepared using apples (Example 3) and carrots (Example 4) respectively, in the same manner as the above preparation example.

[0095] 4. Manufacturing of Comparative Example 1 (Difference in manufacturing process)

[0096] Comparative Example 1 was prepared by performing the hibiscus collagen preparation method of Example 1 under the same conditions, excluding the supercritical CO₂ post-treatment step.

[0098] 5. Preparation of Comparative Example 2 (Difference in proteolytic enzyme)

[0099] Comparative Example 2 was prepared in the same manner as the hibiscus collagen of Example 1, except that the type of enzyme was substituted with "pectinase".

[0102] Experimental Example

[0104] 1. Collagen Component Analysis

[0105] (1) Analysis of amino acid composition

[0106] Powder samples of Example 1 (hibiscus collagen), Example 2 (hibiscus collagen, NF process added), Example 3 (apple collagen), Example 4 (carrot collagen), Comparative Example 1, and Comparative Example 2 were each taken and hydrolyzed with a 6 N HCl solution in a sealed tube at 110 °C for 24 hours. The hydrolysate was concentrated under reduced pressure to remove residual acid.

[0107] Subsequently, the constituent amino acids were analyzed using an automatic amino acid analyzer (Hitachi L-8900, Japan), and the content of glycine (Gly), proline (Pro), and hydroxyproline (Hyp), which are known as indicator amino acids of collagen, was measured with particular focus.

[0109] (2) Results of amino acid composition analysis

[0110] The results of the amino acid composition analysis for each example are as shown in Table 1 below.

[0111] As a result of the analysis, the Hyp content of hibiscus collagen (Example 1) was 1.85 mol% and 0.12 mol%, which was approximately 1.9 to 4.4 times higher than that of Comparative Example 1 and Comparative Example 2. This suggests that low-molecular-weight peptides can be effectively obtained while maintaining collagen-like structural characteristics through enzymatic hydrolysis based on hibiscus extract and a supercritical CO₂ post-treatment process.

[0112] In particular, in the case of Comparative Example 2 (pectinase treatment), protein hydrolysis was not properly carried out due to the characteristics of the pectinase, resulting in significantly lower Hyp production. This proves that the use of a protein-degrading enzyme (protease) is key in the present invention.

[0113] In addition, the Gly ratio was also relatively high, exhibiting characteristics similar to the typical collagen amino acid pattern (Gly-XY repeating structure).

[0115] division Example 1 (Hibiscus Collagen) Example 2 (Hibiscus Collagen (Filtration Process Added)) Example 3 (Apple Collagen) Example 4 (Carrot Collagen) Comparative Example 1 (no SC-CO2 treatment) Comparative Example 2 (Pectinase Treatment) Gly 27.8 mol% 28.1 mol% 26.1 mol% 25.7 mol% 25.4 mol% 24.9 mol% Pro 12.4 mol% 12.9 mol% 10.8 mol% 10.2 mol% 9.6 mol% 9.1 mol% Hyp 1.85 ± 0.12 mol% 2.15 ± 0.10 mol% 1.12 ± 0.09 mol% 0.97 ± 0.08 mol% 0.92 ± 0.08 mol% 0.42 ± 0.05 mol%

[0117] 2. Experiment to confirm skin improvement efficacy

[0118] (1) Cell-level evaluation (in vitro)

[0119] Samples of Example 1, Example 2, Comparative Example 1, and Comparative Example 2 were each treated to human dermal fibroblasts (HDF) at a concentration of 100 Ìg / mL for 48 hours (the control group was untreated). Subsequently, Pro-collagen I C-peptide secretion (ELISA), COL1A1 mRNA expression (RT-qPCR), and MMP-1 protein expression (Western blot) were measured.

[0120] The degree of increase in secretion or expression amount compared to the control group was indicated relatively, and the experimental results are as shown in Table 2 below.

[0121] division Example 1 Example 2 Comparative Example 1 Comparative Example 2 Pro-collagen I C-peptide secretion amount 42% increase 43.5% 28% increase 12% increase COL1A1 mRNA expression level 1.8 times 1.9 times 1.2 times 1.1 times MMP-1 protein expression 18% inhibition 20% inhibition 8% restraint 3% inhibition

[0123] Therefore, it was confirmed that the hibiscus collagen of the example showed the most superior effects in promoting collagen synthesis and inhibiting collagen degradation in fibroblasts.

[0125] (2) Skin elasticity test

[0126] The hibiscus collagen powder of Example 1, Comparative Example 1, and Comparative Example 2 was mixed with 20 parts by weight of maltodextrin, 1 part by weight of citric acid, and 0.2 parts by weight of steviol glycosides to form a powder stick-type food.

[0128] Female volunteers aged 25 to 45 (n=30) were randomly assigned to consume the powder stick-type foods of Example 1, Comparative Example 1, and Comparative Example 2 once a day for 4 weeks. Skin elasticity was measured using a Cutometer (Courage+Khazaka, Germany) to determine R2 (elastic recovery) and R7 (dermal elasticity coefficient).

[0129] The results of summarizing the increase in each measured value based on the time before sample intake (day 0) are as shown in Table 3 below.

[0131] division Example 1 Comparative Example 1 Comparative Example 2 Improvement rate of elastic recovery force (R2) +15.2% improvement +4.6% improvement +2.1% improvement Improvement rate of dermal elasticity coefficient (R7) +18.7% improvement +6.3% improvement +3.5% improvement

[0133] Therefore, it was confirmed that the hibiscus collagen intake group of the example showed a skin elasticity index improved by more than 2.5 times compared to the comparative example.

[0135] (3) Skin tone improvement test

[0136] Each skin tone index was measured using a Colorimeter and a Mexameter on the skin of the forearm of the same subject group (n=30) as described in (2) above.

[0137] The results of summarizing the increase in each measured value based on the time before sample intake (day 0) are as shown in Table 4 below.

[0139] division Example 1 Comparative Example 1 Comparative Example 2 Skin L value (brightness indicator) +3.2 unit increase +0.8 unit increase (low significance) +0.3 unit increase (low significance) Melanin index -9.5% decrease -3.6% decrease -1.2% decrease

[0140] Therefore, the hibiscus collagen intake group of Example 1 showed distinct improvements in both skin brightness and melanin reduction, and a statistically significant difference was confirmed compared to the comparative example (p<0.05).

[0142] 3. Sensory Evaluation

[0143] (1) Experimental method

[0144] Subjects and Assignment: Thirty female subjects aged 25–45 were randomly assigned to three groups (Example 1, Comparative Example 1, Comparative Example 2). Each group consumed the product once a day for four weeks. The study was conducted as a single-blind study, and new skincare procedures and changes in functional cosmetics were restricted during the study period.

[0145] The evaluation points are as follows. A survey was administered at week 0 (baseline) and week 4 under the same conditions (2 hours after washing the face).

[0146] - Measurement item (5-point Likert scale, 1=Not at all, 5=Very much so):

[0147] 1) Skin hydration, 2) Skin elasticity, 3) Skin tone brightening, 4) Relief of skin roughness, 5) Overall satisfaction, 6) Intention to repurchase, 7) Taste and aroma solubility (convenience of consumption).

[0148] - Statistical analysis: Improvement scores were calculated by subtracting baseline scores from the Week 4 scores for each item. Comparisons between groups were performed using one-way ANOVA followed by Tukey's post-hoc test, with a significance level of p<0.05. Safety was assessed using the self-reported adverse event questionnaire.

[0151] (2) Experimental results

[0152] The classification of specific sensory evaluation improvement scores (△, on a 5-point scale, average ± SD) is as shown in Table 5 below.

[0154] division Example 1 Comparative Example 1 Comparative Example 2 Skin moisture +1.10 ± 0.52 a +0.48 ± 0.47 b +0.32 ± 0.41 b skin elasticity +1.08 ± 0.49 a +0.44 ± 0.45 b +0.28 ± 0.39 b Skin tone becomes clearer +0.96 ± 0.51 a +0.35 ± 0.43 b +0.22 ± 0.37 b Relieves skin roughness +0.92 ± 0.50 a +0.38 ± 0.42 b +0.20 ± 0.36 b Overall satisfaction +1.12 ± 0.55 a +0.51 ± 0.46 b +0.34 ± 0.40 b Willingness to repurchase +0.98 ± 0.57 a +0.43 ± 0.45 b +0.25 ± 0.38 b Water-soluble flavor and aroma +0.62 ± 0.48 a +0.31 ± 0.40 b +0.56 ± 0.46 b Note: Different superscripts (?, ?) indicate a statistically significant difference with p<0.05 in the ANOVA-Tukey post-hoc test (within-row comparison).

[0157] The Example 1 (Hibiscus Collagen) group showed significant improvements compared to Comparative Example 1 and Comparative Example 2 in skin hydration, elasticity, skin tone clarity, overall satisfaction, and intention to repurchase (p<0.05). There was no significant difference in taste or scent waterability (convenience of consumption) between Example 1 and Comparative Example 2, but it was significantly higher compared to Comparative Example 1 (p<0.05).

[0158] Specifically, the intake group of Example 1 reported the greatest improvements in moisturization, elasticity, and skin tone in terms of subjective perception, and overall product satisfaction and repurchase intention were significantly higher. This showed a consistent trend with the results of the experiment confirming the efficacy of skin elasticity and skin tone improvement in Section 2.

[0159] In addition, although the taste and aroma waterability of Example 1 and Comparative Example 2 are similar, they are superior to Comparative Example 1, suggesting that Example 1 has commercial advantages in terms of compliance with dosage.

Claims

Claim 1 A method for producing plant-based collagen comprising: (a) a step of preparing a plant extract from a plant raw material; (b) an enzymatic hydrolysis step of treating the plant extract with a proteolytic enzyme; (c) an enzymatic inactivation step of heating the hydrolysate obtained in step (b); (d) a step of fractionating the enzymatic inactivation solution obtained in step (c) through a filtration process to obtain low molecular weight peptides and freeze-drying the recovered solution; and (e) a step of treating the freeze-dried product obtained in step (d) with supercritical carbon dioxide; wherein step (d) comprises fractionating the enzymatic inactivation solution through an ultrafiltration process including 10 kDa and 3 kDa cutoffs and then additionally performing a nanofiltration (NF) process, and step (e) comprises a supercritical carbon dioxide treatment step including static treatment for 10 to 30 minutes and dynamic treatment for 30 to 90 minutes of the freeze-dried product under conditions of 200 to 300 bar and 40 to 50℃. Claim 2 A method according to claim 1, wherein step (a) involves mixing dried plant raw materials with water in a ratio of 1:10 to 1:30 (w / w) and extracting at 90 to 110 ℃ for 20 to 60 minutes to obtain an extract. Claim 3 A method according to claim 1, wherein step (b) involves adjusting the pH of the extract to 6.4 to 7.2, adding 0.1 to 0.3 wt% of a proteolytic enzyme relative to the total weight of the solution, and hydrolyzing by stirring at 50 to 60 ℃ for 2 to 5 hours. Claim 4 A method according to claim 1, wherein step (c) involves heating the hydrolysate at 80 to 110 ℃ for 5 to 15 minutes to inactivate the enzyme. Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 In claim 1, the protein-degrading enzyme is a protease, method. Claim 9 A method according to claim 1, wherein the plant raw material is one or more selected from the group consisting of hibiscus, apple, carrot, spinach, kale, and broccoli. Claim 10 A skin-improving composition comprising hibiscus collagen prepared by the method of claim 1 as an active ingredient, wherein the hibiscus collagen comprises (a) a glycine (Gly) content of 25.5 mol% to 28.5 mol%, (b) a proline (Pro) content of 10.0 mol% to 13.0 mol%, and (c) a hydroxyproline (Hyp) content of 0.9 mol% to 2.2 mol%. Claim 11 delete Claim 12 In item 10, the above skin improvement is a composition for skin moisturization, skin elasticity improvement, skin tone improvement, or skin roughness alleviation. Claim 13 In Clause 10, the above composition is a composition for cosmetic use. Claim 14 A food composition comprising hibiscus collagen prepared by the method of claim 1 as an active ingredient, wherein the hibiscus collagen comprises (a) a glycine (Gly) content of 25.5 mol% to 28.5 mol%, (b) a proline (Pro) content of 10.0 mol% to 13.0 mol%, and (c) a hydroxyproline (Hyp) content of 0.9 mol% to 2.2 mol%. Claim 15 delete Claim 16 In Clause 14, the above food composition is a food composition that is a general food or a health functional food. Claim 17 In claim 14, the food composition is formulated into one or more of tablets, granules, pills, powders, drinks, tea bags, candies, confectionery, beverages, and jellies.

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