WO2017135754A1

WO2017135754A1 - Collagen hydrolysate containing high content of collagen tripeptide and use thereof

Info

Publication number: WO2017135754A1
Application number: PCT/KR2017/001217
Authority: WO
Inventors: 신용철; 표쩌; 최수림; 김이수; 김현아; 조윤영; 정혜진
Original assignee: 아미코젠주식회사
Priority date: 2016-02-05
Filing date: 2017-02-03
Publication date: 2017-08-10
Also published as: CN107531775B; CN107531775A; KR101980361B1; KR20170093694A

Abstract

The present invention relates to a collagen hydrolysate with a high content of collagen tripeptides, and the use thereof and, more particularly, to a collagen hydrolysate prepared by using a collagenase that specifically produces a predominant amount of collagen tripeptides and a composition comprising the same as an effective ingredient for preventing hair loss and/or for promoting hair regrowth. Remarkably rich in collagen tripeptide content compared to previously reported collagen hydrates, the collagen hydrolysate of the present invention has an outstanding absorption rate in the body and exhibits an excellent capability of preventing hair loss and promoting hair regrowth and an excellent effect of improving skin wrinkles and/or moisturizing the skin.

Description

【Specification】

[Name of invention]

Collagen Hydrolyzate Containing High Content of Collagen Tripeptide and Its Soluble

Technical Field

This application claims the priority of Korean Patent Application No. 10-2016-0014896, filed February 5, 2016, the entirety of which is a reference of the present application. The present invention relates to a collagen hydrolyzate containing a high content of collagen tritide and its use, and more particularly, to a collagen hydrolyzate prepared by using a collagen hydrolase that specifically produces a collagen tripeptide, and It relates to a hair growth promoting and skin condition composition comprising this as an active ingredient.

[Background ᅳ technology]

Collagen is an animal's fibrous protein consisting of about 18 amino acids such as plin, oxyplin, glycine, and glutamic acid. In humans, a unique structure that accounts for the most 3W of the 5,000 kinds of proteins that make up the human body. Protein. In particular, the skin, bones and tendons are present in a lot, especially in the dermal layer of the skin 70% collagen is composed of collagen plays a very important role as a skin component. Collagen is a three-stranded strand of amino acids combined in the form of polypeptides and has a molecular weight of about 300, 000. The low molecular collagen is degraded by collagen degrading enzymes and the like and is low molecular weight with a molecular weight of 5, 000. It is also called a collagen peptide. When the low molecular collagen enters the human body, it is further broken down by proteolytic enzymes in the human body and finally the amino acid type. Absorbed by human body with Efl. Most proteins have a molecular weight of 12,000 ~ 70,000, but collagen has a molecular weight of 300, 000, which makes it more difficult to absorb. As a result, low molecular collagen is easily extracted, separated, After the purification process, the decomposition process is made in the form of a peptide having a molecular weight of 5,000 or less. Collagen tripeptide is a small chain of three amino acids (glycine-xy) Collagen (molecular weight is usually 200 ~ 500) refers to the small molecular weight is known to easily penetrate the skin. On the other hand, if more than four amino acids are linked, the molecular structure is large and cannot penetrate the skin. Collagen tritide contributes to shortening the time to repair damaged collagen tissue by reconnecting each other immediately after skin penetration. In addition, even when ingested, the absorption rate in the body is higher than low molecular collagen has the advantage of maximizing the bioefficiency of collagen. Generally, collagen is mainly made of cows, pigs, fish, squids, etc., and can be hydrolyzed by enzymes to obtain collagen peptide compositions. However, it is difficult to obtain a high content tripeptide composition by the proteinase which is generally used, so that the content of tripeptide is very low. Therefore, there is a steady need for an efficient production method for producing a high content of tripeptide collagen hydrolyzate. On the other hand, the hair has different cycles and grows and falls out through the anagen, the catagen, and the telogen. This cycle is repeated over three to six years, with an average of 50 to 100 hairs falling out daily. In general, alopecia refers to an abnormal increase in the number of hairs falling out due to shortening of the growth phase hair and more degenerative or resting hair in this cycle. The causes of hair loss are discussed, such as hyperthermia of male hormone action, excess sebum secretion, hypoglycemia, hypoxic function caused by peroxide, bacteria, genetic factors, aging, and stress. Testosteron, a type of male hormone, is activated by dihydrotestoserone (DHT) by an enzyme called 5 a -reductase, which induces a protein that binds to specific receptors and causes hair loss. Hair loss occurs. In addition, by this mechanism, the sebum may be overproduced, and acne or seborrheic dermatitis may be caused by hair loss accompanied by inflammation of the scalp. Alopecia is known to be caused by disease, malnutrition, aging, and hormonal imbalances. Despite much research, fundamental hair loss The mechanism for this is not well known. Efforts have also been made to treat hair loss. Nevertheless, only two drugs so far (pinasteride)

Only f inaster ide and minoxidil were approved by the Food and Drug Administraton (U.S. A) for hair loss treatment. Finasteride, a 5 -reductase inhibitor, has been used to promote hair growth in androgenetic alopecia in men. Minoxidil, an ant i-hypertensive agent, can promote hair growth by opening ATP-sensitive K-channels. However, the effects of drugs are limited and temporary because of unpredictable effects and side effects. There is a need for better new treatments to prevent hair loss and promote hair growth. In the case of preparations such as minoxidil and tr ichosacchar ide, which are known to prevent hair loss and have an effect on promoting hair growth and hair growth, the absence of obvious efficacy and human stability, As a side effect such as skin irritation is emerging, it is urgent to develop a composition having secured safety and efficacy.

[Detailed Description of the Invention]

[Technical problem]

Therefore, the present inventors endeavored to prepare a collagen hydrolyzate having a high content of collagen tripeptides with excellent body absorption, so that the collagen is hydrolyzed by a novel collagen hydrolase prepared using Bacillus subtilis. It has been found that the hydrolyzate having a high content of tripeptides can be prepared, and it has been found that the effect of promoting hair growth and improving skin condition is remarkably superior to the conventional collagen hydrolyzate, thereby completing the present invention. Accordingly, an object of the present invention is to provide a method for preparing a baby fish comprising: (a) mixing and heat-treating a young (fish scales) with water;

(b) preparing a collagen degrading enzyme by centrifuging the Bacillus subtilis strain culture solution, concentrating the centrifuged supernatant, and purifying by ion exchange chromatography; And

(c) the collagen degrading enzyme is added to the mixture of step (a) to be hydrolyzed at 25 to 45 ^° C. for 5 to 15 hours, and the collagen tripeptide is 20 to 20 relative to the total hydrolyzate. It is to provide a collagen hydrolyzate, which is contained in 70% by weight. Another object of the present invention to provide a food composition for hair loss prevention and / or hair growth promoting comprising the collagen hydrolyzate as an active ingredient. It is another object of the present invention to provide a pharmaceutical composition for preventing hair loss and / or promoting hair growth comprising the collagen hydrolyzate as an active ingredient. It is another object of the present invention to provide a cosmetic composition for preventing hair loss and / or promoting hair growth comprising the collagen hydrolyzate as an active ingredient. It is another object of the present invention to provide a cosmetic composition for improving skin wrinkles, improving skin elasticity, preventing skin aging and / or skin moisturizing comprising the collagen hydrolyzate as an active ingredient. It is another object of the present invention to provide a food composition for improving skin wrinkles, improving skin elasticity, preventing skin aging and / or skin moisturizing comprising the collagen hydrolyzate as an active ingredient. Another object of the present invention is to provide the use of the collagen hydrolyzate for the preparation of a hair loss prevention and / or hair growth promoting agent. Another object of the present invention is to provide a method for preventing hair loss and / or hair growth, characterized in that the effective amount of the collagen hydrolyzate is administered to a subject in need thereof. Another object of the present invention is to provide the use of the collagen hydrolyzate for the preparation of skin wrinkle improvement, skin elasticity improvement, anti-aging or skin moisturizing agent. Another object of the present invention is to provide a method for improving skin wrinkles, improving skin elasticity, preventing skin aging or skin moisturizing, characterized in that the effective amount of the collagen hydrolyzate is administered to a subject in need thereof. Technical Solution

In order to achieve the above object, the present invention comprises the steps of: (a) mixing the young (fish scales) with water and heat treatment;

(b) preparing collagen degrading enzyme by centrifuging the Bacillus subtilis strain culture solution, concentrating the centrifuged supernatant, and purifying by ion exchange chromatography; And

(c) the collagen degrading enzyme is added to the mixture of step (a) to be hydrolyzed at 25 to 45 ^° C. for 5 to 15 hours, and the collagen tripeptide is 20 to 20 relative to the total hydrolyzate. It is to provide a collagen hydrolyzate, characterized in that contained in 70% by weight. Another object of the present invention to provide a food composition for hair loss prevention and / or hair growth promoting comprising the collagen hydrolyzate as an active ingredient. It is another object of the present invention to provide a pharmaceutical composition for preventing hair loss and / or promoting hair growth comprising the collagen hydrolyzate as an active ingredient. In order to achieve the other object of the present invention, the present invention provides a cosmetic composition for preventing hair loss and / or hair growth comprising the collagen hydrolyzate as an active ingredient.

In order to achieve the another object of the present invention, the present invention provides a cosmetic composition for skin wrinkle improvement, skin elasticity improvement, skin anti-aging and / or skin moisturizing including the collagen hydrolyzate all active ingredients. In order to achieve the other object of the present invention, the present invention provides a food composition for skin wrinkle improvement, skin elasticity improvement, skin anti-aging and / or skin moisturizing comprising the collagen hydrolyzate as an active ingredient. In order to achieve another object of the present invention, there is provided a use of the above-described collagen hydrolyzate for preparing a hair loss prevention and / or hair growth promoting agent. To achieve another object of the present invention, an effective amount of the collagen hydrolyzate is administered to a subject in need thereof, thereby providing a method for preventing hair loss and / or promoting hair growth. To achieve another object of the present invention. It provides the use of the collagen hydrolyzate for the preparation of skin wrinkle improvement, skin elasticity improvement, skin anti-aging or skin moisturizing agent. In order to achieve the another object of the present invention, the present invention provides a method for improving skin elasticity, improving skin elasticity, preventing skin aging or moisturizing skin, by administering to the subject in need thereof an effective amount of the collagen hydrolyzate. Hereinafter, the present invention will be described in detail. The present invention comprises the steps of (a) mixing the young (fish scales) with water and heat-treated at 80 to 100 ^° C for 3 to 8 hours;

(c) the collagen degrading enzyme is added to the mixture of step (a) to be hydrolyzed at 25 to 45 ^° C. for 5 to 15 hours, and the collagen tripeptide is 20 to 20 relative to the total hydrolyzate. It provides a collagen hydrolyzate, characterized in that contained in 70% by weight. In the present invention, the step) is a process of heat-treating in silver by mixing the young to be extracted with collagen, and extracting the collagen contained in the young with water. The ratio of young to water added in step (a) is not particularly limited, but preferably young: water may be mixed in a weight ratio of 2: 8. The temperature condition for extracting collagen from the young in step (a) may be 80 to 120 ^° C, preferably 85 to 110 ^° C, more preferably 90 to 100 ^° C, but is not limited thereto. The optimal temperature condition is the time to extract collagen, Depending on the ratio of water and water. The time for heat treatment in step (a) may vary depending on the temperature conditions for extracting collagen, but may be from 1 hour to 10 hours, preferably from 2 hours to 9 hours, preferably from 3 hours to 8 hours. It is not limited to this. The step (b) is to prepare a hydrolase to hydrolyze collagen, to prepare a hydrolyzate having a particularly high content of collagen lipopeptide compared to enzymes used to hydrolyze collagen in the art. This step is to prepare enzymes. The present inventor has filed with the Korean Intellectual Property Office for the method of preparing collagen degrading enzyme of step (b) (Application No. KR10-2015-0124453), which can be referred to in the present invention. The Bacillus subtilis in the present invention is an accession number. KCTC12866BP may be characterized as a strain, but is not limited thereto. According to an embodiment of the present invention, the inventors performed ion exchange chromatography under various conditions in order to separate the collagenase enzyme contained in the Bacillus subtilis strain culture solution. The ion exchange chromatography of step (b) may use cation exchange chromatography, in which case it was confirmed that the recovery of the target enzyme is the best when using sodium chloride (NaCl) in a concentration of 0.1 to 0.3M. Therefore, when the ion exchange chromatography of step (b) is cation exchange chromatography, it is preferable to use 0.1 to 0.3 M sodium chloride (NaCl). According to another embodiment of the present invention, the ion exchange chromatography of step (b) in the present invention may use anion exchange chromatography, in this case sodium chloride (NaCl) concentration of 0.09-0.155M When used as it was confirmed that the recovery rate of the target enzyme is the best. Therefore, when the ion exchange chromatography of step (b) is anion exchange chromatography, it is preferable to use 0.09-0.155M sodium chloride (NaCl). In the present invention, step (c) is a step of hydrolyzing collagen by mixing the collagen extract prepared in step (a) and the collagen degrading enzyme prepared in step (b). In one embodiment of the present invention, it was confirmed that the collagen degrading enzyme prepared in step (b) maintains enzymatic activity for the longest time at 25 to 45 ^° C temperature conditions. If the temperature condition exceeds 45 ^° C, the initial enzyme activity may increase temporarily, but there is a problem that the loss of the activity over time is so large that the effect of hydrolysis of collagen does not appear sufficiently, on the contrary 25 ^° C If less than, there is a problem that the enzyme activity is too low to hydrolyze the collagen. Therefore, the hydrolysis temperature conditions in the step (c) is preferably 25 to 45 ^° C, more preferably 27 to 42 ^° C, even more preferably 30 to 40 ^° C, most preferably 35 to 40 May be ^° C. In the present invention, the collagen hydrolyzate obtained after the step (c) may further include a step of removing foreign substances through centrifugation, ion exchange chromatography, filtration, and concentrating. The process of removing and concentrating foreign matter can be used without limitation the methods commonly used in the art. In the present invention, the collagen hydrolyzate prepared according to the above method is characterized in that the content of the collagen tripeptide is remarkably improved compared to the collagen hydrolyzate prepared using the tagase. Collagen tripeptide is a small collagen (molecular weight of 200 ~ 500) with three amino acids (glycine-xy) linked. It is known to easily penetrate the skin due to its small molecular weight. On the other hand, if more than four amino acids are linked, the molecular structure is large and cannot penetrate the skin. Collagen tritide contributes to shortening the time to repair damaged collagen tissue by reconnecting each other immediately after skin penetration. In addition, even when ingested higher absorption in the body than the general low molecular weight collagen has the advantage of maximizing the bioefficiency of collagen. Therefore, the higher the content of collagen tripeptide in the collagen hydrolyzate, the higher the bioavailability can maximize the physiological activity of collagen. According to one embodiment of the present invention, the collagen hydrolyzate of the present invention is collagen The content of tripeptide was found to be about 53% by weight relative to the total hydrolyzate. The collagen hydrolyzate prepared using the tagase was found to contain little collagen tripeptide (Example 4 and Comparative Example). Since collagen is not easy to be absorbed into the body by itself, it is most preferable to use it by hydrolysis in the form of small peptides. The collagen hydrolyzate of the present invention has been previously reported to have a content of collagen tripeptides that are easily absorbed in the body. It is very good in that it is significantly higher than the collagen hydrolyzate. Specifically, in the present invention, the collagen hydrolyzate has 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 compared to the total hydrolyzate. , 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70% by weight may be included, if the collagen tripeptide to the total hydrolyzate within the range of 20 to 70% by weight limited to the above number It doesn't work. Preferably it may be 30 to 70% by weight, 40 to 70% by weight, 50 to 70% by weight, 30 to 60% by weight, 40 to 60% by weight, and most preferably 50. to 60% by weight. It is not limited to this.

The present invention also provides a composition for preventing hair loss and / or hair growth comprising the collagen hydrolyzate as an active ingredient. As used herein, "hair loss" refers to a phenomenon in which hair falls off the scalp or a condition in which the hair is coarse or thin, and "hair loss prevention" means preventing and suppressing hair loss as described above. "Promoting hair growth" means not only promoting the production of new hair, but also keeping the existing hair healthy. In general, "hair growth" occurs in the growth phase and is promoted by induction from the resting phase to the growth phase and the delay from the growth phase to the degenerative phase. The hair cycle can be divided into three main stages, known as growing, catenary and resting phases. In the growth phase, hair growth occurs as hair follicles grow deep into the skin with rapid proliferation of cells. The next phenomenon is the degenerative phase, which is a transitional period in which the disruption of cell division is prominent, during which hair follicles gradually degenerate and hair growth ceases. In the next phase, the resting phase, the degenerating hair follicles contain germs with densely packed dermal papilla cells. At rest The onset of new growth phase phenomena is induced by rapid cell proliferation in the embryo, expansion of the dermal papilla and synthesis of the underlying membrane elements. Therefore, it is necessary to prevent hair loss, that is, to prevent hair loss or to promote hair regrowth, that is, to promote hair growth, by promoting or extending the growth phase, and the composition according to the present invention has an effect of preventing hair from already falling out. At least one of the effects of thickening already existing hair and the like, and the effect of generating new hair. Compared with the conventional hydrolyzate, the composition of the present invention comprising a collagen hydrolyzate having a significantly higher content of collagen tripeptide as an active ingredient has high absorption and transdermal absorption rate of the collagen hydrolyzate and prevents hair loss due to fast absorption rate. The effect is very good. Specifically, according to one embodiment of the present invention, the in vivo absorption of collagen hydrolyzate prepared using the collagen hydrolyzate according to the present invention and hydrolases generally used to hydrolyze collagen in the art As a result of the evaluation, the collagen hydrolyzate according to the present invention showed that AUClast and Cmax were remarkably high and Tmax was short. This, in the case of collagen hydrolyzate according to the present invention because the content of collagen tripeptide is significantly high, it can be determined that the absorption in the body is very fast and the absorption rate is excellent. According to another embodiment of the present invention, the composition comprising the collagen hydrolyzate according to the present invention as an active ingredient promotes the proliferation of hair follicle cells in the degenerative hair loss animal model, not only inhibits the progression of the degenerative stage but also maintains the growth stage. It was confirmed that the hair growth effect was very excellent. On the other hand, the hair growth promoting effect of the composition according to the present invention was confirmed to be excellent to the same extent as minoxidil (Minoxidil) used as a positive control group. The collagen hydrolyzate according to the present invention is considered to have an excellent effect because the collagen tripeptide content is very high and the amount of collagen-derived peptide absorbed into the body is high. Accordingly, the present invention provides a hair loss preventing and / or hair growth promoting food composition comprising the collagen hydrolyzate as an active ingredient. In addition to containing the collagen hydrolyzate as an active ingredient, the food composition may contain various flavors or natural carbohydrates as additional components, as in the general product composition. Examples of such natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like, and xylyl, sorbitol, erythritol and the like. The aforementioned flavoring agents can advantageously be used natural flavoring agents (tautin), stevia extracts (for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention may be formulated in the same manner as the pharmaceutical composition and used as a functional food, or may be added to various foods. Foods to which the composition of the present invention may be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, ¾, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements. Foods; In addition to the collagen hydrolyzate, which is an active ingredient, the food composition may include various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, flavoring agents such as colorants and neutralizing agents (such as cheese, chocolate), pectic acid and Salts, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the food composition of the present invention may contain fruit flesh for the production of natural fruit juice and fruit juice beverage and vegetable beverage. Collagen hydrolyzate, the active ingredient of the present invention, is a natural substance and has little toxicity and side effects, so it can be used with confidence even when taken for a long period of time for the purpose of preventing hair loss and / or promoting hair growth. The food composition of the present invention may be a health functional food for hair loss prevention and / or hair growth promotion comprising a collagen hydrolyzate as an active ingredient. The health functional food of the present invention may be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing hair loss and / or promoting hair growth. In the present invention, "health functional food" refers to a food product manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6727 of the Health Functional Food Act. It means the ingestion for the purpose of obtaining a beneficial effect for health use such as nutrient control or physiological action. The health functional food of the present invention may include ordinary food additives, and the suitability as a food additive is not applicable unless otherwise specified in accordance with the General Regulations and General Test Act of the Food Additives Approval approved by the Food and Drug Administration. Judgment is made according to the standards and standards. Examples of the items listed in the "Food Additive Revolution" include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid and cinnamic acid; Natural additives such as dark blue pigment, licorice extract, crystalline cellulose, high pigment, guar gum and the like; And mixed preparations such as sodium L-glutamate, alkalescent additives, preservatives and tar dyes. For example, the health functional food in the form of tablets may be prepared by granulating a mixture of the collagen hydrolyzate, the active ingredient of the present invention, with an excipient, a binder, a disintegrant, and other additives in a conventional manner, and then using a lubricant and the like. Compression molding can be carried out, or the mixture can be directly compression molded. In addition, the health functional food in the form of tablets may contain a mating agent or the like as necessary. Hard capsules among the health functional foods in the form of capsules can be prepared by layering a mixture mixed with additives such as excipients and the collagen hydrolyzate, which is the active ingredient of the present invention, in a conventional hard capsule, and the soft capsule agent is a collagen hydrolyzate. It is possible to prepare a mixture mixed with an additive such as an excipient in a capsule base such as gelatin. The soft voxel agent may contain a plasticizer such as glycerin or sorbide, a colorant, a preservative, and the like, as necessary. Health functional foods in cyclic form are the collagen hydrolysates and excipients Formulations, binders, disintegrants, etc., may be prepared by molding known mixtures, and may be avoided with sucrose or other epidermis, or the surface may be coated with a substance such as starch or talc. You may. The health functional food in the form of granules can be prepared into a granular mixture of a collagen hydrolyzate, an active ingredient of the present invention, a brother, a binder, a disintegrant, etc. by a known method, and a flavoring agent, A mating agent and the like. The health functional food may be beverages, meat, chocolate, foods, confectionary, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements. The present invention also provides a pharmaceutical composition for preventing hair loss and / or promoting hair growth comprising the collagen hydrolyzate as an active ingredient. The pharmaceutical compositions according to the invention may contain the collagen hydrolyzate of the invention alone or may further contain one or more pharmaceutically acceptable carriers, excipients or diluents. Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like. In addition, carriers for parenteral administration may include water, suitable oils, saline, aqueous glucose and glycols, and the like, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-parabens and chlorobutanol. Other pharmaceutically acceptable carriers may be referred to those described in the following literature (Remington's Pharmaceut i cal Sciences, 19th ed., Mack Publ i shing Company, East on, PA, 1995). The pharmaceutical compositions of the present invention can be administered to any mammal, including humans. For example, it can be administered orally or parenterally. Parenteral By way of example, but not limited to, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal administration. All. Preferably the pharmaceutical composition of the present invention may be administered orally. For example, the pharmaceutical composition of the present invention may be prepared in an injectable formulation and administered by lightly prying the skin with a 30 gauge thin injection needle, or by directly applying it to the skin. The pharmaceutical composition of the present invention may be formulated into a preparation for oral or parenteral administration according to the route of administration as described above. In the case of preparations for oral administration, the compositions of the present invention are formulated using powders, granules, tablets, pills, sugar tablets, capsulants, solutions, gels, syrups, slurries, suspensions, etc. using methods known in the art. Can be. For example, oral formulations can be obtained by tablets or dragees by combining the active ingredient with a solid brother and then grinding it, adding suitable auxiliaries and processing it into a granular mixture. Examples of suitable excipients include sugars and corn starch, wheat starch, rice starch and potato starch, including lactose, dextrose, sucrose, solbi, manny, xili, erysri and malty. Layered agents such as starch, cellulose, methyl cellulose, sodium carboxymethyl salose and hydroxypropylmethyl-cellulose, such as salose, gelatin, polyvinylpyridone and the like. In some cases, crosslinked polyvinylpyridone, agar, alginic acid or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition of the present invention may further include anticoagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives. Formulations for parenteral administration may be formulated by methods known in the art in the form of injections, creams, lotions, external ointments, oils, humectants, gels, aerosols and nasal inhalants. These formulations are described in Remington ¹ s Pharmaceutical Science, 15th Edion, 1975. Mack Publ i shing Company, East on, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a prescription commonly known for all pharmaceutical chemistry. It is described. Hair loss room when the pharmaceutical composition of the present invention is used as an external skin pharmaceutical composition It is a skin external preparation which has the effect of improving the hair, hair growth and scalp, and is prepared in the form of a pharmaceutical composition in the form of an external skin preparation such as cream, gel, patch, spray, ointment, warning, lotion, linen, pasta or cataplasma. Can be used, but is not limited thereto. The total effective amount of the pharmaceutical composition of the present invention can be administered to a patient in a single dose, and administered by a fract ionated treatment protocol that is administered for a long time in a multiple iple dose. Can be. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the extent of the disease. Preferably the preferred total dose of glycoprotein fraction of the present invention is about O. per kg body weight of the patient per day. Olug to 1,000 mg, most preferably 0.1 to 100 mg. However, the dose of the candidate drug of the present invention may be determined by considering various factors such as the age, weight, health status, sex, severity of disease, diet and excretion rate, as well as the route of administration and the number of treatments of the pharmaceutical composition. Since the effective dosage is determined, those of ordinary skill in the art should consider the appropriate effective dosage according to the specific use of the pharmaceutical composition of the present invention as a pharmaceutical preparation for preventing hair loss and promoting hair growth. You can decide. The pharmaceutical composition according to the present invention is not particularly limited to the formulation, route of administration and method of administration as long as the effect of the present invention is shown. The present invention also provides a cosmetic composition for preventing hair loss and / or hair growth comprising the collagen hydrolyzate as an active ingredient. The cosmetic composition according to the present invention may include not only collagen hydrolyzate, which is an active ingredient, but also components commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and perfumes. Auxiliaries, and carriers. In addition, the composition of the present invention can be used in addition to the above-described collagen hydrolyzate, in addition to the hair loss prevention agent and / or hair regrowth agent that has been used conventionally, so long as it does not impair the action of hair loss prevention and hair growth promotion by collagen hydrolyzate. . In addition, the cosmetic composition of the present invention is any conventionally prepared in the art It can also be prepared as a dosage form, for example, hair tonic, hair conditioner, hair essence, hair lotion, hair nutrition lotion, hair shampoo, hair rinse, hair treatment, hair cream, hair nourishment cream, hair moisturizer cream hair Massage cream, Hair wax, Hair aerosol, Hair pack, Hair nutrition pack, Hair soap, Hair cleansing product, Hair oil, Hair dryer, Hair preservative, Hair dye, Hair wave, Hair gel, Hair gel, Hair glaze, Hair Dressing, hair lacquer, hair moisturizer, hair mousse and hair spray can be prepared in various forms, but is not limited thereto. According to one embodiment of the present invention, the collagen hydrolyzate of the present invention was found to be very excellent in improving the skin condition such as skin wrinkle improvement effect, skin moisturizing effect. On the other hand, the skin condition improvement effect was remarkably superior to the collagen hydrolyzate produced by the previously reported collagen hydrolase. This is because the collagen hydrolyzate according to the present invention is composed of a high content of collagen tripeptide, it was determined that the biophysical activity of collagen is excellent because of its high bioavailability. The skin condition improvement effect of the collagen hydrolyzate according to the present invention can be more clearly identified through specific examples. Accordingly, the present invention provides a cosmetic composition for improving skin wrinkles, improving skin elasticity, preventing skin aging and / or skin moisturizing, including the collagen hydrolyzate as an active ingredient. ^'

In addition, the present invention provides a food composition for skin wrinkle improvement, skin elasticity improvement, skin anti-aging and / or skin moisturizing comprising the collagen hydrolyzate as an active ingredient. The cosmetic composition and the food composition described above may be applied as it is. The present invention provides the use of the above collagen hydrolyzate for the preparation of a hair loss prevention and / or hair growth promoting agent. The present invention provides a method for preventing hair loss and / or promoting hair growth, comprising administering an effective amount of the collagen hydrolyzate to a subject in need thereof. The present invention provides the use of the collagen hydrolyzate for the preparation of skin wrinkle improvement, skin elasticity improvement, anti-aging or skin moisturizing preparation. The present invention provides a method for improving skin wrinkles, improving skin elasticity, preventing skin aging or skin moisturizing, characterized in that the effective amount of the collagen hydrolyzate is administered to a subject in need thereof. The 'effective amount' of the present invention, when administered to an individual, refers to an amount indicating hair growth promotion and / or hair loss improvement, treatment prevention, etc., and improvement of skin wrinkles, skin elasticity, skin aging and / or skin moisturizing. cure. Refers to an amount indicating prophylaxis, etc. The 'individual' may be an animal, preferably a mammal, particularly an animal including a human, or may be a cell, tissue, organ or the like derived from an animal. The subject may be a patient in need of the effect. Advantageous Effects

The present invention relates to a collagen hydrolyzate containing a high content of collagen tritide and its use, the collagen hydrolyzate of the present invention has a significantly higher content of collagen tripeptide compared to the previously reported collagen hydrolyzate, so that the body absorption rate It is excellent and shows excellent hair loss prevention, hair growth promotion, wrinkle improvement and / or moisturizing effect.

【Brief Description of Drawings】

1 is a view showing the results of separation and purification using cationic ion resin of collagen degrading enzyme prepared by the embodiment of the present invention. A is the result of SDS-PAGE analysis of purified collagenase (M: protein si marker, cone: concentrated sample, wash: wash, E1-E5: elut ion samples 1-5). B shows the peak of collagen degrading enzyme purification according to the change of buffer concentration in AKTA pr imer (arrow is indicated for each elut ion samples).

Figure 2 is a view showing the results of separation and purification using negative ion resin of the collagen degrading enzyme prepared by the embodiment of the present invention. A is the purified collagen degrading enzyme Results of SDS-PAGE analysis (M: protein size marker, PI: peak 1). B shows the peak of the purification of collagen degrading enzyme according to the change of buffer concentration in the AKTK pr imer (arrow is indicated for each e hit ion sample).

Figure 3 is a graph showing the results of enzyme activity by temperature of the collagen degrading enzyme prepared by the embodiment of the present invention.

Figure 4 is a graph showing the results of changes in enzyme activity according to the time and temperature of the collagen degrading enzyme prepared by the embodiment of the present invention.

Figure 5 is a graph showing the enzyme activity results for each pH of the collagen degrading enzyme prepared by the embodiment of the present invention.

6 is HPLC data comparing collagen tripeptide productivity of collagenase and general protease prepared according to an embodiment of the present invention (A: CTP standard, B: BP, C: Alcalase 2.4L FG ′ D: Flavorzyme 1000L, E: Col lupul in MG).

7 is a production process for producing collagen hydrolyzate having high collagen tripeptide content using collagen degrading enzyme prepared according to an embodiment of the present invention.

8 is HPLC data analyzing the collagen tripeptide (CTP) content of the hydrolyzate produced using the collagenase produced by the embodiment of the present invention (A: CTP standard, B: BP, C: Alcalase 2.4L FG).

Figure 9 is a graph evaluating the effect of the administration of the test substance on the growth and degenerative rate of hair follicles in the animal model induced degenerative hair loss with Dexamethasone ((A) 9 days hair removal, (B) 11 days hair removal, (C) 14 days of hair removal, (D) 17 days of hair removal, anagen: growth, catagen: degenerative, Nor: normal group without hair removal without dexamethasone treatment, Control: dexamethasone treatment and hair removal after solvent treatment, BP-CH: the group administered with the collagen hydrolyzate of the present invention after the dexamethasone treatment and hair removal, Minox: the positive control group administered minoxidil after the dexamethasone treatment and hair removal).

10 is a result showing the effect of the administration of the test substance on the hair growth progression of the test animal in the animal model induced degenerative hair loss with Dexamethasone.

11 is a result of observing hair follicle growth of animal model skin tissue induced by dexamethasone through tissue staining (Dex: dexamethasone).

12 is a result of observing the effect of wrinkles after the administration of each substance to the animal model induced wrinkles by UV irradiation with a replica plate using silicone and EPON resin (N: Normal, C: Control, P: Posi t ive control, C0L17, C0L34: collagen hydrolysates produced by normal collagenase 17 mg / kg or 34 mg / kg dose group, CTP17, CTP34: of the present invention Collagen hydrolyzate 17 mg / kg or 34 mg / kg dose group).

Figure 13 shows the results of the Masson's Tri chrome staining and the skin of SKH-1 hairless mice after the end of the experiment (N: Normal, C: Control, P: Positive control, C0L17, C0L34: general collagenase 17 mg / kg or 34 mg / kg administration group of collagen hydrolyzate produced by CTP17, CTP34: 17 mg / kg or 34 mg / kg administration group of the collagen hydrolyzate of the present invention).

FIG. 14 shows the results of quantifying the skin expression of SKH-1 hairless mice after the experiment was completed by Western blot for protein expression levels of Collagen 1A (A) and MMP-1 (B) in tissues (N: Normal, C: Control, P: Positive control, C0L17, C0L34: Collagen hydrolyzate produced by normal collagenase 17 mg / kg or 34 mg / kg dose group, CTP17, CTP34: Collagen hydrolyzate 17 mg of the present invention / kg or 34 mg / kg dose group).

15 is a result of checking the difference in the degree of oil (A), moisture (B) of the skin of the mouse at the 5th and 10th weeks of the experiment (N: Normal, C: Control, P: Positive control, C0L17, C0L34: General 17 mg / kg or 34 mg / kg of collagen hydrolyzate produced by collagenase, CTP17, CTP34: 17 mg / kg or 34 mg / kg of collagen hydrolyzate of the present invention).

16 is a result of evaluating the skin moisture content after administration of each substance to Balb-c mice induced skin drying (N: Normal, C: Control, P: Positive control, CTP17: collagen hydrolyzate of the present invention 17 mg / kg, C0L34: 34 mg / kg dose of collagen hydrolyzate prepared using a previously reported hydrolase).

17 is a result of measuring the number of scratches due to itching after administration of each substance to Balb-c mice induced skin drying (N: Normal, C: Control, P: Positive control, CTP17: of the present invention Collagen hydrolyzate 17 mg / kg, C0L34: 34 mg / kg administration group of collagen hydrolyzate prepared using a previously reported hydrolase).

18 is a result of administering each substance to Balb-c mice induced skin drying, and after the experiment was completed, the skin tissue of each mouse was collected, and the amount of skin moisturizing factor protein was confirmed through Western blot and quantified. (A: Collagen 1A, B: AQP3, C: HAS2, N: Normal, C: Control, P: Positive control, CTP17: Collagen hydrolyzate of the present invention 17 mg / kg, C0L34: A previously reported hydrolase Collagen hydrolyzate prepared by using 34 mg / kg administration group). [Form for implementation of invention]

Hereinafter, the present invention will be described in detail.

However, the following examples are merely to illustrate the present invention, the content of the present invention is not limited to the following embodiments.

Preparation of Collagen Hydrolase

For the method of preparing the collagen hydrolyzate in the present invention and the collagen hydrolyzate prepared by the method, Korean Patent Application No. 10—2014-0158019 may be referred. That is, Bacillus subtilis strain (Accession No. KCTC12866BP) in 100 ml of mTB medium (yeast extract 2.4%, tryptone 1.2%, glycerol 1%, KH ₂ P0 ₄ 2.31%, H ₂ HP0 ₄

12.54%) and centrifuged for 15 minutes at 6500 rpm. The supernatant was carefully transferred to a new lyve and concentrated 5 times using 30 kDa f i lter. The concentrated supernatant was purified by ion exchange chromatography using an AKTA primer. The purification conditions used were A buffer (50 mM Tr i s-HCKpH 7.5) as a binding buffer and B buffer (50 mM Tr i s-HCKpH 7.5), 0.5M NaCl) was inclined. The flow rate was flowed at 5 ml / min. Purification was carried out using cation exchange chromatography and anion exchange chromatography. As the ion resin, SP sepharose resin (GE Heal thcare, new-Jersey, USA) as a cation resin and Q sepharosse resin (GE healthcar, new-Jersey) as an anion resin , USA). In the first cation exchange chromatography, the product was inclined to 0.5 M NaCl and purified protein was obtained for each fraction. As shown in FIG. 1B, the protein purity of the fractions (E1 to E5) in which the protein peaks appear in the purification profile was confirmed on SDS-PAGE, and the protein expected to be the target enzyme was included in E1 and E2. In particular, E1 It was confirmed that there is the most collagen degrading enzyme in (arrows of Figure 1A)

NaCl concentrations of E1 to E5 are El: 0.1-0.2M, E2: 0.2-0.3M, E3: 0.3-0.4M, E4: 0.4-0.5M, E5: 0.5M, which is why Minute enzymes The NaCl concentration was found to be 0.1-0.3M and the recovery rate of the target enzyme was the best at the NaCl concentration of 0.1-0.2M. Next, E1 was purified by anion exchange chromatography. The buffer used was 0.25M NaCl and was inclined in the same manner as cation exchange chromatography. As shown in FIG. 2B, the anion exchange resin in the purification profile showed that a protein peak appeared in 0.09-0.115M NaCl (arrow of FIG. 2B). As a result of confirming the purification degree of this fraction on SDS-PAGE, it was confirmed that the target enzyme was mostly purified (P1 of FIG. 2A). Therefore, the anion exchange chromatography showed the best recovery of the target enzyme in NaCl of 0.09 ~ 0.155M. Thereafter, the purified new collagen degrading enzyme was named BP.

BP Enzyme Reaction Condition Experiment

In the optimum temperature experiment, the enzyme reaction was carried out at a temperature of 20 ~ 70 ^° C to measure the titer. As shown in FIG. 3, the experimental results have enzymatic activity at 30-55 ^° C., in particular, the highest specific activity and relative activity at 50 ^° C., and activity from 60 ^° C. It was confirmed to fall sharply. Temperature stabilization experiments were conducted for actual mass production. The temperature was adjusted to 30 ^° C, 35 ^° C, 40 ^° C, and 50 ^° C, and each silver sample was sampled hourly from 0 hours to 12 hours to confirm each residual activity.

As shown in FIG. 4, as a result, the lower the temperature, the more stable the enzyme was observed, and at 35 ^° C. and 40 ^° C., it was confirmed that the activity of the enzyme remained about 59.3% after 12 hours. In view of these results, it is desirable to set the production process of the collagen tripeptide at 35 ^° C.

Next, the optimum pH was examined. Donpi gelatin substrates were prepared for each pH (50 mM citrate-Na ₂ HP0 ₄ (pH 5.0-6.0), 50 mM Tris-HCKpH

6.0-9.0), 50 mM Na ₂ C0 ₃ -N¾HC0 ₃ (pH 9.0 ~ 10.0)) and then determined the optimum pH by measuring the activity of the enzyme. BP showed the highest activity at pH 7.4 and high activity in the region of vocal cords up to pH 6-10. However, as shown in FIG. It was confirmed that the activity of the enzyme was similarly decreased with increasing basicity. Hereinafter, a process of preparing collagen tripeptides using the collagen degrading enzyme (BP) will be described.

BP Collagen Tripeptide Productivity Verification

Prepare the four samples by heat-treating the pre-treated fish (fish scales) in a reaction vessel equipped with a stirrer and uniformly mixing the weight ratio of the fish and water at a ratio of 20:80 for 5 hours at 90 ^° C. It was. BP (manufacturer: Amicogen), Alcalase 2.4L FG (manufacturer: Novozyme, shop: Biosis), Flavorzyme 1,000L (manufacturer: Novozyme, shop: Biosis), Collupulin MG (manufacturer: Novozyme , Collagen tripeptide productivity was compared using an enzyme from Daejong Co., Ltd.). The reaction conditions (temperature, pH, enzyme use, reaction time) of each enzyme are as follows.

-BP: 35 ^° C, pH 7.4, 30 unit / g (young), 12 hours

Alcalase 2.4L FG: 60 ^° C, pH 7.0, 30 unit / g (child), 12 hours lav Flavorzyme 1,000L: 55 ^° C, pH 6.0, 30 unit / g (child), 12 hours

-Collupulin MG: 60 ^° C, pH 7.0, 30 unit / g (young), after 12 hours of enzyme treatment, heat treatment at 80 ^° C for 30 minutes to inactivate the enzyme. Next, the four enzymes were compared and analyzed to confirm the collagen tripeptide production of BP.

Collagen hydrolyzate analysis was performed using HPLCXgilson)

TM

Collagen tripeptide (CTP) was analyzed under conditions of Superdex Peptide 10 / 300GL column, mobile phase (10 mM Tris-CKpH 7.4), 0.15 M NaCl, 5 mM CaCl ₂ , and flow rate: 0.5 i / min. The standard for CTP was glycine-proline-hydroxyproline (GPH) consisting of three amino acids. As shown in FIG. 6, in the case of the CTP standard material, it was confirmed that a peak was formed at about 55 minutes. As a result of comparing the degrading activity of the four enzymes, the peaks of the collagen hydrolysates hydrolyzed using BP were mostly late. It was found that collagen was lowered in time, and there was a large peak at the same time zone in the standard, indicating that the CTP content was high. Collagen hydrolysates hydrolyzed with Alcalase 2.4 L FG had higher molecular weight than collagen hydrolysates hydrolyzed with other Flavorzyme 1000 L or col lupuin MG, but showed little CTP content. As a result, it was found that BP is very effective in producing collagen tripeptide.

Preparation of Collagen Hydrolysates Using BP

In order to produce collagen hydrolyzate containing collagen tripeptide, a manufacturing method using BP was established. The pre-treated fish (fish scales) was heat-treated at about 90 ^° C. for 5 hours by uniformly mixing the weight ratio of the fish and water in a ratio of 20:80 to the reaction vessel equipped with the stirrer.

The heat-treated solution was calibrated to pH 7.4 in solution by adding 10% NaOH at 35 ^° C. BP was added to the calibrated solution and reacted at 35 ^° C. for 12 hours after adding about 30 units / g (young). After enzyme reaction, heat treatment was performed at 80 ^° C. for 30 minutes to inactivate the enzyme. Enzyme-treated collagen peptide solution was removed using ultra-fast serial centrifuge to remove foreign substances such as metal ions through ion column purification.

The solution was concentrated to Brix 35% using a vacuum pressure concentrator, and then decolorized and deodorized through activated carbon tablets. Activated charcoal purified liquid was microfiltration (membrane f ltrat ion) and then powdered with a spray dryer to perform a quality test and through the packaging step to prepare a collagen hydrolyzate containing a high content of the peptide (Fig. 7).

Comparative Example 1

Preparation of Coke Hydrolysates Using General Hydrolase

For comparison with the production process using the new BP, collagen tripeptide productivity was compared using Alcalase 2.4 L FG (manufacturer: Novozyme, place of purchase: Biosis), which is generally used for low molecular weight collagen degradation process. The pre-treated fish (fish scales) was heat-treated at about 90 ^° C. for 5 hours by uniformly mixing the weight ratio of the fish and water in a ratio of 20:80 to the reaction vessel equipped with the stirrer.

The heat-treated solution was calibrated to pH 7.5 in solution by adding 10% NaOH at 55 ^° C. Alcalase enzyme was added to the calibrated solution and about 30 unit / g (young) was added at 35 ^° C. Treatment for 12 hours. After enzymatic reaction, the enzyme was inactivated by heat treatment at 80 ^° C for 30 minutes.

Enzyme-treated collagen peptide solution was removed using an ultra-fast serial centrifuge to remove foreign substances such as metal ions through ion calmpur purification. The solution was concentrated to Br ix 35% using a vacuum depressurizer and then decolorized and deodorized using activated carbon tablets. Activated carbon tablets were filtered by filter press filtration and microfiltration (membrane f i lt ion), and then powdered with a spray dryer for quality testing and analyzed for collagen tripeptide (CTP) content.

CTP content was carried out in the same manner as in Example 2. Samples were analyzed for collagen hydrolyzate before final formulation prepared using BP and Alcalase. As shown in FIG. 8, in the case of the CTP content, it was confirmed that the peak appeared at the same position as the CTP standard in the collagen hydrolyzate prepared using BP, and in the same position in the collagen hydrolyzate prepared using Alcalase. Pick did not appear.

The quantitative comparison of CTP content of collagen hydrolysates prepared using BP and Al cal ase showed that BP was 53.1 ^ Alcalase and 0%. This result shows that BP can be used to prepare collagen hydrolyzate with high content of collagen tripeptides.

Example 5

Comparison of Absorbance of Collagen Hydrolysates Using BP and Collagen Hydrolysates Using Alcalase

After anesthetizing male Sprague-Daw ley (SD) rats with ether, the right carotid artery is cannulat ion with polyethylene tubing, and then the cannula is pulled back and forth using the infusion harness to allow the rats to move freely and the surgical site is sutured. It was. After fasting cannulat ion rats for 12 hours or more and freely supplying water, the collagen hydrolysates (BP-CH) using BP were prepared according to the method for preparing collagen hydrolyzate of Example 4 and Comparative Example 1 in each of 6 rats. ) And Al cal ase Collagen Hydrolyzate (Alcalase-CH) was dissolved in distilled water at a dose of 500 mg / kg (5 mL / kg). After administration, blood was collected into the carotid artery (15, 30, 45, 60, 90, 120, 180, 240, and 360 minutes) and centrifuged to separate plasma. Plasma samples were stored in -20 ^° C freezer until analysis, and quantitatively analyzed by simultaneous analysis of the concentration of hydroxyproline (Hydroxypro line, Hyp) in plasma by LC-MS / MS.

<5-2> LC-MS / MS Analysis

Sample pretreatment: Add 50 uL of internal standard solution (Gabapentin 0.1 ug / mL) to the plasma 50, mix, add 5% trichloroacetic acid 50, mix, and centrifuge for 10 minutes at 13,000 rpm. 2 was analyzed by injection into LC-MS / MS.

Analytical Instruments: Agilentl260 HPLC system and Agilent6460 tr i le-quadrupole mass spectrometer

Column: Zorbax SB-Aq, particle size 1.8μ, 2.0 mm, id 100 mm, 1.Column temperature: 30 ^° C

Mobile phase: 0.1% formic acid (A) acetonitrile (B) gradient elution Flow rate: 0.25 mL / min

Injection volume: 2 _U L

<5-3> Absorbency Evaluation Method

Plasma concentration-time curves were prepared for each target component in each group, and the absorbance was compared by calculating the area under the plasma concentration rat ion-time curve (AUC). AUC was obtained by the linear trapezoidal method. Extrapolation in the terminal phase was difficult, so the total AUC up to infinity was not obtained, but AUClast up to 6 hours, the last time of blood collection, was obtained. The peak blood concentration (Cmax) and the peak blood concentration arrival time (Tmax) were obtained by reading directly on the graph. The AUC, Cmax, and Tmax values of each group were examined by one-way AN0VA analysis and Duncan's post hoc test.

<5-4> Absorption test results of collagen hydrolyzate

AUC and Cmax levels of blood free Hyp were significantly different between BP-CH and Alcalase-CH groups. This means that the absorption rate of Hyp in the blood was high. Also BP-CP administration group Tmax was faster than Alcalase-CH group. This result indicates that Hyp was rapidly absorbed after ingesting BP-CH, and the shorter time spent in the blood resulted in faster absorption of blood from the tissue (Table 1). Through the experiments, it was confirmed that the absorption efficiency of collagen hydrolyzate using BP was higher than that of hydrolyzate by examining the change of collagen-specific amino acid Hyp in blood.

Table 1

Comparison of Absorbency Results for Hyp of BP-Used Collagen Hydrolysates and Alcalase-Used Collagen Hydrolysates

Abbreviation

AUC st, total area under the lasma concent rat iont ime curve from time zero to last measured time, 6 hr; C peak lasma concent rat i on; T _max , ti me to reach C _max

a

SD: Standard deviations b CTP group is significantly (p <0.05) different from Alcalase-CP group.

Median (range) for T _max

Hair loss prevention and hair growth promoting effect of collagen hydrolyzate using BP <6-l> Experimental Animal Diet

Collagen hydrolyzate was prepared by the same method as the method for preparing collagen hydrolyzate of Example 4, but BP-enzyme treated with 17 uni t / g of collagen hydrolyzate (BP-CH) having 28% CTP content. It was prepared and used in the diet.

<6-2> Establishment of experimental and degenerative alopecia animal models

The experimental animal is a C57BL / 6 mouse, a melanin pigment is present in the hair follicles, and the skin color changes depending on the amount of the melanin pigment, and the hair color can be identified by the skin color (growth: black, degenerative and resting period: pink). At -7 weeks of age, it changes to resting period. Because of these characteristics, 35 were selected from Biolink (Lamb North) for 6 week old C57BL / 6 female mice (17 g-20 g). The test group consisted of dexamethasone S.-derived dexamethasone-treated group, dexamethasone-treated group and BP-treated collagen hydrolyzate (BP-CH) -treated group (cont ro l) and positive control group (3% Minox i di l). ) Was set as a treatment group, and composed of 8 animals in each group. The control and collagen hydrolysates were administered orally once every 10 days for 2, 000 mg / 10 ml / kg, and the positive control Minoxi dil was also applied daily.

The back of the 7-week-old test animal was removed from the hair using an animal hair remover and a hair remover niklin (Ildong Pharmaceutical) and quickly washed with water. After 7 days of hair removal, the sample and minoxidil were administered once daily for 10 days. 0.1% Dexamethasone was applied to the back of test animals for 5 days at 1 ml / day starting from day 9 of hair removal. The test animals were regenerated by anesthesia with ether the day after the last sample and Minoxi di administration.

<6-3> Determination of hair loss stage and maintenance of growth stage

The clinical symptoms and death of test animals were checked once daily. Photographs were taken once a week to visually identify the growth-induction effects, and photographed once every 3 days to observe the depressive effects. The photograph was taken to determine the ratio of growth stage and degeneration stage by measuring the rate of change of color from gray to black of the total epilation of the test animals. The results are shown in Table 2, FIG. 9 and FIG. 10.

[Table 2]

Percentage of degeneratives in epileptic restraint model (%)

Dexatoethasone

Orthodox Contrast after Stepmother — ϊ¾1 fll

IP-CH

(coniro 扁)

Day 9 57.0 ± 26.8 67.4 ± 17.0 56.9 ± 2S.3 40.8 15.4 * π 22.6 ± iz. r ^* 100 -0 0.0 29.8 ± 197 "17,8 ± 4.9

14 7.6 + 5, 7 ** 99,3 ± 1.2 32.0 ± ： 23.6 ^s * 9.7 + 6, 2 "

17 0, 8 ± 2, Γ 97,7 ± 2.3 27, 3 ± 26.4 ** 8, 8 ± 6.6

Tlhe al es are me ni.SE,

SSgnificant dsfierene © from Control group by D mist's t-teat ： ^** Ο.ΟΙ.

The numerical value in the above table means the rate of progression of the retrograde stage at the site of induction. In other words, the higher the ratio, the higher the rate of progression of the degenerative stage, and the lower the rate. In the above table, BP-CH means the animal group ingested the collagen hydrolyzate of the present invention. As shown in Table 2, Figure 10 and Figure 11, the ratio of the growth period and the age of hair removal on the 9th day of hair removal did not differ between the normal control group and the test substance administration group, but the minoxidi l application group was statistically significant compared to the solvent control group treated with dexamethasone only. As a result, the growth rate was high and the rate of degeneration was decreased. At 11, 14, and 17 days of hair removal, the solvent control group remained pink until the end of the test. From the 11th day of hair removal, the growth rate of the test group and the minoxidi group was significantly increased.

<6-3> histological examination

After the experiment according to Example <5-1>, the skin of the epilation site of the test animal was The hair follicles were collected and examined.

In other words, the test animals were reestablished with ethyl ether, regenerated, skin tissue was removed, stored in 10% neutral formalin solution, paraffin embedded and cut into 5 ί thicknesses to prepare tissue slides, and stained with Hematoxyl in & Eosin Staining. . The results are shown in FIG. 11.

As shown in FIG. 11, in the solvent control group induced by the dexamethasone treatment, the epidermal layer was thinned and no hair follicles were formed. The epidermis of the group treated with dexamethasone and ingested collagen hydrolyzate (BP-CH) was thickened and developed as hair follicles developed. The minoxidi l-coated group also found that the epidermal layer became thick and hair follicles developed.

That is, the collagen hydrolyzate according to the present invention was found to be effective in promoting hair follicle cell proliferation in the degenerative hair loss state and improving hair growth.

Anti-wrinkle Effect of Hydrolysates of Coke Hydrolysates Using BP

SKH-1 Hair less mouse-based, 6-week-old model (female) was imported and sold from Samtako BIOKOREA (Osan-si, Gyeonggi-do, Korea). Dong-Ae Chemical Research Institute (Animal facility registration certificate: No. 412) No.) set a healthy environment with quarantine and purified breeding for one week, and set the breeding environment to temperature (22223) ^° C, relative humidity (50 士 10)%, lighting time 12 hours (07: 00 ~ 19: 00). It was carried out by. As shown in Table 3, the experiment was divided into a total of seven groups of 10 animals in each group. Other than the normal group, the wrinkle-inducing group was subjected to UVB irradiation stepwise for 10 weeks, and the Cntr group, Posi ive controls (ret inoic acid 2 mg / kg, i.p.), and normal collagen degrading enzyme ( Alcalase) collagen hydrolyzate (C0L) and collagen hydrolyzate (CTP) sample administration according to the present invention were administered orally for 17 mg / kg and 34 mg / kg for 14 weeks, respectively. The feed was used for solid animal feed (Samtako BIOKOREA, Korea) and the drinking water was freely consumed.

[Table 3] Composition of Laboratory Animals

In the case of positive ive control, 0.05% ret inoic acid was intraperitoneally administered using the Bel inda M method (The journal of invest igat ive dermatology, 112 (3); 271-278, 1999.).

UV irradiation device (East-West Science, UV-1000) emits UVB in the cabinet.

The light was emitted by the Sunlamp, and the light source was 302 nm, and the UVB intensity was irradiated at a height of 0.3 mW / cm ² .

The amount of UV irradiation was measured by a UV-radiometer, the mouse was placed in a cage for ultraviolet irradiation, three times a week every other day on the back area, for a total of 10 weeks [0 weeks: 60 mJ / m ² (1 MED), 1 Note: 120 mJ / m ² (2 MED), 2-3 weeks: 180 mj / m ² (3 MED), 4-5 weeks: 240 mJ / m ² (4 MED) 5-10 weeks: 240 mJ / m ² (4 MED)].

<7-1> Morphological Observation of Skin Wrinkles

Skin wrinkles were observed by lightly anesthetizing experimental animals with ether for 5 to 14 weeks, photographing the surface with a digital camera, and then using SILFLO (Flexico developments LTD. Tokyo, Japan) si l icone rubber impression material. A repl ica of the skin wrinkles was prepared and photographed with a microscope. The result of visual observation of the shape of the skin wrinkles is shown in FIG. 12. SKH-1 hair less As a result of observing the skin wrinkle pattern of the mock plate prepared using SILFL0 on the mouse skin, UVB-irradiated Contr group (Group C) had thick wrinkles, wide intervals and deep wrinkles, and administered retinoic acid. Group P and C0L 17 ， 34 mg / kg administered group, CTP 17 ， 34 mg / kg administered group showed less wrinkles or thinner lines and thinner lines than the C group (FIG. 12).

In consideration of this, despite the 10-week UVB irradiation, the collagen hydrolyzate according to the present invention was found to be effective for SKH-1 hairless mouse epidermal keratinocytes to improve and prevent skin wrinkle formation. .

<7-2> Histological observation of the skin

The cut skin tissue was fixed in a 10% thick formalin solution at room temperature for 24 hours, washed, dehydrated, cleared, and infiltrated in a usual manner, then embedded in a paraffin, sliced into 4 iim thick, and overnight in Bouin Yongmec at room temperature. After soaking and Masson 'trichrome staining, the amount and shape of collagen fibers in the dermis were observed under an optical microscope. In addition, put the cut skin tissue in 10% formal in to fix the tissue, and then wash with water and sequentially dehydrate from 60% to 100% alcoh to paraffin _. Embedded and made a block. Tissue sections of 5 μιτι thickness were prepared using rotary microtomeol, stained with Hematoxylin & Eosin, and observed with an optical microscope (Nikon Co., Tokyo, Japan).

When irradiated with UVB excessively, the epidermis becomes rough and the structure of normal collagen is destroyed, resulting in an irregular array of collagen and decreased expression of elastic fibers. As a result of observing the change and loss amount and shape of the elastic fibers in the dermal layer of the skin, the Contr group (C group) was able to identify the denatured elastic fibers, but the positive control group and the normal collagen hydrolyzate (C0P group), according to the present invention , Collagen hydrolyzate administration group (CTP group) was confirmed that the elastic fiber was densified compared to the C group by 14 weeks of sample treatment (Fig. 13).

In addition, as a result of observing the amount and shape of collagen in the dermis, the group C, which was irradiated with UVB only, had an irregular arrangement due to the breakdown of collagen island oil, and the amount of collagen was reduced compared to the NormaKN group). I could see the incense. However, all of the positive control group (P group) treated with retinoic acid (C group), C0L group, and CTP treated group for 14 weeks showed relatively dense and regular collagen density compared to group C. In particular, the CTP-administered group showed better results in the degree and density of collagen in the dermis compared to the C0L group (FIG. 13).

<7-3> Expression of collagen and collagenase protein expression in skin tissue (Collagen 1A,

MMP-1)

Excessive exposure to UV light causes a lot of MMP-1 expression and excess free radicals, which significantly impair antioxidant activity. In addition, it acts on keratinocytes to increase the secretion of inflammatory cytokines, inhibits collagen synthesis in the dermal fibroblasts, promotes enzyme activity, and forms excess wrinkles. Accordingly, the present inventors administered the collagen hydrolyzate (C0L group) and the collagen hydrolyzate (CTP group) according to the present invention to the SKH-1 haireless mouse irradiated with UVB for 14 weeks, and collagen 1A and collagen, a collagen synthesis factor, Protein expression of the degradation enzyme MMP-1 was confirmed by Western blot. For protein analysis, mouse skin tissues collected after the end of the experiment were put in 200 per 20 mg of cell lysis buffer, and then crushed using a homogenizer, and centrifuge at 800 rpm for 10 seconds to use only the supernatant. After 24 hours ice incubation, centrifuge at 14,000 rpm, 4 ^° C, 20 min. The protein was quantified using Bradford assay2) and separated by size using SDS-PAGE (Poly aery 1 amide Gel Electrophoresis). Transfer to PVDF (Polyvinyl idene fluoride) membrane using Semi-dry transfer system (Bio-Rad, USA) and block blocking buffer (5% skim milk, IX TBST buffer) containing 5¾ skim milk for 1 hour. Treated. After 10 rain, 3 washes with lx TBST buffer, after treatment with primary antibody Collagen 1A, MMP-1, MMP-13, 4 ^° C overnight reaction, wash 10 min, 3 times with lx TBST buffer. After the reaction with the membrane using Western Blot detection kit (Abfrontier, WEST SAVE GOLD), the degree of expression was observed using Chemi-Doc (BIORAD XRS system) and calibrated with GAPDH. The results for this are shown in FIG. 14.

Control group (C group) irradiated with UVB is the foot of collagen 1A, a collagen synthesis factor. While the present amount was significantly decreased (p <0.05), it was confirmed that the expression level of Col lagen 1A was increased in the sample administration group. In particular, it was confirmed that the p group and the C0L 34 and CTP 34 administration group to which ret inoic acid was administered increased to a level of p <0.05 (FIG. 14A).

As a result of confirming the protein expression of MMP-1 known as an enzyme that degrades collagen, it was confirmed that the amount of MMP-1 expression increased in the C group irradiated with UVB. On the other hand, the amount of MMP-1 expression was significantly decreased (p <0.05) in the CTP 17 and 34 groups, which was much better than the positive control group and the C0L group (FIG. 14B). Taken together, the results showed that the increase in collagen degrading enzyme and the expression of collagen synthase decreased due to excessive UV irradiation. As a result of treating the collagen hydrolyzate according to the present invention for 14 weeks, compared to the normal collagen hydrolyzate, Col lagen 1A The expression level of was increased, and the expression of MMP-1 was decreased and it was judged that it was excellent in suppressing skin wrinkle formation.

<7-4> Skin moisturizing of skin tissue

When the skin tissue is damaged by UV irradiation, the oil and water content of the skin is reduced. To determine whether the oil and water content in the skin tissue of the mouse irradiated with ultraviolet light for 10 weeks is reduced, and whether the collagen hydrolyzate of the present invention can recover the decrease in oil and water. Corneometer CM 820 (Courage-hazake, Koln, Germany), a skin oil / moisture content measuring device, was administered after 5 weeks and 10 weeks of normal collagen hydrolyzate (C0L group) and collagen hydrolyzate (CTP group) according to the present invention. Oil / moisture content was measured. After rest at least 30 minutes at constant temperature and humidity room 21.5 ± 2 ^° C, relative humidity 40 ± 5%, a certain area was marked to measure the moisture content. The results are shown in FIG. 15.

As can be seen in FIG. 15, the oil and water contents of the control (C) group were significantly reduced compared to the normal control group (N) without irradiating ultraviolet rays due to 10 weeks of ultraviolet irradiation. On the other hand, in weeks 5 and 10, the animal group treated with the collagen hydrolyzate according to the present invention was higher than the normal control group. The oil content and water content were shown, and this effect was increased as the content of collagen hydrolyzate increased. In particular, it was confirmed that the skin moisturization of the rats to which the collagen hydrolyzate (CTP group) according to the present invention was very excellent compared to the normal collagen hydrolyzate (C0L group) (FIG. 15A, B).

Improvement of Skin Moisturizing Effect of Collagen Hydrolyzate Using BP

With reference to the KFDA guidelines (Health functional food test guidelines, beauty-related functional tests, 2004.), the experiment was divided into a total of five groups of 10 animals in each group as shown in Table 4 below. The normal group (Group N) used Balb-C mice, and the skin dry model was set to NC / Nga Tnd mouse which increased water release of epidermis which is useful for moisturizing experiment. To induce dry skin, Okawa et al. Refer to the method (Dermatol Sci. 66 (2): 136-43, 2012.) and a cotton pad (2 X 2cm) was soaked in a solution mixed with acetone and ether 1: 1 for 1 week before dissection, and put on the back for 15 seconds. After releasing it, it was wiped with distilled water for 30 seconds to induce dry skin. The normal control group (Normal group, N group) that did not induce skin dryness and the negative control group (Control group, C group) that induce skin dryness and administered only solvent were orally administered 0.9% sal ine for 4 weeks. Group P used N-acetyl glucosamine (NAG), which has been recognized by the Ministry of Food and Drug Safety to improve skin drying and water retention. Intake dose was orally administered 17 mg / kg of N-acetyl glucosamine.

On the other hand, despite the lower content of CTP17 in Example 7-4 than skin moisturizing than C0L34, compared with the collagen hydrolyzate of the present invention prepared according to Example 4 17 mg / kg (CLT17 group) 34 mg / kg of collagen hydrolyzate (C0L34 group) prepared according to Example 1 were orally administered for 4 weeks each. Feed was fed using the experimental animal solid feed (Samta Ko BI0K0REA, Korea), and drinking water was freely consumed.

TABLE 4

Laboratory animals Dc @ 3

Group h

1 M-1

Sal i ne, p.'o.

2 C-10

5 COL 34 34 Co! l Bgen _a p, o, 10

<8-l> Skin moisture content measurement

Healthy skin keratin contains 15 ~ 20% water, and when the epidermis water drops below 10%, the skin becomes dry, no gloss and elasticity, and wrinkles increase. This soft, moist feeling of the skin is maintained by the moisture present in the stratum corneum of the epidermis.

The moisture content of the stratum corneum is determined by the lipids produced and secreted by the epidermis, the sebaceous membrane which is the adsorbent, and the natural moisture factor (NMF), which is a water-soluble component present in each stratum corneum. It is closely related to the change of content of factor. Effects and characteristics of 4 weeks of administration of collagen hydrolysate 17 mg / kg (CTP 17) or collagen hydrolyzate 34 mg / kg (C0L 34) prepared by a previously reported collagen degrading enzyme according to the present invention on skin moisturizing One week before the end of the experiment, the skin moisture content measuring device, Corneometer CM 820 (Courage-hazake, Koln, Germany), was used to confirm the results. In the constant temperature and humidity room (temperature 21.5 ± 2 ^° C, relative humidity 40 ± 5%), the system was allowed to stabilize for more than 30 minutes, and then a certain area was marked to measure the moisture content. The results are shown in FIG. 16.

Balb-C mice in the normal control group (N) retained the water content of the epidermal layer, but the moisture content of the ContiOl group (group C), which induced skin drying, was lower than that of the normal control group. On the other hand, the positive control group, CTP 17, and C0L 34 group all showed an effect of increasing skin moisture content. Among them, CTP 17 showed the best effect.

In the case of CTP, although it was administered at 17 mg / kg, C0L showed a better skin moisturizing effect than 34 mg / kg, and the collagen hydrolyzate of the present invention, which has a high content of collagen tripeptide, has been reported previously. It was found to be significantly better than the hydrolyzate.

<8-2> Effects on Itching due to Skin Drying

The skin is inherent in various immune substances and is the first place to react to foreign substances. Damage to the skin barrier increases transdermal moisture loss and decreases water binding, resulting in dry skin and itching and scratching. Therefore, itching effect was evaluated by the administration of each substance in a dry animal model.

Scratching behavior was observed to scratch the animals for 10 minutes. The same group was placed in a cage and counted how many times each mouse scratched. If the continuous operation lasting longer than 2 seconds lasted n seconds, it counted n times, and the short operation within 2 seconds was counted once each. The results are shown in FIG. 17.

As a result of confirming the number of scratches for 10 minutes after inducing skin dryness, the number of scratches increased at p <0.05 significance level in the ContiOl group (Group C), which caused dryness, whereas the number of scratches in the group treated with each sample Decreased (ρ <0 · 05).

On the other hand, itching effect from skin dryness was the best in CTP 17, which was superior to the positive control group as well as C0L 34.

<8-3> Skin moisturizing factor protein repair effect (Col lagen 1A, AQP3, HAS2)

For protein analysis, the skin tissue was placed in cel l lysi s buffer, and the tissue was crushed with a homogenizer, followed by centri fuge. Proteins were quantified by Bradford assay, followed by SDS-PAGE (Poly aery 1 amide Gel Electrophoresi s). The semi-dry transfer system (Bio-Rad, USA) was used to transfer polyvinyl idene fluoride (PVDF) membranes, and the expression levels of the Col lagen 1A, AQP3 and HAS2 proteins were measured using Chemi-Doc (BIORAD XRS system, USA). Observations were made using the instrument and calibrated and compared with GAPDH. ^' (i) Collagen is an important component that participates in the regulation of functions such as cell shape, differentiation, migration, and protein synthesis, and in wound repair. Dry skin can have a negative effect on skin physiology, including a decrease in the amount of collagen protein in the skin, resulting in wrinkles.

(ii) Aquaporin-3 (AQP3) is known to play the most important role in the hydration of the skin. Dry skin reduces AQP3 expression and has been reported to affect trans epidermal water loss (TEWL). Increasing aquaporine 3 (AQP3), an aquaglyceroporin present in cell membranes, is known as a factor in preserving skin moisture and eventually preventing aging (J. Lipid Res. 46: 2706, 2005.) Confirmation is one of the important factors to check skin moisture level (Biology of the cell. 97 (7): 479-4862005.)

(iii) Hyaluronic acid (HA) is a high molecular glycosaminoglycan present in extracellular connective tissue and is known to act as a barrier against water evaporation. However, it is known that the content of hyaluronic acid, which acts as a water barrier, decreases rapidly with age). Among the external environmental factors, especially ultraviolet rays have a negative effect on the maintenance of regular hyaluronic acid of the skin. The decrease in the content of hyaluronic acid due to these intrinsic or extrinsic factors is one of the causes of decreased skin elasticity, rough skin and wrinkles. Therefore, maintaining the content of hyaluronic acid in the skin is a very important factor in maintaining the skin while moisturizing and skin. Hyaluronic acid is continuously synthesized by hyaluronic acid synthase (HAS). Therefore, if the gene expression of HAS is increased, the amount of hyaluronic acid in the kiwibu rises to have a positive effect on not only skin moisturizing but also elasticity and wrinkles. Thus, the present inventors attempted to evaluate the effect of collagen hydrolyzate according to the present invention on the reduction of skin moisturizing factors by skin drying through Western blot. The results are shown in FIG. 18.

As can be seen in Figure 18, Collagen 1A, AQP3 and HAS was confirmed that significantly reduced than the normal control by the skin dry induction. Meanwhile, training college In the control group, CTP 17 and COL 34 groups, the amount of each protein was recovered significantly compared to the control group (C). Among them, the CTP 17 group showed significantly better skin moisturizing factor recovery than the positive control group (P group), and showed a significantly better effect than the C0L 34 group despite the half dose of the C0L 34 group. In other words, the collagen hydrolyzate of the present invention containing a high amount of collagen tripeptides was found to have an excellent effect even at a small dose because the absorption rate is very good in the body. As described above, since the collagen hydrolyzate of the present invention exhibits an excellent skin wrinkle improvement and skin moisturizing effect, it may have an effect of preventing skin aging and improving skin elasticity.

Industrial Applicability

The present invention relates to a collagen hydrolyzate contained in a high content of collagen tritide and its use. The collagen hydrolyzate of the present invention has a significantly higher content of collagen tripeptide in the body compared to the previously reported collagen hydrolyzate and thus has an excellent absorption rate in the body. It is very useful for industrial use because it shows excellent hair loss prevention, hair growth promotion, skin wrinkle improvement and / or moisturizing effect.

Receipt of a certificate of deposit in accordance with Rule 7.1 by the International Depository under the Budapest International Treaty for International Form Patent Application: Amikozen Co., Ltd.

Mikogen stock society

(660-852) 64, Dongbu-ro 1259beon-gil, Jinseong-myeon, Jinju-si, Gyeongsangnam-do, Korea

I. Microbial signs

Indications for identification of microorganisms attached by the depositary:

Accession number assigned by the International Depositary:

Bacillus subtilis BP

KCTC 12866BP

II. Explanation of scientific properties and / or taxonomic location

For the microorganisms listed in Section I, the following is included:

[X] Explanation of Scientific Properties

[] Proposed taxonomic position

(X mark when applied)

I II. Deposit and Deposit The International Depositary has deposited the microorganisms indicated in item I deposited on July 13, 2015.

IV. Receipt of the Request for Escalation The International Depositary has deposited the microorganisms indicated in item I of E, and received the request for escalation of the E deposit to the deposit under the Budapest Treaty.

V. International Depositary Name of the person having authority to represent the International Depositary: Korean Collect ion for Type Cultures

Or authorized official signature:

Address: Yuseong-gu, Daejeon, Korea (305—806)

Doo-sang Park, Director, Korea Research Institute of Bioscience and Biotechnology 125

Signature Date: 2015.07.15

BP / 4 Form (KCTC Form 17) Single Page

(I confirm that there is no difference from the original) Representative ： Patent Attorney

Patent Attorney

Claims

[Range of request]

[Claim 1]

(a) mixing the young (fish scales) with water and heat-treating;

(c) hydrolyzing the collagen degrading enzyme in the mixture of step (a) for 5 to 15 hours at 25 to 45 ^° C., wherein the collagen tripeptide is 20 to 20 relative to the total hydrolyzate. Collagen hydrolyzate, characterized in that contained in 70% by weight.

[Claim 2]

According to claim 5, wherein the Bacillus subtilis collagen hydrolyzate, characterized in that the strain of Accession No. KCTC12866BP.

[Claim 3]

The collagen hydrolyzate of claim 1, wherein the ion exchange chromatography of step (b) uses cation exchange chromatography, and sodium chloride (NaCl) is used at a concentration of 0.1 to 0.3 M.

[Claim 4]

The collagen hydrolyzate according to claim 1, wherein the step (b) and the ion exchange chromatography use anion exchange chromatography, and sodium chloride (NaCl) is added at a concentration of 0.09 to 0.155M.

[Claim 5]

A food composition for preventing hair loss or promoting hair growth comprising the collagen hydrolyzate according to any one of claims 1 to 4 as an active ingredient.

[Claim 6]

A pharmaceutical composition for preventing hair loss or promoting hair growth comprising the collagen hydrolyzate according to any one of claims 1 to 4 as an active ingredient.

[Claim 7]

Hair loss prevention or hair growth promoting cosmetic composition comprising a collagen hydrolyzate according to any one of claims 1 to 4 as an active ingredient.

[Claim 8]

A cosmetic composition for improving skin wrinkles, improving skin elasticity, preventing skin aging or skin moisturizing, comprising the collagen hydrolyzate according to any one of claims 1 to 4 as an active ingredient.

[Claim 9]

A food composition for improving skin wrinkles, improving skin elasticity, preventing skin aging or skin moisturizing, comprising the collagen hydrolyzate according to any one of claims 1 to 4 as an active ingredient.

[Claim 10]

Use of the collagen hydrolyzate according to any one of claims 1 to 4 for preparing a hair loss preventing or hair growth promoting agent.

[Claim 11]

A method for preventing hair loss or promoting hair growth, comprising administering an effective amount of the collagen hydrolyzate according to any one of claims 1 to 4 to a subject in need thereof.

[Claim 12]

Use of the collagen hydrolyzate according to any one of claims 1 to 4 for the preparation of skin wrinkle improvement, skin elasticity improvement, anti-aging or skin moisturizing preparation.

[Claim 13]

A method for improving skin wrinkles, improving skin elasticity, preventing skin aging or skin moisturizing, comprising administering to the subject in need thereof an effective amount of the collagen hydrolyzate according to any one of claims U to 4.