Growth-promoting composition containing whey protein-derived peptides

Abstract

The present invention relates to a growth-promoting composition containing a peptide consisting of Asp-Lys-Phe-Leu-Asp. When using the peptide consisting of the peptide Asp-Lys-Phe-Leu-Asp of the present invention, growth is promoted and efficient quality control of whey protein enzyme hydrolysates is possible.

Description

Technical Field

[0001] The present invention was made under the support of the Ministry of Agriculture, Food and Rural Affairs of Korea, project number 314077033SB010. The specialized research management institution for this project is the Rural Development Administration, the name of the research project is "Development of High-Added-Value Food Technologies", the name of the research topic is "Development of Organic Functional Materials Utilizing Organic Farm Cheese By-Products and Development of Organic Powdered Milk Applied Therewith", the managing institution is Neo Creamer Co., Ltd., and the research period is from December 17, 2014 to December 16, 2017.

[0002] This patent application claims priority from Korean Patent Application No. 10-2022-0082797, filed with the Korean Intellectual Property Office on July 5, 2022, and the disclosure of the said patent application is incorporated herein by reference.

[0003] The present invention relates to a growth-promoting composition containing peptides derived from whey protein.

[0004]

Background Art

[0005] Growth generally refers to an increase in height and is promoted by nutrients, growth hormones, etc. In particular, the nervous system, including the brain that secretes growth hormone, grows significantly during childhood and then grows slowly. The growth of the iliac length determines height and skeletal structure, and its growth is regulated by a special mechanism. In particular, the growth of the proximal epiphyseal growth plate of the ilium becomes an important measure in the process of bone length growth.

[0006] Bone growth is carried out by the actions of osteoclasts and osteoblasts, and bone growth occurs when the activity of osteoblasts is higher than that of osteoclasts. The differentiation of such osteoblasts is regulated by hormones, cytokines such as bone morphogenetic protein (BMP), and various enzymes such as alkaline phosphatase (ALP).

[0007] In recent years, with the improvement of nutritional status and changes in eating habits due to economic growth, the growth and development of children and adolescents have also been greatly improved. In addition, due to the social atmosphere that favors tall stature, people's interest in growth has been increasing. In recent years, while the interest in promoting the growth of children with short stature has been increasing, the interest in growth hormone injections has also been increasing, but this has problems such as being difficult to take and causing side effects such as acromegaly and hypothyroidism. Therefore, a plan to promote growth by food along with the supply of nutrients such as vitamins and minerals has been explored.

[0008] On the other hand, whey is the water-soluble part of milk (the part other than casein), is a component of cheese, and is a by-product containing active ingredients such as proteins, lactose, minerals, vitamins, and inorganic compounds. Whey has high nutritional or physiological value and can be used in various foods. Recently, there are research results that whey protein can help prevent colorectal cancer and liver cancer, and improve blood cholesterol, immunity, and the bone growth of children. The milk-based proteins present in whey not only promote the proliferation of osteoblasts and the synthesis of collagen, but also directly act on osteoblasts to suppress the formation and differentiation of osteoblasts. However, whey is difficult to utilize due to its poor digestive structural characteristics, and research has been conducted to overcome the poor digestive characteristics using various digestive enzymes.

[0009] Therefore, there is a need to develop peptides that are easy to digest, have a growth-promoting function, and enable quality control of whey protein enzyme hydrolysates.

[0010] Throughout this specification, a number of papers and patent documents are referenced and their citations are indicated. The disclosed contents of the cited papers and patent documents are hereby incorporated by reference in their entirety into this specification to more clearly explain the level of the technical field to which the present invention pertains and the content of the present invention.

[0011]

Summary of the Invention

Problems to be Solved by the Invention

[0012] The inventors have made intensive research efforts to develop a peptide that is well digested, has a growth-promoting function, and enables quality control of whey protein enzyme hydrolysates. As a result, they have found that a peptide consisting of Asp-Lys-Phe-Leu-Asp has the function of promoting growth, and thus completed the present invention.

[0013] Accordingly, an object of the present invention is to provide a food composition for promoting growth, which contains a peptide consisting of Asp-Lys-Phe-Leu-Asp.

[0014] Another object of the present invention is to provide a pharmaceutical composition for promoting growth, which contains a peptide consisting of Asp-Lys-Phe-Leu-Asp.

[0015] Still another object of the present invention is to provide a whey protein hydrolyzate containing a peptide consisting of Asp-Lys-Phe-Leu-Asp as a peptide.

[0016] Still another object of the present invention is to provide a method for producing a whey protein hydrolyzate containing a peptide consisting of Asp-Lys-Phe-Leu-Asp as a peptide.

[0017] Still another object of the present invention is to provide a method for preventing or treating growth disorders, which includes administering the above-described pharmaceutical composition to a subject in need of treatment.

[0018] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings.

[0019]

Means for Solving the Problems

[0020] According to one aspect of the present invention, the present invention provides a food composition for promoting growth, which contains a peptide consisting of Asp-Lys-Phe-Leu-Asp.

[0021] The inventors have made intensive research efforts to develop peptides that are easily digested, have a growth-promoting function, and enable quality control of whey protein enzyme hydrolysates. As a result, it was found that a peptide containing Asp-Lys-Phe-Leu-Asp (DKFLD) has the function of promoting growth. Asp-Lys-Phe-Leu-Asp (DKFLD) is shown in SEQ ID NO: 1.

[0022] In one embodiment of the present invention, the food composition of the present invention may include a whey protein hydrolyzate containing a peptide consisting of Asp-Lys-Phe-Leu-Asp.

[0023] In one embodiment of the present invention, the whey protein of the present invention may be derived from cheese whey.

[0024] In one embodiment of the present invention, the whey protein of the present invention may be normal whey powder, demineralized whey powder, whey protein concentrate, or whey protein isolate.

[0025] In one embodiment of the present invention, the whey protein hydrolyzate of the present invention may be a water-soluble whey protein hydrolyzate from which insoluble substances have been removed.

[0026] In one embodiment of the present invention, the content of the peptide consisting of Asp-Lys-Phe-Leu-Asp of the present invention may be 0.5 mg / g to 40 mg / g based on the total weight of the whey protein hydrolyzate. More specifically, the content of the peptide consisting of Asp-Lys-Phe-Leu-Asp is 0.5 mg / g to 40 mg / g, 0.5 mg / g to 25 mg / g, 0.5 mg / g to 15 mg / g, 0.5 mg / g to 10 mg / g, 0.5 mg / g to 8 mg / g, 0.5 mg / g to 7 mg / g, 0.5 mg / g to 6 mg / g, 1.0 mg / g to 40 mg / g, 1.0 mg / g to 25 mg / g, 1.0 mg / g to 15 mg / g, 1.0 mg / g to 10 mg / g, 1.0 mg / g to 8 mg / g, 1.0 mg / g to 7 mg / g, 1.0 mg / g to 6 mg / g, 1.5 mg / g to 40 mg / g, 1.5 mg / g to 25 mg / g, 1.5 mg / g to 15 mg / g, 1.5 mg / g to 10 mg / g, 1.5 mg / g to 8 mg / g, 1.5 mg / g to 7 mg / g, 1.5 mg / g to 6 mg / g, 2.0 mg / g to 40 mg / g, 2.0 mg / g to 25 mg / g, 2.0 mg / g to 15 mg / g, 2.0 mg / g to 10 mg / g, 2.0 mg / g to 8 mg / g, 2.0 mg / g to 7 mg / g, 2.0 mg / g to 6 mg / g, 3.0 mg / g to 40 mg / g, 3.0 mg / g to 25 mg / g, 3.0 mg / g to 15 mg / g, 3.0 mg / g to 10 mg / g, 3.0 mg / g to 8 mg / g, 3.0 mg / g to 7 mg / g, 3.0 mg / g to 6 mg / g, 4.0 mg / g to 40 mg / g, 4.0 mg / g to 25 mg / g, 4.0 mg / g to 15 mg / g, 4.0 mg / g to 10 mg / g, 4.0 mg / g to 8 mg / g, 4.0 mg / g to 7 mg / g, or 4.0 mg / g to 6 mg / g based on the total weight of the whey protein hydrolyzate.

[0027] In one embodiment of the present invention, the whey protein hydrolyzate of the present invention may be one obtained by subjecting whey protein to primary hydrolysis with an endo protease derived from Bacillus licheniformis and then subjecting it to secondary hydrolysis with an exo protease derived from Aspergillus oryzae.

[0028] In one embodiment of the present invention, the endoprotease derived from Bacillus licheniformis is Alcalase, Protamex, or a mixed enzyme thereof, and the exoprotease derived from Aspergillus oryzae may be Flavourzyme.

[0029] In one embodiment of the present invention, the mixed enzyme may be a mixture of Alcalase and Protamex mixed at a weight ratio of 1:0.5 to 1:2. More specifically, the mixed enzyme may be a mixture of Alcalase and Protamex at a weight ratio of 1:0.5 to 1:2, 1:0.5 to 1:1.75, 1:0.5 to 1:1.5, 1:0.5 to 1:1.25, 1:0.5 to 1:1, 1:0.75 to 1:2, 1:0.75 to 1:1.75, 1:0.75 to 1:1.5, 1:0.75 to 1:1.25, 1:0.75 to 1:1, 1:1 to 1:2, 1:1 to 1:1.75, 1:1 to 1:1.5, or 1:1 to 1:1.25.

[0030] In one embodiment of the present invention, the growth promotion may be due to the promotion of bone growth. In one embodiment of the present invention, the growth promotion may be the promotion of bone growth.

[0031] In one embodiment of the present invention, the growth promotion may be due to the promotion of the growth of bone length or bone volume.

[0032] The food composition of the present invention may be manufactured in the form of powder, granules, tablets, capsules, beverages, etc. For example, there are various foods such as candies, beverages, gums, teas, vitamin complexes, or health supplements.

[0033] The food composition of the present invention includes any processed form of natural materials such as foods, functional foods, nutritional supplements, health foods, and food additives. The food compositions of the above types may be manufactured in various forms by ordinary methods known in the art. For example, health foods may be manufactured and consumed in the form of tea, juice, and drinks, or may be ingested in the form of granulation, encapsulation, and powdering. Further, as foods, beverages (including alcoholic beverages), fruits and processed foods thereof (for example, canned fruits, bottled fruits, jams, marmalades, etc.), fish, meats and processed foods thereof (for example, hams, sausages, corned beef, etc.), breads, and noodles (for example, udon, cold buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, candies, dairy products (for example, yogurt, fermented milk, butter, cheese, etc.), edible vegetable oils, margarine, vegetable proteins, retort foods, frozen foods, various seasonings (for example, miso, soy sauce, sauce, etc.), etc. may be manufactured by adding the peptide consisting of Asp-Lys-Phe-Leu-Asp of the present invention or a whey protein hydrolyzate containing the peptide consisting of Asp-Lys-Phe-Leu-Asp. Further, in order to use the peptide consisting of Asp-Lys-Phe-Leu-Asp of the present invention or a whey protein hydrolyzate containing the peptide consisting of Asp-Lys-Phe-Leu-Asp in the form of a food additive, it can be manufactured and used in the form of a powder or a concentrate.

[0034] The food composition of the present invention can be taken for a long time.

[0035] The mixing amount of the active ingredient of the food composition of the present invention may be appropriately determined according to its intended use (prevention, improvement, or therapeutic treatment). Generally, the whey hydrolyzate of the present invention may be added in an amount of 0.1 to 70% by weight, preferably 2 to 50% by weight, based on 100% by weight of the raw materials of the food or beverage, during the production of the food or beverage. The effective dosage of the peptide of the present invention can be used according to the effective dosage of the pharmaceutical composition, but may be below the above range in the case of long-term intake for the purpose of health and hygiene or for the purpose of health regulation. Since the active ingredient has no problem in terms of safety, it may also be used in an amount above the above range.

[0036] The food composition of the present invention may contain components usually added during food production, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. Examples of the above-mentioned carbohydrates are monosaccharides such as glucose and fructose; disaccharides such as maltose, sucrose, and oligosaccharides; and polysaccharides such as dextrin and cyclodextrin, as well as ordinary sugars and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [thaumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)] and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is produced as a drink, in addition to the peptide consisting of Asp-Lys-Phe-Leu-Asp or the whey protein hydrolyzate containing the peptide consisting of Asp-Lys-Phe-Leu-Asp of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, kudzu extract, jujube extract, licorice extract, etc. may further be included.

[0037] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating growth disorders containing a peptide consisting of Asp-Lys-Phe-Leu-Asp.

[0038] In one embodiment of the present invention, the growth disorder may be a bone growth disorder.

[0039] In one embodiment of the present invention, the growth disorder may be selected from the diseases consisting of familial short stature, constitutional growth delay, idiopathic short stature, osteochondrodysplasia, short stature due to Down syndrome, short stature due to Turner syndrome, short stature due to Prader-Willi syndrome, short stature due to Russell-Silver syndrome, short stature due to Noonan syndrome, short stature due to chronic systemic disease, short stature due to growth hormone deficiency, short stature due to hypothyroidism, short stature due to precocious puberty, short stature due to Cushing's syndrome, and psychosocial dwarfism.

[0040] The pharmaceutical composition of the present invention contains, in addition to the active ingredient, a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier contained in the pharmaceutical composition of the present invention is one generally used in formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, etc., but is not limited thereto. The pharmaceutical composition of the present invention may further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. in addition to the above components. Appropriate pharmaceutically acceptable carriers and formulations are described in detail in Remington’s Pharmaceutical Sciences (19th ed., 1995).

[0041] The appropriate dosage of the pharmaceutical composition of the present invention can be formulated variously depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reactivity. On the other hand, the dosage of the pharmaceutical composition of the present invention is preferably 0.001 to 1000 mg / kg (body weight) per day.

[0042] The pharmaceutical composition of the present invention can be administered orally or parenterally. When administered parenterally, it can be administered by topical application to the skin, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc.

[0043] The pharmaceutical composition of the present invention can be formulated using pharmaceutically acceptable carriers and / or excipients by a method that can be easily implemented by those having ordinary knowledge in the technical field to which the present invention pertains, and can be manufactured in the form of unit doses or in multiple-dose containers. At this time, the dosage form may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may further contain a dispersant or stabilizer.

[0044] According to another aspect of the present invention, the present invention provides a whey protein hydrolyzate containing a peptide consisting of Asp-Lys-Phe-Leu-Asp.

[0045] In one embodiment of the present invention, the whey protein of the present invention may be derived from cheese whey.

[0046] In one embodiment of the present invention, the whey protein of the present invention may be normal whey powder, demineralized whey powder, whey protein concentrate, or whey protein isolate.

[0047] In one embodiment of the present invention, the whey protein hydrolyzate of the present invention may be a water-soluble whey protein hydrolyzate from which insoluble substances have been removed.

[0048] In one embodiment of the present invention, the content of the peptide consisting of Asp-Lys-Phe-Leu-Asp may be 0.5 mg / g to 40 mg / g based on the total weight of the whey protein hydrolyzate.

[0049] In one embodiment of the present invention, the content of the peptide consisting of Asp-Lys-Phe-Leu-Asp of the present invention may be 0.5 mg / g to 40 mg / g based on the total weight of the whey protein hydrolyzate. More specifically, the content of the peptide consisting of Asp-Lys-Phe-Leu-Asp is 0.5 mg / g to 40 mg / g, 0.5 mg / g to 25 mg / g, 0.5 mg / g to 15 mg / g, 0.5 mg / g to 10 mg / g, 0.5 mg / g to 8 mg / g, 0.5 mg / g to 7 mg / g, 0.5 mg / g to 6 mg / g, 1.0 mg / g to 40 mg / g, 1.0 mg / g to 25 mg / g, 1.0 mg / g to 15 mg / g, 1.0 mg / g to 10 mg / g, 1.0 mg / g to 8 mg / g, 1.0 mg / g to 7 mg / g, 1.0 mg / g to 6 mg / g, 1.5 mg / g to 40 mg / g, 1.5 mg / g to 25 mg / g, 1.5 mg / g to 15 mg / g, 1.5 mg / g to 10 mg / g, 1.5 mg / g to 8 mg / g, 1.5 mg / g to 7 mg / g, 1.5 mg / g to 6 mg / g, 2.0 mg / g to 40 mg / g, 2.0 mg / g to 25 mg / g, 2.0 mg / g to 15 mg / g, 2.0 mg / g to 10 mg / g, 2.0 mg / g to 8 mg / g, 2.0 mg / g to 7 mg / g, 2.0 mg / g to 6 mg / g, 3.0 mg / g to 40 mg / g, 3.0 mg / g to 25 mg / g, 3.0 mg / g to 15 mg / g, 3.0 mg / g to 10 mg / g, 3.0 mg / g to 8 mg / g, 3.0 mg / g to 7 mg / g, 3.0 mg / g to 6 mg / g, 4.0 mg / g to 40 mg / g, 4.0 mg / g to 25 mg / g, 4.0 mg / g to 15 mg / g, 4.0 mg / g to 10 mg / g, 4.0 mg / g to 8 mg / g, 4.0 mg / g to 7 mg / g, or 4.0 mg / g to 6 mg / g based on the total weight of the whey protein hydrolyzate.

[0050] In one embodiment of the present invention, the whey protein hydrolyzate of the present invention may be obtained by first hydrolyzing whey protein with an endo protease derived from Bacillus licheniformis and then secondarily hydrolyzing it with an exo protease derived from Aspergillus oryzae.

[0051] In one embodiment of the present invention, the endo protease derived from Bacillus licheniformis is Alcalase, Protamex, or a mixed enzyme thereof, and the exo protease derived from Aspergillus oryzae may be Flavourzyme.

[0052] In one embodiment of the present invention, the mixed enzyme may be a mixture of Alcalase and Protamex mixed at a weight ratio of 1:0.5 to 1:2. More specifically, the mixed enzyme may be a mixture of Alcalase and Protamex at a weight ratio of 1:0.5 to 1:2, 1:0.5 to 1:1.75, 1:0.5 to 1:1.5, 1:0.5 to 1:1.25, 1:0.5 to 1:1, 1:0.75 to 1:2, 1:0.75 to 1:1.75, 1:0.75 to 1:1.5, 1:0.75 to 1:1.25, 1:0.75 to 1:1, 1:1 to 1:2, 1:1 to 1:1.75, 1:1 to 1:1.5, or 1:1 to 1:1.25.

[0053] According to still another aspect of the present invention, the present invention provides a method for producing a whey protein hydrolyzate containing a peptide consisting of Asp-Lys-Phe-Leu-Asp, comprising: (a) mixing whey protein and water in a weight ratio to dissolve the whey protein; (b) adding an endo protease derived from Bacillus licheniformis to 100 parts by weight of the dissolved whey protein lysate to perform primary hydrolysis; and (c) adding an exo protease derived from Aspergillus oryzae to the primary hydrolyzed product to perform secondary hydrolysis.

[0054] In one embodiment of the present invention, in the step (a), whey protein and water can be mixed at a weight ratio of 1:3 to 1:10, 1:3 to 1:8, 1:3 to 1:6, 1:4 to 1:10, 1:4 to 1:8, 1:4 to 1:6, 1:5 to 1:10, 1:5 to 1:8, or 1:5 to 1:6. Most specifically, they can be mixed at a weight ratio of 1:5, but it is not limited thereto.

[0055] In one embodiment of the present invention, in the step (a), the pH can be adjusted to 7.0 to 7.5 using a base.

[0056] In one embodiment of the present invention, in the step (b), 0.05 to 1, 0.05 to 0.7, 0.05 to 0.4, 0.05 to 0.2, 0.1 to 1, 0.1 to 0.7, 0.1 to 0.4, 0.1 to 0.2, 0.15 to 1, 0.15 to 0.7, 0.15 to 0.4, or 0.15 to 0.2 parts by weight of an endo protease derived from Bacillus licheniformis can be added to 100 parts by weight of the dissolved whey protein lysate to perform primary hydrolysis. Most specifically, primary hydrolysis can be performed by adding 0.2 parts by weight, but it is not limited thereto.

[0057] In one embodiment of the present invention, the step (b) can be carried out under temperature conditions of 30°C to 80°C. More specifically, it can be carried out under temperature conditions of 30°C to 80°C, 30°C to 70°C, 30°C to 60°C, 30°C to 55°C, 40°C to 80°C, 40°C to 70°C, 40°C to 60°C, 40°C to 59°C, 40°C to 58°C, 40°C to 57°C, 40°C to 56°C, 40°C to 55°C, 42°C to 60°C, 42°C to 59°C, 42°C to 58°C, 42°C to 57°C, 42°C to 56°C, 42°C to 55°C, 44°C to 60°C, 44°C to 59°C, 44°C to 58°C, 44°C to 57°C, 44°C to 56°C, 44°C to 55°C, 46°C to 60°C, 46°C to 59°C, 46°C to 58°C, 46°C to 57°C, 46°C to 56°C, 46°C to 55°C, 48°C to 60°C, 48°C to 59°C, 48°C to 58°C, 48°C to 57°C, 48°C to 56°C, 48°C to 55°C, 50°C to 80°C, 50°C to 70°C, or 50°C to 60°C, 50°C to 59°C, 50°C to 58°C, 50°C to 57°C, or 50°C to 56°C, and most specifically 50°C to 55°C, but it is not limited thereto.

[0058] In one embodiment of the present invention, in the step (b), hydrolysis can be carried out for 1 hour to 24 hours. More specifically, it can be carried out for 1 hour to 24 hours, 1 hour to 20 hours, 1 hour to 15 hours, 1 hour to 10 hours, 1 hour to 8 hours, 1 hour to 6 hours, 1 hour to 5 hours, 1 hour to 4 hours, 2 hours to 24 hours, 2 hours to 20 hours, 2 hours to 15 hours, 2 hours to 10 hours, 2 hours to 8 hours, 2 hours to 6 hours, 2 hours to 5 hours, 2 hours to 4 hours, 3 hours to 24 hours, 3 hours to 20 hours, 3 hours to 15 hours, 3 hours to 10 hours, 3 hours to 8 hours, 3 hours to 6 hours, 3 hours to 5 hours, 3 hours to 4 hours, 4 hours to 24 hours, 4 hours to 20 hours, 4 hours to 15 hours, 4 hours to 10 hours, 4 hours to 8 hours, 4 hours to 6 hours, or 4 hours to 5 hours, and most specifically hydrolysis can be carried out for 4 hours, but it is not limited thereto.

[0059] In one embodiment of the present invention, in the step (c), an exo protease derived from Aspergillus oryzae is added to the once-hydrolyzed decomposition product in an amount of 0.05 to 1, 0.05 to 0.7, 0.05 to 0.4, 0.05 to 0.2, 0.1 to 1, 0.1 to 0.7, 0.1 to 0.4, 0.1 to 0.2, 0.15 to 1, 0.15 to 0.7, 0.15 to 0.4, or 0.15 to 0.2 parts by weight to perform secondary hydrolysis. Most specifically, secondary hydrolysis can be performed by adding 0.2 parts by weight, but it is not limited thereto.

[0060] In one embodiment of the present invention, the step (c) can be carried out under temperature conditions of 30°C to 80°C. More specifically, it can be carried out under temperature conditions of 30°C to 80°C, 30°C to 70°C, 30°C to 60°C, 30°C to 55°C, 40°C to 80°C, 40°C to 70°C, 40°C to 60°C, 40°C to 59°C, 40°C to 58°C, 40°C to 57°C, 40°C to 56°C, 40°C to 55°C, 42°C to 60°C, 42°C to 59°C, 42°C to 58°C, 42°C to 57°C, 42°C to 56°C, 42°C to 55°C, 44°C to 60°C, 44°C to 59°C, 44°C to 58°C, 44°C to 57°C, 44°C to 56°C, 44°C to 55°C, 46°C to 60°C, 46°C to 59°C, 46°C to 58°C, 46°C to 57°C, 46°C to 56°C, 46°C to 55°C, 48°C to 60°C, 48°C to 59°C, 48°C to 58°C, 48°C to 57°C, 48°C to 56°C, 48°C to 55°C, 50°C to 80°C, 50°C to 70°C, or 50°C to 60°C, 50°C to 59°C, 50°C to 58°C, 50°C to 57°C, or 50°C to 56°C. Most specifically, it can be carried out under temperature conditions of 50°C to 55°C, but it is not limited thereto.

[0061] In one embodiment of the present invention, in the step (c), hydrolysis can be carried out for 1 hour to 24 hours. More specifically, it can be 1 hour to 24 hours, 1 hour to 20 hours, 1 hour to 18 hours, 1 hour to 16 hours, 1 hour to 15 hours, 5 hours to 24 hours, 5 hours to 20 hours, 5 hours to 18 hours, 5 hours to 16 hours, 5 hours to 15 hours, 10 hours to 24 hours, 10 hours to 20 hours, 10 hours to 18 hours, 10 hours to 16 hours, 10 hours to 15 hours, 13 hours to 24 hours, 13 hours to 20 hours, 13 hours to 18 hours, 13 hours to 16 hours, 13 hours to 15 hours, 14 hours to 24 hours, 14 hours to 20 hours, 14 hours to 18 hours, 14 hours to 16 hours, or 14 hours to 15 hours. Most specifically, hydrolysis can be carried out for 15 hours, but it is not limited thereto.

[0062] In one embodiment of the present invention, the method for producing a whey protein hydrolyzate containing the peptide consisting of Asp-Lys-Phe-Leu-Asp of the present invention may further include, after the step (c), (d) a step of inactivating and filtering the whey protein hydrolyzate. More specifically, it can be inactivated at 90°C for 10 minutes and then cooled, and filtered through a housing filter (1 μm).

[0063] In one embodiment of the present invention, the method for producing a whey protein hydrolyzate containing the peptide consisting of Asp-Lys-Phe-Leu-Asp of the present invention may further include, after the step (c), (d) a step of sterilizing the whey protein hydrolyzate. More specifically, it can be sterilized at 90°C for 30 minutes, but it is not limited thereto. Further, it may further include a step of cooling at room temperature after sterilization.

[0064] In one embodiment of the present invention, the method for producing a whey protein hydrolyzate containing the peptide consisting of Asp-Lys-Phe-Leu-Asp of the present invention may further include, after the step (c), (d) filtering and drying the whey protein hydrolyzate. More specifically, it can be filtered through an 80-mesh filter paper and spray-dried (temperature conditions: inlet: 190 ± 10 °C, outlet: 95 ± 5 °C), but is not limited thereto. Further, after the drying step, a step of removing impurities with a magnetic bar may further be included.

[0065] According to still another aspect of the present invention, the present invention provides a method for preventing or treating growth disorders, which includes administering the above-described pharmaceutical composition to a subject in need of treatment.

[0066] In one embodiment of the present invention, the growth disorder may be selected from the group consisting of familial short stature, constitutional growth delay, idiopathic short stature, osteochondrodysplasia, short stature due to Down syndrome, short stature due to Turner syndrome, short stature due to Prader-Willi syndrome, short stature due to Russell-Silver syndrome, short stature due to Noonan syndrome, short stature due to chronic systemic diseases, short stature due to growth hormone deficiency, short stature due to hypothyroidism, short stature due to precocious puberty, short stature due to Cushing's syndrome, and psychosocial dwarfism.

[0067] The subject of the present invention is a mammal such as a human, chimpanzee, orangutan, non-human primate, dog, cat, horse, hamster, mouse, rat, guinea pig, pig, sheep, cow, etc., and most specifically a human, but is not limited thereto.

[0068] Since the method for preventing or treating growth disorders of the present invention is a method including the step of administering the pharmaceutical composition which is one aspect of the present invention, the description thereof is omitted in order to avoid excessive duplication in this specification for overlapping contents.

[0069]

Advantages of the Invention

[0070] The present invention provides a growth-promoting composition containing a peptide consisting of Asp-Lys-Phe-Leu-Asp, a whey protein hydrolyzate containing a peptide consisting of Asp-Lys-Phe-Leu-Asp, and a method for producing the same. The composition containing the peptide consisting of Asp-Lys-Phe-Leu-Asp of the present invention has a growth-promoting function, and when this is utilized, efficient quality control of whey protein enzymatic hydrolyzates becomes possible.

[0071]

Brief Description of the Drawings

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Figure 6

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Figure 7

[0079]

Figure 8

[0080]

Figure 9

[0081]

Figure 10

[0082]

Figure 11

[0083]

Figure 12

[0084]

Figure 13

[0085]

Figure 14

[0086]

Figure 15

[0087]

Figure 16

[0088]

Figure 17

[0089]

Figure 18

[0090]

Figure 19

[0091]

Figure 20

[0092]

Mode for Carrying Out the Invention

[0093] Hereinafter, the present invention will be described in more detail with reference to examples. It will be apparent to those having ordinary knowledge in the art that these examples are merely for more specifically explaining the present invention, and the scope of the present invention is not limited by these examples along the gist of the present invention.

[0094] Examples

[0095] <Example I> Production of Whey Protein Hydrolysate and Measurement of Constituent Amino Acids

[0096] For the whey of the present invention, whey protein concentrate obtained by adding rennet to milk, removing curds and concentrating was used. The pH of the whey is pH 5 to 6 based on a 10 wt% aqueous solution of whey. WPI (Whey Protein Isolate) was purchased from Meal Heaven, USA and used.

[0097] For the whey protein enzymatic hydrolyzate, a powder product provided by NeoCrem Co., Ltd. was used.

[0098] For the manufacturing process of the enzymatic hydrolyzate, the whey protein enzymatic hydrolyzate produced by the standard manufacturing process of NeoCrem Co., Ltd. was used as a sample for test analysis. The manufacturing process is shown in Figure 1.

[0099] The concentrated whey protein and water were mixed at a weight ratio of 1:5, and sodium bicarbonate (NaHCO3) was added to adjust the pH to 7.0 - 7.5.

[0100] For the enzyme for hydrolysis, 0.2% Alcalase (Alcalase 2.4L FG, Novo Nordisk) and 0.2% Protamex were used as one-step hydrolysis enzymes and hydrolyzed at 50°C - 55°C for 4 hours, and 0.2% Flavourzyme was used as a two-step hydrolysis enzyme and hydrolyzed at 50°C - 55°C for 15 hours. After the hydrolyzate was inactivated at 90°C for 10 minutes, it was cooled to room temperature. The hydrolyzate was filtered through a 1μm housing filter, sterilized at 90°C for 30 minutes, and then spray-dried at an inlet temperature of 190 ± 10°C and an outlet temperature of 95 ± 5°C. The spray-dried product was removed of metal foreign substances with a magnetic bar of 8,000 GAUS and then packaged and used as a sample for analysis.

[0101] The compositional amino acid profile of the whey protein hydrolyzate of Example I was measured with an amino acid analyzer after hydrolyzing the whey protein hydrolyzate by the acid hydrolysis method. That is, after accurately weighing 25 mg of the whey protein hydrolyzate into a cap tube, 2.5 ml of 6N HCl was added and hydrolyzed at 110 °C for 24 hours. The filtrate from which undigested substances were removed with a 3G-4 glass filter was completely evaporated of the solvent at 50 °C using a rotary vacuum evaporator (N-1110, EYELA, Tokyo, Japan), and then made up to 25 ml with 0.01N HCl and used as a sample for amino acid analysis. For amino acid analysis, 40 μl of the sample solution was injected and analyzed with an amino acid analyzer (Biochrom30, Cambridge, UK) (Table 1).

[0102]

Table 1

[0103] As shown in Table 1, the total amino acid content of the whey protein hydrolyzate of Example I was 801.1 ± 24.1 mg / g, slightly higher than 74.09 g / 100 g of bovine whey protein, and much higher than 64.45 g / 100 g, 60.53 g / 100 g, and 61.57 / 100 g of egg, wheat, and soy proteins, respectively (Table 1). In particular, the contents of Lys, Tyr, etc., which promote growth hormone secretion, were higher than those of other proteins. Such results indicate that the whey protein hydrolyzate of Example I has an effective effect on growth functionality compared to the hydrolyzates of other proteins, in light of reports that whey protein rich in Lys and Tyr is correlated with the growth functionality of children. On the other hand, the relatively low quantification of the Cys content is presumably because it was oxidized to cysteic acid.

[0104] <Example II> Comparison of Whey Protein Hydrolyzates and Evaluation of Growth Functionality

[0105] Comparative Example 1

[0106] The whey hydrolysate was produced in the same manner as in Example I, except that only alcalase was used without using flavorzyme. That is, hydrolysis was further carried out for 14 hours after 4 hours of hydrolysis with alcalase.

[0107] Comparative Example 2

[0108] The whey hydrolysate was produced in the same manner as in Example I, except that neutrase was used instead of flavorzyme. That is, hydrolysis was carried out with neutrase for 14 hours after 4 hours of hydrolysis with alcalase.

[0109] Comparative Example 3

[0110] The whey hydrolysate was produced in the same manner as in Example I, except that collupulin was used instead of flavorzyme. That is, hydrolysis was carried out with collupulin for 14 hours after 4 hours of hydrolysis with alcalase.

[0111] Comparative Example 4

[0112] The whey hydrolysate was produced in the same manner as in Example I, except that ficin was used instead of flavorzyme. That is, hydrolysis was carried out with ficin for 14 hours after 4 hours of hydrolysis with alcalase.

[0113] Comparative Example 5

[0114] The whey hydrolysate was produced in the same manner as in Example I, except that protamex was used instead of flavorzyme. That is, hydrolysis was carried out with protamex for 4 hours after 4 hours of hydrolysis with alcalase.

[0115] Experimental Example 1: Comparison of ALP Activity of Whey Protein Hydrolysates by Different Enzymes Used

[0116] The whey protein hydrolysates of Example I and Comparative Examples 1 to 5 were each treated with MC3T3-E1 cells, and the activity of ALP (alkaline phosphatase), which is a growth index, was measured. At this time, each sample was treated at different concentrations of 62.5 μg / ml, 125 μg / ml, 250 μg / ml, and 500 μg / ml.

[0117] As a result, the ALP activity of the whey protein hydrolysate of Example I was the highest (Figure 2). Therefore, hereinafter, in the test, the whey hydrolysate of Example I was used for the test.

[0118] Experimental Example 2: Analysis of gene expression related to osteoblast differentiation

[0119] The sample of Example I was treated with MC3T3-E1 cells at concentrations of 12.5 mg / ml, 25 mg / ml, and 50 mg / ml, and the indicators related to osteoblast differentiation were evaluated.

[0120] As a result, as shown in the Western blot (Figure 3) and gene expression (Figure 4) of the indicators related to osteoblast differentiation, ALP (Alkaline phosphatase), BMP (Bone morphogenetic protein), BSP (bone sialoprotein), and COL2 (Zinc finger protein CONSTANS-LIKE 2) were significantly increased at the protein level when the sample of Example I was treated. Also, at the mRNA level, it was confirmed that the expression of ALP, BMP-4, and BSP increased significantly as the concentration increased. In the case of COL1 (Zinc finger protein CONSTANS-LIKE 1), although not significant, it was confirmed that the gene expression increased as the concentration increased.

[0121] Thus, it was confirmed that the sample of Example I affects the differentiation and growth of MC3T3-E1 cells in a concentration-dependent manner.

[0122] Experimental Example 3: Mouse Experiment

[0123] Three-week-old male Sprague-Dawley white mice were obtained from Central Laboratory Animals (Seoul, Korea). They were divided into four groups of six mice, and three white mice were placed in a plastic cage. During the experiment, the room temperature was maintained at 24 ± 1 °C, the atmospheric humidity was 60 ± 5%, and a light cycle (12 hours / 12 hours) was maintained. Body weight and food intake were monitored twice a week.

[0124] The test results were shown as mean ± standard deviation. If necessary, analysis of variance (ANOVA) was performed for comparison between groups, and the differences between samples were examined by Tukey's multiple range test using SPSS version 12.0 (Statistical Package for Social Sciences version 12.0) (SPSS Inc., Chicago IL, USA).

[0125] After the adaptation period, the Sprague-Dawley male white mice were divided into four experimental groups. (NOR; normal diet group, WPC; whey protein diet therapy group (600 mg / kg); WPH-L: whey protein hydrolysate diet therapy group of Example I (300 mg / kg); WPH-H: whey protein hydrolysate diet therapy group of Example I (600 mg / kg).

[0126] Each sample was orally administered to the animals in a total of four groups daily for 4 weeks. Then, the feed intake, water intake, body weight, and tibia length were measured every 3 days. After 4 weeks, the growth promoting effects of whey protein and whey protein hydrolysate were confirmed by sacrificing with the hemolysis index and tibia as the main indicators.

[0127] At the end of the experimental treatment period, the white mice were fasted for 12 hours before being sacrificed. The white mice were euthanized with carbon dioxide, and blood was collected from the inferior vena cava for hormone level and blood chemical analysis. The collected blood samples were centrifuged at 3,000 rpm for 10 minutes at 4 °C. The supernatant was collected, separated into multiple aliquots in tubes, and stored at -80 °C until analysis.

[0128] The level of IGF-1 (Insulin like Growth Factor-1) in serum was measured with reference to the manufacturer's instructions using an IGF-1 (Insulin like Growth Factor-1) ELISA kit for rats (fine test ER0030, Wuhan Fine Biological Technology Co., KOR). The contents of triglyceride (TG), glucose (Glu), total cholesterol (TCHO) and high density lipoprotein cholesterol (HDL) in serum were measured using a serum analyzer (Dri-chem 3500i, Fuji Photo Co., Osaka, Japan). Total calcium (Ca) and ALP (Alkaline Phosphatase) were measured using a serum analyzer (Dri-chem 3500i, Fuji Photo Co., JPN).

[0129] The dissected tibias were evaluated by the following method. The tibias were scanned using a PIXImus densitometer (GE Lunar, Madison, WI). To confirm the calibration and precision of the equipment, samples provided by the manufacturer were scanned daily during data collection, and the tibias were maintained at room temperature before scanning. All bones of a single animal were examined under the same conditions. The tibias were manually placed in air on a 7-mm thick Plexiglas platform provided by the equipment manufacturer to obtain a similar orientation for the left-side bones. The time taken to scan one sample was approximately 5 minutes. The observation area was manually adjusted and observed to individually analyze each bone, excluding bones misidentified by the software. The position of the bone was explained by multiple linear regression analysis. The adjustment algorithm was developed by scanning sets of bones at various positions within the PIXImus field before ashing, the BMD was modeled by stepwise multiple regression analysis, and the final model included the mineral content due to ashing, the X coordinate within the scanning field, and the Y coordinate of the scanning field as important independent variables. The distance from the center of the scanning field was not an important independent variable, the overall R2 value was 0.95, and the ΔR2 obtained by adjusting the X and Y coordinates was 0.027 and 0.003, respectively.

[0130] The staining of bone tissue and growth plates was performed as follows. First, the tibia was incised, and the incised tibia was fixed with 4% paraformaldehyde for 48 hours and decalcified by immersing it in 10% ethylenediaminetetraacetic acid for 24 hours (Sigma Chemicals Co, USA). Then, it was immersed in a 30% sucrose solution and dehydrated for 2 days. Each sample was longitudinally sectioned to a thickness of 40 μm with a sliding-microtome and prepared as a slide. Thereafter, chondrocytes were stained in the growth plates of the samples using cresyl violet. The slides were immersed in distilled water for 3 minutes, cresyl violet solution for 5 minutes, 50% ethanol for 3 minutes, 75% ethanol for 3 minutes, 90% ethanol for 3 minutes, 100% ethanol for 3 minutes, and xylene for 10 minutes. The 0.5% cresyl violet solution used for the sections was prepared as follows. Cresyl violet was purchased from Sigma (Sigma Chemical Co., St. Louis, USA). 2.5 mg of cresyl violet, 300 ml of distilled water, 1M sodium acetate (13.6 g / 92 ml 30 ml), and 1M acetic acid (170 ml of glacial acetic acid / 170 ml of distilled water) were stirred for 7 days before use. For the permount solution and cover glass, the height of the growth plate was measured in three separate regions per sample using Image J software (NIH, USA) for each of the three sections and the average value was calculated.

[0131] Experimental Example 3-1: Evaluation of Body Weight, Feed Intake, and Water Intake of Growing Mice

[0132] During the experimental period of the experimental group, changes in body weight, food intake, and beverage volume were measured. The white mice in the experimental group had similar average initial body weights.

[0133] As a result, the experimental groups (WPC, WPH-L, and WPH-H) that were treated with whey or whey hydrolysate tended to have a greater weight gain compared to the control group (NOR). However, no significant differences were observed. The mice administered WPH-H (8.24 g / day) obtained significantly (p < 0.05) higher weights than the mice in the control group (7.21 g / day) after 28 days. However, there was no significant difference in weight gain between WPC (7.70 g / day) and WPH-L (8.09 g / day). There were no differences in feed intake and water intake among the four groups. From this, it was confirmed that whey protein and whey protein hydrolysate were effective in the growth of mice (Table 2).

[0134]

Table 2

[0135] Experimental Example 3-2: Evaluation of Organ Weights and Serum Lipids

[0136] The organ weights and serum lipids for each experimental group were measured.

[0137] As a result, the kidney weights of the four groups were somewhat different from those of the other groups, but such differences were not significant. There were no significant differences in the weights of the heart, liver, and spleen among the four groups (Table 3). No abnormal changes such as organ hypertrophy and shrinkage were observed in the groups of WPC, WPH-L, and WPH-H. Such results indicate that the oral administration of the drinking water administered to the groups of WPC, WPH-L, and WPH-H is safe. As a result of measuring the changes in serum lipid values for NOR and the experimental groups (WPC, WPH-L, WPH-H), there were no significant differences in the serum lipid (triglyceride, total cholesterol, HDL-cholesterol, and LDL-cholesterol) levels between NOR and the experimental groups (WPC, WPH-L, and WPH-H) (Table 4). Based on the differences in organ weights and serum lipid levels, it was found that whey protein and whey protein hydrolysate are not toxic to mice.

[0138]

Table 3

[0139]

Table 4

[0140] Experimental Example 3-3: Evaluation of Serum Calcium (Ca) and ALP

[0141] The concentrations of serum calcium (Ca) and ALP were measured for each experimental group of rats.

[0142] As a result, it was confirmed that the sample treatment groups (WPC; 12.67 mg / dL, WPH-L; 12.73 mg / dL, WPH-H; 12.20 mg / dL) tended to have decreased serum calcium concentrations compared to the control group (NOR) (13.60 mg / dL). The serum ALP concentration in the sample treatment groups tended to be higher than that in the control group (NOR), but this difference was not significant. When observing the concentrations of ALP and calcium, no significant difference was found, but it was judged that whey protein and whey protein hydrolysates tended to have a beneficial effect on the growth of rats (Table 5).

[0143]

Table 5

[0144] Experimental Example 3-4: Evaluation of Serum IGF-1 Level

[0145] IGF-I (Serum Insulin like Growth Factor-1) is an insulin growth factor synthesized and secreted by the liver and other organs in response to stimulation by growth hormone. It promotes growth, differentiation, and matrix synthesis activities in osteoblasts and chondrocytes. Also, IGF-I has been reported to play an important role in the growth of long bones and the growth of the mandible.

[0146] As a result of measuring the serum IGF-1 levels in each experimental group, serum IGF-1 was slightly higher in the whey protein administration group and the whey protein hydrolysate administration group (WPC; 1580.21 pg / ml, WPH-L; 1658.61 pg / ml, WPH-H; 1683.28 pg / ml) compared to the control group (NOR) (1575.71 pg / ml). In particular, it was determined that whey protein hydrolysate was effective in the secretion of IGF-1 (Table 6, Figure 5). Figure 5 shows the result value of NOR as 0 and the difference between the result value of NOR and the result of the sample.

[0147]

Table 6

[0148] Experimental Examples 3 - 5: Tibial Growth and Increase in Growth Plate

[0149] In the whey protein (WPC) administration group, whey was administered at a dose of 600 mg / kg / day for 4 weeks. In the whey protein hydrolysate administration group of Example I, the whey protein hydrolysate of Example I was administered for 4 weeks, at a dose of 300 mg / kg / day in the WPH-L group and 600 mg / kg / day in the WPH-H group. After 4 weeks, the differences between the sample treatment group and the control NOR group were evaluated for the increase in longitudinal bone growth for each group.

[0150] As a result, the increased values of tibial growth in the WPC, WPH-L, and WPH-H groups were 39.10 mm, 39.65 mm, and 40.63 mm respectively, while in the control NOR group it was 38.18 mm, showing a significant difference (p < 0.05).

[0151] On the other hand, the growth plate is composed of four distinct histological regions that start from the resting zone and expand through the proliferative and hypertrophic zones. Since the height of the growth plate is related to the body growth rate, the height of the growth plate of the mice was measured.

[0152] As a result, it was found that the heights of the growth plates in the WPC, WPH-L, and WPH-H groups were significantly higher than those in the NOR group, which was the control group (p<0.05). The heights of the growth plates in the WPC, WPH-L, and WPH-H groups were 345.86μm, 353.32μm, and 399.23μm, respectively. In the NOR group, it was 289.66μm (Figure 6, Table 7). Thus, it was confirmed that WPC, WPH-L, and WPH-H promoted the growth of the growth plates in mice, and in particular, it was found that when the whey protein hydrolyzate of Example I was treated at a high content, the growth of the growth plates and tibiae of mice was significantly promoted.

[0153]

Table 7

[0154] Experimental Examples 3-6: Bone Analysis

[0155] For the NOR group, which was the control group, the whey protein administration group (WPC), and the whey protein hydrolyzate administration groups of Example I (WPH-L and WPH-H groups), the tibiae of each group were measured for BMC (Total amount of bone mineral), BMD (Average of bone density), bone area, bone volume, and bone length. BMD is the value obtained by dividing BMC by the bone area value. Bone area, bone volume, and bone length were measured using micro-CT.

[0156] As a result, the WPC, WPH-L, and WPH-H groups tended to show an increase in BMC compared to the NOR group. BMD generally showed the same trend as BMC in the sample treatment groups, but the NOR group showed the highest value.

[0157] BMD is the value obtained by dividing BMC by the bone area value. Since the bone area value is the lowest in NOR, BMD showed the highest numerical value in the NOR group. The BMC values of the WPC, WPH-L, and WPH-H groups were 298.60, 306.13, and 317.35 g, respectively, and the BMC value of the NOR group was 296.55 g. The BMD levels were 176.58, 176.15, and 179.83 g / cm 2 respectively, and the BMD level of the NOR group was 181.25 g / cm 2 However, there was no significant difference.

[0158] However, there was a significant difference in bone length between the NOR group and the sample administration groups (WPC, WPH-L, and WPH-H groups), and more significant results were shown particularly in the WPH-H group (Table 8, Figure 7).

[0159]

Table 8

[0160] Thus, it was confirmed that the whey protein hydrolyzate of Example I grows bone volume and length in a concentration-dependent manner.

[0161] <Example III: Selection, Quantification, and Evaluation of the Functionality of a Peptide Comprising Asp-Lys-Phe-Leu-Asp (DKFLD)>

[0162] Experimental Example 4: Selection, Quantification, and Comparison of the Content of a Peptide Comprising Asp-Lys-Phe-Leu-Asp (DKFLD)

[0163] All reagents used for mass spectrometry and HPLC (High Performance Liquid Chromatography) analysis were HPLC-grade reagents, and other reagents were analytical reagents. The water used for the production of the reagents was secondary distilled water.

[0164] Experimental Example 4-1: Selection of a Peptide Comprising Asp-Lys-Phe-Leu-Asp (DKFLD)

[0165] The peptides of the whey protein hydrolyzate of Example I were separated and purified step by step by performing ion exchange chromatography, size exclusion chromatography, and reverse phase chromatography in sequence as follows.

[0166] Ion exchange chromatography

[0167] Ion exchange chromatography was performed using an AKTA Purifier system (GE Healthcare Life Science, MA, USA) equipped with a HiLoad 16 / 10 Q-Sepharose column. 0.5 g of whey protein was dissolved in 10 ml of 25 mM Tris-Cl (pH 8.0), and the supernatant obtained by centrifuging at 7,500 xg for 20 minutes (Supra 22K, Hanil Scientific Ltd, Gimpo, Korea) was filtered through a 0.45 μm syringe filter, and 2 ml (protein content 65 mg) was injected. The peptides were eluted at a flow rate of 5 ml / min with a concentration gradient of 25 mM Tris-Cl (pH 8.0) containing 0.6 M NaCl. The eluate was fractionated in 5-ml portions, and peaks were detected at 280 nm and 220 nm. The concentration gradient conditions are as shown in Table 9.

[0168] [Table 9]

[0169] As a result of ion exchange chromatography, a total of seven fractions were obtained (Figure 8). The cell activity for each fraction was measured in the following manner. MC3T3-E1 was differentiated using a differentiation medium for 7 days. The samples were treated with 50 μM and photographed under a microscope 48 hours later. The lengths of the thickest 10 cells each were measured from 3 photographs for each group. As a result, as shown in Figures 9 and 10, in fraction EX-1 and fraction EX-4, the myotube diameters were significantly increased by 19% and 17% respectively compared to the induction control group (Control) treated with dexamethasone, indicating higher cell activity.

[0170] Size exclusion chromatography

[0171] Size exclusion chromatography was performed on fraction EX-1 and fraction EX-4, which showed the highest cell activity. Size exclusion chromatography was carried out using an AKTA purifier system (GE Healthcare Life Science, MA, USA) equipped with Superdex peptide 10 / 300. The solvent of the active fraction obtained by ion exchange chromatography was evaporated using a rotary vacuum evaporator, and the concentrate was dissolved in HPLC-grade water and used as a sample. The injected sample volume was 1 ml, and HPLC-grade water was used as the elution solvent to obtain the effect of removing the salts used in ion exchange chromatography. The column was washed with 1 CV at a flow rate of 0.5 ml / min, and the peptides were eluted with 1.5 CV while detecting peaks at 280 nm and 220 nm.

[0172] Fractions EX-1 to S9-10, S13, S19, and S26 were detected (Figure 11). However, the fractions of S13 and S26 were discarded because their protein contents were too low, and the fractions of S9-10 and S19 were taken. From fraction EX-4, S14-15, S16, S19, and S20-21 were fractionated (Figure 12). For each fraction, cell activity (the effect on the root cap cell thickness) was measured by cell experiments in the same manner as ion exchange chromatography. As a result, as shown in Figure 13, the fractions of EX / S9-10, EX1 / S19, EX4 / S16, and EX-4 / S19 showed a significant difference in the growth of the root cap cell thickness, indicating high cell activity (p<0.05).

[0173] Reverse-phase chromatography

[0174] Reverse-phase chromatography was performed on the active fractions EX-1 / S9-10, EX-1 / S19, EX-4 / S16, EX-4 / S19, and EX-4 / S20-21 obtained by size exclusion chromatography. The active fractions obtained by size exclusion chromatography were completely volatilized of the solvent at 2000 rpm using a Speed Vac centrifuge (HyperVAC-MAX, Hanil Scientific Ltd, Gimpo, Korea), and then dissolved in an acetonitrile solvent containing 100 - 200 μl of 0.1% TFA according to the peptide concentration range and used as samples for reverse-phase chromatography fractionation. The reverse-phase chromatography fractionation conditions are as shown in Table 10.

[0175]

Table 10

[0176] Among the patterns of the resulting reverse-phase chromatograms, only the peaks with no overlapping and good separation state were fractionated and used as samples for mass spectrometry for amino acid sequence analysis of peptides (Table 11).

[0177]

Table 11

[0178] Amino acid sequence analysis

[0179] The solvent of the same peak separated by reverse-phase chromatography and fractionated several times was completely evaporated using a Speed Vac. After that, it was dissolved in 80% acetonitrile, combined into one, and the solvent was completely evaporated again using a Speed Vac. Then, 15 μl of distilled water was added to the dried sample and completely dissolved to be used as an analytical sample. 15 μl of the analytical sample was injected into a UHPLC system (Ultimate 3000, Thermo Scientific, San Jose, CA, USA) equipped with ACQUITY U-HPLC@BEH 130 C18 (1.7 μm). Peptides were separated with a uniform concentration of 0.1% formic acid / acetonitrile, and the amino acid sequence of the peptides was determined using the protein pilot (AB SCIEX / peak view) program on a Triple TOF 5600+ system (AB SCIEX, Ontario, Canada) connected on-line. The liquid chromatography conditions and Q-TOF mass spectrometry conditions used for the analysis are as shown in Table 12 and Table 13, respectively.

[0180]

Table 12

[0181]

Table 13

[0182] The analyzed amino acid sequence was compared with the amino acid sequences of bovine and milk proteins in the Uniport database. The Uniprot amino acid sequences of α-lactalbumin and β-lactoglobulin, which are the main constituent proteins of whey protein, are as shown in Table 14.

[0183]

Table 14

[0184] Peptide DKFLD corresponds to the 97th to 101st amino acids of α-lactalbumin. Peptide DKFLD was detected from the fraction and presumed to be usable as an indicator peptide.

[0185] Experimental Example 4-2: Synthesis of a Peptide Composed of Asp-Lys-Phe-Leu-Asp (DKFLD)

[0186] The peptide composed of Asp-Lys-Phe-Leu-Asp (DKFLD) confirmed by amino acid sequence analysis was synthesized by A&pep (Seoul, Korea) using the general solid-phase synthesis method. The molecular weight and purity of the synthesized peptide DKFLD were 636.71 Da and 96.76%, respectively.

[0187] Experimental Example 4-3: Quantification of a Peptide Composed of Asp-Lys-Phe-Leu-Asp (DKFLD)

[0188] Quantification of the peptide composed of Asp-Lys-Phe-Leu-Asp (DKFLD) was performed using a Watchers 120 ODS-BP (4.6 x 250 mm, 5 μm) column with a Waters2695 HPLC system (Milford, MA, USA). The same analysis conditions other than the analytical equipment were applied. The whey protein hydrolysate sample was not pretreated otherwise. 0.5 g of whey protein hydrolysate powder was completely dissolved in HPLC-grade distilled water and made up to a constant volume of 10 ml, and then the supernatant obtained by centrifugation (13,000 rpm, 15 minutes) was filtered through a 0.45 μm syringe filter and used as an analytical sample. The analytical conditions for peptide quantification composed of Asp-Lys-Phe-Leu-Asp in the HPLC system are as shown in Table 15.

[0189]

Table 15

[0190] The peptide DKFLD was detected at a retention time of 10.400 minutes in the same HPLC system and showed a separation ability in a form that could be quantified without interference from adjacent peaks even in whey protein hydrolysates (Figure 14). The sample pretreatment process for whey protein hydrolysates for HPLC analysis was not performed. DKFLD has a cLog value of -0.84 and a solubility value of 10.7 mg / ml, and is predicted to be a hydrophilic peptide, which may be eluted in water with Sep-Pak solid-phase columns in both forward and reverse images. It was expected to save the time required for sample pretreatment during on-site application, so the pretreatment process was omitted for quantification.

[0191] The content at each stage of the manufacturing process of the peptide consisting of Asp-Lys-Phe-Leu-Asp (DKFLD) was measured (Figure 15). The average value and standard deviation value were measured using a total of 9 samples obtained by analyzing 0.5 g of whey protein hydrolysates from 3 lots three times each. The content was 4.94 ± 0.59 mg / g-sample, and the RSD (%) value was 4.51%, having a precision within 5%.

[0192] Experimental Example 4-4: Comparison of Peptide Content of Asp-Lys-Phe-Leu-Asp (DKFLD) in Different Whey Protein Hydrolysates

[0193] The peptide content of Asp-Lys-Phe-Leu-Asp (DKFLD) in the whey protein hydrolysate of the present invention (Example I) and whey protein hydrolysates from other companies was compared. As whey protein hydrolysates from other companies, Arla whey protein hydrolysate (Arla Foods Ingredients, Arla SP-8011, Arla whey protein), Hilmar whey protein hydrolysate (Hilmar Ingredients, Hilmar8010), and Murray Goulburn Co-operative whey protein hydrolysate (Example 2 of Korean Patent Registration No. 1311318) were used.

[0194] As a result, as shown in Table 16, a peptide consisting of Asp-Lys-Phe-Leu-Asp (DKFLD) was detected only from the whey protein hydrolyzate of the present invention, and a peptide consisting of Asp-Lys-Phe-Leu-Asp (DKFLD) was not detected from other whey protein hydrolyzates. It was found that the peptide consisting of Asp-Lys-Phe-Leu-Asp (DKFLD) is produced only by the production method of the present invention.

[0195]

Table 16

[0196] Experimental Example 4-5: Comparison of the content of the peptide consisting of Asp-Lys-Phe-Leu-Asp (DKFLD) depending on the enzyme used and the enzyme addition method

[0197] Various enzyme treatment methods were tried for the maximum yield of the peptide DKFLD (Asp-Lys-Phe-Leu-Asp) which is the active ingredient. As a result, as shown in Table 17, as the primary enzyme reaction, it was confirmed that the yield was the best when Alcalase and Protamex, which are endo-enzymes, were reacted for 4 hours and then Flavourzyme, which is an exo-enzyme, was reacted for 15 hours. All enzymes were added at 0.2% based on the raw material.

[0198]

Table 17

[0199] Experimental Example 5: Confirmation of the growth functionality of the peptide consisting of Asp-Lys-Phe-Leu-Asp (DKFLD)

[0200] Bone is a special tissue in which a calcified and firm surface and an internal cell component called bone marrow are combined. Osteoblasts located on the surface of bone have alkaline phosphatase (ALP), a glycoprotein enzyme, on their cell membranes, and are known to play a role in secreting and calcifying bone matrix substances such as osteocalcin (OCN), osteopontin (OPN), and bone sialoprotein (BSP).

[0201] ALP is particularly involved in the process of osteoblast differentiation, and the differentiation stage of osteoblasts can be confirmed by the relative activity of this enzyme. That is, the ALP present in bone tissue increases in activity when bone growth progresses actively.

[0202] The final stage in the process of bone tissue formation is the calcification stage in which primitive cells ossify. Bone nodules are important labeling factors for osteoblast differentiation and can be confirmed using alizarin, which has a particularly high adsorptive power for calcium. Since this plant dye is stained in the extracellular matrix of calcified cells, it is stained darker as the degree of calcification progresses.

[0203] MC3T3-E1 cells are osteoblasts derived from mouse calvaria, have similar metabolic characteristics such as proliferation, differentiation, and calcification that occur in vivo, and in particular, have ALP, a glycoprotein, on their cell membranes, and were used in the experiment because they are usefully used in research related to bone formation.

[0204] Experimental Example 5-1: Measurement of the cytotoxicity of a peptide consisting of Asp-Lys-Phe-Leu-Asp (DKFLD)

[0205] Cytotoxicity was measured using the WST-1 assay. Cells were cultured in a 96-well plate such that the final culture medium was 100 μl / well. After culturing the cells for 24 hours, the medium was replaced and the samples were treated according to concentration. After treating the samples and culturing for 2 to 4 hours, 10 μl of Premix WST-1 was added per well and reacted for 2 to 4 hours, and then the absorbance was measured at 450 nm.

[0206] Figure 16 is a graph showing the cytotoxicity of a peptide consisting of Asp-Lys-Phe-Leu-Asp (DKFLD) against MC3T3-E1 cells.

[0207] As shown in Figure 16, since there was no test group in which cell viability decreased compared to the growth medium (Con) as the control group (Control), it was confirmed that the peptide consisting of Asp-Lys-Phe-Leu-Asp (DKFLD) had no cytotoxicity up to 250 ppm.

[0208] Experimental Example 5-2: Evaluation of ALP Activity by Treatment with a Peptide Consisting of Asp-Lys-Phe-Leu-Asp (DKFLD)

[0209] Sample (Example I) wells (80 μl) and standard wells (120 μl) were set in a 96-well plate. The samples were added in the same amount by BCA analysis (Bicinchoninic acid assay) and diluted when necessary, and the dilution was carried out using ALP assay buffer. 20 μl of stop solution was added to the 80 μl of the sample wells, and no stop solution was added to the standard wells. Then, 50 μl of 5 mM pNPP solution was added only to the 80 μl of the sample wells, and 10 μl of ALP enzyme solution was added only to the standard wells. Incubation was carried out at 37 °C for 60 minutes in the dark. Then, 20 μl of stop solution was added to both the sample wells and the standard wells to terminate the reaction, and the absorbance was measured at 405 nm.

[0210] As a result, as shown in FIGS. 17 and 18, on both the 3rd and 7th days, there was a significant difference between the differentiation medium (Con) of the positive control group and the growth medium (Nor) of the normal control group (Normal). Thus, it was confirmed that the experiment was valid. Also, the activity increased concentration-dependently among the test groups compared to the positive control group (Con), and it was found that there was a statistically significant difference from the positive control group (Con) at p = 0.05 during Duncan's post hoc analysis starting from 125 ppm.

[0211] Therefore, it was confirmed that the peptide consisting of Asp-Lys-Phe-Leu-Asp (DKFLD) has the function of promoting osteoblast differentiation.

[0212] Experimental Example 5-3: Analysis of the degree of bone mineralization formation by treatment with a peptide consisting of Asp-Lys-Phe-Leu-Asp (DKFLD)

[0213] After washing MC3T3-E1 cells twice with PBS, the cells were fixed with 10% formalin for 30 minutes. After suction, they were sufficiently stained with Alizarin red S (pH 4.2) staining solution. After washing approximately twice with distilled water, 10% cetylpyridinium chloride in 10 mM sodium phosphate buffer (pH 7.0) was added for elution. Then, the absorbance was measured at 550 nm.

[0214] As a result, as shown in FIGS. 19 and 20, on both the 3rd and 7th days, there was a significant difference between the differentiation medium (Con) of the positive control group and the growth medium (Nor) of the normal control group (Normal). Thus, it was confirmed that the experiment was valid. Also, the activity increased concentration-dependently among the test groups compared to the positive control group (Con), and it was found that there was a statistically significant difference from the positive control group (Con) at p = 0.05 during Duncan's post hoc analysis starting from 62.5 ppm.

[0215] Therefore, it was confirmed that the peptide consisting of Asp-Lys-Phe-Leu-Asp (DKFLD) has the function of promoting the progress of bone mineralization.

[0216] As described above, specific parts of the present invention have been described in detail. However, it is obvious to those with ordinary knowledge in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention.

Claims

1. A food composition for growth promotion, comprising a peptide consisting of Asp-Lys-Phe-Leu-Asp.

2. The food composition according to claim 1, wherein the food composition comprises a whey protein hydrolyzate containing a peptide consisting of Asp-Lys-Phe-Leu-Asp.

3. The food composition for growth promotion according to claim 2, wherein the content of the peptide is 0.5 mg / g to 40 mg / g based on the total weight of the whey protein hydrolyzate.

4. The food composition for growth promotion according to claim 2, wherein the whey protein hydrolyzate is obtained by primary hydrolysis of whey protein with an endoprotease derived from Bacillus licheniformis and secondary hydrolysis with an exoprotease derived from Aspergillus oryzae.

5. The food composition for growth promotion according to claim 4, wherein the endoprotease derived from Bacillus licheniformis is Alcalase, Protamex, or a mixed enzyme thereof, and the exoprotease derived from Aspergillus oryzae is Flavourzyme.

6. The food composition for growth promotion according to claim 5, wherein the mixed enzyme is a mixture of Alcalase and Protamex in a weight ratio of 1:0.5 to 1:

2.

7. The food composition for growth promotion according to claim 1, wherein the growth promotion is by promoting the growth of bone length or bone volume.

8. A pharmaceutical composition for preventing or treating growth disorders, comprising a peptide consisting of Asp-Lys-Phe-Leu-Asp.

9. A whey protein hydrolyzate containing a peptide consisting of Asp-Lys-Phe-Leu-Asp.

10. The whey protein hydrolyzate according to claim 9, wherein the content of the peptide is 0.5 mg / g to 40 mg / g based on the total weight of the whey protein hydrolyzate.

11. (a) mixing whey protein and water to dissolve the whey protein; (b) adding an endoprotease derived from Bacillus licheniformis to the dissolved whey protein lysate to perform primary hydrolysis; (c) adding an exoprotease derived from Aspergillus oryzae to the primary hydrolyzed product to perform secondary hydrolysis, and a method for producing a whey protein hydrolyzate containing a peptide consisting of Asp-Lys-Phe-Leu-Asp. (Claim 12) (d-1) After the step (c), further including the step of sterilizing the whey protein hydrolyzate, the method for producing a whey protein hydrolyzate containing a peptide consisting of Asp-Lys-Phe-Leu-Asp according to claim 11. (Claim 13) (d-2) After the step (c), further including the step of filtering and drying the whey protein hydrolyzate, the method for producing a whey protein hydrolyzate containing a peptide consisting of Asp-Lys-Phe-Leu-Asp according to claim 11. (Claim 14) (d-3) The endoprotease derived from Bacillus licheniformis is Alcalase, Protamex, or a mixed enzyme thereof, and the exoprotease derived from Aspergillus oryzae is Flavourzyme, the method for producing a whey protein hydrolyzate containing a peptide consisting of Asp-Lys-Phe-Leu-Asp according to claim 11. (Claim 15) (d-4) The mixed enzyme is a mixture of Alcalase and Protamex mixed at a weight ratio of 1:0.5 to 1:2, the method for producing a whey protein hydrolyzate containing a peptide consisting of Asp-Lys-Phe-Leu-Asp according to claim 14.

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