Composition for increasing body height and bone density derived from yolk protein

Abstract

The present invention relates to a method for producing a composition for increasing height and bone density derived from egg yolk protein, and the composition. The present invention produces an egg yolk protein hydrolysate having a novel composition, and a functional composition having physiological activity and effects such as promoting height growth, increasing bone density, and promoting bone growth can be obtained. The functional composition can be utilized in the development of functional foods and medicines, which can contribute to the promotion of public health.

Description

Technical Field

[0001] The present invention relates to a method for producing a composition comprising a hydrolyzate of egg yolk protein obtained by hydrolyzing a protein present in egg yolk, which has an effect of promoting growth of height and increasing bone density, and to the composition.

Background Art

[0002] Osteoporosis is a disease in which the amount of bone decreases, the strength of bone becomes weak, and fractures are likely to occur even with a small impact. Osteoporosis is a problem because various fractures caused by bone weakening, particularly hip fractures or spinal fractures, limit long-term activities, and it is known to account for 15% of deaths in the elderly population.

[0003] As osteoporosis therapeutic agents, there are hormones, alendronate, calcitonin, raloxifene, Na-F, calcitriol, or bisphosphonate preparations. However, the treatment of osteoporosis is a disease that requires a long treatment period, and when the above therapeutic agents are taken for a long time, side effects such as urinary stones, endometrial cancer, and breast cancer are likely to occur.

[0004] In particular, in the case of hormone therapy containing estrogen or calcitonin, side effects such as breast cancer, myocardial infarction, and venous thrombosis have been reported. Bisphosphonates are used as bone resorption inhibitors that suppress osteoclasts, but have problems such as low absorption rate, and problems such as lesions being observed in the upper airway during incorrect oral administration have been reported, and there is an urgent need to develop therapeutic agents with less toxicity and side effects.

[0005] On the other hand, egg yolk contains a large amount of lipids and proteins and is known to be very effective for health promotion. However, it contains lecithin and neutral fat with excellent emulsifying ability, and directly commercializing it is not preferable in terms of stability and physiological activity.

[0006] Therefore, the present inventors introduced an enzymatic hydrolysis step in order to provide the useful substances contained in egg yolk in an optimal state while eliminating the instability of egg yolk as described above, and completed the present invention by confirming that the egg yolk hydrolyzate thus produced has an effect in increasing bone density, promoting bone growth, and improving osteoporosis.

Summary of the Invention

Problems to be Solved by the Invention

[0007] One object of the present invention is to provide a defatted egg yolk hydrolyzate obtained by treating defatted egg yolk with a combination of an endo-peptidase enzyme and an exo-peptidase enzyme.

[0008] Another object of the present invention is to provide a food composition for promoting body growth or improving bone density containing the defatted egg yolk hydrolyzate as an active ingredient.

[0009] Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating osteoporosis containing the defatted egg yolk hydrolyzate as an active ingredient.

[0010] Still another object of the present invention is to provide a method for producing a defatted egg yolk hydrolyzate, comprising: (a) producing defatted egg yolk by adding an organic solvent capable of dissolving lipids to egg yolk powder, followed by extraction and filtration; (b) obtaining defatted egg yolk powder by drying the remaining egg yolk cake after separation in step (a); (c) producing a defatted egg yolk powder solution by adding water to the defatted egg yolk powder obtained in step (b), and obtaining a primary hydrolyzate by treating with an endo-peptidase; (d) removing insoluble egg yolk cake from the primary hydrolyzate obtained in step (c); and (e) obtaining a secondary hydrolyzate by treating the primary hydrolyzate from which the egg yolk cake has been removed in step (d) with an exo-peptidase.

Means for Solving the Problems

[0011] The present invention provides a defatted egg yolk hydrolyzate obtained by treating defatted egg yolk with a combination of an endopeptidase enzyme and an exopeptidase enzyme.

[0012] At this time, it is preferable to remove insoluble egg yolk meal through filtration during the treatment with the endopeptidase enzyme and the exopeptidase enzyme for the hydrolyzate.

[0013] At this time, it is preferable that the sum of the contents of arginine, phenylalanine, tyrosine, and histidine in the hydrolyzate is 7% (w / w) or more based on the total solid content.

[0014] On the other hand, the present invention provides a food composition for promoting body height growth containing the defatted egg yolk hydrolyzate as an active ingredient.

[0015] On the other hand, the present invention provides a food composition for improving bone density containing the defatted egg yolk hydrolyzate as an active ingredient.

[0016] On the other hand, the present invention provides a pharmaceutical composition for preventing or treating osteoporosis containing the defatted egg yolk hydrolyzate as an active ingredient.

[0017] At this time, the hydrolyzate has the effect of increasing the activity of ALP (Alkaline phosphatase).

[0018] On the one hand, the present invention provides a method for producing a hydrolyzed defatted egg yolk, comprising: (a) producing defatted egg yolk by adding an organic solvent capable of dissolving lipids to egg yolk powder, followed by extraction and filtration; (b) obtaining defatted egg yolk powder by drying the remaining egg yolk meal after separation in step (a); (c) producing a defatted egg yolk powder solution by adding water to the defatted egg yolk powder obtained in step (b), and obtaining a primary hydrolyzate by treating with endopeptidase; (d) removing insoluble egg yolk meal from the primary hydrolyzate obtained in step (c); and (e) obtaining a secondary hydrolyzate by treating the primary hydrolyzate from which the egg yolk meal has been removed in step (d) with exopeptidase.

Advantages of the Invention

[0019] The present invention can produce a hydrolyzed egg yolk protein having a novel composition, and obtain a functional composition having physiological activities and effects such as promoting height growth, increasing bone density, and promoting bone growth. By utilizing this in the development of functional foods, pharmaceuticals, etc., it can contribute to the improvement of the health of the people.

Brief Description of the Drawings

[0020]

Figure 1

Figure 2

Figure 3

Figure 4

Modes for Carrying Out the Invention

[0021] The present invention provides a method for producing a hydrolyzed defatted egg yolk, comprising: (a) producing defatted egg yolk by adding an organic solvent capable of dissolving lipids to egg yolk powder, followed by extraction and filtration; (b) obtaining defatted egg yolk powder by drying the remaining egg yolk residue after separation in step (a); (c) producing a defatted egg yolk powder solution by adding water to the defatted egg yolk powder obtained in step (b), and obtaining a primary hydrolyzate by treating with endopeptidase; (d) removing insoluble egg yolk residue from the primary hydrolyzate obtained in step (c); and (e) obtaining a secondary hydrolyzate by treating the primary hydrolyzate from which the egg yolk residue has been removed in step (d) with exopeptidase.

[0022] Hereinafter, each step of the present invention will be described in detail.

[0023] <Step (a): Production of defatted egg yolk>

[0024] This step is to produce defatted egg yolk by adding an organic solvent capable of dissolving lipids to egg yolk powder, followed by extraction and filtration at 50°C to 70°C for 3 to 5 hours.

[0025] As the solvent, it is preferably ethanol or an acetone dilution, and more preferably ethanol at 50% (v / v) to 99.99% (v / v). At this time, the solvent is preferably added in a weight ratio of 5 to 10 times the dry egg yolk powder. By adding water to the solvent under the corresponding conditions, the fat component containing lecithin can be efficiently removed.

[0026] When the egg yolk extraction step is carried out at less than 50°C, the extraction yield of useful components may be low and the removal of fat components may not be complete, so it is not preferable. When the egg yolk extraction step is carried out at 70°C or higher, the useful components will be denatured, so it is not preferable. For the same reason as above, the extraction time is preferably 3 to 5 hours.

[0027] <Step (b): Obtaining defatted egg yolk powder>

[0028] This step is a process of obtaining defatted egg yolk powder by drying the remaining egg yolk meal at 40°C to 60°C after separation in the previous step (a).

[0029] If the drying is carried out at a temperature lower than 40°C, it is not preferable because the drying will not be completed. If the drying is carried out at a temperature exceeding 60°C, it is not preferable because the active ingredients will be denatured. Through this process, the residual amount of the solvent can be adjusted to 1% (w / w) or less.

[0030] Although the first raw material of the present invention is egg yolk, if it is available for purchase in the state of defatted egg yolk, the processes of the above steps (a) and (b) can be omitted.

[0031] <Step (c): Primary enzymatic hydrolysis>

[0032] This step is a process of producing a defatted egg yolk powder solution by adding water to the defatted egg yolk powder obtained in the above step (b) and obtaining a primary hydrolyzate by treating it with endopeptidase.

[0033] As the endopeptidase in this step, any known enzyme may be used, but it is more preferable to use alcalase. Insoluble components can be removed through this process.

[0034] <Step (d): Removal of insoluble egg yolk meal>

[0035] This process is a process of removing insoluble egg yolk meal from the primary hydrolyzate obtained in the above step (c). By removing insoluble egg yolk meal from the primary hydrolyzate obtained in step (c), a supernatant (so-called "primary hydrolyzate from which egg yolk meal has been removed") is obtained. The removal of insoluble egg yolk meal is preferably carried out through filtration rather than centrifugation.

[0036] When emulsification occurs during the removal of egg yolk meal in this step (d), the following secondary enzymatic hydrolysis reaction is significantly inhibited. Therefore, this process includes a process of assisting in the removal of insoluble egg yolk meal so that emulsification of the residual liquid by lecithin remaining in the defatted egg yolk does not occur.

[0037] In a method for preventing the occurrence of emulsification, in the case of a centrifugal pump with a general motor, since it rotates at a high speed of 1,800 rpm or more, it inevitably causes emulsification. Therefore, in this process, it is more preferable to apply a transfer method that transfers without emulsification by using a diaphragm pump, a mono pump, or a pressure transfer method without using a centrifugal pump equipped with a high-speed rotating motor. This is because it not only improves the progress of the enzymatic reaction itself but also enables easy separation in the separation stage after the reaction is completed.

[0038] <The said step (e): Secondary enzymatic hydrolysis>

[0039] This step is a process of obtaining a secondary hydrolyzate by treating the primary hydrolyzate from which the egg yolk meal obtained in the said step (d) has been removed with exopeptidase.

[0040] As the exopeptidase in this step, any known enzyme may be used, but it is more preferable to use flavourzyme. Through this process, it can be decomposed into more peptides and amino acids.

[0041] As an additional step, the present invention can further include the following steps.

[0042] Specifically, (f) a layer separation and concentration step can be further performed.

[0043] In the layer separation step, the separated aqueous solution layer is recovered and concentrated. During this process, the separated aqueous solution layer is filtered using a microfilter with a pore size of 5 microns or less. This step is applied to remove insoluble substances contained in the oil layer, neutral fat of egg yolk, and some contaminating bacteria during the layer separation process.

[0044] Generally, when the layer separation method of the present invention is not used, a method of adding diatomaceous earth or the like as a filtration aid and filtering with a filter press or the like is used. However, according to this method, each fat component in the reaction solution is directly filtered and enters, resulting in a problem that the content of crude fat in the final powder becomes high. The process for filtration becomes complicated, interfering with the progress of the secondary enzyme reaction, and there are decisive manufacturing process and quality disadvantages of requiring expensive equipment.

[0045] In the case of the present invention, the content of crude fat can finally be produced at 1% (w / w) or less. However, according to general processes, it will have a crude fat content of 2% (w / w) to 3% (w / w). A high crude fat content can be a factor that easily causes a rancid oil odor when exposed to air.

[0046] Thereafter, the residual liquid after the reaction and filtration is subjected to a concentration process. In the concentration process, vacuum concentration is usually carried out in the range of a vacuum degree of 20 Torr to 100 Torr to make the content of solid matter 20% (w / v) to 40% (w / v).

[0047] Before spray drying the concentrated liquid, a microfilter can be used to remove contaminating bacterial cells present in the concentrated liquid. The microfilter to be used must have a pore size of 0.5 microns or less. The reason is that it is difficult to remove contaminating bacteria with a microfilter having a pore size larger than 0.5 microns.

[0048] Accordingly, the present invention provides a defatted egg yolk hydrolyzate obtained by treating defatted egg yolk with a combination of an endopeptidase enzyme and an exopeptidase enzyme.

[0049] At this time, the enzyme preferably consists of each of endopeptidase and exopeptidase or a combination thereof. In the present invention, more preferably, by sequentially treating each of endopeptidase and exopeptidase, a yolk enzyme hydrolyzate containing more peptides and amino acids could be obtained.

[0050] At this time, it is preferable that the sum of the contents of arginine, phenylalanine, tyrosine and histidine in the hydrolyzate is 7% (w / w) or more based on the total solid content.

[0051] Furthermore, it is more preferable that the sum of the contents of arginine, phenylalanine, tyrosine and histidine in the hydrolyzate is 10% (w / w) or more of the total sum of amino acids and is 13% (w / w) to 18% (w / w) of the total sum of amino acids.

[0052] The yolk enzyme hydrolyzate of the present invention contains, among amino acids, leucine, lysine, valine, arginine, glutamic acid, threonine, serine, isoleucine, phenylalanine, tyrosine, asparagine, alanine, aspartic acid, methionine, glutamine, histidine, tryptophane, cystine, glycine and proline.

[0053] Among them, the sum of the contents of arginine, phenylalanine, tyrosine and histidine, which are known to have effects such as growth of height, improvement of bone density, and prevention of osteoporosis, is 7% (w / w) or more based on the total solid content, and it has excellent functionality.

[0054] According to an embodiment of the present invention, it has been confirmed that the egg yolk enzyme hydrolyzate of the present invention increases the activity of ALP (Alkaline phosphatase), and has also confirmed the effect of promoting bone growth and increasing bone density in an animal model.

[0055] Therefore, the present invention provides a food composition for promoting height growth or improving bone density, which contains the defatted egg yolk hydrolyzate as an active ingredient.

[0056] The term "improvement" used in the present invention means all acts in which height growth or bone density improves or is advantageously changed by administration of the composition of the present invention.

[0057] In the food composition of the present invention, the defatted egg yolk hydrolyzate is preferably contained in an amount of 0.00001 wt% to 50 wt% of the food composition. If it is contained in an amount less than 0.00001 wt%, its effect will be negligible, and if it is contained in an amount exceeding 50 wt%, the increase in effect compared to the usage amount will be negligible, which is uneconomical.

[0058] The food composition of the present invention can be, for example, any one selected from noodles, gums, dairy products, ice creams, meats, grains, caffeinated beverages, general beverages, chocolates, breads, snacks, confectioneries, candies, pizzas, jellies, alcoholic beverages, liquors, vitamin complexes, and other health supplements, but is not necessarily limited thereto.

[0059] When the food composition of the present invention is used as a food additive, it can be added as it is or used together with other foods or food ingredients, and can be appropriately used by a normal method.

[0060] In the present invention, the food composition includes health functional foods.

[0061] The "health functional food" means a food manufactured and processed using raw materials and ingredients having functional properties useful for the human body as defined by Law No. 6727 regarding health functional foods. "Functionality" means regulating nutrients with respect to the structure and function of the human body, or ingesting for the purpose of obtaining effects useful for health applications such as physiological effects.

[0062] The food composition and health functional food of the present invention can contain additional ingredients that are commonly used and can improve odor, taste, visual appearance, etc. For example, the food composition and health functional food of the present invention can contain biotin, folate, pantothenic acid, vitamins A, C, D, E, B1, B2, B6, B12, niacin, etc., can contain minerals such as chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), zinc (Zn), iron (Fe), calcium (Ca), etc., and can contain amino acids such as cysteine, valine, lysine, tryptophan, etc. Further, the food composition and health functional food of the present invention can be added with food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), colorants (tar dyes, etc.), color developers (sodium nitrite, sodium acetate, etc.), bleaching agents (sodium sulfite), bactericides (bleaching powder and high - grade bleaching powder, sodium hypochlorite, etc.), swelling agents (alum, potassium hydrogen D - tartrate, etc.), fortifiers, emulsifiers, thickeners (paste), coating agents, antioxidants (butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), etc.), seasonings (sodium glutamate (MSG), etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavors (vanillin, lactones, etc.), gum bases, foam inhibitors, solvents, improvers, etc. The said additives can be selected according to the type of food and used in appropriate amounts.

[0063] In the health functional food of the present invention, the content of the defatted egg yolk hydrolyzate is not particularly limited and can be variously changed depending on the state of the administration subject, the specific type of disease condition, the degree of progression, etc., and can also be the total content of the food if necessary.

[0064] On the one hand, the present invention provides a pharmaceutical composition for preventing or treating osteoporosis containing the defatted egg yolk hydrolyzate as an active ingredient.

[0065] The term "prevention" used in the present invention means all actions that suppress a disease affected by the administration of the pharmaceutical composition according to the present invention, that is, osteoporosis, or delay the onset.

[0066] The term "treatment" used in the present invention means all actions in which the symptoms caused by osteoporosis improve or are favorably changed by the administration of the pharmaceutical composition according to the present invention.

[0067] The composition containing the defatted egg yolk hydrolyzate of the present invention as an active ingredient may additionally contain one or more active ingredients exhibiting the same or similar functions to the above components.

[0068] The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier in addition to the defatted egg yolk hydrolyzate.

[0069] The carrier that can be used in the present invention is not particularly limited in type, and any carrier commonly used in the technical field can be used. Non-limiting examples of the carrier include lactose, dextrose, sucrose, sorbitol, mannitol, saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, and the like. These may be used alone or in combination of two or more.

[0070] In addition, the pharmaceutical composition of the present invention can be used by adding other pharmaceutically acceptable additives such as antioxidants, excipients, diluents, buffers or bacteriostatic agents, if necessary, and surfactants, binders, fillers, extenders, wetting agents, disintegrants, dispersants or lubricants can be additionally added and used.

[0071] In the pharmaceutical composition of the present invention, the defatted egg yolk hydrolyzate may be contained in an amount of 0.00001% to 99.99% by weight, preferably 0.1% to 90% by weight, more preferably 0.1% to 70% by weight, still more preferably 0.1% to 50% by weight, based on the total weight of the pharmaceutical composition, but is not limited thereto, and can be variously changed depending on the state of the administration subject, the type of specific disease condition, the degree of progression, etc., and may also be included as the total content of the pharmaceutical composition if necessary.

[0072] That is, the pharmaceutically effective amount and effective dosage of the pharmaceutical composition of the present invention can vary depending on the formulation method, administration method, administration time and / or administration route of the pharmaceutical composition, etc. Also, the type and degree of reaction to be achieved by the administration of the pharmaceutical composition, the type of individual to be administered, age, body weight, general health condition, symptoms and degree of disease, gender, diet, excretion, and many factors including the components of other compositions of drugs used simultaneously or at different times for the relevant individual, and similar factors well known in the pharmaceutical field can vary. A person having ordinary knowledge in the relevant technical field can easily determine and formulate an effective dosage for the intended treatment. For example, the pharmaceutical composition of the present invention has a daily dosage of about 0.01 mg / kg to 1,000 mg / kg, preferably 150 mg / kg to 300 mg / kg, and can be administered once a day or divided into several doses a day.

[0073] The pharmaceutical composition of the present invention may be administered once a day or divided into several doses a day. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent, may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Considering all of the above elements, it can be administered in an amount that provides the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

[0074] The pharmaceutical composition of the present invention can be additionally used in combination with various methods such as hormone therapy and drug therapy for preventing or treating osteoporosis.

[0075] As used herein, the term "administering" means introducing the pharmaceutical composition of the present invention to a patient by any suitable method, and the route of administration and the mode of administration of the pharmaceutical composition of the present invention may be independent of each other, and any route of administration and mode of administration can be followed without special limitation as long as the pharmaceutical composition can reach the target site.

[0076] The pharmaceutical composition can be administered by oral administration or parenteral administration, and can be formulated into appropriate and various dosage forms for oral administration or parenteral administration and used.

[0077] Non-limiting examples of formulations for oral administration using the pharmaceutical composition of the present invention include oily suspensions, troches, lozenges, tablets, aqueous suspensions, dispensing powders, granules, emulsions, hard capsules, soft capsules, syrups or elixirs.

[0078] In order to formulate the pharmaceutical composition of the present invention for oral administration, binders such as sorbitol, mannitol, starch, amylopectin, cellulose lactose, sucrose or gelatin; lubricating oils such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax; excipients such as dibasic calcium phosphate; disintegrants such as corn starch or sweet potato starch can be used, and flavoring agents, syrup agents, sweetening agents, etc. can also be used. Further, in the case of capsules, in addition to the substances mentioned above, a liquid carrier such as fatty oil can be further used.

[0079] As methods of parenteral administration of the pharmaceutical composition of the present invention, intramuscular administration, transdermal administration, intravenous administration, intraperitoneal administration or subcutaneous administration can be used, and the method of applying the composition to the diseased site, spraying and inhalation can also be used, but is not limited thereto.

[0080] Non-limiting examples of parenteral preparations using the pharmaceutical composition of the present invention include injection solutions, suppositories, ointments, powders for application, oils, powders for respiratory inhalation, aerosol agents for sprays, creams, and the like.

[0081] In order to formulate the pharmaceutical composition of the present invention for parenteral administration, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, external preparations, and the like can be used. Further, as the non-aqueous solvent and suspension, vegetable oils such as olive oil, propylene glycol, polyethylene glycol, injectable esters such as ethyl oleate, and the like can be used.

[0082] When formulating the pharmaceutical composition of the present invention into an injection solution, the pharmaceutical composition of the present invention is mixed with water together with a stabilizer or a buffer to produce a solution or a suspension, and this can be formulated into ampoules or vials for unit administration.

[0083] When formulating the pharmaceutical composition of the present invention into an aerosol agent, a propellant and the like can be blended together with additives so that a water-dispersed concentrate or a wet powder is dispersed.

[0084] When formulating the pharmaceutical composition of the present invention into an ointment, an oil, a cream, a powder for application, an external preparation for skin, and the like, animal oils, vegetable oils, waxes, paraffins, polyethylene glycols, silicones, bentonite, silica, talc, starch, tragacanth, cellulose derivatives, zinc oxide, and the like can be used as carriers for formulation.

[0085] Hereinafter, the present invention will be described in detail with the following examples. However, the scope of the present invention is not limited by the following examples.

Examples

[0086] [Example 1: Production of Degreased Egg Yolk Hydrolysate by Secondary Hydrolysis]

[0087] Domestic eggs were purchased, cracked, and the egg yolks were separated. After that, egg yolk powder was obtained by spray-drying the separated egg yolks. Then, 5,000 L of alcohol was added to 1,000 g of the egg yolk powder, and the mixture was extracted at 60 °C for 2 hours. The extract was separated by filtration. Subsequently, the remaining egg yolk meal was dried in an oven at 50 °C to obtain 550 g of defatted egg yolk powder.

[0088] 100 g of defatted egg yolk powder was completely dissolved in 600 ml of water and then heated at 60 °C. After that, 5 g of Novo's Alcalase, an enzyme for protein hydrolysis, was added, and hydrolysis was carried out for 3 hours. After the reaction was completed, insoluble egg yolk meal was removed so that the reaction solution would not be emulsified, and 410 ml of the remaining solution after the completion of the primary enzyme reaction was obtained.

[0089] To this, 4 ml of Novo's Flavourzyme, an exopeptidase for the secondary reaction, was added, and the reaction was carried out at 55 °C for 4 hours. Then, vacuum concentration was carried out at a vacuum degree of 50 Torr to obtain 82 g of a concentrated solution with a solid content of 25% (w / v). The yield was confirmed to be 20.5% based on the solid content.

[0090] [Comparative Example 1: Production of Defatted Egg Yolk Hydrolysate by Primary Hydrolysis]

[0091] 600 ml of water was added to 100 g of the defatted egg yolk of Example 1, and after the impeller was rotated at 3,000 rpm at 60 °C for 30 minutes, 5 g of Alcalase as a hydrolysis enzyme was added, and an enzyme reaction was carried out for 3 hours.

[0092] In contrast, an attempt was made to obtain an enzyme reaction solution by removing insoluble solids as in Example 1, but since the enzyme reaction solution could not be separated due to emulsification, it was impossible to obtain.

[0093] Therefore, 320 ml of the remaining solution was obtained by filtration. Based on the solid content, the yield was confirmed to be 12.3%.

[0094] [Example 2: Analysis of Amino Acid Composition]

[0095] The amino acid composition analysis was performed using the enzyme reaction solutions obtained in Example 1 and Comparative Example 1. The amino acid analysis conditions and results are described below.

[0096] Amino acid analysis conditions:

[0097] Model: HITACH L-8900

[0098] Type: Post-reaction type (Ninhydrin reagent)

[0099] Detector: Photometer 440, 570 nm

[0100] Injection volume: 20 μl

[0101] Amino acid analysis results: Refer to Table 1 below.

[0102] [Table 1]

[0103] As shown in Table 1 and Figure 1 above, it was confirmed that the egg yolk hydrolyzate produced in the process of Example 1 reached about three times the content of total amino acids when compared with the sample of Comparative Example 1 produced under the conditions where emulsification occurred, and it was also confirmed that the yield of the water-soluble component was 1.7 times higher. Figure 1 is a diagram showing the results of the amino acid analysis chromatogram of the egg yolk hydrolyzate.

[0104] [Example 3: Comparison of Protein Degradation Degree Using SDS-PAGE]

[0105] The molecular weights of the proteins decomposed in the proteolytic sample were compared using the electrophoresis method of Example 1, and the results are shown in Fig. 2. Fig. 2 is a diagram showing the results of an SDS-PAGE photograph of the egg yolk hydrolyzate. M2 shows the sample after treatment with alkaline protease, and M3 shows the sample additionally treated with flavorzyme.

[0106] As in Example 1, when two enzymes were treated and hydrolysis was carried out in two steps, it was confirmed that most proteins were decomposed to a molecular weight of 10 kDa or less and were reduced in molecular weight, deviating from the size of the molecular weight marker by electrophoresis.

[0107] [Example 4: Cell test for verifying the efficacy of height growth and bone density increase]

[0108] To confirm whether the composition of the present invention exhibits the effects of height growth and bone density increase, the influence on the activity of ALP (Alkaline phosphatase) was measured. As the sample, the sample of Example 1 was treated.

[0109] MC3T3-E1 osteoblastic cells derived from mouse bones were purchased and used from ATCC (American Type Culture Collection). For the MC3T3-E1 cells, a medium was used in which 10% (v / v) FBS (Fetal Bovine Serum), 100 U / ml penicillin, and 100 U / ml streptomycin were added to α-MEM medium (Gibco-Invitrogen), and the cells were cultured in a humidified CO2 incubator (5% CO2) at 37°C. When the cells reached about 80% confluence in the culture dish, the cell monolayer was washed out with PBS (Phosphate-buffered saline), treated with 0.25% trypsin-2.65 mM EDTA for subculture, and the medium was changed every two days. When the MC3T3-E1 cells reached about 90% confluence in the culture dish, the cell culture medium was replaced with an osteoblast differentiation culture medium in which 10 mM β-glycerophosphate (β-glycerophosphate; Sigma-Aldrich Co.) and 50 μg / ml ascorbic acid (ascorbic acid; Sigma-Aldrich Co.) were added to α-MEM medium to induce differentiation, and the osteoblast differentiation culture medium was changed every three days.

[0110] To measure the activity of ALP (Alkaline phosphatase), MC3T3-E1 cells were dispensed into a 96-well plate at a density of 1*10 4 cells / well and allowed to stabilize for 24 hours. After treating the samples at various concentrations and culturing for 8 days, the collagen production of the induced-differentiation cells was measured using a Sirius red collagen detection kit (Chondrex) by the method presented by the manufacturer.

[0111] The results are shown in Figure 3. Figure 3 is a diagram showing the results of the increased ALP activity effect in the cell test.

[0112] It was confirmed that administration of Y-PEP, which is the composition of Example 1, has the effect of increasing the activity of ALP in a concentration-dependent manner. Such results serve as evidence indicating that the composition of Example 1 is effective in height growth and bone density increase.

[0113] [Example 5: Verification of height growth and bone growth effects by animal experiments]

[0114] Three-week-old specific pathogen-free SD (Sprague Dawley) rats were purchased from DooYeol Biotech Co., Ltd. After a one-week quarantine and adaptation process, healthy animals were selected and used. Throughout the entire test period, the experimental animals were allowed to freely consume solid feed for experimental animals (Cargill Agri Purina Co., Ltd.) and drinking water.

[0115] After a one-week adaptation period, the animals were classified by sex and then randomly divided into four groups for males and females respectively, namely the control group (AG1), the 100 mg / kg administration group (AG2), the 300 mg / kg administration group (AG3), and the recombinant human growth hormone 200 μg / kg administration group (AG4) as the positive control group. Ten experimental animals were used for each test group. The test substance (TM) was prepared by dissolving the product manufactured in Example 1 in drinking water and orally administered at a fixed time every day for 14 days, while the positive control substance was subcutaneously injected once a day.

[0116] For the evaluation of height growth, the NA-NT (from nose to tail) of the experimental animals was measured on the start day and end day of the test respectively to evaluate the growth of the experimental animals.

[0117] For the measurement of the weight, length, and bone density of the tibia, after blood sampling on the test end date, the tibia was excised, the muscles, ligaments, and fat attached to the skeleton were removed, and then the weight of the tibia was measured. After measuring the length of the tibia using a digital caliper, the bone density was measured using DEXA (dual-energy X-ray absorptiometry) (PIXmus TM, GE Lunar). Thereafter, for the right tibia, tomography was performed using a high-resolution micro-computed tomography system (micro-CT, Quantum GX, Perkinelmer, USA), and for the left tibia, histological observation and immunostaining were performed.

[0118]

Table 2

[0119] **P < 0.01, ***P < 0.001. It can be confirmed that there is a growth-promoting effect at a level similar to that of the recombinant human growth hormone administration group (AG4) when the length of the experimental animals is compared with the control group (AG1) in terms of the error probability for the conclusion of a significant difference from the control group.

[0120]

Table 3

[0121] It can be confirmed that there is a tendency for the promotion of tibia length and an increase in bone density compared with the control group. Based on such results, it is judged that it can be utilized as a functional material and pharmaceutical for increasing bone density and preventing osteoporosis in the future.

[0122]

Table 4

[0123] AG1 - 4: Male experimental animals; AG5 - 8: Female experimental animals; AG1, 5: Negative control groups; AG4, 8: Positive control groups (recombinant human growth hormone). It was confirmed that the length of the growth plate was significantly increased in the test substance groups (AG2, 3, 6, 7) compared to the control groups (AG1, 5).

[0124] Figure 4 shows the results of histological staining and analysis of bone length growth. It was confirmed that the defatted egg yolk hydrolysates of the present invention (AG2, AG3) have a significant level of bone length growth effect when compared to the control group (AG1).

Claims

1. A defatted egg yolk hydrolyzate obtained by sequentially treating defatted egg yolk with an endopeptidase enzyme and an exopeptidase enzyme in this order, wherein the sum of the contents of arginine, phenylalanine, tyrosine, and histidine is 7% (w / w) or more based on the total solid content.

2. During the treatment with the endopeptidase enzyme and the exopeptidase enzyme, The defatted egg yolk hydrolyzate according to claim 1, characterized in that insoluble egg yolk meal is removed through filtration.

3. A food composition for promoting height growth containing the defatted egg yolk hydrolyzate according to claim 1 as an active ingredient.

4. A food composition for improving bone density containing the defatted egg yolk hydrolyzate according to claim 1 as an active ingredient.

5. A pharmaceutical composition for preventing or treating osteoporosis containing the defatted egg yolk hydrolyzate according to claim 1 as an active ingredient.

6. The hydrolyzate The pharmaceutical composition according to claim 5, characterized in that it increases the activity of ALP (Alkaline phosphatase).

7. (a) A step of producing defatted egg yolk by adding an organic solvent capable of dissolving lipids to egg yolk powder, followed by extraction and filtration; (b) A step of obtaining defatted egg yolk powder by drying the remaining egg yolk meal after separation in the step (a); (c) A step of producing a defatted egg yolk powder solution by adding water to the defatted egg yolk powder obtained in the step (b), and obtaining a primary hydrolyzate by treating with an endopeptidase; (d) A step of removing insoluble egg yolk meal from the primary hydrolyzate obtained in the step (c); And (e) A step of obtaining a secondary hydrolyzate by treating the primary hydrolyzate from which the egg yolk meal has been removed in the step (d) with an exopeptidase. A method for producing a defatted egg yolk hydrolyzate, comprising: a sum of the contents of arginine, phenylalanine, tyrosine and histidine is 7% (w / w) or more based on the total solid content.

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