KR20220091277A - Hydrolysate of defatted egg yolk protein, preparing the same, and immunity enhancing composition including the same - Google Patents

Abstract

The present invention relates to a hydrolyzate of defatted egg yolk protein having excellent immune enhancing activity, and more particularly, a hydrolyzate of defatted egg yolk protein obtained by performing complex hydrolysis of skim egg yolk, a food-derived ingredient, using an enzyme in two steps. , to a method for preparing the hydrolyzate of defatted egg yolk protein, and to an immune enhancing composition comprising the hydrolyzate of defatted egg yolk protein. The hydrolyzate of defatted egg yolk protein of the present invention can improve the amount of NO production by increasing iNOS expression in macrophages, and can improve the expression of pro-inflammatory cytokines at the mRNA and protein level, thereby enhancing immunity in food and pharmaceutical fields. It can be usefully used as a functional material for

Description

Defatted egg yolk protein hydrolyzate, method for preparing same, and immune enhancing composition comprising same {Hydrolysate of defatted egg yolk protein, preparing the same, and immunity enhancing composition including the same}

The present invention relates to a hydrolyzate of defatted egg yolk protein having excellent immune enhancing activity, a method for producing the hydrolyzate of defatted egg yolk protein, and an immune enhancing composition comprising the hydrolyzate of defatted egg yolk protein.

Eggs, which are well known as a natural complete food along with milk, have various nutrients such as proteins, carbohydrates, lipids, and minerals, and have been consumed as a cheap protein source since ancient times. In particular, various proteins exist in the egg. For example, as proteins present in egg white, ovalbumin, ovotransferrin, ovomucin, lysozyme, and the like are known representatively. Although egg yolk has a higher proportion of lipids than proteins, various proteins such as IgY (Immunoglobulin Y), phosvitin, and lipovitelin also exist.

Since these proteins and their hydrolysates have high utility value as functional food materials and pharmaceutical materials, various studies on functionality, for example, research on antioxidant, antibacterial, antihypertensive, anticancer, anti-inflammatory, immune enhancing activity, etc. has been actively reported.

In relation to the above-described immune enhancing activity, immune enhancing substances that activate the human immune system have recently attracted attention in many studies. The immune response is a defense system that protects the human body from the external environment, and when foreign substances enter the body, it goes through a series of processes to recognize and defend them.

Therefore, it is very important for such an immune response to occur, and research on functional substances capable of appropriately regulating the generated immune response is being actively conducted.

One of the cells in the body that is representatively known in relation to such an immune response is a macrophage. Macrophages are activated when an immune response is in progress, and pro-inflammatory cytokines, for example, IL-Iβ, IL-6, IL-10, IL-10, interferon-γ, tumor necrosis factor-α (tumor necrosis factor-α) ), etc., removing foreign substances through phagocytosis, and inducing activation of other immune cells. Therefore, research on natural substances that help the production of the above immune substances by appropriately activating these macrophages is being conducted.

On the other hand, it has been reported that the protein hydrolyzate has improved functionality by decomposing the protein using an enzyme and producing small molecular weight peptides. It is known that the functionality of these hydrolysates is affected by factors such as the type of peptides constituting the hydrolysates, the size of the peptides, and the amino acid sequence of the peptides. Therefore, many studies are making efforts to manufacture various peptides using various enzymes, and as part of that, hydrolysates and peptides with better functionality by using two or more complex enzymes instead of using only one single enzyme for hydrolysis. Research to manufacture is in progress.

Therefore, in the present invention, the immune enhancing activity of a new hydrolyzate obtained by using a two-step complex enzymatic hydrolysis of the defatted egg yolk protein obtained after removing the lipid component present in the egg yolk through a defatting process was to be confirmed.

It is an object of the present invention to provide a method for preparing a hydrolyzate of defatted egg yolk protein using a proteolytic enzyme.

Another object of the present invention is to provide a hydrolyzate of defatted egg yolk protein having excellent immune enhancing activity, prepared by using the method for preparing hydrolyzate of defatted egg yolk protein.

Another object of the present invention is to provide a composition for preventing, improving, or treating inflammatory diseases, comprising the hydrolyzate of defatted egg yolk protein having excellent immune enhancing activity as an active ingredient.

In order to achieve the above purpose,

The present invention relates to a protein hydrolyzate of defatted egg yolk, which comprises obtaining a hydrolyzate of defatted egg yolk by treating and reacting one or more proteolytic enzymes selected from the group consisting of neutrase, pancreatin, and combinations thereof. A method for preparing a decomposition product is provided.

The present invention comprises the steps of preparing defatted egg yolk; treating the prepared defatted egg yolk with neutrase or pancreatin as a proteolytic enzyme to perform a primary reaction; terminating the primary reaction by inactivating the protease; and centrifuging the reactant after the first reaction to obtain defatted egg yolk protein.

Specifically, the first reaction may be carried out in a temperature range of 25°C to 60°C for 1 hour to 8 hours.

In the present invention, after the first reaction by treating the prepared defatted egg yolk with neutrase or pancreatin as a proteolytic enzyme, a second reaction step is added by treating nutrase or pancreatin as a proteolytic enzyme once more may include

In the present invention, the protease used in the second reaction may be different from the protease used in the first reaction. Specifically, in the first reaction, pancreatin may be used as a proteolytic enzyme, and in the second reaction, neutrase may be used as a proteolytic enzyme.

Specifically, the secondary reaction may be performed at a temperature range of 25°C to 60°C for 1 hour to 8 hours.

In addition, the present invention provides a hydrolyzate of defatted egg yolk protein, which is produced by the method for producing a hydrolyzate of defatted egg yolk protein.

Specifically, the hydrolyzate of defatted egg yolk protein may increase iNOS expression in macrophages. Preferably, the hydrolyzate of defatted egg yolk protein may exhibit immune enhancing activity by increasing iNOS expression in macrophages to improve NO production.

Specifically, the hydrolyzate of defatted egg yolk protein may exhibit immune enhancing activity by increasing the expression of pro-inflammatory cytokines.

The present invention also provides a composition for preventing, improving, or treating inflammatory diseases, comprising the hydrolyzate of defatted egg yolk protein prepared by the method for producing hydrolyzate of defatted egg yolk protein as an active ingredient.

Specifically, the composition for preventing, improving, or treating the inflammatory disease may be a pharmaceutical composition, a cosmetic composition, or a health functional food.

Specifically, the inflammatory disease is, enteritis, gastritis, hepatitis, bronchitis, pleurisy, arthritis, rhinitis, keratitis, nephritis, peritonitis, osteomyelitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, burn inflammation, dermatitis, periodontitis, gingivitis, It may include one selected from the group consisting of degenerative neuritis, inflammatory pain, inflammatory bowel disease, and combinations thereof.

According to the present invention, it is possible to prepare a hydrolyzate of defatted egg yolk protein exhibiting immune enhancing activity by performing a two-step complex hydrolysis of skim egg yolk, a food-derived ingredient, using an enzyme.

The hydrolyzate of defatted egg yolk protein according to the present invention can increase the amount of NO production by amplifying iNOS expression in macrophages, and can improve the expression of pro-inflammatory cytokines at the mRNA and protein level, so that it can be used in food and pharmaceutical fields. It can be usefully used as a functional material for enhancing immunity.

1 is a graph showing the cell viability of RAW 264.7 macrophages according to the hydrolyzate treatment of defatted egg yolk protein according to an embodiment of the present invention.
2 is a graph showing the effect on NO production in RAW 264.7 macrophages according to the hydrolyzate treatment of defatted egg yolk protein according to an embodiment of the present invention.
3 is a graph showing the effect on iNOS expression in RAW 264.7 macrophages according to the hydrolyzate treatment of defatted egg yolk protein according to an embodiment of the present invention.
4 (a) and (b) are graphs showing the effect on TNF-α and IL-6 mRNA expression in RAW 264.7 macrophages according to the hydrolyzate treatment of defatted egg yolk protein according to an embodiment of the present invention.
5 (a) and (b) are graphs showing the effect on TNF-α and IL-6 production of RAW 264.7 macrophages according to the hydrolyzate treatment of defatted egg yolk protein according to an embodiment of the present invention.
6 is a graph showing the effect on the phagocytic activity of RAW 264.7 macrophages in accordance with the hydrolyzate treatment of defatted egg yolk protein according to an embodiment of the present invention.
7 is a graph showing the amount of NO production in RAW 264.7 macrophages induced by a hydrolyzate of defatted egg yolk protein after treatment with a MAPK pathway inhibitor according to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail with reference to embodiments and drawings of the present invention.

It will be apparent to those of ordinary skill in the art that these examples are merely presented by way of example to explain the present invention in more detail, and that the scope of the present invention is not limited by these examples. .

In addition, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and in case of conflict, this specification, including definitions will take precedence.

In order to clearly explain the invention proposed in the drawings, parts irrelevant to the description are omitted, and similar reference numerals are assigned to similar parts throughout the specification. And, when a part "includes" a certain component, this means that other components may be further included, rather than excluding other components, unless otherwise stated. In addition, "unit" described in the specification means one unit or block that performs a specific function.

In each step, identification numbers (first, second, etc.) are used for convenience of description, and identification numbers do not describe the order of each step, and each step does not clearly describe a specific order in context. It may be performed differently from the order specified above. That is, each step may be performed in the same order as the specified order, may be performed substantially simultaneously, or may be performed in the reverse order.

Hereinafter, embodiments and embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, these embodiments and examples and drawings of the present invention are not limited thereto.

One embodiment of the present invention includes obtaining a hydrolyzate of defatted egg yolk protein by treating and reacting defatted egg yolk with one or more proteolytic enzymes selected from the group consisting of nutrase, pancreatin, and combinations thereof, It relates to a method for producing a hydrolyzate of defatted egg yolk protein.

In one embodiment, the manufacturing method comprises the steps of preparing defatted egg yolk; treating the prepared defatted egg yolk with neutrase or pancreatin as a proteolytic enzyme to perform a primary reaction; terminating the primary reaction by inactivating the protease; and centrifuging the reactant after the first reaction to obtain defatted egg yolk protein.

In one embodiment, the defatted egg yolk may be obtained by separating the egg yolk from the egg, drying and pulverizing it, and then heating and extracting the dried egg yolk in a solvent. For example, the defatted egg yolk may be prepared by separating the yolk from the egg; obtaining dried egg yolk powder by pulverizing the separated egg yolk after vacuum drying; heating and extracting the dried egg yolk powder by adding a solvent corresponding to about 5 times or more by weight of the dry egg yolk powder; and filtering and drying the extract to obtain defatted egg yolk. In the heat extraction step, the solvent may include ethanol, but is not limited thereto.

In one embodiment, the first reaction may be performed for about 1 hour to about 8 hours. If the first reaction is performed for less than about 1 hour, the protein may not be sufficiently hydrolyzed by the protease, and thus the amount of hydrolyzate obtained may be reduced, and if it exceeds about 8 hours, hydrolysis Since the decomposition reaction is excessively performed and unnecessary products may be generated together, the first reaction is preferably performed for about 1 hour to about 8 hours. For example, the first reaction may be from about 1 hour to about 8 hours, from about 1 hour to about 6 hours, from about 1 hour to about 4 hours, from about 1 hour to about 2 hours, from about 2 hours to about 8 hours, about 2 hours to about 6 hours, about 2 hours to about 4 hours, about 4 hours to about 8 hours, about 4 hours to about 6 hours, or about 6 hours to about 8 hours.

In one embodiment, the first reaction may be carried out in a temperature range of about 25 ℃ to about 60 ℃. If the secondary reaction is carried out at less than about 25 °C, the hydrolysis reaction may not be sufficiently performed, and when it exceeds about 60 °C, denaturation of the protease may occur. More specifically, the first reaction may be carried out in a temperature range of about 37 °C to about 60 °C.

In one embodiment, the first reaction may be carried out in the range of about pH 6.5 to about pH 7.5. If the primary reaction is carried out in the range below or exceeding the above-described pH, hydrolysis may not be sufficiently performed, and thus the amount of hydrolyzate finally obtained may decrease.

In one embodiment, after the first reaction by treating the prepared defatted egg yolk with neutrase or pancreatin as a proteolytic enzyme, the second reaction by treating nutrase or pancreatin as a proteolytic enzyme once more may be additionally included. For example, the manufacturing method includes the steps of preparing defatted egg yolk; treating the prepared defatted egg yolk with neutrase or pancreatin as a proteolytic enzyme to perform a primary reaction; Secondary reaction by treating neutrase or pancreatin as a proteolytic enzyme once more; terminating the primary reaction by inactivating the protease; and centrifuging the reactant after the first reaction to obtain defatted egg yolk protein.

In one embodiment, the protease used in the second reaction may be different from the protease used in the first reaction. For example, after the primary reaction is performed using pancreatin as the proteolytic enzyme, the secondary reaction may be performed using neutrase. Alternatively, after the primary reaction is performed using neutrase as the proteolytic enzyme, the secondary reaction may be performed using pancreatin.

In the present invention, after performing a primary reaction of skimmed egg yolk using the protease, a secondary reaction is performed using a protease different from the protease used in the primary reaction, resulting in a two-step complex By hydrolysis, it is possible to prepare a protein hydrolyzate exhibiting excellent immune enhancing activity. For example, after performing the primary reaction using pancreatin as the proteolytic enzyme, the secondary reaction is performed using a neutrase different from the protease used in the primary reaction, so that only the primary reaction It is possible to exhibit more improved immune enhancing activity than the hydrolyzate obtained by performing the hydrolyzate and/or the hydrolyzate obtained by using neutrase in the first reaction and pancreatin in the second reaction.

In one embodiment, the secondary reaction may be performed at the same time, the same temperature range, and the same pH range as the first reaction.

In one embodiment, the inactivation may be performed by heating the protease. For example, the protease used in the first and/or second reaction may be inactivated by heating it to a protein denaturation temperature or higher to terminate the reaction. The heating temperature is not limited as long as it is a temperature at which the protein can be completely denatured, and specifically, it may be about 70° C. or more, more specifically, about 70° C. to about 120° C., but is not limited thereto.

In another aspect, the present invention relates to a protein hydrolyzate of defatted egg yolk prepared by the method for producing a hydrolyzate of protein.

In one embodiment, the defatted egg yolk protein hydrolyzate comprises the steps of: preparing defatted egg yolk; treating the prepared defatted egg yolk with neutrase or pancreatin as a proteolytic enzyme to perform a primary reaction; terminating the primary reaction by inactivating the protease; and centrifuging the reactant after the first reaction to obtain defatted egg yolk protein.

In one embodiment, the defatted egg yolk protein hydrolyzate comprises the steps of: preparing defatted egg yolk; treating the prepared defatted egg yolk with neutrase or pancreatin as a proteolytic enzyme to perform a primary reaction; Secondary reaction by treating neutrase or pancreatin as a proteolytic enzyme once more; terminating the primary reaction by inactivating the protease; and centrifuging the reactant after the first reaction to obtain defatted egg yolk protein. For example, the defatted egg yolk protein hydrolyzate may be read by a two-step complex hydrolysis by performing a second reaction using a protease different from the protease used in the first reaction. have.

In one embodiment, the defatted egg yolk protein hydrolyzate may exhibit immune enhancing activity.

For example, the defatted egg yolk protein hydrolyzate may exhibit immune enhancing activity by increasing iNOS expression in macrophages, and specifically, by increasing iNOS expression in macrophages to improve the amount of NO production, thereby exhibiting immune enhancing activity. can

For example, the hydrolyzate of defatted egg yolk protein may exhibit immune enhancing activity by increasing the expression of pro-inflammatory cytokines.

In one embodiment, the defatted egg yolk protein hydrolyzate increases the iNOS expression of macrophages to improve NO production, and by increasing the expression of pro-inflammatory cytokines, it exhibits immune enhancing activity to prevent, improve, or It may have a therapeutic effect. For example, by increasing the iNOS expression of the macrophages to improve the production of NO, and by including the hydrolyzate of the defatted egg yolk protein, which exhibits immune enhancing activity by increasing the expression of proinflammatory cytokines, as an active ingredient, inflammatory diseases prevent, ameliorate, or treat. Specifically, the inflammatory disease is, enteritis, gastritis, hepatitis, bronchitis, pleurisy, arthritis, rhinitis, keratitis, nephritis, peritonitis, osteomyelitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, burn inflammation, dermatitis, periodontitis, gingivitis, It may include, but is not limited to, those selected from the group consisting of degenerative neuritis, inflammatory pain, inflammatory bowel disease, and combinations thereof.

In another aspect, the present invention relates to a composition for preventing, improving, or treating inflammatory diseases, comprising, as an active ingredient, a hydrolyzate of defatted egg yolk produced by the method for producing a hydrolyzate of protein.

In one embodiment, the composition for preventing, improving, or treating the inflammatory disease may be a pharmaceutical composition, a cosmetic composition, or a health functional food. For example, the composition for preventing, ameliorating, or treating inflammatory diseases is defatted obtained by treating and reacting defatted egg yolk with one or more proteases selected from the group consisting of neutrase, pancreatin, and combinations thereof. It may be a pharmaceutical composition, a cosmetic composition, or a health functional food comprising a hydrolyzate of egg yolk protein as an active ingredient.

In one embodiment, the composition for preventing, improving, or treating the inflammatory disease may exhibit immune enhancing activity. For example, the composition may exhibit immune enhancing activity by increasing the iNOS expression of macrophages to improve the amount of NO produced, or by increasing the expression of pro-inflammatory cytokines.

In one embodiment, the composition for preventing, improving, or treating an inflammatory disease may include the defatted egg yolk protein hydrolyzate in an amount of about 0.00001 to about 50% by weight of the total weight of the composition.

In one embodiment, the inflammatory disease is enteritis, gastritis, hepatitis, bronchitis, pleurisy, arthritis, rhinitis, keratitis, nephritis, peritonitis, osteomyelitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, burn inflammation, dermatitis, periodontitis , gingivitis, degenerative neuritis, inflammatory pain, inflammatory bowel disease, and combinations thereof may include, but is not limited to.

In one embodiment, the pharmaceutical composition comprising the hydrolyzate of defatted egg yolk protein may prevent or treat the inflammatory disease.

In one embodiment, the pharmaceutical composition comprising the defatted egg yolk protein hydrolyzate is a powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. oral dosage forms, external preparations, and suppositories, respectively, according to a conventional method. Alternatively, it may be formulated and used in the form of a sterile injectable solution.

In one embodiment, when formulating a pharmaceutical composition comprising the hydrolyzate of defatted egg yolk protein, it may be prepared using a diluent or excipient such as a generally used filler, extender, binder, wetting agent, disintegrant, or surfactant. can, but is not limited thereto.

In one embodiment, the solid preparation for oral administration includes tablets, pills, powders, granules, or capsules, and the solid preparation includes at least one or more excipients, for example, starch, calcium carbonate , sucrose, lactose, or gelatin may be mixed and prepared. In addition, for example, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used, but the present invention is not limited thereto.

In one embodiment, liquid formulations for oral administration include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, Perfumes, preservatives, etc. may be included, but are not limited thereto.

In one embodiment, preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. For example, the non-aqueous solvent or suspending agent may include, but is not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. For example, as the suppository, witepsol, macrogol, tween 61, cacao butter, laurin oil, glycerogelatin, etc. may be used, but is not limited thereto.

In one embodiment, the health functional food containing the hydrolyzate of the defatted egg yolk protein may prevent or improve the inflammatory disease.

In one embodiment, the type of health functional food containing the hydrolyzate of the defatted egg yolk protein is not particularly limited. For example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, or dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes, etc. However, it is not limited thereto.

In one embodiment, the health functional food containing the defatted egg yolk protein hydrolyzate may be used in the form of pills, powders, granules, needles, tablets, capsules, or beverages, and includes all health functional foods in a conventional sense. may be doing

In one embodiment, the health functional food containing the defatted egg yolk protein hydrolyzate is various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, etc., but are not limited thereto. does not

In one embodiment, the health functional food containing the defatted egg yolk protein hydrolyzate may contain natural fruit juice, and pulp for the production of fruit juice drinks and vegetable drinks, but is not limited thereto. For example, the components may be used independently or in combination.

In one embodiment, the formulation of the cosmetic composition containing the defatted egg yolk protein hydrolyzate is not particularly limited, for example, among skins, lotions, essences, creams, packs, foundations, and makeup bases. It may have any one selected formulation, but is not limited thereto.

In one embodiment, the cosmetic composition including the defatted egg yolk protein hydrolyzate may contain a material arbitrarily selected according to the cosmetic formulation or purpose of use. For example, purified water, oil, surfactant, humectant, higher alcohol, thickener, chelating agent, color, fatty acid, antioxidant, preservative, wax, pH adjuster, fragrance, etc. may be added, but is not limited thereto.

In one embodiment, when the formulation of the cosmetic composition containing the defatted egg yolk protein hydrolyzate is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol as a carrier component , silicon, bentonite, silica, talc, or zinc oxide, but is not limited thereto.

In one embodiment, when the formulation of the cosmetic composition containing the defatted egg yolk protein hydrolyzate is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be included as a carrier component. and, in particular, in the case of a spray, may additionally include a propellant such as chlorofluorohydrocarbon, propane/butane or dimethyl ether, but is not limited thereto.

In one embodiment, when the formulation of the cosmetic composition including the defatted egg yolk protein hydrolyzate is a solution or emulsion, a solvent, solubilizer or emulsifier may be included as a carrier component, for example, water, ethanol , isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan.

In one embodiment, when the formulation of the cosmetic composition including the defatted egg yolk protein hydrolyzate is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and suspending agents such as polyoxyethylene sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth, and the like.

In one embodiment, when the formulation of the cosmetic composition including the defatted egg yolk protein hydrolyzate is surfactant-containing cleansing, fatty alcohol sulfate, fatty alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, Dazolinium derivatives, methyltaurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester, etc. can, but is not limited thereto.

Hereinafter, the configuration of the present invention and its effects will be described in more detail through specific examples and comparative examples. However, these examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these examples.

Reagents and experimental materials used in the examples

The defatted egg yolk was ground by separating the yolk from the egg white and vacuum drying it at 50° C. or higher using a vacuum dryer. After adding 5 times or more alcohol (95% ethanol) to the ground dried egg yolk, extraction was performed at 50° C. or higher for 1 hour or more, followed by filtration with a 1 μm filter. The filtered solid content was dried in a drying oven at 70° C. for 3 hours and then used for the experiment.

The proteolytic enzymes used to prepare the defatted egg yolk protein hydrolyzate were Neutrase and Pancreatin, respectively, purchased from Daejong Trading Co., Ltd. and Vision Biochem Co., Ltd. and used.

DMEM medium, Fetal bovine serum (FBS) required for cell line culture, is supplied by HyClon Laboratories, Inc. (Logan, MI, USA), penicillin-streptomycin, etc. were purchased from Gibco-BRL (Grand Island, NT, USA). An ELISA kit for TNF-α and IL-6 analysis was purchased from AB frontier (Seoul, Korea). MTT (3-[4,5-dimethythiazol-2-yl]-2,5-diphenyl tetrazolium bromide) reagent and N-(1-naphthyl)-ethylenediamine [N-(1-naphthyl) -ethylenediamine], sulfanilamide, lipopolysaccharide (LPS), sodium nitrate, and Natural Red are sold by Sigma Chemical Co., Ltd. (St. Louis, MO, USA) was purchased. Dimethyl sulfoxide (DMSO), ethyl alcohol, and acetic acid were purchased from Samchun (Seoul, Korea). All other solvents and reagents used in the study were of first-class or higher grade.

[Example 1]

Preparation of defatted egg yolk protein hydrolyzate

The defatted egg yolk protein hydrolyzate is a one-step protein hydrolyzate using only one of neutrase and pancreatin, and a two-step hydrolyzate by changing the processing sequence of the protease. 2 types (Neu-Pan, respectively; after primary hydrolysis with neutrase, secondary hydrolysis with pancreatinase, Pan-Neu; after primary hydrolysis with pancreatinase, neutra Secondary hydrolysis with an enzyme degrading enzyme) was prepared.

에서 10 분간 상기 분해효소의 불활성화 단계를 거쳤으며, 3,000 × g에서 20 분간 원심분리를 한 후, 상등액을 동결건조하여 탈지난황 단백질 가수분해물을 수득하고, 이를 하기 실시예 실험에 사용하였다.First, dissolving skim egg yolk in distilled water, adjusting the pH to 7 using 1 M NaOH, and then treating with a proteolytic enzyme at a ratio of 2% of the protein weight of skim egg yolk (for example, 2 g of skim yolk with 40 mg of enzyme) The first hydrolysis was performed by reacting in a 50°C water-bath for 4 hours. In order to proceed with the secondary hydrolysis, a secondary reaction was performed for 4 hours by treating each of the enzymes different from those used in the primary hydrolysis at a ratio of 2%. After the reaction is complete, 100

After inactivation of the enzyme for 10 minutes, centrifugation was performed at 3,000 × g for 20 minutes, and the supernatant was freeze-dried to obtain a hydrolyzate of defatted egg yolk protein, which was used in the following experiment.

In the following examples, all experimental results are expressed as mean and standard deviation. All experiments were repeated 3 times, and 3 repetitions were performed for each experiment.

In addition, in FIGS. 1 to 7 and Tables 1 to 9, 'Neutrase' denotes a protein hydrolyzate obtained by treating neutrase, 'Pancreatin' denotes a protein hydrolyzate obtained by treating pancreatin, ' N+P' is a protein hydrolyzate obtained by primary treatment with neutrase and then secondary treatment with panthreatin, and 'P+N' is a protein obtained by secondary treatment with neutrase after primary treatment with pancreatin means hydrolyzate.

[Example 2]

Determination of the effect of defatted egg yolk protein hydrolyzate on cell viability on macrophages

In order to measure the immune enhancing effect of the obtained defatted egg yolk protein hydrolysates, the RAW 264.7 cell line, a mouse-derived macrophage, was purchased from the Korea Cell Line Bank (Seoul, Korea) and used. For cell culture, 10% FBS (Fetal bovine serum), 100 unit/mL penicillin, and 100 μg/mL streptomycin were added to DMEM medium for cell culture, and 95% humidity was maintained at 37°C, 5% CO2. It was cultured in ₂ incubators (MCO-18AIC, SANYO, Osaka, Japan).

MTT assay was performed to measure the effect of each hydrolyzate of defatted egg yolk protein on macrophage viability.

First, the RAW 264.7 cell line was aliquoted into a 96-well plate at 2 × 10 ⁵ cells/well, respectively, and 4 types of hydrolysates of the defatted egg yolk protein were treated with each concentration (25, 50, 100, 200 μg/mL). Incubate in a 37 °C, 5% CO ₂ incubator for 24 h. Add 50 μL of MTT (2.5 mg/mL) solution dissolved in PBS buffer to each well and incubate for 4 hours to induce formazan formation. After removal, DMSO was added to dissolve, and absorbance was measured at 570 nm using a microplate reader (Model 680, BioRad, Hercules, CA, USA). Cell viability (%) was calculated using the following formula.

) Х 100Cell viability (%) = (

) Х 100

Defatted egg yolk protein hydrolyzate Cell viability (%) 25 μg/mL 50 μg/mL 100 μg/mL 200 μg/mL Neutrase 98.21 ± 0.64 97.16 ± 0.49 95.73 ± 0.76 95.97 ± 1.15 Pancreatin 99.28 ± 0.57 97.91 ± 0.75 98.17 ± 0.56 100.12 ± 0.47 N+P 96.93 ± 1.84 96.55 ± 1.14 96.95 ± 1.42 98.00 ± 1.19 P+N 96.53 ± 1.25 98.08 ± 0.73 96.03 ± 1.81 94.86 ± 1.94

1 is a graph showing the cell viability of RAW 264.7 macrophages as each concentration of each hydrolyzate of defatted egg yolk protein prepared in the above example is treated, and Table 1 is a table showing the cell viability. As can be seen from the experimental results shown in FIG. 1 and Table 1, it was confirmed that all the hydrolyzates of defatted egg yolk protein used in the experiment had no effect on the survival of macrophages (FIG. 1).

[Example 3]

Determination of Effect on NO and iNOS Expression on Macrophages of Degred Egg Yolk Protein Hydrolyzate

To confirm the effect of the obtained hydrolyzate of defatted egg yolk protein on NO (nitric oxide) production in macrophages, a colorimetric assay was performed using a grease reagent.

First, the RAW 264.7 cell line was seeded in a 96-well plate at 2 × 10 ⁵ cells/well and cultured for 2 hours. The obtained 4 kinds of hydrolyzate of defatted egg yolk protein was treated with each concentration (25, 50, 100, 200 μg/mL) and then cultured in an incubator at 37 °C, 5% CO ₂ for 24 hours. As a positive control, 10 ng/mL of LPS was used, and the generated NO was 1% of grease reagent [1% sulfanilamide in 5% phosphoric acid and 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride in distilled water. 1 mixture]. After mixing 100 μL of supernatant and 100 μL of grease reagent, reacting for 15 minutes, absorbance was measured at 540 nm using a microplate reader. A standard curve was prepared using sodium nitrate as a standard material.

In addition, qRT-PCR (quantitative real-time PCR) analysis was performed to confirm the effect on mRNA expression of iNOS (inducible nitric oxide synthase), which is known to be involved in NO production in macrophages.

First, the RAW 264.7 cell line was seeded in a 6-well plate at 1 × 10 ⁶ cells/well and then cultured for 24 hours. The obtained defatted egg yolk protein hydrolysates were treated in cell lines at different concentrations (25, 50, 100, 200 μg/mL), and then further cultured for 24 hours. Total RNA for PCR was isolated using an RNA isolation kit (RNeasy Mini Kit, Qiagen, Milan, Italy) and synthesized as cDNA (Revert Aid First Strand cDNA synthesis kit, Thermo Fisher Scientific, Carlsbad, CA, USA) . qRT-PCR was performed using the synthesized cDNA. SYBR green kit (PhileKorea, Daejeon, Korea) was used, and the primer sequences of iNOS and β-actin used in the experiment are as follows.

iNOS: forward 5’-CCC TTC CGA AGT TTC TGG CAG CAG C-3’, reverse 5’-GGC TGT CAG AGC CTC GTG GCT TTG G-3’;

β-actin: forward 5’-GTG GGC CGC CCT AGG CAC CAG-3’, reverse 5’GGA GGA AGA GGA TGC GGC AGT-3’.

Amplification results were confirmed using the delta-delta Ct method, and β-actin was used as a housekeeping gene.

Defatted egg yolk protein hydrolyzate NO (μM) 25 μg/mL 50 μg/mL 100 μg/mL 200 μg/mL Control 1.88 ± 0.27 LPS 9.24 ± 0.94 Neutrase 1.92 ± 0.65 1.96 ± 0.84 1.80 ± 0.76 1.88 ± 0.65 Pancreatin 1.97 ± 0.62 1.62 ± 0.43 1.35 ± 0.31 1.27 ± 0.40 N+P 1.35 ± 0.30 1.80 ± 0.30 1.75 ± 0.21 1.40 ± 0.27 P+N 5.54 ± 0.83 6.64 ± 0.68 7.56 ± 0.82 9.06 ± 0.92

Figure 2 is a graph showing the NO production amount of RAW 264.7 macrophages as each concentration of each hydrolyzate of defatted egg yolk protein prepared in the above example was treated, and Table 2 is a table showing the amount of NO production. As a result of the experiment, as shown in FIG. 2 and Table 2, it was confirmed that the Pan-Neu hydrolyzate had the effect of accelerating NO production in macrophages among a total of 4 hydrolysates of defatted egg yolk protein prepared in the above Example. (Fig. 2). NO production was promoted at all concentrations used in the experiment, and it was confirmed that the amount of NO production increased in a concentration-dependent manner. NO is a substance produced by macrophages and is known to play various roles such as wound healing, vasodilation, and modulators in innate immunity. In addition, NO is known to be involved in the death of bacteria, infected cells, cancer cells, etc. invading the body. Therefore, it was confirmed that the Pan-Neu hydrolyzate according to the present invention can help improve immunity by promoting NO production in macrophages. In future experiments, only the Pan-Neu hydrolyzate, which showed an effect on NO production, was used.

In addition, qRT-PCR was performed to confirm the effect of the Pan-Neu hydrolyzate prepared in Example above on the mRNA expression of iNOS enzyme, which is known to be involved in NO production in macrophages.

Defatted egg yolk protein hydrolyzate iNOS mRNA expression level (change compared to LPS) 25 μg/mL 50 μg/mL 100 μg/mL 200 μg/mL Control 0.35 ± 0.07 LPS 100.20 ± 6.40 P+N 51.21 ± 6.21 63.09 ± 6.75 66.99 ± 7.39 71.21 ± 7.44

Figure 3 is a graph showing the iNOS expression level of RAW 264.7 macrophages as the hydrolyzate of defatted egg yolk protein (Pan-Neu hydrolyzate) prepared in the above example was treated for each concentration, Table 3 shows the iNOS expression level This is a table (LPS: lipopolysaccharide). As a result of the experiment, as shown in FIGS. 3 and 3, it was confirmed that the expression level of iNOS mRNA in macrophages increased in a concentration-dependent manner as the Pan-Neu hydrolyzate was treated as in NO generation ( FIG. 3 ). NO is known to be produced by NO synthase (nitric oxide synthase, NOS) present in the cell, and among them, it is known to be closely related to iNOS related to the immune response. In conclusion, it was confirmed that the Pan-Neu hydrolyzate according to the present invention promotes NO production by increasing iNOS mRNA expression in macrophages.

[Example 4]

Determination of the effect of defatted egg yolk protein hydrolyzate on TNF-α and IL-6 expression on macrophages

qRT-PCR (mRNA) and qRT-PCR (mRNA) to confirm the effect of the hydrolyzate of defatted egg yolk protein obtained in the above example (Pan-Neu hydrolyzate) on TNF-α and IL-6 production of macrophages at the mRNA and protein level ELISA (protein) analysis was performed.

qRT-PCR was performed in the same manner as described above, and the primer sequences of TNF-α and IL-6 used in the experiment are as follows.

TNF-α: forward 5’-TTG ACC TCA GCG CTG AGT TG-3’, reverse 5’-CCT GTA GCC CAC GTC GTA GC-3’.

IL-6: forward 5’-GTA CTC CAG AAG ACC AGA GG-3’, reverse 5’-TGC TGG TGA CAA CCA CGG CC-3’.

The ELISA assay method for measuring the effect on expression at the protein level is as follows.

First, the RAW 264.7 cell line was seeded in a 12-well plate at 4 × 10 ⁵ cells/well, respectively, and cultured for 24 hours. After treatment with degnan protein hydrolyzate (Pan-Neu hydrolyzate) by concentration (25, 50, 100, 200 μg/mL), it was further cultured for 24 hours. Finally, in order to quantify TNF-α and IL-6 present in the supernatant, the ELISA kit was used according to the manufacturer's method.

Defatted egg yolk protein hydrolyzate TNF-α mRNA expression level (change compared to LPS) 25 μg/mL 50 μg/mL 100 μg/mL 200 μg/mL Control 6.14 ± 0.39 LPS 100.16 ± 5.79 P+N 57.99 ± 6.92 67.31 ± 5.00 70.71 ± 5.80 70.58 ± 5.40

Defatted egg yolk protein hydrolyzate IL-6 mRNA expression level (change compared to LPS) 25 μg/mL 50 μg/mL 100 μg/mL 200 μg/mL Control 0.00 ± 0.00 LPS 100.06 ± 3.65 P+N 31.91 ± 9.51 42.53 ± 5.49 44.17 ± 10.57 60.77 ± 8.03

Defatted egg yolk protein hydrolyzate TNF-α production (ng/mL) 25 μg/mL 50 μg/mL 100 μg/mL 200 μg/mL Control 0.07 ± 0.01 LPS 38.21 ± 5.23 P+N 13.92 ± 1.90 20.68 ± 5.30 38.83 ± 5.85 47.11 ± 3.45

Defatted egg yolk protein hydrolyzate IL-6 production (ng/mL) 25 μg/mL 50 μg/mL 100 μg/mL 200 μg/mL Control 0.00 ± 0.00 LPS 5.87 ± 1.31 P+N 0.68 ± 0.16 2.93 ± 0.46 8.03 ± 1.75 18.59 ± 3.85

4 (a) and (b) are TNF-α and IL-6 mRNA of RAW 264.7 macrophages that are shown by treatment with the hydrolyzate of defatted egg yolk protein (Pan-Neu hydrolyzate) prepared in the above Example by concentration. It is a graph showing the expression level, and Tables 4 and 5 are tables showing the expression levels of TNF-α and IL-6 mRNA, respectively.

와 IL-6 등의 사이토카인의 분비를 유도하는 것으로 알려져 있으며, IL-6의 경우, 면역글로불린의 생산에 영향을 주는 등 면역 반응에 있어 중요한 역할을 하고 있다. 따라서, 본 발명에 따른 탈지난황 단백질 가수분해물 (Pan-Neu 가수분해물)의 처리가 면역 증진에 도움을 줄 수 있음을 확인하였다.In addition, (a) and (b) of FIG. 5 shows that TNF-α and IL- of RAW 264.7 macrophages appearing according to the concentration of the hydrolyzate of the defatted egg yolk protein (Pan-Neu hydrolyzate) prepared in the above example. 6 is a graph showing the production amount (ng / mL), Table 6 and Table 7 is a table showing the TNF-α and IL-6 production amount (ng / mL), respectively. As a result of the qRT-PCR experiment, as shown in FIGS. 4, 5, and 4 to 7, when the Pan-Neu hydrolyzate was treated in macrophages, similarly to the generation of NO, TNF-α, IL- It was confirmed that the mRNA expression level of 6 was increased [Figs. 4 (a) and (b)]. At the same time, as a result of confirming the production amount of TNF-α and IL-6 at the protein level through ELISA, it was confirmed that the concentration-dependent increase of the Pan-Neu hydrolyzate was increased in the same way as the mRNA expression [Fig. 5(a) and (b)]. TNF-α and IL-6 are known as pro-inflammatory cytokines that are initially produced when macrophages are activated. These pro-inflammatory cytokines perform various roles in immune responses, such as movement of cells into tissues to initiate an inflammatory response, promotion of secretion of other inflammatory cytokines, or differentiation of T cells and B cells. For TNF-α, IL-1

It is known to induce the secretion of cytokines such as IL-6 and IL-6, and in the case of IL-6, it plays an important role in the immune response, such as affecting the production of immunoglobulins. Therefore, it was confirmed that the treatment of the hydrolyzate of defatted egg yolk protein (Pan-Neu hydrolyzate) according to the present invention can help to enhance immunity.

[Example 5]

Determination of the effect of defatted egg yolk protein hydrolyzate on phagocytic activity of macrophages

In order to confirm the effect of the hydrolyzate of defatted egg yolk protein (Pan-Neu hydrolyzate) obtained in the above example on the phagocytosis of macrophages, it was measured using the NRU (Neutral Red uptake) method. First, the RAW 264.7 cell line was seeded in a 24-well plate at 2 × 10 ⁵ cells/well and pre-cultured for 4 hours. The defatted egg yolk protein hydrolyzate (Pan-Neu hydrolyzate) obtained in the above example was treated with each concentration (25, 50, 100, 200 μg/mL) and incubated for 24 hours, the supernatant was removed and 0.075% of 400 μL of natural red solution was treated and then incubated for 1 hour. After washing 3 times with PBS, 500 μL of lysis buffer [ethanol: 0.01% glacial acetic acid = 1:1, (v/v)] was treated, and after 15 minutes, absorbance was measured at 540 nm using a microplate reader. As macrophages were activated, more neutral red compound was absorbed, and this was compared and analyzed by colorimetric assay.

Defatted egg yolk protein hydrolyzate Phagocytosis (%) 25 μg/mL 50 μg/mL 100 μg/mL 200 μg/mL Control 100.00 ± 5.70 LPS 128.31 ± 3.15 P+N 125.34 ± 8.61 130.31 ± 4.28 130.53 ± 4.81 142.02 ± 9.34

6 is a graph showing the phagocytosis activity (%) of RAW 264.7 macrophages as the hydrolyzate of defatted egg yolk protein (Pan-Neu hydrolyzate) prepared in the above example was treated by concentration, and Table 8 is phagocytosis This is a table showing the activity (%). As shown in FIGS. 6 and 8, it was confirmed that the phagocytosis of macrophages increased and the absorbance increased as the Pan-Neu hydrolyzate according to the present invention was treated (FIG. 6). Based on the phagocytosis of the untreated control group, an increase in phagocytosis of over 20% was confirmed in all samples, and it was confirmed that the highest concentration of 200 μg/mL used in the experiment showed the highest phagocytosis of 142%. could This was confirmed to be higher than the LPS used as a positive control. Phagocytosis is well known as one of the representative characteristics of activated macrophages. It is known as one of the criteria for judging the activation of macrophages as the first step taken to remove the invasion of foreign substances from the outside. Therefore, it was confirmed that the treatment of the hydrolyzate of defatted egg yolk protein (Pan-Neu hydrolyzate) according to the present invention induces the activation of macrophages.

[Example 6]

Confirmation of macrophage activation mechanism of defatted egg yolk protein hydrolyzate

In order to confirm the macrophage activation mechanism of the defatted egg yolk protein hydrolyzate (Pan-Neu hydrolyzate) obtained in the above example, a MAPK signaling pathway specific inhibitor, which is well known to play an important role in macrophage activity, was used. The experiment was carried out using As inhibitors, MAPK signaling pathway inhibitors SB 202190 (p38 pathway inhibitor), SP600125 (JNK pathway inhibitor), and PD 98059 (ERK pathway inhibitor) were used. First, the RAW 264.7 cell line was seeded in a 12-well plate at 4 × 10 ⁵ cells/well and pre-cultured for 24 hours. After treatment with each specific inhibitor and the hydrolyzate of defatted egg yolk protein obtained in the above example (Pan-Neu hydrolyzate, 200 μg/mL), the reaction was performed for 8 hours, and the medium was replaced with a fresh medium and further cultured for 16 hours. did After the culture was completed, the amount of NO generated in the supernatant was quantified in the same manner as described above.

inhibitor NO (μM) DMSO 40.45 ± 5.83 SB 24.17 ± 6.32 SP 13.29 ± 3.75 PD 40.23 ± 5.40

7 is a graph showing the amount of NO production in macrophages that appears when each MAPK pathway inhibitor is treated with 200 μg/mL of defatted egg yolk protein hydrolyzate (Pan-Neu hydrolyzate) prepared in the above example, Table 9 is a table showing the NO production amount of macrophages. 7 and Table 9, 'DMSO' is a control group using a solvent as an inhibitor, 'SB' is an experimental group treated with SB 202190, a p38 inhibitor, 'SP' is an experimental group treated with a JNK inhibitor, SP600125, ' PD' means an experiment in which the ERK inhibitor, PD 98059, was treated.

As a result of the experiment, as shown in FIGS. 7 and 9, when SB 202190, a p38 pathway inhibitor of the MAPK cell signaling pathway, and SP 600125, a JNK pathway inhibitor, were treated, respectively, the Pan according to the present invention compared to the control not treated with the inhibitor - It was confirmed that the amount of NO produced by the treatment of the -Neu hydrolyzate decreased. However, it was confirmed that there was no change in the amount of NO produced when the ERK pathway inhibitor, PD 98059, was treated. Through this, it was confirmed that the macrophage activation of the Pan-Neu hydrolyzate according to the present invention is achieved through the p38 pathway and the JNK pathway.

As described above, the present invention was to prepare a hydrolyzate having excellent immune enhancing activity by performing hydrolysis from defatted egg yolk using a protease. It was confirmed that the hydrolyzate obtained by only primary hydrolysis using pancreatin or neutrase degrading enzyme did not have any effect on the activation of macrophages, and it was confirmed that the Pan-Neu hydrolyzate (primary hydrolysis with pancreatin) and hydrolyzate, which was subjected to secondary hydrolysis with neutrase), was confirmed to have excellent macrophage activation activity. The Pan-Neu hydrolyzate increased iNOS expression in macrophages to increase NO production, and it was confirmed that the expression of pro-inflammatory cytokines TNF-α and IL-6 was increased at the mRNA and protein levels. Phagocytosis, which is known as a representative characteristic of macrophage activation, increased with the Pan-Neu hydrolyzate treatment, and when the activation mechanism was finally confirmed, the activation of macrophages through the p38 pathway and the JNK pathway among the MAPK cell signaling pathways was could see what was happening. It was confirmed that there was no macrophage activation activity when the hydrolyzate was prepared through the two-step complex hydrolysis, first hydrolyzed using nutrease and secondarily using pancreatin. Therefore, the Pan-Neu hydrolyzate of the present invention obtained by performing primary hydrolysis using pancreatin as a proteolytic enzyme and secondarily hydrolyzing using neutrase is used for enhancing immunity in food and pharmaceutical industries. It was confirmed that it can be used as a functional material.

The description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a dispersed form, and likewise components described as distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the following claims rather than the above detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be interpreted as being included in the scope of the present invention. do.

Claims (16)

A method for preparing a hydrolyzate of defatted egg yolk, comprising obtaining a hydrolyzate of defatted egg yolk by treating and reacting with one or more proteolytic enzymes selected from the group consisting of nutrase, pancreatin, and combinations thereof .
According to claim 1, wherein the manufacturing method,
preparing skimmed egg yolk;
treating the prepared defatted egg yolk with neutrase or pancreatin as a proteolytic enzyme to perform a primary reaction;
terminating the primary reaction by inactivating the protease; and
centrifuging the reactant after the first reaction to obtain defatted egg yolk protein;
A method for producing a hydrolyzate of defatted egg yolk protein, comprising a.
The method according to claim 1, wherein the first reaction is performed for 1 to 8 hours.
According to claim 1, wherein the first reaction is carried out at a temperature range of 25 ℃ to 60 ℃, the method for producing a hydrolyzate of defatted egg yolk protein.
The method according to claim 2, wherein after the first reaction by treating the prepared defatted egg yolk with neutrase or pancreatin as a proteolytic enzyme,
A method for producing a hydrolyzate of defatted egg yolk protein, further comprising the step of performing a secondary reaction by treating neutrase or pancreatin as a proteolytic enzyme once more.
The method according to claim 5, wherein the protease used in the second reaction is different from the protease used in the first reaction.
6. The method of claim 5,
The proteolytic enzyme used in the first reaction is pancreatin, and the proteolytic enzyme used in the second reaction is neutrase.
The method according to claim 5, wherein the secondary reaction is performed for 1 to 8 hours.
The method of claim 5, wherein the secondary reaction is performed at a temperature range of 25°C to 60°C.
10. A hydrolyzate of defatted egg yolk protein produced by the method according to any one of claims 1 to 9.
11. The method of claim 10, wherein the defatted egg yolk protein hydrolyzate,
A hydrolyzate of defatted egg yolk protein, which increases iNOS expression in macrophages.
11. The method of claim 10, wherein the defatted egg yolk protein hydrolyzate,
A hydrolyzate of defatted egg yolk protein that exhibits immune enhancing activity by increasing the iNOS expression of macrophages and improving the amount of NO produced.
11. The method of claim 10, wherein the defatted egg yolk protein hydrolyzate,
A hydrolyzate of defatted egg yolk protein, which exhibits immune enhancing activity by increasing the expression of pro-inflammatory cytokines.
A composition for preventing, improving, or treating inflammatory diseases, comprising, as an active ingredient, a hydrolyzate of defatted egg yolk protein prepared by the method according to any one of claims 1 to 9.
The method of claim 14, wherein the composition is a pharmaceutical composition, a cosmetic composition, or a health functional food, preventing, improving, or treating an inflammatory disease.
15. The method of claim 14, wherein the inflammatory disease is enteritis, gastritis, hepatitis, bronchitis, pleurisy, arthritis, rhinitis, keratitis, nephritis, peritonitis, osteomyelitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, burn inflammation, dermatitis, periodontitis , Gingivitis, degenerative neuritis, inflammatory pain, inflammatory bowel disease, and a composition for preventing, improving, or treating an inflammatory disease, including those selected from the group consisting of combinations thereof.

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