Low-allergenic whey protein hydrolysate and method for preparing same

Abstract

The present invention provides a low-allergenic whey protein hydrolysate produced by hydrolyzing whey protein using an endoproteinase derived from Bacillus licheniformis, an exoproteinase derived from Aspergillus oryzae, or a combination thereof, and a method for preparing same. By utilizing the low-allergenic whey protein hydrolysate of the present invention, whey protein with high nutritional value can be consumed without the risk of causing an allergic reaction to milk.

Description

Hypoallergenic whey protein hydrolysate and method for producing the same

[0001] The present invention was made under the support of the Ministry of Agriculture, Food and Rural Affairs of the Republic of Korea under project number 322011042SB010, and the research management specialized institution of the project is the National Institute of Agricultural Science and Technology Planning and Evaluation, the research project name is “Development of high value-added food technology”, the research project name is “Establishment of mass production technology for milk-derived functional peptides and development of utilization products”, the main institution is Neocrema Co., Ltd., and the research period is from January 1, 2023 to December 31, 2023.

[0002] The present invention relates to a low-allergenic whey protein hydrolysate and a method for producing the same.

[0003]

[0004] Milk allergy occurs when the body's immune system abnormally reacts to allergens found in milk. Milk allergy is relatively common, especially in infants and young children. Symptoms of milk allergy include abdominal pain, diarrhea, vomiting, abdominal bloating, hives, eczema, rash, cough, and nasal congestion. In severe cases, anaphylaxis may also occur.

[0005] The most common allergen found in milk is casein. Casein is a phosphoprotein found in milk, used in cheesemaking, and accounts for approximately 80% of milk protein.

[0006] Meanwhile, whey protein (WP) is also known to contain milk allergens. Whey is a byproduct formed when milk is processed into cheese, and refers to the remaining solution after the solids, fat and casein, are removed from milk. Whey contains proteins, lactose, minerals, and vitamins, and the proteins contained in whey are collectively called whey proteins. Among whey proteins, beta-lactoglobulin (βLG) and alpha-lactalbumin (αLA) act as milk allergens along with the aforementioned casein, causing milk allergy. It is reported that most patients with milk allergy have immunoglobulin E (IgE) antibodies specific for at least two or more allergens among casein, beta-lactoglobulin, and alpha-lactalbumin. In particular, β-lactoglobulin, which accounts for 7% to 12% of milk protein, is known to induce milk allergy when it passes into the intestines due to its strong resistance to digestion in the stomach, despite its very low content compared to casein, which accounts for 80% of milk protein.

[0007] Whey protein, an animal-based protein, is a source of essential amino acids and possesses excellent physiological properties, including boosting immunity and promoting muscle formation and recovery. Therefore, it is used in a variety of foods. Therefore, to safely consume whey protein, which offers high nutritional value, without the risk of allergies, there is a growing need to reduce the antigenicity of allergens present in whey protein.

[0008]

[0009] The present inventors have conducted extensive research efforts to develop a hypoallergenic whey protein hydrolysate that reduces the antigenicity of milk allergens, α-lactalbumin and β-lactoglobulin, and is highly digestible. As a result, the optimal hydrolysis conditions for decomposing α-lactalbumin and β-lactoglobulin were identified, thereby completing the present invention.

[0010] Accordingly, the object of the present invention is to provide a low-allergenic whey protein hydrolysate, which is obtained by hydrolyzing whey protein with an endoprotease derived from Bacillus licheniformis, an exoprotease derived from Aspergillus oryzae, or a combination thereof.

[0011] Another object of the present invention is to provide a method for producing a low-allergenic whey protein hydrolysate, comprising the steps of: preparing a whey protein suspension of 5% (w / v) to 20% (w / v) by mixing whey protein and water; and performing hydrolysis by adding an endoprotease derived from Bacillus licheniformis, an exoprotease derived from Aspergillus oryzae, or a combination thereof, to the whey protein suspension at 0.5% (w / v) to 2% (w / v) of the whey protein suspension.

[0012]

[0013] According to one aspect of the present invention, the present invention provides a low-allergenic whey protein hydrolysate, which is obtained by hydrolyzing whey protein with an endoprotease derived from Bacillus licheniformis, an exoprotease derived from Aspergillus oryzae, or a combination thereof.

[0014] The present inventors have conducted extensive research to develop a hypoallergenic whey protein hydrolysate that reduces the antigenicity of milk allergens, α-lactalbumin and β-lactoglobulin, and exhibits high digestibility. As a result, the optimal hydrolysis conditions for decomposing α-lactalbumin and β-lactoglobulin were identified, and the hypoallergenic whey protein hydrolysate produced under these conditions was demonstrated to reduce milk allergic reactions and exhibit high digestibility.

[0015] As used herein, the term "milk allergy" refers to an abnormal reaction of the body's immune system to allergens present in milk. The most common allergen causing milk allergy is casein, and representative milk allergens in whey proteins include alpha-lactalbumin (α-lactalbumin) and beta-lactoglobulin (β-lactoglobulin).

[0016] α-Lactalbumin is a whey protein that binds to lactose synthase and aids in the synthesis of lactose, the main carbohydrate in milk. α-Lactalbumin is composed of 123 amino acids, has a globular shape, and has four disulfide bonds, making it resistant to heat denaturation.

[0017] β-lactoglobulin (β-lactoglobulin) is a whey protein and is a globular protein composed of 162 amino acids. β-lactoglobulin also exhibits resistance to heat denaturation due to disulfide bonds. Furthermore, β-lactoglobulin contains a single thiol (-SH) group, making it highly acid-stable. This makes it highly resistant to digestion in the stomach and allows it to pass through the intestines, inducing milk allergies. β-lactoglobulin is almost absent from human breast milk, but accounts for 7% to 12% of milk proteins.

[0018] In one embodiment of the present invention, the whey protein of the present invention may be derived from cheese whey.

[0019] In one embodiment of the present invention, the whey protein of the present invention may be normal whey powder, demineralized whey powder, whey protein concentrate, or whey protein isolate.

[0020] In one embodiment of the present invention, the Bacillus licheniformis-derived endoprotease of the present invention may be Alcalase, Protamex, or a mixed enzyme thereof.

[0021] More specifically, the Bacillus licheniformis-derived endoprotease of the present invention may be Alcalase.

[0022] In one embodiment of the present invention, the exoprotease derived from Aspergillus oryzae of the present invention may be Flavorzyme, Prozyme, or a mixed enzyme thereof.

[0023] More specifically, the exoprotease derived from Aspergillus oryzae of the present invention may be a prozyme.

[0024] In one embodiment of the present invention, the combination of the Bacillus licheniformis-derived endoprotease and the Aspergillus oryzae-derived exoprotease of the present invention may be a mixture of Alcalase and Prozyme.

[0025] As demonstrated in the examples described below, the inventors of the present invention confirmed that when whey protein is hydrolyzed using a mixture of Alcalase and Prozyme, both milk allergens, α-lactalbumin and β-lactoglobulin, are decomposed, successfully reducing the allergenicity of whey protein hydrolysate.

[0026] In one embodiment of the present invention, the combination of the Bacillus licheniformis-derived endoprotease and the Aspergillus oryzae-derived exoprotease of the present invention may be a mixture of the Bacillus licheniformis-derived endoprotease and the Aspergillus oryzae-derived exoprotease in a weight ratio of 1:0.2 to 1:5. More specifically, the endoprotease from Bacillus licheniformis and the exoprotease from Aspergillus oryzae are mixed in a ratio of 1:0.2 to 1:5, 1:0.2 to 1:4, 1:0.2 to 1:3, 1:0.2 to 1:2, 1:0.2 to 1:1.5, 1:0.2 to 1:1, 1:0.25 to 1:5, 1:0.25 to 1:4, 1:0.25 to 1:3, 1:0.25 to 1:2, 1:0.25 to 1:1.5, 1:0.25 to 1:1, 1:0.3 to 1:5, 1:0.3 to 1:4, It may be mixed in a weight ratio of 1:0.3 to 1:3, 1:0.3 to 1:2, 1:0.3 to 1:1.5, 1:0.3 to 1:1, 1:0.5 to 1:5, 1:0.5 to 1:4, 1:0.5 to 1:3, 1:0.5 to 1:2, 1:0.5 to 1:1.5, 1:0.5 to 1:1, 1:0.6 to 1:5, 1:0.6 to 1:4, 1:0.6 to 1:3, 1:0.6 to 1:2, 1:0.6 to 1:1.5, or 1:0.6 to 1:1, more specifically, 1:1 or 1:3, but is not limited thereto.

[0027] As demonstrated in the examples described below, the inventors of the present invention confirmed that when Alcalase, an endoprotease derived from Bacillus licheniformis, and exoprotease, an exoprotease derived from Aspergillus oryzae, are mixed in a weight ratio of 1:1 or 1:3, the amino-nitrogen (AN) content and the degree of hydrolysis (DH) of whey protein hydrolysate are significantly higher than when mixed in other weight ratios.

[0028] As used herein, the term "amino-nitrogen (AN) content" refers to a numerical value measuring the amount of free amino acids present in whey protein or whey protein hydrolysate. Free amino acids do not require further digestion and can therefore be rapidly utilized in various metabolic processes, such as protein synthesis and tissue repair. Therefore, the higher the amino-nitrogen content in whey protein or whey protein hydrolysate, the more efficiently it is digested and absorbed upon ingestion, and the more actively metabolism using free amino acids can occur.

[0029] As used herein, the term "Degree of Hydrolysis (DH)" refers to the degree to which whey proteins are broken down into smaller peptides or amino acids through hydrolysis. The degree of hydrolysis can vary depending on the hydrolysis method, time, and enzyme used. The degree of hydrolysis affects the solubility, digestibility, and functional properties of whey proteins. Generally, a higher degree of hydrolysis results in higher solubility and faster digestion compared to unhydrolyzed whey proteins.

[0030] In one embodiment of the present invention, the amino-nitrogen (AN) content of the whey protein hydrolysate of the present invention may be 40 mg / mL to 75 mg / mL. More specifically, the amino-nitrogen (AN) content of the whey protein hydrolysate of the present invention is 40 mg / mL to 75 mg / mL, 40 mg / mL to 75 mg / mL, 40 mg / mL to 65 mg / mL, 40 mg / mL to 62.5 mg / mL, 40 mg / mL to 60 mg / mL, 40 mg / mL to 57.5 mg / mL, 40 mg / mL to 55 mg / mL, 40 mg / mL to 52.5 mg / mL, 40 mg / mL to 50 mg / mL, 45 mg / mL to 75 mg / mL, 45 mg / mL to 65 mg / mL, 45 mg / mL to 62.5 mg / mL, 45 mg / mL to 60 mg / mL, 45 mg / mL to 57.5 mg / mL, 45 mg / mL to 55 mg / mL, 45 mg / mL to 52.5 mg / mL, 45 mg / mL to 50 mg / mL, 48 mg / mL to 75 mg / mL, 48 mg / mL to 65 mg / mL, 48 mg / mL to 62.5 mg / mL, 48 mg / mL to 60 mg / mL, 48 mg / mL to 57.5 mg / mL, 48 mg / mL to 55 mg / mL, 48 mg / mL to 52.5 mg / mL, 48 mg / mL to 50 mg / mL, 50 mg / mL to 75 mg / mL, 50 mg / mL to 65 mg / mL, 50 mg / mL to 62.5 mg / mL, 50 mg / mL to 60 mg / mL, 50 mg / mL to 57.5 mg / mL, 50 mg / mL to 55 mg / mL, 50 mg / mL to 52.5 mg / mL, 52.5 mg / mL to 75 mg / mL, 52.5 mg / mL to 65 mg / mL, 52.5 mg / mL to 62.5 mg / mL, 52.5 mg / mL to 60 mg / mL, 52.5 mg / mL to 57.5 mg / mL, 52.5 mg / mL to 55 mg / mL, 55 mg / mL to 75 mg / mL, 55 mg / mL to 65 mg / mL, 55 mg / mL to 62.5 mg / mL, 55 mg / mL to 60 mg / mL, 55 mg / mL to 57.5 mg / mL, 57.5 mg / mL to 75 mg / mL, 57.5 mg / mL to 65 mg / mL, 57.5 mg / mL to 62.5 mg / mL, 57.5 mg / mL to 60 mg / mL, 60 mg / mL to 75 mg / mL, 60 mg / mL to 65 mg / mL, 60 mg / mL to 62.5 mg / mL, 62.5 mg / mL to 75 mg / mL, 62.5 mg / mL to 65 mg / mL, or 65 mg / mL to 75 mg / mL, but is not limited thereto.

[0031] In one embodiment of the present invention, the free amino acids in the hypoallergenic whey protein hydrolysate of the present invention may be present in an amount of 35 mole% to 45 mole% relative to the total amino acids. More specifically, the free amino acids in the hypoallergenic whey protein hydrolysate of the present invention may be present in an amount of 35 mole% to 45 mole%, 35 mole% to 43 mole%, 35 mole% to 42 mole%, 35 mole% to 41 mole%, 40 mole% to 45 mole%, 40 mole% to 43 mole%, 40 mole% to 42 mole%, 40 mole% to 41 mole%, 41 mole% to 45 mole%, 41 mole% to 43 mole%, 41 mole% to 42 mole%, 42 mole% to 45 mole%, or 42 mole% to 43 mole% relative to the total amino acids, but is not limited thereto.

[0032] In one embodiment of the present invention, free leucine in the hypoallergenic whey protein hydrolysate of the present invention may be present in an amount of 14 mole% to 20 mole% relative to the total free amino acids. More specifically, free leucine in the hypoallergenic whey protein hydrolysate of the present invention may be present in an amount of 14 mole% to 20 mole%, 14 mole% to 18 mole%, 14 mole% to 17 mole%, 16 mole% to 20 mole%, 16 mole% to 18 mole%, or 16 mole% to 17 mole% relative to the total free amino acids, but is not limited thereto.

[0033] In one embodiment of the present invention, the free branched chain amino acid (BCAA) in the low-allergenic whey protein hydrolysate of the present invention may be present in an amount of 35 mole% to 45 mole% relative to the total free amino acids. More specifically, the free branched chain amino acid (BCAA) in the low-allergenic whey protein hydrolysate of the present invention may be present in an amount of 35 mole% to 45 mole%, 35 mole% to 42 mole%, 35 mole% to 41 mole%, 35 mole% to 40 mole%, 37 mole% to 45 mole%, 37 mole% to 42 mole%, 37 mole% to 41 mole%, 37 mole% to 40 mole%, 39 mole% to 45 mole%, 39 mole% to 42 mole%, 39 mole% to 41 mole%, or 39 mole% to 40 mole% relative to the total free amino acids, but is not limited thereto.

[0034] As used herein, the term "branched chain amino acid (BCAA)" refers to three essential amino acids: valine (Val), leucine (Leu), and isoleucine (Ile). Valine, leucine, and isoleucine are branched-chain amino acids that are not synthesized in the human body and are essential amino acids. Unlike other amino acids, which are primarily metabolized in the liver, BCAAs are metabolized in muscles and serve as an energy source for muscles. Accordingly, they are actively being used in muscle supplements, nutritional supplements, and beverages.

[0035] As demonstrated in the examples described below, the inventors of the present invention have confirmed that the major free amino acid in the hypoallergenic whey protein hydrolysate of the present invention is leucine, and that it is also rich in valine and isoleucine. Therefore, the hypoallergenic whey protein hydrolysate of the present invention can be usefully used in compositions for muscle growth, muscle pain relief, exercise fatigue reduction, and muscle loss prevention.

[0036] In one embodiment of the present invention, α-lactalbumin and β-lactoglobulin in the whey protein hydrolysate of the present invention may be decomposed by the hydrolysis described above.

[0037] As described above, the inventors of the present invention have confirmed that when whey protein is hydrolyzed using a combination of an endoprotease derived from Bacillus licheniformis and an exoprotease derived from Aspergillus oryzae, particularly a mixture of Alcalase and Prozyme, both milk allergens, α-lactalbumin and β-lactoglobulin, are decomposed, thereby successfully reducing the allergenicity of whey protein hydrolysate.

[0038] In one embodiment of the present invention, the low-allergenic whey protein hydrolysate of the present invention reduces milk allergic reactions.

[0039] In one embodiment of the present invention, the low-allergenic whey protein hydrolysate of the present invention may reduce allergic reactions by suppressing the production of immunoglobulin E (IgE).

[0040] Immunoglobulin E (IgE) is a type of antibody protein produced by the immune system in response to allergens. When exposed to an allergen, B cells produce IgE antibodies specific to that allergen. These IgE antibodies bind to receptors on the surfaces of mast cells and basophils, immune cells involved in allergic reactions, triggering the release of allergen mediators such as histamine, leukotrienes, and cytokines. These allergen mediators then act in a cascade to induce the characteristic symptoms of an allergic reaction.

[0041] As demonstrated in the examples described below, the inventors of the present invention confirmed that when the hypoallergenic whey protein hydrolysate (HWPH) of the present invention is orally administered, the production of IgE is significantly reduced compared to when whey protein (WP) that has not undergone hydrolysis is orally administered, and thus it was found that the hypoallergenic whey protein hydrolysate (HWPH) of the present invention can reduce milk allergic reactions.

[0042] In one embodiment of the present invention, the low-allergenic whey protein hydrolysate of the present invention may reduce milk allergic reactions by inducing an immune response by Th1 cells (T helper cell 1) and reducing an immune response by Th2 cells (T helper cell 2).

[0043] T helper cells (T helper cells, T H T cells play a central role in the immune response by secreting T cell cytokines. Helper T cells can differentiate into various effector T cells, such as Th1, Th2, Th17, and Tfh (T follicular helper), and each effector T cell exhibits a unique cytokine profile. Th1 cells are typically associated with cell-mediated immunity, and upon activation, they produce cytokines such as interferon (IFN)-γ, tumor necrosis factor (TNF)-β, and interleukin (IL)-2, and induce the synthesis of IgG2a antibodies. In contrast, Th2 cells are generally involved in humoral immunity, and upon activation, they produce cytokines such as IL-4, IL-5, IL-9, IL-10, and IL-13, and induce the synthesis of IgG1 and IgE. IFN-γ and IgG2a antibodies secreted by Th1 cells antagonize IL-4 and IgG1 antibodies secreted by Th2 cells. Disruptions in the homeostasis between Th1 and Th2 immune responses can lead to various immunopathological abnormalities. When Th2 immune responses are relatively dominant, microbial infections such as bacteria and viruses, and the development of allergies, are promoted. When Th1 immune responses are dominant, most autoimmune diseases can be promoted.

[0044] As demonstrated in the examples described below, the IFN-γ value and IgG2a / IgG1 value were higher in the hypoallergenic whey protein hydrolysate (HWPH) of the present invention than in the whey protein (WP) administration group, indicating that the immune response of Th1 cells was relatively stimulated compared to the immune response of Th2 cells, and thus the allergic response induced by the immune response of Th2 cells was reduced in the hypoallergenic whey protein hydrolysate (HWPH).

[0045] In one embodiment of the present invention, the low-allergenic whey protein hydrolysate of the present invention may have superior digestibility and intestinal permeability compared to whey protein that has not undergone hydrolysis.

[0046] As demonstrated in the examples described below, the low-allergenic whey protein hydrolysate of the present invention showed superior digestibility and intestinal permeability compared to whey protein in in vitro experiments using Caco-2 cells and in vivo experiments using SD rats.

[0047]

[0048] According to another aspect of the present invention, the present invention provides a food composition comprising the above-described low-allergenic whey protein hydrolysate of the present invention as an effective ingredient.

[0049] In one embodiment of the present invention, a food composition comprising the above-described low-allergenic whey protein hydrolysate of the present invention as an active ingredient may be a food composition with reduced milk allergenicity.

[0050] Since the low-allergenic whey protein hydrolysate of the present invention has reduced allergenicity by decomposing both α-lactalbumin and β-lactoglobulin, which are milk allergens, when consuming a food composition containing it, the occurrence of milk allergy is prevented or improved, and whey protein with high nutritional value can be safely consumed without the risk of milk allergy.

[0051] The food composition of the present invention can be manufactured in the form of powder, granules, tablets, capsules, or beverages. Examples include various food products such as candies, beverages, gum, tea, vitamin complexes, or health supplements.

[0052] The food composition of the present invention includes processed forms of all natural ingredients, such as foods, functional foods, nutritional supplements, health foods, and food additives. The above-mentioned types of food compositions can be prepared in various forms according to conventional methods known in the art. For example, as health foods, they can be prepared and consumed in the form of tea, juice, and drinks, or can be granulated, encapsulated, or powdered for consumption. In addition, foods such as beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruits, bottled fruits, jams, marmalades, etc.), fish, meats and their processed foods (e.g., ham, sausages, corned beef, etc.), breads and noodles (e.g., udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, taffy, dairy products (e.g., yogurt, fermented milk, butter, cheese, etc.), edible vegetable oils, margarine, vegetable proteins, retort foods, frozen foods, various seasonings (e.g., soybean paste, soy sauce, sauces, etc.) can be produced by adding the low-allergenic whey protein hydrolysate of the present invention. In addition, in order to use the low-allergenic whey protein hydrolysate of the present invention in the form of a food additive, it can be produced in the form of a powder or a concentrate.

[0053] The food composition of the present invention can be taken for a long period of time.

[0054] The mixing amount of the active ingredient of the food composition of the present invention can be suitably determined depending on its intended use. Generally, the whey hydrolysate of the present invention can be added in an amount of 0.1 to 70 wt%, preferably 2 to 50 wt%, based on 100 wt% of the raw material of the food or beverage during the manufacture of the food or beverage. The effective dosage of the peptide of the present invention can be used in accordance with the effective dosage of the pharmaceutical composition, but in the case of long-term intake for the purpose of health and hygiene or health control, it can be below the above range, and since the active ingredient does not have any problems in terms of safety, it can also be used in an amount above the above range.

[0055] The food composition of the present invention may include ingredients commonly added during food manufacturing, such as proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. Examples of the carbohydrates mentioned above include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and common sugars and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)] and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is manufactured as a drink, in addition to the low-allergenic whey protein hydrolysate of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, Eucommia extract, jujube extract, licorice extract, etc. may be additionally included.

[0056] The food composition of the present invention comprises the hypoallergenic whey protein hydrolyzate, which is one embodiment of the present invention described above, as an active ingredient. Therefore, any overlapping content between the two inventions applies equally, and to avoid undue complexity in this specification, such descriptions are omitted.

[0057]

[0058] According to another aspect of the present invention, the present invention provides a method for producing a low-allergenic whey protein hydrolysate comprising the following steps:

[0059] (a) a step of preparing a whey protein suspension of 5% (w / v) to 20% (w / v) by mixing whey protein and water; and

[0060] (b) A step of performing hydrolysis by adding 0.5% (w / v) to 2% (w / v) of an endoprotease derived from Bacillus licheniformis, an exoprotease derived from Aspergillus oryzae, or a combination thereof to the whey protein suspension.

[0061] In one embodiment of the present invention, the concentration of the whey protein suspension of the above-described step (a) may be 5% (w / v) to 20% (w / v), and more specifically, 5% (w / v) to 20% (w / v), 5% (w / v) to 15% (w / v), 5% (w / v) to 12% (w / v), 5% (w / v) to 10% (w / v), 7% (w / v) to 20% (w / v), 7% (w / v) to 15% (w / v), 7% (w / v) to 12% (w / v), 7% (w / v) to 10% (w / v), 10% (w / v) to 20% (w / v), 10% (w / v) to 15% (w / v) or 10% (w / v) It may be 12% (w / v), most specifically 10% (w / v), but is not limited thereto.

[0062] In one embodiment of the present invention, the Bacillus licheniformis-derived endoprotease of step (b) described above may be Alcalase, Protamex, or a mixed enzyme thereof.

[0063] More specifically, the Bacillus licheniformis-derived endoprotease of the present invention may be Alcalase.

[0064] In one embodiment of the present invention, the exoprotease derived from Aspergillus oryzae in the step (b) described above may be Flavorzyme, Prozyme, or a mixed enzyme thereof.

[0065] More specifically, the exoprotease derived from Aspergillus oryzae of the present invention may be a prozyme.

[0066] In one embodiment of the present invention, the combination of the Bacillus licheniformis-derived endoprotease and the Aspergillus oryzae-derived exoprotease of the above-described step (b) may be a mixture of Alcalase and Prozyme.

[0067] As demonstrated in the examples described below, the inventors of the present invention confirmed that when whey protein is hydrolyzed using a mixture of Alcalase and Prozyme, both milk allergens, α-lactalbumin and β-lactoglobulin, are decomposed, successfully reducing the allergenicity of whey protein hydrolysate.

[0068] In one embodiment of the present invention, the Bacillus licheniformis-derived endoprotease, the Aspergillus oryzae-derived exoprotease, or a combination thereof in step (b) described above may be added at 0.5% (w / v) to 2% (w / v) of the whey protein suspension. More specifically, the Bacillus licheniformis-derived endoprotease, the Aspergillus oryzae-derived exoprotease or a combination thereof of the above-described step (b) is present in an amount of 0.5% (w / v) to 2% (w / v), 0.5% (w / v) to 1.5% (w / v), 0.5% (w / v) to 1.2% (w / v), 0.5% (w / v) to 1% (w / v), 0.7% (w / v) to 2% (w / v), 0.7% (w / v) to 1.5% (w / v), 0.7% (w / v) to 1.2% (w / v), 0.7% (w / v) to It can be administered at 1% (w / v), 0.9% (w / v) to 2% (w / v), 0.9% (w / v) to 1.5% (w / v), 0.9% (w / v) to 1.2% (w / v), or 0.9% (w / v) to 1% (w / v), more specifically 1% (w / v), but is not limited thereto.

[0069] As demonstrated in the examples described below, the inventors selected the mixing ratio of Alcalase and Prozyme using response surface methodology (RSM).

[0070] In one embodiment of the present invention, the combination of the Bacillus licheniformis-derived endoprotease and the Aspergillus oryzae-derived exoprotease in the step (b) described above may be a mixture of the Bacillus licheniformis-derived endoprotease and the Aspergillus oryzae-derived exoprotease in a weight ratio of 1:0.2 to 1:5. More specifically, the endoprotease from Bacillus licheniformis and the exoprotease from Aspergillus oryzae are mixed in a ratio of 1:0.2 to 1:5, 1:0.2 to 1:4, 1:0.2 to 1:3, 1:0.2 to 1:2, 1:0.2 to 1:1.5, 1:0.2 to 1:1, 1:0.25 to 1:5, 1:0.25 to 1:4, 1:0.25 to 1:3, 1:0.25 to 1:2, 1:0.25 to 1:1.5, 1:0.25 to 1:1, 1:0.3 to 1:5, 1:0.3 to 1:4, It may be mixed in a weight ratio of 1:0.3 to 1:3, 1:0.3 to 1:2, 1:0.3 to 1:1.5, 1:0.3 to 1:1, 1:0.5 to 1:5, 1:0.5 to 1:4, 1:0.5 to 1:3, 1:0.5 to 1:2, 1:0.5 to 1:1.5, 1:0.5 to 1:1, 1:0.6 to 1:5, 1:0.6 to 1:4, 1:0.6 to 1:3, 1:0.6 to 1:2, 1:0.6 to 1:1.5, or 1:0.6 to 1:1, but is not limited thereto.

[0071] As demonstrated in the examples described below, the inventors of the present invention confirmed that when Alcalase, an endoprotease derived from Bacillus licheniformis, and exoprotease, an exoprotease derived from Aspergillus oryzae, are mixed in a weight ratio of 1:1 or 1:3, the amino-nitrogen (AN) content and the degree of hydrolysis (DH) of whey protein hydrolysate are significantly higher than when mixed in other weight ratios.

[0072] In one embodiment of the present invention, the hydrolysis of step (b) described above may be performed for 5 to 20 hours. More specifically, the hydrolysis of step (b) described above may be performed for 5 to 20 hours, 5 to 15 hours, 5 to 12 hours, 5 to 10 hours, 7 to 20 hours, 7 to 15 hours, 7 to 12 hours, 7 to 10 hours, 9 to 20 hours, 9 to 15 hours, 9 to 12 hours, or 9 to 10 hours, most specifically, 10 hours, but is not limited thereto.

[0073] In one embodiment of the present invention, the hydrolysis of step (b) described above may be performed at a temperature of 30°C to 70°C. More specifically, the hydrolysis of step (b) described above may be performed at a temperature of 30°C to 70°C, 30°C to 60°C, 30°C to 55°C, 30°C to 52°C, 40°C to 70°C, 40°C to 60°C, 40°C to 55°C, 40°C to 52°C, 48°C to 70°C, 48°C to 60°C, 48°C to 55°C, or 48°C to 52°C, most specifically, 50°C, but is not limited thereto.

[0074] In one embodiment of the present invention, the hydrolysis of step (b) described above may be performed under conditions of pH 6.0 to pH 9.0. More specifically, the hydrolysis of step (b) described above may be performed under conditions of pH 6.0 to pH 9.0, pH 6.0 to pH 8.0, pH 7.0 to pH 9.0, pH 7.0 to pH 8.0, pH 7.5 to pH 9.0, or pH 7.5 to pH 8.0, most specifically, pH 7.8, but is not limited thereto.

[0075] In one embodiment of the present invention, the hydrolysis of step (b) described above may be performed at a temperature of 30°C to 70°C for 5 to 20 hours. More specifically, the hydrolysis of step (b) described above may be performed at a temperature of 30°C to 70°C for 5 to 20 hours, at a temperature of 30°C to 70°C for 7 to 20 hours, at a temperature of 30°C to 70°C for 5 to 15 hours, at a temperature of 40°C to 70°C for 5 to 20 hours, at a temperature of 40°C to 70°C for 7 to 20 hours, at a temperature of 40°C to 70°C for 5 to 15 hours, at a temperature of 48°C to 55°C for 5 to 20 hours, at a temperature of 48°C to 55°C for 7 to 20 hours, or at a temperature of 48°C to 55°C for 5 to 15 hours, and most specifically, may be performed at a temperature of 50°C for 10 hours, but is not limited thereto.

[0076] In one embodiment of the present invention, the hydrolysis of step (b) described above may be performed for 5 to 20 hours under conditions of pH 6.0 to pH 9.0. More specifically, the hydrolysis of the above-described step (b) can be performed under conditions of pH 6.0 to pH 9.0 for 5 to 20 hours, under conditions of pH 6.0 to pH 9.0 for 7 to 20 hours, under conditions of pH 6.0 to pH 9.0 for 5 to 15 hours, under conditions of pH 7.0 to pH 9.0 for 5 to 20 hours, under conditions of pH 7.0 to pH 9.0 for 7 to 20 hours, under conditions of pH 7.0 to pH 9.0 for 5 to 15 hours, under conditions of pH 7.5 to pH 8.0 for 5 to 20 hours, under conditions of pH 7.5 to pH 8.0 for 7 to 20 hours, or under conditions of pH 7.5 to pH 8.0 for 5 to 15 hours, and most specifically, under conditions of pH 7.8 for 10 hours. However, it is not limited to these.

[0077] In one embodiment of the present invention, under the temperature condition of 30°C to 70°C and the condition of pH 6.0 to pH 9.0, under the temperature condition of 30°C to 70°C and the condition of pH 7.0 to pH 9.0, under the temperature condition of 30°C to 70°C and the condition of pH 7.5 to pH 8.0, under the temperature condition of 40°C to 70°C and the condition of pH 6.0 to pH 9.0, under the temperature condition of 40°C to 70°C and the condition of pH 7.0 to pH 9.0, under the temperature condition of 40°C to 70°C and the condition of pH 7.5 to pH 8.0, under the temperature condition of 48°C to 55°C and the condition of pH 6.0 to pH 9.0, under the temperature condition of 48°C to 55°C and the condition of pH 7.0 to pH 9.0, or under the temperature condition of 48°C to 55°C and the condition of pH 7.5 It can be performed under conditions of pH 8.0, and most specifically, it can be performed under conditions of temperature of 50°C and pH 7.8, but is not limited thereto.

[0078] In one embodiment of the present invention, α-lactalbumin and β-lactoglobulin in whey protein may be decomposed through the above-described step (b).

[0079] Since the low-allergenic whey protein hydrolysate of the present invention is manufactured through the manufacturing method of the present invention, all overlapping parts between the two inventions are equally applicable, and their description is omitted to prevent complexity of the present specification.

[0080]

[0081] The present invention provides a low-allergenic whey protein hydrolysate and a method for producing the same, wherein whey protein is hydrolyzed using an endoprotease derived from Bacillus licheniformis, an exoprotease derived from Aspergillus oryzae, or a combination thereof.

[0082] When the low-allergenic whey protein hydrolysate of the present invention is used, whey protein with high nutritional value can be safely consumed without the risk of milk allergy.

[0083]

[0084] Figure 1 shows the SDS-PAGE results of the hydrolysate obtained after treating whey protein (WP) with an enzyme mixture. The proteins were loaded onto the gel at concentrations of 50 μg, 60 μg, and 70 μg, respectively.

[0085] Lane 1 shows the SDS-PAGE result of the hydrolysate prepared using a mixture of Alcalase and Prozyme, Lane 2 shows the SDS-PAGE result of the hydrolysate prepared using a mixture of Collupulin and Prozyme, Lane 3 shows the SDS-PAGE result of the hydrolysate prepared using a mixture of Protamex and Prozyme, and Lane 4 shows the SDS-PAGE result of the hydrolysate prepared using a mixture of Prozyme and Flavorzyme.

[0086] Figure 2a shows the amino-nitrogen (AN) content of the hydrolyzate obtained after treatment with the enzyme mixture.

[0087] Figure 2b shows the degree of hydrolysis (DH) of the hydrolyzate obtained after treatment with the enzyme mixture.

[0088] Figure 3a shows the AN content according to the mixing ratio of Alcalase and Prozyme.

[0089] Figure 3b shows the degree of hydrolysis (DH) according to the mixing ratio of Alcalase and Prozyme.

[0090] [Correction pursuant to Rule 91, July 30, 2024]

[0091] [Correction pursuant to Rule 91, July 30, 2024]

[0092] [Revised 30.07.2024 under Rule 91] In the above Figures 2a and 3b, all figures are expressed as the mean ± standard deviation of each group, and n = 3. Different letters indicate significant differences at p<0.05 according to Tukey's multiple test.

[0093] Figure 4 shows the AN content of whey protein hydrolysate optimized using response surface methodology (RSM).

[0094] Figure 5 shows the decomposition of allergens α-lactalbumin (α-LA) and β-lactoglobulin (βLG) when hydrolyzing whey protein (WP) under optimal reaction conditions, as confirmed by gel electrophoresis.

[0095] Figure 6a shows the AN content when whey protein (WP) and low-allergenic whey protein hydrolysate (HWPH) were treated with artificial digestive enzymes, pepsin and pancreatin.

[0096] Figure 6b shows the digestibility of whey protein (WP) and low-allergenic whey protein hydrolysate (HWPH) when treated with artificial digestive enzymes, pepsin and pancreatin.

[0097] Figure 6c shows the intestinal permeability after 20 minutes of treating Caco-2 cells with whey protein (WP) and hypoallergenic whey protein hydrolysate (HWPH) samples prepared with artificial digestive enzymes pepsin and pancreatin.

[0098] Figure 6d shows the intestinal permeability after 40 minutes of treating Caco-2 cells with whey protein (WP) and hypoallergenic whey protein hydrolysate (HWPH) samples prepared with artificial digestive enzymes pepsin and pancreatin.

[0099] Figure 6e shows the intestinal permeability after 60 minutes of treating Caco-2 cells with a whey protein (WP) hypoallergenic whey protein hydrolysate (HWPH) sample prepared with artificial digestive enzymes pepsin and pancreatin.

[0100] Figure 6f shows the intestinal permeability after 120 minutes of treating Caco-2 cells with whey protein (WP) and hypoallergenic whey protein hydrolysate (HWPH) samples prepared with artificial digestive enzymes pepsin and pancreatin.

[0101] In the above Figures 6a to 6d, all values ​​are expressed as the mean ± standard deviation of each group, and n = 3. Different letters indicate a significant difference at p < 0.05 according to Tukey's multiple test, ***p < 0.001 indicates a significant difference between whey protein (WP) and hypoallergenic whey protein hydrolysate (HWPH), and ns indicates no significant difference.

[0102] Figure 7 shows the changes in serum AN content after oral administration of whey protein (WP) and hypoallergenic whey protein hydrolysate (HWPH) to SD rats. All values ​​are expressed as the mean ± standard deviation of each group, n = 4. Different letters indicate significant differences at p < 0.05 according to Tukey's multiple test, ***p < 0.001 indicates a significant difference between whey protein (WP) and hypoallergenic whey protein hydrolysate (HWPH), and ns indicates no significant difference.

[0103] Figures 8a to 8f show the molecular weight distribution of whey protein (WP) and low-allergenic whey protein hydrolysate (HWPH) analyzed by HLPC.

[0104] Figure 8a shows the molecular weight distribution of whey protein (WP), Figure 8b shows the molecular weight distribution of whey protein treated with pepsin (WP-pepsin), Figure 8c shows the molecular weight distribution of whey protein treated with pepsin and then pancreatin (WP-pancreatin), Figure 8d shows the molecular weight distribution of hypoallergenic whey protein hydrolysate (HWPH), Figure 8e shows the molecular weight distribution of hypoallergenic whey protein hydrolysate treated with pepsin and then pancreatin (HWPH-pancreatin).

[0105] Figures 9a to 9c show the results of measuring IgE concentrations in blood, spleen cells, and small intestine, respectively.

[0106]

[0107] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0108]

[0109] Example

[0110]

[0111] Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight / weight) % for solid / solid, (weight / volume) % for solid / liquid, and (volume / volume) % for liquid / liquid, unless otherwise noted.

[0112]

[0113] Experimental materials and methods

[0114] 1. Experimental materials

[0115] Whey protein was used as a whey protein concentrate (WPC; Agri-mark, FL, USA) with a dry basis protein content of 80%. α-Lactalbumin and β-lactoglobulin were 75% α-lactalbumin and β-lactoglobulin extracted from milk (Samyoung Innovation, Pyeongtaek, Korea). Alcalase (Novozymes, Copenhagen, Denmark), Protamex (Novozymes), Flavorzyme (Novozymes), and Collupulin (DSM, Heerlen, Netherlands) were used as endoproteases. Prozyme (DuPont, Delaware, USA) was used as exoprotease. Pepsin and pancreatin (Sigma-Aldrich, St. Louis, USA) were used as artificial digestive enzymes. L-leucine was purchased from Sigma-Aldrich.

[0116]

[0117] 2. Preparation of whey protein hydrolysate

[0118] Whey protein and water were mixed to prepare a 10% (w / v) whey protein suspension as a substrate solution. To prepare a whey protein hydrolysate, a mixture of endoprotease and exoprotease was added at a concentration of 1.0% (w / v) of the substrate solution. This was then hydrolyzed at pH 7.8 and 50°C for 8 hours with shaking at 100 rpm. Each reaction solution was treated at 90°C for 10 minutes to inactivate the proteases, and then centrifuged at 8,000 rpm for 20 minutes to separate the supernatant. A whey protein solution without protease treatment was used as a control. The reaction conditions and enzyme characteristics used for whey protein hydrolysis are shown in Table 1 below.

[0119] EnzymeMajor activityOptimal conditionsTemperature (℃)pHAlcalase® 2.4 L FGEndoprotaseBacillus licheniformis507.8CollupulinMGEndoprotaseCarica papaya50-705.0-7.5ProtamexBroad spectrum endoprotaseBacillus licheniformis35-605.5-7.5Flavourzyme® 500 MGProtase (endo- & exo-)Aspergillus oryzae505.0-7.0Prozyme 2000PExoprotaseAspergillus oryzae50-606.0-9.0

[0120]

[0121] 3. Determination of α-lactalbumin and β-lactoglobulin decomposition in whey protein hydrolysate using gel electrophoresis.

[0122] Whey protein hydrolysate (10 mg) was dissolved in 1 mL of sample buffer [0.5 M trihydrochloric acid (HCl), glycerol, 10% SDS, 0.1% bromophenol blue, pH 6.8], heat-treated at 100°C for 5 min, centrifuged, and the supernatant was collected. 20 μL of the supernatant was subjected to electrophoresis on a 12% polyacrylamide gel at 135 V for approximately 90 min. To confirm the presence of α-lactalbumin and β-lactoglobulin, the gel was stained with Coomassie Brilliant Blue R-250 for 6 h, and then destained using a solution containing 10% methanol and 10% acetic acid until the protein bands were clearly visible.

[0123]

[0124] 4. Calculation of amino-nitrogen (AN) content and degree of hydrolysis (DH)

[0125] The amino nitrogen (AN) content of hypoallergenic whey protein hydrolysates was measured using the 2,4,6-trinitrobenzensulfonic acid (TNBS) method. Briefly, 125 μL of sample, 400 μL of 0.212 M phosphate buffer (pH 8.2), and 0.4 mL of 0.1% TNBS were mixed and incubated in a darkroom at 50°C for 60 min. Next, 0.8 mL of 100 mM HCl was added and incubated for 20 min. After another 10 min, 0.8 mL of distilled water was added, and the absorbance was measured at 340 nm. The AN content was calculated using L-leucine as a standard.

[0126] The degree of protein hydrolysis (DH) after enzyme treatment was calculated using the following equation by estimating the AN content of the whey protein hydrolysate. Total AN content was measured by hydrolyzing whey protein with 6 N HCl at 100°C for 24 h and then neutralizing with sodium hydroxide (NaOH).

[0127] DH = (L t - L0) / (L max - L0) × 100,

[0128] Here L t is the AN content after hydrolysis at time t, L0 is the AN content before hydrolysis, and L max is the AN content after acid hydrolysis with 6 N HCl.

[0129]

[0130] 5. Establishment of optimal mixing ratio and reaction conditions of Alcalase and Prozyme using response surface methodology (RSM).

[0131] The optimal mixing ratio of Alcalase and Prozyme was determined using response surface methodology (RSM). A total of 15 combinations were constructed by repeating the experiment three times at 50°C and pH 7.8 with 10% (w / v) substrate. The mixing ratio of the two enzymes, Alcalase and Prozyme, was used as an independent variable and is shown in Table 2.

[0132] Enzyme Mixing RatioRunSubstrate(w / v%)Alcalase(w / v%)Prozyme(w / v%)1100.50.521030.50.540.750.2550.750.2561070.50.580190.250.7510011110120.250.75130.250.751401150.750.25

[0133] The optimal mixing ratio of the two enzymes was determined by measuring the AN of the hydrolyzate corresponding to the experimental combination. RSM was used to analyze the experimental results, and the regression equation representing the optimal mixing ratio of Alcalase and Prozyme is as follows.

[0134] aX + bY + cXY = 32.29 X + 68.87 Y + 96.58 XY,

[0135] Here, X is Alcalase (w / v%) and Y is Prozyme (w / v%).

[0136]

[0137] Afterwards, the optimal reaction conditions (enzyme addition amount and hydrolysis time) were selected using RSM.

[0138] A total of 39 combinations were constructed by repeating the experiment three times at 50°C and pH 7.8 with 10% substrate. The amount of added enzyme and the hydrolysis time by the mixed enzyme of Alcalase and Prozyme were used as independent variables and are shown in Table 3 below. The optimal mixing ratio of Alcalase and Prozyme was selected.

[0139] Reaction conditions of mixed enzymes RUN Substrate (w / v%) Enzyme (w / v%) Time (h) 110 26 22 6 3 16 4 1.54 5 12 6 110 7 1.58 8 12 9 1.58 100.56 112 6 12 16 13 16 14 16 15 16 16 1.54 17 0.56 18 16 19 0.75 8 20 0.75 4 210.56 22 16 23 110 24 16 25 0.75 8 26 16 27 0.75 4 28 12 29 1.54 30 0.75 4 3 116 32 16 33 16 34 16 35 1.58 36 16 37 110 38 16 39 0.75 8

[0140] The AN content of the hydrolyzate corresponding to the experimental combination was measured to determine the optimal enzyme addition amount and hydrolysis time. The regression equation representing the optimal conditions for enzyme addition amount and hydrolysis time is as follows.

[0141] A + aX + bY + cX 2 + dY 2 + eXY = 29.6286 - 0.9220 2 + 8.3643 Y 2 - 2.0389 XY,

[0142] Here, X is the hydrolysis time and Y is the amount of enzyme added.

[0143]

[0144] 6. Confirmation of amino acid composition of whey protein hydrolysate by HPLC (High-performance liquid chromatography) analysis

[0145] Amino acids were derivatized using AccQ-Tag reagent (Waters, Milford, MA, USA) according to the manufacturer's manual and then analyzed using a high-performance liquid chromatography (HPLC) system (Waters). Analysis was performed using an AccQ-Tag column (3.9 mm × 150 mm, Waters) under the following analytical conditions: temperature 37°C; mobile phase [Water AccQ-Tag Eluent A (acetate-phosphate buffer), acetonitrile, Milli-Q Water], flow rate 1.0 mL / min, fluorescence detector (excitation 250 nm, emission 395 nm).

[0146] Total amino acid analysis was performed after acid hydrolysis. 10 mL of 6 N HCl solution was added to 0.5 g of the sample, which was then purged with nitrogen and hydrolyzed at 110°C for 24 h. The resulting filtrate was centrifuged, and the supernatant was concentrated at 50°C. After complete evaporation of the acid and water, the volume was diluted to 5 mL with 20 mM HCl (pH 2.2). The dissolved solution was filtered through a 0.45 μm membrane, and the filtrate was used as a sample for HPLC analysis.

[0147]

[0148] 7. In vitro digestion rate and intestinal permeability analysis

[0149] 7-1. In vitro digestion rate analysis

[0150] In vitro digestibility was performed using pepsin and pancreatin according to the method of Garrett et al. (Garrett et al., 1999). 1,000 mg of the sample was dissolved in distilled water and the pH was adjusted to 2.0. Pepsin was added in an amount of 2.5% of the substrate and reacted at 37°C for 1 hour. The pH was adjusted to 5.3 with NaHCO3 (sodium bicarbonate) and then to 7.5 with 5 N NaOH. Pancreatin was added in an amount of 4% of the substrate and reacted at 37°C for 2 hours, followed by heating at 90°C for 10 minutes to inactivate the digestive enzymes. AN (mg) was measured in the supernatant obtained by centrifugation (12,000 rpm, 10 minutes), and the digestibility (%) was calculated using the following equation.

[0151] Digestibility (%) = [AN content of sample after digestion (mg) - AN content of sample before digestion (mg)] / [Total AN content of sample before digestion (mg) - AN content of sample before digestion (mg)] × 100.

[0152] (Total AN in sample before digestion: AN content after acid hydrolysis).

[0153]

[0154]

[0155] 7-2. Long-term penetration analysis

[0156] For the intestinal permeability analysis of whey protein and whey protein hydrolysate, Caco-2 Ready 24-well plates (Komabiotech, Seoul, Korea) were used. The plates were incubated in a 5% CO2 incubator (37°C) for 4 h, thawed, and replaced with Dulbecco's Modified Eagle's medium (DMEM) containing 1 g / L glucose, 10% fetal bovine serum, and 1% glutamine. The assay was performed 48 h after the medium replacement. For the intestinal permeability analysis, whey protein and whey protein hydrolysate, prepared using digestive enzymes (pepsin and pancreatin), were diluted 10-fold with Hanks' Balanced Salt Solution (HBSS) buffer. After washing the Caco-2 plate three times with HBSS buffer to a complete monolayer film, 250 μL and 750 μL of the diluted sample and HBSS buffer were dispensed into the insert plate and receiver plate, respectively. The buffer in the receiver plate was withdrawn over time (20, 40, 60, and 120 minutes). The protein content of the sample initially distributed in the insert plate and the samples collected over time were analyzed using the bicinchoninic acid assay (BCA assay), and the intestinal permeability of the sample was analyzed using the following equation:

[0157] Permeability (%) = [Total protein (mg) in receiver plate] / [Initial protein (mg) added to insert plate] × 100.

[0158]

[0159] 8. Evaluation of absorption rate by high-dose single oral administration

[0160] Six-week-old male Sprague-Dawley (SD) rats were purchased from Oriental Bio (Seongnam, South Korea). The animals were housed in a breeding room at 21±1°C, 50% to 55% relative humidity, and a 12-h light-dark cycle. Water and food were provided ad libitum. After acclimation for 1 week, four SD rats were randomly assigned to each experimental group. Whey protein and whey protein hydrolysate were administered orally at 500 mg / kg, and the rats were sacrificed at the appropriate time points (20, 40, 60, and 120 min) for blood collection. Blood samples were collected from the experimental group (control group) at time 0. After centrifugation (3,000 rpm, 15 min, 4℃), serum was separated to measure total AN content. All animal experiments were approved by the Korea University Animal Experiment Ethics Committee (KUIACUC-2022-0095).

[0161]

[0162] 9. Molecular weight distribution of whey protein and whey protein hydrolysate

[0163] To measure the molecular weight distribution of whey proteins and whey protein hydrolysates using HPLC, samples were filtered (microfilter, 0.45 μm) and 20 μL was injected onto a Superdex G-75 column (10 × 300 mm, GE Healthcare, Anaheim, CA, USA). The absorbance was measured at 220 nm, and the column was eluted with 50 mM ammonium formate buffer (pH 5.5) (0.5 mL / min). The molecular weight distribution of whey protein and whey protein hydrolysate was measured according to the molecular weight distribution curve created using glutathione (MW 307), aprotinin (MW 6,512), cytochrome C (MW 12,384), enolase (MW 67,000), and lactate dehydrogenase (MW 142,000).

[0164]

[0165] 10. Evaluation of immunological responses to whey protein and whey protein hydrolysate

[0166] Eight-week-old, specific pathogen-free male BALB / c mice were purchased from Daehan Biolink Co., Ltd. (Chungbuk, South Korea) and maintained aseptically (room temperature: 25°C; humidity: 55%; sterile distilled water supplied). The mice were divided into groups administered 250 mg / kg and 500 mg / kg of whey protein and whey protein hydrolysate, respectively, and a control group administered saline. Eight mice were assigned to each group. Samples were administered orally five times a week for 4 weeks via gastric intubation. Body weights were measured twice a week at 3-day intervals. After administration, blood was collected and centrifuged at 2,800 × g for 10 minutes to isolate serum. After sacrifice, splenocytes were aseptically excised for cell culture, seeded into 24-well plates, and cultured in a CO2 incubator for 48 hours. Afterwards, phytohemagglutinin (Sigma, 5 μg / L × 10) was used as a polyclonal stimulator for in vitro activation. 6 Cells) were added. The concentrations of cytokines (INF-γ and IL4), antibodies (IgG2a and IgG1), and IgE in the cell culture medium were measured using ELISA kits purchased from BD Bioscience (San Diego, CA, USA). For small intestinal cytokines, 2 mL of PBS was added to 0.5 g of duodenum extracted aseptically after sacrifice, stirred, and left at 4°C for 2 days. The supernatant obtained by centrifugation was then analyzed for cytokines and IgE using an ELISA kit.

[0167]

[0168] 11. Statistical Analysis

[0169] Data were statistically analyzed using SPSS (ver. 12.0; SPSS Inc., Chicago, IL, USA), and the means and standard deviations were calculated for all measurements. The significance between experimental groups was tested using ANOVA followed by Tukey's multiple range test at a p<0.05 level. RSM analysis was performed using Minitab 17 software (Minitab Inc., State College, PA, USA) to confirm the appropriateness of the model for understanding the relationship between the independent and dependent variables.

[0170]

[0171] Example 1: Selection of enzyme combinations for the production of hypoallergenic whey protein hydrolysate (HWPH).

[0172] To select enzymes capable of degrading α-lactalbumin and β-lactoglobulin, endoproteases (Alcalase, Collupulin, Protamex) and exoproteases (Prozyme) were mixed and hydrolyzed for 8 hours. The combination of enzymes that degraded α-lactalbumin and β-lactoglobulin was selected by analyzing them using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). As a result, in the hydrolysate prepared using the mixture of Alcalase and Prozyme, no α-lactalbumin or β-lactoglobulin bands were observed, indicating that both allergens were degraded (Fig. 1).

[0173] The results of analyzing the amino nitrogen (AN) and degree of hydrolysis (DH) of the hydrolysate obtained after treatment with the enzyme mixture are shown in Figs. 2a and 2b. As shown in Figs. 2a and 2b, the levels of AN and DH in the whey protein hydrolysate obtained by mixing Alcalase and Prozyme were significantly higher than those in the other hydrolysates. Therefore, it was found that the combination of Alcalase and Prozyme was the most suitable enzyme combination for the hydrolysis of α-lactalbumin and β-lactoglobulin.

[0174]

[0175] Example 2: Establishment of the optimal mixing ratio and optimal reaction conditions of Alcalase and Prozyme for the production of hypoallergenic whey protein hydrolysate (HPWH).

[0176] Example 2-1: Establishing the optimal mixing ratio of Alcalase and Prozyme using RSM

[0177] To select the appropriate mixing ratio of Alcalase and Prozyme for producing hypoallergenic whey protein hydrolysate (HPWH), hydrolysis was performed according to 15 combinations (Table 2) by RSM.

[0178] The AN content of the hydrolyzate and the DH value calculated based on the AN content are shown in Fig. 3a and Fig. 3b, respectively. As shown in Fig. 3a and Fig. 3b, the AN content and DH value of the hydrolyzate obtained by mixing Alcalase and Prozyme at a ratio of 1:1 and 1:3, respectively, were significantly higher than those of the hydrolyzates obtained at other mixing ratios (p<0.05).

[0179] The optimal ratio of the enzyme mixture was predicted using RSM, and it was predicted that the AN content would be the highest at 50.42 mg / mL when Alcalase and Prozyme were mixed in a 1:1 ratio. The AN content of the hydrolysate produced by mixing Alcalase and Prozyme in a 1:1 ratio was 51.52 mg / mL as predicted by RSM, and the actual measured value was 50.42 mg / mL, which was similar to the predicted value.

[0180] Therefore, it was found that the most appropriate mixing ratio of Alcalase and Prozyme for the production of hypoallergenic whey protein hydrolysate (HWPH) was 1:1 or 1:3.

[0181] To establish optimal reaction conditions, a 1:1 mixture of Alcalase and Prozyme was used.

[0182]

[0183] Example 2-2: Establishing optimal reaction conditions for Alcalase and Prozyme using RSM

[0184] In order to select the optimal enzyme concentration and hydrolysis time for producing hypoallergenic whey protein hydrolysate (HWPH), hydrolysis was performed according to 39 combinations (Table 3) suggested by RSM under the conditions of 10% (w / v) substrate, 50℃, pH 7.8, and the AN content of each whey protein hydrolysate was measured, which is shown in Fig. 4. As shown in Fig. 4, the AN content increased as the amount of enzyme added and the hydrolysis time increased, and the AN content was the highest when the enzyme was added at 1.0% (w / v) and the hydrolysis was performed for 10 hours. The expected AN content at the optimal enzyme concentration and hydrolysis time predicted by RSM was 60.00 mg / mL, and the actual measured value was 59.58 mg / mL, which is R 2The value was close to 0.968.

[0185] For the production of hypoallergenic whey protein hydrolysate (HWPH) using an Alcalase-Prozyme mixture, the optimal conditions for hydrolysis were found to be 1.0% enzyme addition and 10 hours of hydrolysis. The regression equation for AN content obtained through multiple regression analysis is as follows.

[0186] AN(mg / mL) = 0.471 X 2 + 8.364 Y 2 - 2.039 XY - 0.922 X + 4.552 Y + 29.62

[0187] Here, X is the hydrolysis time (h), Y is the amount of enzyme added (%), and R 2 =96.80%.

[0188]

[0189] Example 2-3: Analysis of allergen degradation under optimal reaction conditions

[0190] Under the optimal reaction conditions described above, the degradation of the allergens α-lactalbumin and β-lactoglobulin was confirmed through gel electrophoresis when hydrolyzing hypoallergenic whey protein hydrolysate (HWPH) (Fig. 5). As shown in Fig. 5, the bands of the allergens α-lactalbumin and β-lactoglobulin were observed before hydrolysis (0 h), but became faint after 4 h of hydrolysis, and were barely visible after 8 h of hydrolysis. Thus, it was confirmed that α-lactalbumin and β-lactoglobulin were degraded by the mixed enzyme treatment.

[0191]

[0192] Example 3: Determination of the amino acid composition of whey protein and hypoallergenic whey protein hydrolysate (HWPH).

[0193] The amino acid composition of hypoallergenic whey protein hydrolysate (HWPH) treated with Alcalase and Prozyme is shown in Table 4. As shown in Table 4, the free amino acid composition was 2,089.72 ± 76.18 μmole / g, and the total amino acid composition was 4,987.21 ± 93.44 μmole / g. A total of 17 types of free amino acids were detected, with leucine (Leu, leucine) (16.8%, 351.73 ± 4.62 μmole / g) being the most abundant, followed by valine (Val, valine) (13.8%), lysine (Lys, lysine) (11.2%), and isoleucine (Ile, isoleucine) (8.7%). The above amino acids, when present at high levels, were found to induce a bitter taste, which was consistent with the bitter taste of the samples.

[0194] Content Free Amino Acid (μmole / g) Total Amino Acid (μmole / g) Asp 4 6.96 ± 0.33 5 16.35 ± 4.63 Ser 15 1.70 ± 0.86 30 8.41 ± 2.31 Glu 16 9.00 ± 2.30 7 20.73 ± 9.91 Gly 25.27 ± 0.90 18 2.34 ± 3.57 His 6 1.55 ± 4.06 9 5.91 ± 3.63 Arg 7 9.38 ± 0.91 39 4.24 ± 21.16 Thr 5 7.53 ± 28.2 37 2.17 ± 13.46 Ala 4 8.65 ± 35.25 28 8.05 ± 5.65 Pro 10 0.55 ± 2.67 38 8.43 ± 6.84Cys10.91 ± 1.14282.89 ± 8.71Tyr73.76 ± 0.9393.46 ± 2.77Val288.57 ± 6.28396.82 ± 4.46Met66.35 ± 0.2878.32 ± 2.07Lys234.71 ± 1.66308.88 ± 5.78Ile182.55 ± 3.47249.27 ± 5.98Leu351.73 ± 4.62469.35 ± 4.30Phe140.56 ± 3.47141.59 ± 0.99Total2089.72 ± 76.184987.21 ± 93.44The values ​​are the average for each group. ± It is expressed as standard deviation, and n=3.

[0195] Example 4: In vitro digestion rate and intestinal permeability analysis using Caco-2 cells

[0196] Example 4-1: In vitro digestion rate analysis

[0197] When whey protein (WP) and hypoallergenic whey protein hydrolysate (HWPH) were sequentially treated with artificial digestive enzymes, pepsin and pancreatin, the changes in AN content and digestibility were measured (Fig. 6a and Fig. 6b). As shown in Fig. 6a and Fig. 6b, the AN content and digestibility tended to increase as digestion by pepsin and pancreatin progressed. When pancreatin was treated after pepsin, whey protein (WP) and hypoallergenic whey protein hydrolysate (HWPH) were hydrolyzed more efficiently, and the AN content and digestibility were also significantly higher than when pepsin was treated alone. At each stage of digestive enzyme treatment, the AN content was significantly higher in hypoallergenic whey protein hydrolysate (HWPH) than in whey protein (WP). In terms of digestibility, hypoallergenic whey protein hydrolysate (HWPH), which had a high AN content before hydrolysis, showed a lower digestibility than whey protein (WP) in the pepsin treatment step, but showed a higher level of digestibility increase than whey protein (WP) in the pancreatin treatment step.

[0198]

[0199] Example 4-2: Analysis of intestinal permeability

[0200] The intestinal permeability of Caco-2 cells was measured and shown in Fig. 6c to Fig. 6f. At each stage of pepsin and pancreatin treatment at the indicated treatment time points of 20, 40, 60, and 120 min, the intestinal permeability of Caco-2 cells was significantly higher in hypoallergenic whey protein hydrolysate (HWPH) than in whey protein (WP). Therefore, it was confirmed that the digestibility and intestinal permeability of hypoallergenic whey protein hydrolysate (HWPH) were higher than those of whey protein (WP) as measured by Alcalase and Prozyme.

[0201]

[0202] Example 5: Evaluation of absorption rate by high-dose single oral administration in an animal model

[0203] Changes in serum AN levels were measured after oral administration of whey protein (WP) and hypoallergenic whey protein hydrolysate (HWPH) to SD rats (Fig. 7). Serum AN levels peaked 20 minutes after oral administration and then gradually decreased. At 20 and 40 minutes, serum AN levels were significantly higher in the hypoallergenic whey protein hydrolysate (HWPH) group compared to the whey protein (WP) group (p<0.001). The hypoallergenic whey protein hydrolysate produced by Alcalase-Prozyme is composed of smaller peptides than whey protein (WP) and is therefore easily digested and absorbed, suggesting that serum AN levels are high in the early stages of oral administration.

[0204]

[0205] Example 6: Molecular weight distribution of whey protein hydrolysate

[0206] The results of analyzing the molecular weight distribution of whey protein and whey protein hydrolysate are shown in Figs. 8a to 8f. Whey protein (WP) was found to contain several peptides with molecular weights of 16 kDa and 5 kDa. On the other hand, the hypoallergenic whey protein hydrolysate (HWPH) produced by Alcalase and Prozyme was mainly composed of peptides below 5 kDa, with major peaks at approximately 384 Da and 214 Da, respectively. Whey protein (WP) was hydrolyzed into small peptides of 2.6 kDa, 1.3 kDa, and 491 Da when treated with pepsin, and was hydrolyzed into peptides with smaller molecular weights after treatment with pancreatin than when treated with pepsin (Figs. 8b and 8c). The molecular weight distribution of hypoallergenic whey protein hydrolysate (HWPH) was similar before and after pepsin and pancreatin treatment. Because the hypoallergenic whey protein hydrolysate (HWPH) was already enzymatically hydrolyzed (Figures 8e and 8f), the effects of pepsin and pancreatin treatment on the molecular weight distribution appeared to be minimal.

[0207]

[0208] Example 7: Evaluation of immunological responses to whey protein and whey protein hydrolysate

[0209] Example 7-1: Analysis of cytokines and antibodies in animal serum, spleen cells, and small intestine

[0210] The concentrations of cytokines (INF-γ and IL4) and antibodies (IgG2a and IgG1) in the serum of BALB / c mice administered 250 mg / kg and 500 mg / kg of whey protein (WP) and hypoallergenic whey protein hydrolysate (HWPH) and the saline-administered control group are shown in Table 5. The values ​​are expressed as the mean ± standard error of the mean (SEM) for each group, n = 8. * p<0.05, ** p<0.01 and *** p<0.001 indicates a significant difference from the control group according to Tukey's multiple test, and #p<0.05, ## p<0.01 and ### p<0.001 indicates a significant difference between the same concentration groups by Student's t-test. ns indicates no significant difference.

[0211] Concentrations of cytokines (INF-γ and IL4) and antibodies (IgG2a and IgG1) in serum - Control Whey protein 250 mg / kg Whey protein 500 mg / kg Whey protein hydrolysate 250 mg / kg Whey protein hydrolysate 500 mg / kg INF-γ (pg / ml) 8.87 ± 1.50 ns 8.82 ± 2.079.95 ± 1.7110.70 ± 1.8911.66 ± 0.81IL-4(pg / ml)5.30 ± 0.21 ns 7.73 ± 0.7210.53 ± 2.907.64 ± 1.168.94 ± 2.61INF-γ1.65 ± 0.24 ns1.12 ± 0.321.16 ± 0.351.55 ± 0.471.63 ± 0.33IgG2a (ng / ml)52.62 ± 2.18136.70 ± 1.76 *** 103.27 ± 2.98 ** 155.63 ± 5.22 ***,# 339.82 ± 13.33 ***,### IgG1(ng / ml)26.32 ± 0.6569.29 ± 1.57500.69 ± 36.01 *** 64.15 ± 3.3363.96 ± 3.20 ### IgG2a / IgG12.01 ± 0.131.97 ± 0.020.21 ± 0.01 *** 2.43 ± 0.12 # 5.35 ± 0.44 ***,###

[0212] Blood INF-γ and IL-4 levels in mice administered whey protein (WP) and hypoallergenic whey protein hydrolysate (HWPH) showed a tendency to slightly increase compared to the control mice, but this difference was not significant. In addition, INF-γ corresponding to Th1 / Th2 was shown to increase in hypoallergenic whey protein hydrolysate (HWPH) treatment compared to whey protein (WP) treatment, but the difference was not statistically significant. In the case of IgG2a / IgG1, hypoallergenic whey protein hydrolysate (HWPH) treatment significantly increased compared to whey protein (WP) treatment in both the 250 mg / kg and 500 mg / kg administration groups.

[0213] The concentrations of spleen cytokines (INF-γ and IL4) and antibodies (IgG2a and IgG1) in each mouse group are shown in Table 6 below. The statistical processing and display methods are as in Table 5.

[0214] Concentrations of cytokines (INF-γ and IL4) and antibodies (IgG2a and IgG1) in splenocytes - Control Whey protein 250 mg / kg Whey protein 500 mg / kg Whey protein hydrolysate 250 mg / kg Whey protein hydrolysate 500 mg / kg INF-γ (μg / g) 45.37 ± 0.89 44.30 ± 0.85 45.13 ± 0.63 44.30 ± 0.62 52.19 ± 0.92 ***,### IL-4 (μg / g)2.85 ± 0.283.68 ± 0.284.57 ± 0.51 * 3.51 ± 0.983.85 ± 0.16INF-γ16.74 ± 1.7212.37 ± 0.8710.50 ± 1.1513.28 ± 1.21 * 13.67 ± 1.71IgG2a (μg / g)0.36 ± 0.080.40 ± 0.050.48 ± 0.050.70 ± 0.08 *,# 0.77 ± 0.08 **,## IgG1(μg / g)0.32 ± 0.040.82 ± 0.04 *** 1.89 ± 0.08 *** 0.53 ± 0.02 *,### 0.72 ± 0.01 ***,### IgG2a / IgG11.18 ± 0.300.50 ± 0.080.26 ± 0.031.35 ± 0.16 *,## 1.08 ± 0.12 ###

[0215] INF-γ was significantly decreased in the 500 mg / kg whey protein (WP) administration group compared to the control group (p<0.01). IgG2a / IgG1 (Th1:Th2) was similar to or higher in the hypoallergenic whey protein hydrolysate (HWPH) administration group compared to the control group, and the whey protein (WP) administration group showed a lower ratio compared to the control group, but there was no significant difference. There was a significant difference in IgG2a / IgG1 between the hypoallergenic whey protein hydrolysate (HWPH) administration group and the whey protein (WP) administration group.

[0216] The concentrations of cytokines (INF-γ and IL4) and antibodies (IgG2a and IgG1) in the small intestine of each mouse group are shown in Table 7 below.

[0217] Concentrations of cytokines (INF-γ and IL4) and antibodies (IgG2a and IgG1) in the small intestine - Control Whey protein 250 mg / kg Whey protein 500 mg / kg Whey protein hydrolysate 250 mg / kg Whey protein hydrolysate 500 mg / kg INF-γ (μg / g) 1.92 ± 0.34 1.91 ± 0.79 3.77 ± 0.17 4.54 ± 1.04 6.85 ± 0.62 * IL-4(μg / g)3.17±0.0033.97±0.01 *** 5.37±0.02 *** 3.54±0.02 ***,### 4.58±0.01 ***,### INF-γ0.61±0.110.48±0.200.70±0.031.281±0.301.49±0.14 *,## IgG2a (μg / g)0.28±0.010.39±0.01 *** 0.73±0.03 *** 0.94±0.04 ***,### 1.68±0.03 ***,### IgG1(μg / g)6.08±0.2511.18±0.18 *** 14.83±0.13 *** 7.19±0.06 *,### 6.81±0.22 ### IgG2a / IgG10.05±0.0020.04±0.0010.05±0.0020.13±0.01 ### 0.25±0.01 ###

[0218] IL-4 levels were significantly higher in the whey protein (WP) and hypoallergenic whey protein hydrolysate (HWPH) groups compared to the control group (p<0.05). INF-γ, which corresponds to Th1 / Th2, tended to increase as the dose of hypoallergenic whey protein hydrolysate (HWPH) increased, and the 500 mg / kg group showed a significantly higher value than the control group (p<0.05). IgG2a levels in the small intestine of both groups were significantly higher than the control group. IgG2a / IgG1 and the ratio of Th1 to Th2 cells were significantly higher in the hypoallergenic whey protein hydrolysate (HWPH) group compared to the control group (p<0.001). There was a significant difference in IgG2a / IgG1 between the whey protein (WP) group and the hypoallergenic whey protein hydrolysate (HWPH) group in terms of dose (p<0.001).

[0219]

[0220] In summary, oral administration of hypoallergenic whey protein hydrolysate (HWPH) increased Th1 cytokine and antibody levels in blood, spleen cells, and small intestine, whereas Th2 cytokine and antibody levels were lower than those in the whey protein (WP) group. Thus, it was confirmed that the hypoallergenic whey protein hydrolysate of the present invention reduces milk allergic reactions by inducing an immune response mediated by Th1 cells (T helper cell 1) and reducing an immune response mediated by Th2 cells (T helper cell 2).

[0221]

[0222] Example 7-2: Analysis of IgE content in animal serum, spleen cells, and small intestine

[0223] In addition, changes in IgE content in blood, spleen cells, and small intestine were measured and shown in Figures 9a to 9c. There was a significant difference in IgE content between the whey protein (WP) administration group and the hypoallergenic whey protein hydrolysate (HWPH) administration group. Since α-lactalbumin and β-lactoglobulin were decomposed in the hypoallergenic whey protein hydrolysate (HWPH), it was confirmed that the level of intracellular immune response related to allergens was lower than that of whey protein (WP), indicating low allergenicity.

Claims

1. A low-allergenic whey protein hydrolysate obtained by hydrolyzing whey protein using an endoprotease derived from Bacillus licheniformis, an exoprotease derived from Aspergillus oryzae, or a combination thereof.

2. A low-allergenic whey protein hydrolysate according to claim 1, wherein the endoprotease derived from Bacillus licheniformis is Alcalase, Protamex, or a mixed enzyme thereof.

3. A low-allergenic whey protein hydrolysate according to claim 1, wherein the exoprotease derived from Aspergillus oryzae is Flavorzyme, Prozyme, or a mixed enzyme thereof.

4. In the first paragraph, the combination of the Bacillus licheniformis-derived endoprotease and the Aspergillus oryzae-derived exoprotease is a low-allergenic whey protein hydrolysate in which the Bacillus licheniformis-derived endoprotease and the Aspergillus oryzae-derived exoprotease are mixed in a weight ratio of 1:0.2 to 1:

5.

5. A low-allergenic whey protein hydrolysate according to claim 1, wherein the amino-nitrogen (AN) content of the whey protein hydrolysate is 40 mg / mL to 75 mg / mL.

6. In the first paragraph, the low-allergenic whey protein hydrolysate reduces milk allergic reactions.

7. A food composition with reduced milk allergenicity comprising the low-allergenic whey protein hydrolyzate of paragraph 1 as an active ingredient.

8. A method for producing a low-allergenic whey protein hydrolysate comprising the following steps: (a) a step of preparing a whey protein suspension of 5% (w / v) to 20% (w / v) by mixing whey protein and water; and (b) A step of performing hydrolysis by adding 0.5% (w / v) to 2% (w / v) of an endoprotease derived from Bacillus licheniformis, an exoprotease derived from Aspergillus oryzae, or a combination thereof to the whey protein suspension.

9. A method for producing a low-allergenic whey protein hydrolysate in claim 8, wherein the endoprotease derived from Bacillus licheniformis is Alcalase, Protamex, or a mixed enzyme thereof.

10. A method for producing a low-allergenic whey protein hydrolysate in claim 8, wherein the exoprotease derived from Aspergillus oryzae is Flavorzyme, Prozyme, or a mixed enzyme thereof.

11. A method for producing a low-allergenic whey protein hydrolysate, wherein the combination of the Bacillus licheniformis-derived endoprotease and the Aspergillus oryzae-derived exoprotease in step (b) is a mixture of the Bacillus licheniformis-derived endoprotease and the Aspergillus oryzae-derived exoprotease in a weight ratio of 1:0.2 to 1:

5.

12. A method for producing a low-allergenic whey protein hydrolysate, wherein the hydrolysis in step (b) is performed for 5 to 20 hours in the 8th paragraph.

13. A method for producing a low-allergenic whey protein hydrolysate, wherein α-lactalbumin and β-lactoglobulin in whey protein are decomposed through step (b) in paragraph 8.

14. A method for producing a low-allergenic whey protein hydrolysate, wherein the hydrolysis in step (b) is performed at a temperature of 30°C to 70°C.

15. A method for producing a low-allergenic whey protein hydrolysate, wherein the method is performed under conditions of pH 6.0 to pH 9.0 in the 8th paragraph.

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