Method for producing milk protein hydrolysate

Abstract

The present invention addresses the problem of providing a method for obtaining a milk protein degradation product which has good emulsifiability despite a low-molecular-weight peptide. A method for producing a milk protein degradation product comprises a protein degradation step in which a proteolytic enzyme is made to act on the milk protein, wherein: the proteolytic enzyme comprises a trypsin-like endo-type protease derived from microorganisms, an endo-type protease derived from bacteria belonging to the genus Bacillus, and papain; and the number average molecular weight of the milk protein degradation product is 650 or less.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a milk protein hydrolysate. [Background technology]

[0002] In the prevention and treatment of food allergies in infants, antiallergic formulas with reduced antigenicity of proteins are used. Such formulas generally contain milk proteins, such as whey protein and casein, that have been hydrolyzed to reduce their antigenicity. From the viewpoint of digestibility, milk proteins, which serve as nitrogen sources in formulas, are usually formulated as hydrolyzed products.

[0003] In order to improve the absorption of fat in formula, it is desirable that the fat in the formula be emulsified. However, the emulsifying ability of milk protein hydrolysates is generally lower than that of milk proteins, and when formula is produced from milk protein hydrolysates and fat, it is difficult to maintain an emulsified state in which fat globules are completely dispersed. Even when emulsification is performed using a conventional homogenizer (for example, a homogenizer that passes through a homogenizing valve), the fat globules easily aggregate, resulting in a fat-separated state, and the fat separates into the upper layer of the liquid.

[0004] Therefore, various production methods have been devised in the past in search of milk protein hydrolysates with excellent emulsifying properties. For example, Patent Document 1 discloses that a milk protein hydrolysate with excellent emulsifying properties, heat stability, and low antigenicity can be produced by hydrolyzing whey protein using a combination of three enzymes: Bacillus subtilus-derived endoprotease, porcine trypsin, and papain. It has also been reported that trypsin or chymotrypsin derived from microorganisms is used when producing a milk protein hydrolysate by decomposing milk proteins (Patent Documents 2 to 5, etc.). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 3226695 [Patent Document 2] International Publication No. 2012 / 042013 [Patent Document 3] International Publication No. 2010 / 112546 [Patent Document 4] Patent Publication No. 2000-063284 [Patent Document 5] Japanese Patent Publication No. 6-343422 Summary of the Invention [Problem to be solved by the invention]

[0006] As mentioned above, for milk protein hydrolysates to be incorporated into infant formula and the like, there is a demand for the molecular weight of the peptides that are the hydrolysates to be small from the viewpoints of low antigenicity, digestibility, etc. On the other hand, generally, the higher the degree of protein hydrolysis, the poorer the emulsifying ability tends to be. Conventional milk protein hydrolysis methods have not always been able to achieve both the emulsifying ability and low molecular weight of the milk protein hydrolysates at the same time. In view of this situation, an objective of the present invention is to provide a method for obtaining a milk protein hydrolysate that is a low molecular weight peptide but has good emulsifying properties. [Means for solving the problem]

[0007] As a result of intensive research, the present inventors have discovered that the above-mentioned problems can be solved by decomposing milk proteins using a combination of three types of proteolytic enzymes: a trypsin-like endoprotease derived from a microorganism, an endoprotease derived from a bacillus bacterium, and papain, and have completed the present invention.

[0008] That is, the present invention is as follows. [1] A method for producing a milk protein hydrolysate, A proteolysis step of allowing a protease to act on the milk protein, the protease comprises a trypsin-like endoprotease derived from a microorganism, an endoprotease derived from a bacillus bacterium, and papain; A production method in which the number average molecular weight of the milk protein hydrolysate is 650 or less. [2] The method of producing according to [1], wherein the milk protein is whey protein. [3] The method according to [1] or [2], wherein the microorganism is a Fusarium bacterium. [4] The method according to any one of [1] to [3], wherein the protease is substantially free of animal-derived proteases. [Effects of the Invention]

[0009] The present invention provides a method for producing a milk protein hydrolysate that is a highly hydrolyzed, low-molecular-weight peptide with good emulsifying properties. The milk protein hydrolysate produced by this method exhibits excellent emulsion stability when mixed with fat, good heat stability, and low antigenicity, making it suitable for incorporation into infant formula and the like. Furthermore, by not using animal-derived enzymes such as porcine-derived enzymes, it is possible to produce a milk protein hydrolysate that meets the standards for halal food certification, making it possible to provide products that are suitable for a variety of cultures. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, embodiments of the present invention will be described. However, the present invention is not limited to the following preferred embodiments, and can be freely modified within the scope of the present invention. In this specification, when a numerical range is expressed as "lower limit to upper limit," the upper limit may be "equal to or less than" or "less than," and the lower limit may be "equal to or greater than" or "more than."

[0011] The method for producing a milk protein hydrolysate of the present invention includes a proteolysis step in which a protease is allowed to act on a milk protein. The milk protein is not particularly limited as long as it is a protein derived from milk, and examples thereof include whey protein, casein, caseinate, casein rennet, etc., with whey protein being more preferred. Examples of milk include milk from humans, cows, horses, sheep, goats, pigs, etc. Note that when using milk protein hydrolysates in halal foods, it is best to avoid proteins derived from pig milk. The whey protein can be one or more selected from commercially available products, whey isolated from milk, skim milk, etc. by known methods (e.g., cheese whey, acid whey, membrane-separated whey, whey powder, desalted whey powder, etc.), separated and purified whey protein concentrate (WPC), whey protein isolate (WPI), or whey produced by genetic engineering technology, etc. The casein may be one or more selected from commercially available products, caseins isolated from milk or skim milk by known methods, and caseins produced by genetic recombination techniques, etc. Caseins are classified into α-casein, β-casein, and κ-casein, and any of these can be used in the present invention.

[0012] The proteolytic enzymes used in the production method of the present invention include trypsin-like endoproteases derived from microorganisms, endoproteases derived from Bacillus bacteria, and papain.

[0013] The microbial trypsin-like endoprotease is a serine protease, and although its origin is not particularly limited as long as it is a microorganism, it is preferably derived from bacteria of the genus Fusarium, and more preferably from Fusarium oxysporum. One or more types of microbial trypsin-like endoproteases may be used. Commercially available products may also be used. Commercially available trypsin-like endoproteases derived from microorganisms are not particularly limited, but preferred examples include Formea ​​TL 1200 BG (Novozymes) derived from Fusarium oxysporum.

[0014] The amount of microbial trypsin-like endoprotease used can be appropriately set depending on the yield of the desired milk protein hydrolysate, etc. For example, the upper limit is preferably 20 activity units or less, 19 activity units or less, 18 activity units or less, 17 activity units or less, 16 activity units or less, 15 activity units or less, 14 activity units or less, 13 activity units or less, 12 activity units or less, or 11 activity units or less, more preferably 10 activity units or less, 9 activity units or less, 8 activity units or less, 7 activity units or less, 6 activity units or less, and even more preferably 5 activity units or less, per gram of milk protein. The lower limit is not particularly limited, but may be 1 activity unit or more per gram of milk protein. A suitable range may be any combination of the above-mentioned upper and lower limits that are not inconsistent, and preferably 1 to 20 activity units per gram of milk protein, more preferably 1 to 10 activity units per gram of milk protein, and even more preferably 1 to 5 activity units per gram of milk protein. Here, the activity unit of a trypsin-like endoprotease is defined as the amount of enzyme determined according to the method described in WO2021 / 004817A1, etc. Specifically, it refers to the amount of enzyme that produces 1 micromole of p-nitroaniline per minute from the substrate Ac-Arg-p-nitroanilide (Ac-Arg-pNA) and / or Ac-Lys-p-nitroanilide (Ac-Arg-pNA) at 37°C and pH 8.0.

[0015] Examples of endoproteases derived from Bacillus bacteria include those derived from Bacillus subtilus, Bacillus licheniformis, Bacillus amyloliquefaciens, etc. Proteases are classified into alkaline proteases, neutral proteases, and acidic proteases, with neutral proteases being more preferred. One or more types of endo-proteases derived from Bacillus bacteria may be used. Commercially available products may also be used.

[0016] Commercially available endoproteases derived from Bacillus bacteria are not particularly limited, but preferred examples include Bioprase SP-20 (manufactured by Nagase ChemteX Corporation), Protin SD-AY50 (manufactured by Amano Enzyme Inc.), Protease N Amano (manufactured by Amano Enzyme Inc.), and Neutrase (manufactured by Novozymes).

[0017] The amount of endoprotease derived from Bacillus bacteria used can be appropriately set depending on the yield of the desired milk protein hydrolysate, etc. For example, the upper limit is preferably 5,000 or less, 4,500 or less, more preferably 4,000 or less, 3,500 or less, and even more preferably 3,000 or less, per gram of milk protein, and the lower limit is 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, and more preferably 1,000 or more, per gram of milk protein. Suitable ranges may be any combination of the upper and lower limits that are consistent with each other, with 500 to 5,000 activity units per gram of milk protein being preferred, 1,000 to 4,000 activity units per gram of milk protein being more preferred, and 1,000 to 3,000 activity units per gram of milk protein being more preferred. Here, the activity unit of endoprotease derived from Bacillus bacteria refers to the amount of enzyme that causes an increase in the amount of Folin's test solution color substance equivalent to 1 μg of L-tyrosine per minute at 37°C and pH 7, using casein as the substrate.

[0018] Papain is a cysteine ​​protease derived from papaya. One or more types of papain may be used. Commercially available papain products may also be used. Commercially available papain products are not particularly limited, but preferred examples include Papain W-40 (manufactured by Amano Enzyme) and Purified Papain (manufactured by Asahi Breweries, Ltd.).

[0019] The amount of papain used can be appropriately set depending on the yield of the desired milk protein hydrolysate, etc. For example, the upper limit is preferably 5,000 activity units or less per gram of milk protein, more preferably 4,000 activity units or less, 3,500 activity units or less, and even more preferably 3,000 activity units or less, and the lower limit is 500 activity units or more, 600 activity units or more, 700 activity units or more, 800 activity units or more, 900 activity units or more, more preferably 1,000 activity units or more, and even more preferably 1,500 activity units or more per gram of milk protein. Furthermore, a suitable range may be any combination of the above-mentioned upper and lower limits that is not inconsistent, and preferably 500 to 5,000 activity units per gram of milk protein, more preferably 1,000 to 4,000 activity units per gram of milk protein, and even more preferably 1,500 to 3,000 activity units per gram of milk protein. Here, an activity unit of papain refers to the amount of enzyme that can increase the absorbance of the substrate casein at a wavelength of 275 nm, equivalent to 1 μg of tyrosine, in one minute at 38° C. and pH 6.

[0020] In the present invention, a combination of three types of proteases, namely, a trypsin-like endoprotease derived from a microorganism, an endoprotease derived from a bacillus bacterium, and papain, is used, but other proteases may be used as long as the effects of the present invention are not impaired. Examples of other proteases include those derived from animals, microorganisms, and plants, but it is preferable that animal-derived proteases are not substantially used in the present invention. Here, "substantially not used" does not only mean that no animal-derived proteases are used at all, but also means that the milk protein decomposition activity of the animal-derived proteases cannot be measured, or even if the milk protein decomposition activity of the animal-derived proteases can be measured, it is lower than the decomposition activity of the three types of proteases, and the animal-derived proteases are contained to an extent that they do not exert their effect as proteases, and are negligible from the perspective of a person skilled in the technical field to which the present invention pertains.

[0021] For example, "animal-derived proteases are not substantially used" can be said to be "substantially not used" when the milk protease hydrolysis activity of the animal-derived protease is 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.1% or less, 0.05% or less, or 0.01% or less when the milk protease hydrolysis activity of a microbial trypsin-like endoprotease, a Bacillus endoprotease, or papain is taken as 100%.

[0022] As used herein, "derived from a microorganism," "derived from a bacillus bacterium," or "derived from papain" refers to the microorganism or plant that originally possesses the protease, and does not refer to the source of collection. For example, a protease produced by introducing a gene encoding a protease produced by a bacillus bacterium into Escherichia coli and expressing the gene is "derived from" a bacillus bacterium. Furthermore, when "originally possessed by a bacillus bacterium," the bacillus bacterium includes not only wild-type but also recombinant or mutant forms, as long as it maintains the ability to produce a protease with the protease activity and specificity required for the present invention.

[0023] In the proteolysis step of the present invention, a protease is allowed to act on milk proteins. A general procedure is described below, but the present invention is not limited to this. The raw milk protein is dispersed or dissolved in water or warm water. Next, the milk protein liquid is desirably heat-sterilized at 70 to 130°C for 2 seconds to 10 minutes to prevent spoilage due to bacterial contamination. Heat-sterilization conditions include, for example, 85°C for 10 minutes, 90°C for 6 minutes, 121°C for 1 minute, and 130°C for 2 seconds. Furthermore, the heat-sterilized milk protein liquid may be desalted by an ion exchange method using a sodium or potassium type cation exchange resin (preferably a strongly acidic cation exchange resin), an electrodialysis method, an ultrafiltration membrane method, a nanofiltration membrane method, etc. Either a column method or a batch method may be used for desalting. Next, it is preferable to add an alkaline agent or an acid agent to the milk protein solution and adjust the pH to the optimum pH for the hydrolase used or to a pH value close to the optimum pH. The alkaline agent or acid agent used in the production method of the present invention is not particularly limited as long as it is acceptable for food or pharmaceutical use. Specific examples of alkaline agents include sodium hydroxide, potassium hydroxide, potassium carbonate, etc., and examples of acid agents include hydrochloric acid, citric acid, phosphoric acid, acetic acid, etc.

[0024] Next, a predetermined amount of protease is added to the milk protein solution, and the reaction is carried out at a temperature of 10 to 85°C for about 0.1 to 48 hours. The enzyme reactions of the three types of proteases mentioned above may be carried out simultaneously or separately.

[0025] The solution containing the enzyme is maintained at an appropriate temperature depending on the type of enzyme to initiate hydrolysis of the milk protein. The temperature may be, for example, 30°C or higher, 40°C or higher, or 45°C or higher, or 60°C or lower, or 55°C or lower. Any compatible combination of these may also be used. The preferred temperature range is 30 to 60°C, preferably 45 to 55°C. The hydrolysis reaction is continued while monitoring the hydrolysis rate of the enzyme reaction until a desired hydrolysis rate is reached. To obtain hydrolyzed products corresponding to the molecular weights described below, the hydrolysis rate may be 8% or higher, 9% or higher, 10% or higher, 11% or higher, 12% or higher, 13% or higher, 14% or higher, or 15% or higher, and may be 35% or lower, 34% or lower, 33% or lower, 32% or lower, 31% or lower, 30% or lower, 29% or lower, 28% or lower, 27% or lower, 24% or lower, or 25% or lower. Any compatible combination of these may also be used. The preferred range of the decomposition rate is 8 to 35%, 10 to 30%, or 15 to 25%.

[0026] The decomposition rate of milk protein is calculated by measuring the total nitrogen content of the sample using the Kjeldahl method (Food Analysis Methods, edited by the Japan Food Industry Association, p. 102, Korin Co., Ltd., 1984), measuring the formol nitrogen content of the sample using the formol titration method (Food Engineering Experiment Book, edited by Mitsuda et al., Vol. 1, p. 547, Yokendo, 1970), and then calculating the decomposition rate from these measured values ​​using the following formula. Decomposition rate (%) = amount of formol nitrogen ÷ total amount of nitrogen × 100

[0027] The enzymatic reaction can be stopped, for example, by inactivating the enzyme in the hydrolyzed solution, which can be carried out by a conventional heat inactivation treatment. The heating temperature and holding time for the heat inactivation treatment can be appropriately set to ensure sufficient inactivation, taking into account the thermal stability of the enzyme used. For example, the treatment can be carried out at a temperature in the range of 80 to 130°C for a holding time of 2 seconds to 30 minutes.

[0028] After heat inactivation, the product is cooled in a conventional manner and can be used as is, or can be concentrated to obtain a concentrate, if necessary. The concentrate can also be dried to obtain a powder product. The obtained milk protein hydrolysate can also be subjected to separation, purification, and other procedures in a conventional manner.

[0029] Purification of milk protein hydrolysates can be carried out by appropriately combining techniques similar to those typically used for purifying peptides, such as various chromatographic methods such as ion exchange chromatography, adsorption chromatography, reverse phase chromatography, partition chromatography, and gel filtration chromatography, as well as methods such as solvent precipitation, salting out, and partitioning between two liquid phases.

[0030] The upper limit of the number average molecular weight of the milk protein hydrolysate obtained by the production method of the present invention is 650 or less, 640 or less, optionally 630 or less, optionally 620 or less, optionally 610 or less, preferably 600 or less, 590 or less, 580 or less, 570 or less, or 560 or less, more preferably 550 or less, 540 or less, or 530 or less, and the lower limit may be 230 or more, 240 or more, or 250 or more, preferably 260 or more, 270 or more, more preferably 280 or more. Any consistent combination of these may also be used. Here, the average molecular weight of the milk protein hydrolysate is determined based on the concept of number average molecular weight as follows. The number average molecular weight (NAM) is a measure of the average molecular weight of a polymer compound, as described in, for example, "Fundamentals of Polymer Science," edited by the Society of Polymer Science, pp. 116-119, published by Tokyo Kagaku Dojin Co., Ltd. in 1978, based on the following different indices: In other words, since high molecular weight compounds such as milk protein hydrolysates are heterogeneous substances and have a distribution of molecular weights, the molecular weight of milk protein hydrolysates must be expressed as an average molecular weight in order to handle them physicochemically, and the number average molecular weight (hereinafter sometimes abbreviated as Mn) is the average for the number of molecules.

[0031] As used herein, the average molecular weight of a milk protein hydrolysate is measured and calculated by the following method. Specifically, high-performance liquid chromatography was performed using a Poly Hydroxyethyl Aspartamide Column (Poly LC, diameter 4.6 mm, length 200 mm) with 20 mM sodium chloride and 50 mM formic acid at an elution rate of 0.5 mL / min (N. Ui et al., eds., "High-Performance Liquid Chromatography of Proteins and Peptides," Chemical Special Issue No. 102, p. 241, Kagaku Dojin Co., Ltd., 1984). Detection was performed using a UV detector (Shimadzu Corporation), and data analysis was performed using a GPC analysis system (Shimadzu Corporation) to calculate the number-average molecular weight. Proteins and / or peptides with known molecular weights may be used as standard samples for molecular weight calculation.

[0032] In addition, milk protein hydrolysates generally contain free amino acids during the manufacturing process.

[0033] The milk protein hydrolysate obtained by the production method of the present invention is a highly hydrolyzed, low-molecular-weight peptide, yet has good emulsifying properties. Due to its good emulsifying properties, it exhibits excellent emulsion stability when mixed with fat. Furthermore, the milk protein hydrolysate obtained by the production method of the present invention also has good heat stability.

[0034] Furthermore, the milk protein hydrolysates obtained by the production method of the present invention have a high degree of hydrolysis and a low molecular weight, and therefore have reduced antigenicity. Generally, antigens that can be problematic in milk proteins and their hydrolysates include casein and β-lactoglobulin, but the milk protein hydrolysates obtained by the production method of the present invention have low antigenicity to these. Specifically, the residual antigenic activity of casein is preferably 2 ppm or less, more preferably 1.5 ppm or less. Furthermore, the residual antigenic activity of β-lactoglobulin is preferably 150 ppm or less, more preferably 100 ppm or less, and even more preferably 75 ppm or less.

[0035] Furthermore, the milk protein hydrolysates obtained by the manufacturing method of the present invention can be produced as excellent milk protein hydrolysates without using animal-derived milk protease enzymes, and therefore can be suitably used in products that meet the standards for halal food certification.

[0036] The milk protein hydrolysate obtained by the production method of the present invention can be incorporated into pharmaceuticals, foods and beverages, feed, etc. Since the milk protein hydrolysate obtained by the production method of the present invention uses milk-derived raw materials, it is highly safe for living organisms and is suitable for long-term, continuous ingestion. In addition, because it is produced from milk-derived raw materials, which are relatively inexpensive as biological materials, it can be produced stably, easily, and in large quantities, and can be provided to consumers at low cost.

[0037] The milk protein hydrolysate obtained by the production method of the present invention can be preferably contained in foods and beverages. The foods and beverages also include additives to be added to foods and beverages or pharmaceuticals. Examples of such additives include additives to expressed breast milk or formula, and it is expected that the milk after addition will be ingested by newborns or infants. Food and beverages are usually taken orally, but are not limited to this and may be taken nasally, or through a gastrostomy or enterostomy. For example, it is envisioned that newborns and infants will be given formula milk (described below) or breast milk supplemented with a milk protein hydrolysate through a nasogastric feeding tube or the like.

[0038] Food and drink products include, regardless of their form, liquid, paste, gel-like solid, powder, etc., such as tablet sweets; wheat flour products such as bread, macaroni, spaghetti, noodles, cake mix, fried chicken flour, breadcrumbs, etc.; instant noodles, cup noodles, retort / prepared foods, canned foods, microwave foods, instant soup / stew, instant miso soup / cleaning liquid, canned soup, freeze-dried foods, other instant foods, etc.; canned agricultural products, canned fruit, jams / marmalades, pickles, etc. processed agricultural products such as vegetables, boiled beans, dried agricultural goods, and cereals (processed grain products); processed seafood products such as canned seafood, fish ham and sausage, fish paste products, seafood delicacies, and tsukudani (fish stew); processed livestock products such as canned livestock paste, livestock ham and sausage; processed milk, milk drinks, yogurt, lactic acid bacteria drinks, cheese, ice cream, cream, and other dairy products; fats and oils such as butter, margarine, and vegetable oil; soy sauce, miso, sauces, and processed tomato seasonings basic seasonings such as mirin, vinegars, etc.; complex seasonings and foods such as cooking mixes, curry bases, sauces, dressings, noodle soups, spices, and other complex seasonings; frozen foods such as frozen ingredients, semi-cooked frozen foods, and cooked frozen foods; sweets such as caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese sweets, rice snacks, bean snacks, dessert sweets, jelly, and other sweets; beverages such as carbonated drinks, natural fruit juice, fruit juice drinks, soft drinks with fruit juice, fruit pulp drinks, fruit drinks with fruit pieces, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, energy drinks, alcoholic drinks, and other beverages; other commercially available foods such as baby food, furikake, and ochazuke nori seaweed; nutritional compositions such as infant formula (including powdered milk, liquid milk, etc.), liquid foods, and supplements; and functional foods (foods for specified health uses, foods with nutrient claims).

[0039] Of these, nutritional compositions are preferred. In the present invention, the "nutritional composition" is an embodiment of a food or drink, and is not particularly limited, but is preferably a formula, a liquid diet, a supplement, etc., more preferably a formula. The target of intake may be an infant, a small child, a child, or an adult, but is preferably an infant or a small child. Formulated milk includes powdered milk and liquid milk. Formulated milk powder is defined in the Ministerial Ordinance on the Compositional Standards of Milk and Dairy Products (Milk Ministerial Ordinance) as "a powder made by processing raw milk, cow's milk, special milk, or foods made from these ingredients, or using them as the main ingredient, and adding nutrients necessary for infants." The ministerial ordinance defines modified liquid milk as "a liquid product made by processing raw milk, cow's milk, special milk, or foods made from these ingredients, or using these as the main ingredient, and adding nutrients necessary for infants." In addition, modified milk is a mixture of various nutritional components such as proteins, fats and oils, carbohydrates, minerals, and vitamins, and includes those processed into powder or liquid form. Furthermore, prepared milk also includes "powdered infant formula," "liquid infant formula," and "powdered milk for pregnant and nursing women," which are special-use foods defined in the Health Promotion Act, as well as forms such as powdered infant formula, nutritional powder for adults, and nutritional powder for the elderly.

[0040] When in the form of a supplement, it can be formulated into solid preparations such as powders, granules, tablets, capsules, etc.; liquid preparations such as solutions, syrups, suspensions, emulsions, etc. When formulating such preparations, the ingredients, carriers, and methods for formulating pharmaceuticals described below can be followed.

[0041] Furthermore, one aspect of the food and drink product may be feed, such as pet food, livestock feed, and fish feed. The form of the feed is not particularly limited, and may contain, for example, grains such as corn, wheat, barley, rye, milo, etc.; vegetable oil cakes such as soybean oil cake, rapeseed oil cake, palm oil cake, linseed oil cake, etc.; bran such as wheat bran, wheat bran, rice bran, defatted rice bran, etc.; manufacturing residues such as corn gluten meal and corn jam meal; animal feeds such as fish meal, skim milk powder, whey, yellow grease, tallow, etc.; yeasts such as torula yeast and brewer's yeast; mineral feeds such as calcium triphosphate and calcium carbonate; oils and fats; simple amino acids; sugars, etc.

[0042] The milk protein hydrolysate obtained by the production method of the present invention may be contained as an active ingredient in a pharmaceutical product. The pharmaceutical form can be formulated into a desired dosage form depending on the administration method. For example, in the case of oral administration, it can be formulated into solid preparations such as powders, granules, tablets, and capsules; or liquid preparations such as solutions, syrups, suspensions, and emulsions. In addition, in the case of parenteral administration, it can be formulated into suppositories, ointments, injections, and the like. When preparing the formulation, ingredients commonly used in formulations, such as excipients, pH adjusters, colorants, and flavoring agents, can be used. It is also possible to use other medicinal ingredients, known or future prebiotics against Bifidobacterium bacteria, or prebiotics against other bacteria in combination. In addition, formulation can be carried out by a known method as appropriate depending on the dosage form. When formulating, a pharmaceutical carrier may be appropriately blended to form the formulation.

[0043] Examples of excipients include sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, α-starch, dextrin, and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, and carboxymethyl cellulose calcium; gum arabic; dextran; pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, and magnesium aluminometasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; and sulfate derivatives such as calcium sulfate.

[0044] Examples of binders include gelatin, polyvinylpyrrolidone, macrogol, and the like, in addition to the above-mentioned excipients.

[0045] Examples of disintegrants include the above-mentioned excipients as well as chemically modified starch or cellulose derivatives such as croscarmellose sodium, carboxymethyl starch sodium, and cross-linked polyvinylpyrrolidone.

[0046] Examples of lubricants include talc; stearic acid; metal stearates such as calcium stearate and magnesium stearate; colloidal silica; waxes such as veegum and gaelt; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid; sodium carboxylates such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as silicic anhydride and silicic acid hydrate; and starch derivatives.

[0047] Examples of stabilizers include paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; sorbic acid; and the like.

[0048] Examples of flavoring agents include sweeteners, acidulants, and fragrances. In the case of a liquid preparation for oral administration, examples of the carrier to be used include solvents such as water. [Example]

[0049] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples as long as it does not depart from the gist of the invention.

[0050] <Test Example 1> Production of milk protein hydrolysate by enzymatic hydrolysis 1 (1) Enzymatic hydrolysis of whey Various proteases were allowed to act on whey protein to produce milk protein hydrolysates. Specifically, 50 g of bovine milk-derived whey protein (Milei 80, manufactured by Mirai Co., Ltd.) was added to 450 g of water and thoroughly dispersed. Sodium hydroxide was added to adjust the pH of the solution to 9, and the temperature of the solution was adjusted to approximately 50°C. A predetermined amount of the enzyme shown in Table 1 was added to the solution to initiate the hydrolysis reaction. After 5 hours, the solution was heated at 85°C for 10 minutes to inactivate the enzyme and terminate the enzymatic reaction, and then cooled to 5°C. This hydrolyzed solution was concentrated and then freeze-dried to obtain a freeze-dried milk protein hydrolysate.

[0051] (2) Measurement of molecular weight of milk protein hydrolysate The obtained milk protein hydrolysate was subjected to high performance liquid chromatography (HPLC) under the following conditions to measure the molecular weight distribution. Column: Poly Hydroxyethyl Aspartamide Column (Poly LC, diameter 4.6 mm and length 200 mm) Mobile phase: Aqueous solution containing 20 mM sodium chloride and 50 mM formic acid Elution rate: 0.5mL / min Detection: UV detector (Shimadzu Corporation) Analysis: A GPC analysis system (Shimadzu Corporation) was used. From the peak areas of the obtained HPLC chart, the molecular weight was calculated based on [percentage (%) of each molecular weight range] = [area of ​​each molecular weight range in the molecular weight distribution / total area (total area) of the milk protein hydrolysate in the molecular weight distribution]. The number average molecular weight and weight average molecular weight are also shown in Table 1.

[0052] (3) Measurement of the decomposition rate of milk protein hydrolysates The degree of decomposition (decomposition rate) of the milk protein hydrolysate was measured by formol titration. Specifically, the sample powder was dissolved in deionized water at a concentration of 10% w / w. 30 mL of deionized water was added to 4 mL of the solution. While stirring with a stirrer, 0.1 M sodium hydroxide solution or 0.1 M hydrochloric acid solution was added dropwise using a pH meter to adjust the pH to 6.80. 5 mL of 37% formalin solution adjusted to pH 8.0 was then added, and the solution was titrated with 0.1 M sodium hydroxide solution to a pH of 7.90 (x mL). At the same time, the Brix value (z%) of the milk protein hydrolysate solution was measured using a saccharimeter. Based on the above measured values, the decomposition rate of the milk protein hydrolysate was calculated using the following formula, and the results are also shown in Table 1. Decomposition rate (%) = amount of formol nitrogen ÷ total amount of nitrogen × 100 Formol nitrogen (mgN / dL) = 1.4 x x f (sodium hydroxide titer) x 100 / 4 Total nitrogen (mgN / dL) = z × 0.7 × 1000 / 6.38

[0053] (4) Evaluation of emulsion stability of milk protein hydrolysates The emulsion stability of milk protein hydrolysates was evaluated when mixed with water and fats and oils. Specifically, 10 g of sample powder was dissolved in 150 mL of deionized water, and 100 g of soybean oil (Fujifilm Wako Pure Chemical Industries, Ltd.) was added. The mixture was emulsified at 5000 rpm using a homogenizer (TK HOMOMIXER MARK II, formerly Tokushu Kika Kogyo Co., Ltd., now Primix Corporation) passing through a homogenizing valve. The resulting emulsion was placed in a graduated cylinder and stored at room temperature for 24 hours. The heights of the oil, emulsion, and water layers were measured, and their proportions relative to the total liquid were calculated. The emulsions were visually observed after storage and rated as follows: no separation of fats and oils (good), slight separation (good), and separation (poor). The results are shown in Table 1.

[0054] (5) Evaluation of thermal stability The thermal stability of the milk protein hydrolysates was evaluated by a retort sterilization test. Specifically, 200 mL of a solution prepared by dissolving the sample powder in deionized water at a concentration of 5% w / w was sealed in a retort pouch and subjected to heat sterilization (130°C, 90 seconds, 0.3 MPa) equivalent to an F value of 4 using a high-temperature, high-pressure cooking sterilizer (RCS-60 / 10SPXTG-FAM). The solution after heat sterilization was visually inspected for the presence or absence of precipitation. The absence of precipitation or precipitation was evaluated as ◯, and the presence of precipitation was evaluated as ×. The results are also shown in Table 1.

[0055] (6) Evaluation of antigenicity The antigenicity of milk protein hydrolysates was evaluated using a Morinaga FASPEK ELISA II (Morinaga Biological Science Institute). Specifically, the sample powder was diluted 20-fold with the kit's sample diluent I to a total peptide concentration of 1–50 ng / mL. 100 μL of the diluted solution was poured into plastic wells containing the primary antibody and incubated for 1 hour. After washing the plastic wells with the prepared washing solution, the enzyme-labeled antibody was reacted for 30 minutes. The epitopes detected were epitopes contained in the casein and β-lactoglobulin sequences, respectively. The wells were then washed with the prepared washing solution, and the enzyme substrate solution was added to the wells. The reaction was terminated with a reaction stop solution. Within 30 minutes after the reaction was terminated, the absorbance was measured at a dominant wavelength of 450 nm and a secondary wavelength of 620 nm. The measured absorbance was applied to a calibration curve prepared by simultaneous measurements to determine the total protein concentration in the sample diluent. This was then multiplied by the dilution factor to determine the residual antigen activity in the sample. The residual antigen activity was evaluated on a three-point scale according to the following criteria. The results are shown in Table 1. casein; ○: 2 ppm or less (non-antigenic) △: 2-50 ppm (slightly antigenic) ×: 50 ppm or more (antigenic) β-lactoglobulin; ○: 150 ppm or less (non-antigenic) △: 150-2000 ppm (slightly allergenic) ×: 2000 ppm or more (antigenic)

[0056] [Table 1]

[0057] The results in Table 1 show that the milk protein hydrolysates prepared by the production method of the present invention are hydrolyzed to low molecular weights at a high hydrolysis rate, while exhibiting high emulsion stability with little separation of oil and water. They also have excellent thermal stability and antigenicity.

[0058] <Test Example 2> Production of milk protein hydrolysate by enzymatic hydrolysis 2 Using the enzymes and amounts thereof shown in Table 2, milk protein hydrolysates were produced in the same manner as in Test Example 1, and the molecular weight, hydrolysis rate, emulsification stability, thermal stability, and antigenicity were evaluated. The results are also shown in Table 2. It has been found that the milk protein hydrolysate prepared by the production method of the present invention is hydrolyzed to low molecular weights at a high hydrolysis rate, but exhibits high emulsion stability with little separation of oil and water.It has also been found that it has excellent thermal stability and antigenicity.

[0059] [Table 2]

[0060] <Test Example 3> Production of milk protein hydrolysate by enzymatic hydrolysis 3 Using the enzymes and amounts thereof shown in Table 3, milk protein hydrolysates were produced in the same manner as in Test Example 1, and the molecular weight, hydrolysis rate, and emulsion stability were measured. The results are also shown in Table 3. It can be seen that the milk protein hydrolysate prepared by the production method of the present invention is decomposed to low molecular weights at a high decomposition rate, yet has little separation of oil and water, and has high emulsion stability.

[0061] [Table 3]

[0062] <Test Example 4> Production of milk protein hydrolysate by enzymatic hydrolysis 3 Using the enzymes and amounts thereof shown in Table 4, milk protein hydrolysates were produced in the same manner as in Test Example 1, and the molecular weight, hydrolysis rate, and emulsion stability were evaluated. The results are also shown in Table 3. It can be seen that the milk protein hydrolysate prepared by the production method of the present invention is hydrolyzed to low molecular weights at a high hydrolysis rate, yet has little separation of oil and water, and has high emulsion stability.

[0063] [Table 4] [Industrial Applicability]

[0064] The present invention is useful in the fields of pharmaceuticals, foods and beverages, feeds, etc.

Claims

1. A method for producing a milk protein hydrolysate, A proteolysis step of allowing a protease to act on the milk protein, the protease comprises a trypsin-like endoprotease derived from a microorganism, an endoprotease derived from a bacillus bacterium, and papain; the microorganism is Fusarium oxysporum, The production method, wherein the number average molecular weight of the milk protein hydrolysate is 650 or less.

2. The method of claim 1 , wherein the milk protein is whey protein.

3. The method according to claim 1 or 2, wherein the protease is substantially free of animal-derived proteases.

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