WO2023190529A1

WO2023190529A1 - Method for producing milk protein degradation product

Info

Publication number: WO2023190529A1
Application number: PCT/JP2023/012550
Authority: WO
Inventors: 創中田; 智弘椎名
Original assignee: 森永乳業株式会社
Priority date: 2022-03-30
Filing date: 2023-03-28
Publication date: 2023-10-05

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

Method for producing milk protein decomposition product

The present invention relates to a method for producing a milk protein decomposition product.

For the prevention and treatment of food allergies in infants, anti-allergic formula milk with reduced protein antigenicity is used. Such formula milk generally contains milk proteins such as whey protein and casein that have been hydrolyzed to reduce their antigenicity. Furthermore, from the viewpoint of digestibility, milk protein as a nitrogen source in formula milk is usually blended as a decomposed product.

In order to improve the absorption of fat in formula milk, it is considered desirable that the fat in formula milk be emulsified. However, in general, the emulsifying properties of milk protein decomposition products are lower than that of milk proteins, and when preparing milk with milk protein decomposition products and fat, it is difficult to maintain an emulsified state in which fat globules are completely dispersed. Even if emulsification is performed using a normal homogenizer (for example, a homogenizer that passes through a homogenizer valve), fat globules easily aggregate and exhibit a state of fat separation, resulting in fat separation in the upper layer of the liquid.

Therefore, various production methods have been devised in search of milk protein decomposition products with excellent emulsifying properties. For example, Patent Document 1 discloses that whey protein is degraded using a combination of three enzymes: endoprotease derived from Bacillus subtilis, trypsin derived from pigs, and papain, resulting in emulsifying properties, thermostability, and low antigenicity. It is disclosed that an excellent milk protein decomposition product can be produced.
It has also been reported that trypsin or chymotrypsin derived from microorganisms is used when decomposing milk proteins to produce milk protein decomposition products (Patent Documents 2 to 5, etc.).

Patent No. 3226695 International Publication No. 2012/042013 International Publication No. 2010/112546 JP2000-063284 Japanese Patent Application Publication No. 6-343422

As mentioned above, with respect to milk protein decomposition products to be added to formula milk etc., there is a demand for reducing the molecular weight of the peptides that are the decomposition products from the viewpoint of low antigenicity and digestibility. On the other hand, in general, the higher the degree of protein decomposition, the worse the emulsifying property tends to be. Conventional milk protein decomposition methods have not always been able to achieve both emulsifying properties and low molecular weight decomposition of milk protein decomposition products.
In view of this situation, an object of the present invention is to provide a method for obtaining a milk protein decomposition product that is a low molecular weight peptide but has good emulsifying properties.

As a result of extensive research, the present inventors have found that milk proteins can be synthesized using a combination of three types of proteolytic enzymes: trypsin-like endoprotease derived from microorganisms, endoprotease derived from Bacillus bacteria, and papain. It was discovered that the above problems could be solved by disassembling it, and the present invention was completed.

That is, the present invention is as follows.
[1] A method for producing a milk protein decomposition product, comprising:
comprising a proteolytic step of causing a proteolytic enzyme to act on the milk protein,
The proteolytic enzyme includes a trypsin-like endoprotease derived from a microorganism, an endoprotease derived from a Bacillus bacterium, and papain,
The production method, wherein the milk protein decomposition product has a number average molecular weight of 650 or less.
[2] The production method according to [1], wherein the milk protein is whey protein.
[3] The production method according to [1] or [2], wherein the microorganism is a Fusarium bacterium.
[4] The production method according to any one of [1] to [3], wherein the protease is substantially free of animal-derived proteases.

The present invention provides a method for producing a milk protein decomposition product that is a peptide with a high degree of decomposition and a low molecular weight but has good emulsifying properties. The milk protein decomposition product produced by this method shows excellent emulsion stability when mixed with fat, has good thermal stability, and has low antigenicity, so it is suitable for blending into formula milk etc. be able to.
Furthermore, by not using animal-derived enzymes such as pig-derived enzymes, we can create milk protein decomposition products that meet the standards for certification as halal food, providing products that are suitable for a variety of cultures. You can also.

Embodiments of the present invention will be described below. 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 addition, in this specification, when a numerical range is expressed as "lower limit to upper limit", the upper limit may be "less than or equal to" or "less than", and the lower limit may be "more than" or "more than". It may be "super".

The method for producing a milk protein decomposition product of the present invention includes a proteolysis step in which a proteolytic enzyme is allowed to act on milk protein.
The milk protein is not particularly limited as long as it is a milk-derived protein, and examples thereof include whey protein, casein, caseinate, casein rennet, and the like, with whey protein being more preferred. Examples of milk include human, cow, horse, sheep, goat, and pig milk. In addition, when applying milk protein decomposition products to halal foods, it is better to avoid proteins derived from pig milk.
Whey protein is a commercially available product, whey separated from milk or skim milk by a known method (e.g., cheese whey, acid whey, membrane-separated whey, whey powder, desalted whey powder, etc.), or separated and purified whey protein. One or more types selected from concentrates (WPC), whey protein isolates (WPI), those produced by genetic recombination technology, etc. can be used.
As the casein, one or more types selected from commercially available products, casein separated from milk, skim milk, etc. by a known method, and casein produced by genetic recombination technology, etc. can be used. Note that casein is classified into α-casein, β-casein, and κ-casein, and any casein can be used in the present invention.

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

The trypsin-like endoprotease derived from a microorganism is a serine protease, and its origin is not particularly limited as long as it is a microorganism, but those derived from bacteria of the genus Fusarium are preferred, and those derived from Fusarium oxysporum are more preferred. preferable. One or more types of trypsin-like endoproteases derived from microorganisms may be used. Alternatively, commercially available products may be used.
Commercially available trypsin-like endoproteases derived from microorganisms are not particularly limited, but preferred examples include Formea TL 1200 BG (manufactured by Novozymes) derived from Fusarium oxysporum.

The amount of trypsin-like endoprotease derived from microorganisms can be appropriately set depending on the desired yield of milk protein decomposition product, etc., but for example, the upper limit is preferably 20 activity units or less per 1 g of milk protein, 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, 11 activity units or less, more preferably 10 activity units It can be used in an amount of up to 9 active units, up to 8 active units, up to 7 active units, up to 6 active units, more preferably up to 5 active units. Further, the lower limit is not particularly limited, but may be 1 or more activity units per 1 g of milk protein. A suitable range may be any consistent combination of the above upper and lower limits, preferably 1 to 20 activity units per gram of milk protein, more preferably 1 to 10 activity units per gram of milk protein. units, more preferably 1 to 5 active units per gram of milk protein.
Here, the activity unit of trypsin-like endo-protease is defined by the amount of enzyme specified according to the method described in WO2021/004817A1 and the like. Specifically, the substrate Ac-Arg-p-nitro-anilide (Ac-Arg-pNA) and/or Ac-Lys-p-nitro-anilide (Ac-Arg-pNA) was heated at 37°C and at pH 8. 0, the amount of enzyme that produces 1 micromole of p-nitroaniline per minute.

Endo-type proteases derived from bacteria of the genus Bacillus include endo-type proteases derived from Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, etc. Examples include type proteases. Furthermore, proteases are classified into alkaline proteases, neutral proteases, and acidic proteases, with neutral proteases being more preferred.
One or more types of endo-type proteases derived from bacteria of the genus Bacillus may be used. Alternatively, commercially available products may be used.

Commercially available endo-proteases derived from bacteria of the genus Bacillus are not particularly limited; Preferred examples include Neutrase (manufactured by Novozymes) and Neutrase (manufactured by Novozymes).

The amount of endo-protease derived from Bacillus bacteria can be appropriately set depending on the desired yield of milk protein decomposition product, etc., but for example, the upper limit is preferably 5000 activity units or less per 1 g of milk protein, 4500 activity units or less, more preferably 4000 activity units or less, 3500 activity units or less, still more preferably 3000 activity units or less can be used, and the lower limit is 500 activity units or more, 600 activity units or more, 700 activity units per gram of milk protein. Above, 800 or more active units, 900 or more active units, more preferably 1000 or more active units can be used. Further, a suitable range may be any combination of the above-mentioned upper and lower limits that is consistent with each other, preferably 500 to 5000 activity units per gram of milk protein, more preferably 1000 to 5000 activity units per gram of milk protein. 4000 activity units are used, more preferably 1000 to 3000 activity units per gram of milk protein.
Here, the activity unit of endo-type protease derived from bacteria of the genus Bacillus is defined as the amount of enzyme that causes an increase in the colored substance of Folin reagent corresponding to 1 μg of L-tyrosine per minute at 37°C and pH 7 against the substrate casein. say.

Papain is a cysteine protease derived from papaya. One or more types of papain may be used. Alternatively, commercially available products may be used.
Commercial products of papain are not particularly limited, but preferred examples include papain W-40 (manufactured by Amano Enzyme), purified papain (manufactured by Asahi Breweries), and the like.

The amount of papain used can be appropriately set depending on the desired yield of milk protein decomposition products, etc., but for example, the upper limit is preferably 5000 activity units or less, more preferably 4000 activity units or less per 1 g of milk protein. , 3500 activity units or less, more preferably 3000 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, it can be used in an amount of 1000 or more active units, and still more preferably 1500 or more active units. Further, a suitable range may be any combination of the above-mentioned upper and lower limits that is consistent with each other, preferably 500 to 5000 activity units per gram of milk protein, more preferably 1000 to 5000 activity units per gram of milk protein. 4000 activity units are used, more preferably 1500 to 3000 activity units per gram of milk protein.
Here, the activity unit of papain refers to the amount of enzyme that can increase the amount of absorption at a wavelength of 275 nm corresponding to 1 μg of tyrosine per minute at 38° C. and pH 6 with respect to the substrate casein.

In the present invention, a trypsin-like endoprotease derived from a microorganism, an endoprotease derived from a Bacillus bacterium, and papain are used in combination, but other proteolytic enzymes may be used as long as they do not impair the effects of the present invention. It's okay. Examples of other proteolytic enzymes include those derived from animals, microorganisms, and plants; however, in the present invention, it is preferable not to use substantially animal-derived proteolytic enzymes.
Here, "substantially not used" does not mean not only that no animal-derived protease is used at all, but also that the milk proteolytic activity of the animal-derived protease cannot be measured or that the milk protein degradation activity of the animal-derived protease is not used. Even if the proteolytic activity can be measured, it is lower than the degrading activity of the three types of proteolytic enzymes mentioned above, and the animal-derived protease is contained to the extent that it is not effective as a protease, and the technology to which the present invention belongs This also means that it is negligible from the perspective of a person skilled in the art.

For example, "substantially no animal-derived proteases are used" means that when the milk proteolytic activity of trypsin-like endoprotease derived from microorganisms, endoprotease derived from Bacillus bacteria, or papain is taken as 100%, The milk proteolytic 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. If it is less than 0.01%, it can be said that it is "substantially not used."

In this specification, "origin" when referring to "derived from a microorganism," "derived from a Bacillus bacterium," or "derived from papain" means that the microorganism or plant originally retains the protease. It doesn't mean anything. For example, a protease produced by introducing a gene encoding a protease produced by a bacterium of the genus Bacillus into Escherichia coli and expressing the same gene is "derived from" a bacterium of the genus Bacillus. In addition, the Bacillus bacteria that are referred to as "originally possessed by Bacillus bacteria" include not only wild type bacteria, but also those that maintain the ability to produce proteases that have the protease activity and specificity required for the present invention. Recombinants or mutants are included as long as they exist.

In the proteolytic step in the present invention, a proteolytic enzyme is allowed to act on milk proteins.
A general procedure will be explained below, but it is not particularly limited thereto.
Milk protein as a raw material is dispersed or dissolved in water or hot water.
Next, it is desirable to heat sterilize the milk protein liquid at 70 to 130° C. for about 2 seconds to 10 minutes from the viewpoint of preventing deterioration 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 milk protein liquid after heat sterilization is subjected to ion exchange method using a sodium type or potassium type cation exchange resin (preferably a strongly acidic cation exchange resin), electrodialysis method, ultrafiltration membrane method, or nanofiltration method. Desalination may be performed using a membrane method or the like. For desalting, either a column method or a batch method may be employed.
Next, it is preferable to add an alkaline agent or an acidic agent to the milk protein solution to adjust the pH to the optimum pH of the hydrolyzing enzyme used or around it. 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 foods or medicines. Specifically, 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.

Next, a predetermined amount of proteolytic enzyme is added to the milk protein solution, and a reaction is carried out at a temperature of 10 to 85°C for about 0.1 to 48 hours.
Note that the enzymatic reactions of the three types of proteolytic enzymes described above may be performed simultaneously or separately.

The solution containing the enzyme is maintained at an appropriate temperature depending on the type of enzyme to begin hydrolysis of milk proteins. The temperature may be, for example, 30°C or higher, 40°C or higher, 45°C or higher, or 60°C or lower, or 55°C or lower. Further, any combination of these that does not contradict each other may be used. Note that the suitable temperature range is 30 to 60°C, preferably 45 to 55°C. The hydrolysis reaction time is such that the reaction is continued until a desired decomposition rate is reached while monitoring the decomposition rate of the enzymatic reaction. In order to obtain decomposition products corresponding to the molecular weights described below, the decomposition rate must be 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, or 15% or more. and 35% or less, 34% or less, 33% or less, 32% or less, 31% or less, 30% or less, 29% or less, 28% or less, 27% or less, 24% or less, 25% or less. Good too. Further, any combination of these that is not contradictory may be used. Note that the preferable range of decomposition rate is 8 to 35%, 10 to 30%, and 15 to 25%.

The decomposition rate of milk protein is calculated by measuring the total nitrogen content of the sample using the Kjeldahl method (edited by the Japan Society of Food Industry, "Food Analysis Methods", p. 102, Korin Co., Ltd., 1982), and by measuring the total nitrogen content of the sample. The amount of formol nitrogen in the sample was measured by the titration method (edited by Mitsuda et al., "Food Engineering Experiment Book", Vol. 1, p. 547, Yokendo, 1970), and the decomposition rate was calculated from these measured values using the following formula: do.
Decomposition rate (%) = formol nitrogen amount ÷ total nitrogen amount x 100

The enzymatic reaction is stopped, for example, by deactivating the enzyme in the hydrolysis solution, and can be carried out by heat deactivation treatment using a conventional method. The heating temperature and holding time of the heat inactivation treatment can be appropriately set to conditions that can sufficiently inactivate the enzyme, taking into consideration the thermal stability of the enzyme used. This can be done with a holding time of ~30 minutes.

After heat inactivation, it can be cooled by a conventional method and used as it is, or if necessary, it can be concentrated to obtain a concentrated liquid. Furthermore, it is also possible to dry the concentrate to obtain a powder product. Further, the obtained milk protein decomposition product may be subjected to operations such as separation and purification using conventional methods.

Purification of milk protein digests is usually carried out using various techniques similar to those used for peptide purification, such as ion-exchange chromatography, adsorption chromatography, reversed-phase chromatography, partition chromatography, and gel filtration chromatography. This can be carried out by appropriately combining methods such as chromatography, solvent precipitation, salting out, distribution between two liquid phases, and the like.

The number average molecular weight of the milk protein decomposition product obtained by the production method of the present invention is 650 or less as an upper limit, may be 640 or less, may be 630 or less, may be 620 or less, may be 610 or less, Preferably it may be 600 or less, 590 or less, 580 or less, 570 or less, 560 or less, more preferably 550 or less, 540 or less, 530 or less, and the lower limit is 230 or more, 240 or more, or 250 or more. It may be preferably 260 or more, 270 or more, and more preferably 280 or more. Further, any combination of these that is not contradictory may be used.
The average molecular weight of the milk protein decomposition product is determined based on the concept of number average molecular weight below.
The number average of Molecular Weight is described, for example, in the literature (edited by The Society of Polymer Science, "Basics of Polymer Science", pp. 116-119, Tokyo Kagaku Dojin Co., Ltd., 1978). The average value of the molecular weight of a polymer compound is shown based on the following different indicators.
In other words, high molecular compounds such as milk protein decomposition products are heterogeneous substances and have a distribution of molecular weights, so the molecular weight of milk protein decomposition products must be expressed as an average molecular weight in order to treat them physicochemically. The number average molecular weight (hereinafter sometimes abbreviated as Mn) is the average number of molecules.

In this specification, the average molecular weight of a milk protein decomposition product is measured and calculated by the following method. That is, using high performance liquid chromatography, using a Poly Hydroxyethyl Aspartamide Column (manufactured by Poly LC, diameter 4.6 mm and length 200 mm), the elution rate was adjusted using 20 mM sodium chloride and 50 mM formic acid. Elutes at 0.5 mL/min (Nobuo Ui et al., ed., "High Performance Liquid Chromatography of Proteins and Peptides", Kagaku Special Edition No. 102, p. 241, Kagaku Dojin Co., Ltd., 1984). Detection is performed using a UV detector (manufactured by Shimadzu Corporation), and data is analyzed by a GPC analysis system (manufactured by Shimadzu Corporation) to calculate the number average molecular weight. In addition, as a sample for calculating the molecular weight, a protein and/or a peptide with a known molecular weight may be used as appropriate.

Note that milk protein decomposition products generally contain free amino acids during the manufacturing process.

The milk protein decomposition product obtained by the production method of the present invention is a peptide with a high degree of decomposition and a low molecular weight, but has good emulsifying properties. Due to its good emulsifying properties, it exhibits excellent emulsion stability when mixed with fat.
Furthermore, the milk protein decomposition product obtained by the production method of the present invention also has good thermal stability.

In addition, the milk protein decomposition product obtained by the production method of the present invention has a high degree of decomposition and a low molecular weight, and therefore has reduced antigenicity.
In general, casein, β-lactoglobulin, and the like are examples of antigens that can be a problem with milk proteins and their decomposition products, but the milk protein decomposition products obtained by the production method of the present invention have low antigenicity against these. Specifically, the antigen residual activity of casein is preferably 2 ppm or less, more preferably 1.5 ppm or less. Further, the antigen residual activity of β-lactoglobulin is preferably 150 ppm or less, more preferably 100 ppm or less, and still more preferably 75 ppm or less.

In addition, the milk protein decomposition product obtained by the production method of the present invention can be produced as an excellent milk protein decomposition product without using animal-derived milk protein degrading enzymes, so it is a product that meets the standards to be certified as a halal food. It can be used suitably.

The milk protein decomposition product obtained by the production method of the present invention can be used by blending it into medicines, food and drink products, feeds, etc. Since the milk protein decomposition product obtained by the production method of the present invention uses raw materials derived from milk, it is highly safe for living organisms and is suitable for continuous intake over a long period of time. In addition, because it is produced from milk-derived raw materials, which are relatively inexpensive as biomaterials, it can be produced stably and easily in large quantities, and it can also be provided to users at low cost. .

The milk protein decomposition product obtained by the production method of the present invention can be preferably included in foods and drinks. Note that foods and drinks also include the form of additives added to foods and drinks and medicines. Such embodiments include, for example, additives added to expressed breast milk or formula milk, and it is assumed that newborns and infants ingest the added milk.
Foods and drinks are usually ingested orally, but are not limited to this, and may be ingested nasally, or through a gastrostomy or intestinal fistula. For example, it is envisaged that newborns and infants will be fed formula milk, which will be described later, or breast milk to which milk protein decomposition products have been added, through a nasogastric feeding tube or the like.

Foods and beverages may be in the form of liquids, pastes, solid gels, powders, etc., such as tablets; bread, macaroni, spaghetti, noodles, cake mixes, flour products such as fried flour and bread crumbs; instant noodles; instant foods such as cup noodles, retort/cooked foods, cooked canned foods, microwave foods, instant soups/stews, instant miso soup/suimono, canned soups, freeze/dried foods, and other instant foods; canned agricultural products, canned fruits, and jams.・Processed agricultural products such as marmalade, pickles, boiled beans, dried agricultural products, and cereals (processed grain products); Processed marine products such as canned seafood, fish ham/sausages, fish paste products, seafood delicacies, and boiled fish; Canned livestock products - Processed livestock products such as pastes, meat hams and sausages; Milk and dairy products such as processed milk, milk drinks, yogurts, lactic acid bacteria drinks, cheese, ice creams, creams, and other dairy products; Butter, margarine , fats and oils such as vegetable oil; basic seasonings such as soy sauce, miso, sauces, processed tomato seasonings, mirin, vinegars; cooking mixes, curry ingredients, sauces, dressings, noodle soups, spices, Complex seasonings and foods such as other complex seasonings; frozen foods such as raw frozen foods, semi-cooked frozen foods, and cooked frozen foods; caramels, candies, chewing gums, chocolates, cookies, biscuits, cakes, pies, snacks, Confectionery such as crackers, Japanese sweets, rice sweets, bean sweets, dessert sweets, jellies, and other sweets; carbonated drinks, natural fruit juices, fruit juice drinks, soft drinks with fruit juice, pulp drinks, fruit drinks with fruit granules, vegetable drinks, Soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutritional drinks, alcoholic drinks, other drinks such as other drinks, baby food, furikake, other commercially available foods such as ochazuke nori, etc. Nutrient compositions such as formula milk (including powdered milk, liquid milk, etc.), liquid foods, and supplements; Functional foods (foods for specified health uses, foods with nutritional function functions), and the like.

Among these, nutritional compositions are preferred.
In the present invention, the "nutritional composition" is not particularly limited as an aspect of food or drink, but preferably includes formula milk, liquid food, supplements, etc., and more preferably formula milk. Ingestion targets may be infants, toddlers, children, or adults, but infants and young children are preferred.
Formulated milk includes powdered milk and liquid milk.
According to the Ministerial Ordinance Concerning Ingredient Standards for Milk and Dairy Products (Ministerial Ordinance on Milk, etc.), infant formula is defined as ``processed raw milk, cow's milk, special milk, or foods manufactured using these as raw materials, or as a main ingredient, It is defined as a powdered product with added nutrients.
Prepared liquid milk is defined in the above ministerial ordinance as "a product made by processing raw milk, cow's milk, special milk, or food products made from these as raw materials, or by adding nutrients necessary for infants and infants to liquid form as a main ingredient." .
In addition, formula milk contains nutritional ingredients such as various proteins, fats and oils, carbohydrates, minerals, and vitamins, and includes those processed into powder or liquid form.
In addition, formula milk further includes ``infant formula'', ``infant formula liquid milk'', and ``powdered milk for pregnant women and lactating mothers'', which are foods for special dietary uses stipulated by the Health Promotion Act. Embodiments such as nutritional powder for adults and nutritional powder for the elderly are also included.

When in the form of a supplement, it can be formulated into solid preparations such as powders, granules, tablets, and capsules; liquid preparations such as solutions, syrups, suspensions, and emulsions; and the like. Such formulation can be carried out in accordance with the explanations of components, carriers, and methods for formulating pharmaceuticals, which will be described later.

Moreover, it can also be used as feed as one aspect of food and drink products. Examples of the feed include pet food, livestock feed, and fish feed.
The form of the feed is not particularly limited and includes, for example, grains such as corn, wheat, barley, rye, and milo; vegetable oil cakes such as soybean oil meal, rapeseed oil meal, coconut oil meal, and linseed oil meal; bran, wheat bran, rice bran, Bran such as defatted rice bran; Manufactured lees such as corn gluten meal and corn jam meal; Animal feed such as fish meal, skim milk powder, whey, yellow grease, and tallow; Yeast such as Torula yeast and brewer's yeast; It may contain mineral feeds such as calcium phosphate and calcium carbonate; oils and fats; simple amino acids; sugars, etc.

The milk protein decomposition product obtained by the production method of the present invention may be included in pharmaceuticals as an active ingredient.
The pharmaceutical form can be appropriately 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; liquid preparations such as solutions, syrups, suspensions, and emulsions. In the case of parenteral administration, it can be formulated into suppositories, ointments, injections, etc.
In formulation, components such as excipients, pH adjusters, coloring agents, and flavoring agents that are commonly used in formulation can be used. It is also possible to use other medicinal ingredients, prebiotics against Bifidobacterium bacteria that are known or will be discovered in the future, prebiotics against other bacteria, and the like.
In addition, formulation can be carried out by appropriately known methods depending on the dosage form. When formulating, a pharmaceutical carrier may be added as appropriate.

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; crystalline cellulose, hydroxypropyl cellulose, Cellulose derivatives such as hydroxypropyl methylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium; gum arabic; dextran; pullulan; silicate derivatives such as light silicic anhydride, synthetic aluminum silicate, magnesium aluminate metasilicate; phosphate derivatives such as calcium phosphate; carbonic acid Examples include carbonate derivatives such as calcium; sulfate derivatives such as calcium sulfate.

Examples of the binder include, in addition to the excipients mentioned above, gelatin; polyvinylpyrrolidone; macrogol, and the like.

Examples of the disintegrant include, in addition to the above excipients, chemically modified starch or cellulose derivatives such as croscarmellose sodium, sodium carboxymethyl starch, and crosslinked polyvinylpyrrolidone.

Examples of lubricants include talc; stearic acid; stearic acid metal salts such as calcium stearate and magnesium stearate; colloidal silica; waxes such as vegum and gay wax; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid. ; carboxylic acid sodium salts 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; starch derivatives, etc. It will be done.

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

Examples of flavoring agents include sweeteners, acidulants, fragrances, and the like.
In addition, examples of carriers used in the case of liquid preparations for oral administration include solvents such as water.

Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples unless the gist thereof is exceeded.

<Test Example 1> Production of milk protein decomposition product by enzymatic degradation 1
(1) Enzymatic decomposition of whey Various proteases were allowed to act on whey protein to produce milk protein decomposition products. Specifically, 450 g of water was added to 50 g of bovine milk-derived whey protein (Milei 80, manufactured by Mirai), and the solution was well dispersed. Sodium hydroxide was added to adjust the pH of the solution to 9, and the temperature of the solution was adjusted. The temperature was adjusted to about 50°C. A predetermined amount of the enzyme shown in Table 1 was added thereto to start the decomposition reaction. After 5 hours, the solution was heated at 85°C for 10 minutes to inactivate the enzyme to stop the enzyme reaction, and then cooled to 5°C. This decomposition solution was concentrated and freeze-dried to obtain a freeze-dried milk protein decomposition product.

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

(3) Measurement of decomposition rate of milk protein decomposition product The degree of decomposition (degradation rate) of milk protein decomposition product was measured by formol titration method. 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, and 0.1M hydroxide was added using a pH meter while stirring with a stirrer. The pH was adjusted to 6.80 by dropping sodium solution or 0.1M hydrochloric acid solution. After adding 5 mL of 37% formalin solution adjusted to pH 8.0, the mixture was titrated with 0.1 M sodium hydroxide solution to adjust the pH to 7.90 (x mL). At the same time, the Brix value (25° C.) of the milk protein decomposition solution was measured using a saccharometer (z%).
Based on the above measurement values, the decomposition rate of the milk protein decomposition product was calculated using the following formula, and is shown in Table 1.
Decomposition rate (%) = formol nitrogen amount ÷ total nitrogen amount x 100
Amount of formol nitrogen (mgN/dL) = 1.4 x x x f (potency of sodium hydroxide) x 100/4
Total nitrogen amount (mgN/dL) = z x 0.7 x 1000/6.38

(4) Evaluation of emulsion stability of milk protein decomposition product Emulsion stability when milk protein decomposition product was mixed with water and fats and oils was evaluated. Specifically, 10 g of sample powder was dissolved in 150 mL of deionized water, 100 g of soybean oil (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was added, and the mixture was passed through a homogenizer (T.K. ) Emulsified at a pressure of 5000 rpm using Tokushu Kika Kogyo Co., Ltd. (currently Primix Co., Ltd.). The resulting emulsion was placed in a graduated cylinder and stored at room temperature for 24 hours, and then the heights of the oil layer, emulsion layer, and water layer were measured, and the ratio to the entire liquid was calculated. In addition, the emulsion after storage was visually observed and evaluated as ◯ if there was no separation of oil or fat, △ if there was some separation, and × if there was separation. The results are also shown in Table 1.

(5) Evaluation of thermal stability The thermal stability of the milk protein decomposition product was evaluated by a retort sterilization test. Specifically, 200 mL of a solution obtained by dissolving the sample powder in deionized water at a concentration of 5% w/w was sealed in a retort pouch, and the F value was determined using a high temperature and high pressure cooking sterilizer (RCS-60/10SPXTG-FAM). Heat sterilization equivalent to 4 (130° C., 90 seconds, 0.3 MPa) was performed. The solution after heat sterilization was visually observed to confirm the presence or absence of precipitation or precipitation. The case where there was no precipitation or precipitation was evaluated as 〇, and the case where it was evaluated as ×. The results are also shown in Table 1.

(6) Evaluation of antigenicity The antigenicity of the milk protein decomposition product was evaluated. Specifically, using Morinaga FASPEK Elizer II (manufactured by Morinaga Bioscience Institute), the sample powder was diluted 20 times with sample diluent I from the kit, and the total peptide concentration in the diluted solution was 1 to 50 ng/mL. It was prepared so that 100 μL of the diluted solution was poured into each plastic well on which the primary antibody had been immobilized, and incubated for 1 hour. After washing the plastic well with the prepared washing solution, the enzyme-labeled antibody was reacted for 30 minutes. The detection targets were epitopes contained in the sequences of casein and β-lactoglobulin, respectively. Thereafter, it was washed with a prepared washing solution, an enzyme substrate solution was added to react, and the reaction was stopped with a reaction stop solution. After the reaction was stopped, the absorbance was measured at a main wavelength of 450 nm and a sub wavelength of 620 nm within 30 minutes. The measured absorbance was applied to a calibration curve prepared by measuring at the same time to determine the total protein concentration in the sample diluted solution, and further multiplied by the dilution factor to determine the residual antigen activity in the sample. Antigen residual activity was evaluated in three stages based on the following criteria. The results are also shown in Table 1.
casein;
○: 2 ppm or less (no antigenicity)
△: 2 to 50 ppm (slightly antigenic)
×: 50 ppm or more (antigenic)
β-lactoglobulin;
○: 150 ppm or less (no antigenicity)
△: 150-2000ppm (slightly antigenic)
×: 2000 ppm or more (antigenic)

From the results in Table 1, it can be seen that the milk protein decomposition product prepared by the production method of the present invention is degraded to a low molecular weight with a high decomposition rate, but has high emulsion stability with little separation of fats and oils and water. It is also found that it has excellent thermal stability and antigenicity.

<Test Example 2> Production of milk protein decomposition product by enzymatic degradation 2
A milk protein decomposition product was produced in the same manner as in Test Example 1 using the enzymes and their usage amounts shown in Table 2, and the molecular weight measurement, degradation rate measurement, emulsion stability evaluation, thermal stability evaluation, and antigenicity were determined. We conducted an evaluation. The results are also shown in Table 2.
It can be seen that although the milk protein decomposition product prepared by the production method of the present invention is decomposed to a low molecular weight with a high decomposition rate, there is little separation of fats and water from oil and water, and the emulsion stability is high. It is also found that it has excellent thermal stability and antigenicity.

<Test Example 3> Production of milk protein decomposition product by enzymatic degradation 3
A milk protein decomposition product was produced in the same manner as in Test Example 1 using the enzymes and their usage amounts shown in Table 3, and the molecular weight, decomposition rate, and emulsion stability were evaluated. The results are also shown in Table 3.
It can be seen that although the milk protein decomposition product prepared by the production method of the present invention is decomposed to a low molecular weight with a high decomposition rate, there is little separation of fats and water from oil and water, and the emulsion stability is high.

<Test Example 4> Production of milk protein decomposition product by enzymatic degradation 3
Milk protein decomposition products were produced in the same manner as in Test Example 1 using the enzymes and their usage amounts shown in Table 4, and the molecular weight, decomposition rate, and emulsion stability were evaluated. The results are also shown in Table 3.
It can be seen that although the milk protein decomposition product prepared by the production method of the present invention is decomposed to a low molecular weight with a high decomposition rate, there is little separation of fats and water from oil and water, and the emulsion stability is high.

The present invention is useful in the fields of pharmaceuticals, food and drink, feed, etc.

Claims

A method for producing a milk protein decomposition product, the method comprising:
comprising a proteolytic step of causing a proteolytic enzyme to act on the milk protein,
The proteolytic enzyme includes a trypsin-like endoprotease derived from a microorganism, an endoprotease derived from a Bacillus bacterium, and papain,
The production method, wherein the milk protein decomposition product has a number average molecular weight of 650 or less.
The manufacturing method according to claim 1, wherein the milk protein is whey protein.
The manufacturing method according to claim 1 or 2, wherein the microorganism is a Fusarium bacterium.
The production method according to any one of claims 1 to 3, wherein the proteolytic enzyme is substantially free of animal-derived proteases.