JP2018057346A - Casein enzyme treated product and production method therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently producing a casein enzyme treated product containing high levels of a tripeptide consisting of Met-Lys-Pro and having emulsifying properties.SOLUTION: The method for producing a casein enzyme treated product containing a tripeptide consisting of Met-Lys-Pro is characterized by using a proline-specific endoprotease concentration of 0.1 to 3 U/g to casein as a raw material. The content of the tripeptide consisting of Met-Lys-Pro (mg/1 g of casein enzyme treated product) is 0.9 to 1.3 mg/g in the casein treated product. From the casein enzyme treated product, a fraction is obtained having a molecular weight of 1,000 Da or less containing the tripeptide consisting of Met-Lys-Pro.SELECTED DRAWING: None

Description

  The present technology relates to a casein-treated product containing a tripeptide composed of Met-Lys-Pro and a method for producing the same.

In recent years, the search for oligopeptides and research on their functionality have progressed, and many oligopeptides having physiological activity such as blood pressure lowering action and blood glucose rise inhibiting action have been found.
For example, a tripeptide consisting of Met-Lys-Pro (SEQ ID NO: 1) has an angiotensin converting enzyme inhibitory action, dipeptidyl peptidase-IV inhibitory action, and cerebral dysfunction caused by amyloid beta protein such as Alzheimer-type dementia. Prevention or treatment has been reported (Patent Documents 1 to 3).

In general, as a method for producing an oligopeptide, for example, a chemical synthesis method, a method for hydrolyzing a protein, and the like are known. As this hydrolysis method, for example, acid treatment, alkali treatment, enzyme treatment and the like are known.
However, when the desired oligopeptide is produced on an industrial basis, in the chemical synthesis method, in addition to establishing a mass synthesis method, it is necessary to consider the removal of the solvent used in the chemical reaction and the by-products of the chemical reaction, etc. . On the other hand, when a hydrolysis method using a known protein as a raw material is adopted, it is easy to ensure the amount of the raw material for mass production, but after hydrolysis, the target oligopeptide is not intended. Polypeptides and oligopeptides are mixed in various ways, and therefore, a larger number of separation / purification steps are often required.

International Publication No. 2003/044044 International Publication No. 2013/125622 Japanese Patent Laid-Open No. 2006-69343

  The main object of the present technology is to provide a method for efficiently producing an enzyme-treated product of casein having a high content of a tripeptide (SEQ ID NO: 1) comprising Met-Lys-Pro and having emulsifying properties.

In general, when a protein is enzymatically degraded to obtain a high concentration of a target oligopeptide, it is important to properly combine a plurality of proteolytic enzymes having different properties. Because protein has a complex structure, it cuts out the target oligopeptide while reducing the molecular weight of the protein by using not only one kind of enzyme but also one or more kinds of enzymes having different properties. This is necessary.
However, when the present inventors selected casein as a raw material and hydrolyzed this casein using a specific amount of proline-specific endoprotease, a tripeptide consisting of Met-Lys-Pro (SEQ ID NO: 1; Hereinafter, a casein enzyme-treated product containing a high concentration of “tripeptide MKP” was obtained, and it was found that this casein enzyme-treated product also has emulsifying properties.

Furthermore, as a result of studying the molecular weight distribution of the dry powder of this casein-treated product, the fraction containing undegraded casein that has not undergone enzymatic degradation or hardly undergoes enzymatic degradation is present in the casein-treated product. It was found that the fraction of more than half of the total and more than 1,000 Da accounted for about 80% of the total.
Thus, it was surprisingly found that a fraction containing the tripeptide MKP (375 Da) at a higher concentration can be obtained from this casein-treated product.
In this way, the inventors have completed the present invention.

In other words, this technology
Provided is a method for producing a casein enzyme-treated product containing a tripeptide consisting of Met-Lys-Pro, wherein a proline-specific endoprotease concentration of 0.1 to 3 U / g is used for casein as a raw material. be able to.

Moreover, in the manufacturing method which concerns on this technique, the usage-amount of the said proline specific endoprotease can be made into the density | concentration of 0.4 U / g or more and 2 U / g or less.
In the manufacturing method according to the present technology, the MKP molar yield can be at least 70%.
Moreover, in the manufacturing method which concerns on this technique, although reaction pH is not specifically limited, It can be set as a neutral area | region.

In addition, this technology
After carrying out the method for producing the casein-treated product, the undegraded casein fraction or the fraction having a molecular weight of more than 1,000 Da is removed from the casein-treated product, and the molecular weight containing a tripeptide consisting of Met-Lys-Pro A method for obtaining a fraction of 1,000 Da or less can be provided.

In addition, this technology
A casein enzyme-treated product having a tripeptide MKP content (mg / casein enzyme-treated product 1 g) composed of Met-Lys-Pro of 0.9 to 1.3 mg / g can be provided.
In the casein-treated product according to the present technology, the content of tripeptide MKP composed of Met-Lys-Pro (mg / fraction 1 g of molecular weight of 1,000 Da or less) can be 3 mg / g or more.
The casein-treated product according to the present technology can contain undegraded casein.

According to the present technology, it is possible to efficiently produce a casein enzyme-treated product having a high content of a tripeptide composed of Met-Lys-Pro and having emulsifying properties.
In addition, the effect described here is not necessarily limited, and may be any effect described in the present technology.

  Hereinafter, preferred embodiments for carrying out the present technology will be described. In addition, embodiment described below shows an example of typical embodiment of this technique, and, thereby, the scope of this technique is not interpreted narrowly.

The production method of the present technology produces a casein enzyme-treated product containing a tripeptide (SEQ ID NO: 1) comprising Met-Lys-Pro, which uses a proline-specific endoprotease for casein as a raw material. Is the method.
The tripeptide composed of Met-Lys-Pro is an amino acid sequence (SEQ ID NO: 1) composed solely of Met-Lys-Pro, and is a tripeptide cleaved from casein as a raw material.

(1) Raw material casein Casein, which is a raw material, is mainly composed of milk-derived protein. Although the casein is not particularly limited, a commercially available product may be used as the casein, or a purified product separated from milk may be used.
As said casein, various casein, caseinate, rennet casein etc. are mentioned, for example, These can be used 1 type or in combination of 2 or more types.
Specifically, acid casein such as lactate casein, sulfate casein, and casein hydrochloride; caseinate such as sodium caseinate, potassium caseinate, calcium caseinate, magnesium caseinate, or a mixture of two or more of these It is done.
In addition, bovine casein and the like separated and purified from cow's milk, skim milk, whole milk powder, skim milk powder and the like by a conventional method can also be used.

  Here, it is known that the structural components in general casein are not a single protein but are roughly classified into three types: α-casein, β-casein, and κ-casein. Among them, the present inventors efficiently perform the tripeptide MKP from the amino acid sequence portion consisting of -Met-Lys-Pro-(-MKP-) present in the amino acid sequence of α-casein. It was confirmed that it could be cut out. Therefore, in the method for producing a casein-treated product according to the present technology, α-casein is preferably used as a main substrate source when obtaining the tripeptide MKP.

  Since casein used as a raw material is derived from milk, it has the advantage that it is relatively inexpensive, stable, convenient and available in large quantities. Therefore, by the production method of the present technology using casein, the casein enzyme-treated product of the present technology can be obtained relatively inexpensively, safely, stably, simply, and in large quantities.

(2) Proline-specific endoprotease The proline-specific endoprotease used in the production method of the present technology is an endoprotease that can cleave at the carboxy terminal side of the proline residue of the peptide.
The origin of the proline-specific endoprotease is not particularly limited, and may be any of animal origin, plant origin, and microorganism origin. From these animals, proline-specific endoprotease may be separated and purified by a conventional method, or commercially available products that can be generally used in pharmaceuticals and foods may be used.
Of these, microbial origin is preferred.
Examples of microorganisms include Aspergillus, Flavobacterium, Aeromonas, Xanthomonas, and Bacteroides, which can be used alone or in combination. can do.
Of these microorganisms, those derived from the genus Aspergillus are preferred, and those derived from Aspergillus niger are more preferred.

In the proline-specific endoprotease, the physicochemical properties of this enzyme are preferably as follows.
As for the action of the enzyme, it is preferable to specifically cleave the C-terminus of proline present in the substantially soluble animal or plant derived protein.
The action of the enzyme is preferably to cleave between peptide bonds present in the protein and polypeptide; without the bitterness formation or excessive hydrolysis, the size, shape and chain length of the protein and polypeptide can be reduced. It is preferable to decrease.
The properties of the enzyme are preferably such that the enzyme is a serine protease; it is preferred that the enzyme is not inhibited by a regular protease inhibitor.
The enzyme is preferably derived from a fungus.
The optimum pH of the enzyme is preferably in a neutral region to an acidic region (pH around 8 to around 1.5).
In the case of an endoprotease derived from the genus Aspergillus, the optimum pH is preferably in the acidic region (pH around 1.5 to around 7), and the more specific optimum pH of this endoprotease is in the acidic region lower than 7. It is preferable that it is 3.5 to 6.5.
The pH range on which the enzyme acts is preferably 1.5-7.
The optimum temperature of the enzyme is preferably around 55 ° C (50-60 ° C).
The temperature range at which the enzyme acts is preferably 0 to 75 ° C.
Regarding the active state of the enzyme, the enzyme is inactivated by holding at 80 to 85 ° C. for 5 to 10 minutes; the inactivity of the enzyme is significantly affected by pH and substrate concentration; It is preferable to measure the residual activity to ensure conversion.

(3) Method for producing casein enzyme-treated product The method for producing a casein enzyme-treated product according to the present technology uses a proline-specific endoprotease for casein as a raw material, and a tripeptide consisting of Met-Lys-Pro (SEQ ID NO: As long as the casein-treated product containing 1) can be obtained, the detailed steps are not particularly limited.
In the production method according to the present technology, various processes used in a known method for producing a casein enzyme-treated product can be freely selected and adopted.
Hereinafter, an example of the manufacturing method will be specifically described.

[Preparation of raw material solution]
A raw material containing casein is dissolved or dispersed in a solvent such as water to prepare a casein-containing solution.

The casein used for the raw material is as described above. The casein is used as a substrate for a proline-specific endoprotease.
Examples of raw materials other than gazein include a pH adjuster, a surfactant, and the like, and among these, one or more can be selected.
Although it does not specifically limit as a solvent, It is preferable to use distilled water etc. as water.
Moreover, the casein concentration in the casein-containing solution is not particularly limited, but it is usually preferable from the viewpoint of efficiency and operability that the concentration range is about 5 to 15% by mass in terms of protein.

Moreover, it is preferable that pH of the said casein containing solution shall be pH 4-8, it is more preferable to set it as a neutral area | region (pH 6-8), and it is further more preferable to set it as pH 6.2-7. The pH is particularly preferably 6.5-7.
For adjusting the pH, inorganic acids such as hydrochloric acid and sulfuric acid; organic acids such as acetic acid and the like can be used. For alkali adjustment of pH, alkali metal salts such as sodium hydroxide; alkaline earth metal salts such as calcium hydroxide can be used. These can be used alone or in combination.

[Enzymatic reaction]
Next, a proline-specific endoprotease is added to the casein-containing solution.
In the present technology, the proline-specific endoprotease is characterized by being used alone without being used in combination with other types of protein enzymes.
Conventionally, it has been considered that a large number of target oligopeptides cannot be efficiently produced unless a plurality of proteolytic enzymes are used in combination. However, in this technology, when used in combination with a proteolytic enzyme other than proline-specific endoprotease, the content of the target tripeptide MKP in the casein enzyme-treated product is lowered, and the target product is recovered in a high content fraction. It becomes difficult to do. Normally, when we continue to stick to and examine combinations of different enzymes, the present inventors accidentally used the propeptide-specific endoprotease alone, so that the tripeptide MKP is highly efficient against casein. Was cut out and the concentration could be increased in the casein-treated product.

  The enzyme-treated product of the present technology was easily soluble in water, had emulsifying properties, and had little bitterness. The enzyme-treated product of the present technology having such properties has high value in itself, but also has high utility value as a food additive.

It is preferable that the usage-amount of the proline specific endoprotease of this technique is 0.1-3 U / g density | concentration with respect to casein which is a raw material. When the concentration is 0.1 U / g or more, the tripeptide MKP can be efficiently cut out from the casein as the raw material, and the tripeptide MKP can be highly contained in the casein-treated product. In addition, it is preferable that the enzyme concentration is 3 U / g or less, because the tripeptide MKP can be efficiently cut out, while many undegraded products of casein can be obtained.
Further, the amount of the proline-specific endoprotease used is preferably 0.3 U / g concentration or more, more preferably 0.4 U / g concentration or more, further preferably 0.5 U / g concentration or more as a lower limit. . Moreover, as an upper limit, Preferably it is 2.5 U / g density | concentration or less, More preferably, it is 2 U / g density | concentration or less, More preferably, it is 1.5 U / g density | concentration or less.
Further, the amount of the proline-specific endoprotease used is preferably 0.4 to 2 U / g concentration, and more preferably 0.5 to 1 U / g concentration. Within this concentration range, this amino acid sequence (MKP) portion can be efficiently excised as a tripeptide MKP from the amino acid sequence including the amino acid sequence of -MKP- present in the casein that is the raw material. In addition, the tripeptide MKP content (mg / fraction having a molecular weight of 1,000 Da or less, 1 g) can be increased.

In the present technology, the progress of the enzyme treatment can be appropriately adjusted by adjusting reaction conditions such as reaction duration, reaction temperature, and initial pH.
For example, by monitoring the enzyme reaction, the reaction duration can be determined so that the physicochemical properties of the casein enzyme-treated product have a desired value.
Examples of the enzyme reaction monitoring method include a method of collecting a part of the reaction solution and measuring the protein degradation rate, MKP molar yield, tripeptide MKP content, and the like. The reaction duration may be determined by measuring the collected reaction solution.

  In the present technology, the decomposition rate is preferably adjusted to 10% or less, more preferably 9% or less, and further preferably 8% or less.

  In the present technology, the MKP molar yield is preferably adjusted to 20% or more, more preferably 24% or more, still more preferably 70% or more, and particularly preferably 95% or more.

In the present technology, the reaction time is preferably 1 hour or more, more preferably 2 hours or more, further preferably 4 hours or more, particularly preferably 6 hours or more, and most preferably 9 hours or more as a lower limit. Moreover, as an upper limit, Preferably it is 24 hours or less, More preferably, it is 20 hours or less, More preferably, it is 16 hours or less, Most preferably, it is 15 hours or less.
The reaction time is preferably 1 to 24 hours, more preferably 1 to 20 hours, further preferably 2 to 16 hours, particularly preferably 4 to 15 hours, and most preferably 6 to 15 hours. More preferably, it is 9 to 15 hours.

  In the present technology, the reaction pH is preferably in the neutral region (pH 6 to 8), more preferably 6.2 to 7.5. A more preferable upper limit value is 7.0. A more preferred lower limit is pH 6.3. The reaction pH is preferably 6 or more in order to suppress acid precipitation of casein.

In the present technology, the reaction temperature is preferably 20 ° C. or higher, more preferably 40 ° C. or higher, and further preferably 50 ° C. or higher as the lower limit. As an upper limit, Preferably it is 65 degrees C or less, More preferably, it is 60 degrees C or less, More preferably, it is 55 degrees C or less.
Moreover, reaction temperature becomes like this. Preferably it is 20-65 degreeC, More preferably, it is 40-60 degreeC, More preferably, it is 50-60 degreeC, Especially preferably, it is 50-55 degreeC. The reaction temperature is preferably 60 ° C. or lower in order to suppress heat denaturation of casein.

Next, the enzyme reaction is stopped.
The enzyme reaction is stopped by deactivating the enzyme in the hydrolyzed solution. The deactivation treatment can be performed by a conventional method such as a heat deactivation treatment.
Conditions for the heat deactivation treatment (heating temperature, heating time, etc.) can be appropriately set under conditions that allow sufficient deactivation in consideration of the thermal stability of the enzyme used.
In the present technology, for example, the enzyme can be inactivated by maintaining at 80 to 85 ° C. for 5 to 10 minutes.

[Separation / Purification]
The enzyme reaction product subjected to the above-described enzyme reaction may be separated and purified by a conventional separation / purification method. The enzyme-treated product may be in a solution state.
Thereby, impurities may be removed from the enzyme-treated product of the present technology. A desired fraction or a desired peptide may be recovered from the enzyme-treated product of the present technology.

Examples of the separation / purification include precipitation, membrane separation, HPLC separation / purification, ion exchange chromatography, and crystallization.
Examples of the separation / purification method include filtration methods, microfiltration methods, membrane separation treatment methods such as ultrafiltration membranes, resin adsorption separation methods, column chromatography methods, HPLC separation and purification methods, precipitation methods, crystal methods, and the like. One or two or more combinations can be selected from these.

  The said filtration method can be implemented by a well-known method, for example, can be implemented with a well-known apparatus using diatomaceous earth.

  The membrane separation treatment method can be performed using a known apparatus. Although it does not specifically limit as a well-known apparatus, For example, a microfiltration module etc., ultrafiltration module Pellicon2 Cassettes (the Merck Millipore company make, molecular weight cut off 1,000 Da), ultrafiltration module SEP1053 (Asahi Kasei company make, molecular weight cut off) 3,000 Da), SIP 1053 (manufactured by Asahi Kasei Co., Ltd., molecular weight cut-off 6,000 Da), SLP 1053 (manufactured by Asahi Kasei Co., Ltd., molecular weight cut-off 10,000 Da) and the like.

The resin adsorption separation method can be performed by a known method, for example, by bringing an enzyme-treated product into contact with a resin. Although it does not specifically limit as resin, For example, ion exchange resin, chelate resin, affinity adsorption resin, synthetic adsorption agent, resin for high performance liquid chromatography, etc. are mentioned. More specifically, for example, trade names: Diaion, Sepabeads (manufactured by Mitsubishi Chemical Corporation), Amberlite XAD (manufactured by Organo Corporation), KS-35 (manufactured by Ajinomoto Fine Techno Co., Ltd.) and the like can be mentioned.
The resin adsorption separation method can be performed in a continuous manner by filling these resins into a column and continuously flowing in and out of the enzyme-treated product, or by charging the resin into the enzyme-treated product. Alternatively, the contact may be performed in a batch system in which the enzyme-treated product and the resin are separated after contacting for a certain period of time.

  The said precipitation method can be implemented by a well-known method, for example, an acid precipitation method, the centrifugation method, etc. are mentioned. These can be used alone or in combination of two or more.

[Method for obtaining each fraction after carrying out the method for producing a casein-treated product]
In the present technology, after performing the above-described method for producing a casein enzyme-treated product, a polymer fraction containing undegraded casein or the like or a fraction having a molecular weight exceeding 1,000 Da is obtained from the enzyme-treated product obtained by the production method. And a fraction containing a tripeptide MKP and having a molecular weight of 1,000 Da or less can be obtained.
Thereby, the fraction which made tripeptide MKP higher content can be obtained.

When recovering a polymer fraction containing undegraded casein and the like, it is desirable to perform acid precipitation. By the acid precipitation method, the fraction containing the tripeptide MKP is contained in the supernatant, and the fraction containing the protein and the polypeptide is contained in the acid precipitate. Since the separation of the supernatant and the acid precipitate is easy, each can be recovered efficiently.
Examples of the polymer fraction containing undegraded casein in the present technology generally include proteins and polypeptides having a molecular weight exceeding 10,000 Da.

  In the acid precipitation method, the pH of the solution containing the enzyme reaction product is preferably 6 or less, and more preferably 5.5 or less. Thereby, protein and polypeptide can be precipitated and protein etc. can be removed or collect | recovered from an enzyme processed material. When the acid-precipitated fraction at this time is collected and used, the lower limit pH is preferably 4 or more in order to suppress protein denaturation.

Furthermore, when removing or recovering the acid precipitate, it is desirable to carry out by, for example, a centrifugal separation method or a membrane separation method.
Centrifugation is convenient because of its simplicity and workability and cost. By collecting the supernatant after centrifugation, a fraction containing the tripeptide MKP at a higher concentration can be obtained.

Further, the membrane separation treatment method is preferable because each fraction is easily collected efficiently by molecular weight classification. A fraction containing the tripeptide MKP at a higher concentration can be obtained by lowering the molecular weight category to be collected (preferably a molecular weight of 1,000 Da or less).
Furthermore, it is preferable in terms of work efficiency and cost to remove the polymer fraction containing undegraded casein before performing the membrane separation treatment method. Specifically, the acid precipitation method, the centrifugal separation method, and the membrane separation method are preferably performed in this order. Thereby, a fraction containing the high concentration of the tripeptide MKP can be obtained efficiently.

[Sterilization treatment]
The obtained casein enzyme-treated product may be sterilized.
As the sterilization method, a conventional heat treatment method or the like can be used.
The heating temperature and holding time at the time of the heat treatment may be set appropriately under conditions that can be sufficiently sterilized, and can be sterilized by, for example, heat treatment at 70 to 140 ° C. for 2 seconds to 30 minutes.
The heat sterilization method can be either a batch method or a continuous method, and a plate heat exchange method, an infusion method, an injection method, or the like can also be used in the continuous method.

[Concentration treatment / Dry treatment / Powder treatment / Secondary treatment]
Furthermore, the obtained casein enzyme-treated product can be used in the state of a solution as it is, and if necessary, the solution can be used as a concentrated solution by a known method. Further, the concentrated liquid can be dried by a known method and used as a powder.

(4) Casein enzyme-treated product The casein enzyme-treated product according to the present technology can be obtained by the above-described method for producing a casein enzyme-treated product. The casein-treated product of the present technology is a novel one that has a high content of the tripeptide MKP and has emulsifying properties under mild degradation conditions.

The value (OD500) representing the emulsifiability of the casein enzyme-treated product of the present technology is preferably 1 or more, more preferably 2 or more.
Casein is hardly soluble in water, but the casein-treated product of the present technology has an undegraded product fraction, but has water solubility. Furthermore, the casein enzyme-treated product of the present technology can maintain a high emulsifying property of the protein. For this reason, the casein enzyme-treated product of the present technology can also be used as various food additives or emulsifiers.

In general, it is said that when a protein is treated with an enzyme and an oligopeptide having a hydrophobic amino acid becomes high, bitterness is promoted. However, in this technique, the casein-treated product using a proline-specific endoprotease does not feel bitterness or slightly bitterness.
Such casein enzyme-treated products that do not feel bitterness are low in cost because a step for removing bitterness is not required. Furthermore, although removal of bitterness may lack nutritionally important amino acids, the casein enzyme-treated product of the present technology does not need to be subjected to a bitterness removal step, so this deficiency can also be improved.

  The MKP molar yield in the casein-treated product of the present technology is preferably 20% or more, more preferably 24% or more, further preferably 70% or more, particularly preferably 73% or more, and extremely preferably 96% or more.

  The degradation rate of the casein-treated product of the present technology is preferably 10% or less, more preferably 9% or less, and still more preferably 8% or less.

  The tripeptide MKP content (mg / casein enzyme-treated product 1 g (hereinafter also referred to as “treated product 1g”)) in the casein-treated product is preferably 0.3 mg / treated product 1 g or more, and 0.9 mg / 1 g or more of the treated product is more preferable, and 1.2 mg / treated product or more is more preferable. Moreover, the upper limit of the content of tripeptide MKP in the casein-treated product is 1.3 (mg / treated product 1 g) or less from the logical value.

  The tripeptide MKP content (mg / molecular weight of 1,000 Da or less fraction 1 g (hereinafter also referred to as “fraction 1 g”)) in the casein enzyme-treated product is preferably 1.4 mg / fraction 1 g or more. 0.0 mg / fraction 1 g or more is more preferred, 4.0 mg / fraction 1 g or more is more preferred, 4.6 mg / fraction 1 g or more is particularly preferred. The upper limit of the tripeptide MKP content in the casein enzyme-treated product is preferably as high as possible, but is, for example, 5.2 mg / fraction 1 g or less.

Furthermore, since various physiological activity actions are recognized for peptides, the casein enzyme-treated product of the present technology contains a higher amount of tripeptide MKP as a secondary treated product by appropriately removing proteins and peptides. The secondary casein-treated product (for example, a fraction having a molecular weight of 1,000 Da or less, a separated / purified fraction such as a supernatant fraction) can be used.
The tripeptide MKP content in 1 g of the solid content of the fraction separated and purified from the casein enzyme-treated product (hereinafter also referred to as “separated / purified fraction”) Peptide MKP content (mg / fraction 1 g) "or more can be adjusted.
For example, a high molecular fraction such as protein and polypeptide is removed to obtain a fraction containing a higher content of the tripeptide MKP, which can be used as a functional food or the like. In addition, since this tripeptide MKP-rich fraction is water-soluble even under acidic conditions, it can be used for various foods.

(5) Use of casein enzyme-treated product and each separated / purified fraction The casein enzyme-treated product and each separated / purified fraction obtained by the production method according to the present technology are used for various pharmaceuticals, foods and drinks, feeds, etc. can do.
Since the casein enzyme-treated product or each separated / purified fraction obtained by the production method according to the present technology uses milk-derived raw materials, it is highly safe to the living body and can be continuously administered or ingested for a long period of time. Also suitable for. In addition, since it is produced from a relatively inexpensive milk-derived raw material as a biomaterial, it can be stably and easily produced in large quantities, and can be provided to consumers at low cost. .

  When the casein enzyme-treated product or each separated / purified fraction is used as a pharmaceutical product, the pharmaceutical product may be administered orally or parenterally, but oral administration is preferred. Examples of parenteral administration include injection (blood, skin, muscle, etc.), rectal administration, inhalation and the like. Examples of the dosage form for oral administration include tablets, capsules, troches, syrups, granules, powders, ointments and the like.

  In addition, in addition to the casein enzyme-treated product and each separated / purified fraction, components such as excipients, pH adjusters, colorants, and corrigents that are commonly used for formulation can be used for formulation. it can. Furthermore, a component having an effect of preventing or treating a known or future disease can be used in combination according to the purpose.

  Furthermore, depending on the administration method, it can be appropriately formulated into a desired dosage form. 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. For parenteral administration, it can be formulated into suppositories, sprays, ointments, patches, injections and the like.

  In addition, formulation can be appropriately performed by a known method according to the dosage form. At the time of formulation, only the casein enzyme-treated product or each separated / purified fraction may be formulated, or may be formulated by blending a formulation carrier as appropriate.

  In addition, as the preparation carrier, various organic or inorganic carriers can be used depending on the dosage form. Examples of the carrier in the case of a solid preparation include excipients, binders, disintegrants, lubricants, stabilizers, and flavoring agents.

  Examples of the excipient include sugar derivatives such as lactose, sucrose, glucose, mannitol and sorbit; starch derivatives such as corn starch, potato starch, α-starch, dextrin and carboxymethyl starch; crystalline cellulose, hydroxypropyl cellulose, Cellulose derivatives such as hydroxypropylmethylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium; gum arabic; dextran; pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, magnesium magnesium magnesium silicate; phosphate derivatives such as calcium phosphate; And carbonate derivatives such as calcium; sulfate derivatives such as calcium sulfate and the like.

  Examples of the binder include gelatin, polyvinyl pyrrolidone, macrogol and the like in addition to the above excipients.

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

  As the lubricant, for example, talc; stearic acid; stearic acid metal salts such as calcium stearate and magnesium stearate; colloidal silica; waxes such as pea gum and geirow; 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 anhydrous silicic acid and silicic acid hydrate; starch derivatives and the like It is done.

  Examples of the stabilizer include paraoxybenzoates such as methyl paraben and propyl paraben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; sorbic acid and the like.

Examples of the flavoring agent include sweeteners, acidulants, and fragrances.
In addition, as a carrier used in the case of a liquid for oral administration, a solvent such as water, a flavoring agent and the like can be mentioned.

  When using casein enzyme-treated product or each separated / purified fraction for food or drink for humans or animals, it can be prepared by adding it to known food or drink, or it can be mixed in the raw material of food or drink to produce new food or drink. The product can also be manufactured.

  The foods and drinks may be in the form of liquids, pastes, solids, powders, etc., in addition to tablet confections, liquid foods, feeds (including for pets), etc., for example, flour products, instant foods, processed agricultural products, marine products Processed products, processed livestock products, milk / dairy products, fats and oils, basic seasonings, compound seasonings / foods, frozen foods, confectionery, beverages, and other commercial products.

Examples of flour products include bread, macaroni, spaghetti, noodles, cake mix, fried flour, bread crumbs and the like.
Examples of instant foods include instant noodles, cup noodles, retort / cooked food, cooked canned food, microwave food, instant soup / stew, instant miso soup / soup, canned soup, freeze-dried food, other instant foods, etc. It is done.
Examples of processed agricultural products include canned agricultural products, canned fruits, jams and marmalades, pickles, boiled beans, dried agricultural products, cereals (cereal processed products), and the like.
Examples of processed fishery products include canned fishery products, fish hams and sausages, marine products, marine delicacies, and tsukudani.
Examples of livestock processed products include canned livestock, pastes, livestock meat ham, sausage and the like.
Examples of milk / dairy products include processed milk, milk beverages, yogurts, lactic acid bacteria beverages, cheese, ice creams, prepared powdered milks, creams, and other dairy products.
Examples of the fats and oils include butter, margarine, vegetable oil and the like.
Basic seasonings include, for example, soy sauce, miso, sauces, tomato processed seasonings, mirins, vinegars, etc., and the above mixed seasonings and foods include cooking mix, curry ingredients, sauces, Examples include dressings, noodle soups, spices, and other complex seasonings.
Examples of the frozen food include raw material frozen food, semi-cooked frozen food, cooked frozen food, and the like.
Examples of the confectionery include caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pie, snacks, crackers, Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery, and other confectionery.
Examples of beverages include carbonated beverages, natural fruit juices, fruit juice drinks, soft drinks with fruit juice, fruit drinks, fruit drinks with fruits, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks Sports drinks, nutritional drinks, alcoholic drinks, other taste drinks, and the like.
Examples of commercially available foods other than the above include baby foods, sprinkles, and tea paste.

In addition, the food and drink defined in the present technology can be provided and sold as a food and drink displaying health applications.
The “display” act includes all acts for informing the consumer of the use, and if the expression can remind the user of the use, the purpose of the display, the content of the display, the display Regardless of the target object / medium, etc., all fall under the “display” act of this technology.

  Moreover, it is preferable that the “display” is performed by an expression that allows the consumer to directly recognize the above-described use. Specifically, it is the act of transferring, displaying, importing, displaying, or importing products that are related to food or drinks or products that describe the use, on advertisements, price lists, or transaction documents. For example, an act of describing and displaying the above uses or distributing them, or describing the above uses in information including the contents and providing them by an electromagnetic (Internet or the like) method can be given.

  On the other hand, the display content is preferably a display approved by the government or the like (for example, a display that is approved based on various systems determined by the government and performed in a mode based on such approval). Moreover, it is preferable to attach such display contents to advertising materials at sales sites such as packaging, containers, catalogs, pamphlets, POPs, and other documents.

  “Indication” also includes indication as health food, functional food, enteral nutrition food, special purpose food, health functional food, food for specified health, functional nutrition food, quasi drug, etc. Among these, in particular, there are indications approved by the Consumer Affairs Agency, for example, indications approved under the food system for specific health use, a similar system, and the like. Examples of the latter include a display as a food for specified health use, a display as a conditionally specified food for specified health use, a display indicating that it affects the structure and function of the body, and a display for reducing disease risk. More specifically, indications as foods for specified health (especially indications for health use) and regulations stipulated in the Ordinance for Enforcement of the Health Promotion Act (Ministry of Health, Labor and Welfare Ordinance No. 86 of April 30, 2003) and A similar display is a typical example.

  When using the casein enzyme-treated product and each separated / purified fraction for feed, it can be prepared by adding to a known feed, or can be mixed in the feed raw material to produce a new feed.

  Examples of raw materials for the feed include cereals such as corn, wheat, barley, and rye; bran such as bran, wheat straw, rice bran, and defatted rice bran; Animal feeds such as whey, fish meal and bone meal; yeasts such as beer yeast; mineral feeds such as calcium phosphate and calcium carbonate; fats and oils; amino acids; In addition, examples of the form of the feed include pet animal feed (pet food, etc.), livestock feed, fish feed, and the like.

  In addition, the said food-drinks or feed using the casein enzyme processed material produced by the manufacturing method which concerns on this invention, or each isolation | separation and refinement | purification fraction can also be pasteurized or sterilized.

  In addition, the tripeptide MKP of the present technology can be expected to have the following effects. For this reason, the casein enzyme-treated product containing a high amount of this tripeptide MKP and each separated / purified fraction can also be expected to have the following.

The tripeptide MKP can be used as an active ingredient of an angiotensin converting enzyme inhibitor. This peptide has an angiotensin converting enzyme inhibitory action and bradykinin inactivation inhibitory action, and exhibits a blood pressure lowering action.
Therefore, the tripeptide MKP is used as a preventive or therapeutic agent (specifically, a hypotensive agent, etc.) for various diseases derived from high blood pressure (eg, cerebral hemorrhage, cerebral infarction, angina pectoris, myocardial infarction, renal failure, etc.). can do.
Angiotensin converting enzyme inhibitors are also known to have an effect on essential hypertension of unknown cause, and the tripeptide MKP is also expected to show a therapeutic or preventive effect on essential hypertension. In addition, the tripeptide MKP can be used as a therapeutic or prophylactic agent for diseases such as cardiac hypertrophy and angina disease for which an angiotensin converting enzyme inhibitor is known to be effective (see Patent Document 1). ). The tripeptide MKP is also considered useful for the reserves of these diseases.

The tripeptide MKP can be used as an active ingredient of a dipeptidyl peptidase-IV inhibitor. This peptide has dipeptidyl peptidase-IV inhibition, hyperglycemic inhibitory action, hypoglycemic action, vascular endothelial function decline inhibitory action, vascular endothelial disorder inhibitory action, vascular disorder inhibitory action, vascular endothelial cell protective action and appetite suppressant The action etc. are shown. In addition, the tripeptide MKP is considered to be capable of suppressing appetite and protecting vascular endothelial cells.
Therefore, the tripeptide MKP is a prophylactic or therapeutic agent (specifically, hypoglycemia, for various diseases derived from hyperglycemia (for example, hyperglycemia, diabetes, diabetic complications, vascular endothelial disorder, vascular disorder, etc.) Inhibition of vascular endothelial injury and anti-diabetes) can also be used.
The tripeptide MKP can be said to be a very useful compound that has both an angiotensin converting enzyme inhibitory action and a dipeptidyl peptidase-IV inhibitory action, that is, has two effects of a blood glucose lowering action and a blood pressure lowering action (Patent Document 2).
The tripeptide MKP may also be useful for reserves of these diseases such as hypertension and / or hyperglycemia.
Tripeptide MKP has the effect of improving brain dysfunction caused by amyloid / beta protein, so it should be used as a preventive or therapeutic agent for brain dysfunction caused by amyloid / beta protein such as Alzheimer's dementia. (Patent Document 3).

Accordingly, the casein enzyme-treated product containing a high content of the tripeptide MKP and the separated and purified fractions containing the tripeptide MKP of the present technology are a food composition for inhibiting angiotensin converting enzyme, a food composition for inhibiting dipeptidyl peptidase-IV, and an amyloid. -It can be used as a food composition or the like for improving brain dysfunction caused by beta protein, or it can be used by being contained in these food compositions.
In addition, the casein enzyme-treated product containing a high content of tripeptide MKP and the separation / purification fraction containing tripeptide MKP according to the present technology can be used to prevent, improve, or treat various diseases (including the reserve army) that can be expected to be effective. Can be used as an ingredient for prevention of these various diseases, etc., or can be used as a component of drugs and foods for prevention of these various diseases, or It can also be used to produce drugs and foods for these various diseases.

  Thus, the present technology can be used in a wide range of fields such as pharmaceuticals, foods, food compositions, skin preparations, and functional foods.

  The present technology can also employ the following configurations.

[1] A method for producing a casein-treated product containing a tripeptide composed of Met-Lys-Pro, wherein a proline-specific endoprotease concentration of 0.1 to 3 U / g is used for casein as a raw material. .
[2] A method for producing a casein-treated product according to [1], wherein the amount of the proline-specific endoprotease used is 0.4 to 2 U / g.
[3] The method for producing a casein enzyme-treated product according to the above [1] or [2], wherein the MKP molar yield is at least 70%.
[4] A method for producing a casein enzyme-treated product according to any one of [1] to [3], wherein the reaction pH is in a neutral region.
[5] The method for producing a casein enzyme-treated product according to any one of [1] to [4], wherein the reaction time is 4 to 15 hours. The reaction time is preferably 6 to 15 hours, more preferably 9 to 15 hours.
[6] After performing the method for producing a casein-treated product according to any one of [1] to [5], from the casein-treated product, an undegraded casein fraction or a fraction having a molecular weight of more than 1,000 Da To obtain a fraction having a molecular weight of 1,000 Da or less including a tripeptide consisting of Met-Lys-Pro.
[7] Casein enzyme-treated product having a tripeptide MKP content (mg / casein enzyme-treated product 1 g) consisting of Met-Lys-Pro of 0.9 to 1.3 mg / treated product 1 g.
[8] The casein-treated product according to [7], wherein the content of tripeptide MKP comprising Met-Lys-Pro (mg / fraction 1 g of molecular weight of 1,000 Da or less) is 3 mg / fraction 1 g or more.
[9] The casein-treated product according to [7] or [8], comprising undegraded casein.
[10] A fraction having a molecular weight of 1,000 Da or less, including a tripeptide composed of Met-Lys-Pro obtained from the casein-treated product according to any one of [7] to [9].

  Hereinafter, the present technology will be described in more detail based on examples. In addition, the Example described below shows an example of a typical example of the present technology, and the scope of the present technology is not interpreted narrowly.

The measurement method used in this technique is shown below. Moreover, in the present Example, the following measuring methods were performed and each measurement result was obtained.
(1) Measurement of decomposition rate Sample powder was dissolved in deionized water at a concentration of 10% w / w, 30 mL of deionized water was added to 8 mL of the solution, and 0.1 M was added using a pH meter while stirring with a stirrer. Sodium hydroxide solution or 0.1M hydrochloric acid solution is added dropwise to adjust the pH to 6.80. To this was added 5 mL of 37% formalin solution adjusted to pH 8.0, and titrated with 0.1 M sodium hydroxide solution to pH 7.90. (XmL) At the same time, the Brix value (25 ° C.) of the peptide solution is measured using a saccharimeter. (Z%)
Based on the measured value, the peptide degradation rate is quantified by the following formula.
Decomposition rate (%) = formol nitrogen amount ÷ total nitrogen amount × 100
Formol-type nitrogen amount (mgN / dl) = 1.4 × xx × f (potency of sodium hydroxide) × 100/8
Total nitrogen amount (mgN / dl) = z × 0.7 × 1000 / 6.38

(2) Measurement of tripeptide MKP content The sample powder was diluted and dissolved in 0.2% formic acid aqueous solution so as to be 1.0 mg / mL, ultrasonically crushed for 10 minutes, and then a PVDF filter having a 0.22 μm aperture. (Millipore) to prepare a powder solution. These adjustment sample, C ¹³ labeled chemically synthesized internal standard peptide (manufactured by Thermo Fisher Scientific) was added at a concentration of 0.5 [mu] g / mL, to implement the LC / MS analysis under the following measuring conditions. On the other hand, a solution of tripeptide MKP chemically synthesized standard peptide (manufactured by Peptide Laboratories) is prepared at concentrations of 0.2, 0.6, and 1.0 μg / mL. Them to C ¹³ labeled chemically synthesized internal standard peptide was added at a concentration of 0.5 [mu] g / mL, performed LC / MS analysis under the following measuring conditions, a calibration curve is prepared. Among the peaks in the analysis of the powder solution, those having the same molecular weight and retention time as the standard peptide are identified as the same sequence as the standard peptide. The content of tripeptide MKP in the powder solution is determined by comparing the peak of the standard peptide with the peak area.

(3) LC / MS
<LC / MS equipment>
Mass spectrometer: TSQ Quantum Discovery MAX (manufactured by Thermo Fisher Scientific).
High-performance liquid chromatograph configuration: System controller: CBM-20A, Autoinjector: SIL-20AC, Liquid feed pump: LC-20AD × 2, Column oven: CTO-20AC, UV / VIS detector: SPD-20A, Online degasser: DGU-20A5 (manufactured by Shimadzu Corporation), column: TSKgel ODS-100V 5 μm (2.0 mm ID × 250 mm L) (manufactured by Tosoh Corporation).

<LC / MS measurement conditions>
Mobile phase A: 0.2% by weight Formic acid-aqueous solution Mobile phase B: 0.2% by weight Formic acid-acetonitrile solution Time program: 2% B (0.0 minutes) -25% B (5.0 minutes) -65% B (5.1 min) -65% B (10 min) -85% B (10.1 min) -85% B (13.0%)-2% B (13.1 min) -STOP (30. 0 minutes).
Sample injection volume: 10 μL,
Column temperature: 40 ° C
Ionization mode: ESI (+),
Liquid flow rate: 200 μL / min,
Spray voltage: + 4.5kV
Capillary temperature: 320 ° C.
Sheath gas pressure: 30 Arb,
Auxiliary gas pressure: 4 Arb
Analysis mode: SRM measurement,
Product Mass: m / z = 260.10 (Parent m / z = 375.21),
^{(C 13} internal standard Product Mass: m / z = 260.10 (Parent m / z = 381.22))

(4) MKP molar yield (%)
MKP molar yield (%) = (Peptide MKP measured value / Peptide MKP logical value) × 100
[Measured value of peptide MKP] is a value obtained by measuring peptide MKP in a sample by the above (3) “LC / MS”.
[Theoretical value of peptide MKP] = [per 1000 mg of casein enzyme-treated product] × [content of casein protein in enzyme-treated product (86.9 g / 100 g)] × [content of αs2-casein in casein protein (10 g / 100 g)] × [Mass ratio of peptide MKP in αs2-casein (375: 25200)]
= 1000 × 0.869 × 0.1 × 375 ÷ 25200
= 1.293 mg / g

(5) MKP content (mg / casein enzyme treated product 1 g)
MKP content (mg / casein enzyme-treated product 1 g) = [measured value of tripeptide MKP in the obtained casein enzyme-treated product (mg)] / [mass of the obtained casein enzyme-degraded product (g)]
[Measured value of tripeptide MKP (mg) in the obtained casein-treated product] is a measured value of tripeptide MKP in the sample according to (3) “LC / MS”.

(6) MKP content (mg / fraction 1 g of molecular weight 1,000 Da or less)
MKP content (mg / fraction 1 g of molecular weight of 1,000 Da or less) = [measured value of tripeptide MKP in the obtained casein-treated product (mg)] / [obtained casein-treated product (g) × molecular weight The fraction ratio of 1,000 Da or less]
[Fraction ratio of molecular weight of 1,000 Da or less] = [Fraction area of molecular weight of 1,000 Da or less in molecular weight distribution] / [Total area of casein enzyme treated product in molecular weight distribution]
[Measured value (mg) of tripeptide MKP in the obtained casein-treated product] is a measured value (mg) of tripeptide MKP in the sample according to (3) “LC / MS”.
The molecular weight distribution is determined by the following “(7) Measurement of molecular weight distribution”.

(7) Measurement of molecular weight distribution The molecular weight distribution is measured by high performance liquid chromatography (edited by Nobuo Ui, et al., “High Performance Liquid Chromatography of Proteins and Peptides”, Chemical Special Issue No. 102, page 241, Kagaku Dojin, (1984).
Specifically, a polyhydroxyethyl aspartamide column [Poly Hydroxyethyl Aspartamide Column: Poly LC (manufactured by Poly LC)]. Eluting with 20 mM sodium chloride and 50 mM formic acid at an elution rate of 0.5 mL / min.
Detection uses a UV detector (manufactured by Shimadzu Corporation), and data analysis uses a GPC analysis system (manufactured by Shimadzu Corporation).
[Ratio of each molecular weight range (%)] = [Area of each molecular weight range in molecular weight distribution / Total area of casein enzyme treated product in molecular weight distribution (all areas)]

(8) Measurement of bitterness The sample powder is dissolved in deionized water at room temperature at a concentration of 5% w / w, and a sensory evaluation of bitterness is performed by six flavor panelers. The evaluation of bitterness intensity is quantified according to the following criteria, and an average value is calculated.
・ Number 1: No bitterness (equivalent to water)
・ Number 2: Feels slightly bitter (equivalent to 0.05% aqueous caffeine solution)
・ Number 3: Feel bitter (equivalent to 0.10% caffeine aqueous solution)
・ Number 4: Bitterness is felt slightly stronger (equivalent to 0.15% caffeine aqueous solution)
・ Number 5: Feel bitterness quite strongly (equivalent to 0.20% caffeine aqueous solution)

(9) Measurement of emulsifiability The sample powder was dissolved in deionized water at a concentration of 1% w / w, 0.6 mL of soybean oil was added to 1.4 mL, and 20 KHz, 40 W, 1 minute with an ultrasonic generator. Emulsify to prepare the emulsion.
To the prepared emulsion, 8 mL of deionized water was added to make a total volume of 10 mL. 0.1 mL of this is taken and further mixed with 10 mL of 0.1% SDS aqueous solution, and OD500 is measured with a spectrophotometer. The higher the turbidity value, the higher the emulsifiability. (According to the method described in the Journal of the Japan Food Industry Association, Vol. 27, No. 12, December 1980, "Method for micro-measurement of soy protein emulsifiability by ultrasonic treatment and turbidity")

[Example 1: product of the present invention 1: single degradation of proline-specific endoprotease]
After adding 90 g of distilled water adjusted to about 60 ° C. to 10 g of milk-derived lactate casein (manufactured by Fontera Co., Ltd.), sodium hydroxide was added to adjust the pH to 6.2 to 7 to prepare a substrate solution. . Proline-specific protease (manufactured by DSM) is added to the obtained substrate solution at a concentration of 0.3 to 2 U / g (0.3, 0.4, 0.5, 1.0, 2.0) with respect to casein. [U / g]) and incubated at 55 ° C. for 15 hours. Subsequently, the enzyme was inactivated by heating at 85 ° C. for 10 minutes, and then the obtained enzyme-treated product solution was freeze-dried and powdered.
The MKP content in each powder was measured by LC / MS. The results are shown in Table 1.

[Comparative Example 1: Comparative product 1: Combined degradation of proline-specific endoprotease and serine endoprotease]
90 g of distilled water adjusted to about 60 ° C. was added to 10 g of milk-derived lactic casein (manufactured by Fontera Co., Ltd.) and stirred sufficiently, and then sodium hydroxide was added to adjust the pH to 9.0 to prepare a substrate solution. Serine endoprotease (manufactured by Amano Enzyme) was added to the obtained substrate solution at a concentration of 1,500 U / g with respect to casein. Immediately thereafter, proline-specific protease (manufactured by DSM) was added to casein. Added at a concentration of 0.5 U / g and incubated at 55 ° C. for 15 hours. Subsequently, the enzyme was inactivated by heating at 85 ° C. for 10 minutes, and then the obtained enzyme-treated product solution was freeze-dried and powdered.

[Comparative Example 2: Comparative product 2: Degradation of serine endoprotease]
90 g of distilled water adjusted to about 60 ° C. was added to 10 g of milk-derived lactic casein (manufactured by Fontera Co., Ltd.) and stirred sufficiently, and then sodium hydroxide was added to adjust the pH to 9.0 to prepare a substrate solution. Serine endoprotease (Amano Enzyme) was added to the obtained substrate solution at a concentration of 1,500 U / g with respect to casein and incubated at 55 ° C. for 15 hours. Subsequently, the enzyme was inactivated by heating at 85 ° C. for 10 minutes, and then the obtained enzyme-treated product solution was freeze-dried and powdered.

Table 2 shows the evaluation results of the decomposition rate, tripeptide MKP content, MKP molar yield, bitterness, and emulsification in each powder.
Table 3 shows the molecular weight distribution of each powder.

Compared with comparative product 1 and comparative product 2, the product 1 of the present invention has an MKP molar yield close to almost 100% despite the fact that the decomposition has progressed only to the minimum necessary, while the comparative product using the enzyme in combination. 1 thought that the MKP molar yield would be close to 100%, but the tripeptide MKP content was rather low.
Further, it can be seen that the product 1 of the present invention has more polymer fractions that provide good flavor and emulsification. Casein present in the product 1 of the present invention is considered to be undegraded or hardly decomposed from the molecular weight distribution and is easily water-soluble.
Furthermore, since the product 1 of the present invention is about 60% of the high molecular fraction having a molecular weight of more than 10,000 Da in terms of the molecular weight distribution, it is easy to remove the high molecular fraction and the low molecular weight fraction having a molecular weight of 1,000 Da or less. Easy to collect minutes. And compared with the comparative products 1 and 2, in the product 1 of the present invention, the content of the tripeptide MKP in the low molecular fraction having a molecular weight of 1,000 Da or less is four times or more. On the other hand, according to the molecular weight distribution, the comparative product 1 has a low molecular fraction of about 80% of a molecular weight of 1,000 Da or less, and a large number of peptides are miscellaneous, so it is difficult to recover the tripeptide MKP from this, It is also difficult to increase the concentration.

Thus, by using a proline-specific endoprotease alone, it is possible to easily obtain a casein enzyme-treated product with a high content of tripeptide MKP, and it is also easy to increase the content of tripeptide MKP.
In addition, since the casein-treated product obtained has little or no bitter taste and high emulsifying properties, it can be used as food (for example, functional food) and the like, and further food additives (for example, flavor improvement) Agent, emulsifier, etc.).

  In addition, an aqueous solution containing the product 1 of the present invention was prepared, acid precipitation was performed by adjusting the pH of the solution to 4.5, and the precipitate was separated into an acid precipitate and a supernatant by centrifugation, and the supernatant was recovered. . In this supernatant, since the tripeptide MKP is water-soluble, the tripeptide MKP is present. The measured value of the tripeptide MKP contained in the supernatant was 7.16 mg / solid content of the supernatant 1 g. In acid precipitates, there are proteins and polypeptides that have been acid precipitated.

Claims (8)

  A method for producing a casein-treated product containing a tripeptide consisting of Met-Lys-Pro, wherein a proline-specific endoprotease concentration of 0.1 to 3 U / g is used for casein as a raw material.   The method for producing a casein enzyme-treated product according to claim 1, wherein the amount of the proline-specific endoprotease used is 0.4 to 2 U / g.   The method for producing a casein enzyme-treated product according to claim 1 or 2, wherein the MKP molar yield is at least 70%.   The method for producing a casein enzyme-treated product according to any one of claims 1 to 3, wherein the reaction pH is in a neutral region.   After performing the method for producing a casein enzyme-treated product according to any one of claims 1 to 4, an undegraded casein fraction or a fraction having a molecular weight of more than 1,000 Da is removed from the casein enzyme-treated product, A method for obtaining a fraction having a molecular weight of 1,000 Da or less containing a tripeptide comprising Met-Lys-Pro.   Casein enzyme-treated product having a tripeptide MKP content (mg / casein enzyme-treated product 1 g) consisting of Met-Lys-Pro of 0.9 to 1.3 mg / g.   The casein enzyme-treated product according to claim 6, wherein the content of tripeptide MKP comprising Met-Lys-Pro (mg / fraction 1 g of molecular weight of 1,000 Da or less) is 3 mg / g or more. The casein enzyme-treated product according to claim 6 or 7, comprising undegraded casein.