JP2021164423A - Composition for growth of lachnospiraceae bacteria - Google Patents

Abstract

To provide a composition that can grow Lachnospiraceae bacteria and can be used for anxiolytic or antidepressive purposes.SOLUTION: A composition for the growth of Lachnospiraceae bacteria contains whey protein hydrolysate. The Lachnospiraceae bacteria are one or more selected from Blautia and Dorea.SELECTED DRAWING: Figure 1

Description

The present invention relates to a composition for growing Lachnospiraceae bacteria, which comprises a whey protein hydrolyzate. It also relates to compositions for the treatment, amelioration or prevention of diseases or conditions that correlate with Lachnospiraceae bacteria, which contain whey protein hydrolysates.

Lachnospiraceae bacteria are present in the intestinal flora and have recently been reported to be associated with various diseases.
Non-Patent Document 1 describes that colitis mice treated with Lachnospiraceae bacteria were protected from colitis-related death.
Regarding the occupancy rate in the intestinal flora, it has been reported that depressive patients have more Bacteroidales bacteria and less Lachnospiraceae bacteria than non-depressive patients (Non-Patent Document 2).
The occupancy rate of Lachnospiraceae in the gut microbiota is lower in patients with non-alcoholic fatty liver, type 2 diabetes, type 1 diabetes, and rheumatoid arthritis than in healthy subjects, and is increased in autism spectrum syndrome. It is known that this is done (Non-Patent Document 3).
It has also been reported that the degree of predominance of the bacterium in the intestinal flora differs depending on the genus Lachnospiraceae in relation to obesity (Patent Document 1, Non-Patent Documents 4 to 6).
Furthermore, in Non-Patent Document 7, when the intestinal flora of patients after allogeneic bone marrow transplantation was evaluated, the higher the occupancy rate of Lachnospiraceae Blautia in the intestinal bacterial flora, the more death due to acute graft-versus-host disease. It has been reported that the rate has declined.

By the way, whey protein hydrolyzate is known to have an effect of improving the intestinal flora by growing bifidobacteria and lactic acid bacteria and suppressing the overgrowth of intestinal bacteria. (Patent Documents 2 to 4).
However, it is not known how whey protein hydrolysates affect Lachnospiraceae bacteria in the gut microbiota.

International Publication No. 2012/024638 Re-table 2013/08911 Special Table 2004-536143 Special Table 2001-516570

N. K. Surana et al., Nature Vol. 552, 244-247 (2017) A. Naseribafrouei et al., Neurogastroenterol. Motil. Vol. 26, 1155-1162 (2014) Kunitomo Watanabe, Modern Media Vol. 60 No. 6 356-368 (2014) Keishi Kameyama et al., Microbes Environ. Vol. 29, No. 4, 427-430, (2014) A. Andoh et al., J. Clin. Biochem. Nutr., Vol. 59, No. 1, 65-70 (2016) https://www.cnn.co.jp/fringe/35125905.html R. Jenq et al., Bio Blood Marrow Transplant vol. 21, 1373-1383 (2015)

An object of the present invention is to provide a composition capable of growing Lachnospiraceae bacteria.

As a result of diligent research to solve the above problems, the present inventors have found that a whey protein hydrolyzate causes a specific genus of Lachnospiraceae bacteria to grow in the intestinal flora, and completes the present invention. It came to.

That is, one aspect of the present invention is a composition for growing Lachnospiraceae bacteria containing a whey protein hydrolyzate, wherein the Lachnospiraceae bacteria are one or more selected from the genus Blautia and the genus Drea. , The composition.
In this embodiment, the whey protein hydrolyzate preferably has the following properties (a) to (c).
(A) The number average molecular weight is 200 Da or more and 600 Da or less. (B) The proportion of peptides having a molecular weight of 1000 Da or less is 40% by mass or more and 95% by mass or less. (C) The amino acid release rate is preferably 30% by mass. The composition of this embodiment, which is as follows, is preferably for growth of Blautian spp.
The composition of this embodiment is preferably for the growth of bacteria of the genus Drea.

Another aspect of the invention is an anxiolytic or antidepressant composition containing a whey protein hydrolyzate.
In this embodiment, the whey protein hydrolyzate preferably has the following properties (a) to (c).
(A) The number average molecular weight is 200 Da or more and 600 Da or less. (B) The proportion of peptides having a molecular weight of 1000 Da or less is 40% by mass or more and 95% by mass or less.

According to the present invention, bacteria belonging to the genus Blautia or the genus Drea of the family Lachnospiraceae can be propagated in the intestinal flora. It can also be expected to treat, ameliorate or prevent diseases or conditions that correlate with Lachnospiraceae bacteria.

The graph which shows the intestinal flora occupancy rate of Clostridiales Lachnospiraceae bacteria in a rat after ingestion of a whey protein or a hydrolyzate thereof. The graph which shows the intestinal flora occupancy rate of Clostridiales Lachnospiraceae Blautia bacterium in rat after ingestion of whey protein or its hydrolyzate. The graph which shows the intestinal flora occupancy rate of Clostridiales Lachnospiraceae Drea bacterium in a rat after ingestion of whey protein or a hydrolyzate thereof. The graph which shows the intestinal flora occupancy rate of Akkermansia muciniphila bacterium in rat after ingestion of whey protein or its hydrolyzate.

Next, the present invention will be described in detail. However, the present invention is not limited to the following embodiments, and can be freely changed within the scope of the present invention.

The composition of the present invention contains a whey protein hydrolyzate.

The composition of the present invention contains a whey protein hydrolyzate as an active ingredient.

The number average molecular weight of the whey protein hydrolyzate in the present invention is preferably 200 Dalton (hereinafter, “Da”) or more and 600 Da or less, more preferably 300 Da or more and 500 Da or less, and further preferably 330 Da or more and 480 Da or less. Particularly preferably, it is 360 Da or more and 450 Da or less.
Further, in the whey protein hydrolyzate in the present invention, the proportion of peptides having a molecular weight of 1000 Da or less is preferably 40% by mass or more and 95% by mass or less with respect to the total amount of peptides contained in the whey protein hydrolyzate, which is more preferable. Is 50% by mass or more and 90% by mass or less, more preferably 60% by mass or more and 85% by mass or less, and particularly preferably 64% by mass or more and 82% by mass or less.

The average molecular weight of the whey protein hydrolyzate in the present invention is determined by the following concept of number average molecular weight.
The Number Average of Molecular Weight is described in, for example, the literature (edited by the Society of Polymer Science, "Basics of Polymer Science", pp. 116-119, Tokyo Chemical Co., Ltd., 1978). As shown, the average value of the molecular weights of the polymer compounds is shown based on the following different indexes.
That is, since a high molecular weight compound such as a protein hydrolyzate is a heterogeneous substance and has a distribution in molecular weight, the molecular weight of the protein hydrolyzate must be indicated by an average molecular weight in order to handle it physicochemically. Yes, the number average molecular weight (hereinafter, may be abbreviated as Mn) is an average of the number of molecules.

In the present specification, the average molecular weight of whey protein hydrolyzate refers to those measured and calculated by the following methods. That is, using high performance liquid chromatography, using a Poly Hydroxyethyl Aspartamide Column (Poly LC; diameter 4.6 x 200 mm), 20 mM sodium chloride, 50 mM formic acid. (Elution rate of 0.4 mL / min) (edited by Nobuo Ui et al., "High Performance Liquid Chromatography of Proteins and Peptides", Chemistry Special Edition No. 102, p. 241; Chemistry Dojin Co., Ltd., 1984). The detection is performed using a UV detector (manufactured by Shimadzu Corporation), and data analysis is performed by a GPC analysis system (manufactured by Shimadzu Corporation) to calculate the number average molecular weight. As a standard for calculating the molecular weight, a protein and / or a peptide having a known molecular weight may be appropriately used.

Protein hydrolysates generally contain free amino acids during the manufacturing process. The amino acid release rate of the whey protein hydrolyzate in the present invention is preferably 30% by mass or less, more preferably 20% by mass or less, still more preferably 15% by mass or less, and particularly preferably 10% by mass or less. Is.

In the present specification, the amino acid release rate is the ratio of the amount of free amino acid to the whole whey protein hydrolyzate, and can be measured and calculated by the following method.
For amino acids other than tryptophan, cysteine and methionine, the sample was hydrolyzed with 6N hydrochloric acid at 110 ° C for 24 hours, for tryptophan, alkaline decomposition with barium hydroxide at 110 ° C for 22 hours, and for cysteine and methionine. After the treatment with methionine, it is hydrolyzed at 110 ° C. for 18 hours with 6 specified hydrochloric acid, and each is analyzed by an automatic amino acid analyzer (manufactured by Hitachi, Ltd., type 835), and the mass of amino acids is measured.
The composition of each amino acid in the sample is measured by the above method, and the total is calculated to calculate the mass of all amino acids in the sample. Next, the sample is deproteinized with sulfosalicylic acid, the mass of each remaining free amino acid is measured by the above method, and the total is calculated to calculate the mass of all free amino acids in the sample. From these values, the free amino acid content in the sample is calculated by the following formula.
Amino acid release rate (mass%) = (mass of all free amino acids / mass of all amino acids) x 100

The whey protein hydrolyzate as described above can be preferably obtained by usually decomposing the whey protein, preferably hydrolyzing it. The whey protein decomposition treatment is not particularly limited, and examples thereof include enzyme treatment, acid treatment, alkali treatment, heat treatment, and the like, and two or more of these treatments may be appropriately combined.

Whey protein, which is a starting material for obtaining whey protein hydrolyzate, is, for example, whey (for example, cheese whey, acid whey, membrane-separated whey, whey) separated from commercially available products or milk, defatted milk, etc. by a known method. (Powder, desalted whey powder, etc.), or one or a mixture of two or more selected from separated and purified whey protein concentrate (WPC), whey protein isolate (WPI), and the like. The mixture may be mixed at an arbitrary ratio.
Further, the whey protein may be derived from a plant such as soybean (so-called "soybean whey" or the like) in addition to a general milk-derived protein, but it is more preferably derived from milk, and more preferably. It is derived from bovine milk.

The general method for obtaining a whey protein hydrolyzate will be described below, but the method is not particularly limited thereto.
The raw material whey protein is dispersed in water or warm water and dissolved to prepare an aqueous whey protein solution.
Further, the whey protein aqueous solution is subjected to an ion exchange method using a sodium-type or potassium-type cation exchange resin (preferably a strongly acidic cation exchange resin), an electrodialysis method, a limit filtration membrane method, a loose reverse osmosis membrane method, etc. It is preferable to demineralize with and adjust the pH and calcium concentration as appropriate. At the time of desalting, either a column type or a batch type may be adopted. In addition, the whey protein aqueous solution may be appropriately heat sterilized before desalting.

Next, the whey protein aqueous solution is hydrolyzed. Examples of the hydrolysis treatment include enzyme treatment, acid treatment, alkali treatment, heat treatment and the like, and two or more of these treatments may be appropriately combined.
Examples of the protein hydrolase of the present disclosure include plant-derived, animal-derived, and microbial-derived, and one or a combination of two or more of these can be used. As the protein hydrolase, endoprotease is suitable.
Examples of the endoprotease include serine protease, metalloprotease, cysteine protease, and aspartic protease, and one or more of these can be selected and used. Of these, it is preferable to use serine protease and / or metalloprotease.
In addition, proteases are classified into alkaline proteases, neutral proteases and acidic proteases. Of these, it is preferable to use a neutral protease.

As the protein hydrolase, a commercially available product can be used. Examples of the protein hydrolase include bioplase (manufactured by Nagase Seikagaku Corporation), proleather (manufactured by Amano Enzymes), protease S (manufactured by Amano Enzymes), PTN6.0S (manufactured by Novozymes), and sabinase (manufactured by Novozymes). Made by), GODO B. A. P (manufactured by Joint Sake Seisha), Protease N (manufactured by Amano Enzyme), GODO B. N. P (manufactured by Joint Sake Seisha), Neutrase (manufactured by Novozymes), Alcalase (manufactured by Novozymes), Trypsin (manufactured by Novozymes), Chymotrypsin (manufactured by Novozymes), Subtilisin (manufactured by Novozymes), Papain (manufactured by Amano Enzymes) ), Bromelain (manufactured by Amano Enzyme Co., Ltd.) and the like, and one or more enzymes may be selected and used from these.
Of these, one or more neutral proteases selected from subtilisin (eg, bioplase), trypsin (eg, PTN6.0S), and bachillolysin (eg, protease N) are preferred and more preferred. Uses these three types in combination.

The amount of the endoprotease used for the whey protein is not particularly limited, and may be appropriately adjusted depending on the substrate concentration, enzyme titer, reaction temperature, reaction time, and the like.
By appropriately adjusting the hydrolysis conditions by the protein hydrolase, a whey protein hydrolyzate in a specific average molecular weight range according to the present invention can be obtained.
Prior to hydrolysis by the protein hydrolase, the pH of the raw material milk protein solution can be adjusted to the optimum pH of the enzyme used by using food-available salts such as potassium carbonate and sodium hydroxide. ..
The reaction temperature of the protein hydrolase is preferably in the optimum temperature range of the enzyme used.
The reaction retention time of the protein hydrolase may be appropriately adjusted so as to have the specific non-protein nitrogen ratio.
Hydrolysis by the protein hydrolase may be terminated by heating the enzyme to inactivate it. For example, it is preferable to inactivate at 100 ° C. or higher (preferably 110 to 130 ° C.) for 1 to 3 seconds, and to inactivate at 60 ° C. or higher below 100 ° C. for 3 to 40 minutes. ..

In the production of the whey protein hydrolyzate according to the present invention, when the solution whose calcium concentration has not been adjusted is hydrolyzed, the obtained decomposition solution is desalted as described above to adjust the calcium concentration. You may. Then, it is heated by a conventional method to inactivate the enzyme. The reaction heating temperature and the reaction holding time can be appropriately set under conditions that allow sufficient deactivation in consideration of the thermal stability of the enzyme used. After heat deactivation, it can be cooled by a conventional method and used as it is, and if necessary, it can be concentrated to obtain a concentrated solution, and the concentrated solution can be further dried to obtain a powder product.

Further, when the whey protein aqueous solution is hydrolyzed by acid treatment or alkali treatment, the pH of the whey protein aqueous solution may be adjusted for the treatment. In the case of the treatment by the pH adjustment, the pH of the whey protein aqueous solution is preferably pH 5 or less or pH 9 or more, and more preferably pH 4 or less or pH 10 or more. The pH-treated aqueous solution can be left or stirred at room temperature for several minutes or longer, preferably 5 minutes to 1 hour to obtain an acid-treated or alkaline-treated hydrolyzate. Here, "room temperature" is about 4 to 40 ° C, but 10 to 30 ° C is preferable.
Further, the whey protein aqueous solution may be hydrolyzed by heat treatment. The whey protein aqueous solution may be pH-adjusted, or may be pH-adjusted (specifically, acidic (pH 5 or less), neutral (pH 6 to 8), alkaline (pH 8 or more)). The heat treatment may be performed at about 4 to 100 ° C. under the conditions such as the above acid-alkali treatment.

The obtained whey protein hydrolyzate can exert its efficacy even when used in an unpurified state, but further, known separation and purification may be carried out as appropriate. For example, the obtained whey protein hydrolyzate can be subjected to molecular weight fractionation to obtain a whey protein hydrolyzate that satisfies the range of the average molecular weight and the amino acid release rate according to the present invention.
As the molecular weight fraction, for example, methods such as ultrafiltration and gel filtration can be adopted, whereby the removal rate of peptides and free amino acids having an unnecessary molecular weight can be increased.
In the case of ultrafiltration, a desired ultrafiltration membrane may be used, and in the case of gel filtration, a gel filter agent used for desired size exclusion chromatography may be used.
Further, a known separation and purification method (for example, ion exchange resin or the like) may be used in order to remove desalting, remove impurities, and increase the purity.

The whey protein hydrolyzate according to the present invention can be produced and obtained as described above, but a commercially available product may be used for the composition of the present invention.
Commercially available products include "W800", "W1500HC", "W1600", "WU1800", "W2500H", "W2500HC", "WU2900", "C800", "C2000H", "CU2500A" manufactured by Morinaga Milk Industry Co., Ltd. , "MCH-30", "C3500Ca", "CU5000", "MKP" and the like can be used.
Further, the whey protein hydrolyzate suitable for growing Blautia bacteria is selected from "hydrolyzed product A", "hydrolyzed product B" and "hydrolyzed product C" produced by the method described later. One or more of the above are particularly preferred.
In addition, examples of the whey protein hydrolyzate suitable for growing Drea bacteria are particularly preferably one or more selected from "hydrolyzer A" and "hydrolyzate C".

"Hydrolyzed product A" can be produced by the method disclosed in Japanese Patent No. 3059595.
Specifically, the solution containing whey protein is treated with an ion exchange resin or desalted to adjust the calcium concentration per 100 g of protein to 350 mg or less, endoprotease is added, and non-protein nitrogen with respect to the total amount of nitrogen is added. It can be produced by hydrolyzing the whey protein in the range of 50% (weight) or less, adjusting the pH to 7.0 ± 0.5, and then heating to inactivate the protease.
Alternatively, an endoprotease is added to a solution containing whey protein, the whey protein is hydrolyzed in a range where the ratio of non-proteinaceous nitrogen to the total amount of nitrogen is 50% (weight) or less, and the obtained hydrolyzed solution is used. Produced by ion-exchange resin treatment or desalting, adjusting the calcium concentration per 100 g of protein to 350 mg or less, adjusting the pH to 7.0 ± 0.5, and then heating to inactivate the protease. Can be done.
Here, the whey protein raw material conforms to the above-mentioned general description.

In any of the above production methods, it is essential to adjust the calcium concentration to 350 mg or less with respect to 100 g of protein in the solution, more preferably 250 mg or less from the viewpoint of preventing precipitation, before the enzyme is deactivated by heating. The adjustment process can be performed before, after, or both of the enzymatic decomposition. The calcium concentration is adjusted by desalting by an ion exchange method using a potassium-type cation exchange resin (preferably a strongly acidic cation exchange resin), an electrodialysis method, an extrapolation method, a loose reverse osmosis method, or the like. Just do it.

Endoproteases are used for enzymatic degradation of whey protein solutions. At that time, if the whey protein concentration of the whey protein solution is less than 5%, the production efficiency decreases, and if the concentration exceeds 15%, the viscosity increases at the time of heat deactivation. It is desirable that the whey protein concentration is in the range of 5% or more and 15% or less.
Examples of the end-type protease include proteases derived from microorganisms such as microorganisms belonging to the genus Bacillus and microorganisms belonging to the genus Aspergillus, proteases derived from plants such as papain derived from papain and bromelain derived from pineapple, and pancreatin derived from animals. Trypsin or the like, or a mixture thereof in any proportion, can be used.
Before enzymatic decomposition, the pH of the whey protein solution can be adjusted to the optimum pH of the enzyme used with salts that are harmless to food hygiene such as potassium carbonate and sodium hydroxide.

After completion of hydrolysis, the pH of the decomposition solution is adjusted to 7.0 ± 0.5, preferably 7.0 ± 0.3. If the pH of the obtained decomposition liquid is within the above range, it is not necessary to adjust the pH.
When the solution prepared at the calcium concentration powder is hydrolyzed, the obtained decomposition solution is treated with an ion exchange resin or desalting as described above to adjust the calcium concentration.

Then, it is heated by a conventional method to inactivate the enzyme. The heating temperature and holding time may be appropriately set under conditions that allow sufficient deactivation in consideration of the thermal stability of the enzyme used. After heat deactivation, it can be cooled by a conventional method and used as it is, and if necessary, it can be concentrated to obtain a concentrated solution, and the concentrated solution can be further dried to obtain a powder product.

"Hydrolyzed product B" can be produced by the method disclosed in Japanese Patent No. 29597747.
Specifically, whey protein having a purity of at least 70% by weight is dissolved in water at a concentration of 15% by weight or less, the pH of the aqueous solution is adjusted to 7.5 to 10, and Bacillus subtilis is added to the aqueous solution. )-Derived endopeptidase and lactic acid bacteria-derived exopeptidase are added to start hydrolysis, and the amount of free lysine in the degradation solution is measured over time and contained in the whey protein as the starting material. Proteolysis is stopped when the ratio of the amount of free lysine to the total amount of lysine is 12 to 20% by weight, and exopeptidase is performed to completely remove the fraction having a molecular weight of 10,000 Da or more. Can be done.

Here, the whey protein raw material conforms to the above-mentioned general description as long as the purity is at least 70% by weight.
First, the whey protein raw material is dissolved in water at a concentration of 15% or less, preferably 8 to 12%, and the pH is adjusted to 7.5 to 10, preferably 8 to 9 with an alkaline aqueous solution.

Next, two types of proteolytic enzymes, endopeptidase derived from Bacillus satiris and exopeptidase derived from lactic acid bacteria, are added to the whey protein solution. In addition, a very small amount of endopeptidase such as trypsin or papain can be added. However, the addition of exopeptidase (including pancreatin, etc.) other than that derived from lactic acid bacteria should be avoided because it deteriorates the flavor. As the endopeptidase derived from Bacillus satiris, a commercially available product or the like can be used, and it is added at a ratio of 1,000 to 7,500 PUN units, preferably 2,000 to 3,000 PUN units per 1 g of whey protein.

The exopeptidase derived from lactic acid bacteria can be produced, for example, by the method described in the section "(3) Enzyme to be used" in column 6, line 4 of Japanese Patent Publication No. 54-36235, as follows. Lactic acid bacteria (including bifidobacteria) are cultivated by a known method (for example, the method described in Japanese Patent Publication No. 48-43878), and the obtained culture solution is centrifuged to collect lactic acid bacteria cells and put them in sterile water. The operation of suspending the cells, centrifuging and collecting the lactic acid bacteria cells was repeated twice, the cells were washed, the cells were suspended in sterile water at a concentration of 20%, and a cell disruptor [for example, dynomil (for example, Willy Bachnfen Engineering Works). The cells are crushed by KDL type] and freeze-dried to obtain exopeptidase powder derived from lactic acid bacteria. This enzyme is added in a proportion of 20-200 active units, preferably 60-90 active units per gram of whey protein.

The enzyme-added solution is held at 30-60 ° C, preferably 45-55 ° C to initiate hydrolysis of whey protein. When the hydrolysis reaction proceeds and the pH drops, it is desirable to keep the pH at 6 or more, preferably 6 to 7. After starting hydrolysis, the amount of free lysine in the decomposition solution is measured over time using a device capable of measuring the amount of free lysine in the decomposition solution over time, for example, a biotech analyzer (manufactured by Asahi Kasei Kogyo Co., Ltd.). When the ratio of the amount of free lysine to the total amount of lysine contained in the starting material whey protein reaches the range of 12 to 20%, preferably 14 to 17%, the reaction solution is immediately heated (for example, 85). Inactivate the enzyme and stop hydrolysis.
The obtained reaction solution can be concentrated by a known method to obtain a concentrated solution, and the concentrated solution can be further dried by a known method to obtain a powder.

The "hydrolyzate B" produced in this manner has the following physicochemical properties a) to h).
a) The fraction with a molecular weight of 5,000 to 10,000 Da is less than 1% by weight of the total hydrolyzate.
b) The residual antigen activity measured by the Eliza suppression test method using anti-whey protein serum is 10-5 or less.
c) The ratio of the amount of free amino acids to the total amount of amino acids in the hydrolyzate is 10 to 15% by weight.
d) The ratio of the amount of free lysine to the amount of total lysine contained in whey protein is 12 to 20% by weight.
e) The ammonia content is 0.2% by weight or less.
f) The transmittance of a 10 wt% solution measured in a 1 cm cell at 540 nm is 98% or more.
g) A 5 wt% solution of pH 4-7 is heated at 120 ° C. for 10 minutes to prevent precipitation.
h) Has an antioxidant effect.

"Hydrolyzed product C" can be produced by the method disclosed in Japanese Patent No. 4444450.
Specifically, a step of performing the first hydrolysis reaction of the whey protein raw material with an endoprotease in a decomposition rate range of 20 to 30%, and using a film having a pore size of 1 nm to 5 μm for the obtained protein hydrolyzate. In the step of membrane fractionation, a protease containing an exoprotease in the permeation fraction increases the degradation rate by 2 to 8%, and the final hydrolysis rate is 25 to 35%. It is produced by a method including a step of carrying out a decomposition reaction and a step of filtering the obtained protein hydrolyzate using an ultrafiltration membrane having a fraction molecular weight of 6000 Da or less and recovering the membrane-permeated fraction. be able to.
According to this method, it can be produced so that the final product, whey protein hydrolyzate, does not have residual antigenicity.

Here, the whey protein raw material conforms to the above-mentioned general description.
First, the whey protein raw material is dispersed in water or warm water and dissolved. The concentration of the solution is not particularly limited, but usually the protein concentration may be about 5 to 15%.
Next, the protein solution is sterilized by heating at 65 to 90 ° C. for about 1 to 30 minutes.

Then, the first hydrolysis reaction is carried out.
The endoprotease (endopeptidase) used for the first hydrolysis of the raw material protein of the present invention may be any proteolytic enzyme having no exoprotease activity and only endoprotease activity. Examples of endoproteases are animal-derived (eg, trypsin, chymotrypsin, etc.), plant-derived (eg, papain, etc.), or microbial-derived (eg, lactic acid bacteria, yeast, mold, bacillus, actinomycetes, etc.).

The first hydrolysis reaction is not particularly limited as long as it is a method in which endoprotease is used as a proteolytic enzyme and the protein degradation rate can be adjusted to 20 to 30%, and the proteolytic enzyme method is performed according to a conventional method. Do by. Specifically, the hydrolysis conditions by the proteolytic enzyme method such as the type, amount, temperature, pH, and hydrolysis time of the enzyme capable of adjusting the protein degradation rate to 20 to 30% are set in a preliminary experiment, and then the protein is hydrolyzed. Prepare the degradation product.

The amount of the proteolytic enzyme used with respect to the raw material protein varies depending on the substrate concentration, enzyme titer, reaction temperature and reaction time, but generally, the enzyme is used alone or at a ratio of 50 to 10000 active units per 1 g of the raw material protein. Hydrolysis is performed by adding a plurality of combinations. It should be noted that the addition of the enzyme may be carried out collectively, or may be divided into small amounts or types, and added sequentially.

The pH of the protein hydrolysis reaction is selected from the range of pH 2 to 10 according to the optimum pH of the enzyme used. Specifically, before adding the enzyme to the protein solution, the pH is adjusted to a desired pH by adding an acid or an alkaline agent within the range of pH 2 to 10 depending on the type of enzyme used. In this case, hydrochloric acid, citric acid, phosphoric acid and the like can be exemplified as the acid, and sodium hydroxide, potassium hydroxide, potassium carbonate and the like can be exemplified as the alkaline agent.

The temperature of the protein hydrolysis reaction is not particularly limited and may be selected from a range that can be put into practical use including an optimum temperature range in which the enzymatic action is exhibited, that is, usually in the range of 30 to 70 ° C. It is also possible to prevent spoilage during the protein hydrolysis reaction by keeping the temperature lower or higher than the optimum temperature of the enzyme, for example, in the range of 50 to 60 ° C.

The time of the protein hydrolysis reaction varies depending on the reaction conditions such as the type and combination of enzymes used, the reaction temperature, and the initial pH. Therefore, as described above, the protein decomposition rate set in the preliminary experiment is set to 20 to 30. The reaction duration is determined within the range that can be adjusted to%.

To stop the enzyme reaction, inactivate or remove the enzyme when the degree of hydrolysis is within the range of 20 to 30% of the protein degradation rate based on the hydrolysis conditions set in the preliminary experiment. To be done by. The deactivation operation can be performed by heat treatment (for example, at 85 ° C. for 15 minutes). Further, the removal operation can be carried out by an ultrafiltration membrane or the like.

The obtained solution containing the whey protein hydrolyzate, which is an intermediate product, can be used as it is in the next second hydrolysis reaction step, and if necessary, this solution is used by the reverse osmosis membrane method. It is also possible to concentrate by a known method such as, etc., and use it as a concentrated solution. Further, this concentrated solution is dried by a known method to form a powder, and at a predetermined concentration before the subsequent membrane fractionation. It can also be dissolved in water and used in the next second hydrolysis reaction step.
The membrane used in the membrane fractionation step may be any membrane having a pore diameter of 1 nm to 5 μm, a microfiltration membrane having a pore diameter of 0.5 to 5 μm, and a pore diameter of 1 nm to 5 μm. An example can be exemplified of an extrafiltration membrane having a molecular weight cut off of 10 to 100,000 Da, which corresponds to the above.

In the membrane fractionation step, the protein hydrolyzate, which is an intermediate product in which the decomposition rate of the prepared protein is in the range of 20 to 30%, is adjusted to a solution having a concentration of 5 to 20%, and the pore size is 1 nm to 1 nm. A 5 μm membrane is used to perform a hydrolyzed membrane fractionation treatment (so-called hydrolyzed treatment) to remove an antigenic substance and collect a membrane permeation fraction.

The obtained membrane permeation fraction solution can be used as it is in the next second hydrolysis reaction step, and if necessary, this solution is concentrated by a known method such as a reverse osmosis membrane method. , It is also possible to use it as a concentrated solution, and further, this concentrated solution is dried by a known method to form a powder, and dissolved in water at a predetermined concentration before the next second hydrolysis reaction step. It is also possible to do.

Then, a second hydrolysis reaction is carried out.
The protease containing an exopeptidase (exopeptidase) used in the second hydrolysis reaction may be any proteolytic enzyme having at least exoprotease activity, and is derived from carboxypeptidase, aminopeptidase, or microorganism. Exopeptases (for example, lactic acid bacteria, Aspergillus spp., Resorpus spp.), Pancreatin, pepsin, etc., which also have endoprotease activity, can be exemplified.

The second hydrolysis reaction uses a protease containing an exoprotease as a proteolytic enzyme, increases the rate of protein degradation by 2-8% in addition to the rate of degradation obtained by the first hydrolysis, and The method is not particularly limited as long as the final decomposition rate can be adjusted to 25 to 35%, and the method is carried out by a proteolytic enzyme method according to a conventional method. Specifically, a proteolytic enzyme method such as the type, amount, temperature, pH, and hydrolysis time of an enzyme that can increase the protein degradation rate by 2 to 8% and adjust the final degradation rate to 25 to 35%. Hydrolysis conditions according to the above are set in a preliminary experiment, and then a protein hydrolyzate is prepared.
Each condition of the proteolytic enzyme method in the second hydrolysis reaction is the same as that of the first hydrolysis reaction described above.

Further, the resulting solution containing the whey protein hydrolyzate, which is the final product, is subjected to a treatment for removing a solution having a molecular weight higher than 6000 Da. Specifically, it is preferable to carry out an overtreatment such as extrapolation, and more specifically, an extrapolation membrane having a molecular weight cut off at 6000 Da or less, that is, 1000 to 6000 Da on the market is used. It is carried out by performing a passing treatment, removing a part of the polymer substance that may remain, and recovering the membrane permeation fraction.

The solution containing the protein hydrolyzate, which is the final product obtained, can be used as it is, and if necessary, this solution is used as a concentrated solution concentrated by a known method such as a reverse osmosis membrane method. Further, the concentrated solution can be dried by a known method and used as a powder.

In the present specification, the "PUN unit" of the enzyme is casein [Hammerstein. Merck] is allowed to act on endopeptidase derived from Bacillus saturis, and the enzyme activity showing a color reaction of an allyl amino acid corresponding to 1 μg of tyrosine with a Folin reagent at 30 ° C. for 1 minute is defined as 1 PUN unit.

Further, in the present specification, the "active unit" of an enzyme is defined as being measured by the following method.
The enzyme solution is prepared by dispersing or dissolving the enzyme-containing powder in 0.1 mol of phosphate buffer (pH 7.0) at a ratio of 0.2 g / 100 mL. On the other hand, leucylparanitroanilide (manufactured by Kokusan Kagaku Co., Ltd.) is dissolved in 0.1 mol of phosphate buffer (pH 7.0) to prepare a 2 mM substrate solution. Add 1 mL of the substrate solution to 1 mL of the enzyme solution, react at 37 ° C. for 5 minutes, and then add 2 mL of a 30% acetic acid solution to stop the reaction. The reaction solution is filtered through a membrane filter, and the absorbance of the filtrate is measured at a wavelength of 410 nm. The "active unit" is calculated by the following formula, defining the amount of enzyme required to decompose 1 μmol of leucylparanitroanilide per minute as 1 active unit.
Active unit (per 1 g of powder) = 20 x (A / B)
However, in the above formula, A and B indicate the absorbance of the sample and the absorbance of 0.25 mM para-nitroaniline at a wavelength of 410 nm, respectively.

The composition of the present invention can grow one or more bacteria selected from the genera Blautia and Drea of the family Lachnospiraceae.
As used herein, "proliferation" of a bacterium includes an increase in the abundance of the bacterium in the intestinal flora. That is, it includes increasing the abundance ratio of one or more kinds of bacteria selected from the genus Blautia and the genus Drea of the Lachnospiraceae family present in the digestive tract of animals such as humans ingesting the composition of the present invention. Here, the "presence rate" can also be rephrased as the "occupancy rate" with respect to the entire bacterial group detected in the intestinal flora. "Increased abundance" is defined in the gut flora of other bacteria as long as the abundance of one or more bacteria selected from the genus Blautia and Drea of the Lachnospiraceae family increases. The abundance ratio may increase or decrease at the same time. The composition of the present invention has not been confirmed to have a proliferative effect on Ruminococcaceae bacteria of the order Clostridiales, but the Ruminococcaceae bacteria grow or decrease in association with the effect of the composition of the present invention. That is not hindered.
The degree of increase in the abundance ratio is not particularly limited, but is preferably 2% or more, more preferably 5% or more, still more preferably 9 with respect to the abundance ratio of the bacterium when the composition of the present invention is not applied. It means that it is larger than%.

Also, "proliferation" may include an increase in the absolute number of bacteria. That is, when the composition of the present invention is applied in vivo or in vitro, the number of one or more bacteria selected from the genus Blautia and the genus Drea of the Lachnospiraceae family is increased as compared with the case where the composition is not applied. Say that. The degree of such increase in the number of bacteria is not particularly limited, but is preferably 20% or more, more preferably 50% or more, still more preferably 90% or more, based on the number of bacteria of the bacterium when the composition of the present invention is not applied. It means that it is larger than%.
Such an increase in the number of bacteria is not only a direct measurement of the number of bacteria, but also a short period of turbidity (absorbency) of the digestive tract contents of animals such as humans who ingested a medium or composition in which bacteria are cultured, or short acetic acid. It can be confirmed by measuring the amount of chain fatty acid to increase the value, measuring the pH in the medium and decreasing the value, and the like. In particular, since bacteria of the genus Lachnospiraceae produce short-chain fatty acids (lactic acid, acetic acid, butyric acid, etc.), it is preferable to measure the amount thereof.

The species of Lachnospiraceae that the composition of the present invention can grow is not particularly limited, and examples thereof include bacteria having the OTU sequence of any of SEQ ID NOs: 1 to 8 on the 16S rRNA gene.

In addition to the above-mentioned Lachnospiraceae bacteria, the composition of the present invention can also grow Verrucomicrobia phylum Akkermansia bacterium. Examples of Akkermansia bacterium include Musinifira species, Glycanifira species and the like.
Bacteria of the genus Akkermansia are known to assimilate mucin, which is a polysaccharide, but since a proliferative effect in vivo was confirmed as shown in Examples described later, any of the following mechanisms was confirmed. It is presumed that this is due to. That is, it is presumed that Akkermansia bacterium assimilate and proliferate the peptide remaining digested in the digestive tract of the whey protein hydrolyzate. Alternatively, it is presumed that the residual digested peptide suppresses the growth of any bacterium other than Akkermansia bacterium. Alternatively, it is speculated that whey protein hydrolysates stimulate the secretion of mucin in the gastrointestinal tract, and Akkermansia bacteria assimilate the mucin and proliferate.

The composition of the present invention can be preferably used for the treatment, amelioration or prevention of a disease or condition that correlates with a bacterium belonging to the genus Lachnospiraceae or a bacterium belonging to the genus Drea. The disease or pathological condition preferably corresponds to a disease or pathological condition in which the Lachnospiraceae Blautia bacterium or the Drea genus bacterium decreases in the intestinal flora. In addition, it may be a disease or pathological condition that can be prevented or ameliorated by the growth of Lachnospiraceae Blautia bacteria or Drea bacteria in the intestinal flora, or Lachnospiraceae Blautia bacteria or Drea bacteria are intestinal flora. It may be a disease or pathological condition caused by the reduction in.

Here, "prevention" of a disease or pathological condition includes preventing the occurrence of the disease or pathological condition in the application target, delaying the occurrence of the disease or pathological condition, and reducing the risk of the occurrence of the disease or pathological condition. Further, even when the subject to be applied is already affected (onset), suppressing the further progression of the disease or pathological condition or maintaining the state may be included in "prevention".

Such diseases or conditions include anxiety disorders and depression.
It has been reported that the occupancy rate of Lachnospiraceae bacteria is lower in the intestinal flora of depressed patients than in non-depressed patients (Non-Patent Document 2). Therefore, it is expected that the symptoms of anxiety disorder and depression will be prevented or ameliorated by multiplying Lachnospiraceae bacteria. Therefore, the composition of the present invention can be preferably applied to anxiolytic or antidepressant applications.
Depression and anxiety disorder are similar but different. Depression is a psychiatric disorder that presents with decreased motivation, unreasonable anxiety, malaise, sadness, insomnia, and autonomic symptoms, and anxiety disorder is anxiety. It is a general term for symptoms that are strong and cause behavioral and mental disorders, and refer to panic disorder, compulsive disorder, social anxiety disorder (depression), etc.

In addition, as the disease or pathological condition, colitis disease can also be mentioned. This is because colitis mice administered with Lachnospiraceae bacteria were protected from colitis-related death (Non-Patent Document 1), and it is expected that the growth of Lachnospiraceae bacteria will prevent or improve colitis disease. NS.
In addition, it has been reported that the intestinal flora of patients with non-alcoholic fatty liver, type 2 diabetes, type 1 diabetes, and rheumatoid arthritis has a lower occupancy rate of Lachnospiraceae bacteria than healthy subjects (non-patent). Document 3). In particular, the intestinal flora of children with type 1 diabetes has a reduced number of Lachnospiraceae Blautia bacteria (Murri et al., BMC Medicine, 2013, 11:46). for that reason,
Proliferation of Lachnospiraceae bacteria is expected to prevent or ameliorate these diseases.
Regarding obesity, administration of hydroxypropylmethylcellulose, which is known to improve hyperglycemia and insulin resistance, to high-fat diet mice increases the number of Lachnospiraceae Drea bacteria in the intestinal flora (Patent Document 1). ), Proliferation of Lachnospiraceae Drea bacteria is also expected to have a preventive or ameliorating effect on obesity.
In addition, after allogeneic bone marrow transplantation, patients with a high occupancy rate of the genus Blautia of the family Lacnospirae had a reduced mortality rate due to the onset of acute graft-versus-host disease (Non-Patent Document 7). It is also expected to have a preventive or ameliorating effect on acute graft-versus-host disease.

The compositions of the present invention can also be preferably used for the treatment or amelioration of diseases or conditions that correlate with Akkermansia bacteria. The disease or pathological condition preferably corresponds to a disease or pathological condition in which Akkermansia bacterium is reduced in the intestinal flora. In addition, it may be a disease or pathological condition that can be prevented or ameliorated by the growth of Akkermansia bacterium in the intestinal flora, or a disease or pathological condition caused by the reduction of Akkermansia bacterium in the intestinal flora. May be.

Such diseases or conditions include obesity and diabetes.
It is said that humans with high body weight, obesity index, blood cholesterol level, and fasting blood glucose level have less Akkermansia muciniphila in the intestinal flora than normal humans, and Akkermansia muciniphila in the intestine. It has been reported that predominant people are more likely to obtain the effect of improving insulin resistance by dietary therapy for anti-obesity (https://institute.yakult.co.jp/bacteria/5463/). Therefore, the growth of Akkermansia bacterium is expected to have a preventive or ameliorating effect on obesity and diabetes.

The composition of the present invention has a track record of use as food and is highly safe. Moreover, even if it is continuously ingested (administered) for a long period of time, there is little concern about side effects. Furthermore, it is highly safe when used in combination with other drugs.

The subject to which the composition of the present invention is administered (administrator) and the subject to be ingested (ingestor) are not particularly limited as long as they are animals, but are usually humans. In addition, it may be any of adults, children, infants, newborns (including low-weight babies), etc., but it is usually an adult. The gender is not particularly limited.

Another aspect of the present invention is the use of a whey protein hydrolyzate in the production of a composition for the growth of Lachnospiraceae bacteria, wherein the Lachnospiraceae bacterium is one or more selected from the genus Blautia and the genus Drea. be.
Another aspect of the present invention is the use of a whey protein hydrolyzate in the growth of Lachnospiraceae bacteria, wherein the Lachnospiraceae bacterium is one or more selected from the genus Blautia and the genus Drea.
Another aspect of the present invention is a whey protein hydrolyzate used for the growth of Lachnospiraceae bacteria, wherein the Lachnospiraceae bacterium is one or more selected from the genus Blautia and the genus Drea.
Another aspect of the present invention is a method of growing Lachnospiraceae bacteria, which comprises administering a whey protein hydrolysate to an animal, wherein the Lachnospiraceae bacterium is one or two selected from the genus Blautia and the genus Drea. More than a seed.

Another aspect of the invention is the use of whey protein hydrolysates in the production of anxiolytic or antidepressant compositions.
Another aspect of the invention is the use of whey protein hydrolysates in anxiolytics or antidepressants.
Another aspect of the invention is a whey protein hydrolyzate used for anxiolytics or antidepressants.
Another aspect of the invention is a method of preventing or ameliorating anxiety disorders or depression, which comprises administering a whey protein hydrolyzate to an animal.

In addition, in this specification, "administering a whey protein hydrolyzate to an animal" may be synonymous with "ingesting a whey protein hydrolyzate into an animal". Ingestion is usually voluntary (free ingestion), but may be compulsory (compulsory ingestion). That is, specifically, the administration step may be, for example, a step of blending a whey protein hydrolyzate into food or drink or feed and supplying it to a subject, thereby allowing the subject to freely ingest the whey protein hydrolyzate. good.

The ingestion (administration) timing of the composition of the present invention is not particularly limited, and can be appropriately selected according to the state of the ingestion (administration) target.

The ingestion (administration) amount of the composition of the present invention is appropriately selected depending on the age, sex, condition, other conditions, etc. of the ingestion (administration) subject.
The intake (administration) amount of the composition of the present invention is preferably in the range of 5 g / day to 60 g / day, for example, 5 g / day to 5 g / day as the intake (administration) amount of the whey protein hydrolyzate according to the present invention. The range of 30 g / day is more preferable, and 5 g / day to 10 g / day is even more preferable.
The above-mentioned ingestion (administration) amount may be an amount to be ingested (administered) in addition to the amount of protein to be ingested (administered) in a normal diet or the like.
The composition of the present invention can be ingested (administered) once or in a plurality of times a day regardless of the amount and duration of ingestion (administration).

The ingestion (administration) period of the composition of the present invention is not particularly limited. In addition, there is no particular upper limit on the intake (administration) period, and continuous and long-term intake (administration) is possible.

The route of ingestion (administration) of the composition of the present invention may be oral or parenteral, but oral is preferable. In addition, parenteral ingestion (administration) includes transdermal, intravenous injection, rectal administration, inhalation and the like.

When the composition of the present invention is to be orally ingested (administered), it is preferably in the form of food and drink.

The form and properties of the food and drink are not particularly limited as long as they can be orally ingested (administered) without impairing the effects of the present invention, and are usually used for food and drink except that they contain a whey protein hydrolyzate. It can be produced by a usual method using raw materials.

Foods and drinks may be in the form of liquid, paste, gel solid, powder, etc., for example, nutritional supplements (supplements), tablets; liquid foods (nutrient foods for tube intake); bread, macaroni, spaghetti. , Noodles, cake mix, fried flour, bread flour and other wheat flour products; instant noodles, cup noodles, retort / cooked foods, canned cooking, microwave foods, instant soups / stews, instant miso soups / soups, canned soups, freeze-dried foods , Other instant foods such as instant foods; canned agricultural products, canned fruits, jams and marmalades, pickles, boiled beans, dried agricultural products, processed agricultural products such as cereals (processed grain products); canned marine products, fish hams and sausages , Seafood paste products, fishery delicacies, boiled fish and other marine products; canned livestock and pastes, processed livestock products such as meat ham and sausage; , Prepared milk powder, cream, other dairy products such as dairy products; butter, margarines, vegetable oils and other fats and oils; soy sauce, miso, sauces, tomato processing seasonings, mirins, vinegars, etc. Ingredients: Combined seasonings / foods such as cooking mixes, curry bases, sauces, dressings, noodles, spices, and other complex seasonings; Frozen foods; caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery, jelly, and other confectionery; carbonated beverages, natural fruit juice , Fruit juice drinks, soft drinks with fruit juice, fruit meat drinks, fruit drinks with fruit grains, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powder drinks, concentrated drinks, sports drinks, nutritional drinks, alcoholic drinks, etc. Favorite beverages such as favorite beverages, baby foods, sprinkles, other commercially available foods such as Ochazuke paste; prepared powdered milk for childcare; enteral nutritional foods; functional foods (foods for specified health use, nutritionally functional foods), etc. ..

It can also be used as feed as one aspect of food and drink. Examples of the feed include pet food, livestock feed, fish feed and the like.
The form of the feed is not particularly limited, and in addition to the whey protein hydrolyzate, for example, grains such as corn, wheat, barley, rye, and mylo; vegetable oil cakes such as soybean oil cake, rapeseed oil cake, palm oil cake, and flaxseed oil cake. ; Bran such as bran, wheat bran, rice bran, defatted rice bran; production cake such as corn gluten meal, corn jam meal; animal feed such as fish flour, defatted milk powder, casein, yellow grease, tallow; tolla yeast, beer Yeasts such as yeast; mineral feeds such as tertiary calcium phosphate and calcium carbonate; fats and oils; simple amino acids; sugars and the like may be contained.

When the composition of the present invention is in the form of a food or drink (including feed), it is provided and sold as a food or drink indicating an application related to the growth of a specific bacterium or an application for prevention or improvement of a predetermined disease or pathological condition. It is possible to be done. In addition, the whey protein hydrolyzate according to the present specification can be used for producing these foods and drinks.

Such "display" act includes all acts for informing the consumer of the use, and if it is an expression that can recall or analogize the use, the purpose of the display, the content of the display, etc. Regardless of the object or medium to be displayed, all of them fall under the act of "displaying" in the present invention.
Further, it is preferable that the "display" is performed by an expression that allows the consumer to directly recognize the above-mentioned use. Specifically, the act of transferring a product related to food and drink or the packaging of the product with the above-mentioned use, displaying it for delivery, transfer or delivery, and importing it, advertising on the product, price list or transaction documents Examples include the act of describing the above-mentioned use and displaying or distributing it, or describing the above-mentioned use in the information containing these and providing it by an electromagnetic (Internet, etc.) method.

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

In addition, "labeling" includes health foods, functional foods, enteral nutrition foods, special purpose foods, health functional foods, specified health foods, nutritional functional foods, functionally labeled foods, non-medicinal products, etc. The display is also mentioned. Among these, in particular, labeling approved by the Consumer Affairs Agency, for example, a system related to foods for specified health use, nutritionally functional foods, or functionally labeled foods, or labeling approved by a system similar to these, and the like can be mentioned. Specifically, labeling as a food for specified health use, labeling as a food for specified health use with conditions, labeling to the effect that it affects the structure and function of the body, labeling for reducing the risk of illness, and functionality based on scientific evidence. Indications, etc. can be mentioned, and more specifically, the Cabinet Office Ordinance on Permits for Special Use Labeling, etc. stipulated in the Health Promotion Act (August 31, 2001 Cabinet Office Ordinance No. 57) Labeling as food for specified health use (particularly labeling for health use) and similar labeling specified in the above are typical examples.

The composition of the present invention can also be in the form of a pharmaceutical product.
The route of ingestion (administration) of the drug may be oral or parenteral, but oral is preferable. In addition, parenteral ingestion (administration) includes transdermal, intravenous injection, rectal administration, inhalation and the like.
As the form of the drug, it can be appropriately formulated into a desired dosage form according to the ingestion (administration) method. For example, in the case of oral ingestion (administration), it can be formulated into a solid preparation such as a powder, a granule, a tablet or a capsule; a liquid preparation such as a solution, a syrup, a suspension or an emulsion. In the case of parenteral ingestion (administration), it can be formulated into a suppository, an ointment, an injection, or the like.
In the formulation, in addition to the whey protein hydrolyzate, components such as excipients, pH adjusters, colorants, and flavoring agents that are usually used in the formulation can be used. It is also possible to use other medicines such as other medicinal ingredients and ingredients having a known or future specific bacterial growth effect or a preventive or ameliorating effect on a predetermined disease or pathological condition.
In addition, the formulation can be carried out by a known method as appropriate depending on the dosage form. At the time of formulation, a carrier usually used for formulation may be blended and formulated as appropriate. Examples of such carriers include excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents and the like.

Examples of excipients include sugar derivatives such as lactose, sucrose, glucose, mannit, and sorbit; starch derivatives such as corn starch, horse bell starch, α-starch, dextrin, and carboxymethyl starch; crystalline cellulose, hydroxypropyl cellulose, and the like. Cellulous derivatives such as hydroxypropylmethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium; gum arabic; dextran; purulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, magnesium aluminometasilicate; phosphate derivatives such as calcium phosphate; carbonic acid Carbonate derivatives such as calcium; sulfate derivatives such as calcium sulfate can be mentioned.

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

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

Examples of the lubricant include talc; stearic acid; metal stearate salts such as calcium stearate and magnesium stearate; colloidal silica; waxes such as pea gum and gay wax; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid. Sodium carboxylic acid 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 acid anhydride and silicate hydrate; starch derivatives and the like. 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 acid anhydride; and sorbic acid.

Examples of the flavoring agent include sweeteners, acidulants, and flavors.
Examples of the carrier used in the case of a liquid preparation for oral ingestion (administration) include a solvent such as water.

The timing of ingesting (administering) the drug of the present invention is not particularly limited, for example, before meals, after meals, between meals, and before bedtime.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

(1) Acquisition of whey protein hydrolyzate (1-1) Production of hydrolyzate A 1.5 kg of commercially available milk WPC (75% protein, manufactured by Mirai Co., Ltd.) is dissolved in 8.5 kg of purified water and sodium is used. A type cation exchange resin (Diaion SK-1B, manufactured by Mitsubishi Kasei Co., Ltd.) was passed through 1 L to adjust the calcium content to 50 mg per 100 g of protein. The obtained IE-WPC was adjusted to a concentration of 10%, 1.0% bromelain (manufactured by Amano Pharmaceutical Co., Ltd.) was added per protein, and the mixture was hydrolyzed at 45 ° C. for 10 hours. Since the pH of the decomposition solution was 6.45, sodium hydroxide was added to adjust the pH to 6.7, and the mixture was heated at 80 ° C. for 10 minutes to inactivate the enzyme. Then, it was concentrated by a conventional method and dried to obtain about 1 kg of powdered whey protein hydrolyzate.
The hydrolyzate A is produced by the method disclosed in "Patent No. 3059595".

(1-2) Production of Hydrolyzed Product B 1 kg of commercially available milk WPC (75% protein, manufactured by Mirai Co., Ltd.) was dissolved in 9 kg of deionized water and sterilized by holding at 75 ° C. for 15 seconds to adjust the pH to 9. Adjusted to 0. Here, Protease N Amano (manufactured by Amano Pharmaceutical Co., Ltd.) 1.8 million PUN units (2400 PUN units per 1 g of whey protein), and Lactobacillus herbeticus bacterium prepared by the same method as Reference Example 1 of Japanese Patent No. 29597747. 68,000 active units of crushed body (90 active units per 1 g of whey protein) were added, and the mixture was kept at 50 ° C. and hydrolyzed. The amount of free lysine was measured over time using a biotech analyzer (manufactured by Asahi Kasei Kogyo Co., Ltd.), and when the amount of free lysine reached 14%, it was heated at 80 ° C. for 6 minutes to inactivate the enzyme and cooled. Then, about 10 kg of a solution containing 10% of whey protein hydrolysate B was obtained. Further, it was concentrated by a conventional method and dried to obtain about 1 kg of powdered whey protein hydrolyzate.
The hydrolyzate B is produced by the method disclosed in "Patent No. 29597747".

(1-3) Production of hydrolyzate C 1.3 kg of commercially available milk WPC (75% protein, manufactured by Mirai) is dissolved in 9 kg of deionized water to prepare 10.3 kg of a whey protein aqueous solution having a protein concentration of about 10%. bottom. The whey protein aqueous solution is heat sterilized at 70 ° C. for 1 minute using a plate heat exchanger, the liquid temperature is adjusted to 53 ° C., and the pH is adjusted using a 10% sodium hydroxide aqueous solution and a 20% potassium carbonate aqueous solution. Was adjusted to 9.5. To this, 2.4 L of alcalase (manufactured by Novo Industry Co., Ltd.), which is an endoprotease, was added at a ratio of 1000 active units per 1 g of protein equivalent to initiate the first protein hydrolysis reaction. When 13 hours had passed and the decomposition rate reached 25%, the enzyme was inactivated by heating at 120 ° C. for 15 seconds using a plate heat exchanger, the enzyme reaction was stopped, and the mixture was cooled to 10 ° C. ..
This hydrolyzate is membrane-fractionated by an ultrafiltration membrane (manufactured by Asahi Kasei Kogyo Co., Ltd., molecular weight cut off: 6000) having a pore size of 0.5 μm, the permeated fraction is freeze-dried, and about 1000 g of a powdered protein hydrolyzate (about 1000 g). As a protein equivalent, 770 g) was obtained.
Next, the entire amount of the obtained protein hydrolyzate was dissolved in 9000 g of deionized water to prepare 10 kg of an aqueous protein hydrolyzate solution having a concentration of about 10%. The temperature of the aqueous protein hydrolyzate solution was adjusted to 53 ° C., and the exoprotease Protease A Amano (manufactured by Amano Pharmaceutical Co., Ltd.) was added at a ratio of 500 active units per 1 g of protein equivalent to perform a second protein hydrolysis reaction. Started. After 8 hours, when the decomposition rate increased by 5% and the final decomposition rate reached 30%, the enzyme was inactivated by heating at 120 ° C. for 15 seconds using a plate heat exchanger. , The enzymatic reaction was stopped and cooled to 10 ° C.
This hydrolyzate was freeze-dried to obtain about 940 g (720 g of protein equivalent) of a powdered protein hydrolyzate.
The hydrolyzate C is produced by the method disclosed in "Patent No. 4444450".

The molecular weights and free amino acid amounts of the obtained hydrolysates A, B and C were measured by the above-mentioned methods, and the results were as shown in Table 1.

(2) Administration of whey protein hydrolysate For rats using either whey protein hydrolysates A to C prepared in (1) or commercially available WPC (Milei80 (manufactured by Mirai)) as the only protein source. Feeds (feeds A, B, C, W) were prepared and the following breeding tests were conducted. The protein concentration in the feed was 10% by weight.
Six-week-old male SD rats were acclimated to a normal diet for 12 days and then divided into 4 groups so that the body weight was not biased (n = 5). Then, the feed of each group was switched to any of feeds A, B, C, and W, and the breeding was continued for one week, and the cecal contents of each rat were collected on the final day. Rat feeding and body weight were recorded daily, confirming that there was no difference between the groups (mean intake was 20 g / day).

DNA was extracted from the contents of the cecum by the following method, and intestinal bacterial flora analysis was performed.
DNA was extracted from 20 mg of the collected cecal contents using the bead crushing method. After centrifugation at 20400 xg for 10 minutes, 400 μL of the supernatant was extracted with phenol / chloroform, 250 μL of the supernatant was precipitated with isopropanol, and DNA was recovered.
The V3-V4 region contained in the 16s rRNA sequence of bacterial DNA among the extracted DNA was subjected to TaKaRa Ex Taq HS Kit (manufactured by Takara), Tru357F primer (SEQ ID NO: 9), and Tru806R primer (SEQ ID NO: 10). P
It was amplified by the CR method.
After quality checking of the obtained DNA using QIAxcel system (manufactured by QIAGEN), barcode primers Fwd (SEQ ID NO: 11) and Rev (SEQ ID NO: 12) (N is a different bar for each sample) for measurement with Illumina Miseq. PCR was performed using the coding sequence (A, C, G or T). After quality-checking the PCR product with the QIAxcel system, GeneRead size se
Purified using lection Kit (manufactured by Qiagen), Miseq v3 Rea
Sequence analysis was performed on Miseq using a gent Kit (manufactured by Illumina).

After removing the human genome sequence and the PhixX 174 sequence added to the control from the obtained pair read sequence information, QIME2 (Version 2017.10) and DAD
Quality filtering, sequence merging, and chimera removal were performed using A2 to obtain sequence data. In quality filtering, the forward read was trimmed to a length of 290 bp and the reverse read was trimmed to a length of 230 bp, and sequences having a length shorter than that were excluded.
For the analysis and classification of the genus, a naive Bayes classifier machine-learned based on a database (Greengenes databese 13.8) was used. In addition, one-way ANOVA and Dunnett's test were used for statistical processing.

The results are shown in FIGS. 1 to 4.
In the whey protein hydrolyzate (feed A, B, C) intake group, the intestinal flora occupancy rate of Pharmacutes phylum Clostridiales Lachnospiraceae bacteria tended to be higher than in the whey protein (feed W) intake group. (Fig. 1). Among the Lachnospiraceae, the intestinal flora occupancy of Bactia spp. Was significantly higher in the hydrolyzate-intake group than in the whey protein-intake group (Fig. 2). In addition, the intestinal flora occupancy rate of Lachnospiraceae bacteria of the genus Drea also tended to be higher in the hydrolyzate intake group than in the whey protein intake group (Fig. 3).
Verrucomicrobia phylum Akkermansia muciniphila species bacteria also tended to have a higher intestinal flora occupancy in the hydrolyzate intake group than in the whey protein intake group (Fig. 4).

Claims (6)

A composition for growing Lachnospiraceae bacteria containing a whey protein hydrolyzate, wherein the Lachnospiraceae bacteria are one or more selected from the genus Blautia and the genus Drea. The composition according to claim 1, wherein the whey protein hydrolyzate has the following properties (a) to (c).
(A) The number average molecular weight is 200 Da or more and 600 Da or less. (B) The proportion of peptides having a molecular weight of 1000 Da or less is 40% by mass or more and 95% by mass or less. (C) The amino acid release rate is 30% by mass or less. be The composition according to claim 1 or 2, which is used for growing bacteria of the genus Blautia. The composition according to claim 1 or 2, which is used for growing bacteria of the genus Drea. Anxiolytic or antidepressant compositions containing whey protein hydrolysates. The composition according to claim 5, wherein the whey protein hydrolyzate has the following properties (a) to (c).
(A) The number average molecular weight is 200 Da or more and 600 Da or less. (B) The proportion of peptides having a molecular weight of 1000 Da or less is 40% by mass or more and 95% by mass or less. (C) The amino acid release rate is 30% by mass or less. be

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