JP7724204B2 - Composition for improving the intestinal or oral environment - Google Patents

Description

REFERENCE TO RELATED APPLICATIONS

This application benefits from the priority of an earlier Japanese application, Patent Application No. 2020-67855 (filing date: April 3, 2020), the entire disclosure of which is incorporated herein by reference.

The present invention relates to a composition for improving the intestinal and/or oral environment. The present invention also relates to a composition for improving the intestinal and/or oral flora, a composition for promoting short-chain fatty acid production in the intestines and/or oral cavity, a composition for regulating immunity in the intestinal tract and/or oral cavity, and a composition for promoting improvement of mood states.

An estimated 40 trillion bacteria reside in the intestines, a group known as the intestinal flora. Similar to the intestines, bacteria also reside in the oral cavity, a group known as the oral flora. The types and amounts of bacteria that make up the intestinal or oral flora vary from person to person. These bacteria have diverse functions, and recent research has shown that the state of the intestinal or oral flora is related to many diseases. Furthermore, it has become clear that much of human immune function depends on the state of the intestinal flora, and that human immune function is related to the state of the oral flora. It has also become clear that maintaining a proper balance of the intestinal or oral flora can maintain normal immune function. In other words, improving the intestinal and/or oral environment is expected to maintain health and prevent various diseases, including lifestyle-related diseases.

Against this background, various ingredients and materials have been developed that are effective in improving the intestinal and/or oral environment. For example, Patent Document 1 discloses a composition for improving the intestinal environment that contains milk-derived glycopeptides.

International Publication No. 2019/044960

The present invention aims to provide novel compositions for improving the intestinal and/or oral environment and agents for improving the intestinal and/or oral environment. The present invention also aims to provide novel compositions for improving the intestinal and/or oral flora and agents for improving the intestinal and/or oral flora, novel compositions for promoting short-chain fatty acid production in the intestines and/or oral cavity and agents for promoting short-chain fatty acid production, novel compositions for modulating the immune system in the intestines and/or oral cavity and agents for modulating the immune system in the intestines and/or oral cavity, and compositions for promoting improvement of mood states and agents for promoting improvement of mood states.

In recent animal model and human studies, the inventors have found that when mice or humans ingested peptides having specific sequences or whey protein hydrolysates containing said peptides, they were effective in improving the intestinal and oral flora, as well as the intestinal and oral environment, including promoting the production of short-chain fatty acids. The inventors have also found that peptides having specific sequences and whey protein hydrolysates containing said peptides have an immunomodulatory effect mediated by the immune system in the intestinal tract and oral cavity, and an effect of promoting the improvement of mood. The present invention is based on these findings.

According to the present invention, the following inventions are provided.
[1] A composition for improving the intestinal and/or oral environment, a composition for improving the intestinal and/or oral bacterial flora, or a composition for promoting short-chain fatty acid production in the intestinal and/or oral cavity, comprising as an active ingredient a whey protein enzymatic hydrolysate containing a peptide having the amino acid sequence of GTWY (SEQ ID NO: 1) and/or a peptide having the amino acid sequence of WY, as well as an agent for improving the intestinal and/or oral environment, an agent for improving the intestinal and/or oral bacterial flora, or an agent for promoting short-chain fatty acid production in the intestinal and/or oral cavity.
[2] A composition for regulating intestinal and/or oral immunity, and an intestinal and/or oral immunity regulator, comprising, as an active ingredient, a whey protein enzymatic hydrolysate containing a peptide having the amino acid sequence of GTWY (SEQ ID NO: 1) and/or a peptide having the amino acid sequence of WY.
[3] A composition for promoting improvement of mood state, and an agent for promoting improvement of mood state, comprising as an active ingredient a whey protein enzymatic hydrolysate containing a peptide having the amino acid sequence of GTWY (SEQ ID NO: 1) and/or a peptide having the amino acid sequence of WY.
[4] The composition and agent according to any one of [1] to [3] above, wherein the GTWY content (in terms of solid content) in the whey protein enzymatic hydrolysate is 0.5 to 5 mg/g.
[5] The composition and agent according to any one of [1] to [4] above, wherein the content of whey protein in the enzymatic hydrolysate (converted to solid content) is 0.05 to 2 mg/g.
[6] The composition and agent according to any one of [1] to [5] above, wherein the whey protein enzymatic hydrolysate is ingested by a human at a dose of 1 to 50,000 mg (in terms of solid content) per day.
[7] A composition for improving the intestinal and/or oral environment, a composition for improving the intestinal and/or oral bacterial flora, or a composition for promoting short-chain fatty acid production in the intestinal and/or oral cavity, comprising as an active ingredient a peptide having the amino acid sequence of GTWY (SEQ ID NO: 1) and/or a peptide having the amino acid sequence of WY, as well as an agent for improving the intestinal and/or oral environment, an agent for improving the intestinal and/or oral bacterial flora, or an agent for promoting short-chain fatty acid production in the intestinal and/or oral cavity.
[8] A composition for regulating immunity in the intestinal tract and/or oral cavity, and an immunoregulator for the intestinal tract and/or oral cavity, comprising as an active ingredient a peptide having the amino acid sequence of GTWY (sequence number 1) and/or a peptide having the amino acid sequence of WY.
[9] A composition for promoting improvement of mood state, and an agent for promoting improvement of mood state, comprising as an active ingredient a peptide having the amino acid sequence of GTWY (SEQ ID NO: 1) and/or a peptide having the amino acid sequence of WY.
[10] The composition and agent according to any one of [1] to [9] above, wherein GTWY (SEQ ID NO: 1) is ingested by a human at a dose of 0.001 to 1000 mg (in terms of solid content) per day.
[11] The composition and agent according to any one of [1] to [10] above, wherein WY is ingested by a human at a dose of 0.001 to 500 mg (in terms of solid content) per day.
[12] The composition and agent according to any one of [1] to [11] above, which is a food composition.
[13] The composition and agent according to any one of [1] to [12] above, which is in a unit-packaged form per serving.
[15] The composition and agent according to any one of [1] to [13] above, for administration to a healthy person.
[16] The composition and agent according to any one of [1] to [14] above, for administration to middle-aged and elderly people.

The compositions [1], [2], [3], [7], [8] and [9] above may be referred to herein as "compositions of the present invention," and the pharmaceutical preparations [1], [2], [3], [7], [8] and [9] above may be referred to herein as "pharmaceutical preparations of the present invention."

The whey protein enzymatic hydrolysate, the active ingredient of the present invention, is derived from a food material that has been consumed by humans for many years. Therefore, the compositions and preparations of the present invention are advantageous in that they can be used as functional foods that improve the intestinal and/or oral environment, and are safe for mammals, including humans.

Figure 1 shows the quantitative results (at the end of the intake period) of short-chain fatty acids in 6-month-old (at the start of intake) C57BL/6J male mice using WY peptide (intake period: 1.5 months, non-intake group: n = 7, intake group: n = 7). * indicates P < 0.05%, and error bars indicate standard error. Figure 2 shows the quantitative results (at the end of the intake period) of short-chain fatty acids in 6-month-old (at the start of intake) C57BL/6J male mice using WY peptide (intake period: 3 months, non-intake group: n = 8, intake group: n = 8). * indicates P < 0.05%, and error bars indicate standard error. 3 shows the results of bacterial flora analysis (at the end of the ingestion period) in 6-month-old C57BL/6J male mice treated with WY peptide (ingestion period: 3 months, non-ingestion group: n=8, ingestion group: n=8). * indicates P<0.05%. 4 shows the results of bacterial flora analysis (at the end of the ingestion period) in 6-month-old (at the start of ingestion) C57BL/6J male mice treated with WY peptide, comparing the results of ingestion periods of 1.5 months (1.5M, WY non-ingestion group: n = 7, WY ingestion group: n = 7) and 3 months (3M, WY non-ingestion group: n = 8, WY ingestion group: n = 8). CTL indicates the WY non-ingestion group, WY indicates the WY ingestion group, * indicates P < 0.05%, and error bars indicate standard error. 5 shows the results of bacterial flora analysis (at the end of the ingestion period) in 6-month-old (at the start of ingestion) C57BL/6J male mice treated with WY peptide, comparing the results when the ingestion period was 1.5 months (1.5M, non-ingestion group: n = 7, ingestion group: n = 7) and 3 months (3M, non-ingestion group: n = 8, ingestion group: n = 8). CTL indicates the WY non-ingestion group, WY indicates the WY ingestion group, * indicates P < 0.05%, and error bars indicate standard error. 6 shows the quantitative results (at the end of the ingestion period) of short-chain fatty acids in 66-week-old (at the start of ingestion) C57BL/6J male mice using WY peptide (ingestion period: 4.5 months, non-ingestion group: n=10, ingestion group: n=9). CTL indicates the WY non-ingestion group, WY indicates the WY ingestion group, * indicates P<0.05%, and error bars indicate standard error. 7 shows the results of lymphocyte analysis (at the end of the ingestion period) in 3-month-old (at the start of ingestion) C57BL/6J male mice using WY peptide (ingestion period: 3 months, non-ingestion group: n = 10, ingestion group: n = 10). CTL indicates the WY non-ingestion group, WY indicates the WY ingestion group, * indicates P < 0.05%, and error bars indicate standard error. 8 shows the quantitative results (at the end of the ingestion period) of short-chain fatty acids in 62-week-old (at the start of ingestion) C57BL/6J male mice using GTWY peptide (ingestion period: 8.5 months, non-ingestion group: n=12, ingestion group: n=8). CTL indicates the GTWY non-ingestion group, GTWY indicates the GTWY ingestion group, * indicates P<0.05%, and error bars indicate standard error. 9 shows the quantitative results (at the end of the intake period) of short-chain fatty acids in 6.5-month-old Crl:CD1(ICR) male mice (at the start of intake) using whey hydrolysate (intake period: 3 months, non-intake group: n=10, intake group: n=7). * indicates P<0.05%, and error bars indicate standard error. 10 shows the results of bacterial flora analysis (at the end of the intake period) in 6.5-month-old Crl:CD1 (ICR) male mice (at the start of intake) using whey hydrolysate (intake period: 3 months, non-intake group: n=10, intake group: n=7). * indicates P<0.05%. 11 shows the results of bacterial flora analysis (at the end of the intake period) in 6.5-month-old Crl:CD1 (ICR) male mice (at the start of intake) treated with whey hydrolysate (intake period: 3 months). CTL is from the group that did not take whey hydrolysate (n=10), and HW-3 is from the group that took whey hydrolysate (n=7). * indicates P<0.05%, and error bars indicate standard error. 12 shows the results of bacterial flora analysis (at the end of the intake period) in 6.5-month-old Crl:CD1 (ICR) male mice (at the start of intake) treated with whey hydrolysate (intake period: 3 months). CTL is from the group that did not take whey hydrolysate (n=10), and HW-3 is from the group that took whey hydrolysate (n=7). * indicates P<0.05%, and error bars indicate standard error. 13 shows the quantitative results (at the end of the intake period) of short-chain fatty acids in 62-week-old (at the start of intake) C57BL/6J male mice using whey hydrolysate (intake period: 8.5 months). CTL represents the group that did not take whey hydrolysate (n=12), and HW-3 represents the group that took whey hydrolysate (n=15). * indicates P<0.05%, and error bars indicate standard error.

Specific Description of the Invention

The compositions and preparations of the present invention comprise a whey protein enzymatic hydrolysate as an active ingredient. In the present invention, "whey" is also referred to as whey serum, whey plasma, or whey, and refers to an aqueous solution obtained by removing milk fat, casein, and other components from milk. Whey is composed of proteins such as β-lactoglobulin, α-lactalbumin, serum albumin, and immunoglobulins. The whey used in the present invention can be derived from any animal or plant, but it is preferable to use whey derived from cow's milk. The whey protein enzymatic hydrolysate (sometimes simply referred to as "whey hydrolysate" in this specification), which is the active ingredient in the compositions and preparations of the present invention, is not limited as long as it is an enzymatic hydrolysate of whey.

The content (in terms of solids) of whey hydrolysate in the compositions and preparations of the present invention can be determined arbitrarily depending on the purpose, use, form or dosage form, etc., and the present invention is not limited thereto. For example, in the case of liquid compositions and liquid preparations, it is 0.001 to 100 mg/100 mL (preferably 0.005 to 50 mg/100 mL, more preferably 0.01 to 10 mg/100 mL) of the total amount, and in the case of solid compositions and solid preparations, it is 0.1 to 90% by mass (preferably 0.5 to 80% by mass, more preferably 1 to 70% by mass) of the total amount.

The compositions and agents of the present invention can be specified as comprising, as active ingredients, at least one or both of a peptide having the amino acid sequence of GTWY (SEQ ID NO: 1) and a peptide having the amino acid sequence of WY (hereinafter, these may be referred to as "peptides of the present invention"). Therefore, the whey hydrolysate of the present invention can contain one or both of a peptide having the amino acid sequence of GTWY and a peptide having the amino acid sequence of WY. Here, "peptide having an amino acid sequence" refers to a peptide whose sequence is specified by the amino acid sequence. Furthermore, GTWY refers to a tetrapeptide consisting of four amino acids, and WY refers to a dipeptide consisting of two amino acids.

The GTWY content (in terms of solids) in the whey hydrolysate of the present invention is, for example, 0.01 to 1% by mass (preferably 0.05 to 0.5% by mass, more preferably 0.1 to 0.2% by mass) relative to the total amount of whey hydrolysate, and the WY content (in terms of solids) is, for example, 0.005 to 0.5% by mass (preferably 0.01 to 0.1% by mass, more preferably 0.03 to 0.09% by mass) relative to the total amount of whey hydrolysate.

The content of the peptide of the present invention can be measured using known peptide content analysis methods such as liquid chromatography tandem mass spectrometry (LC-MS/MS), fluorescence methods, or colorimetric methods. For example, LC-MS/MS can be used according to the method described in Example 2 below. When using LC-MS/MS, a peptide with the purity suitable for LC-MS/MS measurement can be used as the standard peptide during measurement; for example, AQUA Peptide (SIGMA ALDRICH) can be used.

Methods for producing the whey hydrolysate of the present invention (particularly whey hydrolysate containing the peptide of the present invention) are known and can be produced, for example, according to the description in International Publication No. 2017/086303. Alternatively, commercially available whey hydrolysates (e.g., HW-3 (Megmilk Snow Brand), HWP-205 (Tatua), Thermax 690 (Glanbia), or Protherma (Glanbia)) may also be used as the whey hydrolysate.

Whey hydrolysates containing the peptides of the present invention can be produced, for example, by treating whey protein with an enzyme preparation containing whey-degrading enzymes.

The concentration of whey protein used in the enzyme reaction is not limited as long as the protein is soluble, but from the perspective of suppressing gelation and aggregation and eliminating the need for concentration, it is preferably 1 to 30 w/v%, more preferably 1 to 20 w/v%, and even more preferably 5 to 15 w/v%.

Whey can be subjected to the enzymatic reaction either directly or after concentration or dilution, and the pH can be adjusted as necessary. Furthermore, when the whey protein raw material is a solid, such as a powder, it can be dissolved in any aqueous solvent as long as the enzymatic reaction proceeds. However, considering its use as a food product, it is preferable to dissolve it in water or a food additive-grade buffer solution. A buffer solution is preferably used to prevent the pH of the reaction solution from changing due to the amino acids produced in the enzymatic reaction. The type of buffer solution is optional and can be determined based on the subsequent use, flavor, taste, mineral content, etc.; however, a composition that maintains the pH of the reaction solution between 4 and 9, preferably between 5 and 8, and more preferably between 7 and 8, is preferred. Examples of buffer solutions include sodium citrate buffer, sodium carbonate buffer, sodium acetate buffer, sodium phosphate buffer, and potassium phosphate buffer, with potassium phosphate buffer being preferred. The buffer concentration can be any range that provides a buffering effect. However, considering flavor, taste, and mineral content, it is typically between 0.01 and 0.5 M, preferably between 0.05 and 0.2 M, and more preferably approximately 0.1 M.

The enzyme used in the enzymatic reaction can be any protease or a composition, enzyme preparation, or enzyme preparation containing a protease, but an enzyme preparation containing a neutral protease is preferred, and one or more types can be used in combination. Enzyme preparations can be derived from microorganisms such as Bacillus subtilis, Aspergillus oryzae, or Aspergillus melleus, among others. Of these, enzyme preparations derived from Aspergillus oryzae or Aspergillus melleus are preferred, and enzyme preparations derived from Aspergillus melleus are even more preferred.

Commercially available enzyme preparations can be used as the enzyme for the enzymatic reaction. For example, enzyme preparations are available from Amano Enzyme, Shin-Nihon Chemical Industry, DSM, Danisco, Novozymes, and HBI. The amount of enzyme preparation added is optional, but considering factors such as an appropriate hydrolysis reaction rate and cost, examples include 0.01 to 5 w/v%, preferably 0.05 to 4 w/v%, and more preferably 0.1 to 0.5 w/v%.

The enzyme reaction temperature and time can be set so that the raw material protein is sufficiently hydrolyzed and the quality of the enzymatic hydrolysate is maintained. The enzyme reaction temperature is, for example, 30 to 70°C, preferably 40 to 70°C, and more preferably 45 to 65°C. The enzyme reaction time is, for example, 1 to 12 hours, preferably 2 to 10 hours, and more preferably 4 to 5 hours. The enzyme reaction temperature and time can be adjusted as appropriate while checking the amount of the peptide of the present invention produced.

The enzyme reaction can also be carried out while increasing the temperature. For example, a method in which the reaction is carried out while increasing the temperature from 30°C to 75°C over 4 to 10 hours is exemplified. A method in which the reaction is carried out while increasing the temperature from 35°C to 75°C over 5 to 8 hours is preferred, and a method in which the reaction is carried out while increasing the temperature from 35°C to 75°C over 6 to 8 hours is more preferred. The temperature increase speed and program are optional, but a preferred method is to add the enzyme, hold the temperature between 45°C and 55°C for a longer period (e.g., 5 to 7 hours), then quickly raise the temperature to 60°C, and hold the temperature between 60°C and 75°C for a longer period (e.g., 1 to 3 hours). The most preferred method is to add the enzyme at 50°C, hold the temperature for 5 to 7 hours, then raise the temperature at a desired rate, and hold the temperature at the target temperature of 60 to 65°C or 65 to 75°C for 1 to 3 hours.

From the perspective of reaction efficiency, it is preferable to stir the reaction solution during the enzymatic reaction. A fast stirring speed is preferable to ensure good contact between the substrate and the enzyme, but stirring too fast can cause the reaction solution to splash. Therefore, a stirring speed of 100 to 500 rpm is recommended, preferably 200 to 400 rpm, and more preferably approximately 250 rpm.

After the desired peptide is produced as a result of the enzymatic reaction, the reaction solution containing the desired peptide is preferably subjected to a step of terminating the enzymatic reaction. The enzymatic reaction terminating step can be performed by inactivating the enzyme by treating the reaction solution at high temperature or by adding a chelating agent to change the chemical structure of the enzyme, or by removing the enzyme through membrane treatment. A preferred method is a deactivation treatment in which the reaction solution is subjected to high temperature. Such a high-temperature treatment method involves, for example, maintaining the reaction solution at 80-90°C for 5-30 minutes, preferably at 80-90°C for 20-30 minutes. Furthermore, if the temperature is high during the concentration step described below, this can also be performed as a concentration step.

The reaction liquid (whey hydrolysate) that has undergone the above-mentioned enzyme reaction and enzyme reaction termination steps may further be subjected to a sterilization step. Examples of sterilization steps include the membrane treatment step or heat sterilization step described below. The heat sterilization step can also serve as the above-mentioned enzyme reaction termination step, which is advantageous in terms of simplifying the manufacturing process.

The reaction solution (whey hydrolysate) that has undergone the enzyme reaction and enzyme reaction termination steps may be further subjected to a purification step. Examples of purification steps include membrane treatment steps using membrane filtration methods such as coarse filtration, microfiltration, ultrafiltration, or reverse osmosis, with ultrafiltration being preferred. The molecular weight cutoff in ultrafiltration can be selected as desired depending on the desired peptide and the enzyme used, but is preferably 3 to 100 kDa, and more preferably 5 to 50 kDa. The purification step is advantageous in that it can improve the flavor of the peptide composition compared to when the purification step is not performed. Furthermore, the purification step can also serve as the enzyme reaction termination step and sterilization step, which is advantageous in terms of simplifying the production process.

The reaction liquid (whey hydrolysate) that has undergone the enzyme reaction and enzyme reaction termination steps may be further subjected to a concentration step for storage and transportation reasons. Any method can be selected for the concentration step, but vacuum concentration, freeze-drying, spray-drying, or membrane concentration (e.g., using a reverse osmosis membrane) is preferred, with freeze-drying or spray-drying being more preferred. Spray-drying is particularly preferred for efficient mass concentration.

When whey hydrolysates are included in the compositions and preparations of the present invention, they can be included using the peptides of the present invention as an indicator, and for example, 0.001 to 1000 mg (preferably 0.01 to 500 mg, more preferably 0.05 to 5 mg) of GTWY and 0.001 to 500 mg (preferably 0.01 to 100 mg, more preferably 0.05 to 3 mg) of WY can be included per serving.

When the compositions and preparations of the present invention contain the peptides of the present invention, they can contain 0.001 to 1000 mg (preferably 0.01 to 500 mg, more preferably 0.05 to 5 mg) of GTWY and 0.001 to 500 mg (preferably 0.01 to 100 mg, more preferably 0.05 to 3 mg) of WY per serving.

The peptides of the present invention can be derived from food materials such as enzymatic hydrolysates of whey protein. Furthermore, because the peptide chains are short and easy to chemically synthesize, synthetic products, their salts, or solvates can also be used. In other words, the peptides of the present invention have the advantage of being easily available.

According to a first aspect of the present invention, the compositions and preparations of the present invention can be used for the purpose of improving the intestinal and/or oral environment. Here, "improving the intestinal and/or oral environment" refers to a relative increase in beneficial bacteria (e.g., short-chain fatty acid-producing bacteria such as lactic acid bacteria, acetic acid bacteria, propionic acid bacteria, and butyric acid bacteria, and bifidobacteria; particularly bifidobacteria) present in the lower gastrointestinal tract and/or oral cavity, or a relative decrease in harmful bacteria (e.g., Clostridium perfringens, Staphylococcus aureus, Escherichia coli; particularly Clostridium bacteria). "Relative increase" refers to an increase in the proportion of beneficial bacteria in the intestinal and/or oral flora, and "relative decrease" refers to a decrease in the proportion of harmful bacteria in the intestinal and/or oral flora. In other words, "improving the intestinal and/or oral environment" is used to mean improving the intestinal and/or oral flora.

Therefore, according to a second aspect of the present invention, the compositions and preparations of the present invention can be used for the purpose of improving the intestinal and/or oral flora. Here, "improving the intestinal and/or oral flora" refers to increasing the proportion of beneficial bacteria in the intestinal and/or oral flora, or decreasing the proportion of harmful bacteria in the intestinal and/or oral flora. The proportion of various bacteria in the intestinal and/or oral flora can be assessed, for example, by collecting a sample such as feces from a subject in the case of the intestine, or a sample such as saliva from a subject in the case of the oral flora, and analyzing the various bacteria in the sample using a method combining next-generation sequencing/amplicon analysis of 16S rDNA or genome and database search, or FISH-flow cytometry. The lower gastrointestinal tract includes the ileum, cecum, colon, and rectum. The improvement of the intestinal and/or oral environment or the improvement of the intestinal and/or oral flora according to the present invention increases the production of short-chain fatty acids in the intestine and/or oral cavity.

According to a third aspect of the present invention, the compositions and preparations of the present invention can be used for the purpose of promoting the production of short-chain fatty acids in the intestine and/or oral cavity. Short-chain fatty acids are typical metabolites derived from intestinal and/or oral bacteria, which are produced by fermentation of intestinal and/or oral bacteria using dietary fiber as a substrate. Short-chain fatty acids are known not only to serve as a simple energy source for the body but also to play an important role in regulating the body's energy metabolism. In recent years, it has been elucidated that promoting the production of short-chain fatty acids in the intestine is effective in preventing and ameliorating metabolic diseases such as diabetes and obesity (Nihon Nai Kai Zasshi 2015, 104, 57-65; Jpn. J. Clin. Immunol., 2017, 40(6), 408-415). Furthermore, promoting the production of short-chain fatty acids in the intestine has been reported to be effective in preventing and improving conditions such as hypertension or chronic kidney disease (Jpn. J. Clin. Immunol., 2017, 59(4), 562-567), inflammatory bowel disease, atopic dermatitis, or autoimmune diseases (Jpn. J. Clin. Immunol., 2017, 40(6), 408-415), mood disorders such as depressive disorder or bipolar disorder, or stress-induced behavioral abnormalities or brain changes (Clinical Psychopharmacology, 2019, 22, 1045-1052). Furthermore, promoting the production of short-chain fatty acids in the oral cavity is known to be involved in maintaining periodontal tissue homeostasis or immune responses, and is effective in preventing and improving dental caries, periodontal disease, and infectious diseases (Journal of Intestinal Microbiology, 2014, 28, 111-120). Therefore, the compositions and preparations of the present invention are advantageous in that they can be expected to prevent and improve the aforementioned diseases.

In the present invention, short-chain fatty acids are fatty acids having 6 or less carbon atoms, preferably 2 to 6 carbon atoms, such as acetic acid, propionic acid, n-butyric acid, iso-butyric acid, n-valeric acid, iso-valeric acid, and n-caproic acid.

The amount of short-chain fatty acid production in the intestine and/or oral cavity can be assessed, for example, by collecting a sample such as feces from the subject in the intestine or saliva from the subject in the oral cavity and analyzing the sample using an analytical device such as gas chromatography to determine the amount of each short-chain fatty acid produced in the sample, or the total amount. Furthermore, the improvement of the intestinal and/or oral environment, the improvement of the intestinal and/or oral bacterial flora, or the promotion of short-chain fatty acid production in the intestine and/or oral cavity according to the present invention is expected to result in immune regulation via intestinal immunity and/or oral immunity.

According to a fourth aspect of the present invention, the compositions and preparations of the present invention can be used for the purpose of regulating intestinal and/or oral immunity. It is known that short-chain fatty acids, which are metabolic products of intestinal and/or oral bacteria, regulate the immune status of the intestinal and/or oral cavity by promoting the differentiation and induction of immune cells such as dendritic cells, B cells, T cells, or regulatory T cells and/or suppressing cell death of immune cells, and that these immune cells are present in intestinal Peyer's patches, mesenteric lymph nodes, periodontal tissue, etc. (Jpn. J. Clin. Immunol., 2017, 40(6), 408-415; Journal of Intestinal Microbiology, 2014, 28, 111-120). That is, in the present invention, "intestinal and/or oral immunomodulation" refers to immunomodulation mediated by intestinal and/or oral immunity, and refers to modulation of the immune system mediated by immune cells (e.g., T cells or regulatory T cells) present in the intestinal tract (particularly intestinal Peyer's patches and/or mesenteric lymph nodes) and/or the oral cavity (particularly periodontal tissues). Modulation of the immune system mediated by immune cells is achieved, for example, by promoting the differentiation and induction of T cells or regulatory T cells and/or by suppressing cell death of immune cells.

The degree of intestinal and/or oral immunomodulation can be assessed, for example, by collecting peripheral blood or saliva from a subject and measuring the concentrations of factors involved in immunomodulation, such as inflammatory cytokines, anti-inflammatory cytokines, or immunoglobulins. For example, if the concentration of inflammatory cytokines is high and the concentration of anti-inflammatory cytokines (anti-inflammatory cytokines) is low, the immune state can be evaluated as activated, and vice versa, the immune state can be evaluated as controlled or suppressed. Furthermore, if the concentration of immunoglobulin (Ig) A is high, the immune state can be evaluated as activated. The degree of intestinal immunomodulation can also be assessed by analyzing lymphocytes collected from the subject. For example, an increase in the number of CD86-positive cells can be evaluated as sufficient and appropriate activation of the immune system, and an increase in the number of T cells or regulatory T cells can be evaluated as suppression of an excessive immune system response.

According to a fifth aspect of the present invention, the composition and agent of the present invention can be used for the purpose of improving and promoting mood state. In the present invention, the mood state includes, for example, the following seven factors comprised in the Profile of Mood States 2nd Edition (POMS2), which is a known method for measuring or evaluating mood state.
(1) Anger-Hostility: A state of anger or antipathy toward others; (2) Confusion-Embarrassment: Bewilderment or poor cognitive performance; (3) Depression-Depressed: A depressed mood accompanied by a lack of confidence; (4) Fatigue-Apathy: Fatigue, lethargy, and low energy; (5) Tension-Anxiety: Increased tension in the musculoskeletal system; (6) Vitality-Energy: High energy, vitality, and energy; and (7) Friendliness: Feeling positive emotions toward others.

In addition, in the present invention, "promoting improvement" includes both phenomena, such as improving from a negative state to a standard or normal state compared to a standard or normal state, and promoting from a standard or normal state to a positive state. That is, examples of promoting improvement of mood states in the present invention include improvement of lethargy, lethargy, depression, depressed feelings, decreased energy, decreased motivation, decreased motivation, anxious mood, anxiety sensitivity, long-term anxiety, subjective stress, stress perception, perceived stress, daily stress, or bodily pain in subjects who often experience irritability or anxiety in their daily lives, as well as enhancement of energy, vitality, motivation, anxiety tolerance, or stress tolerance in mentally healthy subjects. The promotion of improvement of a subject's mood state by the compositions and preparations of the present invention can be measured or evaluated by comparing the state before and after intake or administration of the compositions and preparations of the present invention to the subject using known methods for measuring or evaluating mood states. Examples of known methods for measuring or assessing mood states include, for example, POMS2, Beck Depression Inventory 2nd Edition (BDI-II), State-Trait Anxiety Inventory (STAI), Perceived Stress Scale (PSS), Motivation Score, Sukemune-Hiew (S-H) Resilience Test, Five Facet Mindfulness Questionnaire (FFMQ), or health-related quality of life (HQOL), or a combination of these scales. Also, known comprehensive methods for measuring or assessing mood states, such as Total Mood Disturbance (TMD) in POMS2 or MOS 36-Item Short-Form Health Survey (SF-36 (registered trademark)), can be used.

As mentioned above, the compositions and agents of the present invention can be used for the purposes of improving the intestinal and/or oral environment, improving the intestinal and/or oral flora, promoting intestinal and/or oral short-chain fatty acid production, regulating the intestinal and/or oral immune system, or promoting improvement of mood. Therefore, subjects to whom the compositions and agents of the present invention are ingested or administered include both subjects with the diseases described above and healthy subjects, although healthy subjects (healthy individuals when the subject is human) are preferred. Hereinafter, in this specification, the terms "administer" and "ingest" may be used interchangeably.

In general, the intestinal and/or oral environment tends to change with age (particularly senescence) (Odamaki et al. BMC Microbiology (2016) 16:90). Therefore, while there are no particular limitations on the age of the subject to whom the compositions and preparations of the present invention are to be administered, the compositions and preparations of the present invention are preferably intended for middle-aged or elderly subjects (e.g., humans aged 50 or older), and particularly preferably for elderly subjects (e.g., humans aged 65 or older). In other words, the compositions and preparations of the present invention are preferably intended for middle-aged or elderly subjects. The compositions and preparations of the present invention can also be administered to subjects whose intestinal environment has changed due to reasons other than aging, such as diet, antibiotics, or stress.

The compositions and agents of the present invention can be provided in the form of pharmaceuticals (e.g., pharmaceutical compositions), quasi-drugs, foods (e.g., food compositions), feed (including pet food), etc., and can be implemented as described below.

The active ingredients of the present invention can be administered to humans and non-human animals, preferably orally. Typical dosage forms are pharmaceuticals or quasi-drugs. Formulations containing the active ingredients of the present invention are not particularly limited as long as they are suitable for oral administration, and examples include granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, emulsions, and suspensions. These formulations can be formulated using pharmaceutically acceptable carriers using techniques commonly practiced in the art. Examples of pharmaceutically acceptable carriers include excipients, binders, diluents, additives, flavorings, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, and preservatives. Such formulations typically contain an effective amount of the active ingredient of the present invention.

The active ingredients of the present invention can be administered to humans and non-human animals, preferably orally. A typical oral intake form is food. When providing the active ingredients of the present invention as food, they can be incorporated into food. Foods provided in this manner contain an effective amount of the active ingredients of the present invention. As used herein, "containing an effective amount" of an active ingredient in a food product of the present invention refers to a content such that the active ingredients of the present invention are ingested in a range described below when the individual food product is ingested in a normally consumed amount. Furthermore, the term "food" encompasses not only foods consumed on a daily basis, but also health foods, functional foods, nutritional supplements, health-promoting foods (e.g., foods for specified health uses, foods with nutrient functions, foods with functional claims, etc.), foods for special dietary uses (e.g., foods for infants, foods for pregnant women, foods for the sick, etc.), and supplements. When providing the active ingredients of the present invention to non-human animals, the foods referred to in the present invention are, of course, used as feed.

Because the active ingredients of the present invention have the effects of improving the intestinal and/or oral environment as described above, they can be incorporated into foods consumed daily or provided as supplements. That is, the compositions and agents of the present invention can be provided in the form of food. In this case, the compositions and agents of the present invention can be provided in unit packaging in which the amount to be taken per meal is predetermined. Examples of unit packaging per meal include packs, wrappings, cans, bottles, etc., which specify a fixed amount. To better utilize the various effects of the compositions and agents of the present invention, the intake amount per meal can be determined according to the daily intake amount of the active ingredients of the present invention, as described below. The foods of the present invention may be provided with instructions regarding the intake amount displayed on the packaging or accompanied by a document or other document containing such instructions.

The predetermined intake amount per serving in the unit package form may be the effective daily intake amount, or the effective daily intake amount divided into two or more (preferably two or three) doses. Therefore, the unit package form of the composition and preparation of the present invention may contain the active ingredient of the present invention at the daily human intake amount described below, or may contain the active ingredient of the present invention in an amount that is one-half to one-twentieth of the daily human intake amount described below. For convenience of ingestion, the composition and preparation of the present invention are preferably provided in a "unit package form per serving," in which the intake amount per serving is the effective daily intake amount.

The form of the "food" is not particularly limited, and may be, for example, a beverage, a semi-liquid or gel form, or a solid or powder form. Furthermore, "supplements" include tablets produced by kneading the active ingredients of the present invention with excipients, binders, etc., and then compressing them into tablets, as well as capsules encapsulated in capsules. When provided as a supplement, it is suitable to provide it in the unit packaging form per meal as described above, or in unit packaging forms per day, per week, or per month.

The foods provided by the present invention are not particularly limited as long as they contain the active ingredient of the present invention, but examples thereof include soft drinks, carbonated drinks, fruit juice drinks, vegetable juice drinks, fruit and vegetable juice drinks, livestock milk such as cow's milk, soy milk, dairy drinks, drink-type yogurt, drink-type or stick-type jellies, coffee, cocoa, tea drinks, nutritional drinks, energy drinks, sports drinks, mineral water, near-water or non-alcoholic beer-flavored drinks and other non-alcoholic beverages; carbohydrate-containing foods and beverages such as rice, noodles, bread or pasta; cheese-containing foods such as natural cheeses such as Camembert cheese or processed cheese; hard or soft yogurt, fresh cream made from livestock milk or other fat and oil ingredients; Examples of suitable confectioneries include dairy products such as ice cream or cream; Western confectioneries such as cookies, cakes, or chocolates; Japanese confectioneries such as manju or yokan; tablet confectioneries (refreshing confectioneries) such as ramune (ramune), candies, chewing gum, frozen desserts such as jellies or puddings, frozen desserts, rice crackers, or snacks; alcoholic beverages such as whiskey, bourbon, spirits, liqueurs, wine, fruit wine, sake, Chinese alcohol, shochu, beer, non-alcoholic beer with an alcohol content of 1% or less, happoshu (low-malt beer), other miscellaneous alcoholic beverages, or chuhai; processed foods such as egg products, processed seafood or meat products (including liver and other offal) (including delicacies), and soups such as miso soup; condiments such as miso, soy sauce, furikake (a type of seasoning), or other seasonings; and liquid foods such as concentrated liquid foods. Mineral water includes both sparkling and non-sparkling mineral water. The foods provided by the present invention also include both food manufacturing ingredients and food additives.

Tea beverages include all fermented, semi-fermented, and unfermented teas, such as black tea, green tea, barley tea, brown rice tea, sencha, gyokuro tea, roasted green tea, oolong tea, turmeric tea, pu-erh tea, rooibos tea, rose tea, chrysanthemum tea, ginkgo leaf tea, and herbal tea (specifically, mint tea or jasmine tea, etc.).

Fruits used in fruit juice beverages or beverages containing fruit and vegetable juices include, for example, apples, mandarin oranges, grapes, bananas, pears, peaches, mangoes, acai, blueberries, and plums. Vegetables used in vegetable juice beverages or beverages containing fruit and vegetable juices include, for example, tomatoes, carrots, celery, pumpkins, cucumbers, and watermelons.

The intake (administration) amount of the active ingredient of the present invention can be determined depending on the recipient's gender, age, and weight, symptoms, intake (administration) timing, intake (administration) time, dosage form, intake (administration route), and other drugs to be combined. The daily intake (in terms of solid content) of whey hydrolysates for an adult human being, intended for improving the intestinal and/or oral environment, improving the intestinal and/or oral bacterial flora, promoting production of short-chain fatty acids in the intestinal and/or oral cavity, regulating the intestinal and/or oral cavity immune system, or promoting improvement of mood, is, for example, 1 to 50,000 mg (preferably 10 to 10,000 mg, more preferably 100 to 5,000 mg), the daily intake (in terms of solid content) of GTWY for an adult human being is, for example, 0.001 to 1,000 mg (preferably 0.01 to 500 mg, more preferably 0.05 to 5 mg), and the daily intake (in terms of solid content) of WY for an adult human being is, for example, 0.001 to 500 mg (preferably 0.01 to 100 mg, more preferably 0.05 to 3 mg). The above-mentioned intake amount of the active ingredient of the present invention and the following intake timing and intake period are applicable to both non-therapeutic and therapeutic uses of the active ingredient of the present invention, and in the case of therapeutic uses, "intake" can be read as "administration." The active ingredient of the present invention can also be administered to mammals other than humans (e.g., mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, dolphins, sea lions, etc.), and the intake amount, intake timing, and intake period can be determined with reference to the above-mentioned description for humans.

It is preferable to continue taking the active ingredient of the present invention for the period during which the effects of improving the intestinal and/or oral environment, improving the intestinal and/or oral flora, promoting short-chain fatty acid production in the intestinal and/or oral cavity, regulating the immune system in the intestinal tract and/or oral cavity, or promoting improvement of mood state are expected. The intake period of the active ingredient of the present invention is, for example, at least 7 days (preferably at least 14 days, more preferably at least 42 days) at the above-mentioned daily dose. Furthermore, the intake interval of the active ingredient of the present invention can be, for example, once every 3 days, once every 2 days, or once a day, and the daily dose can also be divided into two or more doses per day (preferably 2 or 3 times a day).

The compositions, agents, and foods of the present invention may be labeled to indicate that they have the effect of improving the intestinal and/or oral environment, improving the intestinal and/or oral bacterial flora, promoting the production of short-chain fatty acids in the intestinal and/or oral cavity, or modulating the immune system in the intestinal tract and/or oral cavity. In this case, some or all of the following labeling may be attached to the compositions, agents, and foods of the present invention to make the labeling easy for consumers to understand. It goes without saying that in the present invention, "the effect of improving the intestinal and/or oral environment, improving the intestinal and/or oral bacterial flora, promoting the production of short-chain fatty acids in the intestinal and/or oral cavity, or modulating the immune system in the intestinal tract and/or oral cavity" is used in a sense that includes the following labeling:
・For those who want to improve their intestinal flora ・For those who want to regulate their stomach ・For those who want to improve bowel movements ・For those who want to improve their oral flora ・For those who want to prevent and improve bad breath ・For those who want to improve stickiness in the mouth

Furthermore, the compositions, preparations, and foods of the present invention may be labeled to indicate that they have the effect of promoting improvement of mood states. In this case, to make the labeling easier for consumers to understand, the compositions, preparations, and foods of the present invention may be labeled with some or all of the following labels. It goes without saying that in the present invention, the term "effect of promoting improvement of mood states" is used in a sense that includes the following labels.
・For those who are worried about various things ・For those who easily lose motivation ・For those who are concerned about a decline in enthusiasm, motivation or energy ・For those who easily get depressed ・For those who want to stay positive ・For those who easily feel depressed ・For those who easily feel stress ・For those who easily feel anxious ・For those who are often irritated

According to another aspect of the present invention, there is provided a method for improving the intestinal and/or oral environment, a method for improving the intestinal and/or oral microflora, a method for promoting short-chain fatty acid production in the intestinal and/or oral cavity, a method for regulating the intestinal and/or oral cavity immune system, or a method for promoting improvement of a mood state, comprising ingesting or administering to a subject in need thereof a peptide having the amino acid sequence of GTWY (SEQ ID NO: 1) and/or a peptide having the amino acid sequence of WY. According to another aspect of the present invention, there is provided a method for improving the intestinal and/or oral environment, a method for improving the intestinal and/or oral microflora, a method for promoting short-chain fatty acid production in the intestinal and/or oral cavity, a method for regulating the intestinal and/or oral cavity immune system, or a method for promoting improvement of a mood state, comprising ingesting or administering to a subject in need thereof a peptide having the amino acid sequence of GTWY (SEQ ID NO: 1) and/or a peptide having the amino acid sequence of WY. The methods of the present invention can be carried out according to the description of the compositions and agents of the present invention.

According to yet another aspect of the present invention, there is provided use of a whey protein enzymatic hydrolysate containing a peptide having the amino acid sequence of GTWY (SEQ ID NO: 1) and/or a peptide having the amino acid sequence of WY as an intestinal and/or oral environment improver, an intestinal and/or oral flora improver, an intestinal and/or oral short-chain fatty acid production promoter, an intestinal and/or oral immunomodulator, or a mood state improver for the manufacture of an intestinal and/or oral environment improver, an intestinal and/or oral flora improver, an intestinal and/or oral short-chain fatty acid production promoter, an intestinal and/or oral immunomodulator, or a mood state improver, or in a method for improving the intestinal and/or oral environment, a method for improving the intestinal and/or oral flora, a method for promoting short-chain fatty acid production in the intestinal and/or oral cavity, a method for promoting immunity in the intestinal and/or oral cavity, or a mood state improver of the present invention. Another aspect of the present invention provides use of a peptide having the amino acid sequence of GTWY (SEQ ID NO: 1) and/or a peptide having the amino acid sequence of WY as an agent for improving the intestinal and/or oral environment, an agent for improving the intestinal and/or oral flora, an agent for promoting short-chain fatty acid production in the intestinal and/or oral cavity, an agent for modulating the intestinal and/or oral cavity immune system, or an agent for promoting improvement of mood states for the manufacture of an agent for improving the intestinal and/or oral environment, an agent for improving the intestinal and/or oral flora, an agent for promoting short-chain fatty acid production in the intestinal and/or oral cavity, an agent for modulating the intestinal and/or oral cavity immune system, or an agent for promoting improvement of mood states, or in a method for improving the intestinal and/or oral environment, a method for improving the intestinal and/or oral flora, a method for promoting short-chain fatty acid production in the intestinal and/or oral cavity, a method for modulating the intestinal and/or oral cavity immune system, or a method for promoting improvement of mood states of the present invention. The use of the present invention can be carried out as described for the composition and agent of the present invention.

According to yet another aspect of the present invention, there is provided a whey protein enzymatic hydrolysate containing a peptide having the amino acid sequence of GTWY (SEQ ID NO: 1) and/or a peptide having the amino acid sequence of WY, for use in a method for improving the intestinal and/or oral environment, improving the intestinal and/or oral flora, promoting short-chain fatty acid production in the intestinal and/or oral cavity, regulating the immune system in the intestinal tract and/or oral cavity, or promoting improvement of mood state.

Another aspect of the present invention provides a peptide having the amino acid sequence of GTWY (SEQ ID NO: 1) and/or a peptide having the amino acid sequence of WY for use in improving the intestinal and/or oral environment, improving the intestinal and/or oral flora, promoting short-chain fatty acid production in the intestinal and/or oral cavity, regulating the immune system in the intestinal tract and/or oral cavity, or promoting improvement of mood. The above-mentioned whey protein enzymatic hydrolysate and two types of peptides can be prepared according to the descriptions for the composition and preparation of the present invention, respectively.

The methods and uses of the present invention may be used in mammals, including humans, and both therapeutic and non-therapeutic uses are intended. As used herein, "non-therapeutic" does not include surgical, therapeutic, or diagnostic procedures on humans (i.e., medical procedures on humans), and specifically does not include methods of surgical, therapeutic, or diagnostic procedures on humans performed by a physician or a person under the direction of a physician.

The present invention will be explained in more detail based on the following examples, but the present invention is not limited to these examples.

Example 1: Improvement of intestinal environment by WY peptide (1)
In Example 1, the effect of WY peptide on improving the intestinal environment was evaluated using an animal model.

(1) Method A: Experimental Procedure: C57BL/6J male mice (Charles River Japan, hereafter referred to simply as "the mice") were used as the animal model. According to Charles River Japan's materials (https://www.crj.co.jp/cms/cmsrs/img/usr/top/B6-Aged.pdf), the mice were developed by the Jackson Laboratory and are widely used in aging research. Observation of 32 mice revealed that some died at 100 weeks of age, and all died by 170 weeks of age (see the Mouse Phenome Database (https://www.phenome.jax.org/)). The mouse's lifespan corresponds to 20-30 years of age in humans, 3-6 months of age in mice (mature individuals), 38-47 years of age in humans, 10-14 months of age in mice (middle-aged individuals), and 56-69 years of age in humans, 18-24 months of age in mice (elderly individuals).

In Example 1, after acclimation, the mice were divided into a WY-fed group (15 mice) and a non-fed group (15 mice) at 6 months of age, with weight distribution evenly distributed. The WY-fed group was fed a purified diet (AIN-93M, Oriental Yeast Co., Ltd.) containing 0.05% (w/w) WY peptide in dry weight equivalent. The non-fed group was fed AIN-93M without WY peptide ad libitum. After the 3-month feeding period, cecal contents and feces were collected, and quantification of short-chain fatty acids in the cecal contents and analysis of the fecal bacterial flora were performed at Techno Suruga Lab, as described in (b) and (c) below. The same procedures were also used to quantify short-chain fatty acids when the feeding period was extended to 1.5 months.

B. Quantification of Short-Chain Fatty Acids Quantification of short-chain fatty acids was performed by gas chromatography (GC) according to standard methods. Specifically, 100 mg of sample was accurately weighed into a bead tube, and 9 volumes of 0.5% phosphoric acid solution was added and mixed, followed by heat treatment at 85°C for 15 minutes. The sample was crushed and cooled, and centrifuged (14,000 rpm, 10 minutes). The supernatant was transferred to a new tube, an equal volume of ethyl acetate was added, mixed, and centrifuged again (14,000 rpm, 10 minutes). The ethyl acetate layer was transferred to a vial, and an internal standard (4-methylvaleric acid) was added to prepare the measurement sample. Measurements were performed using a GC-FID separation and detection system (7890B, Agilent Technologies) under the conditions shown in Table 1. Concentration conversion was performed using the absolute calibration curve method using a standard product.

C. Bacterial flora analysis
Sequencing
For bacterial flora analysis, the 16S rDNA partial base sequence of the bacteria contained in the samples was determined by next-generation sequencing amplicon analysis and searched against a microbial identification database. Specifically, the sample was pretreated and crudely extracted using the method of Takahashi et al. (PLoS One 2014;9:e105592.), and DNA was purified using an automated DNA isolation system (GENE PREP STAR PI-480, Kurabo Industries) and a tissue DNA isolation reagent kit (NR-201, Kurabo Industries). After confirming that the DNA concentration and purity were appropriate, the 16S rDNA was amplified by PCR and its sequence was determined. PCR was performed using 34lf-R806 and Dual-index (8-bp barcode) primers (Appl Environ Microbiol 1993;59:695-700, Proc Natl Acad Sci USA 2011;108 Suppl 1:4516-4522) according to the methods described by Takahashi et al. Sequencing was performed by next-generation sequencing amplicon analysis. Next-generation sequencing was performed using the MiSeq (Illumina) instrument with the MiSeq Reagent Kit v3 (600 cycles) (Illumina) according to the protocol provided with the instrument. FastQ paired-end ligation was performed using fastQ-join (default conditions), and quality filtering was performed to ensure that 99% or more of the sequences met a Quality Value of 20 or higher.

Database search: Using the microbial identification database DB-BA13.0 (TechnoSuruga Laboratory) and Metagenome@KIN (World Fusion) software, taxonomic groups (kingdom to species) with a homology rate of 97% or higher were extracted (Appl Environ Microbiol 1993;59:695-700, Proc Natl Acad Sci USA 2011;108 Suppl 1:4516-4522, Arch Microbiol 2015;197:19-934, BMC Gastroenterol 2015;15:100).

D. Statistical analysis The percentage of reads corresponding to a specific taxonomic group relative to the total number of 16S rDNA sequences (total number of reads) was calculated and reported as the mean ± standard error for each group. Evaluation between groups was performed using a t-test, and a significant difference was considered to exist between the two groups when the risk rate was P<0.05%.

(2) Results A. Quantitation of Short-Chain Fatty Acids Seven short-chain fatty acids were analyzed: acetic acid, propionic acid, n-butyric acid, isobutyric acid, n-valeric acid, isovaleric acid, and n-caproic acid. However, n-caproic acid was excluded from evaluation because its concentration was below the lower limit of quantitation in most samples. As shown in Figure 1, the WY intake group (intake period: 1.5 months) showed significant increases in acetic acid and the total amount of the six short-chain fatty acids compared to the non-intake group. Furthermore, as shown in Figure 2, the 3-month WY intake group (intake period: 3 months) showed significant increases in acetic acid, the total amount of the six short-chain fatty acids, and n-butyric acid compared to the non-intake group.

B. Microbial Flora Analysis As shown in Figure 3, the proportion of Actinobacteria relative to the total number of leads in the WY intake group (intake period: 3 months) was significantly increased, and the proportion of Firmicutes relative to the total number of leads was significantly decreased, compared with the non-intake group. Furthermore, as shown in Figure 4, the proportion of Bifidobacterium relative to the total number of leads in the WY intake group (intake period: 3 months) was significantly increased, and the proportions of Clostridium and Romboutsia relative to the total number of leads were significantly decreased, compared with the non-intake group. Furthermore, as shown in Figure 5, the proportion of Bifidobacterium pseudolongum relative to the total number of leads in the 3-month WY intake group was significantly increased compared with the non-intake group.

C. Summary These results indicate that administration of WY peptide changes the intestinal flora and increases the amount of short-chain fatty acids.

Example 2: Improvement of intestinal environment with WY peptide (2)
In Example 2, the effect of WY peptide on improving the intestinal environment was evaluated using 66-week-old C57BL/6J male mice.

(1) Method: 66-week-old C57BL/6J male mice were used, and the test diet intake period was 4.5 months. The mice were divided into a WY intake group (12 mice) and a non-intake group (13 mice) so that weight distribution was evenly balanced. Excluding mice that died naturally during rearing, 9 mice in the WY intake group and 10 mice in the non-intake group were evaluated. Other conditions and methods were the same as those described in Example 1(1).

(2) Results As shown in Figure 6, the WY intake group showed significantly increased levels of acetic acid and the total amount of six short-chain fatty acids compared to the non-intake group, and n-butyric acid also tended to increase. Here, the mice were converted to middle-aged humans at the start of test diet intake and elderly humans at the end of intake. Therefore, WY intake was shown to increase short-chain fatty acid levels and improve the intestinal environment even in middle-aged and elderly individuals.

Example 3: Effect of WY peptide on intestinal immunity In Example 3, immune cells in the mesenteric lymph nodes of mice receiving the test substance WY peptide were compared with those of mice not receiving the test substance.

(1) Method A: Experimental Procedure: Male C57BL/6J mice were acclimatized and then divided into a WY-fed group (10 mice) and a non-fed group (10 mice) at 3 months of age, with weight distribution evenly distributed. The WY-fed group was fed a purified diet (AIN-93M, Oriental Yeast Co., Ltd.) containing 0.05% (w/w) WY peptide in dry mass equivalent. The non-fed group was fed AIN-93M without WY peptide ad libitum. After the 3-month feeding period, mesenteric lymph nodes were excised. The excised mesenteric lymph nodes were treated with collagenase 4 (Roche) and filtered through a 70 μm cell strainer to recover lymphocytes. The recovered lymphocytes were then analyzed using the method described in A below. Reagents not listed in A below were obtained from eBioscience.

B. Lymphocyte analysis
Analysis of dendritic cells
The collected lymphocytes were stained with FITC-IA/IE, PE-PDL-1, PE-CD80, FITC-CD86, CD11b-APC-Cy7 (BD Pharminge), and CD11c-PE-Cy7 according to standard methods, and dendritic cells were analyzed using a flow cytometer (BD CANTO2).

Analysis of T cells: The collected lymphocytes were treated with Leukocyte Activation Cocktail with BD GolgiPlug (BD Pharminge) for 4.5 hours according to standard methods, stained with CD4-APC, TNF-α-FITC, and IFN-γ-PE-Cy7 antibodies using a Cell Fixation/Permeabilization Kit, and analyzed using a flow cytometer.

Analysis of Regulatory Dendritic Cells The collected lymphocytes were stained with CD3e-PerCP, CD4-APC, CD25-APC-Cy7, and Foxp3-PE-Cy7 antibodies using a Foxp3 staining kit according to standard methods, and analyzed using a flow cytometer.

C. Statistical analysis Measurement values were recorded as mean ± standard error. Evaluation between groups was performed using a t-test, and a significant difference was considered to exist between the two groups when the risk rate was P<0.05%.

(2) Results As shown in Figure 7, the WY intake group showed significant increases in I-A/I-E and CD86 in CD11b ⁺ CD11c ⁺ myeloid dendritic cells compared to the non-intake group, and PDL-1 and CD80 also tended to increase. Furthermore, while there was no change in the proportion of TNF-α-positive CD4 ⁺ T cells among CD3e ⁺ CD4 ⁺ T cells, the proportion of IFN-γ-positive CD4 ⁺ T cells tended to increase. This indicates that the immune system was sufficiently and appropriately activated. Meanwhile, the proportion of CD4 ⁺ CD25 ⁺ Foxp3 ⁺ regulatory T cells among CD3e ⁺ CD4 ⁺ T cells significantly increased, suggesting that excessive immune responses were suppressed. These results demonstrate that WY intake regulates intestinal immunity.

Example 4: Improvement of intestinal environment by GTWY peptide In Example 4, the effect of improving the intestinal environment by the GTWY peptide was evaluated using C57BL/6J male mice.

(1) Method: 62-week-old C57BL/6J male mice were used, and the test diet GTWY peptide (Bachem) was administered for 8.5 months. Eight mice were administered GTWY and 12 mice were administered GTWY. The mice were divided into a GTWY-administered group and a non-administered group, with the weight distribution evenly distributed. Other conditions and methods were the same as those described in Example 1(1).

(2) Results As shown in Figure 8, the GTWY-intake group showed significant increases in propionic acid, n-butyric acid, and n-valeric acid compared to the non-intake group, and isobutyric acid also tended to increase. These results indicate that administration of GTWY increases the amount of short-chain fatty acids.

Example 5: Improving the intestinal environment with whey hydrolysate (1)
In Example 5, the effect of whey hydrolysate (Megmilk Snow Brand, HW-3) on improving the intestinal environment was evaluated using Crl:CD1 (ICR) male mice (Charles River Japan).

(1) Method After acclimation and rearing of Crl:CD1 (ICR) male mice, 6.5 months of age were divided into a whey hydrolysate intake group (7 mice) and a non-intake group (10 mice) so that weight distribution was evenly balanced. The feeding period was 3 months, and the whey hydrolysate intake group was fed ad libitum a purified diet (AIN-93M, Oriental Yeast Co., Ltd., protein content adjusted to 14%) containing 5% (w/w) whey hydrolysate in dry mass equivalent. The non-intake group was fed ad libitum AIN-93M (casein content 14%) without whey hydrolysate. Other conditions and methods were the same as those described in Example 1(1).

(2) Results A. Quantification of short-chain fatty acids As shown in Figure 9, the whey hydrolysate intake group showed significant increases in acetic acid, n-butyric acid, and the total amount of six short-chain fatty acids compared to the non-intake group, and propionic acid also tended to increase. These results indicate that administration of whey hydrolysate increases the amount of short-chain fatty acids.

B. Microbial flora analysis As shown in Figure 10, the whey hydrolysate intake group showed a significant decrease in the proportion of Bacteroidetes relative to the total number of leads, and a non-significant increase in the proportion of Actinobacteria relative to the total number of leads, compared with the non-intake group. Also, as shown in Figure 11, the whey hydrolysate intake group showed a significant decrease in the proportion of Bacteroides relative to the total number of leads, a non-significant decrease in the proportion of Romboutsia , and a non-significant increase in the proportions of Bifidobacterium and Lactobacillus relative to the total number of leads, compared with the non-intake group. Furthermore, as shown in Figure 12, the whey hydrolysate intake group showed a significant decrease in the proportion of Clostridium scindens relative to the total number of leads, a non-significant decrease in the proportion of Romboustia ilealis , and a non-significant increase in the proportion of Bifidobacterium pseudolongum relative to the total number of leads, compared with the non-intake group.

C. Summary These results show that administration of whey hydrolysate changes the intestinal flora and increases the amount of short-chain fatty acids.

Example 6: Improving the intestinal environment with whey hydrolysate (2)
In Example 6, the effect of whey hydrolysate on improving the intestinal environment was evaluated using 62-week-old C57BL/6J male mice.

(1) Method: 62-week-old C57BL/6J male mice were used, and the test sample was whey hydrolysate. The mice were fed for 8.5 months. They were divided into a whey hydrolysate-fed group (15 mice) and a non-fed group (12 mice) so that there was no weight imbalance. Other conditions and methods were the same as those described in Example 1(1) and Example 5(1).

(2) Results As shown in Figure 13, the whey hydrolysate intake group showed significant increases in propionic acid, n-butyric acid, and n-valeric acid compared to the non-intake group, and isobutyric acid also tended to increase. Here, the mice were converted to middle-aged humans at the start of test diet intake and elderly humans at the end of intake. Therefore, it was demonstrated that whey hydrolysate intake increased short-chain fatty acid levels and improved the intestinal environment even in elderly subjects (56-69 years old).

Example 7: Measurement of the content of tetrapeptide GTWY and dipeptide WY in whey hydrolysate (1) Preparation of analytical sample A whey hydrolysate (HW-3, Megmilk Snow Brand Co., Ltd.) was diluted appropriately with sterilized water and filtered to prepare a measurement sample. The whey hydrolysate (HW-3) was a product obtained by treating whey protein with an enzyme preparation containing a protease, followed by membrane treatment to remove undigested matter, and then drying. As described below, the whey hydrolysate (HW-3) contains the tetrapeptide GTWY and dipeptide WY.

(2) Analytical Method: The concentrations of the tetrapeptide GTWY and dipeptide WY in the measurement samples obtained in (1) above were quantified by LC-MS/MS under the following analytical conditions. The concentrations of GTWY and WY in the measurement samples were calculated by a calibration curve method using AQUA Peptide (Sigma Aldrich) as the standard sample.

<Analysis conditions>
Mass spectrometer: 4000Q TRAP (AB Sciex)
HPLC device: Agilent 1200 Series (Agilent Technologies)
Column: TSK gel ODS-100V 3 μm 2.0 mm ID × 150 mm (Tosoh Corporation)
Column temperature: 70°C
Mobile phase A: 0.1% formic acid aqueous solution Mobile phase B: 0.1% formic acid acetonitrile solution Gradient conditions: The gradient conditions shown in Table 4 were applied.

Flow rate: 0.2mL/min Sample injection amount: 2μL
Ionization method: ESI (positive ion detection mode)
Curtain gas: 40 psi
Nebulizer gas: 50 psi
Dry gas: 80 psi
Drying gas temperature: 600°C
Collision gas: nitrogen Ionization voltage: 5000 V

<Analysis conditions for tetrapeptide GTWY>
Set mass number (m/z)/collision energy (eV): 526.4 → 159.2/47, 526.4 → 368.3/23
DP voltage (V): 36

<Analysis conditions for dipeptide WY>
Set mass number (m/z)/collision energy (eV): 368.2 → 351.1/19, 368.2 → 159.2/33
DP voltage (V): 51

(3) Analysis Results It was confirmed that 1 g of whey hydrolyzate (HW-3) contained 1.62 mg of tetrapeptide GTWY and 0.60 mg of dipeptide WY.

Example 8: Test to verify the effects of whey hydrolysate on mood state, health-related QOL, saliva immune index and intestinal environment improvement in humans In Example 8, a clinical trial was conducted on humans to verify the effects of whey hydrolysate (Megmilk Snow Brand, HW-3) on mood state, health-related QOL (Quality of Life), saliva immune index and intestinal environment improvement.

(1) Study Overview This study was a placebo-controlled, randomized, double-blind, parallel-group comparative study. The study period was 6 weeks, during which participants ingested the test food or the control food described below. Specifically, healthy men and women aged 45 to 64 who frequently felt irritability or anxiety in their daily lives ingested a "whey hydrolysate-containing tablet" as the test food and a "whey hydrolysate-free tablet" as the control food, and the effects of the whey hydrolysate on mood state, health-related QOL, salivary immune index, and intestinal environment were examined.

(2) Subjects: Subjects who had low psychological health in the pre-test and were judged healthy by a doctor were randomly assigned to a test food group (30 subjects) and a control food group (30 subjects). The subjects analyzed were 28 in the test food group (19 men, 9 women) and 28 in the control food group (20 men, 8 women). The ages of the subjects analyzed (mean ± standard deviation) were 54.3 ± 4.8 years for the test food group and 53.6 ± 5.5 years for the control food group. During the study period, subjects were instructed to continue their lifestyle as before the study period.

(3) Test Food During the test period (6 weeks), the test food group was instructed to take 6 tablets of the test food, and the control food group was instructed to take 6 tablets of the control food once a day with water or lukewarm water.

A. Preparation of test food: As the test food, whey hydrolyzate (HW-3, Megmilk Snow Brand) was mixed with excipients and binders, kneaded, and then tableted to produce whey hydrolyzate-containing tablets (300 mg/tablet). The whey hydrolyzate content per tablet was 168 mg. As a control food, a whey hydrolyzate-free tablet was used, in which 168 mg of maltodextrin was blended instead of the whey hydrolyzate.

B. Analysis of Test Foods: Thirty tablets (9 g) prepared in (A) above were thoroughly ground in a mortar and pestle, and 0.01 or 0.02% (w/v) solutions were prepared using sterile water. The solutions were centrifuged (15,000 rpm, room temperature, 3 minutes), and the resulting supernatant was ultrafiltered (0.2 μm filter). The filtrate was diluted 5-fold with 20% acetonitrile containing 0.1% (w/v) formic acid to prepare the measurement samples. The amounts of tetrapeptide GTWY and dipeptide WY in the measurement samples were measured using the analytical method described in Example 7(2).

C. Analysis results of test foods: It was confirmed that one tablet (300 mg) containing whey hydrolysate contained 0.27 mg (0.090% by mass) of tetrapeptide GTWY and 0.11 mg (0.036% by mass) of dipeptide WY. It was confirmed that the amounts of tetrapeptide GTWY and dipeptide WY in tablets not containing whey hydrolysate were both below the detection limit.

(4) Measurement A Measurement items The measurement items were as follows.
(i) Mood State/State-Trait Anxiety Inventory (sometimes referred to as "STAI" in this specification)
Perceived Stress Scale (sometimes referred to as "PSS" in this specification)
Motivation score (ii) Health-related QOL
SF-36v2 Japanese Acute Edition (sometimes referred to as "SF-36" in this specification)
(iii) Salivary immune index: Amount of immunoglobulin A in saliva (sometimes referred to as “IgA” in this specification)
(iv) Intestinal environment and fecal short-chain fatty acid content (acetic acid, propionic acid, n-butyric acid, iso-butyric acid, n-valeric acid, iso-valeric acid, n-caproic acid)

(i) Measurement period (i) Mood state STAI, PSS and motivation scores were measured once before the start of test food intake (week 0) and once when the subject visited the hospital 6 weeks after the start of test food intake (a total of 2 times).
(ii) Health-related quality of life
SF-36 was administered to the subjects at home once each (4 times in total) before starting the intake of the test food (week 0), and at 2, 4, and 6 weeks after starting the intake of the test food.
(iii) Saliva immune index Saliva samples were collected once before the start of test food intake (week 0) and once when the subjects visited the hospital 6 weeks after starting test food intake (a total of two times), and these were used as samples taken at week 0 and week 6. The supernatants obtained by centrifugation after collection were stored frozen, and after collection of the 6-week sample, the 0-week sample and the 6-week sample were simultaneously measured.
(iv) Intestinal environment Feces were collected twice in total, once from 7 days before the planned start date of test food intake until the day before, and once from 7 days before the planned end date of test food intake until the day before. These samples were used as samples at week 0 and week 6 of intake. The fecal samples were collected in a refrigerated state and stored frozen. After collection of the week 6 sample, the week 0 and week 6 samples were measured simultaneously.

Measurement Method (i) Mood State Upon arrival, subjects completed questionnaires for the STAI, PSS, and motivation score. The STAI is a questionnaire that assesses anxiety divided into state anxiety (anxiety felt at the time of answering, short-term anxiety) and trait anxiety (respondent's personality susceptibility to anxiety, long-term anxiety). It consists of 20 state anxiety items and 20 trait anxiety items. Higher scores indicate higher levels of anxiety (Reference: "Handbook of Mental and Psychological Function Assessment," pp. 238-239, Toshio Yamauchi, Haruo Kashima et al., Nakayama Shoten). The PSS is a questionnaire that assesses subjective stress; higher scores indicate higher levels of subjective stress (Reference: "Reliability and Validity of the Japanese Version of the Perceived Stress Scale," Health Psychology Research, Vol. 19 (2006) No. 2, Katsunori Sumi). The Motivation Score is a questionnaire consisting of 14 items used to assess apathy (a state of decreased motivation). A higher score indicates a more decreased state of motivation (Reference: "Handbook of Mental and Psychological Function Assessment," pp. 462-463).

(ii) Health-related quality of life
Subjects completed the SF-36v2 Japanese acute version at home. The SF-36 is an internationally widely used method for assessing health-related quality of life (QOL). It consists of 36 items measuring eight health concepts: physical function, role-related functioning (physical), bodily pain, general health, vitality, social functioning, role-related functioning (mental), and mental health. Two component summary scores (physical QOL and mental QOL) were calculated from the eight subscales. Higher scores indicate better QOL (Reference: "Handbook of Mental and Psychological Function Assessment," pp. 205-206).

(iii) Salivary Immune Indicators After arriving at the hospital, subjects gargled with water and rested in a seated position for 15 minutes before saliva collection. Saliva collection was performed using Sarisoft (Sarstedt). The sponge included with the Sarisoft was held in the mouth for 2-3 minutes to allow saliva to soak into the sponge, and after 2-3 minutes, the sponge was returned to the Sarisoft. The resulting Sarisoft was centrifuged (2,000 rpm, room temperature, 5 minutes), and the resulting saliva (approximately 1 g) was dispensed into microtubes and frozen at -80°C. The frozen samples were thawed overnight at 4°C before measurement. Salivary IgA was quantified using the Secretory Immunoglobulin A Salivari Immunoassay Kit (Salimetrics) according to the protocol included with the kit.

(iv) Intestinal Environment The short-chain fatty acids in the fecal samples taken 0 and 6 weeks after ingestion were quantified using gas chromatography in the same manner as in Example 1(1).

(5) Evaluation and Analysis A. Mood State After obtaining actual measured values for STAI, PSS, and motivation scores at week 0 and week 6 of intake, the change from week 0 to week 6 of intake was calculated. Motivation scores were analyzed by gender, and evaluation was conducted on women. The actual measured values and changes in each evaluation item at week 6 of intake were compared between the test food group and the control food group using the Mann-Whitney U test. Furthermore, the actual measured values at week 0 and week 6 of intake between the test food group and the control food group were compared using the Wilcoxon signed-rank test.

B. Health-related QOL
The changes in SF-36 from week 0 to week 2, week 4, and week 6 in the test food group and the control food group were compared using a two-sample t-test. Furthermore, the actual measured values at week 2, week 4, and week 6 in the test food group and the control food group were compared using a two-sample t-test. Furthermore, the actual measured values at week 2, week 4, and week 6 in each group were compared with the actual measured value at week 0 in each group using a one-sample t-test.

C. Salivary immune index After measuring salivary IgA levels at week 0 and week 6, the amount of change from week 0 to week 6 was calculated. The actual measured values and changes at week 6 for the test food group and the control food group were evaluated using a two-sample t-test. In addition, the actual measured values at week 0 and week 6 for the test food group and the control food group were compared using a one-sample t-test.

D. Intestinal environment Analysis of short-chain fatty acids was performed using a stratified analysis targeting older subjects (median age 54.5 years or older). Fecal short-chain fatty acid levels were measured at week 0 and week 6, and the change from week 0 to week 6 was calculated. The measured values and changes at week 6 for the test food group and the control food group were evaluated using a two-sample t-test. Furthermore, the measured values at week 0 and week 6 for the test food group and the control food group were compared using a one-sample t-test. For subjects who showed values below the lower limit of quantitation (0.3 μmol/g) for the short-chain fatty acids iso-butyric acid, n-valeric acid, iso-valeric acid, and n-caproic acid at either week 0 or week 6, the corresponding item was recorded as missing.

(6) Results A. Mood State The results of the STAI are shown in Table 3. In the STAI, the change in trait anxiety score in the test food group was statistically significantly reduced (improved) compared to the control food group (p = 0.046). This result indicated that whey hydrolysate improved trait anxiety (susceptibility to anxiety, long-term anxiety).

The results of the PSS are shown in Table 4. The change in score for the test food group on the PSS was statistically significant (p=0.043) compared to the control food group. These results indicate that whey hydrolysate improves subjective stress.

The results of the motivation scores are shown in Table 5, and the results of the stratified analysis of motivation scores for women are shown in Table 6. Motivation scores for the test food group at 6 weeks of intake were statistically significantly lower (improved) compared to week 0 (p=0.004). Meanwhile, no statistically significant change was observed in the scores for the control food group at 6 weeks of intake compared to week 0 (p=0.796). Furthermore, motivation scores for women were statistically significantly lower (improved) in the test food group compared to the control food group (p=0.047). These results demonstrate that whey hydrolysate improves motivation, with a particularly pronounced improvement in women.

B. Health-related QOL
The results of the SF-36 are shown in Table 7. For "body pain," the test food group's score at 6 weeks of intake was statistically significantly increased (improved) compared to week 0 (p = 0.043). On the other hand, no statistically significant change was observed in the control food group's score (p = 0.674). Furthermore, the test food group showed a tendency for the amount of change at 6 weeks to increase (improve) compared to the control food group (p = 0.060). For "vitality," the test food group's score at 6 weeks of intake was significantly increased (improved) compared to week 0 (p = 0.011). On the other hand, no statistically significant change was observed in the control food group's score (p = 0.194). Furthermore, the test food group showed a statistically significant increase (improvement) in the amount of change at 6 weeks compared to the control food group (p = 0.033). In "Mental Health," the test food group's scores at weeks 4 and 6 of intake were statistically significantly increased (improved) compared to week 0 (p = 0.030, p = 0.019, respectively). Meanwhile, no statistically significant changes were observed in the control food group's scores (p = 0.461, p = 0.998, respectively). Furthermore, the test food group showed a tendency for the amount of change at week 6 to increase (improve) compared to the control food group (p = 0.079). In "Mental Health (Summary Score)," the test food group's scores at weeks 4 and 6 of intake were statistically significantly increased (improved) compared to week 0 (p = 0.045, p = 0.013, respectively). Meanwhile, no statistically significant changes were observed in the control food group's scores (p = 0.606, p = 0.850, respectively). The change in the test food group at week 6 was statistically significantly greater (improved) than the control food group (p=0.039). These results indicate that whey hydrolysate improves health-related QOL, including "body pain,""vitality,""mentalhealth," and "spiritual health."

C. Salivary Immune Indices The results of measuring salivary IgA are shown in Table 8. At 6 weeks of intake, the IgA levels in the control food group were statistically significantly lower (worsened) compared to week 0 (p=0.018). On the other hand, no significant change was observed in the IgA levels in the test food group (p=0.927). The change in salivary IgA levels in the test food group at 6 weeks was significantly smaller than that in the control food group (p=0.045). These results demonstrate that whey hydrolysates improve salivary immune indices.

D. Intestinal Environment Measurement results for short-chain fatty acid levels in older subjects (above the median age) are shown in Table 9. For acetic acid and propionic acid, there was a statistically significant decrease (worsening) in the control food group at 6 weeks of intake compared to week 0 (p<0.001, p=0.045, respectively). On the other hand, no statistically significant change was observed in the test food group (p=0.964, p=0.392, respectively). For n-butyric acid and n-valeric acid, there was a statistically significant increase (improvement) in the test food group at 6 weeks of intake compared to week 0 (p=0.024, p=0.031, respectively). On the other hand, no significant change was observed in the control food group (p=0.459, p=0.348, respectively). The changes in acetic acid (p=0.027), propionic acid (p=0.028), iso-butyric acid (p=0.026), n-valeric acid (p=0.045), and iso-valeric acid (p=0.030) at week 6 in the test food group were significantly increased (improved) compared to the control food group. Furthermore, the changes in n-butyric acid at week 6 in the test food group tended to increase compared to the control food group (p=0.094). These results indicate that whey hydrolysate increases the amount of short-chain fatty acids in feces in older subjects.

(7) Summary These results indicate that whey hydrolysates containing the tetrapeptide GTWY or the dipeptide WY improve mood states such as long-term anxiety or depression, subjective stress, or loss of motivation, improve health-related QOL such as vitality or mental health, improve immune status as indicated by IgA, and increase the amount of short-chain fatty acids in feces, thereby improving the intestinal environment.

Claims (12)

A composition for improving the intestinal and/or oral flora and/or promoting short-chain fatty acid production in the intestines and/or oral cavity, comprising as an active ingredient a whey protein enzymatic hydrolysate containing a tetrapeptide consisting of the amino acid sequence of GTWY (sequence number 1) and/or a dipeptide consisting of the amino acid sequence of WY. A composition for regulating intestinal and/or oral immunity, comprising as an active ingredient a whey protein enzymatic hydrolysate containing a tetrapeptide having the amino acid sequence GTWY (sequence number 1) and/or a dipeptide having the amino acid sequence WY. The composition according to claim 1 or 2, wherein the GTWY content (in terms of solid content) in the whey protein enzymatic hydrolysate is 0.5 to 5 mg/g. The composition according to any one of claims 1 to 3, wherein the whey protein enzymatic hydrolysate contains 0.05 to 2 mg/g of whey protein (in terms of solid content). The composition according to any one of claims 1 to 4, wherein the whey protein enzymatic hydrolysate is ingested by a human at a daily dose of 1 to 50,000 mg (solid content equivalent). A composition for improving the intestinal and/or oral flora and/or promoting short-chain fatty acid production in the intestines and/or oral cavity, comprising as active ingredients a tetrapeptide consisting of the amino acid sequence of GTWY (sequence number 1) and/or a dipeptide consisting of the amino acid sequence of WY. A composition for modulating intestinal and/or oral immunity, comprising as an active ingredient a tetrapeptide consisting of the amino acid sequence GTWY (SEQ ID NO: 1) and/or a dipeptide consisting of the amino acid sequence WY. The composition according to any one of claims 1 to 7, wherein the tetrapeptide consisting of the amino acid sequence of GTWY (SEQ ID NO: 1) is ingested by a human in an amount of 0.001 to 1000 mg (in terms of solid content) per day. The composition according to any one of claims 1 to 8, wherein the dipeptide consisting of the amino acid sequence of WY is ingested by a human in an amount of 0.001 to 500 mg (in terms of solid content) per day. The composition according to any one of claims 1 to 9, which is a food composition. The composition described in any one of claims 1 to 10, which is in a unit-packaged form per serving. A composition according to any one of claims 1 to 11, intended for consumption by middle-aged and elderly people.