Composition for improving the intestinal or oral environment

Abstract

The purpose of the present invention is to provide a composition for improving the intestinal environment or the oral environment, a composition for improving the intestinal flora or the oral flora, a composition for promoting production of short-chain fatty acids in the intestine or the oral cavity, an immunoregulatory composition for the intestinal tract and the oral cavity, and a composition for promoting improvement of mood. The present invention provides a composition for improving the intestinal environment or the oral environment, for improving the intestinal flora or the oral flora, and / or for promoting production of short-chain fatty acids in the intestine or the oral cavity, an immunoregulatory composition for the intestinal tract and the oral cavity, and a composition for promoting improvement of mood which contain, as an active ingredient, an enzymatic decomposition product of whey protein containing a peptide having the amino acid sequence GTWY (SEQ ID NO:1) and / or a peptide having the amino acid sequence WY.

Description

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority from an earlier Japanese application, Patent Application No. 2020-67855 (filing date: April 3, 2020), the entire disclosure of which is incorporated herein by reference. [Technical Field]

[0002] 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 intestinal 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. [Background technology]

[0003] An estimated 40 trillion bacteria reside in the intestine, a group known as the intestinal flora (gut flora). Similar to the intestine, bacteria also reside in the oral cavity, a group known as the oral flora (oral flora or oral microbiota). The types and amounts of bacteria that make up the intestinal or oral microbiota vary from person to person. These bacteria have diverse functions, and recent research has shown that the state of the intestinal or oral microbiota is related to many diseases. Furthermore, it has become clear that much of human immune function depends on the state of the intestinal microbiota, and that human immune function is related to the state of the oral microbiota. Maintaining a healthy balance of the intestinal or oral microbiota can maintain healthy immune function. Therefore, improving the intestinal and / or oral environment is expected to maintain health and prevent various diseases, including lifestyle-related diseases.

[0004] Against this background, various ingredients or materials effective in improving the intestinal and / or oral environment have been developed. For example, Patent Document 1 discloses a composition for improving the intestinal environment that contains milk-derived glycopeptides. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2019 / 044960 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a novel composition for improving the intestinal and / or oral environment and an agent for improving the intestinal and / or oral environment. Another object of the present invention is to provide a novel composition for improving the intestinal and / or oral flora and an agent for improving the intestinal and / or oral flora, a novel composition for promoting short-chain fatty acid production in the intestine and / or oral cavity and an agent for promoting short-chain fatty acid production, a novel composition for modulating the intestinal and / or oral cavity immune system and an agent for modulating the intestinal and / or oral cavity immune system, and a composition for promoting improvement of mood state and an agent for promoting improvement of mood state. [Means for solving the problem]

[0007] The present inventors have now conducted animal model and human studies in which mice or humans were administered a peptide having a specific sequence or a whey protein hydrolysate containing the peptide, and found that this peptide had the effect of improving the intestinal and oral flora, as well as promoting the production of short-chain fatty acids and other intestinal and oral environments. The present inventors have also found that the peptide having the specific sequence and the whey protein hydrolysate containing the peptide have an immune-modulating effect via 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.

[0008] 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 (sequence number 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 content of GTWY in the whey protein enzymatic hydrolysate (converted to solid content) 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 WY in the whey protein 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 in an amount 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 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 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 active ingredients a peptide having the amino acid sequence of GTWY (sequence number 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.

[0009] The compositions of [1], [2], [3], [7], [8] and [9] above may be referred to herein as "compositions of the present invention," and the preparations of [1], [2], [3], [7], [8] and [9] above may be referred to herein as "preparations of the present invention."

[0010] The whey protein enzymatic hydrolysate, which is the active ingredient of the present invention, is derived from a food material that has been consumed by humans for many years. Therefore, the composition and agent of the present invention can be used as a functional food that improves the intestinal and / or oral environment and is advantageous in that it can be used as a functional food that is safe for mammals, including humans. [Brief explanation of the drawings]

[0011] [Figure 1] 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] 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. [Figure 3] Figure 3 shows the results of bacterial flora analysis (at the end of the intake period) in 6-month-old (at the start of intake) C57BL / 6J male mice treated with WY peptide (intake period: 3 months, non-intake group: n = 8, intake group: n = 8). * indicates P < 0.05%. [Figure 4] Figure 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 for 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. [Figure 5] Figure 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 for ingestion periods of 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. [Figure 6]Figure 6 shows the quantitative results (at the end of the intake period) of short-chain fatty acids in 66-week-old (at the start of intake) C57BL / 6J male mice using WY peptide (intake period: 4.5 months, non-intake group: n = 10, intake group: n = 9). CTL indicates the WY non-intake group, WY indicates the WY intake group, * indicates P < 0.05%, and error bars indicate standard error. [Figure 7] Figure 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). CTLs are from the WY non-ingestion group, WY is from the WY ingestion group, * indicates P < 0.05%, and error bars indicate standard error. [Figure 8] Figure 8 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 GTWY peptide (intake period: 8.5 months, non-intake group: n = 12, intake group: n = 8). CTL indicates the non-GTWY intake group, GTWY indicates the GTWY intake group, * indicates P < 0.05%, and error bars indicate standard error. [Figure 9] Figure 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. [Figure 10] Figure 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%. [Figure 11] Figure 11 shows the results of microbiota analysis (at the end of the intake period) in Crl:CD1 (ICR) male mice aged 6.5 months (at the start of intake) treated with whey hydrolysate (intake period: 3 months). CTL is the group that did not take whey hydrolysate (n = 10), and HW-3 is the group that took whey hydrolysate (n = 7). * indicates P < 0.05%, and error bars indicate standard error. [Figure 12]Figure 12 shows the results of microbiota analysis (at the end of the intake period) in Crl:CD1 (ICR) male mice aged 6.5 months (at the start of intake) treated with whey hydrolysate (intake period: 3 months). CTL is the group that did not take whey hydrolysate (n = 10), and HW-3 is the group that took whey hydrolysate (n = 7). * indicates P < 0.05%, and error bars indicate standard error. [Figure 13] Figure 13 shows the quantitative results of short-chain fatty acids (at the end of the intake period) in 62-week-old (at the start of intake) C57BL / 6J male mice using whey hydrolysate (intake period: 8.5 months). CTL is the group that did not take whey hydrolysate (n = 12), and HW-3 is the group that took whey hydrolysate (n = 15). * indicates P < 0.05%, and error bars indicate standard error. Specific Description of the Invention

[0012] 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 the like from milk. Whey is composed of proteins such as β-lactoglobulin, α-lactalbumin, serum albumin, and immunoglobulin. 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 of the compositions and preparations of the present invention, is not limited as long as it is an enzymatic hydrolysate of whey.

[0013] The content (in terms of solid content) of the whey hydrolysate in the compositions and preparations of the present invention can be determined arbitrarily depending on the purpose, use, form, dosage form, etc., and the present invention is not limited thereto. For example, in the case of liquid compositions and liquid preparations, the content 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, the content is 0.1 to 90 mass% (preferably 0.5 to 80 mass%, more preferably 1 to 70 mass%) of the total amount.

[0014] Furthermore, 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, "a 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.

[0015] The content of GTWY (in terms of solid content) in the whey hydrolysate of the present invention is, for example, 0.01 to 1 mass% (preferably 0.05 to 0.5 mass%, more preferably 0.1 to 0.2 mass%) relative to the total amount of whey hydrolysate, and the content of WY (in terms of solid content) is, for example, 0.005 to 0.5 mass% (preferably 0.01 to 0.1 mass%, more preferably 0.03 to 0.09 mass%) relative to the total amount of whey hydrolysate.

[0016] 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), fluorometry, or colorimetry, and can be carried out, for example, using LC-MS / MS according to the method described below in Example 2. When LC-MS / MS is used, a peptide having the purity suitable for LC-MS / MS measurement is used as the standard peptide during measurement, and for example, AQUA peptide (SIGMA ALDRICH) can be used.

[0017] 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), Thermax690 (Glanbia), or Protherma (Glanbia)) may be used as the whey hydrolysate.

[0018] The whey hydrolysate containing the peptide of the present invention can be produced, for example, by allowing an enzyme preparation containing a whey-degrading enzyme to act on whey protein.

[0019] The concentration of whey protein to be subjected to the enzyme reaction is not limited as long as the protein is soluble. However, from the viewpoint of suppressing gelation and aggregation and eliminating the need for concentration, the concentration is preferably 1 to 30 w / v%, more preferably 1 to 20 w / v%, and even more preferably 5 to 15 w / v%.

[0020] 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 can be selected based on the subsequent use, flavor, taste, mineral content, etc., but a composition that maintains the pH of the reaction solution at 4 to 9, preferably 5 to 8, and more preferably 7 to 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 concentration within the range that provides a buffering effect. Considering flavor, taste, and mineral content, the concentration can be, for example, 0.01 to 0.5 M, preferably 0.05 to 0.2 M, and more preferably approximately 0.1 M.

[0021] 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 that can be used include those derived from microorganisms such as Bacillus subtilis, Aspergillus oryzae, and Aspergillus melleus. Of these, an enzyme preparation derived from Aspergillus oryzae or Aspergillus melleus is preferred, and an enzyme preparation derived from Aspergillus melleus is more preferred.

[0022] Commercially available enzyme preparations can be used as the enzymes to be subjected to the enzymatic reaction, and are available from, for example, Amano Enzyme, Shin-Nippon Chemical Industry, DSM, Danisco, Novozymes, and HBI. The amount of enzyme preparation added is optional, but considering an appropriate hydrolysis reaction rate, cost, and the like, it can be, for example, 0.01 to 5 w / v%, preferably 0.05 to 4 w / v%, and more preferably 0.1 to 0.5 w / v%.

[0023] The enzyme reaction temperature and time can be set so that the starting protein is sufficiently hydrolyzed and the quality of the enzymatic hydrolyzate 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 appropriately while checking the amount of the peptide of the present invention produced.

[0024] 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 preferred method is to carry out the reaction while increasing the temperature from 35°C to 75°C over 5 to 8 hours, and more preferably from 35°C to 75°C over 6 to 8 hours. 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 rapidly increase 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 increase 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.

[0025] From the viewpoint of reaction efficiency, it is preferable to stir the reaction solution during the enzymatic reaction. The stirring speed should be high so that the substrate comes into contact with the enzyme well, but if the stirring speed is too high, the reaction solution may splash, so the stirring speed is, for example, 100 to 500 rpm, preferably 200 to 400 rpm, and more preferably about 250 rpm.

[0026] 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 subjecting the reaction solution to high temperature or by adding a chelating agent to change the chemical structure of the enzyme, or by removing the enzyme by membrane treatment. A preferred method is a deactivation treatment in which the reaction solution is subjected to high temperature. The high-temperature treatment method is, for example, a method in which the reaction solution is maintained at 80 to 90°C for 5 to 30 minutes, preferably 80 to 90°C for 20 to 30 minutes. Furthermore, if the temperature is high in the concentration step described below, this can also be performed as the concentration step.

[0027] The reaction solution (whey hydrolyzate) that has undergone the above-mentioned enzyme reaction step and enzyme reaction stopping step may further be subjected to a sterilization step. Examples of the sterilization step include the membrane treatment step or heat sterilization step described below. The heat sterilization step can also serve as the above-mentioned enzyme reaction stopping step, which is advantageous in terms of simplifying the production process.

[0028] The reaction solution (whey hydrolyzate) that has undergone the enzyme reaction step and the enzyme reaction termination step may further be subjected to a purification step. Examples of the purification step 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 the sterilization step, which is advantageous in terms of simplifying the production process.

[0029] The reaction solution (whey hydrolyzate) that has undergone the enzyme reaction step and the enzyme reaction termination step may be further subjected to a concentration step from the viewpoint of storage and transportation. Any method can be selected for the concentration step, but vacuum concentration, freeze-drying, spray-drying, or membrane concentration (e.g., a method using a reverse osmosis membrane) is preferred, and freeze-drying or spray-drying is more preferred. Spray-drying is particularly preferred from the viewpoint of efficient mass concentration.

[0030] When whey hydrolysates are contained in the compositions and preparations of the present invention, they can be contained 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 contained per serving.

[0031] When the composition and agent of the present invention contain the peptide of the present invention, 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 contained per meal.

[0032] The peptides of the present invention can be derived from food materials such as enzymatic hydrolysates of whey protein, or, because the peptide chains are short and chemical synthesis is easy, synthetic products, salts, or solvates thereof can also be used. In other words, the peptides of the present invention are advantageous in that they are easily available.

[0033] According to a first aspect of the present invention, the compositions and agents 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" means relatively increasing the number of beneficial bacteria (e.g., short-chain fatty acid-producing bacteria such as lactic acid bacteria, acetic acid bacteria, propionic acid bacteria, butyric acid bacteria, and bifidobacteria; particularly bifidobacteria) present in the lower gastrointestinal tract and / or oral cavity, or reducing the number of harmful bacteria (e.g., Clostridium perfringens, Staphylococcus aureus, Escherichia coli; particularly Clostridium "Relative increase" refers to increasing the proportion of beneficial bacteria in the intestinal and / or oral flora, while "relative decrease" refers to decreasing 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.

[0034] 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 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.

[0035] 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 short-chain fatty acid production 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 energy metabolism in the body. In recent years, it has been elucidated that promoting short-chain fatty acid production in the intestine is effective in preventing and ameliorating metabolic diseases such as diabetes and obesity (Nippon 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 ameliorating 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 ameliorating dental caries, periodontal disease, or 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 ameliorate the aforementioned diseases.

[0036] In the present invention, a short-chain fatty acid is a fatty acid having 6 or less carbon atoms, preferably 2 to 6 carbon atoms, and examples thereof include acetic acid, propionic acid, n-butyric acid, iso-butyric acid, n-valeric acid, iso-valeric acid, and n-caproic acid.

[0037] The amount of short-chain fatty acid production in the intestine and / or oral cavity can be evaluated, for example, by collecting a sample such as feces from a subject in the intestine or saliva from a 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 its total amount. It is expected that 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 will result in immune regulation via intestinal immunity and / or oral immunity.

[0038] 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 the immune status of the intestinal tract and / or oral cavity. 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 tract and / or oral cavity by promoting the differentiation and induction of immune cells such as dendritic cells, B cells, T cells, and 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 via 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 via immune cells is achieved, for example, by promoting the induction of differentiation of T cells or regulatory T cells and / or by suppressing cell death of immune cells.

[0039] 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, a high concentration of inflammatory cytokines and a low concentration of anti-inflammatory cytokines (anti-inflammatory cytokines) can be considered to indicate an activated immune state, while the reverse can be considered to indicate a regulated or suppressed immune state. Furthermore, a high concentration of immunoglobulin (Ig) A can be considered to indicate an activated immune state. 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 considered to indicate sufficient and appropriate activation of the immune system, and an increase in the number of T cells or regulatory T cells can be considered to indicate an excessive immune system response has been suppressed.

[0040] According to a fifth aspect of the present invention, the compositions and agents of the present invention can be used for the purpose of improving and promoting mood states. In the present invention, the mood states include, 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 states. (1) Anger-hostility: A state of anger or hostility toward others (2) Confusion-perplexity: Perplexity or poor cognitive performance (3) Depression: A depressed mood accompanied by a sense of loss of confidence. (4) Fatigue-Lethargy: Feeling tired, lethargic, and low energy (5) Tension-anxiety: Increased tension in the musculoskeletal system (6) Vibrancy - Vitality: high vitality, dynamism, and energy (7) Friendship: Feeling positive emotions toward others

[0041] In the present invention, "promoting improvement" includes both phenomena, for example, improving a negative state to a standard or normal state compared with a standard or normal state, and promoting a standard or normal state to a positive state. Specifically, examples of promoting improvement of mood states in the present invention include, for example, improvement of lethargy, lethargy, depression, depressed mood, 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 feel irritation or anxiety in their daily lives, and 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. Known methods for measuring or evaluating 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 (SH) Resilience Test, Five Facet Mindfulness Questionnaire (FFMQ), or health-related QOL, or a combination of these scales. Also, known comprehensive methods for measuring or evaluating 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.

[0042] As described 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 intestinal and / or oral immunity, or promoting improvement of mood states, and therefore subjects to whom the compositions and agents of the present invention are ingested or administered include both subjects with the above-mentioned diseases and healthy subjects, although healthy subjects (healthy individuals when the subject is human) are preferred. Hereinafter, in this specification, administration and ingestion may be used interchangeably.

[0043] In general, the intestinal and / or oral environment tends to change with age (particularly senescence) (Odamaki et al. BMC Microbiology (2016) 16:90). Therefore, the age of the subject to be ingested with the compositions and preparations of the present invention is not particularly limited. However, the compositions and preparations of the present invention are preferably intended for middle-aged or elderly subjects (e.g., if the subject is a human, those aged 50 or older), and particularly preferably for elderly subjects (e.g., if the subject is a human, those aged 65 or older). That is, 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 intended for subjects whose intestinal environment has changed due to reasons other than aging, such as diet, antibiotics, or stress.

[0044] 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), feeds (including pet foods), etc., and can be implemented as described below.

[0045] The active ingredient 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 ingredient of the present invention are not particularly limited as long as they are suitable for oral administration, and include, for example, granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, emulsions, and suspensions. These formulations can be formulated using pharmaceutically acceptable carriers according to techniques commonly used 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.

[0046] Furthermore, the active ingredient of the present invention can be administered to humans and non-human animals, preferably orally. A typical oral intake form is food. When the active ingredient of the present invention is provided as a food, it can be incorporated into the food. The food thus provided contains an effective amount of the active ingredient of the present invention. As used herein, the phrase "containing an effective amount" of the active ingredient in the food of the present invention refers to a content such that the active ingredient of the present invention is ingested within the range described below when the individual food is ingested in an amount typically consumed. Furthermore, the term "food" is used to encompass 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 the active ingredient of the present invention is administered to animals other than humans, the food referred to in the present invention is, of course, used as feed.

[0047] 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 daily foods 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 a unit package form in which the amount to be ingested per meal is predetermined. Examples of unit package forms per meal include packs, wrappings, cans, bottles, etc., in which a fixed amount is specified. To better exert 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 the like containing such instructions.

[0048] 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) intake amounts. 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 of one-half to one-twentieth of the daily human intake amount described below. For convenience of intake, 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.

[0049] 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. Examples of "supplements" include tablets produced by kneading the active ingredient of the present invention with an excipient, a binder, etc., and then tableting, and capsules encapsulated in capsules. When provided as a supplement, it is preferable to provide it in the unit package form per meal as described above, or in a unit package form per day, per week, or per month.

[0050] The foods provided by the present invention are not particularly limited as long as they contain the active ingredient of the present invention, and 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 jelly, coffee, cocoa, tea drinks, nutritional drinks, energy drinks, sports drinks, mineral water, near-water or non-alcoholic beer-flavored drinks and other non-alcoholic drinks; carbohydrate-containing foods and drinks 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, etc. Examples of suitable confectioneries include dairy products such as ice cream and other dairy products; Western confectioneries such as cookies, cakes, and chocolates; Japanese confectioneries such as manju and yokan; tablet confectioneries (refreshing confectioneries) such as ramune (ramune), candies, chewing gum, frozen desserts such as jellies and puddings, frozen confections, rice crackers, and other 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, and 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 paste, soy sauce, furikake (a type of seasoning), and 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.

[0051] Tea beverages include all types of fermented tea, semi-fermented tea, and non-fermented tea, 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, jasmine tea, etc.).

[0052] Examples of fruits used in fruit juice beverages or beverages containing fruit and vegetable juices include apples, mandarin oranges, grapes, bananas, pears, peaches, mangoes, acai, blueberries, and plums. Examples of vegetables used in vegetable juice beverages or beverages containing fruit and vegetable juices include tomatoes, carrots, celery, pumpkins, cucumbers, and watermelons.

[0053] The intake (administration) amount of the active ingredient of the present invention can be determined depending on the recipient's sex, age, and weight, symptoms, intake (administration) timing, intake (administration) time, dosage form, intake (administration route), and drugs to be combined, etc. The daily adult intake (in terms of solid content) of whey hydrolysates 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 cavity immune system, or promoting improvement of mood states is, for example, 1 to 50,000 mg (preferably 10 to 10,000 mg, more preferably 100 to 5,000 mg), the daily adult intake (in terms of solid content) of GTWY 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 adult intake (in terms of solid content) of WY 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 whether the active ingredient of the present invention is used for non-therapeutic purposes or for therapeutic purposes, and in the case of therapeutic purposes, "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.

[0054] 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 intestinal and / or oral cavity immune system, 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 be taken twice or more times a day (preferably 2 or 3 times a day).

[0055] 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 phrase "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 improve their stomach condition 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

[0056] 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 lose motivation easily - For those who are concerned about a decline in motivation or drive For those who tend to get depressed For those who want to stay positive · For those who are prone to depression For those who are easily stressed For those who are prone to anxiety For those who often get irritated

[0057] Another aspect of the present invention provides 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 and improving 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. Another aspect of the present invention also provides 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 and improving 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.

[0058] 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 agent for promoting short-chain fatty acid production in the intestinal and / or oral cavity, an immunomodulator in the intestinal tract and / or oral cavity, 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 immunomodulator in the intestinal tract and / or oral cavity, 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 promoting immunity in the intestinal and / or oral cavity, or a method for promoting improvement of mood states 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, 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 compositions and agents of the present invention.

[0059] 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 bacterial flora, promoting short-chain fatty acid production in the intestinal and / or oral cavity, regulating the intestinal tract and / or oral cavity, or promoting improvement of mood state.

[0060] 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 intestinal and / or oral cavity immune system, 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.

[0061] 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" means not including surgical, therapeutic, or diagnostic procedures for humans (i.e., medical procedures for humans), and specifically does not include methods of surgical, therapeutic, or diagnostic procedures for humans performed by a physician or a person under the supervision of a physician. [Example]

[0062] The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples.

[0063] Example 1: Improving the intestinal environment with WY peptide (1) In Example 1, the effect of WY peptide on improving the intestinal environment was evaluated using an animal model.

[0064] (1) Method A. Experimental Procedure C57BL / 6J male mice (Charles River Japan, hereafter referred to simply as "the mouse") were used as the animal model. According to the information from Charles River Japan (https: / / www.crj.co.jp / cms / cmsrs / img / usr / top / B6-Aged.pdf), the mouse was developed by the Jackson Laboratory and is 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 (mature mice), 10-14 months of age in humans (middle-aged mice), and 56-69 years of age in humans (elderly mice) (18-24 months of age).

[0065] In Example 1, after acclimation, 6-month-old mice were divided into a WY-fed group (15 mice) and a non-fed group (15 mice), with even weight distribution. 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 short-chain fatty acids in the cecal contents and fecal bacterial flora were analyzed 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.

[0066] B. Quantitation of short-chain fatty acids The amount of short-chain fatty acids was quantified by gas chromatography (GC) according to standard methods. Specifically, 100 mg of sample was accurately weighed into a bead tube, and nine volumes of 0.5% phosphoric acid solution were added and mixed. The mixture was then heat-treated at 85°C for 15 minutes. The sample was crushed, 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 standards.

[0067] [Table 1]

[0068] C. Bacterial flora analysis Sequencing For bacterial flora analysis, the 16S rDNA partial base sequence of the bacteria contained in the samples was determined using next-generation sequencing and amplicon analysis, and then searched against a microbial identification database. Specifically, the sample was pretreated and crudely extracted using the method described by 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. Next-generation sequencing was performed using 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 settings), and quality filtering was performed to ensure that 99% or more of the sequences met a quality value of 20 or higher.

[0069] 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).

[0070] D. Statistical analysis The percentage of reads corresponding to a specific taxonomic group relative to the total number of 16S rDNA sequences (total 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 between the two groups was considered to exist when the significance level was P<0.05%.

[0071] (2) Results A. Quantitation of short-chain fatty acids Seven short-chain fatty acids were analyzed: acetic acid, propionic acid, n-butyric acid, iso-butyric acid, n-valeric acid, iso-valeric acid, and n-caproic acid. However, n-caproic acid was excluded from the 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.

[0072] B. Bacterial flora analysis As shown in Figure 3, the WY intake group (intake period: 3 months) had a significantly higher percentage of total leads compared to the non-intake group. Actinobacteria The proportion of phyla increased significantly, Firmicutes As shown in Figure 4, the percentage of total leads in the WY intake group (intake period: 3 months) was significantly reduced compared to the non-intake group. Bifidobacterium The proportion of genera increased significantly, Clostridium Genus and Romboutsia Furthermore, as shown in Figure 5, the proportion of genera in the 3-month WY intake group was significantly lower than that in the non-intake group. Bifidacterium pseudolongum The proportion of patients with HIV increased significantly.

[0073] C. Summary These results indicate that administration of WY peptide alters the intestinal flora and increases the amount of short-chain fatty acids.

[0074] Example 2: Improving the 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.

[0075] (1) Method Male C57BL / 6J mice, 66 weeks old, were used and fed the test diet for 4.5 months. The mice were divided into a WY-fed group (12 mice) and a non-fed group (13 mice) with even weight distribution. Excluding mice that died naturally during rearing, 9 mice in the WY-fed group and 10 mice in the non-fed group were evaluated. Other conditions and methods were the same as those described in Example 1 (1).

[0076] (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 equivalent 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.

[0077] 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.

[0078] (1) Method A. Experimental Procedure After acclimation, male C57BL / 6J mice were used at 3 months of age and divided into a WY-fed group (10 mice) and a non-fed group (10 mice), 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, 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.

[0079] 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).

[0080] T cell analysis 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 by flow cytometry.

[0081] 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.

[0082] C. Statistical analysis Measurement values are shown as mean ± standard error. Evaluation between groups was performed using a t-test, and a significant difference between the two groups was considered to exist when the risk level was P<0.05%.

[0083] (2) Results As shown in Figure 7, the WY-intake group showed significantly higher levels of CD11b than the non-intake group. + CD11c + IA / IE and CD86 of myeloid dendritic cells were significantly increased, and PDL-1 and CD80 also showed an increased tendency. + CD4 + TNF-α positive CD4 in T cells + Although there was no change in the proportion of T cells, IFN-γ-positive CD4 + The proportion of T cells showed an increasing trend. This indicates that the immune system was activated sufficiently and appropriately. + CD4 + CD4 in T cells + CD25 + Foxp3 + The significant increase in the proportion of regulatory T cells suggests that excessive immune responses were suppressed. These results suggest that WY intake modulates intestinal immunity.

[0084] Example 4: Improving the intestinal environment with GTWY peptide In Example 4, the effect of the GTWY peptide on improving the intestinal environment was evaluated using C57BL / 6J male mice.

[0085] (1) Method Male C57BL / 6J mice aged 62 weeks 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 no GTWY, with the weights of each group being evenly balanced. Other conditions and methods were the same as those described in Example 1(1).

[0086] (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 GTWY administration increases the amount of short-chain fatty acids.

[0087] 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).

[0088] (1) Method After acclimation, male Crl:CD1 (ICR) mice were divided at 6.5 months of age into a whey hydrolysate intake group (7 mice) and a non-intake group (10 mice) with even weight distribution. 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 on a dry weight basis. 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).

[0089] (2) Results A. Quantitation of short-chain fatty acids As shown in Figure 9, the whey hydrolysate intake group had significantly higher levels of 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.

[0090] B. Bacterial flora analysis As shown in Figure 10, the whey hydrolysate intake group had a significantly higher percentage of total leads compared to the non-intake group. Bacteroidetes The proportion of phyla was significantly reduced, Actinobacteria As shown in Figure 11, the percentage of total leads in the whey hydrolysate intake group was significantly higher than in the non-intake group. Bacteroides The proportion of genera significantly decreased, Romboutsia The proportion of genera decreased, but not significantly, Bifidobacterium genus, Lactobacillus Furthermore, as shown in Figure 12, the whey hydrolysate intake group had a significantly higher percentage of total reads compared to the non-intake group. Clostridium scinders The proportion of Romboustia ilealis The proportion of Bifidacterium pseudolongum The proportion of patients with HIV increased, although not significantly.

[0091] C. Summary These results indicated that administration of whey hydrolysate altered the intestinal flora and increased the amount of short-chain fatty acids.

[0092] 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.

[0093] (1) Method 62-week-old C57BL / 6J male mice were used, and the test sample was whey hydrolysate. The test was conducted for 8.5 months. The mice were divided into a whey hydrolysate intake group (15 mice) and a non-intake group (12 mice) so that there was no imbalance in weight. Other conditions and methods were the same as those described in Example 1(1) and Example 5(1).

[0094] (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 considered middle-aged humans at the start of test diet intake and elderly humans at the end of intake. Therefore, whey hydrolysate intake was shown to increase short-chain fatty acid levels and improve the intestinal environment even in elderly subjects (56-69 years old).

[0095] Example 7: Determination of the content of tetrapeptide GTWY and dipeptide WY in whey hydrolysates (1) Preparation of analytical samples 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 obtained by treating whey protein with an enzyme preparation containing a protease, then removing undigested material through membrane treatment, and then drying. As described below, it contains the tetrapeptide GTWY and dipeptide WY.

[0096] (2)Analysis 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 using a calibration curve method with AQUA Peptide (Sigma Aldrich) as the standard sample.

[0097] <Analysis conditions> Mass spectrometer: 4000Q TRAP (AB Sciex) HPLC equipment: Agilent 1200 Series (Agilent Technologies) Column: TSK gel ODS-100V 3 μm 2.0 mm ID x 150 mm (Tosoh Corporation) Column temperature: 70℃ Mobile phase A: 0.1% formic acid in water Mobile phase B: 0.1% formic acid in acetonitrile Gradient conditions: The gradient conditions shown in Table 4 were applied.

[0098] [Table 2]

[0099] Flow rate: 0.2mL / min Sample injection volume: 2 μL Ionization method: ESI (positive ion detection mode) Curtain gas: 40psi Nebulizer gas: 50 psi Drying gas: 80 psi Drying gas temperature: 600℃ Collision gas: Nitrogen Ionization voltage: 5000V

[0100] <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

[0101] <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

[0102] (3)Analysis results It was confirmed that 1 g of whey hydrolysate (HW-3) contained 1.62 mg of tetrapeptide GTWY and 0.60 mg of dipeptide WY.

[0103] Example 8: Verification test of the effects of whey hydrolysate on mood state, health-related QOL, salivary immune index, and intestinal environment improvement in humans In Example 8, a clinical trial was conducted on humans to examine the effects of whey hydrolysate (Megmilk Snow Brand, HW-3) on mood state, health-related QOL (Quality of Life), salivary immune index, and intestinal environment improvement.

[0104] (1) Overview of the test This study was a placebo-controlled, randomized, double-blind, parallel-group comparative study. The study period was 6 weeks, during which participants ingested either 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 "tablet containing whey hydrolysate" as the test food and a "tablet not containing whey hydrolysate" as the control food, and the effects of whey hydrolysate on mood state, health-related QOL, salivary immune indicators, and intestinal environment were examined.

[0105] (2) Subjects Subjects who had low psychological health in a pre-test and were judged healthy by a doctor were randomly assigned to a test food group (30 people) and a control food group (30 people). The subjects analyzed were 28 people (19 men, 9 women) in the test food group and 28 people (20 men, 8 women) in the control food group, and the ages of the subjects analyzed (mean ± standard deviation) were 54.3 ± 4.8 years in the test food group and 53.6 ± 5.5 years in the control food group. During the test period, subjects were instructed to continue living the same lifestyle as before the test period.

[0106] (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.

[0107] A. Preparation of test food The test food was whey hydrolyzate (HW-3, Megmilk Snow Brand) mixed with excipients and binders, kneaded, and then compressed to produce whey hydrolyzate-containing tablets (300 mg / tablet). Each tablet contained 168 mg of whey hydrolyzate. The control food was a whey hydrolyzate-free tablet containing 168 mg of maltodextrin instead of whey hydrolyzate.

[0108] 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 sample. The amounts of tetrapeptide GTWY and dipeptide WY in the measurement sample were measured using the analytical method described in Example 7(2).

[0109] C. Analysis results of test food 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.

[0110] (4) Measurement A. Measurement items The measurement items were as follows: (i) Mood states 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 quality of life SF-36v2 Japanese Acute Version (sometimes referred to as "SF-36" in this specification) (iii) Salivary immunity index Salivary immunoglobulin A level (sometimes referred to as "IgA" in this specification) (iv) Intestinal environment Fecal short-chain fatty acids (acetic acid, propionic acid, n-butyric acid, iso-butyric acid, n-valeric acid, iso-valeric acid, n-caproic acid)

[0111] B. Measurement period (i) Mood states The STAI, PSS, and motivation scores were measured once before the start of test food intake (week 0) and once when the subjects visited the clinic six weeks after starting test food intake (a total of two times). (ii) Health-related quality of life The SF-36 was administered at the subjects' homes once each (4 times in total) before starting the test food intake (week 0), and once each at weeks 2, 4, and 6 after starting the test food intake. (iii) Salivary immunity index Saliva samples were collected twice in total, once before the start of test food intake (week 0) and once when the subjects visited the hospital six weeks after starting test food intake, and these were designated as week 0 and week 6 samples. After collection, the supernatant was centrifuged and stored frozen, and after the week 6 sample was collected, the week 0 and week 6 samples were measured simultaneously. (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, to serve as samples at week 0 and week 6 of intake. Fecal samples were collected in a refrigerated state and stored frozen, and after the week 6 sample was collected, the week 0 and week 6 samples were measured simultaneously.

[0112] C. Measurement method (i) Mood states Upon arrival, subjects completed the STAI, PSS, and motivation score questionnaires. 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 subjective stress (Reference: "Reliability and Validity of the Japanese Version of the Perceived Stress Scale," Health Psychology Research, Vol. 19 (2006) No. 2, Katsunori Washimi). The motivation score is a 14-item questionnaire used to assess apathy (a state of decreased motivation). The higher the score, the more depressed the state of motivation (Reference: Handbook of Mental and Psychological Function Assessment, pp. 462-463).

[0113] (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 functioning, role-functioning (physical), bodily pain, general health, vitality, social functioning, role-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).

[0114] (iii) Salivary immunity index Upon arrival at the clinic, subjects gargled with water and rested in a seated position for 15 minutes before saliva collection. Saliva collection was performed using a 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, after which 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 a Secretory Immunoglobulin A Salivary Immunoassay Kit (Salimetrics) according to the protocol included with the kit.

[0115] (iv) Intestinal environment The short-chain fatty acids in the fecal samples taken at week 0 and week 6 of ingestion were quantified using gas chromatography in the same manner as in Example 1(1).

[0116] (5) Evaluation and analysis A. Mood state The STAI, PSS, and motivation scores were measured at week 0 and week 6, and the change from week 0 to week 6 was calculated. Motivation scores were stratified by gender, and evaluation was conducted on women. The measured values and changes in each evaluation item at week 6 were compared between the test food group and the control food group using the Mann-Whitney U test. The measured values at week 0 and week 6 were also compared between the test food group and the control food group using the Wilcoxon signed-rank test.

[0117] B. Health-related QOL The changes in SF-36 scores from week 0 to week 2, week 4, and week 6 in the test and control food groups were compared using a two-sample t-test. Additionally, the actual measured values at week 2, week 4, and week 6 in the test and control food groups were compared using a two-sample t-test. Additionally, 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 using a one-sample t-test.

[0118] C. Saliva immune index After measuring salivary IgA levels at week 0 and week 6, 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. The measured values at week 0 and week 6 for the test food group and the control food group were also compared using a one-sample t-test.

[0119] 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 and control food groups were evaluated using a two-sample t-test. Additionally, the measured values at week 0 and week 6 for the test and control food groups were compared using a one-sample t-test. For subjects whose short-chain fatty acid levels of iso-butyric acid, n-valeric acid, iso-valeric acid, and n-caproic acid were below the lower limit of quantitation (0.3 μmol / g) at either week 0 or week 6, the corresponding items were recorded as missing values.

[0120] (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). These results indicated that whey hydrolysate improves trait anxiety (ease of anxiety, long-term anxiety).

[0121] [Table 3]

[0122] The results of the PSS are shown in Table 4. The change in the PSS score in the test food group was statistically significant compared to the control food group (p=0.043). These results indicated that whey hydrolysate improved subjective stress.

[0123] [Table 4]

[0124] 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 control food group at 6 weeks of intake compared to week 0 (p=0.796). Furthermore, the change in motivation scores for women in the test food group was statistically significantly lower (improved) compared to the control food group (p=0.047). These results demonstrate that whey hydrolysate improves motivation, with a particularly pronounced improvement in women.

[0125] [Table 5]

[0126] [Table 6]

[0127] 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 week 6 increased (improved) statistically significantly compared to week 0 (p = 0.043). On the other hand, no statistically significant change was observed for the control food group's score (p = 0.674). 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 = 0.060). For "vitality," the test food group's score at week 6 increased (improved) significantly compared to week 0 (p = 0.011). On the other hand, no statistically significant change was observed for the control food group's score (p = 0.194). Furthermore, the test food group showed a statistically significant increase (improvement) at week 6 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 change was 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 change was 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 significant (p=0.039) compared to the control food group. These results indicated that whey hydrolysate improves health-related QOL, including "physical pain," "vitality," "mental health," and "spiritual health."

[0128] [Table 7]

[0129] C. Saliva immune index 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 indicate that whey hydrolysate improves salivary immune indicators.

[0130] [Table 8]

[0131] D. Intestinal environment The results of measuring 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 week 6 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 week 6 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 showed a tendency 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.

[0132] [Table 9]

[0133] (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 lack of motivation, improve health-related quality of life such as vitality or mental health, improve immune status as measured by IgA, and increase the amount of short-chain fatty acids in feces, thereby improving the intestinal environment.

Claims

1. 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.

2. 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.

3. 3. The composition according to claim 1, wherein the content of GTWY in the whey protein enzymatic hydrolysate (converted to solid content) is 0.5 to 5 mg / g.

4. The composition according to any one of claims 1 to 3, wherein the content of whey protein in the enzymatic hydrolysate (converted to solid content) is 0.05 to 2 mg / g.

5. The composition according to any one of claims 1 to 4, wherein the whey protein enzymatic hydrolysate is ingested by a human in an amount of 1 to 50,000 mg (in terms of solid content) per day.

6. 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.

7. 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.

8. 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.

9. 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.

10. The composition according to any one of claims 1 to 9, which is a food composition.

11. The composition according to any one of claims 1 to 10, which is in a unit package form per serving.

12. The composition according to any one of claims 1 to 11, for ingestion by middle-aged and elderly people.

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