JP7616528B2 - Composition for improving cognitive function - Google Patents

Description

The present invention relates to a composition for use in maintaining, enhancing or improving cognitive function.

Maintaining, enhancing and improving cognitive function is required by a wide range of generations, from young people to the elderly. Maintaining, enhancing and improving memory and learning ability is important not only for students and working people studying for exams and qualification examinations, but also for daily work and life. Furthermore, for the elderly, a decline in memory and concentration affects the quality of life, so there is a demand to prevent the decline in cognitive function and to maintain, enhance and improve it.

Peptides with specific amino acid sequences (Patent Document 1) and hop oxidation products (Non-Patent Document 1) are known to have cognitive function improving effects.

International Publication No. 2015/194564

T. Ayabe et al., Scientific Reports volume 8, Article number: 15372 (2018)

The present invention aims to provide novel compositions and agents that maintain, enhance or improve cognitive function.

The present inventors have now found in an animal model study that administering a whey protein enzymatic hydrolysate in combination with a hop oxidation reaction product has a significant effect in improving cognitive function compared to administering either of them alone. The present invention is based on this finding.

According to the present invention, the following inventions are provided.
[1] A composition for maintaining, enhancing and/or improving cognitive function (hereinafter sometimes referred to as "the composition of the present invention"), and an agent for maintaining, enhancing and improving cognitive function (hereinafter sometimes referred to as "the agent of the present invention"), which comprises a hop oxidation reaction product and an enzymatic hydrolysate of whey protein as active ingredients.
[2] The composition and agent described in [1] above, wherein the cognitive function is a memory function.
[3] The composition and agent described in [2] above, wherein the memory function is a spatial memory function and/or a working memory function.
[4] The composition or agent according to any one of [1] to [3] above, wherein the hop oxidation reaction product is an aqueous medium extract.
[5] The composition or agent according to any one of the above [1] to [4], wherein the hop oxidation reaction product is a water extract.
[6] The composition and agent according to any one of the above [1] to [5], wherein the hop oxidation reaction products include tricyclooxyisohumulone, tricyclooxyisocohumulone, scorpiofumulinol and scorpiocohumulinol.
[7] The composition or agent according to any one of [1] to [6] above, wherein the concentration of bitter acid in the hop oxidation reaction product is 1 to 20 mass%.
[8] The composition and agent according to any one of [1] to [7] above, wherein the whey protein enzymatic hydrolysate comprises a peptide having the amino acid sequence of GTWY (SEQ ID NO: 1) and a peptide having the amino acid sequence of WY.
[9] The composition and agent described in [8] above, wherein the content of GTWY in the whey protein enzymatic hydrolysate (converted into solid content) is 0.01 to 1 mass% based on the total amount, and the content of WY in the whey protein enzymatic hydrolysate (converted into solid content) is 0.005 to 0.5 mass% based on the total amount.
[10] The composition and agent according to any one of [1] to [9] above, wherein the ratio of the hop oxidation reaction product to the whey protein enzymatic hydrolysate in the composition (mass ratio converted into solid content) is 0.1 to 2.
[11] The composition according to any one of [1] to [10] above, which is a food composition.
[12] The composition and agent according to any one of [1] to [11] above, which is in a unit package form per meal.
[13] The composition or agent according to any one of [1] to [12] above, wherein the hop oxidation reaction product is ingested by a human in an amount of 10 to 5,000 mg (in terms of solid content) per meal.
[14] The composition and agent according to any one of [1] to [13] above, wherein the whey protein enzymatic hydrolysate is ingested by a human in an amount of 1 to 50,000 mg (converted into solid content) per meal.
[15] The composition or agent according to the above [13] or [14], which contains 0.01 to 10 mg of GTWY and 1 to 100 mg of a bitter acid.

The active ingredients of the present invention, hop oxidation reaction products and whey protein enzymatic hydrolysates, are both derived from food materials that humans have been ingesting for many years. Therefore, the compositions and preparations of the present invention are advantageous in that they can be used as functional foods that improve cognitive function and are safe for mammals, including humans.

FIG. 1 shows the results of HPLC analysis (HPLC chromatogram) of the hop oxidation reaction products in Reference Example 1. 2 is a diagram showing waveform processing during area calculation of bitter acids derived from hop oxidation reaction products in Reference Example 2. In the figure, A shows a caffeine analysis chromatogram, B shows an analysis chromatogram for confirming elution position (a: trans-isocohumulone, b: trans-isohumulone, c: trans-isoadhumulone), and C shows a sample analysis chromatogram. In the sample analysis chromatogram of C, after background correction, a baseline is drawn at an intensity of 0 between caffeine and trans-isocohumulone to define the base of the peak, and the area of that section (gray portion) is calculated. FIG. 3 is a graph showing the total number of entries into the arms of mice orally administered with each sample in Example 3. 4 is a graph showing the spontaneous alternation behavior fluctuation rate of mice orally administered with each sample in Example 3. * indicates p<0.05, ** indicates p<0.01 (Tukey-Kramer test).

Description of the Invention

<Compositions and preparations>
According to the present invention, there is provided a composition and a preparation comprising a hop oxidation reaction product and a whey protein enzymatic hydrolysate. The hop oxidation reaction product and the whey protein hydrolysate (hereinafter, simply referred to as "whey hydrolysate") will be described below.

In the present invention, the hop oxidation reaction product refers to a product obtained by oxidizing hops or processed products thereof (hop pellets, hop extract, etc.). The hop oxidation reaction product provided by the present invention can be obtained, for example, by contacting hops with oxygen in the air to oxidize them.

The hop oxidation reaction product can be produced by subjecting hops to an oxidation treatment, for example, according to the method described in International Publication No. 2012/081675. The oxidation treatment is preferably carried out by heating hops in air. The heating temperature is not particularly limited, but a preferred upper limit is 100°C, and a more preferred upper limit is 80°C. A heating temperature of 100°C or lower is advantageous in that oxidation proceeds preferentially over isomerization. A preferred lower limit for the heating temperature is 60°C. A heating temperature of 60°C or higher is advantageous in that the oxidation reaction proceeds efficiently. The reaction period is also not particularly limited, and can be appropriately determined depending on the variety of hops and the reaction temperature. For example, 48 to 120 hours at 60°C and 8 to 24 hours at 80°C are preferred. The form of hops when subjected to the oxidation reaction is not particularly limited as long as it can come into contact with oxygen in the air, but the reaction time can be shortened by making the hops into a powder form.

In the present invention, hops may be in any form as long as they contain lupulin, and may be harvested and dried before drying, harvested and dried, compressed, crushed, or processed into pellets, but are preferably in the form of hop pellets. Hop pellets may be commercially available products, such as hop cones compressed into pellets (Type 90 pellets), pellets in which the lupulin portion is selectively concentrated (Type 45 pellets), or isomerized hop pellets (e.g., Isomerized Pellets (HopSteiner)).

A hop extract oxidation reaction product produced by subjecting a hop extract to an oxidation treatment may be provided as the hop oxidation reaction product of the present invention. The hop extract oxidation reaction product can be produced by subjecting a hop extract to an oxidation treatment, for example, according to the method described in WO 2012/081675.

The content (solid content equivalent) of hop oxidation reaction products in the compositions and preparations of the present invention can be arbitrarily determined depending on the purpose, application, form, dosage form, etc., and the present invention is not limited thereto. For example, in the case of a liquid composition, the content can be 0.1 to 50,000 mg/100 mL (preferably 1 to 5,000 mg/100 mL, more preferably 10 to 600 mg/100 mL) relative to the total amount, and in the case of a solid composition, the content can be 1 to 99% by mass (preferably 10 to 80% by mass, more preferably 40 to 75% by mass) relative to the total amount.

Hops contain acidic resin components such as alpha acids (humulones), beta acids (lupulones), and iso-alpha acids (isohumulones). In the present invention, "humulones" is used to include humulone, adhumulone, cohumulone, posthumulone, and prehumulone. In the present invention, "lupulones" is used to include lupulone, adlupulone, colupulone, postlupulone, and prelupulone. Furthermore, in the present invention, "isohumulones" is used to include isohumulone, isoadhumulone, isocohumulone, isoposthumulone, isoprehumulone, Rho-isohumulone, Rho-isoadhumulone, Rho-isocohumulone, Rho-isoposthumulone, Rho-isoprehumulone, tetrahydroisohumulone, tetrahydroisoadhumulone, tetrahydroisocohumulone, tetrahydroisoprehumulone, tetrahydroisoposthumulone, hexahydroisohumulone, hexahydroisoadhumulone, hexahydroisocohumulone, hexahydroisoposthumulone, and hexahydroisoprehumulone. Isohumulones exist as cis and trans stereoisomers, and unless otherwise specified, the term is used to include both.

By subjecting hops to oxidation treatment, the content of alpha acids, beta acids, and iso-alpha acids is reduced, and the content of other components is increased. Examples of such hop oxidation reaction products include those in which, when HPLC analysis is performed in the same manner as in Example 1, the ratio of the peak area of alpha acids, beta acids, and iso-alpha acids to the total HPLC peak area is 20% or less, preferably 10% or less, of the oxidation reaction product.

Components other than α acids, β acids, and iso-α acids contained in the oxidation reaction product of the present invention can be easily detected by known analytical means such as HPLC. For example, a hop oxidation reaction product prepared by a procedure similar to that of Example 1 of WO 2012/081675 contains components other than α acids, β acids, and iso-α acids, and the peaks corresponding to these components (also referred to as "S-fraction (S-Fr)" in this specification) can exhibit physiological activity. The peaks in the range indicated by the arrow in Figure 1A of Example 1 of WO 2012/081675 (excluding the peaks of α acids and β acids) correspond to the S-fraction.

HPLC analysis of hop oxidation products prepared under the same conditions as those described in WO 2012/081675 and the results (HPLC chromatogram) are shown in Reference Example 1 and FIG. 1 of the present specification. The peaks in the range indicated by the arrows A1 and A2 (excluding the α-acid and β-acid peaks) correspond to the S-fraction. In FIG. 1, the peak area value in the range fractionated by the arrows A1 and A2 is the sum of the peak area value of A1 from 3 to 25 minutes of retention time and the peak area value of A2 from 32 to 39 minutes of retention time (excluding the α-acid and β-acid peaks). In this case, "up to 25 minutes of retention time" in A1 means up to the appearance of the peak identified as trans-isocohumulone. In addition, characteristic peaks were observed in the range fractionated by A1 in FIG. 1 at retention times of around 9.7 minutes, 11.8 minutes, and 12.3 minutes. Additionally, a shoulder peak was observed in the range fractionated by A2 in Figure 1, with its start point at approximately 32 minutes retention time, its top point (excluding the α acid and β acid peaks) at approximately 35 to 36 minutes retention time, and its end point at approximately 39 minutes retention time.

The hop oxidation reaction product preferably contains α-acid oxides, iso-α-acid oxides, and β-acid oxides, and examples of such oxides include "tricyclooxyisohumulones." Here, "tricyclooxyisohumulones" include tricyclooxyisocohumulone A (TCOIcoH A: see formula 1 below, IUPAC name: (3aS,5aS,7S,8aS)-3,3a-dihydroxy-7-(1-hydroxy-1-methylethyl)-6,6-dimethyl-2-(2-methylpropanoyl)-5a,6,7,8-tetrahydro-3aH,5H-cyclopenta[c]pentalene-1,4-dione), tricyclooxyisohumulone A (TCOIH A: See formula 2 below, IUPAC name: (3aS,5aS,7S,8aS)-3,3a-dihydroxy-7-(1-hydroxy-1-methylethyl)-6,6-dimethyl-2-(3-methylbutyryl)-5a,6,7,8-tetrahydro-3aH,5H-cyclopenta[c]pentalene-1,4-dione, and tricyclooxyisoadhumulone A (TCOIadH A: See formula 3 below, IUPAC name: (3aS,5aS,7S,8aS)-3,3a-dihydroxy-7-(1-hydroxy-1-methylethyl)-6,6-dimethyl-2-(2-methylbutanoyl)-5a,6,7,8-tetrahydro-3aH,5H-cyclopenta[c]pentalene-1,4-dione). In the present specification, TCOIcoH A, TCOIH A, and TCOIadH A may be collectively referred to as TCOIHsA. The content of TCOIHsA is measured by the method described in Example 1 below.

Examples of oxidation products other than "tricyclooxyisohumulones" contained in hop oxidation products (preferably S-fraction) include scorpiofumulinol A and scorpiocohumulinol A.

In the present invention, the hop oxidation reaction product may be provided as an aqueous medium extract. The aqueous medium is not particularly limited as long as it is one that is normally used in food production, but is preferably water or ethanol, and more preferably water (an extract using water is specifically referred to as a water extract). The extraction temperature is not particularly limited, but is preferably 60°C or lower, and taking into account the extraction efficiency, 50 to 60°C is more preferable.

The hop oxidation reaction product (preferably an aqueous medium extract of a hop oxidation reaction product) used in the present invention can be characterized by the ratio (on a dry mass basis) of the total amount of tricyclooxyisohumulone A and tricyclooxyisocohumulone A to the total amount of scorpiofumulinol A and scorpiocohumulinol A; for example, an aqueous medium extract of a hop oxidation reaction product in which this ratio is in the range of 1 to 30, preferably an aqueous medium extract of a hop oxidation reaction product in which this ratio is in the range of 2 to 20, can be used.

The hop oxidation reaction product used in the present invention can be characterized by the TCOIHsA content (converted to dry mass). For example, a hop oxidation reaction product (preferably an aqueous medium extract of a hop oxidation reaction product) having a TCOIHsA content of 5 to 15 mass%, preferably 5 to 12 mass%, can be used. The content of TCOIHsA can be measured by the same method as that for mature hop bitter acids described in Biosci., Biotechnol., Biochem., 2015 (79):1684-1694.

The Brix value of the aqueous medium extract of the hop oxidation reaction product is not particularly limited, but is, for example, 3 or less, and preferably 1.5 to 3. The aqueous medium extract of the hop oxidation reaction product may have insoluble components removed by decantation or filter paper, etc. The aqueous medium extract of the hop oxidation reaction product may also be treated with activated carbon.

（上記式中、Ｒ、Ｒ_１、Ｒ_２、Ｒ_３およびＲ_４はそれぞれ互いに同一または異なっていてもよく、Ｒは水素原子または炭素数１～１０のアルキルを表す。Ｒ_１、Ｒ_２、Ｒ_３およびＲ_４はそれぞれ任意の置換基を表し、それぞれが独立していても、互いが結合していてもよい。） Hop oxidation reaction products can also be characterized by the total amount of a group of compounds (sometimes referred to as "bitter acids" in this specification) having a β-tricarbonyl structure represented by formula 4 as a common structure. The bitter acid content is measured by the method described in Reference Example 2. The bitter acid content in the water extract of the hop oxidation reaction product is 0.1 to 90% by mass (preferably 0.5 to 50% by mass, more preferably 1 to 20% by mass).

(In the above formula, R, R ₁ , R ₂ , R ₃ and R ₄ may be the same or different from each other, and R represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. R ₁ , R ₂ , R ₃ and R ₄ each represent any substituent, and each may be independent or may be bonded to each other.)

Whey Protein Enzymatic Decomposition Product In the present invention, "whey" is also called milk serum, milk plasma or whey, and refers to an aqueous solution obtained by removing milk fat, casein and the like from milk. Whey is composed of β-lactoglobulin, α-lactalbumin, serum albumin, immunoglobulin and the like. The whey used in the present invention may be derived from any animal or plant, but it is preferable to use whey derived from cow's milk. The whey decomposition product, which is an active ingredient of the composition and preparation of the present invention, is not limited as long as it is an enzymatic decomposition product of whey.

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

The whey hydrolyzate of the present invention can preferably contain 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 "the peptide of the present invention"). Here, "a peptide having an amino acid sequence" means a peptide whose sequence is specified by the amino acid sequence.

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

The content of the peptide of the present invention is measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) according to the method described in Example 2 below. In this case, it goes without saying that a standard peptide with a purity suitable for LC-MS/MS measurement is used during the measurement, and for example, AQUA peptide manufactured by SIGMA ALDRICH can be used.

The method for producing the whey hydrolysate of the present invention (particularly the whey hydrolysate containing the peptide of the present invention) is known, and can be produced, for example, according to the description in WO 2017/086303. Alternatively, commercially available whey hydrolysates (e.g., HW-3 (Snow Brand Megmilk), HWP-205 (Tatua), Thermax690 (Glanbia), Protherma (Glanbia)) may be used as the whey hydrolysate.

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

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

Whey may be subjected to the enzyme reaction as is, or after being concentrated or diluted, and the pH may be adjusted as necessary. When the raw protein is a solid such as a powder, it may be dissolved in any aqueous solvent as long as the enzyme reaction proceeds, but considering its use as a food, it is preferable to dissolve it in water or a food additive grade buffer. It is preferable to use a buffer so that the pH of the reaction solution does not change due to the amino acids generated by the enzyme reaction. The type of buffer is arbitrary and may be determined taking into consideration the subsequent use, flavor, taste, and mineral content, but a composition that can maintain the pH of the reaction solution at 4 to 9, preferably 5 to 8, and more preferably 7 to 8 is preferable. Potassium phosphate buffer is most preferable. The concentration of the buffer is arbitrary as long as it is within a range in which a buffering effect can be obtained, but considering the flavor, taste, and mineral content, it can be 0.01 to 0.5 M, preferably 0.05 to 0.2 M, and more preferably about 0.1 M.

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

In the present invention, commercially available enzyme preparations can be used, and are available from, for example, Amano Enzyme, Shin Nippon Chemical Industry, DSM, Danisco, Novozymes, HBI, etc. The amount of enzyme preparation added is arbitrary, but taking into consideration an appropriate hydrolysis reaction rate and cost, 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%.

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

The enzyme reaction can also be carried out while increasing the temperature. For example, the reaction can be carried out while increasing the temperature from 30°C to 75°C over 4 to 10 hours. Preferably, the reaction can be carried out while increasing the temperature from 35°C to 75°C over 5 to 8 hours, and more preferably, the reaction can be carried out while increasing the temperature from 35°C to 75°C over 6 to 8 hours. The temperature can be increased at any speed, but it is preferable to hold the temperature between 45°C and 55°C for a long time (5 to 7 hours), then quickly increase the temperature to 60°C, and then hold the temperature between 60°C and 75°C for a long time (for example, 1 to 3 hours). The most preferred method is to add the enzyme at 50°C, hold for 5 to 7 hours, then raise the temperature at any speed, and hold at the target temperature of 60 to 65°C or 65 to 75°C for 1 to 3 hours.

From the viewpoint of reaction efficiency, it is preferable to stir the reaction solution during the reaction. The faster the stirring speed, the better so that the substrate comes into contact with the enzyme. However, if the stirring speed is too fast, the reaction solution may splash, so the stirring speed can be set to, for example, 100 to 500 rpm, preferably 200 to 400 rpm, and more preferably about 250 rpm.

Once the desired peptide is obtained, the reaction solution is preferably subjected to a step of terminating the enzyme reaction. In the enzyme reaction terminating step, a method of increasing the temperature of the reaction solution, changing the chemical structure of the enzyme by adding a chelating agent, or removing the enzyme by membrane treatment can be used. A preferred method is inactivation treatment at high temperature. This method can be carried out by holding the reaction solution at 80-90°C for 5-30 minutes, preferably at 80-90°C for 20-30 minutes. In addition, if the temperature is increased in the concentration step described below, this can also be carried out as the concentration step.

The reaction liquid (milk protein hydrolysate) that has undergone the enzyme reaction process and the enzyme reaction stopping process may further be subjected to a sterilization process. Examples of the sterilization process include the membrane treatment process and high-temperature sterilization process described below. The heat sterilization process can also serve as the enzyme reaction stopping process, which is advantageous in terms of simplifying the manufacturing process.

The reaction liquid (milk protein hydrolysate) that has undergone the enzyme reaction step and the enzyme reaction stopping step may be further subjected to a purification step. An example of the purification step is a membrane treatment step, and a preferred membrane treatment is ultrafiltration. The molecular weight cutoff for ultrafiltration 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 it is not performed. In addition, the purification step can also serve as the enzyme reaction stopping step and the sterilization step, which is also advantageous in terms of simplifying the manufacturing process.

The reaction liquid (whey hydrolyzate) that has undergone the enzyme reaction step and the enzyme reaction stopping step may be further subjected to a concentration step from the viewpoint of storage and transportation. Any method may be selected for the concentration step, but vacuum concentration, freeze drying, spray drying, and concentration by membrane processing (e.g., a method using a reverse osmosis membrane) are preferred, and freeze drying and spray drying are more preferred. Spray drying is particularly preferred from the viewpoint of performing concentration in large quantities and efficiently.

The composition and preparation of the present invention may contain a specific ratio of hop oxidation reaction products to whey protein enzymatic hydrolysates. In order to better maintain, enhance, or improve cognitive function, the ratio of hop oxidation reaction products to whey protein enzymatic hydrolysates in the composition and preparation (mass ratio in terms of solid content) can be 0.1 to 2, and preferably 0.5 to 1.5.

When the composition and preparation of the present invention contain a hop oxidation reaction product, bitter acids can be used as an indicator, and 0.1 to 1000 mg (preferably 0.5 to 500 mg, more preferably 1 to 100 mg) of bitter acids can be contained per meal. When the composition and preparation of the present invention contain an enzymatic hydrolyzate of whey protein, the peptide of the present invention can be used as an indicator, and 0.001 to 100 mg (preferably 0.005 to 50 mg, more preferably 0.01 to 10 mg) of GTWY and 0.0001 to 30 mg (preferably 0.005 to 15 mg, more preferably 0.001 to 3 mg) of WY can be contained per meal.

<Applications>
As described in the Examples below, hop oxidation reaction products and whey hydrolysates (hereinafter, these may be referred to as "active ingredients of the present invention") have the effect of maintaining, enhancing and/or improving cognitive functions including memory function. Therefore, the combination of hop oxidation reaction products and whey hydrolysates can be used as an active ingredient for maintaining, enhancing and/or improving cognitive function, and can be used as an active ingredient in a method for maintaining, improving and improving cognitive function. The combination of hop oxidation reaction products and whey hydrolysates can also be used as an active ingredient in an agent for maintaining, improving and improving cognitive function. In the present invention, "cognitive function" is a concept that encompasses all information processing processes from perception to judgment, and various higher brain functions such as memory function (including memory acquisition function, memory consolidation function, memory retrieval function and memory reproduction function), attention function and judgment function are involved in this process. That is, in the present invention, "cognitive function" is used in a sense that includes these higher brain functions.

In the present invention, "maintaining cognitive function" includes, for example, suppressing or preventing a decline in cognitive function. Furthermore, "improving cognitive function" includes, for example, raising cognitive function from its current level. Furthermore, "improving cognitive function" includes, for example, restoring cognitive function that has declined or symptoms that show signs of decline. The terms "maintaining, enhancing, and/or improving cognitive function" are used to mean including enhancing cognitive function and suppressing a decline in cognitive function.

In the present invention, the maintenance, enhancement, and/or improvement of cognitive function can be evaluated using higher brain functions involved in cognitive function as indicators. Model animal tests for evaluating higher brain functions involved in cognitive function are known, and for example, the maintenance, enhancement, and/or improvement of cognitive function can be evaluated by a Y-maze test (see Example 3 below) for evaluating short-term memory and spatial memory/working memory.

In the present invention, "memory function" refers to functions including spatial cognitive function, working memory function, episodic memory function, visual memory function, and learning function, and is used to mean medium- to long-term memory function. Working memory includes linguistic working memory, task working memory, and spatial working memory. In the present invention, the memory function is preferably long-term memory function.

In the present invention, "maintaining memory function" includes, for example, suppressing or preventing a decline in memory function. Furthermore, "improving memory function" includes, for example, increasing memory function from its current level, promoting the consolidation of medium- to long-term memories, and ultimately promoting brain development. Furthermore, "improving memory function" includes, for example, restoring memory function that has declined or symptoms that show signs of decline. Examples of maintaining, enhancing, and/or improving memory function include enhancing memory function and suppressing a decline in memory function.

In the present invention, the "spatial memory function" refers to the function of memorizing spatial information required for searching for an object, heading to a destination, returning home, etc.

In the present invention, "maintaining spatial memory function" includes, for example, inhibiting or preventing a decline in spatial memory function. Furthermore, "improving spatial memory function" includes, for example, enhancing spatial memory function from its current state. Furthermore, "improving spatial memory function" includes, for example, restoring a spatial memory function that has declined or a symptom that shows signs of decline. Examples of maintaining, enhancing, and/or improving spatial memory function include enhancing spatial memory function and inhibiting a decline in spatial memory function.

In the present invention, "working memory function" refers to the function of working while temporarily retaining information.

In the present invention, "maintaining working memory function" includes, for example, suppressing or preventing a decline in working memory function. Furthermore, "improving working memory function" includes, for example, increasing working memory function from its current level. Furthermore, "improving working memory function" includes, for example, restoring a working memory function that has declined or symptoms that show signs of decline. Examples of maintaining, enhancing, and/or improving working memory function include enhancing working memory function and suppressing a decline in working memory function.

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

The active ingredient of the present invention can be orally administered to humans and non-human animals. Oral preparations include granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, emulsions, and suspensions. These preparations can be formulated using pharma- ceutical acceptable carriers by methods commonly used in the art. Pharmaceutically acceptable carriers include excipients, binders, diluents, additives, flavorings, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, etc.

The active ingredient of the present invention can be orally ingested by humans and non-human animals, and a typical form of ingestion is food. When the active ingredient of the present invention is provided as food, it can be provided by being contained in food. The food provided in this way is a food containing an effective amount of the active ingredient of the present invention. In this specification, "containing an effective amount" of the active ingredient of the present invention refers to a content such that the active ingredient of the present invention is ingested in the range described below when the amount normally consumed in each food is ingested. In addition, "food" is used to mean health food, functional food, nutritional supplement, health functional food (e.g., specific health food, nutritional functional food, functional food), special purpose food (e.g., food for infants, food for pregnant women, food for sick people) and supplements. It goes without saying that when the active ingredient of the present invention is ingested by animals other than humans, the food referred to in the present invention is used as feed.

The active ingredient of the present invention has the effect of improving cognitive function as described above, and therefore can be included in foods that are consumed daily, or can be provided as a supplement. That is, the composition and agent of the present invention can be provided in the form of a food. In this case, the composition and agent of the present invention can be provided in a unit package form in which the amount to be taken per meal is predetermined. Examples of the unit package form per meal include a form in which a certain amount is specified in a pack, package, can, bottle, etc. In order to better exert the various effects of the composition and agent of the present invention, the intake amount per meal can be determined according to the daily intake amount of the active ingredient of the present invention, which will be described later. The food of the present invention may be provided with an explanatory matter regarding the intake amount displayed on the package, or together with a document or the like in which the explanatory matter is written.

The predetermined intake amount per meal in the unit package form may be the effective intake amount per day, or the effective intake amount per day divided into two or more portions (preferably two or three portions). Thus, 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 meal" in which the intake amount per meal is the effective intake amount per day.

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. In addition, examples of "supplements" include tablets produced by kneading the active ingredient of the present invention with excipients, binders, etc., and then compressing, 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 the unit package form per day, per week, or per month.

The foods provided by the present invention are not particularly limited as long as they contain the active ingredient of the present invention, but include, for example, non-alcoholic beverages such as soft drinks, carbonated drinks, drinks containing fruit juice, drinks containing vegetable juice, drinks containing fruit juice and vegetable juice, livestock milk such as cow's milk, soy milk, dairy drinks, drink-type yogurt, drink-type or stick-type jellies, coffee, cocoa, tea drinks, nutritional drinks, energy drinks, sports drinks, mineral water, near-water, and non-alcoholic beer-flavored drinks; carbohydrate-containing foods and beverages such as rice, noodles, bread, and pasta; cheeses, hard or soft yogurt, fresh cream made from livestock milk or other fats and oils, ice cream, etc. Examples of the mineral water include dairy products; Western confectionery such as cookies, cakes, and chocolates, Japanese confectionery such as manju and yokan, tablet confectionery (refreshing confectionery) such as ramune, candies, gums, cold and frozen desserts such as jellies and puddings, snacks, and other confectionery; alcoholic beverages such as whiskey, bourbon, spirits, liqueurs, wine, fruit wine, sake, Chinese wine, shochu, beer, non-alcoholic beer with an alcohol content of 1% or less, sparkling wine, other miscellaneous alcohol, and chuhai; processed foods such as processed foods using eggs, processed foods (including delicacies) made from seafood and meat (including liver and other organ meats), soups such as miso soup, seasonings such as miso, soy sauce, furikake, and other seasonings, and liquid foods such as thick liquid foods. Mineral water includes both sparkling and non-sparkling mineral water. The food provided by the present invention also includes both food manufacturing ingredients and food additives.

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

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

The intake amount of the active ingredient of the present invention can be determined depending on the recipient's sex, age, and body weight, symptoms, intake time, dosage form, intake route, and drugs to be combined, etc. The daily intake amount (converted into solid content) for an adult of a hop oxidation reaction product for the purpose of maintaining, enhancing, or improving cognitive function is, for example, 10 to 5000 mg (preferably 50 to 2000 mg, more preferably 100 to 1000 mg) as a water extract, which, when converted into bitter acid, is, for example, 0.1 to 1000 mg (preferably 1 to 100 mg, more preferably 10 to 60 mg). In addition, the daily intake (in terms of solids) of whey hydrolysates for adults for the purpose of maintaining, improving or improving cognitive function is, for example, 1 to 50,000 mg (preferably 10 to 10,000 mg, more preferably 100 to 5,000 mg), which is converted to GTWY, for example, 0.1 to 5,000 mg (preferably 1 to 1,000 mg, more preferably 10 to 500 mg), and converted to WY, for example, 0.01 to 1,000 mg (preferably 0.1 to 300 mg, more preferably 3 to 100 mg). The above-mentioned intake amount of the active ingredient of the present invention and the following intake timing and intake period are applicable to the case where the active ingredient of the present invention is used for both non-therapeutic and 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 dosage, timing, and duration of administration can be determined with reference to the descriptions for humans.

It is preferable to continue taking the active ingredient of the present invention for the period during which the maintenance, improvement or improvement of cognitive function is expected. The period of intake of the active ingredient of the present invention can be, for example, at least one day (preferably three days, more preferably five days) at the above daily dose, and can be one week or more, preferably one month or more, particularly when maintenance of cognitive function is expected. In addition, it can be at least one day when maintenance, improvement or improvement of memory function, in particular, of cognitive function is expected. The interval of intake of the active ingredient of the present invention can be once every three days, once every two days, or once a day at the above daily dose.

The active ingredient of the present invention can also be started to be taken before the date and time when the effect of maintaining, improving or improving cognitive function is expected. Examples of taking the active ingredient before the date and time when the effect of maintaining, improving or improving cognitive function is expected include before studying for an exam in which one hopes to improve cognitive function (particularly memory function), or before cognitive function begins to decline with age.

The compositions, preparations, and foods of the present invention may be labeled to the effect of maintaining, enhancing, or improving cognitive function. In this case, the labeling can be made easy for consumers to understand.

According to another aspect of the present invention, there is provided a combination of a hop oxidation reaction product and a whey protein enzymatic hydrolysate for use in maintaining, enhancing and/or improving cognitive function. An example of the combination of the present invention is a kit for use in maintaining, enhancing and/or improving cognitive function, comprising a hop oxidation reaction product and a whey protein enzymatic hydrolysate in separate forms (as independent entities). The combination of the present invention can be implemented according to the description of the composition, preparation and food of the present invention.

According to another aspect of the present invention, there is provided a composition for maintaining, enhancing and/or improving cognitive function, and an agent for maintaining, enhancing and improving cognitive function, comprising a hop oxidation reaction product, for use in combination with a whey protein enzymatic hydrolysate. According to another aspect of the present invention, there is provided a composition for maintaining, enhancing and/or improving cognitive function, and an agent for maintaining, enhancing and improving cognitive function, comprising a whey protein enzymatic hydrolysate, for use in combination with a hop oxidation reaction product. The above-mentioned compositions and agents can be used in accordance with the description of the compositions, agents and foods of the present invention.

According to another aspect of the present invention, there is provided a method for maintaining, enhancing or improving cognitive function, comprising ingesting or administering to a subject in need thereof a combination of a hop oxidation reaction product and a whey protein enzymatic hydrolysate or a composition containing the same. The method of the present invention can be carried out according to the description of the composition and preparation of the present invention.

According to yet another aspect of the present invention, there is provided use of a combination of a hop oxidation reaction product and a whey protein enzymatic hydrolysate or a composition containing the same for the manufacture of an agent for maintaining, improving or improving cognitive function, as an agent for maintaining, improving or improving cognitive function, or in a method for maintaining, improving or improving cognitive function of the present invention. The use of the present invention can be carried out in accordance with the description of the composition and preparation of the present invention.

According to yet another aspect of the present invention, there is provided a combination of a hop oxidation reaction product and a whey protein enzymatic hydrolysate for use in maintaining, enhancing or improving cognitive function. The combination of the hop oxidation reaction product and the whey protein enzymatic hydrolysate can be carried out according to the description of the composition and preparation of the present invention.

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

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Reference Example 1: Preparation of hop pellet oxidation reaction product Pellet-shaped Hallertau perle (HPE) hops were used. The hops were pulverized in a mill and heated at 80° C. for 24 hours. The product obtained was pretreated as follows and then subjected to HPLC analysis.

[Pretreatment of reactant analysis]
The collected product was added to ethanol to a concentration of 10% w/v, and extraction was carried out for 1 hour at 50° C. The resulting extract was diluted 10-fold with ethanol.
[HPLC configuration device]
Pump: LC-10ADvp x 3 (SHIMADZU)
Degasser: DGU-20A5 (SHIMADZU)
System controller: CBM-20A (SHIMADZU)
Autosampler: SIL-20ACHT (SHIMADZU)
Column oven: CTO-20AC (SHIMADZU)
Photodiode array detector: SPD-M20A (SHIMADZU)
Waveform analysis software: LCSolution (SHIMADZU)

[HPLC conditions]
Column: Alltima C18 2.1mm ID x100mm Particle size 3μm
Flow rate: 0.6mL/min Elution solvent A: Water/phosphoric acid, 1000/0.2, (v/v) + EDTA(free) 0.02%(w/v)
Elution solvent B: acetonitrile Elution solvent C: water Injection volume: 3 μL
Column temperature: 40°C
Detection wavelength: 270 nm (oxidation reaction products, iso-α acids, α acids, β acids)
Gradient conditions: Table 1

Under the above analytical conditions, the ratio (%) of the area values of the α-acid, β-acid, and iso-α-acid peaks to the total area value (mAU·min) of all peaks detected at a detection wavelength of 270 nm was calculated. In the waveform analysis, areas where solvent peaks and negative peaks due to injection shock occurred were excluded from the analysis. The HPLC chromatogram obtained during the analysis of the above product is shown in Figure 1.

Reference Example 2: Measurement of bitter acids (1) Measurement of bitter acids contained in hop oxidation reaction products Measurement is performed using a high performance liquid chromatograph. For details, follow the method published in the notification of functional food labeling by the Consumer Affairs Agency (https://www.fld.caa.go.jp/caaks/cssc04/bunsekihoho?bunsekihohoFile=E522%255CE522_bunsekihoho.pdf).

(i) Preparation of the measurement sample: The sample is water with an appropriate amount of hop oxidation products dissolved in it. Dichloromethane and 1N hydrochloric acid are added to the sample, and the mixture is stirred and centrifuged. The bottom layer is collected and evaporated to dryness at room temperature under a nitrogen stream. This is then redissolved in ethanol and centrifuged to obtain the measurement sample.

(ii) Conditions for high-performance liquid chromatography Column: Alltima C18 2.1 mm ID x 100 mm Particle size 3 μm
Mobile phase A: water/phosphoric acid 1000/0.2 (v/v), 0.02% w/v EDTA
Mobile phase B: acetonitrile Mobile phase C: water Injection volume: 3 μL
Column temperature: 40°C
Autosampler temperature: 4°C
Detection: 270 nm
Gradient conditions: Table 2

(iii) Waveform processing The elution time of the end point of the caffeine peak (Figure 2A) is set as the start time of the calculation of the area of bitter acids, and the peak of trans-isocohumulone (peak a in Figure 2B) is set as the end time. If necessary, background processing is performed to calculate the peak area of bitter acids (Figure 2C).

(iv) Calculation of the amount of bitter acid
Using the calibration curve prepared with the TCOIHA standard solution, the concentration of bitter acid in the sample is calculated as a TCOIHA equivalent value according to the following formula.
Bitter acid concentration in sample (mg/100 mL) = (bitter acid peak area in hop oxidation products - intercept of calibration curve prepared with TCOIHA standard solution) / slope of calibration curve prepared with TCOIHA standard solution x 1/5 x 100/1000

(2) Preparation of specimens for measuring bitter acids in compositions containing hop oxidation products The specimens in (1) above are prepared by the same procedure as in (1) for solid compositions, and as liquid compositions either as is or after appropriate dilution. It has been concluded that all compounds detected in the bitter acid elution region have a β-tricarbonyl skeleton (Biosci., Biotechnol., Biochem., 2015 (79): 1684-1694, J., Agric., Food Chem., 2015: 63: 10181-10191).

Example 1: Preparation of hop oxidation reaction product and component analysis (1) Hop oxidation treatment step Hallertauperle (HPE) hops were ground in a pellet mill, and the resulting ground hops were heated at 60° C. for about 120 hours while stirring in the air. Water was added to the resulting heated hops (matured hop pellets: one embodiment of a hop oxidation reaction product) to a solids concentration of 5 w/v%, and an extraction treatment was carried out for 30 minutes at 50° C. The resulting extract was subjected to solid-liquid separation by decantation to obtain a solid-liquid separated liquid (Brix about 2).

(2) Activated carbon treatment step Activated carbon (Y180C, manufactured by Ajinomoto Fine Chemicals Co., Ltd.; 0.5 w/v% relative to the solid-liquid separated liquid) and polyvinyl polypyrrolidone (Polyclar 10, manufactured by ISP Japan Co., Ltd.; 0.4 w/v% relative to the solid-liquid separated liquid) were added to the solid-liquid separated liquid obtained in (1) above, and the mixture was allowed to stand for 2 hours. A filter aid (diatomaceous earth) was added to the resulting mixture, and the mixture was filtered to obtain a filtrate (Brix value of about 1.5). The resulting filtrate was used in the following examples as a water extract of a hop oxidation reaction product.

(3) Analysis of components of hop oxidation reaction products The filtrate obtained in (2) above (aqueous extract of hop oxidation reaction products) was subjected to HPLC-MS/MS analysis under the following conditions to measure the contents of various components contained in the hop oxidation reaction products. It is known that hop oxidation reaction products contain scorpiofumulinol A, scorpiocohumulinol A, tricyclooxyisohumulone A, and tricyclooxyisocohumulone A as α-acid oxides, iso-α-acid oxides, and β-acid oxides (Biosci., Biotechnol., Biochem., 2015 (79): 1684-1694, J., Agric., Food Chem., 2015: 63: 10181-10191). In addition, the standards used in the analysis were prepared according to the methods described in J., Agric., Food Chem., 2015:63:10181-10191 and J. Nat. Prod. 2014, 77, 1252-1261.

[HPLC conditions]
Column: Unison UK-C18 100 x 2 mm i.d. particle size 3 μm
Flow rate: 0.25 mL/min Column temperature: 40° C.
Mobile phase A: 1% formic acid in water Mobile phase B: 1% formic acid in acetonitrile Injection volume: 3 μL
Gradient conditions: 0 → 30 min, 15 → 31% B
30 → 40 minutes, 31 → 80%B
40 → 43 minutes, 80%B
After that, washing and equilibration steps

[MSMS conditions]
Mass spectrometer: AB SCIEX 4000Q Trap
Ion source: ESI - negative ion mode Ion spray voltage: -4500V
Analysis parameters:

The above (1) and (2) were carried out three times to obtain a filtrate, and the contents of scorpiofumulinol A, scorpiocohumulinol A, tricyclooxyisohumulone A and tricyclooxyisocohumulone A were measured. As a result, the ratio of the total amount of tricyclooxyisohumulone A and tricyclooxyisocohumulone A to the total amount of scorpiofumulinol A and scorpiocohumulinol A ((tricyclooxyisohumulone A + tricyclooxyisocohumulone A)/(scorpiofumulinol A + scorpiocohumulinol A)) was as follows:
Lot 1: 3.2
Lot 2: 6.8
Lot 3: 12.1

From these results, it was found that in hop oxidation reaction products, the ratio of the total amount of tricyclooxyisohumulone and tricyclooxyisocohumulone to the total amount of scorpiofumulinol and scorpiocohumulinol is approximately 2 to 20.

Example 2: Measurement of the content of tetrapeptide GTWY and dipeptide WY in whey hydrolysate (1) Preparation of analysis sample Whey hydrolysate (HW-3, Megmilk Snow Brand) was diluted appropriately with sterilized water and filtered to prepare a measurement sample . The above whey hydrolysate (HW-3) is a product obtained by treating whey protein with an enzyme preparation containing a protease, then removing undecomposed matter by membrane treatment, and drying, and contains the tetrapeptide GTWY and dipeptide WY as described below.

(2) Analysis Method The concentrations of 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 GTWY and WY concentrations in the measurement samples were calculated by the calibration curve method using AQUA Peptide (Sigma Aldrich) as the standard sample.

<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 I.D. × 150 mm (Tosoh)
Column temperature: 70 ° C.
Mobile phase A: 0.1% formic acid aqueous solution Mobile phase B: 0.1% formic acid acetonitrile solution Gradient conditions: The gradient conditions shown in Table 4 were applied.

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

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

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

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

(4) Measurement of the peptide of the present invention in a composition containing a whey hydrolysate When the composition is solid, the analysis sample is prepared from the composition instead of the whey hydrolysate of (1). When the composition is liquid, the analysis sample is prepared by appropriate dilution and filtration.

Example 3: Effects of hop oxidation reaction products and whey hydrolysate on cognitive function (particularly short-term memory, spatial memory, and working memory) (1) Method A Experimental procedure Six-week-old male CD-1 mice (Japan Oriental Yeast Co., Ltd.) were acclimated for one week, raised on crudely purified feed CE-2 (Japan CLEA), and divided into five groups to ensure uniformity. The samples shown in Table 5 were forcibly administered intragastrically to each group. The samples were prepared by adding and dissolving the dried hop oxidation reaction product extract (prepared in Example 1), whey hydrolysate (prepared in Example 2), and donepezil hydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd.) in distilled water immediately before administration. The concentrations of the samples were 0.03 mg/mL for the whey hydrolysate and hop oxidation reaction products, and 0.1 mg/mL for donepezil hydrochloride. 40 minutes after administration, scopolamine hydrochloride (SCP) (Sigma) was administered intraperitoneally at 0.85 mg/kg body weight to induce memory impairment, and amnesic model mice were prepared. 20 minutes after intraperitoneal administration of SCP, the Y-maze test described below was performed. The experiment was performed with 10 animals per group, and the average and standard error of each group were calculated. The dose of donepezil hydrochloride was, for example, the amount described in Behav Brain Res. 2009 Dec 1;204(1):217-25.

B. Evaluation of cognitive function When mice are placed in a space, if they remember the route they previously chose, they will choose a different route from the one they previously chose due to their desire to explore novelty. Therefore, when mice are placed in a Y-maze with three arms of equal width and length, they will usually enter an arm different from the one they previously entered. The Y-maze test utilizes this property of mice to evaluate short-term memory, spatial memory, and working memory, which are indicators of cognitive function.

The Y-maze apparatus used had three arms, each 25 cm long, with walls 20 cm high and a floor 5 cm wide, connected at a 120-degree angle. Mice were placed at the end of one of the arms of the Y-maze and allowed to explore freely for eight minutes, and the order of the arms they moved through was recorded. Spontaneous alternation behavior was defined as a case in which a mouse selected and entered a different arm three times in a row. The total number of entries into the arms and the number of spontaneous alternation behaviors within the time period were counted, and the spontaneous alternation behavior variability (%) was calculated using the following formula (1). A higher spontaneous alternation behavior variability indicates that short-term memory, spatial memory, and working memory are better retained.

(2) Results The results are shown in Fig. 3 (total number of entries) and Fig. 4 (rate of spontaneous alternation behavior fluctuation). Although cognitive function was improved when hop oxidation reaction products or whey hydrolysates were administered alone, it was confirmed that the combined administration of hop oxidation reaction products and whey hydrolysates had a more remarkable effect on improving cognitive function than the single administration.

Claims (12)

A composition for maintaining, enhancing and/or improving cognitive function, comprising a hop oxidation reaction product and a whey protein enzymatic hydrolysate as active ingredients, wherein the hop oxidation reaction product includes tricyclooxyisocohumulone A, tricyclooxyisohumulone A, scorpiofumulinol A and scorpiocohumulinol A, the whey protein enzymatic hydrolysate includes a peptide having the amino acid sequence of GTWY (sequence number 1) and a peptide having the amino acid sequence of WY, the cognitive function is a memory function, and the memory function is a spatial memory function and/or a working memory function . The composition of claim 1 , wherein the hop oxidation reaction product is an aqueous medium extract. 3. The composition of claim 1 or 2 , wherein the hop oxidation reaction product is a water extract. The composition according to any one of claims 1 to 3, wherein the ratio (in terms of dry mass) of the total amount of tricyclooxyisohumulone A and tricyclooxyisocohumulone A to the total amount of scorpiofumulinol A and scorpiocohumulinol A is in the range of 2 to 20 . The composition according to any one of claims 1 to 4 , wherein the concentration of bitter acid in the hop oxidation reaction product is 1 to 20% by mass. The composition according to claim 5, wherein the content of GTWY in the enzymatic hydrolyzate of whey protein (converted into solid content) is 0.01 to 1 mass% based on the total amount, and the content of WY in the enzymatic hydrolyzate of whey protein (converted into solid content) is 0.005 to 0.5 mass% based on the total amount. 7. The composition according to claim 1 , wherein the ratio of the hop oxidation reaction product to the whey protein enzymatic hydrolysate in the composition (mass ratio converted into solid content) is 0.1 to 2. The composition according to any one of claims 1 to 7 , which is a food composition. The composition according to any one of claims 1 to 8 , which is in a unit package form per serving. The composition according to any one of claims 1 to 9 , wherein the hop oxidation reaction product is ingested by a human in an amount of 10 to 5,000 mg (based on solid content) per meal. The composition according to any one of claims 1 to 10 , 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 meal. The composition according to claim 10 or 11 , comprising 0.01 to 10 mg of GTWY and 1 to 100 mg of a bitter acid.

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