JP2019043956A - Composition for promoting increase in amount of in vivo bdnf - Google Patents

Abstract

To provide a composition for promoting an increase in an amount of in vivo, particularly, intramuscular brain-derived neurotrophic factor (BDNF).SOLUTION: A composition according to the present invention is a composition for promoting an increase in an amount of in vivo BDNF, containing a whey protein hydrolysate as an active ingredient. The whey protein hydrolysate contains at least one dipeptide selected from the group consisting of Ile-Leu, Leu-Leu, and Val-Leu.SELECTED DRAWING: None

Description

  The present invention relates to a composition having an action of promoting an increase in the amount of Brain Derived Neurotrophic Factor (BDNF) (hereinafter simply referred to as “BDNF” in the present invention) in the body. More particularly, the present invention relates to compositions capable of promoting an increase in the amount of BDNF in the body, in particular in muscle, with or without exercise.

  At present, in Japan, an increase in mental diseases such as depression and dementia (including Alzheimer's disease, vascular dementia and the like) has become a social problem. The number of elderly people with dementia has doubled in the 10 years from 1.49 million in 2002, and is said to have reached more than 3 million in 2012. In addition, according to a presentation by the Ministry of Health, Labor and Welfare research team of Japan (2012), about 4 million elderly people with mild dementia are noted as the onset onset group of dementia, and 4 people aged 65 and over It will be calculated that one person will become dementia or dementia reserve group. Dementia is, at present, once treated, it can not be eradicated by only suppressing its progression even if treated. For this reason, once the onset of dementia occurs, not only the patient's life changes significantly but also the family to be cared with an excessive burden and forced changes in life design, such as having to leave the company for reasons of care . The impact of labor losses on working generations due to such leave of absence on the national economy is by no means negligible.

  From these things, it is an urgent task not only for individuals but also for modern society to prevent and control dementia.

  In the past, milk and dairy products have been considered to be useful for preventing such dementia. Specifically, calcium and magnesium contained in large amounts in milk and milk products have a preventive effect against dementia (Non-Patent Document 1), and whey contained in milk and milk products has Alzheimer's disease. It has been reported or suggested to improve insulin resistance which is a risk factor of (Non-patent Document 2).

  On the other hand, it is known that exercise significantly increases BDNF, which is one of the neurotrophic factors involved in neural cell development, growth, maintenance, or regeneration, and enhances performance such as memory and learning (non- Patent Document 3). Moreover, it is known by many epidemiological studies and experiments that exercise can suppress the decline in cognitive function of elderly people (Non-patent documents 4 to 6). Also, recently, it has been confirmed by animal experiments that hippocampal BDNF is increased by forced exercise load through transient electrical stimulation (Non-patent Document 7). Furthermore, it has been reported that the expression level of BDNF in the brain is decreased in patients with depression and Alzheimer's disease (Non-patent documents 8 and 9).

  Thus, it has been suggested that BDNF, which is increased by exercise, is effective for the prevention of mental diseases such as depression and dementia.

  Also, it has been reported that BDNF suppresses the increase in body weight and improves glucose metabolism by administering it intracerebroventricularly, subcutaneously or intraperitoneally (Non-patent Document 10).

J Am Geriatr Soc. 2012, 60 (8): 1515-1520. Nutrition. 2014, 30 (9): 1076-1080. Neeper SA et al. Brain Res, 726: 49-56 (1996). Trends Neurosci. 2002, 25 (6): 295-301. Nature. 1999, 29; 400 (6743): 418-419. Cell Mol Neurobiol. 2010, 30 (4): 493-503. The 31st Health Science Research Grant for Young Researchers Achievement Report 2014, pp. 104-109. Chen B et al, Biol Psychiatry. 50: 260-265 (2001). Phillips HS et al. Neuron. 7: 695-702 (1991). Nakagawa T et al, Diabetes, 49: 436-444 (2000)

  As mentioned above, although research on exercise and cognitive function is active and results suggesting that BDNF is related to cognitive function improvement by exercise, the influence of food intake on BDNF, especially exercise and food There are many unexplained effects of the combination of substance intake. For this reason, the present invention focuses on the effects of food intake on BDNF, particularly the effects of exercise and food intake. Specifically, the object of the present invention is to efficiently increase the amount of BDNF in the body involved in improvement of cognitive function and glucose metabolism by food intake or exercise in combination with food intake. It was developed.

  That is, the object of the present invention is to provide a composition used to promote an increase in the amount of BDNF in the body, in particular in the muscle, in other words a composition for promoting the increase in the amount of BDNF in the body, in particular in the muscle. . Another object of the present invention is to provide a method for promoting an increase in the amount of BDNF in the body, particularly in muscle.

  The present inventors have intensively studied to solve the above-mentioned problems, and it has been found that a whey protein hydrolyzate containing at least one dipeptide selected from the group consisting of Ile-Leu, Leu-Leu and Val-Leu. It was found that intake significantly increases the amount of BDNF in muscle. Since such effects are not significantly observed in whey protein before hydrolysis, it is considered that dipeptide which is a component unique to whey protein hydrolyzate not included in whey protein contributes. As mentioned above, BDNF has been suggested to be effective in preventing mental disorders such as depression and dementia, and improving cognitive function, and also effective in suppressing weight gain and improving glucose metabolism deterioration. Has also been reported. For this reason, whey protein hydrolyzate containing the dipeptide is considered to be useful for preventing mental diseases such as depression and dementia, improving cognitive function, suppressing weight gain, and / or improving glucose metabolism.

  The present invention has been completed based on such findings, and has the following embodiments. Hereinafter, in the present specification, whey protein hydrolysate (Whey Protein Hydrolysate) is simply abbreviated as "WPH". Moreover, the dipeptide describes the amino acid sequence which comprises the said dipeptide as "Xxx-Yyy" using the three-letter symbol of an amino acid.

(I) Composition for promoting BDNF amount increase in the body (I-1) A composition for promoting BDNF amount increase in the body, preferably muscle, comprising WPH as an active ingredient,
The BDNF amount, wherein the WPH contains at least one dipeptide selected from the group consisting of Ile-Leu (isoleucyl-leucine), Leu-Leu (leucyl-leucine), and Val-Leu (valyl-leucine). Composition for promoting increase.
(I-2) The composition for promoting BDNF amount increase described in (I-1), wherein the WPH contains Ile-Leu, Leu-Leu, and Val-Leu.
(I-3) From the group consisting of Ile-Val (isoleucyl-valine), Ile-Ile (isoleucyl-isoleucine), Leu-Val (leucyl-valine), and Leu-Ile (leucyl-isoleucine) as described above. The composition for promoting the increase in the amount of BDNF described in (I-1) or (I-2), which contains at least one dipeptide selected.
(I-4) described in (I-1) or (I-2), wherein the WPH further contains a dipeptide consisting of Ile-Val, Ile-Ile, Leu-Val, and Leu-Ile Composition for promoting BDNF amount increase.
(I-5) The composition for promoting the increase in the amount of BDNF according to any one of (I-1) to (I-4), which is orally administered or ingested to a mammal including a human before or during exercise object.
(I-6) The composition for promoting the increase in the amount of BDNF described in (I-5), which promotes an increase in the amount of BDNF in muscle during or after exercise.
(I-7) A composition for promoting BDNF amount increase described in any of (I-1) to (I-4), which is orally administered or ingested to a mammal including a non-exercise human.
(I-8) The composition for promoting the increase in the amount of BDNF according to any one of (I-1) to (I-7), which is orally administered or ingested to a cognitively impaired person or a preparatory group thereof.
(I-9) The composition for promoting BDNF increase described in (I-8), which is a composition for suppressing or improving the decrease in cognitive function.
(I-10) The amount of BDNF described in any of (I-1) to (I-7), which is orally administered or ingested to an obese person and / or a person having impaired glucose metabolism, or a preparatory group thereof Composition for promoting increase.
(I-11) The composition for promoting BDNF increase described in (I-10), which is a composition for suppressing or improving obesity, and / or a composition for suppressing or improving reduction in glucose metabolism.
(I-12) A unit package form per one dose (intake), wherein the WPH is contained in an amount of 0.1 to 40 g in terms of total protein as a single dose (intake) in the unit package ((I-12) -1) The composition for promoting the increase in the amount of BDNF described in any one of (I-11).
(I-13) The composition for promoting BDNF amount increase described in any of (I-1) to (I-12) which is food and drink.
(I-14) Health food, functional food, dietary supplement, functional food for indication, food for specified health use, food for sickness, infant formula, milk powder for pregnant or lactating women, or body BDNF content by exercise The composition for promoting the increase in the amount of BDNF described in (I-13), which is a food and drink marked to be used to promote the increase.

(II) Method for increasing the amount of BDNF in vivo (II-1) A composition comprising WPH containing at least one dipeptide selected from the group consisting of Ile-Leu, Leu-Leu, and Val-Leu is administered to a subject A method of increasing or reducing the amount of BDNF in the subject's body, preferably in the muscle, or by ingestion.
(II-2) The method described in (II-1), wherein the WPH contains Ile-Leu, Leu-Leu, and Val-Leu.
(II-3) The above WPH further contains at least one dipeptide selected from the group consisting of Ile-Val, Ile-Ile, Leu-Val, and Leu-Ile, (II-1) or The method described in (II-2).
(II-4) described in (II-1) or (II-2), wherein the WPH further contains a dipeptide consisting of Ile-Val, Ile-Ile, Leu-Val, and Leu-Ile Method.
(II-5) The method according to any one of (II-1) to (II-4), wherein the subject is a mammal including a human before or during exercise.
(II-6) The method described in (II-5), which is a method of increasing the amount of intramuscular BDNF during or after exercise.
(II-7) The method according to any one of (II-1) to (II-4), wherein the subject is a mammal including a non-exercise human.
(II-8) The method according to any one of (II-1) to (II-7), wherein the subject is a cognitive decliner or a preparatory group thereof.
(II-9) The method described in (II-8), which is a method for suppressing or improving the decrease in cognitive function of the subject.
(II-10) The method according to any one of (II-1) to (II-8), which is a method for enhancing the memory or learning performance of a mammal including human.
(II-11) The method according to any one of (II-1) to (II-7), wherein the subject is an obese or / and a person who is impaired in glucose metabolism, or a preliminary group thereof.
(II-12) The method according to (II-11), which is a method for suppressing or improving obesity in the subject, and / or suppressing or improving hypoglycemia.
(II-13) The composition is in a unit package form per single dose (intake), and in the unit package form, WPH is used as a single dose (intake) in an amount of 0.1 to 40 g in terms of total protein The method according to any one of (II-1) to (II-12), which is included.
(II-14) The composition is a food or drink, preferably a health food, functional food, dietary supplement, functional display food, food for specified health use, food for sick persons, infant formula, milk powder for pregnant women or lactating women The method according to any one of (II-1) to (II-13), which is milk powder, or a food and drink marked to be used to promote an increase in the amount of BDNF in the body by exercise.

(III) WPH used to promote the increase in the amount of BDNF in the body
(III-1) A composition used for promoting the increase in the amount of BDNF in the subject's body, preferably in the muscle,
The composition as described above, wherein the composition comprises WPH containing at least one dipeptide selected from the group consisting of Ile-Leu, Leu-Leu, and Val-Leu as an active ingredient.
(III-2) The composition described in (III-1), wherein the WPH contains Ile-Leu, Leu-Leu, and Val-Leu.
(III-3) The above WPH further contains at least one dipeptide selected from the group consisting of Ile-Val, Ile-Ile, Leu-Val, and Leu-Ile, (III-1) or The composition described in (III-2).
(III-4) described in (III-1) or (III-2), wherein the WPH further contains a dipeptide consisting of Ile-Val, Ile-Ile, Leu-Val, and Leu-Ile Composition.
(III-5) The composition according to any one of (III-1) to (III-4), wherein the subject is a mammal including a human before or during exercise.
(III-6) The composition described in (III-5), which is used to promote an increase in the amount of intramuscular BDNF during or after exercise.
(III-7) The composition according to any one of (III-1) to (III-4), wherein the subject is a mammal including a non-exercise human.
(III-8) The composition according to any one of (III-1) to (III-7), wherein the subject is a person with cognitive decline or a preparatory group thereof.
(III-9) The composition described in (III-8), which is used to suppress or improve the decrease in cognitive function.
(III-10) The composition according to any one of (III-1) to (III-7), wherein the subject is an obese or / and a person who is impaired in glucose metabolism, or a preliminary group thereof.
(III-11) The composition described in (III-10), which is used to suppress or ameliorate obesity and / or to suppress or ameliorate hypoglycemia.
(III-12) A composition comprising a unit package form per one dose (intake), wherein the above WPH is contained in a single dose (intake) in an amount of 0.1 to 40 g in terms of total protein in the unit package form The composition according to any one of (III-1) to (III-11).
(III-13) food and drink, preferably health food, functional food, dietary supplement, functional food for indication, food for specified health use, food for sick people, infant formula, infant formula, infant formula or milk powder, or exercise A composition described in any one of (III-1) to (III-12), which is a food and drink marked to be used to promote an increase in the amount of BDNF in the body due to

(IV) Use of WPH for Producing a Composition for Promoting BDNF Amount Increase in Body (IV-1) Use of WPH for Producing a Composition for Promoting BDNF Amount Increase in a Body of a Subject, preferably an Muscle The above use, wherein the WPH contains at least one dipeptide selected from the group consisting of Ile-Leu, Leu-Leu, and Val-Leu.
(IV-2) The use according to (IV-1), wherein the WPH contains Ile-Leu, Leu-Leu and Val-Leu.
(IV-3) The above WPH further contains at least one dipeptide selected from the group consisting of Ile-Val, Ile-Ile, Leu-Val, and Leu-Ile, (IV-1) or The use described in (IV-2).
(IV-4) described in (IV-1) or (IV-2), wherein the WPH further contains a dipeptide consisting of Ile-Val, Ile-Ile, Leu-Val, and Leu-Ile use.
(IV-5) The use according to any one of (IV-1) to (IV-4), wherein the subject is a mammal including a human before or during exercise.
(IV-6) The use according to (IV-5), wherein the composition is for promoting an increase in the amount of intramuscular BDNF during or after exercise of a subject.
(IV-7) The use according to any of (IV-1) to (IV-4), wherein the subject is a mammal including a non-exercise human.
(IV-8) The use according to any one of (IV-1) to (IV-7), wherein the subject is a person with cognitive decline or a preparatory group thereof.
(IV-9) The use according to (IV-8), wherein the composition is for suppressing or improving the decline in cognitive function.
(IV-10) The use according to any one of (IV-1) to (IV-7), wherein the subject is an obese or / and a person who is impaired in glucose metabolism, or a preliminary group thereof.
(IV-11) The use according to (IV-10), wherein the composition is for suppressing or improving obesity or / and suppressing or improving hypoglycemia.
(IV-12) The composition is in a unit package form per single dose (intake), and the above WPH in a unit package form is a single dose (intake) in an amount of 0.1 to 40 g in terms of total protein The use described in any of (IV-1) to (IV-11), which is a composition comprising.
(IV-13) The composition is preferably a food or drink, preferably a health food, a functional food, a nutritional supplement, a functional display food, a food for specified health use, a food for a sick person, an infant formula, a pregnant woman or a lactating woman Powdered milk powder, or the use described in any of (IV-1) to (IV-12), which is a food and drink marked to be used to promote an increase in the amount of BDNF in the body by exercise.

  According to the present invention, there is provided a composition for promoting the increase in the amount of BDNF in a body, particularly an muscle, comprising WPH as an active ingredient (hereinafter sometimes referred to simply as "the composition of the present invention").

  By exercising after receiving the composition of the present invention, or by taking the composition of the present invention during exercise, it is possible to promote an increase in the amount of BDNF in the body, particularly in the muscle, by exercise, and as a result, The amount of BDNF in muscle can be efficiently increased (see Test Example 1). As mentioned above, it has been known that exercise can suppress the decline in cognitive function of the elderly by exercise, and BDNF improves performance such as memory and learning, and also for prevention of mental diseases such as depression and dementia. It is suggested to be effective. From this, it is possible to further enhance the above effect by ingesting the composition of the present invention during exercise.

  Moreover, the increase in BDNF is significantly recognized not only when exercising with WPH but also when not exercising (see Test Example 2). From this, it is considered that the increase in the amount of BDNF is a specific effect obtained by taking WPH regardless of whether or not exercise is performed. In addition, since such effects are not significantly observed in WPH before hydrolysis, a contribution is made from the dipeptide which is an inherent component of WPH which is not contained in whey protein (it is contained in trace amounts). It is thought that

  BDNF has also been reported to have an effect of suppressing weight gain and improving glucose metabolism. For this reason, according to this invention, the said effect can be anticipated by ingesting the composition of this invention.

  The WPH contained in the composition of the present invention utilizes milk protein which has been used as a raw material of food for many years. Therefore, the composition of the present invention is advantageous in that it has high safety because there is no concern of side effects even when taken continuously for a long period of time. In addition, whey protein is usually discharged after curdling of raw material milk in the process of producing natural cheese, and is discarded to become industrial waste. The WPH contained as an active ingredient in the composition of the present invention can be used as a raw material by reusing the whey as the industrial waste, so that the present invention is useful also in terms of environment and economy.

Test animals (male SD rats) are divided into (a) "dipeptide non-administration / non-exercise group", (b) "dipeptide non-administration / exercise group", and (c) "dipeptide administration / exercise group", (a) The amount of BDNF in muscle (ng / g) (average value ± standard error, the same applies hereinafter) in the case of not giving exercise load without taking WPH in and giving exercise load without taking WPH in (b) The result of having compared the same BDNF amount in case WPH was ingested, and the same BDNF amount after giving an exercise load in the case of (c) is shown. In the figure, * means that there is a significant difference of p <0.05 with respect to (a) by Dunnett's test. Test animals (male SD rats) are divided into (1) “water administration / exercise group”, (2) “WPC administration / exercise group”, and (3) “WPH administration / exercise group”, and water is divided into (1) Give exercise load by ingesting WPC (whey protein concentrate) to BDNF amount (ng / g) (mean ± standard error, the same applies hereinafter) in muscle after taking exercise and giving exercise load The results of measuring the same BDNF amount after the administration and the same BDNF amount after exercise load by taking WPH in (3) are respectively shown. In the figure, * means that there is a significant difference of p <0.05 with respect to (1) by Dunnett's test. Test animals (male SD rats) are divided into (i) "water administration / non-exercise group", (ii) "WPC administration / non-exercise group", and (iii) "WPH administration / non-exercise group", all of which are exercise BDNF amount (ng / g) after taking water in (i) without applying load (mean ± standard error, the same applies hereinafter), the same BDNF amount after taking WPC in (ii), and (Iii) shows the results of measurement of the same BDNF amount after intake of WPH. In the figure, * means that there is a significant difference of p <0.05 with respect to (i) by Dunnett's test.

  The composition for promoting BDNF dose increase in the present invention (the composition of the present invention) is a WPH containing at least one dipeptide selected from the group consisting of Ile-Leu, Leu-Leu, and Val-Leu. It is characterized in that it contains (hereinafter sometimes referred to as "the WPH of the present invention") as an active ingredient.

  In general, WPH is obtained by hydrolyzing all or part of whey protein as a raw material, and is also referred to as whey peptide.

  Whey protein is a mixture of globular proteins contained in whey (whey) which is a liquid portion obtained by removing casein and fat from milk, and α-lactalbumin (α-La) and β-lactoglobulin (β-Lg) And proteins such as immunoglobulin, lactoferrin and bovine serum albumin. In the present invention, whey protein used as a raw material for preparation of WPH may be whey protein purified from milk or whey, or crude whey protein (whey protein-containing substance separated from milk or whey) ) May be. Furthermore, food and drink containing whey protein and its raw materials (for example, a composition containing other components in addition to milk protein) can also be used. The food and drink or the raw material thereof may contain casein in addition to whey protein.

  Examples of the purified whey protein include α-lactalbumin (α-La), β-lactoglobulin (β-Lg), immunoglobulin, lactoferrin, and a mixture of proteins containing at least one of them, and purified whey protein (WPI) Etc. can be mentioned. The purified whey protein (whey protein-containing material) may be prepared from milk, for example, a stock solution of whey (sweet whey, acid whey etc.), its concentrate, its dried product (whey flour etc.), its frozen And reduced products thereof. They can also include desalted whey, whey protein concentrate (WPC), and their reductants. In addition, as food and drink containing whey protein or raw materials thereof, for example, raw milk, pasteurized milk (such as milk), skimmed milk, component-modified milk, processed milk, dairy products (concentrated milk, powdered milk, condensed milk, fermented milk (such as yoghurt etc.) ), Lactic acid bacteria beverages, processed cheeses, ice creams, creams) and milk protein concentrates (MPC), and their concentrates, their dried products, their frozen products and the like. At this time, the raw materials and food and drink containing the whey protein may be obtained from commercial products and used, or may be prepared and used by themselves.

  The WPH of the present invention can be prepared by hydrolyzing the above-mentioned whey protein one or more times. The means and / or method is not particularly limited. For example, during the process of separating or purifying whey protein from milk or whey, whey protein may be partially hydrolyzed and then further hydrolyzed to prepare. The hydrolysis can be carried out by a known hydrolysis method, but preferably, a hydrolysis method by a proteolytic enzyme can be used.

  The proteolytic enzyme is not particularly limited as long as it hydrolyzes whey protein to generate WPH that exerts the effects of the present invention. For example, referring to the description of WO 2007/123200 etc., the type and reaction conditions can be set arbitrarily. For example, a proteolytic enzyme derived from a microorganism belonging to Bacillus genus or Aspergillus (Aspergillus); papain derived from papaya, bromelain derived from pineapple, plant derived protease such as actinidin derived from kiwi; pancreatin, trypsin, Animal-derived proteolytic enzymes such as pepsin and chymotrypsin. These can be used individually by 1 type or in combination of 2 or more types arbitrarily. Moreover, commercially available products can be used as these proteolytic enzymes. Commercially available proteolytic enzymes include, for example, Protease M Amano (Amano Enzyme), Protease N Amano (Amano Enzyme), Protease P Amano (Amano Enzyme), Protease A Amano (Amano Enzyme), Trypsin (Novo) , Pepsin (Wako Pure Chemical Industries, Ltd.), umamizyme (Amanoenzyme), flavor enzyme (Novo) and the like, but not limited thereto. According to a preferred embodiment of the present invention, the proteolytic enzyme used to hydrolyze whey protein comprises a Bacillus-derived proteolytic enzyme, an Aspergillus-derived proteolytic enzyme, trypsin, pepsin and flavor enzyme (Novo) One or two or more selected from the group.

  The WPH of the present invention is a hydrolysis that is rich in at least one dipeptide selected from the group consisting of Ile-Leu, Leu-Leu, and Val-Leu by using a proteolytic enzyme that hydrolyzes whey protein. It can be prepared as a product. For example, in order to hydrolyze whey protein to produce an Ile-Leu dipeptide, it is preferable to perform hydrolysis by combining whey protein with a protease derived from Bacillus and a protease derived from Aspergillus. Moreover, in order to hydrolyze whey protein and to produce the dipeptide of Leu-Leu, it is preferable to make whey protein react with protease derived from Aspergillus, after reacting with pepsin. In addition, according to the said method, whey protein may be hydrolyzed and the dipeptide of Leu-Val or Gly-Leu may also be produced | generated. Furthermore, in order to hydrolyze whey protein to generate a Val-Leu dipeptide, it is preferable to hydrolyze whey protein using a protease derived from Aspergillus. According to the method, whey proteins may be hydrolyzed to produce Ile-Val, Ile-Ile, and Leu-Ile dipeptides. In addition, in order to hydrolyze whey protein to produce a dipeptide of Ala-Leu, it is preferable to react whey protein with trypsin and then react with a protease derived from Bacillus.

  The reaction temperature for hydrolyzing whey protein with a proteolytic enzyme to produce a desired dipeptide is preferably 20 to 80 ° C., more preferably 25 to 75 ° C., still more preferably 30 to 70 ° C., still more preferably 35 It is -65 ° C, more preferably 40-60 ° C, particularly preferably 45-55 ° C. When the reaction temperature is 20 ° C. or more, whey protein can be efficiently hydrolyzed by a proteolytic enzyme. Moreover, if reaction temperature is 80 degrees C or less, the inactivation by modification | denaturation of a proteolytic enzyme can be suppressed. The reaction temperature may be set to a constant temperature (one step) or may be set to two or more different temperatures (two or more steps) in view of the optimum temperature of the protein hydrolase used. . When setting the reaction temperature of two or more steps, the order is arbitrary.

  Although the reaction time to hydrolyze whey protein by a protein hydrolyzing enzyme to produce a desired dipeptide is not limited, it is preferably 2 to 48 hours, more preferably 2 to 36 hours, still more preferably 2 to 24 hours. More preferably, it is 2 to 18 hours, more preferably 2 to 14 hours, further preferably 4 to 12 hours, further preferably 6 to 10 hours, and particularly preferably 7 to 9 hours. By setting the reaction time of the proteolytic enzyme to 2 hours or more, more whey proteins can be hydrolyzed. In order to hydrolyze whey protein efficiently, it is preferable to keep the reaction time within 48 hours. In addition, when setting reaction temperature by 2 or more steps, reaction temperature of 2 or more steps can be arbitrarily set in the range of the said reaction time.

  The amount of proteolytic enzyme added to hydrolyze whey protein by proteolytic enzyme to produce a desired dipeptide is not particularly limited as long as it can produce WPH exhibiting the effects of the present invention. For example, preferably 0.5 to 10 g, more preferably 0.7 to 5 g, still more preferably 0.8 to 4 g, particularly preferably 1 to 2 g can be mentioned per 100 g of whey protein.

  The reaction pH for hydrolyzing whey protein with a proteolytic enzyme to produce a desired dipeptide is desirably in the pH range in which the proteolytic enzyme used is reacted, and more preferably at or near its optimum pH Can be mentioned. For example, when using a protease derived from Bacillus sp. And a protease derived from Aspergillus sp. In combination, preferably pH 5-9, more preferably pH 5.5-8.5, still more preferably pH 6-8, particularly preferably Is pH 6.5-7.5.

  The reaction pH may be set to a constant pH condition (one step) or in two or more different pH conditions (two or more steps) in view of the optimum pH of the protein hydrolase used. You can also. When setting two or more steps of pH conditions, the order is arbitrary.

  As described above, the WPH of the present invention is characterized by comprising at least one dipeptide selected from the group consisting of Ile-Leu, Leu-Leu, and Val-Leu. Preferably, two or more of these dipeptides are contained, and more preferably, three of these dipeptides are contained. As an aspect of the combination in the case of containing 2 or more types, Il-Leu and Leu-Leu, Ile-Leu and Val-Leu, Leu-Leu and Val-Leu, or Ile-Leu, Leu-Leu and Val-Leu are exemplified. can do. The WPH of the present invention can be an indicator that it contains the above specific dipeptide.

  These dipeptides contained in the WPH of the present invention can be measured by LC / MS or LC / MS / MS. Specifically, it can be measured by the method described in Production Example 1 described later. The WPH of the present invention containing these dipeptides can be produced by referring to the examples described in the following Production Examples 1 to 11 in addition to the hydrolysis reaction conditions as described above.

The content (mg) of these dipeptides per gram of WPH can be set as follows.
Ile-Leu: 0.1 to 25 mg / g WPH, preferably 0.5 to 15 mg / g WPH, more preferably 1 to 10 mg / g WPH.
Leu-Leu: 0.5 to 50 mg / g WPH, preferably 1 to 25 mg / g WPH, more preferably 2 to 10 mg / g WPH.
Val-Leu: 0.5 to 50 mg / g WPH, preferably 1 to 25 mg / g WPH, more preferably 2 to 10 mg / g WPH.

The WPH of the present invention may contain at least one of the dipeptides described above at a ratio of 1 g of protein content of WPH shown below.
Ile-Leu: 0.09-22.5 mg / g protein, preferably 0.45-13.5 mg / g protein, more preferably 0.9-9 mg / g protein.
Leu-Leu: 0.45 to 45 mg / g protein, preferably 0.9 to 22.5 mg / g protein, more preferably 1.8 to 9 mg / g protein.
Val-Leu: 0.45 to 45 mg / g protein, preferably 0.9 to 22.5 mg / g protein, more preferably 1.8 to 9 mg / g protein.

  Here, the “protein content of WPH” means the total amount of amino acids contained in WPH, and amino acids constituting proteins and peptides contained in WPH. Such protein content can be measured and calculated by the Kjeldahl method. In fact, in the case of the Kjeldahl method, nitrogen contained in the target WPH can be measured, and the value can be calculated by multiplying the value by a nitrogen-protein conversion coefficient (usually 6.25). For example, since the WPH of the present invention is obtained by hydrolyzing whey protein, the protein content of WPH calculated by the above method is theoretically equal to the protein content of whey protein before hydrolysis. It becomes the same.

  Furthermore, in addition to including at least one, preferably two or more, more preferably three dipeptides selected from the group consisting of Ile-Leu, Leu-Leu, and Val-Leu described above, the WPH of the present invention Thus, it may further contain at least one dipeptide selected from the group consisting of Ile-Val, Ile-Ile, Leu-Val, and Leu-Ile. Preferably, it contains two or more of these dipeptides, more preferably, contains three or more of these dipeptides, and still more preferably, contains four of these dipeptides. As an embodiment of the combination in the case of containing two or more species, Ile-Val and Ile-Ile, Ile-Val and Leu-Val, Ile-Val and Leu-Ile, Ile-Ile and Leu-Val, Ile-Ile and Leu- Ile, Leu-Val and Leu-Ile, Ile-Val and Ile-Ile and Leu-Val, Ile-Val, Ile-Ile and Leu-Val, Ile-Ile and Leu-Val and Leu-Ile, Ile-Val and There may be mentioned Ile-Ile, Leu-Val and Leu-Ile. Such WPH of the present invention can be indicated as including the above specific dipeptide.

These dipeptides contained in the WPH of the present invention can also be measured by LC / MS or LC / MS / MS. The content per WPH of these dipeptides can be set as follows.
Ile-Val: 0.1 to 10 mg / g WPH, preferably 0.2 to 5 mg / g WPH, more preferably 0.5 to 1.5 mg / g WPH,
Ile-Ile: 0.05 to 10 mg / g WPH, preferably 0.1 to 5 mg / g WPH, more preferably 0.2 to 1 mg / g WPH
Leu-Val: 0.1 to 10 mg / g WPH, preferably 0.2 to 5 mg / g WPH, more preferably 0.5 to 1.5 mg / g WPH
Leu-Ile: 0.01 to 10 mg / g WPH, preferably 0.02 to 5 mg / g WPH, more preferably 0.05 to 0.2 mg / g WPH.

The WPH of the present invention may contain at least one of the dipeptides described above at a ratio of 1 g of protein content of WPH shown below. The meaning of "the protein content of WPH" is as described above.
Ile-Val: 0.09-9 mg / g protein, preferably 0.18-4.5 mg / g protein, more preferably 0.45-1.35 mg / g protein Ile-Ile: 0.045-9 mg / G protein, preferably 0.09 to 4.5 mg / g protein, more preferably 0.18 to 0.9 mg / g protein Leu-Val: 0.09 to 9 mg / g protein, preferably 0. 18 to 4.5 mg / g protein, more preferably 0.45 to 1.35 mg / g protein Leu-Ile: 0.009 to 9 mg / g protein, preferably 0.018 to 4.5 mg / g protein, More preferably, 0.045 to 0.2 mg / g protein.

  The WPH of the present invention, as the total amount of Ile-Leu, Leu-Leu, and Val-Leu dipeptides, is preferably 1.1 to 125 mg / g WPH, preferably 2.5 to 65 mg / g WPH, per gram of WPH. Those containing 5 to 30 mg / g WPH can be mentioned. The total amount of these dipeptides contained in the WPH is 0.99 to 112.5 mg / g protein, preferably 2.25 to 58.5 mg / g protein, more preferably It can be 4.5-27 mg / g protein.

  Furthermore, the WPH of the present invention is 0.26 to 40 mg / g WPH, preferably 0.52 to 20 mg / g WPH per gram of WPH, as the total amount of Ile-Val, Ile-Ile, Leu-Val, and Leu-Ile dipeptides. More preferably, those containing at a ratio of 1.25 to 4.2 mg / g WPH can be mentioned. These dipeptides contained in the WPH are 0.234 to 36 mg / g protein, preferably 0.468 to 18 mg / g protein, and more preferably 1.125 to 3.3, in terms of the ratio of WPH per 1 g of protein content. It can be 8 mg / g protein.

  The WPH of the present invention is liquid immediately after preparation, but in order to enhance its storage property, it can be preserved by freezing, drying (spray drying, freeze drying), concentration, heat sterilization (plate heat sterilization, tube heat sterilization, Batch-type heat sterilization, electric current heat sterilization, heat sterilization with microwave waves, indirect heat sterilization such as retort heating, direct heat sterilization such as steam injection, steam infusion, etc., or non-heat sterilization (light irradiation sterilization, radiation irradiation sterilization, High voltage pulse sterilization). Among them, from the viewpoint of storage stability and easiness of handling, it is preferable to prepare WPH and then carry out drying (spray drying, lyophilization) to obtain a dry solid form.

  As shown in Test Examples 1 and 2 below, the composition of the present invention containing WPH can be administered to or taken from a mammal (including human and non-human animals) and exercise-loaded, particularly in the body, in particular. The amount of BDNF in muscle can be increased (see FIGS. 1 and 2). Also, the amount of increase is higher than the amount of BDNF in the body, especially in muscle, which increases when exercised without administration or intake of the composition of the present invention to a mammal. Therefore, the composition of the present invention containing the WPH is useful as a composition for promoting the increase in the amount of BDNF in the body, particularly in the muscle (a composition for promoting the increase in the amount of BDNF in the body, especially in the muscle). In addition, the composition for promoting the increase in the amount of BDNF is preferably administered or consumed by the mammal before or during exercise of the mammal to increase the amount of BDNF in the body, particularly in the muscle after exercise. It can be used for Here, “promoting increase in the amount of BDNF” means BDNF in the body, especially in the muscle after exercise when the composition of the present invention is administered (intake) to the mammal before or during exercise of the mammal. The amount is meant to be higher than the amount when the composition of the present invention is not administered (ingested). Here, "when the composition of the present invention is not administered (ingested)", when the composition of the present invention is not administered (ingested) or exercise-loaded, and when the composition of the present invention is not administered (ingested) Includes both when loaded. More specifically, administration (intake) of the composition of the present invention before or during exercise increases the amount of intramuscular BDNF when exercised without administration (intake) of the composition of the present invention And increase the amount of BDNF in muscle more than the increase when exercised without administration (intake) of the composition of the present invention.

  In the present invention, “exercise” may be any one that involves energy consumption more than at rest, ie, exerts a load on muscles, by contraction of skeletal muscle. Although there is no particular limitation in this regard, specifically, activities to maintain and improve the physical strength, such as walking, fast-footing, jogging, running, dancing, underwater exercise, skating, cycling, gymnastics, yoga, stretching, weight training, adding Pressure training, swimming, mountain climbing, climbing, various sports (for example, ball games, badminton, ballet etc.) can be illustrated.

  Also, as shown in Test Example 2 below, the composition of the present invention containing WPH can increase the amount of BDNF in the body, particularly in the muscle, by administering or ingesting to a mammal even when not exercised. Can be done (see Figure 3). In this case, the term "increase in BDNF amount" means that the BDNF amount in the body, especially in the muscle when the composition of the present invention is administered (ingested) to a mammal without exercise load is the composition of the present invention without exercise load. It means that it is high compared with the amount concerned when not administered (ingested). Therefore, the composition of the present invention containing the WPH is useful as a composition that promotes an increase in the amount of BDNF in the body, in particular in the muscle, by simply taking it into the body even when exercise is not given.

  Here, "administration or intake" may be any method as long as it is put into the body of a mammal (including human and non-human animals), preferably, but not limited to, oral administration or intake, enteral administration, and gastric fistula. For example, it is possible to illustrate an embodiment in which it is digested and absorbed in the intestine, and can be appropriately selected depending on the condition and use of the target mammal. Preferably it is oral intake. Therefore, the composition of the present invention has a form for oral administration or ingestion, a form for enteral administration, and a form for gastric fistula, and can be prepared in the form.

  As described above, BDNF is one of the neurotrophic factors involved in the development, growth, maintenance, and regeneration of neurons, and is known to enhance memory and learning performance (Non-patent Document 3). From this, the composition of the present invention is a composition for assisting the improvement of memory or learning performance, those who need to improve memory ability, learning ability or learning efficiency (for example, student, memory ability, learning ability, etc. It can be taken or administered to those who are concerned about the decrease in learning efficiency.

  In addition, in patients with depression or Alzheimer's disease, it is known that the expression level of BDNF in the brain decreases (Non-patent Documents 8 and 9). From this, is the composition of the present invention taken as a composition for cognitive function improvement, cognitive function depression suppression, or cognitive function improvement to patients with dementia, particularly with Alzheimer's disease, or a preparatory group thereof? Or can be administered. Here, "cognitive function" means a function to correctly understand things properly and to execute appropriately, and memory ability, language ability, judgment (including orientation, attentiveness), calculation ability, and / or Performance is included. The dementia reserve group also includes people with mild cognitive impairment. In addition, since one in four elderly people aged 65 or older is considered to have dementia or mild cognitive impairment, elderly people aged 65 years or older may also be included in the dementia reserve group in the present invention. In addition, the composition of the present invention can be taken or administered as a composition for ameliorating depression to patients with depression (including new-type depression), or a depression group that is depressed or prone to depression.

  Furthermore, BDNF has been reported to suppress weight gain and improve glucose metabolism by administering it intracerebroventricularly, subcutaneously or intraperitoneally (Non-patent Document 10). From this, the composition of the present invention can be taken or administered to an obese person, a person who is obese, or a preparatory group thereof as a composition for suppressing or improving obesity. The composition of the present invention can also be taken or administered to diabetic patients, borderline diabetic patients, or a preparatory group thereof as a composition for suppressing glucose metabolism reduction or improving glucose metabolism.

  In addition to the WPH of the present invention, other food ingredients and / or food additive raw materials can be added to the composition of the present invention as long as the effects of the present invention can be exhibited. For example, in order to impart palatability to the composition of the present invention, sweeteners (sugars, liquid sugars, fructose, maltose, three-point sugar, etc.), sugar alcohols (eg erythritol), high sensitivity sweeteners (stevia, aspartame) Sucralose, potassium acesulfame, etc., acidulants, flavors, fruit juices, vegetable juices, polysaccharide thickeners, emulsifiers, flavoring agents, flavoring agents, etc. can be added. In addition, minerals (calcium, iron, manganese, magnesium, zinc, etc.), vitamins, functional materials, probiotic lactic acid bacteria, etc. for the purpose of imparting additional functions (nutritional function, physiological function) to the composition of the present invention Can be added to make the composition of the present invention.

  The compositions of the present invention can be in a containerized form for market delivery. As a form in a container, the aspect which prepared the composition of this invention in the solid state, and filled the container can be mentioned. Such solid forms may include powders and granules. In this case, known containers such as paper, bags, boxes, cans and bottles can be used. The internal volume of the container at this time is not particularly limited, and, for example, 1 to 5000 g, 2 to 4000 g, 3 to 3000 g, 4 to 2000 g, 4 to 1000 g, 4 to 500 g, 4 to 250 g, 4 to 200 g, 4 to 100 g, 4 to 50 g, 4 to 30 g. Among them, in the case of family size (plural doses), for example, 300 to 5000 g, 300 to 4000 g, 300 to 3000 g, 300 to 2000 g, 300 to 1500 g, 300 to 1400 g, 300 to 1300 g, 300 to 1200 g, 300 to 1100 g 1 to 500 g, 2 to 250 g, 3 to 200 g, 4 to 100 g, 4 to 50 g, 4 to 40 g, 4 to 30 g, 5 to 30 g, 10 to 30 g, 15 to 5 for personal size (individual dose) It is 30g.

  Also, the composition of the present invention can be in the form of a containerized beverage for the purpose of market provision. Here, the container may be any container that can be filled with a beverage and can be consumed as it is, and known containers such as paper containers, soft bags, plastic bottles, cans and bottles can be used. The beverage can be prepared by dispersing and dissolving the above powder or granules in drinking water. The inner volume of the container is not particularly limited, and is, for example, 1 to 5000 g, 2 to 4000 g, 3 to 3000 g, 4 to 2000 g, 5 to 1000 g, 6 to 800 g, 7 to 1300 g, 80 to 1200 g, 90 to 1 100 g. . Among them, in the case of family size (for multiple use), for example, 300 to 5000 g, 300 to 4000 g, 300 to 3000 g, 300 to 2000 g, 300 to 1500 g, 300 to 1400 g, 300 to 1300 g, 300 to 1200 g, 300 to 1100 g 10 to 500 g, 20 to 500 g, 30 to 500 g, 40 to 500 g, 50 to 500 g, 60 to 500 g, 70 to 500 g, 80 to 500 g, 90 to 500 g in the case of personal size (for individual use).

  According to one embodiment of the present invention, for example, in the composition of the present invention, the total protein content of WPH is ingested so as to be 0.1 g or more (preferably 1 g or more) per single dose (intake) Administration). Preferably, the total protein content per single dose (intake) is 0.1 to 40 g, more preferably 0.1 to 30 g, and still more preferably 1 to 30 g.

  The “single dose (intake) amount” means a single dose of a condition requiring promotion of increase in the amount of BDNF in the body. For example, when performing a series of exercises at a predetermined time of the day, it means the total dose to be administered or taken before or during the exercise, not in one bite (at a time) but in several doses In the case of these, the amount administered (ingested) in these several times is totaled as “single administration (ingested)”.

  The timing at which the composition of the present invention is administered (ingested) to a subject (mammal) requiring an increase in the amount of BDNF in the body, especially in muscle, is from the viewpoint of promoting the increase in the amount of BDNF due to exercise. Before or during exercise. Preferably, it can be taken 15 minutes to 240 minutes before exercise, more preferably 30 minutes to 180 minutes before exercise, and still more preferably 45 minutes to 135 minutes before exercise. Also, if you do not exercise, you can be administered (ingested) at any time, but because it is easier to be absorbed when administered (ingested) when you are relatively hungry, such as before meals or between meals, you get a higher effect. Can.

  According to another embodiment of the present invention, for example, the composition of the present invention may be provided in unit package form per single dose (intake). In the composition of the present invention having the unit package form, the WPH is prepared in the unit in an amount of 0.1 g or more (preferably 1 g or more) in terms of total protein content as a single dose (intake) In addition, it is desirable that one dose (intake) amount be adjusted to 40 g or less (preferably 30 g or less) in terms of total protein content. That is, the content of WPH of the present invention per unit package is 0.1 to 40 g, preferably 0.1 to 30 g, more preferably 1 to 30 g, in terms of total protein content as single dose (intake) can do. Here, “consisting of“ unit packaging form per single dose (intake) ”” means that the amount per single dose (intake) is previously determined, and, for example, food and drink that can orally ingest a specific amount As products, it means not only general foods but also forms such as beverages (drinks etc.), health supplements, health functional foods, supplements and the like. In "unit packaging form per single dose (intake)", for example, liquid beverages, gel-like, paste-like, paste-like jelly, powder-like, granular-like, capsule-like, block-like solid food etc. In the case where a specific amount (dose) can be specified in packaging containers such as metal cans, glass bottles (such as bottles), plastic containers (such as plastic bottles), packs, pouches, film containers, paper boxes, etc. The form which can prescribe | regulate a specific amount by displaying the amount (usage, dosage) per per on a packaging container, a website, etc. is mentioned. Since the composition of the present invention uses WPH as an active ingredient, gelation upon heating is less compared to whey protein, and therefore, it is possible to maintain physical properties suitable for the process of producing beverages, jellies and the like. In addition, the composition of the present invention exerts the effects of the present invention even if ingested in a small amount, and therefore it may be in any form such as a block shape of a bite size or a mini can beverage which can be ingested by one drink. It is easy to take and swallow, and it can improve the convenience for children and the elderly.

  The composition of the present invention can be provided in the form of a composition containing a plurality of components. The form of the composition of the present invention is not particularly limited as long as the effects of the present invention are exhibited, but for example, solid (powder, granule, capsule, block, etc.), liquid, gel, glue It can be in the form of paste, paste, etc., and is preferably in the form of powder. The compositions include food compositions and pharmaceutical compositions.

  According to a preferred embodiment of the present invention, there is provided a food and drink for promoting the increase in the amount of BDNF, which comprises WPH as an active ingredient. In the present invention, the form of the food is not particularly limited as long as it is an orally ingestible form such as a solution, a suspension, an emulsion, a powder, a solid molding and the like. Specifically, for example, instant foods such as instant noodles, retort foods, canned foods, microwave foods, instant soups and miso soups, freeze-dried foods, etc .; soft drinks, fruit juices, vegetable drinks, soy milk drinks, coffee drinks, tea Beverages, Powdered beverages, Concentrated beverages, Beverages such as alcoholic beverages; Bread products such as pasta, noodles, cake mix, bread crumbs; Strawberries, caramel, chewing gum, chocolate, cookies, biscuits, biscuits, cakes, pies, snacks, crackers, Sweets such as Japanese sweets and dessert sweets; Sauces, processed tomato seasonings, flavored seasonings, cooked mixes, sauces, dressings, soups, seasonings such as ingredients of curry and stew; Processed fats and oils, butter, margarine, Oils such as mayonnaise; Milk drinks, yogurts, lactic acid bacteria drinks, ice creams, creams How dairy products; agricultural canning, jam, marmalade such, processed agricultural products such as serial; such as frozen food, and the like. Preferably, the food or drink is a dairy product, more preferably a milk drink or a lactic acid bacteria drink.

  Foods and beverages are classified into health food, functional food, dietary supplement, functional labeling food, food for specified health use, food for the sick, adjusted powdered milk for infants, powdered milk for pregnant women or lactating women Is also included.

  The food and drink product of the present invention may be labeled as "increase BDNF in the body", "promote increase in BDNF in the body", "help increase BDNF in the body", etc. . The term "in vivo" also includes the meaning "intramuscularly".

  According to another aspect of the present invention, there is provided a method of promoting the increase in the amount of BDNF in the body, in particular in a muscle, comprising ingesting or administering WPH to a mammal (including human or non-human animals) Is provided. The method for promoting the increase of the BDNF amount of the present invention can be practiced according to the description of the composition of the present invention.

  Here, in the method for promoting the increase of the BDNF amount of the present invention, it may be used in mammals and samples derived therefrom (muscle tissue, muscle cells), and both of therapeutic use and non-therapeutic use Intended. Here, "non-therapeutic" means that it does not include an operation of surgery, treatment or diagnosis of a human (ie, a medical procedure for a human), and more specifically, a doctor or a person who has been instructed by a doctor Means not include methods of performing surgery, treatment or diagnosis on humans.

  Hereinafter, the present invention will be described in more detail by way of production examples and experimental examples. However, these production examples and experimental examples are only examples used for the description for facilitating the understanding of the present invention, and the present invention is not limited thereto. Unless otherwise stated, the following production examples and experimental examples were performed under the conditions of room temperature (25 ± 5 ° C.) and atmospheric pressure. Moreover, the protein content contained in a test sample was measured using the Kjeldahl method.

Production Example 1
(1) Production of whey protein hydrolyzate (dry powder) 50 g of a commercially available whey protein concentrate (hereinafter simply referred to as "WPC") is dissolved in 1 L of water to prepare a WPC aqueous solution, and this is adjusted to pH The pH was adjusted to 7 with an agent. To this, 500 mg of a protease derived from Bacillus (Protease M Amano, Amano Enzymes) and 500 mg of a protease derived from Aspergillus (Protease N Amano, Amano Enzymes) are added, and the whey protein is treated at 50 ° C. for 8 hours Was hydrolyzed. After heating this to inactivate the protease, it was subjected to lyophilization treatment to be powdered, and this was made into whey protein hydrolyzate (WPH) (dry powder).

(2) Component analysis The whey protein hydrolyzate (dry powder) obtained above is diluted and dissolved 1000 times (volume ratio) with a 0.1 (w / w)% aqueous solution of trifluoroacetic acid, Under the conditions of LC / MS / MS under the following conditions, the dipeptides contained in WPH (Ile-Leu, Leu-Leu, Val-Leu, Ile-Asn, Leu-Ala, Leu-Glu, Ala-Leu, and Lys- The amount of Ile) (unit: mg / g protein: reduced to solid, hereinafter the same) was measured.

[Analytical conditions of LC / MS / MS]
Column: ACQUITY UPLC BEH C18 Column, 130 Å, 1.7 μm, 2.1 × 50 mm (Waters)
Mobile phase: A solution: 0.1 w / w aqueous solution containing 0.1% (w / w) trifluoroacetic acid B solution: 0.1 w / w ratio of trifluoroacetic acid containing acetonitrile solution ratio of A solution and B solution in mobile phase Start of phase flow → 0 minutes: 100% A solution A (0% B solution B)
0 min → 9 min: A solution 100 v / v% → A solution 60 v / v%-B solution 40 v / v%
9 minutes → 10 minutes: A solution 60 v / v% -B solution 40 v / v% → A solution 20 v / v% -B solution 80 v / v%
10 to 11 minutes: A liquid 20 v / v%-B liquid 80 v / v% → A liquid 100 v / v% (B liquid 0 v / v%)
Column temperature: 40 ° C
Flow rate: 0.3 mL / min.

[MS / MS conditions]
ESI positive mode capillary voltage: 3000 V
Source temperature: 120 ° C
Desolvation temperature: 400 ° C
Desolvation gas flow rate: 850 L / hour cone gas flow rate: 50 L / hour cone voltage: 25 V
Collision voltage: 15mV.

The content of the dipeptide contained in the WPH (dry powder) prepared in the above Preparation Example 1 is shown in Table 1.

Production Example 2
An aqueous WPC solution was prepared by dissolving 50 g of a commercially available WPC (same as the WPC used in Production Example 1) in 1 L of water, and this was adjusted to pH 7 with a pH adjuster. To this was added 500 mg of a protease derived from Bacillus (Protease M Amano, Amano Enzyme Inc.), and the mixture was treated at 50 ° C. for 8 hours to hydrolyze whey protein in the WPC aqueous solution. Next, this was heated to inactivate the protease, and then subjected to lyophilization treatment to be powdered, and this was used as WPH (dry powder) of Production Example 2.

The amount of dipeptide contained in WPH (dry powder) prepared above was measured according to the analysis method described in the column (2) of Preparation Example 1. The results are listed in Table 2.

Production Example 3
An aqueous WPC solution was prepared by dissolving 50 g of a commercially available WPC (same as the WPC used in Production Example 1) in 1 L of water, and this was adjusted to pH 7 with a pH adjuster. To this was added 500 mg of a protease derived from Aspergillus (Protease N Amano, Amano Enzyme Co., Ltd.), and the mixture was treated at 50 ° C. for 8 hours to hydrolyze whey protein in the WPC aqueous solution. Next, this was heated to inactivate the protease, and then subjected to lyophilization to make a powder, which was used as WPH (dry powder) of Production Example 3.

The amount of dipeptide contained in WPH (dry powder) prepared above was measured according to the analysis method described in the column (2) of Preparation Example 1. The results are listed in Table 3.

Production Example 4
An aqueous WPC solution was prepared by dissolving 50 g of a commercially available WPC (same as the WPC used in Production Example 1) in 1 L of water, and this was adjusted to pH 7 with a pH adjuster. To this, 500 mg of trypsin (Novo) is added and reacted at 37 ° C for 4 hours, 500 mg of protease derived from Bacillus (Protease M Amano, Amano Enzyme) is added, and the mixture is further reacted at 50 ° C for 4 hours. In the whey protein was hydrolyzed. Next, this was heated to inactivate the protease, and then subjected to lyophilization treatment to be powdered, and this was used as WPH (dry powder) of Production Example 4.

The amount of dipeptide contained in WPH (dry powder) prepared above was measured according to the analysis method described in the column (2) of Preparation Example 1. The results are set forth in Table 4.

Production Example 5
An aqueous WPC solution was prepared by dissolving 50 g of a commercially available WPC (same as the WPC used in Production Example 1) in 1 L of water, and this was adjusted to pH 7 with a pH adjuster. To this, 500 mg of trypsin (Novo) is added and reacted at 37 ° C for 4 hours, 500 mg of protease derived from Aspergillus (Protease N Amano, Amano Enzyme) is added, and the mixture is further reacted at 50 ° C for 4 hours. Of whey protein was hydrolyzed. Next, this was heated to inactivate the protease, and then subjected to lyophilization to make a powder, which was used as WPH (dry powder) of Production Example 5.

The amount of dipeptide contained in WPH (dry powder) prepared above was measured according to the analysis method described in the column (2) of Preparation Example 1. The results are set forth in Table 5.

Production Example 6
An aqueous WPC solution was prepared by dissolving 50 g of a commercially available WPC (same as the WPC used in Production Example 1) in 1 L of water, and this was adjusted to pH 7 with a pH adjuster. To this, 500 mg of pepsin (Wako Pure Chemical Industries, Ltd.) is added and reacted at 37 ° C. for 4 hours, and then 500 mg of protease derived from Bacillus (Protease M Amano, Amano Enzyme) is added, and the reaction is further performed at 50 ° C. for 4 hours. The Whey protein in WPC aqueous solution was hydrolyzed. Subsequently, this was heated to inactivate the protease, and then subjected to lyophilization treatment to be powdered, and this was used as WPH (dry powder) of Production Example 6.

The amount of dipeptide contained in WPH (dry powder) prepared above was measured according to the analysis method described in the column (2) of Preparation Example 1. The results are set forth in Table 6.

Production Example 7
An aqueous WPC solution was prepared by dissolving 50 g of a commercially available WPC (same as the WPC used in Production Example 1) in 1 L of water, and this was adjusted to pH 7 with a pH adjuster. To this, 500 mg of pepsin (Wako Pure Chemical Industries, Ltd.) is added and reacted at 37 ° C. for 4 hours, and then 500 mg of a protease derived from Aspergillus (Protease N Amano, Amano Enzyme) is added and further reacted at 50 ° C. for 4 hours. Then, whey protein in whey protein aqueous solution was hydrolyzed. Next, this was heated to inactivate the protease, and then subjected to lyophilization to make a powder, and this was used as WPH (dry powder) of Production Example 7.

The amount of dipeptide contained in WPH (dry powder) prepared above was measured according to the analysis method described in the column (2) of Preparation Example 1. The results are listed in Table 7.

Production Example 8
An aqueous WPC solution was prepared by dissolving 50 g of a commercially available WPC (same as the WPC used in Production Example 1) in 1 L of water, and this was adjusted to pH 7 with a pH adjuster. To this was added 500 mg of flavorzyme (Novo) and reacted at 50 ° C. for 6 hours to hydrolyze whey protein in aqueous whey protein solution. Next, this was heated to inactivate the protease, and then subjected to lyophilization to make a powder, and this was made into WPH (dry powder) of Production Example 8.

The amount of dipeptide contained in WPH (dry powder) prepared above was measured according to the analysis method described in the column (2) of Preparation Example 1. The results are set forth in Table 8.

Production Example 9
An aqueous WPC solution was prepared by dissolving 50 g of a commercially available WPC (same as the WPC used in Production Example 1) in 1 L of water, and this was adjusted to pH 7 with a pH adjuster. To this was added 500 mg of a protease derived from Aspergillus (Umamizyme, Amano Enzymes Co., Ltd.), and the mixture was reacted at 50 ° C. for 6 hours to hydrolyze whey protein in the WPC aqueous solution. Next, this was heated to inactivate the protease, and then subjected to lyophilization to make a powder, and this was designated as WPH (dry powder) of Production Example 9.

The amount of dipeptide contained in WPH (dry powder) prepared above was measured according to the analysis method described in the column (2) of Preparation Example 1. The results are set forth in Table 9.

Production Example 10
An aqueous WPC solution was prepared by dissolving 50 g of a commercially available WPC (same as the WPC used in Production Example 1) in 1 L of water, and this was adjusted to pH 7 with a pH adjuster. To this was added 500 mg of a protease derived from Aspergillus (Protease A Amano, Amano Enzyme Inc.), and the mixture was reacted at 50 ° C. for 6 hours to hydrolyze the whey protein in the aqueous WPC solution. Next, this was heated to inactivate the protease, and then subjected to lyophilization treatment to be powdered, and this was made to be WPH (dry powder) of Production Example 10.

The amount of dipeptide contained in WPH (dry powder) prepared above was measured according to the analysis method described in the column (2) of Preparation Example 1. The results are set forth in Table 10.

Production Example 11
An aqueous WPC solution was prepared by dissolving 50 g of a commercially available WPC (same as the WPC used in Production Example 1) in 1 L of water, and this was adjusted to pH 7 with a pH adjuster. To this was added 500 mg of a protease derived from Aspergillus (Protease P Amano, Amano Enzyme Inc.), and the mixture was reacted at 50 ° C. for 6 hours to hydrolyze whey protein in aqueous whey protein solution. Next, this was heated to inactivate the protease, and then subjected to lyophilization to make a powder, which was used as WPH (dry powder) of Production Example 11.

The amount of dipeptide contained in WPH (dry powder) prepared above was measured according to the analysis method described in the column (2) of Preparation Example 1. The results are set forth in Table 11.

Test Example 1
This test was conducted to evaluate the influence on the cognitive function by combining the intake of whey protein hydrolyzate, which is a milk material, with exercise, using BDNF concentration in muscle as an index. In the test, when exercise was exercised by taking whey protein hydrolyzate using rats, exercise was exercised without taking WPH, and neither intake nor exercise stress of whey protein hydrolyzate was exercised. BDNF concentrations contained in skeletal muscle were measured and compared.

(1) Test method (1-1) Preparation of test sample (test food) The test food of Test Example 1 is obtained by hydrolyzing a commercially available WPC (the same as WPC used in Production Example 1) with a protease WPH (dry powder) was used. The WPH contains about 89.3% by mass of protein per 100% by mass of solid content. The said WPH was melt | dissolved in distilled water so that solid content concentration might be 10 w / v%, and this was made into the test sample (test food).

The amount (mg / g WPH, mg / g protein) of the dipeptide contained in WPH (dry powder) prepared above was measured according to the analysis method described in the column (2) of Production Example 1. The results are set forth in Table 12.

(1-2) Preparation of Test Animal As a test animal, 8-week-old male SD rat was used. Pre-breeding for 4 to 5 days was performed beforehand, and after the pre-breeding, the lower right thigh was removed under isoflurane anesthesia by the day before the test. In addition, fasting was performed for 16 hours from the day before the test.

On the day of the test, grouping was carried out according to the following, so that the weight was uniform (about 290 to 320 g).
[Test group (all 3 groups)]
(A) “Dipeptide non-administration / non-exercise group” (n = 6): no exercise load, test sample non-administration (b) “dipeptide non-administration / exercise group” (n = 6): exercise load 50 times, Test sample non-administration (c) “dipeptide administration / exercise group” (n = 3): exercise load 50 times, test sample administration.

(1-3) Test Among the test animals in each group described above, the test sample (test food) prepared above is 10 ml / kg body weight (WPH containing dipeptide) for the test animals in the “dipeptide administration / exercise group” A single oral dose of 1.0 g / kg body weight was given. Thirty minutes after administration, the right hind limb was forcibly given an exercise load by electrical stimulation (details will be described later) under isoflurane anesthesia. In addition, for test animals in the "dipeptide non-administration / exercise group", a single oral administration of distilled water at a rate of 10 ml / kg body weight instead of the test sample (test food) was performed 30 minutes after administration. Similarly, the right hind limb was forcefully exercised with electrical stimulation under isoflurane anesthesia. Furthermore, for test animals in the "dipeptide non-administration / non-exercise group", a single oral administration of distilled water at a rate of 10 ml / kg body weight instead of the test sample (test food) is followed by resting under isoflurane anesthesia. I let it go.

  3 hours after the end of exercise load for test animals of "dipeptide administration / exercise group" and "dipeptide nonadministration / exercise group", and 3 hours after distilled water administration for test animals of "dipeptide nonadministration / nonexercise group" Thirty minutes later, whole blood was drawn from the abdominal descending vena cava under isoflurane anesthesia and sacrificed.

  Thereafter, the lower extremity skeletal muscle was quickly collected and weighed. BDNF concentration (ng / g) which is an evaluation index of cognitive function was measured using collected skeletal muscle.

[The exercise load method]
Under isoflurane anesthesia, rats were fixed in a prone position on a dedicated fixed base of a device that gives exercise load by electrical stimulation. The knees of the right hind leg were fixed by compression and the ankle joint was fixed to the foot plate. A skin electrode was attached to the calf and forced to exercise by electrical stimulation. Forced exercise was performed at a pace of electrical stimulation once every 8 seconds, and a total of 5 repetitions were made into one set, and 10 sets (50 electrical stimulations in total) were performed in the exercise group. The exercise load was isometric contraction load (ISO).

(2) Results The results are shown in FIG. From the comparison between (b) “dipeptide non-administration / exercise group” and (a) “dipeptide non-administration / non-exercise group” in FIG. 1, it was confirmed that the exercise raises the BDNF concentration in the muscle. In addition, from the comparison between (b) "dipeptide non-administration / exercise group" and (c) "dipeptide administration / exercise group", taking test sample (WPH) in addition to exercise compared with exercise alone It was confirmed that the BDNF concentration in muscle was further increased. Furthermore, from the comparison between (a) "dipeptide non-administration / non-exercise group" and (c) "dipeptide administration / exercise group", the subject sample is taken and exercised, and neither intake nor exercise is performed. In comparison, it was confirmed that the BDNF concentration in the muscle was significantly higher (Student's t test: P <0.05).

  From this, it was shown that WPH containing the dipeptide targeted by the present invention exerts an effect of promoting BDNF production in skeletal muscle by combining its internal use and exercise. BDNF is known to pass through the blood-brain barrier (Pardridge WM et al. Pharm Res. 11 (5): 738-46 (1994). Therefore, BDNF produced in muscle is a blood brain. It is thought that it moves into the brain through the barrier and contributes to the suppression of cognitive decline, that is, expecting the effect of suppressing the cognitive decline by ingesting the whey protein hydrolyzate of the present invention and exercising it. Can.

Test example 2
The present study was conducted to evaluate the influence of WPH intake on cognitive function using BDNF concentration in muscle as an index. In the test, when WPH is ingested in rats, the concentration of BDNF in skeletal muscle is measured in the case of ingesting water or whey protein before hydrolyzate instead of whey protein hydrolyzate Then compared. Also, similarly, when exercise is exercised by taking WPH, the concentration of BDNF contained in skeletal muscle is measured when exercise is exercised by taking whey protein before hydrolyzate instead of WPH and taking exercise. We also compared and confirmed the effects of combined use with exercise.

(1) Test method (1-1) Preparation of test sample (test food, comparative test food) In the test food of Test Example 2, similarly to Test Example 1, a commercially available WPC (WPC used in Production Example 1) and The same WPH (dry powder) obtained by hydrolysis with protease was used (see Table 12). The said WPH was melt | dissolved in distilled water so that solid content concentration might be 10 w / v%, and this was made into the test sample (test food). On the other hand, whey protein before hydrolysis (commercially available WPC, same as WPC used in Production Example 1) was used as a comparative test food. The test food (WPH) and the comparative test food (whey protein) were all dosed in such a way that the dose converted to protein content was 1.0 g protein / kg body weight.

(1-2) Preparation of Test Animal As a test animal, 8-week-old male SD rat was used. Pre-breeding for 4 to 5 days was performed beforehand, and after the pre-breeding, the lower right thigh was removed under isoflurane anesthesia by the day before the test. In addition, fasting was performed for 16 hours from the day before the test.

On the day of the test, grouping was performed according to the following, so that the weight was uniform (about 290 to 310 g).
[Test group (all 6 groups)]
<Exercise group>
(1) "Water administration / exercise group" (n = 5): exercise load 50 times, water administration (2) "WPC administration / exercise group" (n = 5): exercise load 50 times, comparative test food (whey Protein) at a dose of 1.0 g protein / kg body weight (3) “WPH administration / exercise group” (n = 5): 50 exercise loads, 1.0 g protein / kg body weight of test food (WPH).
<Non-motor group>
(I) "water administration / non-exercise group" (n = 5): no exercise load, water administration (ii) "WPC administration / non-exercise group" (n = 5): no exercise load, comparative test food (whey protein A) 1.0 g protein / kg body weight administration (iii) “WPH administration / non-exercise group” (n = 5): no exercise load, 1.0 g protein / kg body weight administration of test diet (WPH).

(1-3) Test The test animals in the above exercise group were given a single oral administration of water or a test sample, and after 15 minutes, exercise stress by electrical stimulation was forcibly given to the right lower leg under isoflurane anesthesia. The exercise load method is as described in Test Example 1. The test animals in the non-exercise group were orally administered water or a test sample once, and after 15 minutes, were subjected to isoflurane anesthesia for the same time as the exercise group. One hundred and eighty minutes after the end of exercise load for the exercise group, and 180 minutes after the end of anesthesia for the non-exercise group, all blood was collected from the abdominal descending vena cava under isoflurane anesthesia and sacrificed. Thereafter, the lower extremity skeletal muscle was quickly collected and weighed. BDNF concentration (ng / g) which is an evaluation index of cognitive function was measured using collected skeletal muscle.

(2) Results The results of the exercise group and the non-exercise group are shown in FIGS. 2 and 3, respectively. As shown in FIG. 2, (1) the test animals in the “WPC administration / exercise group” show a slight increase in the BDNF concentration in muscle, compared with the test animals in the “water administration / exercise group” (1) There was no difference. On the other hand, it is confirmed that (3) the test animals in the "WPH administration / exercise group" significantly increase the BDNF concentration in muscle as compared to the test animals in the (1) "water administration / exercise group" It was done. Moreover, as shown in FIG. 3, the same applies to the non-exercise group, and (i) the test animal of the “WPC administration / non-exercise group” compared to the test animals of the “water administration / non-exercise group” Although, although the BDNF concentration in muscle increased, no significant difference was observed, (iii) the test animals in the "WPH administration / non-exercise group" were (i) in the "water administration / non-exercise group" It was confirmed that the muscle BDNF concentration was significantly increased as compared to the test animals.

  From the results of Test Examples 1 and 2 described above, it was confirmed that a significant increase in the BDNF concentration in muscle due to intake of the test sample (dipeptide-containing WPH) is caused regardless of the presence or absence of exercise. Such remarkable effects can be achieved by dipeptides (Ile-Leu, Leu-Leu, Val-Leu, Ile-Val, Ile-Ile, Leu-Val, Leu-Ile, Ile-Asn, Leu-Ala, Leu-Glu, Ala- Uniquely recognized by WPH uptake containing Leu, or / and Lys-Ile), not uptake of whey proteins not containing these dipeptides. That is, the dipeptide-containing WPH of the present invention can be taken either in combination with exercise (eg, before or during exercise) or by taking it without exercise (intake under exercise), It was confirmed that BDNF production in muscle is promoted and the body content of BDNF is increased. As described above, it is suggested that BDNF is effective in suppressing cognitive decline, improving memory and learning performance, suppressing obesity, glucose metabolism reduction, and the like. Therefore, the dipeptide-containing WPH of the present invention is considered to be effective for suppressing cognitive decline, improving memory and learning performance, suppressing obesity, or suppressing glucose metabolism depression through the action of promoting BDNF production in the body.

Claims (13)

  A composition for promoting the increase in brain-derived neurotrophic factor (BDNF) amount in the body, comprising whey protein hydrolyzate as an active ingredient, wherein the whey protein hydrolyzate is prepared from Ile-Leu, Leu-Leu and Val-Leu In addition to at least one dipeptide selected from the group consisting of Ile-Leu, Leu-Leu, and Val-Leu, which contains at least one dipeptide selected from the group consisting of A composition for promoting BDNF increase, which comprises at least one dipeptide selected from the group consisting of Ile-Ile, Leu-Val, and Leu-Ile.   The whey protein hydrolyzate is one containing the Ile-Leu, Leu-Leu, and Val-Leu dipeptides, or in addition to the Ile-Leu, Leu-Leu, and Val-Leu dipeptides, -A composition for promoting the increase in the amount of BDNF, which contains the dipeptide of Val, Ile-Ile, Leu-Val, and Leu-Ile.   The composition for promoting BDNF dose increase according to claim 1 or 2, which is orally administered or ingested to a mammal including a human before or during exercise.   The composition for promoting BDNF level increase according to claim 3, which promotes an increase in the level of intramuscular BDNF during or after exercise.   The composition for promoting the increase in the amount of BDNF according to claim 1 or 2, which is orally administered or ingested to a mammal including a non-exercise human.   The composition for promoting increase in the amount of BDNF according to any one of claims 1 to 5, which is orally administered or ingested to a person with cognitive impairment or a preparatory group thereof.   The composition for promoting BDNF amount increase according to claim 6, which is a composition for suppressing or improving the decrease in cognitive function.   The composition for promoting the increase in the amount of BDNF according to any one of claims 1 to 7, which is orally administered or ingested to an obese person and / or a person having impaired glucose metabolism, or a preparatory group thereof.   The composition for promoting BDNF amount increase according to claim 8, which is a composition for suppressing or improving obesity, and / or a composition for suppressing or improving reduction in glucose metabolism.   The composition for promoting BDNF amount increase according to any one of claims 1 to 9, which is food and drink.   A composition used for promoting the increase in the amount of BDNF in the body of a subject, wherein the composition contains at least one dipeptide selected from the group consisting of Ile-Leu, Leu-Leu, and Val-Leu. Whey protein hydrolyzate or at least one dipeptide selected from the group consisting of Ile-Leu, Leu-Leu, and Val-Leu, as well as Ile-Val, Ile-Ile, Leu-Val, and Leu- The above composition, wherein whey protein hydrolyzate containing at least one dipeptide selected from the group consisting of Ile is used as an active ingredient.   The composition for promoting the increase in the amount of BDNF is a composition used to suppress or improve the decrease in cognitive function, or to suppress or improve obesity or / and suppress or improve the decrease in glucose metabolism The composition for promoting BDNF amount increase according to claim 11, which is a composition used for   Whey protein hydrolysate containing at least one dipeptide selected from the group consisting of Ile-Leu, Leu-Leu and Val-Leu, or selected from the group consisting of Ile-Leu, Leu-Leu and Val-Leu A composition comprising a whey protein hydrolyzate containing at least one dipeptide selected from the group consisting of Ile-Val, Ile-Ile, Leu-Val, and Leu-Ile in addition to at least one dipeptide to be Is administered or taken to the subject to suppress or improve the cognitive function of the subject, to improve memory or learning performance, to suppress or improve obesity, or / and to reduce glucose metabolism How to suppress or improve.