Agent for enhanced pumping up or enhanced motivation

Abstract

This agent for enhanced pumping up or for enhanced motivation contains a casein hydrolysate as an active ingredient, the hydrolysate containing a tripeptide of at least one of Ile-Pro-Pro and Val-Pro-Pro. The agent for enhanced pumping up contains at least one tripeptide of Ile-Pro-Pro or Val-Pro-Pro as an active ingredient.

Description

Agents for improving pumping or motivation

[0001] This disclosure claims the priority of Japanese Patent Application No. 2024-220364, the content of which is incorporated by reference into the description of this application.

[0002] This disclosure relates to agents for improving pumping or motivation.

[0003] Patent Document 1 discloses that an enzyme group containing various peptidases and proteases can be used to hydrolyze casein contained in mammalian milk (for example, cow's milk) to 2.1 or less in terms of the number of amino acid residues to obtain a casein hydrolyzate. It is also disclosed that this casein hydrolyzate contains a dipeptide having an Xaa-Pro sequence and a tripeptide having an Xaa-Pro-Pro sequence (for example, Ile-Pro-Pro and Val-Pro-Pro, etc.). Patent Document 1 explains that these peptides are expected to have high resistance to degradation by in vivo peptidases due to having a Pro residue at the C-terminus, and there is a high possibility of fully exerting the function of the peptide in vivo.

[0004] Also, generally, each of the two types of tripeptides, Ile-Pro-Pro and Val-Pro-Pro, is known to have biological regulatory functions such as angiotensin-converting enzyme (ACE) inhibitory activity, blood pressure lowering effect, NO (nitric oxide) production promoting effect, and vasodilatory effect, and is also referred to as "lactotripeptides (LTP)". In general, a biological regulatory function is a function that can regulate some physiological action in a living body and is also called the third function of food.

[0005] Japanese Patent No. 5341300

[0006] An object of this disclosure is to find a new biological regulatory function in lactotripeptides and casein hydrolyzates and provide an agent for use based on the found biological regulatory function.

[0007] To provide a basic understanding of some of the features of this disclosure, a brief overview of the disclosure is provided below. This overview is not intended to outline the scope of the disclosure. It is not intended to identify or limit the main or important elements of the disclosure. Its purpose is solely to present some of the fundamental concepts of the disclosure in a simplified form as an introduction to the more detailed explanations that follow.

[0008] An agent according to one embodiment contains a hydrolyzed casein as an active ingredient, wherein the hydrolyzed casein contains at least one of the tripeptides Ile-Pro-Pro and Val-Pro-Pro, and is an agent for improving pump-up or motivation.

[0009] Furthermore, another embodiment of the agent is an agent for improving pump-up, containing at least one of the tripeptides Ile-Pro-Pro and Val-Pro-Pro as an active ingredient.

[0010] The aforementioned and other features of this disclosure will become clear from the description and drawings illustrating the embodiments of this disclosure, as follows. Figure 1 is a flow chart showing each step of the method for producing the agent according to one embodiment. Figure 2 is a graph showing the thigh circumference immediately before exercise load for test group A (2.0 mg of Ile-Pro-Pro and 1.4 mg of Val-Pro-Pro ingested daily, n=12) and control group C (placebo, n=12) on the first day of test food intake (0w) and four weeks later (4w). Figure 3 is a graph showing lean body mass for test group A (2.0 mg of Ile-Pro-Pro and 1.4 mg of Val-Pro-Pro ingested daily, n=12) and control group C (placebo, n=12) on the first day of test food intake (0w) and four weeks later (4w). Figure 4A is a graph showing the change in body weight (Δ0-4w) from the start of test food intake (0w) to four weeks later (4w) for test group A (2.0 mg of Ile-Pro-Pro and 1.4 mg of Val-Pro-Pro were taken daily, n=12) and control group C (placebo, n=12). Figure 4B is a graph showing the change in BMI (Δ0-4w) from the start of test food intake (0w) to four weeks later (4w) for test group A (2.0 mg of Ile-Pro-Pro and 1.4 mg of Val-Pro-Pro were taken daily, n=12) and control group C (placebo, n=12). Figure 5 is a graph showing the change (Δ0 - 4w) in thigh circumference measurements taken immediately after exercise load from the start of test food intake (0w) to four weeks later (4w) for test group A (2.0 mg Ile-Pro-Pro and 1.4 mg Val-Pro-Pro taken daily, n=12) and control group C (placebo, n=12). Figures 6A, 6B, and 6C are graphs showing the aggregated results of questionnaire responses scores from the start of test food intake (0w) to four weeks later (4w) for test group A (2.0 mg Ile-Pro-Pro and 1.4 mg Val-Pro-Pro taken daily, n=12) and control group C (placebo, n=12). Of these, Figure 6A is a graph showing the score for motivation to perform strength training immediately before exercise load.Figure 6B is a graph showing the score of motivation to perform strength training immediately after exercise. Figure 6C is a graph showing the score of motivation to perform strength training at the time of waking up the day after exercise. Figure 7 is a graph showing the change in score (Δ0 - 4w) from the start of test food intake (0w) to four weeks later (4w) for the score of motivation to perform strength training at the time of waking up the day after exercise for test group A (2.0 mg of Ile-Pro-Pro and 1.4 mg of Val-Pro-Pro ingested daily, n=12) and control group C (placebo, n=12). Figure 8 is a graph showing the aggregated scores of questionnaire responses regarding the bump-up effect immediately after exercise load, at the start of the test diet intake (0w) and four weeks later (4w), for test group B (1.0 mg of Ile-Pro-Pro and 0.7 mg of Val-Pro-Pro were ingested daily, n=10) and control group C (placebo, n=12). Figure 9 is a graph showing the correlation between the change in questionnaire response scores regarding the bump-up effect immediately after exercise load from the start of the test diet intake (0w) to four weeks later (4w) (Δ0-4w, vertical axis of the graph) for 34 test subjects, and the change in the value obtained by subtracting the thigh circumference measurement immediately before exercise load from the thigh circumference measurement immediately after exercise load (Δ0-4w, horizontal axis of the graph) from the start of the test diet intake (0w) to four weeks later (4w). Figure 10 is a graph showing the correlation between the change in the thigh circumference measurement obtained by subtracting the thigh circumference measurement immediately before exercise load from the thigh circumference measurement immediately before exercise load for 34 test subjects, from the start date of test food intake (0w) to four weeks later (4w) (Δ0 - 4w, vertical axis of the graph), and the change in the FMD measurement value at the time immediately before exercise load from the start date of test food intake (0w) to four weeks later (4w) (Δ0 - 4w, horizontal axis of the graph).Figures 11A and 11B are two graphs showing the correlation between the change in thigh circumference (Δ0 - 4w, vertical axis of the graph) from the start of test food intake (0w) to four weeks later (4w) for 34 test subjects, and the change in blood pressure measured immediately before exercise from the start of test food intake (0w) to four weeks later (4w), respectively. Figure 11A shows the case where the blood pressure measured on the horizontal axis is systolic blood pressure. Figure 11B shows the case where the blood pressure measured on the horizontal axis is diastolic blood pressure.

[0011] The embodiments will be described below with reference to the drawings. The items depicted in each figure are for illustrative purposes only. The embodiments are not limited to those shown in the figures. In this specification, the tripeptide of Ile-Pro-Pro or Val-Pro-Pro will also be referred to as "LTP". In this specification, an agent containing hydrolysate of casein as an active ingredient will also be referred to as "the first agent", and an agent containing LTP as an active ingredient will also be referred to as "the second agent". When "content" as described in this specification refers to the content of two or more components, it refers to the total content of those two or more components. For example, "content of free amino acids and peptides" means the total content of free amino acids and peptides. In this specification, "peptides" such as LTP will refer to peptides and their salts. In this specification, "free amino acids" will refer to free amino acids and their salts. Here, the salt may be any water-soluble salt that is pharmacologically acceptable for oral intake, such as sodium salts or potassium salts.

[0012] An agent according to one embodiment contains a hydrolyzed casein product as an active ingredient, and this hydrolyzed product contains LTP, and is an agent for improving pump-up or motivation, and is hereinafter also referred to as "the first agent".

[0013] The first agent may be obtained, for example, by the manufacturing method S1 illustrated in Figure 1. Manufacturing method S1 may include a raw material preparation step S2, an enzyme treatment step S3, an inactivation step S4, and a crude purification step S5.

[0014] In the raw material preparation step S2, casein, which will be the raw material for the first agent, and a group of enzymes to be used for enzymatic treatment are prepared. Casein is the main protein contained in animal milk. As casein, for example, animal milk casein, which is casein contained in cow's milk, mare's milk, goat's milk, or sheep's milk, can be used, and it is preferable to use cow's milk casein.

[0015] The enzyme group can be any combination of various peptidases and proteases that can be appropriately combined so that a large number of free amino acids, dipeptides, and tripeptides can be produced by the hydrolysis of casein. Preferably, the enzyme group includes a protease capable of cleaving the Pro-Xaa sequence at the C-terminal end of the Xaa-Pro-Pro-Xaa sequence. Preferably, the protease capable of cleaving the Pro-Xaa sequence has an isoelectric point in the acidic range. Furthermore, preferably, the enzyme group includes a serine-type proteinase having a serine residue in its active site, or a metal proteinase having a metal element in its active site. Examples of metal proteinases include neutral protease I, neutral protease II, or leucine aminopeptidase. The enzyme group preferably includes, for example, a combination of an extracellular enzyme derived from Aspergillus oryzae that is capable of cleaving Pro-Xaa, a peptidase, a leucine aminopeptidase, at least one neutral protease selected from neutral protease I and neutral protease II, a metalloprotease, and a serine protease.

[0016] As the enzyme group, for example, commercially available enzyme preparations derived from Aspergillus oryzae may be used. Examples of such enzyme preparations include Sumizyme FP, LP, or MP manufactured by Shin Nippon Chemical Co., Ltd., Umamizyme manufactured by Amano Enzyme Co., Ltd., Sternzyme B11024 or PROHIDROXY AMPL manufactured by Higuchi Shokai Co., Ltd., Orientase ONS manufactured by Hankyu Bioindustry Co., Ltd., or Denatzyme AP manufactured by Nagase Biochemical Co., Ltd. Note that "Sumizyme," "Orientase," and "Denazyme" are registered trademarks. The enzyme group is preferably one whose isoelectric point is in the acidic range. A person skilled in the art can appropriately adjust the conditions such as the amount of enzyme used, reaction time, pH, and temperature so that a large number of free amino acids, dipeptides, and tripeptides can be produced, referring to the optimal conditions published by the manufacturer for the enzyme preparation used.

[0017] In the enzyme treatment step S3, an enzyme reaction solution is prepared by mixing a suspension of casein in water with a group of enzymes, and an enzyme treatment is performed to hydrolyze the casein. The casein concentration in the enzyme reaction solution is not particularly limited as long as it does not contradict the purpose of this disclosure, but from an efficiency standpoint, it is preferably 3% by mass or more and 19% by mass or less. Furthermore, the enzyme reaction solution may be mixed so that the content ratio of the pure mass of the enzyme group to the mass of casein (pure mass of the enzyme group / mass of casein) is, for example, 0.001 or more and 0.1 or less, preferably 0.01 or more and 0.1 or less, and more preferably 0.025 or more and 0.1 or less. The conditions for the enzyme treatment can be adjusted as appropriate as described above, and the liquid temperature of the enzyme reaction solution may be, for example, 25°C or more and 60°C or less, preferably 45°C or more and 55°C or less. Similarly, the pH of the enzyme reaction solution may be, for example, 3 or more and 10 or less, 5 or more and 9 or less, preferably 5 or more and 8 or less. Similarly, the enzyme reaction time may be, for example, 2 hours or more and 48 hours or less, and preferably 7 hours or more and 15 hours or less.

[0018] In the deactivation step S4, the enzyme reaction is terminated by deactivating the enzyme in the enzyme reaction solution that has undergone the enzyme treatment step S3. For example, the enzyme reaction solution may be heated to a liquid temperature higher than 60°C and lower than 110°C to deactivate the enzyme. The reaction solution from which the enzyme has been deactivated may be treated as the first agent as is, and in this case, the first agent may be added to a food composition such as a beverage for consumption. Alternatively, the reaction solution from which the enzyme has been deactivated may be subjected to the crude purification step S5 described below.

[0019] In the crude purification step S5, the reaction solution that has gone through the deactivation step S4 may be subjected to any treatment as necessary to remove any undesirable components or to relatively increase the LTP content. For example, the LTP content may be relatively increased by concentrating the reaction solution that has gone through the deactivation step S4. Examples of concentration methods include vacuum concentration, freeze-drying, spray drying, or centrifugal drying. In addition, precipitates, bitter components, and odor components may be removed from the reaction solution that has gone through the deactivation step S4. The precipitate can be removed, for example, by centrifugation or filtration using various filters. The bitter components and odor components can be removed, for example, by using activated carbon or hydrophobic resin. When using activated carbon, an amount of activated carbon of 1% to 20% by mass of the casein used as the raw material can be added to the hydrolysate, and the added activated carbon can be removed along with the bitter components and odor components by treatment such as centrifugation or filtration after 1 to 10 hours. The reaction solution obtained through these processes may be used as the first agent as is, or the first agent in this case may be added to a food composition such as a beverage and made edible.

[0020] In the crude purification step S5, it is preferable to concentrate and dry the reaction solution that has gone through the deactivation step S4, or the reaction solution that has gone through the above-mentioned centrifugation or filtration, and treat the resulting powder as the first agent. When the first agent is in powder form, it may be used as is for food consumption, or it may be used as an additive to various agents or as an additive to pharmaceutical compositions. The first agent may be mixed with auxiliary additives to improve nutritional balance or flavor, etc. Examples of auxiliary additives include various carbohydrates, lipids, vitamins, minerals, sweeteners, flavorings, colorings, texture improvers, etc. The food composition to which the first agent is added is not particularly limited as long as it does not contradict the purpose of this disclosure, but examples include beverages, yogurt, liquid foods, jellies, candies, retort foods, tablets, cookies, sponge cakes, bread, or supplements. When the first agent is in powder form, it may be molded into any form that can be taken orally, such as tablets, pills, capsules, microcapsules, powders, granules, or liquids. Furthermore, the first agent may, if necessary, contain at least one of the following: excipients, adjuvants, preservatives, stabilizers, binders, pH adjusters, buffers, thickeners, gelling agents, preservatives, or antioxidants.

[0021] As illustrated in the description of the manufacturing method S1 above, the hydrolyzed casein contained in the first agent may be, for example, the reaction solution itself obtained by enzymatically hydrolyzing casein to produce LTP and then inactivating the enzyme, or it may be a concentrate of this reaction solution, preferably a powder obtained by concentrating and drying this reaction solution.

[0022] The LTP contained in the first agent may be, for example, only a tripeptide having an Ile-Pro-Pro sequence, or only a tripeptide having a Val-Pro-Pro sequence. Casein's amino acid sequence contains many Xaa-Pro-Pro sequences (e.g., Ile-Pro-Pro sequences or Val-Pro-Pro sequences). Therefore, the first agent containing hydrolysate of casein usually contains both Ile-Pro-Pro and Val-Pro-Pro tripeptides. Generally, the Ile-Pro-Pro tripeptide is also called L-isoleucyl-L-prolyl-L-proline or IPP. The Val-Pro-Pro tripeptide is also called L-valyl-L-prolyl-L-proline or VPP.

[0023] In this specification, "pump-up" refers to the physiological phenomenon in which skeletal muscles, subjected to load by exercise, temporarily expand and increase in volume after the exercise. Furthermore, in this specification, "improved pump-up" refers to an increase in the degree of temporary volume increase of skeletal muscles due to pump-up. Pump-up is a term that has traditionally been mainly used in fields related to strength training, and it is said to originate from "PUMP UP". There are still many aspects of the mechanism by which pump-up occurs that have not been precisely elucidated. In terms of mechanism, it is thought that when skeletal muscles are subjected to a heavy load by exercise, metabolic byproducts such as lactic acid accumulate in the skeletal muscle cells, increasing the intracellular osmotic pressure, and at the same time, blood flow in the skeletal muscles temporarily increases to make it easier to supply nutrients to the cells, causing the skeletal muscles to appear swollen (as if they were being inflated by a pump) due to the water and blood, that is, a temporary pump-up occurs after exercise. To explain in more detail, when skeletal muscles contract strongly during exercise, the veins that carry blood from the skeletal muscles are compressed, while the arteries continue to supply blood to the skeletal muscles as before exercise. As a result, the blood volume in the skeletal muscles increases, and plasma leaks from the capillaries of the skeletal muscles into the interstitial space. The extracellular pressure increased by the accumulation of plasma in the interstitial space triggers the return of plasma to the skeletal muscle cells. Furthermore, metabolic byproducts accumulated in the skeletal muscle cells due to exercise increase the intracellular osmotic pressure, making the aforementioned return to the cells even more likely, leading to phenomena such as cell swelling or pump-up. However, the mechanism by which blood flow temporarily increases in localized skeletal muscles where intracellular metabolic byproducts have accumulated after exercise, rather than in the heart or the whole body during exercise, is not adequately explained, and much of this mechanism of action remains unclear from a physiological and molecular biological perspective. Furthermore, it has been observed that the volume expansion of skeletal muscle due to pump-up generally peaks around 10 to 20 minutes after strength training, then rapidly decreases, and disappears within a few hours. On the other hand, muscle hypertrophy is a physiological phenomenon in which, when muscles are subjected to a heavy load through exercise, the muscles themselves grow in an adaptable manner to that load, resulting in the muscles becoming permanently thicker.Pump-up can occur in skeletal muscles during the process from exercise to muscle hypertrophy, but since it is merely a temporary phenomenon after exercise, those skilled in the art clearly distinguish it as a physiological phenomenon distinct from permanent muscle hypertrophy (muscle tissue growth).

[0024] The exercises that cause a pump-up are not particularly limited as long as they do not contradict the purposes of this disclosure, as long as they place a load on the skeletal muscles that causes a temporary volume expansion of the skeletal muscles after the exercise. In the daily lives of humans, for example, activities such as walking or cycling for walking, commuting, going to school, going to work or shopping, or activities that require relatively strong muscles such as climbing stairs, carrying luggage or doing heavy labor, may in some cases be considered exercises that place a load on the skeletal muscles that causes a temporary volume expansion of the skeletal muscles. Preferably, the exercises here include strength training or athletic competitions, and more preferably strength training. Examples of athletic competitions include walking, jogging, running, marathon, swimming, aerobics, rowing, baseball, tennis, table tennis, soccer, basketball, volleyball, dancing, archery, cycling, bicycle racing, skiing, skating, skateboarding, trekking, mountain climbing, diving or skydiving. Examples of strength training include bodyweight training, which uses the exerciser's own body weight as a load, free weight training, which uses equipment such as barbells or dumbbells to apply a load, or machine training, which uses machines to apply a load. Examples of bodyweight training include push-ups, squats, or planks. Examples of free weight training include dumbbell squats, deadlifts, or bench presses. Examples of machine training include chest presses, lat pulldowns, or leg presses. The exercise that causes the pump may be one of the candidates exemplified here, or a combination of two or more. Furthermore, the exercise that causes the pump may be either anaerobic or aerobic.

[0025] The subjects who will benefit from the continuous oral intake of the first agent (those who take the first agent) are not particularly limited as long as it does not contradict the purposes of this disclosure, but are preferably those who perform strength training aimed at muscle hypertrophy at a frequency of at least once every two weeks.

[0026] The mechanism by which continuous oral intake of the first agent improves muscle pump is unknown, but it is thought to be as follows. First, LTP contained in the first agent is conventionally known to be a functional component that can help in the primary prevention of hypertension (making it less likely to develop) due to its ACE inhibitory activity and suppression of blood pressure elevation (see Patent Document 1). It is also conventionally known that LTP is a functional component that has NO production promoting effect and vasodilatory effect. These known functions of LTP can be exerted to keep blood pressure at a relatively low level throughout the body at a constant rate over a long period of several months to several years. However, based on the inventors' idea that LTP and hydrolysates containing LTP may have a latent function of improving specific blood flow in localized skeletal muscles that have been stressed by exercise, for example, for a short period of time such as 20 minutes after exercise, the following tests were conducted and the effect of improving muscle pump after exercise was confirmed.

[0027] Regarding the indication that a product is for enhancing muscle pump, examples of phrases that may be used for the first agent include, but are not limited to, phrases such as "it pumps up," "makes muscles plump," "makes muscles look bigger after exercise," "makes muscles bulge during exercise," "makes muscles swell during exercise," "pushes muscles to their limits during exercise," "it's effective for pumping up," and "ideal for bodybuilding."

[0028] In this specification, "increased motivation" refers to the physiological phenomenon in which a person who continuously takes the first agent orally experiences an increased motivation to engage in any activity involving the active contraction of skeletal muscles (moving at least a part of one's own body of one's own volition). The activities targeted for increased motivation here, from the perspective of involving the active contraction of skeletal muscles, may be, for example, the aforementioned locomotion activities or the aforementioned activities requiring relatively high muscle strength, but are preferably strength training or athletic competitions, and more preferably strength training. The subjects whose motivation is increased by continuous oral intake of the first agent (those who take the first agent) are not particularly limited as long as it does not contradict the purposes of this disclosure, but are preferably those who perform strength training aimed at muscle hypertrophy at a frequency of at least once every two weeks. Furthermore, the increased motivation here is preferably an increased willingness to perform strength training or athletic competitions on a daily basis, and more preferably an increased willingness to perform strength training on a daily basis.

[0029] The mechanism by which the first agent improves motivation through continuous oral intake is unknown, but it is thought to be as follows. First, as mentioned above, the previously known functionality of LTP is related to the primary prevention of hypertension, and it is unlikely that it could significantly influence the phenomenon of motivation improvement. Also, generally, exercise that puts a load on the muscles that causes a pump often results in muscle soreness, which leads to a decrease in the motivation to engage in any kind of activity on a daily basis. In contrast, it is thought that oral intake of the first agent may contribute to improved motivation by having the LTP and various oligopeptides (e.g., dipeptides, tripeptides other than LTP, etc.) contained in the first agent exert some effect that contributes to the improvement of brain function (e.g., increased cerebral blood flow).

[0030] Regarding the indication that a product is intended to improve motivation, examples of phrases that may be used for the first agent include, but are not limited to, phrases such as "to inspire enthusiasm," "to boost motivation," "to get motivated," "to switch on motivation," "to give a pep talk," "to improve enthusiasm," "to enhance motivation," "to ignite the fuse," "to inspire," and "to arouse interest."

[0031] The first agent is preferably intended to improve pump-up and also to improve motivation. In this case, the labeling of the first agent may be a combination of the aforementioned statement indicating that it is for improving pump-up and the statement indicating that it is for improving motivation.

[0032] From the viewpoint of easily exhibiting the functionality of LTP, in the first agent, when the content of peptides and free amino acids contained in the hydrolyzed casein is 100 parts by mass, the content of LTP is preferably 0.3 parts by mass or more, more preferably 0.5 parts by mass or more, and even more preferably 1.0 part by mass or more. When the first agent contains only Ile-Pro-Pro or Val-Pro-Pro as LTP, the "LTP content" in this specification refers to the content of only Ile-Pro-Pro or Val-Pro-Pro. When the first agent contains both Ile-Pro-Pro and Val-Pro-Pro as LTP, the "LTP content" in this specification refers to the total content of Ile-Pro-Pro and Val-Pro-Pro.

[0033] In this specification, the terms "ingestion of Ile-Pro-Pro and Val-Pro-Pro tripeptides" and "LTP intake" refer to the intake of only one tripeptide if the first agent contains only one of Ile-Pro-Pro or Val-Pro-Pro, and the total intake of both tripeptides if the first agent contains both Ile-Pro-Pro and Val-Pro-Pro tripeptides. The first agent may be administered so that the daily LTP intake of the inhaler is, for example, 1.0 mg / day or more, but from the viewpoint of easily exerting the biological regulatory function by LTP intake, it is preferably administered at a rate of 1.7 mg / day or more, more preferably at a rate of 2.6 mg / day or more, and even more preferably at a rate of 3.4 mg / day or more. As a specific example, the first agent may be taken by the inhaler in such a way that, for example, the daily intake of Val-Pro-Pro is 0.4 mg / day or more and the daily intake of Ile-Pro-Pro is 0.6 mg / day or more, preferably the daily intake of Val-Pro-Pro is 0.7 mg / day or more and the daily intake of Ile-Pro-Pro is 1.0 mg / day or more, more preferably the daily intake of Val-Pro-Pro is 1.1 mg / day or more and the daily intake of Ile-Pro-Pro is 1.5 mg / day or more, and even more preferably the daily intake of Val-Pro-Pro is 1.4 mg / day or more and the daily intake of Ile-Pro-Pro is 2.0 mg / day or more. Furthermore, the first agent may be taken so that the daily intake of LTP by the inhaler is, for example, 1,000 mg / day or less, 100 mg / day or less, or 10 mg / day or less.

[0034] From the viewpoint of facilitating the expression of LTP and the functionality of various oligopeptides (dipeptides and tripeptides, etc.) contained in casein hydrolysates in addition to LTP, it is preferable that the average chain length of the peptides and free amino acids contained in the hydrolysate of the first agent is 2.1 or less in terms of the number of amino acid residues. Here, "average chain length" is the ratio of the number of moles of total amino acids in the same mass of acid hydrolysate of casein to the total number of moles of peptides and free amino acids produced when casein is enzymatically hydrolyzed. Note that "acid hydrolysate of casein" is obtained by breaking down casein protein into free amino acids.

[0035] In this specification, the value of "average chain length" is calculated from the measured value obtained by measuring the mol concentration of free amino groups in the hydrolysate using the OPA method, which uses o-phthalaldehyde (OPA) that reacts with amino groups to produce color, as described below. In the OPA method, an OPA solution is prepared by dissolving 40 mg of o-phthalaldehyde (special grade reagent for fluorescence analysis, manufactured by Nacalai Tesque) in 1 ml of methanol and adding 100 μl of β-mercaptoethanol. In addition, an OPA reagent is prepared by diluting the above OPA solution to 25 ml using a solution prepared in advance by adding 2.5 ml and 20% by mass of sodium dodecyl sulfate to 25 ml of 100 mM sodium tetraborate solution, and then adding distilled water to adjust the total volume to 50 ml. Next, as preparation for the OPA method, the sample containing the casein hydrolysate is dried into a powder using a spray dryer (this drying process can be omitted if the sample is already in powder form). A suitable solvent is added to the powdered sample, and an amount of the powdered sample equivalent to the amount at which the pre-decomposition casein concentration becomes 1% by mass is dissolved. The mixture is then centrifuged at 15,000 rpm for 10 minutes, and 50 μl of the supernatant is collected. 1 ml of the above OPA reagent is added to the collected supernatant, and the mixture is thoroughly mixed. After standing at room temperature for 5 minutes, the absorbance at a wavelength of 340 nm is measured using a spectrophotometer (product name Ubest-35, manufactured by JASCO Corporation). The calibration curve is obtained by preparing a 1% by mass casein acid hydrolysate, diluting it appropriately, and measuring the same results to determine the relationship between absorbance and molar concentration. Then, the average chain length is calculated according to the following formula. Average chain length = (molar concentration of 1% by mass of casein acid hydrolysate) / (molar concentration of 1% by mass of casein enzyme hydrolysate for each sample)

[0036] From the viewpoint of easily exhibiting the functionality of LTP and the functionality of various oligopeptides (dipeptides and tripeptides, etc.) contained in casein hydrolysates in addition to LTP, when the total content of peptides and free amino acids contained in the hydrolysate of the first agent is 100 parts by mass, the content of tripeptides having an Xaa-Pro-Pro sequence is preferably 1 part by mass or more, and more preferably 3 parts by mass or more. Examples of tripeptides having an Xaa-Pro-Pro sequence include tripeptides such as Ser-Pro-Pro, Val-Pro-Pro, Ile-Pro-Pro, Leu-Pro-Pro, or Phe-Pro-Pro.

[0037] In this specification, the values ​​for LTP content, LTP intake, and the content of the tripeptide having the Xaa-Pro-Pro sequence described above are calculated based on measurements obtained by liquid chromatography-mass spectrometry (LC-MS / MS) as described below. A sample containing hydrolyzed casein is dried to a powder state using a spray dryer (this drying process can be omitted if the sample is already in powder form). Distilled water is added to the powdered sample to prepare a sample solution by diluting and dissolving it so that the powder mass concentration per unit of solution mass is 50 μg / ml. This sample solution is analyzed by LC-MS / MS under the measurement conditions described below. In addition, five types of solutions are prepared in which the total concentrations of various chemically synthesized standard peptides having the sequences shown in Table 1 below are 30 ng / ml, 60 ng / ml, 120 ng / ml, 240 ng / ml, and 480 ng / ml, respectively, and these are analyzed by LC-MS / MS under the measurement conditions described below. Among the multiple peaks detected by LC-MS / MS, the peak in the sample that matches the molecular weight and retention time of the standard peptide is identified as the peptide having the same sequence as the standard peptide. Based on the comparison results with such standard peptide peaks and the dilution ratio from the original sample to the sample solution for LC-MS / MS measurement, the LTP content or Xaa-Pro-Pro content in the original sample is calculated. <Equipment used> High-performance liquid chromatograph mass spectrometer: ACQUITY UPLC, XeVo-TQD (manufactured by Waters Japan Ltd.), Column: ACQUITY UPLC HSS T3 (particle size 1.8 μm, 2.1 mm I.D. × 100 mm L, manufactured by Waters Japan Ltd.).<Measurement Conditions> Mobile phase A: 0.1% by mass formic acid aqueous solution, Mobile phase B: 0.1% by mass formic acid methanol solution, Time program: 0.1% B (0 min) - 0.1% B (2 min) - 12.0% B (10 min) - 80% B (13 min) - 80% B (14.5 min) - 0.1% B (14.6 min) - 0.1% B (16 min) - STOP (16 min), Sample injection volume: 5 μl, Column temperature: 55°C, Ionization mode: ESI (+), Atomization gas flow rate: 50 L / hour, Cone voltage: 24-30 V, Collision voltage: 25-35 V V, Desolvent temperature: 400°C, Source temperature: 130°C, Analysis mode: MRM measurement, Analysis range: Ser-Pro-Pro (m / z: -299.92 → 70.00), Phe-Pro-Pro (m / z: 359.92 → 70.00), Leu-Pro-Pro (m / z: 326.10 → 70.00), Val-Pro-Pro (m / z: -312.21 → 70.00), Ile-Pro-Pro (m / z: -326.10 → 70.00), Acquisition time: 0.1 sec / Ch.

[0038]

[0039] Preferably, the first agent has the effect of increasing muscle mass when continuously ingested orally by humans. Here, "increased muscle mass" refers to the fact that the aforementioned muscle hypertrophy effect is likely to occur when the agent is continuously ingested orally by a person who exercises regularly. Examples of statements indicating that the first agent has the effect of increasing muscle mass include, but are not limited to, the use of phrases such as "supports the ability to build muscle," "suppresses muscle breakdown," "enhances muscle strength," "suppresses, maintains, improves, and enhances the decline in walking ability," "improves metabolism through muscle strengthening," "suppresses metabolic decline," "prevents and recovers from muscle fatigue," "suppresses and maintains the decline in balance ability," "shapes up," "muscular," "lean and muscular," "beautiful body," "prevents being bedridden," "prevents being bedridden," "prevents falls," "improves metabolism through muscle strengthening," and "maintains muscle mass and strength."

[0040] The duration of intake of the first agent is not particularly limited; for example, it may be taken only once. However, from the viewpoint of easily exerting pump-up-enhancing or motivation-enhancing effects, it is preferable to take it orally at least once a day for a long period of time. The long period of intake may be, for example, 7 days or more, preferably 14 days or more, more preferably 20 days or more, and even more preferably 28 days or more.

[0041] The timing for orally ingesting the first agent can be, for example, before, during, or after exercise during the day. However, from the viewpoint of maximizing the pump-up effect or motivation-enhancing effect, it is preferable to ingest it before exercise during the day, and even more preferably within one hour before exercise during the day. The amount, duration, and timing of intake of the first agent may be determined by the individual taking it. If the individual taking the agent belongs to a sports club or gym, it is preferable to base the decision on the guidance or instructions of a coach, supervisor, trainer, or instructor at the club or gym, from the viewpoint of being able to calmly observe and judge the individual's situation.

[0042] The agent according to the other embodiment is an agent for improving pump-up, containing at least one of the tripeptides Ile-Pro-Pro and Val-Pro-Pro as an active ingredient, and is hereinafter referred to as the "second agent." The second agent may be, for example, a composition of the fraction containing LTP obtained by enzymatically hydrolyzing casein in the same manner as the first agent described above, and fractionating the reaction solution containing LTP by any method. The second agent may also be prepared by enzymatically hydrolyzing any protein source other than casein that has an Ile-Pro-Pro sequence or a Val-Pro-Pro sequence in its amino acid sequence. The second agent may be the first agent, or a composition obtained by recovering LTP from a similar hydrolysate or a composition obtained by concentrating this composition may be used. The second agent may also be prepared by synthesizing LTP by a known peptide synthesis method.

[0043] The subjects (intake subjects of the second agent) to be improved in pump-up by continuous oral intake of the second agent are not particularly limited as long as they do not conflict with the object of the present disclosure, but are preferably those who perform muscle strength training for the purpose of muscle hypertrophy at least once every two weeks. Also, the second agent may be ingested so that the daily LTP intake amount of the intake subject is, for example, 1.0 mg / day or more, preferably 1.7 mg / day or more, more preferably 2.6 mg / day or more, and even more preferably 3.4 mg / day or more. Also, the second agent may be ingested so that the daily LTP intake amount of the intake subject is, for example, 1,000 mg / day or less, 100 mg / day or less, or 10 mg / day or less. In addition, the second agent may have the same matters as those of the first agent described above.

[0044] The pump-up improvement method according to another embodiment uses a person performing exercise as a subject to be improved in pump-up, and causes this subject (intake subject) to ingest the above-described first agent containing a hydrolyzate of casein as an active ingredient and the hydrolyzate containing at least one of Ile-Pro-Pro and Val-Pro-Pro, or the above-described second agent containing at least one of Ile-Pro-Pro and Val-Pro-Pro as an active ingredient, so as to obtain the above-described pump-up improvement effect in this subject (intake subject). In the above pump-up improvement method, as a means for causing the subject (intake subject) to ingest the first agent or the second agent, for example, when selling the first agent or the second agent as a product, a product explanation to the effect that the effect of pump-up improvement can be exhibited by continuous oral intake is given to customers who are candidates for the subject (intake subject) (for example, writing the product explanation text on the packaging of the product, or using the product explanation text in the promotional advertisement of the product, etc.). Also, as a means for causing ingestion, for example, when the subject (intake subject) belongs to various sports clubs or sports gyms, etc., a coach, supervisor, trainer, instructor, etc. of the club or gym guides or instructs the subject (intake subject) to orally ingest the first agent or the second agent.

[0045] The above method for improving muscle pump may involve administering either the first or second agent to the subject (intaker) such that their daily LTP intake is, for example, 1.0 mg / day or more, preferably 1.7 mg / day or more, more preferably 2.6 mg / day or more, and even more preferably 3.4 mg / day or more. Alternatively, the above method for improving muscle pump may involve administering either the first or second agent to the intaker such that their daily LTP intake is, for example, 1000 mg / day or less, 100 mg / day or less, or 10 mg / day or less.

[0046] The above pump-up enhancement method preferably has the effect of increasing muscle mass by continuously taking either the first or second agent orally. In the above pump-up enhancement method, the subject (intaker) who is to be given either the first or second agent orally and whose pump-up is enhanced is not particularly limited as long as it does not contradict the purpose of this disclosure, but preferably is a person who performs strength training for the purpose of muscle hypertrophy at a frequency of at least once every two weeks. The timing of the subject (intaker) taking either the first or second agent orally may be, for example, before, during, or after exercise during the day, but from the viewpoint of easily exerting the pump-up enhancement effect, it is preferably before, and more preferably within one hour before the start of exercise during the day.

[0047] The pump-up enhancement agent or motivation enhancement agent and pump-up enhancement method according to the embodiments described above are as described above, but this disclosure is not limited to the embodiments described above, and various modifications are possible without departing from the gist of this disclosure. Furthermore, the pump-up enhancement agent or motivation enhancement agent and pump-up enhancement method according to this disclosure are not limited by the effects of the embodiments described above. That is, the embodiments disclosed herein should be considered in all respects to be illustrative and not restrictive. The scope of this disclosure is indicated by the claims rather than the above description. Furthermore, the scope of this disclosure is intended to include all modifications in the sense and scope equivalent to the claims.

[0048] This disclosure includes the following embodiments: (1) An agent for improving muscle pump or motivation, comprising a hydrolysate of casein as an active ingredient, wherein the hydrolysate comprises at least one tripeptide of Ile-Pro-Pro and Val-Pro-Pro. (2) The agent according to paragraph 1, wherein the hydrolysate contains 0.3 parts by mass or more of the tripeptide when the total content of peptides and free amino acids is 100 parts by mass. (3) The agent according to paragraph 1 or paragraph 2, wherein the average chain length of the peptides and free amino acids contained in the hydrolysate is 2.1 or less in terms of the number of amino acid residues. (4) The agent according to any one of paragraphs 1 to 3, wherein the hydrolysate contains 1 part by mass or more of the tripeptide having the Xaa-Pro-Pro sequence when the total content of peptides and free amino acids is 100 parts by mass. (5) An agent for improving muscle pump, comprising at least one tripeptide of Ile-Pro-Pro and Val-Pro-Pro as an active ingredient. (Item 6) An agent described in any one of the above items 1 to 5, which has the effect of increasing muscle mass. (Item 7) An agent described in any one of the above items 1 to 6, which is taken so that the total daily intake of Ile-Pro-Pro and Val-Pro-Pro tripeptides by the person taking the agent is 1.0 mg / day or more. (Item 8) An agent described in any one of the above items 1 to 7, which is taken orally by a person who exercises at least once every two weeks. (Item 9) An agent described in any one of the above items 1 to 8, which is taken orally by a person who exercises within one hour of taking the above agent orally. (Item 10) A method for improving muscle pump, comprising orally taking an agent containing a hydrolyzed casein as an active ingredient, wherein the hydrolyzed casein contains at least one of the tripeptides Ile-Pro-Pro and Val-Pro-Pro, to a person who is to be targeted for improved pump. (Archive 11) A method for improving muscle pump, comprising orally administering a drug containing at least one of the tripeptides Ile-Pro-Pro and Val-Pro-Pro as an active ingredient to a person who is to be targeted for improved muscle pump.(Paragraph 12) The pump-up enhancement method described in Paragraph 10 or 11 above, wherein the person to be targeted for pump-up enhancement is a person who exercises at a frequency of at least once every two weeks. (Paragraph 13) The pump-up enhancement method described in any one of Paragraphs 10 to 12 above, wherein the person to be targeted for pump-up enhancement is a person who exercises within one hour of orally ingesting the agent.

[0049] Furthermore, the inventors of this disclosure believe that if they can discover new bioregulatory functions for lactotripeptides and hydrolysates containing lactotripeptides, in addition to ACE inhibitory activity and blood pressure lowering effects, it will lead to the development of new applications for lactotripeptides and casein hydrolysates, and thus they have created the agents and methods of this disclosure. Through diligent research by the inventors of this disclosure, it was found that, with regard to the agent described in paragraph 1 above, when continuously ingested by individuals who regularly engage in exercise that puts a load on skeletal muscles, the degree of temporary muscle expansion related to "pump-up," a physiological phenomenon in which muscles that have been subjected to exercise temporarily increase in volume after exercise, is surprisingly greater. In addition, it was found that, with regard to the agent described in paragraph 1 above, when continuously ingested by the aforementioned individuals, it is surprisingly effective in improving motivation. For this reason, the agent described in paragraph 1 above can be used for the purpose of improving pump-up or improving motivation. Furthermore, the agent described in paragraph 5 above, like the agent described in paragraph 1 above, contains Ile-Pro-Pro or Val-Pro-Pro tripeptides and can be used for the purpose of improving pump-up. Therefore, this disclosure makes it possible to discover novel bioregulatory functions in lactotripeptides and casein hydrolysates, and to provide agents for improving muscle pump or motivation based on the discovered bioregulatory functions.

[0050] The following describes examples of tests related to this disclosure, but this disclosure is not limited to these examples. In order to evaluate the effects of continuous intake of the agent related to this disclosure on improving muscle bulk, motivation, and muscle mass, the following tests were conducted.

[0051] First, a powdered agent consisting of casein hydrolysate was prepared according to manufacturing method S1, as explained with reference to Figure 1, in accordance with the method described in S. Mizuno et.al “Release of Short and Proline-Rich Antihypertensive Peptides from Casein Hydrolysate with an Aspergillus oryzae Protease”, Journal of Dairy Science, 2004, vol.87, No.10, pp.3183-3188 and Patent Document 1. Specifically, a reaction solution was prepared by adding an enzyme group containing peptidase and protease derived from Aspergillus oryzae to a suspension of milk casein in water, allowing the enzymes to react for a predetermined time, and then inactivating the enzymes. This reaction solution was then concentrated and dried to prepare a powdered agent consisting of casein hydrolysate.

[0052] The composition of the prototype powdered agent was analyzed using the various measurement methods described above. The analysis revealed that the powdered agent contained both Ile-Pro-Pro and Val-Pro-Pro as LTPs, and the average chain length of the peptides and free amino acids contained in the agent was 2.1 or less. Furthermore, when the total content of peptides and free amino acids contained in the powdered agent is taken as 100 parts by mass, the total content of Ile-pro-Pro and Val-Pro-Pro was 0.3 parts by mass or more, and the content of tripeptides having the Xaa-Pro-Pro sequence, including Ile-pro-Pro and Val-Pro-Pro, was 1 part by mass or more.

[0053] Furthermore, the prototype powder was mixed with known excipients and lubricants according to a predetermined formulation, and compressed into tablets. These tablets were then coated with a known coating agent to produce prototype tablets A and B, each containing casein hydrolysate with LTP. Tablets A and B differ in the proportion of the powdered agent consisting of casein hydrolysate. In addition, a placebo tablet C was produced under the same conditions as tablet A, except that instead of incorporating casein hydrolysate, sodium caseinate (sodium salt of unhydrolyzed casein protein) was added so that the total amino acid content was equal to that of tablet A. Tablets A, B, and C were produced in such a way that there were no significant differences in energy and sodium content.

[0054] The LTP content was measured for each of the prototype tablets A, B, and placebo tablet C as described above, using the aforementioned LC-MS / MS method. As a result, three tablets (722.4 mg) of tablet A contained 1.4 mg of Val-Pro-Pro tripeptide and 2.0 mg of Ile-Pro-Pro tripeptide. Three tablets (722.4 mg) of tablet B contained 0.7 mg of Val-Pro-Pro tripeptide and 1.0 mg of Ile-Pro-Pro tripeptide. No LTP was detected in placebo tablet C. Furthermore, in the tests described below, the daily intake for each of tablets A, B, and placebo tablet C was set to three tablets. Therefore, the LTP intake was 3.4 mg / day for three tablets of tablet A, 1.7 mg / day for three tablets of tablet B, and 0 mg / day for three placebo tablets C.

[0055] Furthermore, in the trial described below, there were 111 applicants who wished to participate. After conducting pre-examinations on each individual, 50 did not meet the exclusion criteria described later. In addition, considering the FMD (flow-dependent vasodilation) test values ​​at the time of pre-examination and the frequency of daily leg muscle training, 36 were selected from the 50 applicants to participate in the trial described below. These 36 included one person who had taken a drug that reduced blood flow during the trial period, and one person who had not performed full-effort exercise four weeks after consuming the test diet. After further excluding these two individuals, the remaining 34 were selected as subjects for data analysis.

[0056] The 34 study participants did not include any individuals who met the following exclusion criteria: individuals who regularly use supplements or pre-workouts that may have vasodilatory effects or affect blood flow; individuals who plan to newly take supplements or foods that claim to reduce exercise volume or fatigue; individuals who are currently participating in or have completed within four weeks of completing a clinical trial of another drug or health food; individuals who plan to participate in another clinical trial after consenting to participate in the study disclosed herein; heavy alcohol drinkers; individuals with extremely irregular eating habits; individuals with irregular lifestyles such as shift workers or night shift workers; and individuals with a history of or current history of serious diseases of the heart, liver, kidneys, or digestive system, etc. Individuals who have any of the following conditions: allergies to pharmaceuticals or food, diarrhea from dairy products (including those who are aware of having lactose intolerance), individuals who have donated 200 ml of whole blood or apheresis within one month prior to the start of the study disclosed herein, individuals who have donated 400 ml of whole blood within three months prior to the start of the study disclosed herein, individuals whose total blood volume collected within 12 months prior to the start of the study disclosed herein exceeds 1,200 ml, and individuals whom the principal investigator or co-investigator of the study disclosed herein has deemed unsuitable for participation in the study disclosed herein.

[0057] Each of the 34 study participants was a male between the ages of 21 and 49, who had been performing leg strength training for muscle hypertrophy at least once every two weeks for at least three months. The mean ± standard error profiles (age 35.2 ± 1.6 years, height 172.1 ± 0.9 cm, weight 67.3 ± 1.3 kg, BMI 22.7 ± 0.43, and body fat percentage 19.5 ± 0.9%) of the 34 study participants on the start date of the study.

[0058] The 34 test subjects were randomly assigned to one of the following groups: Test Group A, Test Group B, and Control Group C, based on their pre-test exercise capacity (anaerobic power test using an ergometer, described below), thigh circumference, age, and whether or not they consumed any of the ingredients on the restricted food list. Test Group A consisted of 12 test subjects who received three tablets A per day as their test food. Test Group B consisted of 10 test subjects who received three tablets B per day as their test food. Control Group C was a placebo group consisting of 12 test subjects who received three placebo tablets C per day as their test food. In addition, each test subject performed leg strength training aimed at muscle hypertrophy on a daily basis throughout the four-week study period. No statistically significant differences were observed in the frequency of strength training among the groups.

[0059] As part of the study design, each participant consumed three tablets of the test food daily for four consecutive weeks. Each participant maintained their normal lifestyle while keeping a diary of their medication intake, exercise habits, and type of exercise. Each participant visited the clinic twice: on the first day of test food intake (0w) and again four weeks later (4w). There, they underwent a medical interview, physical measurements, blood tests, urine tests, FMD tests, and exercise stress tests. Whether each participant consumed the test food as designed was confirmed by their diary entries and the number of remaining test food bags collected after the four-week trial.

[0060] In the two exercise stress tests conducted on the first day of test diet intake (0w) and four weeks later (4w), each test subject underwent an anaerobic power test using a stationary bicycle-type ergometer (POWER MAX V3 Pro, manufactured by Konami Sports & Life Co., Ltd.). This test involved each test subject pedaling a weight-loaded pedal at maximum effort for 10 seconds for three sets, with a 120-second rest between sets. The weight load on the pedals increased with each set.

[0061] Furthermore, the thigh circumference of each subject was measured at two exercise stress tests: on the day the test diet intake began (0w) and four weeks later (4w). The measurement was taken immediately before exercise and immediately after exercise (within one minute after the end of the third set of exercise). For each subject's thigh circumference, the circumference was measured using a measuring tape at the midpoint of 50% of the total length of the femur (length from the greater trochanter to the femoral condyle). The thigh circumference was measured two or three times at each timing point immediately before and after exercise, and the arithmetic mean of two measurements with an accuracy of less than 0.3 cm difference was adopted as the measured value of the thigh circumference. Then, the value of "(thigh circumference immediately before exercise at four weeks later (cm)) - (thigh circumference immediately before exercise on the day the test diet intake began (cm))" was used as an evaluation index for muscle mass increase. Furthermore, the value of "(Thigh circumference immediately after exercise load (cm)) - (Thigh circumference immediately before exercise load (cm))" was used as an evaluation index for pump-up. In addition, the value of "(Thigh circumference immediately after exercise load at four weeks later (cm)) - (Thigh circumference immediately after exercise load on the first day of test food intake (cm))" was used as an evaluation index for improved pump-up and increased muscle mass.

[0062] Furthermore, in order to evaluate the physical condition of each test subject, for each of the two exercise tests—on the first day of intake of the test food (0w) and four weeks later (4w)—each test subject was asked the following questions at the time immediately before exercise, immediately after exercise, and upon waking the day after exercise. The questions were answered using a five-point scale where lower scores indicate higher evaluation (1 point: strongly felt, 2 points: slightly felt, 3 points: no change from before, 4 points: not felt much, 5 points: not felt at all). - Question immediately before exercise: Motivation to perform training - Question immediately after exercise: Pump-up effect immediately after training, motivation to perform training - Question upon waking the day after exercise: Motivation to perform training

[0063] The details of the physical measurements, blood tests, urine tests, and FMD tests performed on each test subject before two exercise tests: on the day of the start of test food intake (0w) and four weeks later (4w). Physical measurements included height, weight, BMI, body fat percentage, lean body mass, blood pressure (systolic and diastolic), and pulse rate. Blood tests included TP, Alb, T-Bil, ALP / IFCC, LD / IFCC, AST, ALT, γ-GT, CK, T-Cho, TG, HDL-C, LDL-C, UN, CRE, UA, Na, K, Cl, Ca, DHEA-S, and GLU. Urine tests included protein, glucose, urobilinogen, bilirubin, and occult blood. FMD test values ​​were measured using a clinical-grade FMD testing device (UNEX EF-18VG, manufactured by UNEX Corporation). No values ​​were found in these measurements for any of the test subjects that would fall under the aforementioned exclusion criteria.

[0064] The measured values ​​obtained from the tests in each group are shown below as mean ± standard error. For the statistical analysis of the test results, Dunnett tests were used to evaluate between test group A and control group C, and between test group B and control group C, using the measured values ​​and the values ​​of change from the start of test food intake (0w) to four weeks later (4w). In addition, a t-test was used to evaluate the comparison between the start of test food intake (0w) and four weeks later (4w) using the corresponding measured values. Furthermore, Steel tests were used to evaluate between test group A and control group C, and between test group B and control group C, using the scores obtained from questionnaire responses. A Wilcoxon signed-rank test was used to evaluate the temporal comparison between the start of test food intake (0w) and four weeks later (4w) using the scores obtained from questionnaire responses. The correlation between measured values ​​and changes in each parameter from the start date of test food intake (0w) to four weeks later (4w) was evaluated using Pearson's product-moment correlation coefficient. The correlation between parameters including scores and changes obtained from questionnaire responses was evaluated using Spearmand's rank correlation coefficient. The comparison of correlation coefficients was evaluated using Fisher's Z-transform for the test of differences in correlation coefficients. The statistical analysis software used was Microsoft Excel (Microsoft Corporation), IBM SPSS Statistics (IBM Japan, Ltd.), and Excel Statistics (Social Information Service Co., Ltd.).

[0065] The results of thigh circumference measurements immediately before exercise are shown in Figure 2 and Table 2 below. In test group A, which ingested tablet A for four weeks and had a daily LTP intake of 3.4 mg / day, the thigh circumference measured four weeks later (4w) was significantly increased compared to the measurement taken on the first day of test food intake (0w) (p < 0.05). On the other hand, in control group C, which ingested a placebo for four weeks, no statistically significant change in the measured values ​​was observed.

[0066]

[0067] Table 3 below shows the measured values ​​of body weight, BMI, body fat mass, and lean body mass for each group. The results of the lean body mass measurements are also shown in Table 3 and Figure 3. Lean body mass is the total weight of muscles, bones, and internal organs, excluding body fat, and was calculated from the measured body weight and body fat percentage of each subject. Furthermore, the changes in body weight and BMI (Δ0-4w) from the start of test food intake (0w) to four weeks later (4w) for each of the test group A and control group C are shown in Table 3, Figure 4A, and Figure 4B. In test group A, who ingested tablet A for four weeks and had a daily LTP intake of 3.4 mg / day, lean body mass (muscle mass) increased significantly (p < 0.05). On the other hand, in control group C, who ingested a placebo for four weeks, no statistically significant change in lean body mass was observed. Therefore, it was suggested that in test group A, the values ​​of weight change and BMI change over four weeks were significantly increased compared to control group C (p < 0.05) due to the increase in lean body mass.

[0068]

[0069] Table 4 below shows the measured thigh circumference immediately after exercise load in each group. Figure 5 shows the change in thigh circumference immediately after exercise load (Δ0-4w) between the start of test food intake (0w) and four weeks later (4w) in each of the test group A and control group C. In test group A, which consumed tablet A for four weeks and had a daily LTP intake of 3.4 mg / day, the thigh circumference immediately after exercise load was significantly increased compared to control group C (p < 0.05).

[0070]

[0071] The aggregated results (mean ± standard error) of the responses to the aforementioned questionnaire are shown in Table 5, Figures 6A, 6B, 6C, and 7 below. As is clear from Table 5, Figures 6A, 6B, and 6C below, in the timing immediately before exercise load and upon waking the day after exercise load, the score four weeks later (4w) was significantly lower (p < 0.05) compared to the score on the first day of test food intake (0w), indicating an improvement in motivation to perform strength training. On the other hand, no decrease in score was observed in the control group C, so no particular improvement in motivation was observed. Furthermore, in the timing immediately after exercise load, in the test group A, a tendency for the score to decrease four weeks later (4w) was observed compared to the score on the first day of test food intake (0w) (a tendency for improved motivation was observed), whereas in the control group C, no statistically significant decrease in score (improvement in motivation) was observed. As is clear from Table 5 and Figure 7 below, regarding the timing of waking up the day after exercise, the score change value "(score after four weeks) - (score on the first day of test food intake)" was significantly lower in test group A compared to control group C (p < 0.05), indicating an improvement in motivation to perform strength training.

[0072]

[0073] Regarding the evaluation of improved muscle pump, the measurement results of the change in thigh circumference immediately after exercise compared to the thigh circumference immediately before exercise are shown in Table 6 and Figure 8 below. In addition, the results of the questionnaire regarding muscle pump (mean ± standard error) are shown in Table 7 below. Compared with control group C, a significant decrease in score (p < 0.05) was observed in test group B, suggesting improved muscle pump. Furthermore, in test group B, which consumed tablet B for four weeks and had a daily LTP intake of 1.7 mg / day, a downward trend in the score after four weeks compared to the score on the start of the test diet was observed (p < 0.1), suggesting a tendency toward improved muscle pump. As a result of the statistical analysis, as shown in Figure 9, a significant negative correlation was found between the change in thigh circumference (Δ0 - 4w) immediately before and immediately after exercise, and the change in the questionnaire score (Δ0 - 4w), which were treated as two variables. In other words, it was found that in the subjective questionnaire, participants who reported an improvement in the pump-up effect also showed an increase in the objective changes in thigh circumference immediately before and after exercise.

[0074]

[0075]

[0076] We decided to investigate the relationship between the vasodilatory effect of LTP (FMD and blood pressure) and muscle pump-up through statistical analysis. FMD was used as an indicator to evaluate the vasodilatory effect of LTP due to NO production, and systolic and diastolic blood pressure were used as indicators to evaluate the blood pressure lowering effect due to the inhibition of vasoconstriction. Since none of the test subjects had any problems with vascular function or hypertension, no significant differences were found in the changes in FMD and blood pressure as a result of the statistical analysis. On the other hand, as shown in Figure 10, the statistical analysis revealed a positive correlation trend between the change in FMD over four weeks ("(FMD value at four weeks post-start of test food intake)") and the change in thigh circumference over four weeks immediately before and after exercise load, which is an objective indicator of improved muscle pump-up, as two variables. In other words, among the test subjects who showed an increase in FMD values ​​four weeks later (4w) compared to the start date of test food intake (0w), there was a tendency (p < 0.1) for increased muscle pump immediately after exercise four weeks later (4w) compared to the start date of test food intake (0w).

[0077] Furthermore, as shown in Figures 11A and 11B, the four-week fluctuations in systolic and diastolic blood pressure, and the four-week fluctuations in thigh circumference immediately before and after exercise (an objective indicator of muscle pump), showed inverse correlations between the study levels (between study group A and control group C, and between study group B and control group C), with significantly different correlation coefficients (p < 0.05). Therefore, as shown in Table 8 below, it was unexpectedly discovered that subjects who experienced a long-term decrease in blood pressure by taking tablet A or tablet B containing LTP for four weeks (subjects in study group A or study group B) tended to experience a greater degree of temporary muscle pump improvement immediately after exercise. Conversely, as shown in Table 8 below, subjects in control group C who took a placebo for four weeks were more likely to experience suppressed muscle pump.

[0078]

Claims

1. An agent for improving muscle pump or motivation, comprising a hydrolyzed casein product as an active ingredient, wherein the hydrolyzed product contains at least one tripeptide of Ile-Pro-Pro and Val-Pro-Pro.

2. The agent according to claim 1, wherein the hydrolyzed product contains 0.3 parts by mass or more of the tripeptide when the total content of peptides and free amino acids is 100 parts by mass.

3. The agent according to claim 1, wherein the average chain length of the peptides and free amino acids contained in the hydrolysate is 2.1 or less in terms of the number of amino acid residues.

4. The agent according to claim 1, wherein the hydrolyzed product contains 1 part by mass or more of a tripeptide having an Xaa-Pro-Pro sequence, when the total content of peptides and free amino acids is 100 parts by mass.

5. An agent for improving muscle pump, comprising at least one of the tripeptides Ile-Pro-Pro and Val-Pro-Pro as an active ingredient.

6. An agent according to any one of claims 1 to 5, which has the effect of increasing muscle mass.

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