JP6524176B2 - Prolyl oligopeptidase inhibitor - Google Patents

Description

  The present technology relates to a prolyl oligopeptidase inhibitor, and a brain function improving agent, a central nervous system function improving agent, or a swallowing function improving agent.

  Prolyl oligopeptidase (EC. 3.4.21.26, also referred to as prolyl endopeptidase (PEP) but hereinafter also referred to as "POP") is a chymase, elastase, cathepsin G, kallikrein, Similar to tPA, thrombin, dipeptidyl aminopeptidase IV and the like, it is a serine protease. Each of these enzymes has different characteristics.

  For example, chymase is a chymotrypsin-like serine protease present in mast cell granules. Elastase degrades cytokines, cleaves IgA and IgG, and cleaves complement C3bi and receptor CR1. Cathepsin G is present in azure granules of polymorphonuclear leukocytes and is involved in pathogen killing and digestion. Kallikrein is a proteolytic enzyme involved in blood pressure drop, and its expression in many tumor tissues has been confirmed. tPA degrades fibrin by activating plasminogen and is used as a thrombolytic agent for treatment of embolism and thrombotic diseases. Thrombin is involved in the coagulation of blood and catalyzes the reaction of fibrinogen to fibrin. Dipeptidyl aminopeptidase IV is a causative enzyme of periodontal disease, and inactivates glucagon-like peptide 1 involved in biosynthesis and secretion of insulin. Prolyl oligopeptidase (POP) is thought to be involved in the metabolism of physiologically active peptides such as substance P, vasopressin, oxytocin and the like.

  Among the above-mentioned serine proteases, prolyl oligopeptidase (POP) is widely distributed in the mammalian body and can be found in various organs. For example, they are present at high levels in the brain, testis and skeletal muscle (Non-patent document 1), especially in the hippocampus of the brain (non-patent documents 2 to 4) and in particularly high concentrations in the central nervous system (non-patent document) 5).

  Although neurotransmitters such as substance P, neurotensin, vasopressin related to memory, and proline-containing peptides such as oxytocin function to keep the brain normal, prolyl oligopeptidase (POP) is a neurotransmitter for these neurotransmitters. It is known to specifically recognize and cleave the peptide bond on the carboxyl side of a proline residue in the peptide chain of By the action of such POP, the neurotransmitter peptide is reduced, resulting in disturbance of brain function and impaired memory retention.

  In fact, patients with dementia (dementia) are known to have lower than normal amounts of vasopressin. In addition, it is known that in brain tissue of Alzheimer's disease, the activity of POP is increased, and the neurotransmitter peptide is degraded more than necessary to cause the brain function to be impaired. In addition, neurotransmitter peptides act on the central nervous system to alter animal and human responses in learning and memory tasks (Non-patent Documents 6 and 7).

For example, treatment of dementia is being studied by inhibiting the activity of POP against memory retention disorder due to excessive degradation of vasopressin (Patent Document 1, Patent Document 2).
And, as a compound (inhibitor) that inhibits the POP activity, Patent Document 1 discloses a peptide derived from rice protein, and shows that the POP inhibitory activity (IC50) of the peptide is 24.3 μM / L. There is.
Further, Patent Document 2 discloses two types of peptides derived from wine, and shows that the POP inhibitory activity (IC50) of the two types of peptides is 17.0 μM and 87.8 μM, respectively.
It is disclosed that these peptides are applied to the prevention and treatment of dementia etc. by utilizing such a POP inhibitory activity (Patent Document 1, Patent Document 2).

  Further, as described above, since prolyl oligopeptidase (POP) is involved in the metabolism of oxytocin, it is expected that the inhibition of POP inhibits the metabolism of oxytocin and improves the in vivo function to which oxytocin acts. .

  For example, oxytocin is best known for induction of labor pain (uterine contraction at delivery) and mammary stimulation during lactation (non-patent document 8), but it is also known as “reliable hormone”, “love hormone”, “happiness” It is also known that it plays an important role in building trust as it is called "hormone" and the like (Non-patent Document 9), and has a stress alleviating action (Non-patent document 10).

  Furthermore, in recent research, oxytocin has been known to have a muscle stem cell proliferation promoting action (Non-patent Document 11), to act on osteoblasts to promote bone formation (Non-patent Document 12), etc. The

  Thus, since oxytocin is involved in a large number of functions in vivo, inhibiting the POP involved in the metabolism of oxytocin increases the amount of oxytocin in vivo, and as a result, oxytocin acts It is considered possible to improve the in vivo function.

  In addition, dopamine, which is present in the central nervous system and is a neurotransmitter, regulates the amount of substance P secreted from the vagal sensory branch to the pharynx and trachea. Substance P plays an important role in the perception of the pharynx and airways, and it is known that when this secretion is reduced, dysphagia is impaired in the pharynx and cough reflex in the airways (Patent Document 3). Therefore, to improve dysphagia, it is considered effective to increase substance P, which is a neurohormone, but it is possible to use an inhibitor of prolyl endopeptidase (PEP) that degrades substance P to swallow function It is suggested that it is effective in the improvement of (Patent Document 3).

JP-A-9-40693 Japanese Patent Laid-Open No. 2002-80497 JP 2004-107285 A

T. Yoshimoto, K. et al. Ogita, R .; Walter, M .; Koida and D.K. Tsuru: Biochim. Biophys. Acta, 569, (1979), p. 184-192 Science, Vol. 173, p. 827, 1971 Molecular & Cellular Biochemistry, Vol. 30, p. 111, 1980 Journal of Japanese Society of Agricultural Chemistry, Vol. 58, p. 1147, 1984 Wilk, et al. , Journal of Neurochemistry, vol. 41, no. 1, p. 69-75, 1983 K. Toide, Z. Iwamoto, T .; Fujiwara and H. Abe: J.A. Pharm. Exp. Therapeutics, Vol. 274, (1995), p. 1370-1378 W. Riedel and J.A. Jolles, Drugs & Aging, Vol. 8, (1996), p. 245-274 Japanese Journal of Endocrine Society Vol. 69, p. 520-529, 1993 Nature reviews of neuroscience, Vol. 12, p. 524-538, 2011 Psychoneuroendocrinology, Vol. 38, p. 399-407, 2013 Nature communications, Vol. 5, article, No. 4082, 2014 Endocrinology, Vol. 155, p. 1340-1352, 2014

  The present technology uses a prolyl oligopeptidase (POP) inhibitor that uses a peptide having high inhibitory activity against prolyl oligopeptidase (POP) as an active ingredient, and a brain function improving agent, a central nervous system function improving agent, or a swallowing function improving agent Intended to provide.

  As a result of intensive studies to solve the above problems, the present inventors have found that a protein derived from milk is degraded by a specific enzyme, and a peptide having high prolyl oligopeptidase (POP) inhibitory activity is found in the degraded product. , Has completed the present technology.

That is, the present technology provides a prolyl containing as an active ingredient one or more kinds of peptides selected from the group consisting of peptides consisting of the amino acid sequences shown in the following (d) to (l): An oligopeptidase inhibitor is provided.
(D) Ala-Val-Pro-Tyr-Pro-Gln (SEQ ID NO: 4)
(E) Val-Leu-Pro-Val-Pro-Gln (SEQ ID NO: 5)
(F) Glu-Met-Pro-Phe-Pro-Lys (SEQ ID NO: 6)
(G) Val-Ile-Pro-Tyr (SEQ ID NO: 7)
(H) Thr-Lys-Val-Ile-Pro-Tyr (SEQ ID NO: 8)
(I) Val-Ala-Pro-Phe-Pro-Glu (SEQ ID NO: 9)
(J) Phe-Phe-Val-Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly (SEQ ID NO: 10)
(K) Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn (SEQ ID NO: 11)
(L) Ala-Met-Lys-Pro-Trp-Ile-Gln-Pro-Lys (SEQ ID NO: 12)

  Furthermore, the present technology contains, as an active ingredient, any one or two or more peptides selected from the group consisting of peptides consisting of the amino acid sequences represented by (d) to (l) above. An agent for improving a central nervous system function, an agent for improving swallowing function, an agent for reducing uterine contraction, a agent for stimulating mammary gland, an agent for promoting muscle stem cell proliferation, and an agent for improving osteoporosis are provided.

  In addition, the present technology provides a peptide consisting of the amino acid sequence shown in (j) above.

  The present technology relates to a food or drink containing as an active ingredient one or more kinds of peptides selected from the group consisting of peptides consisting of the amino acid sequences shown in (d) to (l) above. provide. These food and drink and feed are food and drink for brain function improvement, food and drink for central nervous system function improvement or food and drink for swallowing function improvement, and feed for brain function improvement, feed for central nervous system function improvement or swallowing It can be used as a feed for functional improvement.

  The present technology provides a method for producing any one or more peptides selected from the group consisting of peptides consisting of the amino acid sequences shown in (d) to (l) above.

  The present technology relates to prolyl oligopeptidase, which causes any one or two or more peptides selected from the group consisting of peptides consisting of the amino acid sequences shown in (d) to (l) above to act on prolyl oligopeptidase Methods of inhibiting oligopeptidases are provided. The present method can be performed in vitro, ex vivo, in vivo or in situ.

The milk protein-derived peptide according to the present technology can be produced or obtained from natural protein as a starting material, so it is highly safe and easy to apply to medicines, foods, feeds.
In addition, the milk protein-derived peptide according to the present technology has excellent inhibitory activity against prolyl oligopeptidase (POP) involved in brain function and central nervous system, so that symptoms such as dementia and depression are improved. -A POP inhibitor used for treatment and prevention, a brain function improving agent, a central nervous system function improving agent, or a swallowing function improving agent can be provided.
Furthermore, since the milk protein-derived peptide according to the present technology has excellent inhibitory activity against prolyl oligopeptidase (POP) involved in the metabolism of oxytocin involved in many functions in vivo, oxytocin acts It is possible to improve in vivo functions.

  Hereinafter, preferred embodiments for implementing the present technology will be described. Note that the embodiment described below shows an example of a representative embodiment of the present technology, and the scope of the present technology is not narrowly interpreted by this. In the present specification, with regard to those expressing the numerical range as “lower limit to upper limit”, the upper limit may be “below” or “less than”, and the lower limit may be “above” even if “above”. It may be "super".

(1) Peptide The peptide used in the present technology comprises the amino acid sequence represented by the following (a) to (l) (SEQ ID NOS: 1 to 12), and each peptide has a prolyl oligopeptidase (POP) inhibitory activity Have. In the present technology, any one selected from the group consisting of the following peptides (a) to (l) may be selected and used, or two or more may be selected and used.
(A) Leu-Lys-Pro-Thr-Pro-Glu (SEQ ID NO: 1)
(B) Leu-Lys-Pro-Thr-Pro-Glu-Gly-Asp (SEQ ID NO: 2)
(C) Leu-Lys-Pro-Thr-Pro-Glu-Gly-Asp-Leu-Glu (SEQ ID NO: 3)
(D) Ala-Val-Pro-Tyr-Pro-Gln (SEQ ID NO: 4)
(E) Val-Leu-Pro-Val-Pro-Gln (SEQ ID NO: 5)
(F) Glu-Met-Pro-Phe-Pro-Lys (SEQ ID NO: 6)
(G) Val-Ile-Pro-Tyr (SEQ ID NO: 7)
(H) Thr-Lys-Val-Ile-Pro-Tyr (SEQ ID NO: 8)
(I) Val-Ala-Pro-Phe-Pro-Glu (SEQ ID NO: 9)
(J) Phe-Phe-Val-Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly (SEQ ID NO: 10)
(K) Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn (SEQ ID NO: 11)
(L) Ala-Met-Lys-Pro-Trp-Ile-Gln-Pro-Lys (SEQ ID NO: 12)

  The (a) to (l) peptides used in the present technology (hereinafter referred to as "the peptides of SEQ ID NOs: 1 to 12" in the present specification) may be salts of these peptides as salts Examples thereof include alkali metals such as potassium and sodium, and alkaline earth metals such as calcium magnesium.

(2) POP Inhibitory Activity The POP inhibitory activity of the peptide of SEQ ID NO: 1 to 12 used in the present technology is not particularly limited. (Biochim. Biophys. Acta, 1979, Aug. 15, 569, No. 2, page 184-192).
The IC50 of the POP inhibitory activity thus measured is preferably 30 μg / mL or less, more preferably 10 μg / mL or less, and still more preferably 5 μg / mL or less. Alternatively, the IC 50 is preferably 50 μM or less, more preferably 20 μM or less, and still more preferably 10 μM or less.

(3) Method for Producing the Peptide The outline of the method for producing the peptide of SEQ ID NOs: 1 to 12 is that milk-derived proteins such as whey protein and casein are dispersed, suspended or dissolved in water and The reaction is stopped when the decomposition proceeds appropriately, and the obtained hydrolyzate is concentrated by ultrafiltration or the like, and the peptide is fractionated and eluted by chromatography or the like. About the eluted fraction liquid, a POP inhibitory activity is measured by said method, The component containing the target peptide can be manufactured by collect | recovering the fraction which has POP inhibitory activity.
For the purpose of further isolating the peptide used in the present technology, ion exchange chromatography, adsorption chromatography, reverse phase chromatography, partition chromatography, gel filtration chromatography, solvent precipitation, salting out, etc. It may be purified.
Hereinafter, although an example of a manufacturing method is demonstrated concretely, it is not limited to this. In the present technology, various steps used in hydrolysis of known whey protein and casein can be freely selected and adopted.

[material]
The peptides of SEQ ID NO: 1-12 used in the present technology can be produced by hydrolysis of natural proteins. As a natural protein which becomes a raw material, whey protein and casein contained in milk of a mammal (a cow, a goat, a sheep, a pig, a human, etc.) are preferred.

  As whey protein derived from milk, whey (for example, whey powder, desalted whey powder etc.) separated by a known method from commercial products or milk, skimmed milk etc., whey protein concentrate separated or purified, whey Protein isolates, or mixtures of any of these may be used.

  In addition, milk-derived casein contains α-casein, β-casein, κ-casein and the like, and any casein and a mixture thereof can be used in the present technology. It is preferable to use bovine milk α-casein and β-casein, which are easily available.

[Preparation of substrate solution]
First, a raw material (protein derived from milk) is dissolved or dispersed in a solvent such as water to prepare a protein solution.
The solvent is not particularly limited, but preferably distilled water is used.
Further, the concentration of the solution is not particularly limited, but in general, it is preferable in terms of efficiency and operability that the concentration range is approximately 5 to 15% by mass in terms of protein. By setting the dissolution concentration to 5% or more, the manufacturing efficiency can be improved. Further, by setting the dissolution concentration to 20% or less, it is possible to prevent the degradation of the decomposition efficiency, the sticking at the time of heat treatment, the viscosity increase at the time of cooling and the like.

  Next, a substrate solution is prepared by adjusting the pH of the above-mentioned solution to around the optimum pH of the enzyme to be used. For example, adjustment to pH 5 to 10 is preferable, and adjustment to pH 7 to 8 is more preferable.

  Although the alkaline agent used for pH adjustment is not specifically limited, For example, sodium hydroxide, potassium hydroxide, potassium carbonate etc. are mentioned.

  In the present technology, heat treatment, ion exchange treatment, and the like can also be appropriately carried out before and / or after pH adjustment as the decomposition pretreatment step.

[Enzymatic reaction]
Next, a proteolytic enzyme is added to the substrate solution. The proteolytic enzyme is not particularly limited, and known endoproteases can be used alone or in combination of two or more. Examples of endoprotease include bioplase (manufactured by Nagase Seikagaku Kogyo Co., Ltd.), Protin SD-AY10 (manufactured by Amano Enzyme Inc.), Protin NY 100 (manufactured by Amano Enzyme Inc.), Protease N Amano (manufactured by Amano Enzyme Inc.), Neutrase (manufactured by Amano Enzyme Inc.) Novo Nordisk, Alcalase (Novo Nordisk), trypsin (Novo Nordisk), chymotrypsin (Novo Nordisk), papain (Amano Enzyme), bromelain (Amano Enzyme) Commercial products such as those manufactured by

  In addition, exoproteases may be combined as needed. Examples of the exoprotease include commercially available products such as Protease A Amano (manufactured by Amano Enzyme), Sumiteam LP50D (manufactured by Shin Nippon Kagaku Kogyo), and Flavorzyme (manufactured by Novo Nordisk).

The addition amount of the proteolytic enzyme can be appropriately determined and used depending on the substrate concentration, enzyme titer, reaction temperature, reaction time and the like. For example, when using a protease derived from Bacillus bacteria, it is preferably 1000 or more, more preferably 1250 or more, and preferably 5000 or less, more preferably 3000 or less, per gram of protein. It is. When a protease derived from animal pancreas is used, it is preferably 3000 activity units or more, more preferably 5000 activity units or more, and preferably 10000 activity units or less, more preferably 8000 activity units or less, per 1 g of protein.
The activity unit can be measured according to the type of other proteolytic enzyme used.

  The proteolytic enzyme is preferably dispersed and dissolved in cold water of 4 to 10 ° C. and then used, from the viewpoint of efficiency and operability. In addition, proteolytic enzymes can be added all at once or at appropriate intervals, and furthermore, immobilized enzymes can be used.

  In the present technology, the temperature of the reaction system during the enzyme reaction can be appropriately determined within the practically usable range including the optimum temperature range in which the enzyme action is developed. For example, the temperature of the reaction system is preferably 30 to 60 ° C., and more preferably 40 to 55 ° C.

Further, in the present technology, the reaction duration time differs depending on the reaction conditions such as the reaction temperature and the initial pH. For example, if the reaction duration of the enzyme reaction is constant, there is a problem that degradants having different physicochemical properties may be generated for each production batch, so it can not be determined.
Thus, by monitoring the enzyme reaction, the reaction duration is determined so as to obtain a peptide used in the present technology.
In the present technology, for example, the reaction duration is preferably determined between 1 and 48 hours, more preferably between 4 and 18 hours.

  In addition, as a monitoring method of an enzyme reaction, the method etc. which extract | collect a part of said reaction solution, and measure the decomposition rate of protein etc. are mentioned, for example.

  Also, in order to maintain the optimum pH of the proteolytic enzyme, the pH of the solution may be appropriately adjusted during the enzymatic degradation.

Next, stop the enzyme reaction.
The termination of the enzyme reaction is carried out by inactivating the enzyme in the hydrolyzate. The inactivation treatment can be carried out by a conventional method, for example, heat inactivation treatment and the like.
The conditions (heating temperature, heating time, etc.) of the heat inactivation treatment can be appropriately set in consideration of the thermal stability of the used enzyme and the conditions that can be sufficiently inactivated.
In the present technology, for example, the enzyme can be inactivated in a temperature range of 80 to 130 ° C. with a holding time of 30 minutes to 2 seconds.

[Purification]
After termination of the enzyme reaction, the obtained hydrolysis inactivation solution is subjected to (a) filtration, (b) microfiltration, membrane separation treatment such as ultrafiltration membrane, (c) resin adsorption separation, (d) from column chromatography Purification by any one or a combination of two or more thereof selected from the group consisting of

  By performing the above-mentioned purification, removal of insolubles contained in the above-mentioned hydrolysis and inactivation solution, reduction of fat and lactose, and other unnecessary components can be performed. As a result, it is possible to obtain a so-called peptide having excellent storage stability, which is transparent in the solution state, and in which turbidity, precipitation, aggregation, browning and the like do not occur even in long-term storage in the solution state.

  In addition, by performing the above-mentioned purification, the taste, appearance and the like of the obtained peptide can be improved.

The filtration of (a) can be carried out by a known method, for example, using diatomaceous earth and can be carried out by a known device.
By carrying out filtration, it is possible to remove the insoluble matter generated at the time of the hydrolysis reaction and / or at the time of the enzyme heat inactivation present in the above-mentioned hydrolysis and inactivation liquid.
In addition, gel filtration chromatography using gel filtration resin which has the effect of molecular sieve is also included in the method of filtration.

The membrane separation treatment (b) can be performed using a known device. The known device is not particularly limited. For example, a microfiltration module or the like, ultrafiltration module SEP1053 (manufactured by Asahi Kasei Corp., molecular weight cut off 3,000), SIP1053 (manufactured by Asahi Kasei Corp., molecular weight cut off 6,000), SLP1053 (Manufactured by Asahi Kasei Corporation, molecular weight cut off: 10,000) and the like.
In this case, a solution containing a peptide is obtained as a membrane-permeable fraction after membrane separation treatment.
By carrying out the membrane separation treatment, as in the filtration of (a), it is possible to remove the insoluble matter generated during the hydrolysis reaction and / or the enzyme heating and deactivation present in the hydrolysis and deactivation liquid.

The resin adsorption and separation of (c) can be carried out by a known method, for example, can be carried out by packing a resin in a column and letting the above-mentioned hydrolysis solution pass through the column. Examples of the resin include, but are not particularly limited to, ion exchange resins, chelate resins, affinity adsorption resins, synthetic adsorbents, resins for high performance liquid chromatography, and the like. For example, trade names: Diaion, Sepabeads (manufactured by Mitsubishi Chemical Corporation), Amberlight XAD (manufactured by Organo Corporation), KS-35 (manufactured by Ajinomoto Fine Techno Co., Ltd.), and the like.
The resin adsorption separation can also be performed in a continuous system by packing these resins in a column, continuously letting the hydrolysis inactivating solution flow in and out, and letting the resin in the hydrolysis inactivating solution flow. It can also be carried out in a batch mode in which the hydrolysis inactivation liquid and the resin are separated after being charged and brought into contact for a certain period of time.
In the hydrolysis and inactivation solution, factors that cause turbidity, precipitation, aggregation, browning, etc. (eg, a peptide containing a large amount of hydrophobic amino acids) may remain during storage, so resin adsorption and separation These factors can be removed by

[Sterilization treatment]
In addition, after purification, the solution containing the obtained peptide may be sterilized.
As a sterilization method, a heat treatment method by a conventional method can be used.
The heating temperature and the holding time at the time of the heat treatment may be set appropriately as long as sufficient conditions can be sterilized. For example, the heat treatment can be performed at 70 to 140 ° C for 2 seconds to 30 minutes.
The heat sterilization method may be either a batch method or a continuous method, and a plate heat exchange method, an infusion method, an injection method or the like may be used in the continuous method.

[Concentration treatment / drying treatment / pulverization treatment / secondary treatment]
Furthermore, the solution containing the obtained peptide can be used as it is, or, if necessary, the solution can be used as a concentrated solution concentrated by a known method. The concentrate can also be dried and powdered according to known methods.

  In addition, the peptide of sequence number 1-12 can also be manufactured also by chemical synthesis, for example, the liquid phase method or solid-phase method normally used for the synthesis | combination of oligopeptide is mentioned. The synthesized peptide is optionally deprotected, and it is possible to remove unreacted reagents, byproducts and the like to isolate and purify the peptide used in the present technology. Such peptide synthesis can be performed using a commercially available peptide synthesizer.

(4) POP inhibitor The peptide of sequence number 1-12 can be used as a POP inhibitor. The POP inhibitor may be used in the form of liquid or concentrated, or after being processed into solid, granular or powder.
Moreover, the POP inhibitor in the present technology can be used not only as a research reagent but also as a diagnostic agent and the like. For example, a research kit for researching functions and oxytocin metabolism to the brain, a diagnostic kit for brain functional diseases and central nervous system diseases involving prolyl oligopeptidase, various diseases involving oxytocin metabolism, and dysphagia are exemplified. Be done.
Furthermore, the POP inhibitor in the present technology can be used as a preventive or therapeutic agent for brain functional diseases and central nervous system diseases involving prolyl oligopeptidase, various diseases involving oxytocin metabolism, dysphagia and the like. . That is, the POP inhibitor in the present technology can be used as a prophylactic or therapeutic agent for various diseases involving prolyl oligopeptidase.
Examples of preventive or therapeutic agents for such various diseases include brain function improving agents, central nervous system function improving agents, swallowing function improving agents, uterine contraction agents, mammary stimulants, muscle stem cell proliferation promoters, Agents such as

(5) Various agents The brain function improving agent of the present technology may be used, for example, to improve symptoms, such as dementia, amnesia, decline in judgment and thinking ability, decline in cognitive ability, and intellectual impairment, treatment and prevention. it can.
In addition, the central nervous system function improving agent of the present technology can be used, for example, to improve, treat, prevent symptoms such as Alzheimer's disease, Parkinson's disease, depression, affective disorder, schizophrenia, anxiety neurosis and the like.
In the present specification, the above indications of the brain function improving agent and the central nervous function improving agent of the present technology are exemplification, and the indications of the two prophylactic or therapeutic agents are not strictly distinguished.
Furthermore, the agent for improving swallowing function of the present technology can be used, for example, for improvement, treatment, prevention of aspiration, prevention of aspiration, and the like for dysphagia and dysphagia associated with aging and other diseases.

Here, as the POP inhibitor, a large number of compounds have already been synthesized as its active ingredient, and a component having POP inhibitory activity has been found also from microorganisms and animal tissues.
For some of these POP inhibitors, in vivo effects have been confirmed, for example, intraperitoneal injection of mice actually reached the brain and inhibited POP, and its amine derivative was induced with scopolamine Recovery of memory impairment in mice, oral administration to reach the brain and oral administration to inhibit POP, oral administration to increase substance P in the cerebral cortex of aged rats (23 to 24 months old) (protein nucleic acid enzyme, Vol. 42, No. 6, p. 857-864 (1997), having anti-amnestic effects in rat and mouse models (Yoshimoto et al., J. Pharmacobio-Dyn. 10, 730 (1983). J. Enz. Inhib. 3: 163 (1990); Uchida, I. et al. International Publication No. WO 90/12005) has become clear.

  The peptides of SEQ ID NOs: 1 to 12, which are active ingredients in the prophylactic or therapeutic agents for various diseases according to the present technology, are selected from the group consisting of these peptides and any one of them is selected for various diseases. It may be contained in the prophylactic or therapeutic agent, or two or more may be selected and contained. In addition, the peptides of SEQ ID NOs: 1 to 12, which are active ingredients of the brain function improving agent, central nervous system function improving agent, and swallowing function improving agent according to the present technology, a medicine for improving brain function containing these peptides, central It is also possible to use it as a medicine for improving nervous system function and a medicine for improving swallowing function.

The dose of the peptide of SEQ ID NO: 1 to 12 as an active ingredient is not particularly limited, but when it is used for the brain function improving agent, central nervous system function improving agent and swallowing function improving agent of the present technology, 0.001 to 3000 mg / Day, preferably 0.01 to 30 mg / day, and is determined according to age, sex, degree of symptoms and the like. Also, the daily dose may be divided into once to three times a day.
The administration route includes, for example, oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, transmucosal administration, intranasal administration, intrarectal administration and the like.
The subject of administration is usually a human but also mammals other than human, for example, pet animals such as dogs and cats, and domestic animals such as cattle, sheep and pigs.

  The administration form (or preparation) may be any form of solid preparation and liquid preparation, and examples thereof include tablets, pills, capsules, powders, granules, solutions, injections, powders, spray preparations and the like. Be

  Pharmaceutically acceptable carriers include excipients or diluents, for example dextrans, sucrose, lactose, maltose, xylose, trehalose, mannitol, sorbitol, gelatin, carboxymethylcellulose, carboxymethylcellulose, hydroxypropyl methylcellulose, Gum arabic, guar gum, tragacanth, acrylic acid copolymer, ethanol, physiological saline, Ringer's solution and the like.

  In addition to the above-mentioned carrier, additives such as preservatives, stabilizers, binders, pH adjusters, buffers, thickeners, gelling agents, antioxidants and the like can be added if necessary. These additives are preferably those used in pharmaceuticals.

  When manufacturing a pharmaceutical containing the peptide of SEQ ID NO: 1 to 12 as an active ingredient, it can be manufactured according to a conventional method in the field of pharmaceutical technology, for example, a method described in Japanese Pharmacopoeia or a method according thereto.

  The prophylactic or therapeutic agents for various diseases according to the present technology may be used in combination with other pharmaceutical agents. For example, medicines used in combination with brain function improving agents and central nervous system function improving agents include agents for treating dementia, such as acetylcholine esterase inhibitors (donepezil, galantamine, rivastigmine, tacrine etc.), NMDA receptor antagonists (memantine Etc), anti-anxiety agents such as benzodiazepine anti-anxiety agents, anti-depressants such as selective serotonin reuptake inhibitors (SSRI), serotonin norepinephrine (noradrenaline), reuptake inhibitors (SNRI), tricyclics Antidepressant (TCA), tetracyclic antidepressant, triazolopyridine antidepressant (SARI), monoamine oxidase inhibitor (MAO inhibitor), noradrenergic specific serotonergic antidepressant (NaSSA) ), Norepinephrine-dopamine reuptake inhibitors (NDR) ), And the like, anti-psychotic drugs, like sleep-inducing agents and the like.

  The pharmaceutical agents used in combination may be administered simultaneously with, before or at any time after administration of the prophylactic or therapeutic agents for various diseases of the present technology. The dosage is not particularly limited, but in the case of a commercially available drug, it is preferably a dosage indicated by a pharmaceutical manufacturer.

(6) Foods and Beverages / Feeds The POP inhibitors, brain function improving agents, central nervous system function improving agents, uterine contraction agents, mammary stimulants, muscle stem cell proliferation promoting agents, and swallowing function improving agents in the present technology include It can be used as a form of feed or added to food and drink or feed.
The food and drink may be in any form such as liquid, paste, solid, powder, etc., in addition to tablets, liquid food, feed (including for pets), etc., for example, flour products, ready-to-eat foods, processed agricultural products, marine products Examples thereof include processed products, livestock products, milk and dairy products, fats and oils, basic seasonings, combined seasonings and foods, frozen foods, confectioneries, beverages, and commercially available foods other than these.

Examples of wheat flour products include bread, macaroni, spaghetti, noodles, cake mix, fried flour and bread crumbs.
Instant foods include, for example, instant noodles, cup noodles, retorts, cooked foods, cooked cans, microwave oven foods, instant soups and stews, instant miso soup and soup, soup cans, freezes and dry foods, and other instant foods etc. Be
Examples of processed agricultural products include canned agricultural products, canned fruits, jams and marmalades, pickles, boiled beans, dried agricultural products, cereals (processed cereal products) and the like.
Examples of processed fish products include stuffed fish cans, fish ham / sausages, fish paste products, fish delicacies, and sweetfish.
Examples of livestock products include livestock cans and pastes, livestock ham and sausages, and the like.
Examples of milk and milk products include processed milk, milk drinks, yogurts, lactic acid bacteria drinks, cheese, ice creams, prepared milk powders, creams, and other dairy products.
As fats and oils, butter, margarines, vegetable oil etc. are mentioned, for example.
Examples of basic seasonings include soy sauce, miso, sauces, tomato processed seasonings, mirins, vinegars, etc. As the combined seasonings / foods, cooking mixes, elements of curry, sauces, Dressings, noodle soups, spices, other compound seasonings, etc. may be mentioned.
Examples of frozen food include raw frozen food, partially cooked frozen food, and cooked frozen food.
Examples of confectionery include caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese sweets, rice sweets, bean sweets, dessert sweets, and other sweets.
Examples of beverages include carbonated beverages, natural fruit juices, fruit juice beverages, soft drinks with fruit juices, fruit pulp beverages, fruit beverages with fruit grains, vegetable beverages, soy milk, soy milk beverages, coffee beverages, tea beverages, powdered beverages, concentrated beverages Sports beverages, nutritional beverages, alcoholic beverages, other favorite beverages, etc.
Examples of commercially available food other than the above include baby food, sprinkles, simmered tea, and the like.

Moreover, the food and drink defined by the present technology can be provided and sold as a food and drink in which a specific use (especially for health use) and a function are displayed.
"Display" acts include all acts to inform consumers of the application, and if it is an expression that can recall or analogize the application, the purpose of the display, the content of the display, the display Regardless of the target object, medium, etc., they all correspond to the “display” act of the present technology.

  Moreover, it is preferable that "display" is performed by the expression which a consumer can recognize the said application directly. Specifically, the goods or goods packaging pertaining to food and drink are transferred to those which describe the use, and displayed for delivery, transfer or delivery, and the act of importing, advertisement for goods, price list or transaction documents The act of describing and displaying the application, or disseminating the information, or describing the application in information containing the information and providing the information by an electromagnetic (Internet etc.) method may be mentioned.

  On the other hand, it is preferable that the display content is a display approved by the administration or the like (for example, a display etc. which is approved on the basis of various systems defined by the administration and performed in a mode based on such authorization). In addition, it is preferable to attach such display contents to advertising materials at sales sites such as packaging, containers, catalogs, brochures, POPs (Point of Purchase advertising), and other documents.

  In addition, "indication" includes health food, functional food, food for sick people, enteral nutrition food, special purpose food, health food, food for specified health food, functional display food, nutrition food, medicine department The display as an external item etc. is also mentioned. Among these, the display approved by the Consumer Agency, for example, the food system for specified health, the food system for functional indication, the display approved by a system similar to these, and the like can be mentioned. More specifically, the indication as food for specific health, the indication as food for conditional specific health, the indication as functional indication food, the indication that the structure or function of the body is affected, the disease risk reduction indication, etc. Can be mentioned. Among these, as a typical example, the indication as food for specified health defined in the Health Promotion Act Enforcement Regulations (April 30, 2003, Ministry of Health, Labor and Welfare Ordinance No. 86 of Japan) (especially indication of the use of health) The display as a functional display food defined in the Food Display Act (Law No. 70 of 2013) and the display similar thereto are typical examples.

  When using POP inhibitor, brain function improving agent, central nervous system function improving agent, uterine contraction agent, mammary stimulant, muscle stem cell growth promoting agent, and swallowing function improving agent as feed, it is prepared by adding to known feed It is possible to produce fresh feed by mixing it in feed ingredients. For example, it can be mixed with dog food, feed such as cattle, sheep, goats and pigs.

  Examples of feed materials include grains such as corn, wheat, barley and rye; brans such as bran, wheat bran, rice bran and defatted rice bran; manufactures such as corn gluten meal and corn jam meal; Animal feeds such as whey, fish meal and bone meal; yeasts such as brewer's yeast; mineral substance feeds such as calcium phosphate and calcium carbonate; oils and fats; amino acids; Moreover, as a form of the said feed, the feed for pet animals (pet food etc.), livestock feed, fish feed etc. are mentioned, for example.

  Although the compounding quantity of the peptide of sequence number 1-12 is not specifically limited, It is preferable that it is at least 0.001 mass% with respect to the final food-drinks product.

When using a POP inhibitor, brain function improving agent, central nervous system function improving agent, uterine contraction agent, mammary stimulant, muscle stem cell proliferation promoting agent, and swallowing function improving agent as food and drink, leave it as liquid or concentrate it Alternatively, it may be used after being processed into a solid, granular or powdery state. In addition, additives such as excipients, binders, disintegrants, lubricants, coloring agents, flavoring agents, buffers, stabilizers and the like may be added to these food and drink.
As such an additive, those generally used in the relevant field may be used.

For example, as the excipient, lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, boric acid and the like can be mentioned.
As the binder, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, calcium phosphate, polyvinylpyrrolidone and the like can be mentioned.
Disintegrants include dry starch, sodium alginate, agar powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, lactose and the like.
The lubricant includes purified talc, stearate, borax, polyethylene glycol and the like.
Examples of coloring agents include caramel dyes, paprika dyes, cochineal dyes, indigo carmine and the like.
Examples of the flavoring agent include sucrose, orange peel, citric acid and tartaric acid.
Examples of the buffer include sodium citrate and the like.
As a stabilizer, tragacanth, gum arabic, gelatin etc. are mentioned.

  Furthermore, when a POP inhibitor, a brain function improving agent, a central nervous system function improving agent, a uterine contraction agent, a mammary stimulant, a muscle stem cell proliferation promoting agent, and a swallowing function improving agent are used as food or drink or a supplement, other brain functions Materials and compounds said to have an improving effect can be combined and blended. For example, Ginkgo biloba leaf extract, arachidonic acid (ARA), gaba (GABA), theanine, ceramide, caffeine, carnitine, α-glyceryl phosphoryl choline (α-GPC), bacopa monniera, DHA-bound phospholipid, phosphatidyl serine (PS), phosphatidyl choline , St. John's wort, astaxanthin, niacin, pyrroloquinoline quinone (PQQ), coenzyme Q10 (CoQ10), unsaturated fatty acids such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), etc. polyphenols such as resveratrol etc, chlorogen Acids, catechins and the like can be mentioned.

  Hereinafter, the present technology will be described in more detail based on examples. Note that the embodiments described below show an example of a representative embodiment of the present technology, and the scope of the present technology is not narrowly interpreted.

[Production example 1: Production of peptide of SEQ ID NO: 1 to 3]
Preparation of Whey Hydrolyzate 1
A commercially available WPC (whey protein concentrate, Milei 80 (manufactured by Mirai)) is dissolved in water at a concentration of 10%, sodium hydroxide is added to adjust the pH of the solution to 8.0, and an aqueous whey solution is prepared. did. To this aqueous whey solution, 5,000 units of Protease N Amano (manufactured by Amano Enzyme Inc.) were added per 1 g of protein, and the mixture was decomposed at 50 ° C. for 7 hours. Then, the enzyme was inactivated by heating at 90 ° C. for 10 minutes, and powdered with a spray dryer to obtain a dried whey hydrolyzate.

Preparation of Whey Hydrolyzate 2
A commercially available whey powder (NZ-GLP (manufactured by Fontela)) was dissolved in water at a concentration of 20%, and sodium hydroxide was added to adjust the pH of the solution to 8.5 to prepare an aqueous whey solution. To this aqueous whey solution, 7,000 units of Protin NY 100 (manufactured by Amano Enzyme) and 1,000 units of Alcalase (manufactured by Novo Nordisk) were added per 1 g of protein, and the mixture was decomposed at 55 ° C. for 5 hours. Then, the enzyme was inactivated by heating at 80 ° C. for 10 minutes, clarified by a microfiltration membrane, and then powdered by a spray dryer to obtain a dried product of whey hydrolyzate.

<Separation of peptide by HPLC>
About the whey hydrolyzate prepared above, separation of the peptide by HPLC was performed under the following HPLC condition 1.

[HPLC condition 1]
Column: Cadenza CD-C18 10 mm I. D. × 250 mm (Intact Corporation)
Detection: UV 215 nm
Flow rate: 3 mL / min Eluent A: aqueous solution containing 0.1% TFA Eluent B: acetonitrile solution containing 0.1% TFA

<Method of measuring POP inhibitory activity>
Measurement of POP inhibition is described by Yoshimoto, T. et al. Act “Biochim. Biophys. Acta, 1979, Aug. 15, 569, No. 2, page 184-192”.
Specifically, the enzyme (POP) was subjected to enzyme reaction using Recombinant Human Prolyl Oligopeptidase / PREP (R & D Systems, Inc.), and the substrate was Z-Gly-Pro-AMC (BACHEM). To each well of a 96-well microplate (nunc137101) is added water or an aqueous solution of the test substance at each concentration, or a fraction fraction of HPLC, Buffer (Tris-HCl (1 M, PH 8.0, 1.25 mL), DTT The total volume was adjusted to 80 μL by adding 10 μL of (1 M, 125 μL), NaCl (5 M, 2.5 mL), water (6.125 mL)).
After stirring, the plate was warmed in a 37 ° C. incubator for about 10 minutes, and 10 μL of POP solution and 10 μL of substrate solution were added (total liquid volume 100 μL), and the reaction was initiated by stirring.
A well to which water was added instead of the enzyme served as a control.

  The measurement of the enzyme reaction was performed using a microplate reader (SH-9000, Corona Electric Co., Ltd.) under the condition that the inside temperature was kept at 37 ° C. (interval of 2 minutes, ex360 nm / em 460 nm). The inhibition activity was calculated by the following formula from the value of the fluorescence intensity during a period in which the increase in fluorescence intensity is linear (within 30 minutes from the start of the reaction).

Inhibition rate (%) = 100%-[(Y-b) / (X-a)] x 100%
X: water + enzyme + substrate Y: test substance + enzyme + substrate a: water + substrate b: test substance + substrate

<How to determine the concentration of IC50>
The concentration of the test substance was serially diluted (0.1 to 2000 μg / mL), and the inhibition rate was determined. Based on the results, the relationship between the logarithm (log 10) of the added concentration of the test substance and the inhibition rate was determined. And IC50 was computed by back-calculating the density | concentration from which the inhibition rate of an enzyme will be 50% from this relational expression.

<POP inhibitory activity of isolated peptide>
Separate the hydrolyzate under the gradient conditions of 75% after 30 minutes, 50% after 40 minutes, 50% after 40 minutes, and 20% after 43 minutes based on the condition of HPLC condition 1 The eluate was fractionated every 0.75 mL.
The prolyl oligopeptidase inhibitory activity was measured for the eluted fraction, and the fractions eluted in (1) and (2) at the time described in the retention time (RT (min)) of Analysis 1 in Table 1 below. A strong inhibitory activity was observed.
From the fraction (2) obtained in Analysis 1, fractions in which two inhibitory activities were observed were obtained.

Moreover, about the compound contained in the fraction in which these inhibitory activity is recognized, mass spectrometry was performed by ThermoQuest mass spectrometer LTQ.
In mass spectrometry, parent ions and daughter ions were measured, and peptides were identified by analysis software (manufactured by ThermoQuest, BioWorks). The results are shown in Table 1 below.

  In Table 1, the peptide [LKTPTE] of elution fraction No. (1) is the peptide of SEQ ID NO: 1 and the peptide of elution fraction No. (2) [LKPTPEGD] is the peptide of SEQ ID NO: 2, elution fraction No. (2 The peptide of [LKPTPEGDLE] was used as the peptide of SEQ ID NO: 3.

Preparation Example 2: Preparation of Peptides of SEQ ID NOS: 4 to 7, 9 and 11
<Preparation of casein hydrolyzate>
90 g of water is added to 10 g of commercially available casein (manufactured by New Zealand Dailyboard), dispersed well, and the pH of the solution is adjusted to 7.0 by adding sodium hydroxide, and the casein is completely dissolved to a concentration of about 10% An aqueous casein solution was prepared.
The casein aqueous solution is heat-sterilized at 85 ° C. for 10 minutes, temperature-adjusted to 50 ° C., sodium hydroxide is added to adjust pH to 9.5, and then bioplase sp-20 (manufactured by Nagase Seikagaku Kogyo Co., Ltd.) 10 20,000 active units (1,250 active units per gram of protein), Protease N (Amano Enzyme Inc.) 17,000 active units (2,000 active units per gram of protein), and PTN 6.0 S (Novozymes Japan Ltd.) The hydrolysis reaction was initiated by adding 60,000 active units (7,000 active units per gram of protein). When the decomposition rate of casein reached 24.5%, the enzyme reaction was stopped by heating at 80 ° C. for 7 minutes to stop the enzyme reaction and cooled to 10 ° C.
The hydrolyzate was subjected to ultrafiltration using a ultrafiltration membrane with a molecular weight cut off of 10,000 (manufactured by Asahi Kasei Corp.), concentrated and freeze-dried to obtain 8 g of a lyophilized product (CN-degraded product A).

<Separation of peptide by HPLC>
The separation of the peptide by HPLC was performed under the above-mentioned HPLC condition 1 for CN degradation product A (casein hydrolyzate) prepared above. Further, in order to further increase the degree of purification, the fraction showing the POP inhibitory activity obtained under HPLC condition 1 was separated under the following HPLC condition 2.

[HPLC condition 2]
Column: Cadenza CD-C18 10 mm I. D. × 250 mm (Intact Corporation)
Detection: UV 215 nm
Flow rate: 3 mL / min Eluent A: aqueous solution containing 0.2% formic acid Eluent B: acetonitrile solution containing 0.2% formic acid

<POP inhibitory activity of isolated peptide>
Separate the hydrolyzate under the gradient conditions of 75% after 30 minutes, 50% after 40 minutes, 50% after 40 minutes, and 20% after 43 minutes based on the condition of HPLC condition 1 The eluate was fractionated every 0.75 mL.
With respect to the eluted fraction, the prolyl oligopeptidase inhibitory activity was measured, and it was determined that the fraction (1) to (5) eluted at the time described in the retention time (RT (min)) of Analysis 1 in Table 1 above. A strong inhibitory activity was observed.
Furthermore, separation was performed under HPLC condition 2 in order to increase the purity of the eluted fractions (1) to (5). At this time, the eluents A and B of the condition 1 were changed to the eluents A and B of the condition 2 respectively, and the other conditions were the same as those of the condition 1.

The hydrolyzate was separated under a gradient condition of HPLC condition 2 and the eluate was fractionated every 0.75 mL. With respect to the eluted fraction, when the POP inhibitory activity was measured by the above-mentioned method for measuring the POP inhibitory activity, a strong inhibitory activity was observed in the fraction obtained at the retention time of Analysis 2 in Table 2 below.
From the fraction (3) obtained in Analysis 1, fractions in which two inhibitory activities were observed were obtained.

  Further, with respect to the compounds contained in the fractions in which these inhibitory activities were observed, mass spectrometry and peptide identification were carried out in the same manner as in Production Example 1 above. The results are shown in Table 2 below.

  In Table 2, the peptide [AVPYPQ] of elution fraction No. (1) is the peptide of SEQ ID NO: 4 and the peptide of elution fraction No. (2) [VLPVPQ] is the peptide of SEQ ID NO: 5, elution fraction No. (3 The peptide [SEQ ID NO: 6], the peptide of elution fraction No. (3) [VIPY] the peptide of SEQ ID NO: 7, the peptide of elution fraction No. (4) [VAPFPE] the peptide of SEQ ID NO: 9) The peptide of elution fraction number (5) [VYPFPGPIPN] was used as the peptide of SEQ ID NO: 11.

[Production example 3: Production of peptide of SEQ ID NO: 8, 10, 12]
<Preparation of casein hydrolyzate>
Casein hydrolyzate was obtained in the same manner as in Production Example 2 above.

<Separation of peptide by HPLC>
About the casein hydrolyzate prepared above, the separation of the peptide by HPLC was performed under the following HPLC condition 3.

[HPLC condition 3]
Column: Intrada WP-RP 10 mm I. D. × 250 mm (Intact Corporation)
Detection: UV 215 nm
Flow rate: 3 mL / min Eluent A: aqueous solution containing 0.1% TFA Eluent B: acetonitrile solution containing 0.1% TFA

<POP inhibitory activity of isolated peptide>
Based on the conditions of HPLC condition 3, the hydrolyzate is separated under gradient conditions such that the ratio of eluent A is 80%, 50% after 40 minutes, and 20% after 43 minutes, and the eluate is 0. It fractionated every 75 mL.
The prolyl oligopeptidase inhibitory activity was measured for the eluted fraction, and the fractions of (1) to (3) eluted at the time described in retention time (RT (min)) of Analysis 1 in Table 3 below A strong inhibitory activity was observed.

  Further, with respect to the compounds contained in the fractions in which these inhibitory activities were observed, mass spectrometry and peptide identification were carried out in the same manner as in Production Example 1 above. The results are shown in Table 3 below.

  In Table 3, the peptide [TKVIPY] of elution fraction No. (1) is the peptide of SEQ ID NO: 8 and the peptide of elution fraction No. (2) [AMKPWIQPK] is the peptide of SEQ ID NO: 12, elution fraction No. (3 The peptide [FFVAPFPEVFG] was used as the peptide of SEQ ID NO: 10.

Preparation Example 4: Chemical Synthesis of Peptides of SEQ ID NOS: 1 to 12
Using a peptide synthesizer (Model 433A, Applied Biosystems), Fmoc-AA-Wang-PEG Resin (Watanabe Chemical Industry Co., Ltd.), Fmoc-AA (Ala, Val, Pro, Tyr, Gln, The peptide of sequence number 1-12 was synthesize | combined by the solid-phase synthesis method using Leu, Glu, Met, Phe, Lys, Ile, Gly, Asn, Thr, Trp or Asp (Peptide Laboratories).
The procedure was performed according to the Applied Biosystems manual and then deprotected.
These peptides were separated and purified under the HPLC conditions 1 to 3 described in Example 1.
The IC50 (μg / mL) was determined for the obtained purified product (synthetic peptide) by the method for measuring the POP inhibitory activity. In addition, mass spectrometry of the synthetic peptide was performed by the same method as the mass spectrometry described in Example 1. The results are shown in Table 4.

  According to this example, peptides consisting of the amino acid sequences of SEQ ID NOs: 1 to 9 can be obtained, and these peptides have high IC 50 of about 100 μg / mL to about 1 μg / mL (about 120 μM to about 1 μM) high POP It was confirmed to have inhibitory activity.

  In addition, when the peptides of SEQ ID NO: 1 to 3 are compared, they have the same sequence [LKTPTE], but two amino acids [GD] are added to the C-terminal side compared to the peptide of SEQ ID NO: 1 The POP inhibitory activity of 2 peptides was low. However, the POP inhibitory activity of the peptide of SEQ ID NO: 3 in which two amino acids [LE] were added to the C-terminal side was higher than that of the peptide of SEQ ID NO: 2.

  Furthermore, when the peptides of SEQ ID NOS: 7 and 8 are compared, they have the same sequence [VIPY] but have two amino acids [TK] added at the N-terminal side as compared to the peptide of SEQ ID NO: 7 The POP inhibitory activity of 8 peptides was low.

  In addition, when comparing the peptides of SEQ ID NO: 9 and 10, they have the same sequence [VAPFPE] and the peptide of SEQ ID NO: 10 has two amino acids [FF] compared to the peptide of SEQ ID NO: 9 [FF] It is a sequence in which three amino acids [VFG] are added to the C-terminal side on the terminal side, but the POP inhibitory activities of both were similar.

  From the above results, even if one amino acid sequence of the peptide changes, the POP inhibitory activity may change significantly, and no rule is found in the relationship between the amino acid sequence and the intensity of the POP inhibitory activity. It is believed to be a peptide-specific effect.

  According to the present technology, a POP inhibitor can be produced from easily available casein. When this POP inhibitor is administered to animals (including humans), it reaches the central nerve and brain and inhibits the degradation of neurotransmitters by POP, so it is possible to improve brain function, central nervous system function, and swallowing. A function improving agent, a uterine contraction agent, a breast stimulating agent, a muscle stem cell proliferation promoting agent, an osteoporosis improving agent and the like can be provided.

Claims (5)

The prolyl oligopeptidase inhibitor which contains as an active ingredient one or 2 types or more types of peptide selected from the group comprised by the peptide which consists of an amino acid sequence shown by the following (d)-(l).
(D) Ala-Val-Pro-Tyr-Pro-Gln (SEQ ID NO: 4)
(E) Val-Leu-Pro-Val-Pro-Gln (SEQ ID NO: 5)
(F) Glu-Met-Pro-Phe-Pro-Lys (SEQ ID NO: 6)
(G) Val-Ile-Pro-Tyr (SEQ ID NO: 7)
(H) Thr-Lys-Val-Ile-Pro-Tyr (SEQ ID NO: 8)
(I) Val-Ala-Pro-Phe-Pro-Glu (SEQ ID NO: 9)
(J) Phe-Phe-Val-Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly (SEQ ID NO: 10)
(K) Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn (SEQ ID NO: 11)
(L) Ala-Met-Lys-Pro-Trp-Ile-Gln-Pro-Lys (SEQ ID NO: 12) The brain function improvement agent which contains any one type or two or more types of peptide selected from the group comprised by the peptide which consists of an amino acid sequence shown by the following (d)-(l) as an active ingredient.
(D) Ala-Val-Pro-Tyr-Pro-Gln (SEQ ID NO: 4)
(E) Val-Leu-Pro-Val-Pro-Gln (SEQ ID NO: 5)
(F) Glu-Met-Pro-Phe-Pro-Lys (SEQ ID NO: 6)
(G) Val-Ile-Pro-Tyr (SEQ ID NO: 7)
(H) Thr-Lys-Val-Ile-Pro-Tyr (SEQ ID NO: 8)
(I) Val-Ala-Pro-Phe-Pro-Glu (SEQ ID NO: 9)
(J) Phe-Phe-Val-Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly (SEQ ID NO: 10)
(K) Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn (SEQ ID NO: 11)
(L) Ala-Met-Lys-Pro-Trp-Ile-Gln-Pro-Lys (SEQ ID NO: 12) The central nervous system function improving agent which contains as an active ingredient one or more types of peptide selected from the group comprised by the peptide which consists of an amino acid sequence shown by the following (d)-(l).
(D) Ala-Val-Pro-Tyr-Pro-Gln (SEQ ID NO: 4)
(E) Val-Leu-Pro-Val-Pro-Gln (SEQ ID NO: 5)
(F) Glu-Met-Pro-Phe-Pro-Lys (SEQ ID NO: 6)
(G) Val-Ile-Pro-Tyr (SEQ ID NO: 7)
(H) Thr-Lys-Val-Ile-Pro-Tyr (SEQ ID NO: 8)
(I) Val-Ala-Pro-Phe-Pro-Glu (SEQ ID NO: 9)
(J) Phe-Phe-Val-Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly (SEQ ID NO: 10)
(K) Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn (SEQ ID NO: 11)
(L) Ala-Met-Lys-Pro-Trp-Ile-Gln-Pro-Lys (SEQ ID NO: 12) An agent for improving swallowing function comprising, as an active ingredient, any one or more peptides selected from the group consisting of peptides consisting of the amino acid sequences shown in (d) to (l) below.
(D) Ala-Val-Pro-Tyr-Pro-Gln (SEQ ID NO: 4)
(E) Val-Leu-Pro-Val-Pro-Gln (SEQ ID NO: 5)
(F) Glu-Met-Pro-Phe-Pro-Lys (SEQ ID NO: 6)
(G) Val-Ile-Pro-Tyr (SEQ ID NO: 7)
(H) Thr-Lys-Val-Ile-Pro-Tyr (SEQ ID NO: 8)
(I) Val-Ala-Pro-Phe-Pro-Glu (SEQ ID NO: 9)
(J) Phe-Phe-Val-Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly (SEQ ID NO: 10)
(K) Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn (SEQ ID NO: 11)
(L) Ala-Met-Lys-Pro-Trp-Ile-Gln-Pro-Lys (SEQ ID NO: 12) Food and drink for improvement of brain function, central nervous system, comprising any one or two or more peptides selected from the group consisting of peptides consisting of the amino acid sequences shown in (d) to (l) below: Food and drink for function improvement or food and drink for swallowing function improvement.
(D) Ala-Val-Pro-Tyr-Pro-Gln (SEQ ID NO: 4)
(E) Val-Leu-Pro-Val-Pro-Gln (SEQ ID NO: 5)
(F) Glu-Met-Pro-Phe-Pro-Lys (SEQ ID NO: 6)
(G) Val-Ile-Pro-Tyr (SEQ ID NO: 7)
(H) Thr-Lys-Val-Ile-Pro-Tyr (SEQ ID NO: 8)
(I) Val-Ala-Pro-Phe-Pro-Glu (SEQ ID NO: 9)
(J) Phe-Phe-Val-Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly (SEQ ID NO: 10)
(K) Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn (SEQ ID NO: 11)
(L) Ala-Met-Lys-Pro-Trp-Ile-Gln-Pro-Lys (SEQ ID NO: 12)