JP2015189672A - Circadian rhythm improver - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a circadian rhythm improver which has high safety to a living body and is inexpensive.SOLUTION: A circadian rhythm improver has casein hydrolysate having physical and chemical properties of (a) to (c) below as an active ingredient: (a) ratio of fractions having molecular weight of 1000u or less is 80 mass% or more; (b) ratio of fractions having molecular weight of 3500u or more is less than 1 mass%; and (c) percentage of a total mass of free amino acid occupied in a total mass of total amino acid contained in casein hydrolysate is less than 10 mass%.

Description

  The present technology relates to a circadian rhythm improving agent. More specifically, the present invention relates to a circadian rhythm improving agent comprising a casein hydrolyzate as an active ingredient, and a preventive or therapeutic agent for diseases caused by circadian rhythm disorders using the circadian rhythm improving agent.

  Circadian rhythm (circadian rhythm) is a physiological phenomenon that fluctuates in a cycle of about 24 hours. Circadian rhythms are also present in vital activities such as brain waves, hormone secretion, and cell regeneration, and play an important role in determining sleep and eating patterns. The circadian rhythm disorder causes various modulations such as sleep / wake rhythm disorder, hypersomnia disorder, autonomic dysfunction, and endocrine disorder.

  For example, Delayed Sleep Phase Syndrome (hereinafter referred to as “DSPS”) is known as sleep / wake rhythm disorder caused by circadian rhythm disorder (Non-patent Document 1). Symptoms of DSPS include chronic insomnia and excessive daytime sleepiness, and have a great influence on work, social life, mood and learning (Non-patent Document 2).

  Moreover, in almost all mammals, it is clear that physiological and behavioral circadian rhythms are regulated by the body clock (Non-patent Document 3).

Furthermore, it is known that abnormal rhythms of the body clock are induced by exposure to blue light, stress, etc. (Non-patent Documents 4 and 5).
It is also known that abnormal rhythms in the biological clock are induced by jet lag, obesity, etc., and abnormal rhythms in the biological clock due to jet lag are promoted by obesity (Non-Patent Documents 6 and 7). .

  From this point of view, substances that are effective in rhythm abnormality of the biological clock are extremely useful. So far, several substances have been reported as effective substances for abnormal rhythm of the body clock. For example, cryptoxanthin, fucoxanthin, and the like, which are types of carotenoids, have been reported (for example, Patent Documents 1 and 2). In addition, progesterone which is a kind of steroid hormone has been reported (for example, Patent Document 3).

JP 2009-84192 A JP2013-133295A JP 2010-77081 A

Regestein, Q.R. et al., General hospital psychiatry 17, 335-345 (1995) Czeisler, C.A. et al., Sleep, 4, 1-21 (1981) Pando, M.P. et al., Sci STKE, 107, re16 (2001) Berson D.M. et al., Science, 295, 1070-1073 (2002) Stewart K.T. et al., Physiol. Behav., 48, 149-155 (1990) Kohsaka A. et al., Cell Metabolism, 6, 414-421 (2007) Mendoza J. et al., J Physiol., 586, 5901-5910 (2008)

  As described above, many studies have been conducted so far as substances that are effective in abnormal rhythms of biological clocks. Reported substances include plant-derived extract components and chemicals synthesized by artificial methods such as chemical synthesis, and further development is desired from the viewpoint of safety and effectiveness. Is the actual situation.

  Therefore, the main object of the present technology is to provide an inexpensive circadian rhythm improving agent that is highly safe to living bodies.

  As a result of intensive studies to solve the above problems, the present inventors have found that casein hydrolyzate, which is a milk-derived component, has a good circadian rhythm improving effect, and completes the present technology. It came to.

That is, the present technology is a circadian rhythm improving agent containing a casein hydrolyzate having the following physicochemical properties (a) to (c) as an active ingredient.
(A) The ratio of the fraction having a molecular weight of 1000 Daltons or less is 80% by mass or more,
(B) The ratio of the fraction having a molecular weight of 3500 daltons or more is less than 1% by mass,
(C) The ratio of the total mass of free amino acids to the total mass of all amino acids contained in the casein hydrolyzate is less than 10% by mass.
The circadian rhythm improving agent according to the present technology is characterized by containing a casein hydrolyzate, which is a milk-derived component, which has not been known as a substance effective for rhythm abnormality of the body clock until now. .
The circadian rhythm improving agent according to the present technology can be used for the purpose of improving sleep / wake rhythm disorder and / or improving hypersomnia disorder. The present technology also provides a preventive or therapeutic agent for diseases caused by circadian rhythm disorders, including the circadian rhythm improving agent according to the present technology.

  According to the present technology, a component derived from milk, which has been used for many years as a food, is used as an effective component of a circadian rhythm improving agent. Can do. In addition, the effect described here is not necessarily limited, and may be any effect described in the present technology.

It is a figure which shows the circadian rhythm of the rotation behavior of the mouse | mouth which ingested normal food. It is a figure which shows the daily rhythm of the rotation behavior of the mouse | mouth which ingested the high fat diet. It is the graph which digitized the daily rhythm of the rotation behavior of the mouse | mouth of FIG.1 and FIG.2. It is a figure which shows the circadian rhythm of the rotation behavior of the mouse | mouth which continues ingesting a high fat diet and the internal clock rhythm remains abnormal. It is a figure which shows the daily rhythm of the rotation behavior of the mouse | mouth which switched to normal food. It is a figure which shows the circadian rhythm of the rotation behavior of the mouse | mouth switched to normal meal + 0.25% casein. It is a figure which shows the circadian rhythm of the rotation behavior of the mouse | mouth switched to normal meal + 0.25% casein hydrolyzate. It is the graph which digitized the daily rhythm of the rotation behavior of the mouse | mouth of FIGS.

  Hereinafter, preferred embodiments for carrying out the present technology will be described. In addition, embodiment described below shows an example of typical embodiment of this technique, and, thereby, the scope of this technique is not interpreted narrowly.

<1. Circadian rhythm improving agent>
The circadian rhythm improving agent according to the present technology is characterized by containing a casein hydrolyzate, which is a milk-derived component, as an active ingredient.

(1) Casein hydrolyzate The casein, which is a raw material, contains a protein derived from milk as a main component, and the casein is not particularly limited. For example, commercially available various caseins and caseinates can be used. More specifically, lactate casein, sulfate casein, casein hydrochloride, sodium caseinate, potassium caseinate, calcium caseinate, magnesium caseinate, or any mixture thereof can be used. Moreover, casein etc. refine | purified by the conventional method from cow's milk, skim milk, whole fat milk powder, and skim milk powder can also be utilized.

  Casein hydrolyzate, which is the active ingredient of this technology, is manufactured by hydrolyzing casein, which is a raw material derived from milk that is relatively inexpensive as a biomaterial. Therefore, it should be manufactured stably and simply in large quantities. Can do.

(2) Method for Producing Casein Hydrolyzate The method for producing casein hydrolyzate is not particularly limited, and examples thereof include a method for producing using a protease such as a protease. Hereinafter, a method for producing a casein hydrolyzate using a proteolytic enzyme will be specifically described.

First, a raw material (casein) is dispersed and dissolved in water.
The concentration of the lysate is not particularly limited, but it is usually preferable from the viewpoint of efficiency and operability to make the concentration range around 5 to 15% by mass in terms of protein.

Next, a raw material aqueous solution is prepared by adjusting the pH of the solution to be close to the optimum pH of the protease used. Specifically, it is preferable to adjust the pH of the solution to pH 7 to 10 in which the optimum pH of many proteolytic enzymes is included in the range using an alkaline solution.
Although the alkali agent used for pH adjustment is not specifically limited, For example, sodium hydroxide, potassium hydroxide, potassium carbonate etc. are mentioned.

Next, a proteolytic enzyme is added to the prepared raw material aqueous solution.
Examples of proteolytic enzymes include bacterial, animal and plant derived proteolytic enzymes, and any of them can be used.

The proteolytic enzyme derived from bacteria is not particularly limited. For example, as an endoprotease derived from Bacillus, Alcalase (manufactured by Novozymes), Neutase (manufactured by Novozymes), Protin A (manufactured by Daiwa Kasei), Protin P (Yamato) Kasei Co., Ltd.), Pro Leather (Amano Enzyme), Protease N (Amano Enzyme), Corolase 7089 (Higuchi Trading Co.), Bioprase (Nagase Chemtech), Orientase 90N (Hibiai) Examples include orientase 22BF (manufactured by Hibiai).
The animal-derived proteolytic enzyme is not particularly limited, and examples thereof include PTN mainly composed of trypsin (manufactured by Novozymes), trypsin V (manufactured by Nippon Biocon).
The plant-derived proteolytic enzyme is not particularly limited, and examples thereof include papain (manufactured by Amano Enzyme) and bromelain (manufactured by Amano Enzyme).
Moreover, the proteolytic enzyme mentioned above can also be used individually or in combination of 2 or more types.

  The proteolytic enzyme is preferably used after being dispersed in cold water at 4 to 10 ° C. and dissolved. The concentration of the proteolytic enzyme solution is not particularly limited, but it is usually desirable from the viewpoint of efficiency and operability that the enzyme concentration is about 3 to 10%.

  The amount of proteolytic enzyme used for casein hydrolysis varies depending on the substrate concentration, enzyme titer, reaction temperature, reaction time, etc., but generally 1000 to 20000 units (activity unit) per 1 g protein equivalent mass of casein. ) Is a desirable embodiment.

  The proteolytic enzyme activity unit can be measured according to the type of proteolytic enzyme used.

  In adding the proteolytic enzyme, it is desirable to dissolve and add them one by one, but the order of addition is not particularly limited.

During the enzyme reaction, the temperature of the reaction system is not particularly limited, and is selected from a range that can be put to practical use including the optimum temperature range in which the enzyme action is exhibited, and is usually selected from the range of 30 to 60 ° C.
The reaction duration varies depending on the reaction conditions such as reaction temperature and initial pH.For example, if the reaction duration of the enzyme reaction is constant, a decomposition product having different physicochemical properties may be generated for each production batch. Because there is a problem, it cannot be decided. Therefore, by monitoring the enzyme reaction, the reaction duration is determined so that the physicochemical properties of the casein hydrolyzate have a desired value.

  Here, in the casein hydrolyzate according to the present technology, the ratio of the fraction having a molecular weight of 1000 daltons or less is 80% by mass or more, and the ratio of the fraction having a molecular weight of 3500 daltons or more is less than 1% by mass. It is characterized by that. Therefore, in the present technology, the casein hydrolyzate contained in the filtrate after removing the insoluble matter generated by the hydrolysis by filtration according to the degree of hydrolysis satisfies the conditions (a) and (b). Reaction conditions such as reaction temperature and reaction duration are determined so as to satisfy the conditions.

  Examples of the enzyme reaction monitoring method include a method of collecting a part of the reaction solution and measuring the protein degradation rate and the like.

Next, the enzyme reaction is stopped.
The enzyme reaction is stopped by deactivating the enzyme in the hydrolyzed solution. The deactivation treatment can be performed by a conventional method such as a heat deactivation treatment.
The conditions (heating temperature, heating time, etc.) for the heat deactivation treatment can be set as appropriate in consideration of the thermal stability of the enzyme used, for example, a temperature range of 80 to 130 ° C. In a holding time of 30 minutes to 2 seconds.

  After the enzymatic reaction is stopped, the hydrolysis deactivation liquid obtained is selected from the group consisting of (a) filtration, (b) membrane separation treatment such as microfiltration, ultrafiltration membrane, and (c) resin adsorption separation. It is preferable to purify by any one or a combination of two or more thereof.

  By performing the above-described purification, it is possible to remove insoluble matters contained in the inactivated liquid, reduce fat, lactose, and other unnecessary components. As a result, it is possible to obtain a casein hydrolyzate that is transparent in a solution state and that does not cause turbidity, precipitation, agglomeration, browning or the like even during long-term storage in a solution state, and has excellent storage stability.

  Moreover, the flavor of the casein hydrolyzate which concerns on this technique, an external appearance, etc. can be improved by performing the refinement | purification mentioned above.

The filtration of (a) can be performed by a known method, for example, using diatomaceous earth and a known apparatus.
By performing filtration, it is possible to remove the insoluble matter produced during the hydrolysis reaction and / or during the enzyme heating inactivation in the hydrolysis deactivation liquid.

The membrane separation treatment (b) can be performed using a known apparatus. Although it does not specifically limit as a well-known apparatus, For example, ultrafiltration modules etc., ultrafiltration module SEP1053 (Asahi Kasei company make, molecular weight cut off 3,000), SIP1053 (Asahi Kasei company make, molecular weight cut off 6,000), SLP1053 (Manufactured by Asahi Kasei Co., Ltd., fractional molecular weight 10,000).
In this case, a solution containing a casein hydrolyzate is obtained as a membrane permeation fraction after membrane separation treatment.
By performing the membrane separation treatment, the insoluble matter generated during the hydrolysis reaction and / or the enzyme heating inactivation existing in the hydrolysis deactivation liquid can be removed as in the filtration of (1).

The resin adsorption separation of (c) can be carried out by a known method. For example, it can be carried out by filling a column with resin and allowing the hydrolysis and deactivation liquid to pass through the column. Although it does not specifically limit as resin, For example, brand name: Diaion, Sepa beads (made by Mitsubishi Chemical Corporation), Amberlite XAD (made by Organo Corporation), KS-35 (made by Ajinomoto Fine Techno Co., Ltd.), etc. are mentioned.
Resin adsorption separation can also be performed in a continuous manner by filling these resins into a column and continuously flowing in and out of the hydrolysis deactivation liquid. Can be carried out in a batch system in which the hydrolytic deactivation liquid and the resin are separated after being put in contact for a certain period of time.
Factors that cause turbidity, precipitation, agglomeration, browning, etc. during the storage period (for example, peptides containing a large amount of hydrophobic amino acids) may remain in the hydrolyzed deactivation solution. By performing the above, these factors can be removed.

Moreover, you may sterilize the solution containing the obtained casein hydrolyzate after refinement | purification.
As the sterilization method, a conventional heat treatment method or the like can be used.
The heating temperature and holding time at the time of the heat treatment may be set appropriately under conditions that can be sufficiently sterilized.
The heat sterilization method can be either a batch method or a continuous method, and a plate heat exchange method, an infusion method, an injection method, or the like can also be used in the continuous method.

  Furthermore, the solution containing the obtained casein hydrolyzate can be used as it is, and if necessary, the solution can also be used as a concentrated concentrate by a known method. Further, the concentrated liquid can be dried by a known method and used as a powder.

  In addition, after removing the insoluble components of the casein hydrolyzate of the present technology, for the purpose of improving flavor or improving physical properties, etc., endoprotease or exoprotease is added to perform secondary hydrolysis, Subsequent processing can also be performed.

(3) Ratio of the total mass of free amino acids The casein hydrolyzate according to the present technology has the above-described (a) fraction having a molecular weight of 1000 daltons or less and a fraction having a molecular weight of 3500 daltons or more. In addition to being less than 1% by mass, the ratio of the total mass of free amino acids to the total mass of all amino acids contained in (c) casein hydrolyzate is less than 10% by mass. . In the present technology, the ratio of the total mass of free amino acids can be determined, for example, by the following procedure.

(A) Measurement of amino acid composition For amino acids other than tryptophan, cysteine and methionine, the sample was hydrolyzed with 6N hydrochloric acid at 110 ° C. for 24 hours, and for tryptophan, alkaline decomposition was performed with barium hydroxide at 110 ° C. for 22 hours to obtain cysteine. And methionine are treated with performic acid, hydrolyzed with 6N hydrochloric acid at 110 ° C. for 18 hours, and analyzed with an amino acid analyzer (manufactured by Hitachi, Ltd., type 835) to measure the mass of the amino acid.
In this method, the amount of glutamine and glutamic acid in the sample is quantified as an analytical value of glutamic acid, which is the total amount of both.

(B) Calculation of the ratio of the total mass of free amino acids Each amino acid composition in the sample is measured by the method of (a) above, and the total is calculated to calculate the mass of all amino acids in the sample. Next, the sample is deproteinized with sulfosalicylic acid, the mass of each remaining free amino acid is measured by the method (a) above, and the total is calculated to calculate the mass of all free amino acids in the sample. From these values, the ratio of the total mass of free amino acids in the sample is calculated by the following formula.

  The ratio of the total mass of free amino acids should be adjusted according to the type of enzyme used to hydrolyze casein, the amount of enzyme added, the reaction time, and / or the purification conditions after hydrolysis (membrane separation, resin adsorption separation), etc. Can do.

(4) Features and effects of circadian rhythm improving agent The casein hydrolyzate used in the present technology obtained as described above is the following (a) to (c), as is apparent from the examples described later. ) Physicochemical properties.
(A) The ratio of the fraction having a molecular weight of 1000 Daltons or less is 80% by mass or more,
(B) The ratio of the fraction having a molecular weight of 3500 daltons or more is less than 1% by mass,
(C) The ratio of the total mass of free amino acids to the total mass of all amino acids contained in the casein hydrolyzate is less than 10% by mass.

  The casein hydrolyzate used in the present technology reduces peptides having a molecular weight exceeding 1000 daltons with a sufficient degradation rate, (a) the proportion of the fraction having a molecular weight of 1000 daltons or less is 80% by mass or more, and (b) the molecular weight is 3500 daltons. By setting the ratio of the above fractions to less than 1% by mass, the digestive absorbability from the intestinal tract is excellent. Therefore, the circadian rhythm improving agent using this casein hydrolyzate as an active ingredient has an excellent circadian rhythm improving effect.

  The casein hydrolyzate used in the present technology suppresses the release of amino acids, and (c) the ratio of the total mass of free amino acids to the total mass of all amino acids contained in the casein hydrolyzate is less than 10% by mass. Therefore, the unpleasant taste derived from free amino acids is suppressed, and the flavor is almost tasteless and odorless. Therefore, the circadian rhythm improving agent containing the casein hydrolyzate as an active ingredient can be used in various medicines, foods and drinks, feeds and the like.

  The circadian rhythm improving agent according to the present technology can be used for the purpose of improving sleep / wake rhythm disorder and / or improving hypersomnia disorder. The “sleep / wake rhythm disorder” is one of the disorders caused by the circadian rhythm disorder, and specifically includes the above-mentioned DSPS, advanced sleep phase syndrome, and the like. In addition, “hypnosis disorder” is one of the disorders caused by circadian rhythm disorders, and specific examples include recurrent hypersomnia, periodic somnolence, Klein-Levin syndrome, and narcolepsy. .

  In addition, the dosage of the circadian rhythm improving agent according to the present technology can be appropriately set according to the usage, patient age, sex, degree of disease, other conditions, and the like.

  The circadian rhythm improving agent according to the present technology described above has high safety to the living body because it uses an ingredient derived from milk that has been used for many years as a food. Therefore, there is no risk of side effects and dependence, and it can be taken continuously for a long time.

  In addition, the casein hydrolyzate, which is an active ingredient of the circadian rhythm improving agent according to the present technology, can be stably and easily produced in large quantities from a relatively inexpensive milk-derived raw material as a biomaterial, It is possible to provide a circadian rhythm improving agent at low cost. Therefore, the circadian rhythm improving agent that can be easily ingested over a long period of time can be provided from the aspect of cost burden.

<2. Drugs for preventing or treating diseases caused by circadian rhythm disorders>
As described above, the casein hydrolyzate, which is an active ingredient of the circadian rhythm improving agent according to the present technology, is excellent in digestibility and absorption from the intestinal tract, is almost tasteless and odorless, and as described later, Since it has a rhythm improving effect, it can be used as a medicine for humans or animals for the prevention or treatment of diseases caused by circadian rhythm disorders.

  Moreover, since the circadian rhythm improving agent which concerns on this technique uses the component derived from the milk used for many years as an active ingredient, there is a high possibility that it can be safely administered to patients suffering from various diseases. . Moreover, there is little need to worry about side effects even if administered continuously for a long period of time. Furthermore, it is highly safe when used in combination with other drugs.

  When the circadian rhythm improving agent according to the present technology is used as a medicine, the medicine may be administered orally or parenterally, but oral administration is preferred. Examples of parenteral administration include intravenous injection, rectal administration, and inhalation. Examples of the dosage form for oral administration include tablets, capsules, troches, syrups, granules, acid agents, ointments and the like.

  Moreover, in formulating, in addition to the casein hydrolyzate according to the present technology, components such as an excipient, a pH adjuster, a colorant, and a corrigent that are usually used for formulation can be used. In addition, as long as it contains a circadian rhythm improving agent according to the present technology, a component having the effect of preventing or treating a disease caused by a known or future circadian rhythm disorder is included in the present technology. It can also be used in combination with a circadian rhythm improving agent.

  Furthermore, depending on the administration method, it can be appropriately formulated into a desired dosage form. For example, in the case of oral administration, it can be formulated into solid preparations such as powders, granules, tablets and capsules; liquid preparations such as solutions, syrups, suspensions and emulsions. For parenteral administration, it can be formulated into suppositories, sprays, ointments, patches, injections and the like.

  In addition, formulation can be appropriately performed by a known method according to the dosage form. At the time of formulation, only the casein hydrolyzate which is an active ingredient may be formulated, or may be formulated by appropriately blending a formulation carrier.

  In addition, when blending a pharmaceutical carrier, the content of casein hydrolyzate, which is an active ingredient of a circadian rhythm improving agent, is not particularly limited, and is appropriately determined based on the daily intake or dose according to the dosage form. You can choose. The daily intake or dose of the casein hydrolyzate is preferably in the range of 100 mg / day to 5 g / day.

  In addition, as the preparation carrier, various organic or inorganic carriers can be used depending on the dosage form. Examples of the carrier in the case of a solid preparation include excipients, binders, disintegrants, lubricants, stabilizers, and flavoring agents.

  Examples of the excipient include sugar derivatives such as lactose, sucrose, glucose, mannitol and sorbit; starch derivatives such as corn starch, potato starch, α-starch, dextrin and carboxymethyl starch; crystalline cellulose, hydroxypropyl cellulose, Cellulose derivatives such as hydroxypropylmethylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium; gum arabic; dextran; pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate and magnesium magnesium aluminosilicate; phosphate derivatives such as calcium phosphate; And carbonate derivatives such as calcium; sulfate derivatives such as calcium sulfate and the like.

  Examples of the binder include gelatin, polyvinyl pyrrolidone, macrogol and the like in addition to the above excipients.

  Examples of the disintegrant include, in addition to the above excipients, chemically modified starch or cellulose derivatives such as croscarmellose sodium, carboxymethyl starch sodium, and crosslinked polyvinylpyrrolidone.

  As the lubricant, for example, talc; stearic acid; stearic acid metal salts such as calcium stearate and magnesium stearate; colloidal silica; waxes such as pea gum and geirow; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid Carboxylic acid sodium salts such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as anhydrous silicic acid and silicic acid hydrate; starch derivatives and the like It is done.

  Examples of the stabilizer include paraoxybenzoates such as methyl paraben and propyl paraben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; sorbic acid and the like.

Examples of the flavoring agent include sweeteners, acidulants, and fragrances.
In addition, as a carrier used in the case of a liquid for oral administration, a solvent such as water, a flavoring agent and the like can be mentioned.

<Food and drink, feed>
As described above, the casein hydrolyzate, which is an active ingredient of the circadian rhythm improving agent according to the present technology, is excellent in digestibility and absorption from the intestinal tract, is almost tasteless and odorless, and as described later, Since it has a rhythm-improving effect, it can be used in combination with these as active ingredients such as food or drink for humans or animals and feed for the prevention or treatment of diseases caused by circadian rhythm disorders.

(1) Food and drink When using the circadian rhythm improving agent according to the present technology for food and drink, it can be added to known food and drink to prepare a food and drink having a circadian rhythm improving effect. It is also possible to produce a new food or drink having a circadian rhythm improving effect by mixing in raw materials.

  The foods and drinks may be in the form of liquids, pastes, solids, powders, etc., in addition to tablet confections, liquid foods, feeds (including for pets), etc., for example, flour products, instant foods, processed agricultural products, marine products Processed products, processed livestock products, milk / dairy products, fats and oils, basic seasonings, compound seasonings / foods, frozen foods, beverages, and other commercial products.

Examples of flour products include bread, macaroni, spaghetti, noodles, cake mix, fried flour, bread crumbs and the like.
Examples of instant foods include instant noodles, cup noodles, retort / cooked food, cooking canned food, microwave food, instant soup / stew, instant miso soup / soup, canned soup, freeze-dried food, other instant foods, etc. It is done.
Examples of processed agricultural products include canned agricultural products, canned fruits, jams and marmalades, pickles, boiled beans, dried agricultural products, cereals (cereal processed products), and the like.
Examples of processed fishery products include canned fishery products, fish hams and sausages, marine products, marine delicacies, and tsukudani.
Examples of livestock processed products include canned livestock, pastes, livestock meat ham, sausage and the like.
Examples of milk / dairy products include processed milk, milk beverages, yogurts, lactic acid bacteria beverages, cheese, ice creams, prepared powdered milks, creams, and other dairy products.
Examples of the fats and oils include butter, margarine, vegetable oil and the like.
Basic seasonings include, for example, soy sauce, miso, sauces, tomato processed seasonings, mirins, vinegars, etc., and the above mixed seasonings and foods include cooking mix, curry ingredients, sauces, Examples include dressings, noodle soups, spices, and other complex seasonings.
Examples of the frozen food include raw material frozen food, semi-cooked frozen food, cooked frozen food, and the like.
Examples of the confectionery include caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pie, snacks, crackers, Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery, and other confectionery.
Examples of beverages include carbonated beverages, natural fruit juices, fruit juice drinks, soft drinks with fruit juice, fruit drinks, fruit drinks with fruits, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks Sports drinks, nutritional drinks, alcoholic drinks, other taste drinks, and the like.
Examples of commercially available foods other than the above include baby foods, sprinkles, and tea paste.

In addition, the food and drink defined in the present technology can be provided and sold as a food and drink displaying the health use for prevention or treatment of circadian rhythm disorder.
The “display” act includes all acts for informing the consumer of the use, and if the expression can remind the user of the use, the purpose of the display, the content of the display, the display Regardless of the target object / medium, etc., all fall under the “display” act of this technology.

  Moreover, it is preferable that the “display” is performed by an expression that allows the consumer to directly recognize the above-described use. Specifically, it is the act of transferring, displaying, importing, displaying, or importing products that are related to food or drinks or products that describe the use, on advertisements, price lists, or transaction documents. For example, an act of describing and displaying the above uses or distributing them, or describing the above uses in information including the contents and providing them by an electromagnetic (Internet or the like) method can be given.

  On the other hand, the display content is preferably a display approved by the government or the like (for example, a display that is approved based on various systems determined by the government and performed in a mode based on such approval). Moreover, it is preferable to attach such display contents to advertising materials at sales sites such as packaging, containers, catalogs, pamphlets, POPs, and other documents.

  “Indication” also includes indication as health food, functional food, enteral nutrition food, special purpose food, health functional food, food for specified health, functional nutrition food, quasi drug, etc. Among these, in particular, there are indications approved by the Consumer Affairs Agency, for example, indications approved under the food system for specific health use, a similar system, and the like. Examples of the latter can include indications for food for specified health use, indications for conditional health foods, indications that affect the structure and function of the body, indications for reducing disease risk, etc. Specifically, indications as food for specified health (especially indications for health use) and similar indications stipulated in the Enforcement Regulations of the Health Promotion Act (Ministry of Health, Labor and Welfare Ordinance No. 86 of April 30, 2003) Is a typical example.

  In addition, content of the casein hydrolyzate which is an active ingredient of the circadian rhythm improving agent added when manufacturing foods and drinks is not specifically limited, It can select suitably based on the intake per day. The daily intake of the casein hydrolyzate is preferably in the range of 100 mg / day to 5 g / day.

(2) Feed When the circadian rhythm improving agent according to the present technology is used in feed, it can be added to a known feed to prepare a feed having a circadian rhythm improving effect, or mixed in the feed raw material. A new feed having an effect of improving the circadian rhythm can also be produced.

  Examples of the raw material of the feed include cereals such as corn, wheat, barley, and rye; bran such as bran, wheat straw, rice bran, and defatted rice bran; and manufactured porridges such as corn gluten meal and corn jam meal; Animal feeds such as whey, fish meal and bone meal; yeasts such as beer yeast; mineral feeds such as calcium phosphate and calcium carbonate; fats and oils; amino acids; In addition, examples of the form of the feed include pet animal feed (pet food, etc.), livestock feed, fish feed, and the like.

  In addition, content of the casein hydrolyzate which is an active ingredient of the circadian rhythm improving agent added when manufacturing feed is not specifically limited, It can select suitably based on the daily food intake. The daily intake of the casein hydrolyzate is preferably in the range of 1 mg / kg body weight / day to 100 mg / kg body weight / day.

  Hereinafter, the present invention will be described in more detail based on examples. In addition, the Example demonstrated below shows an example of the typical Example of this invention, and, thereby, the range of this invention is not interpreted narrowly.

  In Example 1, casein hydrolyzate, which is an active ingredient of the present technology, was produced by two methods.

(1) Production Example 1
9 kg of water is added to 1 kg of commercially available casein (manufactured by New Zealand Daily Board), uniformly dispersed, 10% aqueous sodium hydroxide solution is added, the pH of the solution is adjusted to 7.0, and casein is completely dissolved A casein aqueous solution having a concentration of about 10% was prepared. The casein aqueous solution was sterilized by heating at 85 ° C. for 10 minutes, adjusted to a temperature of 50 ° C., sodium hydroxide was added to adjust the pH to 9.5, and Bioplase sp-20 (manufactured by Nagase Seikagaku Corporation) 1,008,000 active units (1,200 active units per gram of protein), Protease N (Amano Pharmaceutical Co., Ltd.) 1,680,000 active units (2,000 active units per gram of protein) and PTN 6.0S ( Novo Nordisk Co., Ltd.) 5,880,000 active units (7,000 active units per gram of protein) were added to start the hydrolysis reaction. Over time, the enzymatic reaction was monitored by the rate of protein degradation, When the rate reached 24.1%, the enzyme was inactivated by heating at 80 ° C. for 6 minutes, the enzyme reaction was stopped, and the mixture was cooled to 10 ° C. To this hydrolyzate, standard supercell (manufactured by Tokyo Kamochi Co., Ltd.) is added as a filter aid, suction filtered, and the resulting filtrate is then used as an ion exchange resin [Amberlite XAD-7 (manufactured by Organo Corporation). respect], SV (space velocity) = 2.5 h ^-1, and contact treatment at a temperature of 10 ° C., the solution containing the resulting casein hydrolyzate is concentrated by a conventional method, spray drying, spray 0.90 kg of a dried product (powdered casein hydrolyzate) was obtained.

  The casein hydrolyzate obtained by the method shown in Production Example 1 has a ratio of the fraction having a molecular weight of 1000 daltons or less of 83.1%, the ratio of the fraction of 3500 daltons or more is 0.1%, and the total mass of free amino acids. Was 6.7%, and the tryptophan contained in 1 g was 3.4 mg and the transmittance was 99.4%. Further, the casein hydrolyzate was a substance having almost no taste and odor, and excellent storage stability (no precipitation and no coloring).

(2) Production Example 2
9 kg of water was added to 1 kg of commercially available sodium caseinate (protein content 85%, manufactured by Uni-Re France), and mixed well with stirring to completely dissolve sodium caseinate to prepare an aqueous casein solution having a concentration of about 10%. The aqueous casein solution was sterilized by heating at 80 ° C. for 10 minutes, adjusted to 50 ° C., sodium hydroxide was added to adjust the pH to 9.0, and Bromelain (Amano Pharmaceutical Co., Ltd.) 13,600,000 active units (16,000 active units per gram of protein), Neutase (manufactured by Novo Nordisk) 1,770,000 active units (2,000 active units per gram of protein) and Protease S (manufactured by Amano Pharmaceutical) 1 , 020,000 active units (1,200 active units per gram of protein) are added to start the hydrolysis reaction. Over time, the enzymatic reaction is monitored by the degradation rate of the protein, and the degradation rate reaches 25.6%. At that time, the enzyme was inactivated by heating at 80 ° C. for 6 minutes, the enzyme reaction was stopped, and the mixture was cooled to 10 ° C. This hydrolyzed solution was filtered with an insoluble material by a membrane separation method (microfiltration) using Microza EMP-313 (pore size 0.25 μm, manufactured by Asahi Kasei Co., Ltd.), and then the obtained filtrate was treated with a hydrophobic resin. The solution containing the casein hydrolyzate obtained by subjecting the filtrate to contact treatment under the conditions of SV (space velocity) = 2h ⁻¹ and temperature of 10 ° C. against [KS-35 (made by Hokuetsu Carbon Co., Ltd.)]. Was concentrated by a conventional method and spray-dried to obtain 0.74 kg of a spray-dried product (powdered casein hydrolyzate).

  The casein hydrolyzate obtained by the method shown in Production Example 2 has a fraction of a molecular weight of 1000 daltons or less of 82.5%, a fraction of 3500 daltons or more of a fraction of 0.2%, and the total mass of free amino acids. Was 5.3%, the tryptophan contained in 1 g was 3.5 mg, and the transmittance was 99.4%. Further, the casein hydrolyzate was a substance having almost no taste and odor, and excellent storage stability (no precipitation and no coloring).

  In Example 2, the effect of the circadian rhythm improving agent according to the present technology was examined.

(1) Induction of abnormal biological rhythm in mice by high fat diet (a) Test method C57BL according to the method of Non-Patent Document 6 (Kohsaka A. et al., Cell Metabolism, 6, 414-421 (2007)) / 6J (5-week-old moth, Charles River Japan, Inc.) was fed a diet with a lipid content of 45% fat / kcal to induce abnormal clock rhythm.

(B) Test result FIG. 1 is a diagram showing a daily rhythm of a rotating behavior of a mouse ingesting a normal diet (lipid content 10% fat / kcal). Moreover, FIG. 2 is a figure which shows the circadian rhythm of the rotation behavior of the mouse | mouth which ingested the high fat diet (lipid content 45% fat / kcal). 1 and 2, the vertical axis represents the number of times the mouse has rotated the rotating basket, and the horizontal axis represents time.

  Mice were bred under a light / dark cycle of 12 hours light and 12 hours dark. More specifically, a light / dark cycle was set with the light period from 8 am to 8 pm and the dark period from 8 pm to 8 am (see the black and white bar in the figure). The horizontal axis in FIGS. 1 and 2 plots two days. The data on the left side of the row is the same as the one-day data on the right side of the row above it. The activity pattern can be easily seen by plotting two days.

(C) Discussion Mice are nocturnal, and if they are kept under a light-dark cycle, the activity of the day is concentrated in the dark period. As shown in FIG. 1, the animals reared on a normal diet had a clear circadian rhythm that ceased in the light period and activated in the dark period. On the other hand, as shown in FIG. 2, animals raised on a high-fat diet increase the amount of activity during the light period, and the amount of activity during the dark period decreases, so that the sharpness of light and dark becomes ambiguous. It was confirmed that abnormalities were induced in the circadian rhythm.

  Table 1 below shows a table in which the daily rhythm of the rotating behavior of the mouse in FIGS. 1 and 2 is quantified. FIG. 3 shows a table in which the daily rhythm of the rotating behavior of the mouse in FIGS. 1 and 2 is quantified. That is, comparing the amount of exercise during the light period (resting period) with the average value of 5 animals / group divided by the amount of daily exercise, intake of a high fat diet significantly increased the rate of exercise during the light period, and the daily rhythm It became clear that abnormalities were induced.

(2) Examination of effect of circadian rhythm improving agent according to the present technology on mice with abnormal rhythm of body clock (a) Test method As shown in (1) above, C57BL / 6J (5 weeks old mouse, Charles River Japan) Intraday rhythm abnormalities were induced by ingesting a diet with a lipid content of 45% fat / kcal for 5 months. Thereafter, three groups were set in which the diet was switched to a normal diet, a normal diet + 0.25% casein, and a normal diet + 0.25% casein hydrolyzate. In addition, as a target group, one group that continued to ingest a high fat diet as it was was set, and a total of four groups were compared.

(B) Test results FIG. 4 shows mice that continue to ingest a high-fat diet and the body clock rhythm remains abnormal, FIG. 5 shows mice that have switched to normal diets, and FIG. 6 shows normal diets plus 0.25% casein. FIG. 7 is a diagram showing the circadian rhythm of the rotating behavior of the mouse that has been switched, FIG. 7, the mouse that has been switched to the normal diet + 0.25% casein hydrolyzate. 4-7, the vertical axis | shaft represents the frequency | count that the mouse rotated the rotation basket, and the horizontal axis represents time.

  Mice were bred under a light / dark cycle of 12 hours light and 12 hours dark. More specifically, a light / dark cycle was set with the light period from 8 am to 8 pm and the dark period from 8 pm to 8 am (see the black and white bar in the figure). In addition, the horizontal axis of these FIGS. 4-7 has plotted two days worth. The data on the left side of the row is the same as the one-day data on the right side of the row above it. The activity pattern can be easily seen by plotting two days.

(C) Consideration As shown in FIG. 4, the animal that continued to be fed with a high-fat diet is ambiguous in the light and dark period, and as shown in FIG. 5, the light and dark period is recovered by switching to the normal diet. . In addition, as shown in FIG. 6, even in mice switched to normal diet + 0.25% casein, the sharpness of the light and dark period was recovered, similar to the animals that received the normal diet. Furthermore, as shown in FIG. 7, in the mouse switched to the normal diet + 0.25% casein hydrolyzate, sharpness in the light and dark period became clearer.

  Table 2 below shows a table in which the daily rhythm of the rotating behavior of the mice shown in FIGS. FIG. 8 shows a graph in which the daily rhythm of the rotating behavior of the mice in FIGS. That is, comparing the amount of exercise during the light period (resting period) with the average value of 5 animals / group divided by the amount of exercise per day, compared to animals that continued to consume high fat diet, In animals switched to food + 0.25% casein, the light momentum decreased and sharpness was induced in the light-dark behavioral rhythm, but the difference was not significant. On the other hand, in the animals switched to the normal diet + 0.25% casein hydrolyzate, the momentum rate during the light period was further reduced, and the difference from the animals that continued to ingest the high fat diet became significant.

(3) Summary From the above results, it has been clarified that by administering the circadian rhythm improving agent according to the present technology, the momentum during the active period of the mouse is improved and the momentum during the rest period is decreased. These effects are considered to be very effective for improving sleep / wake rhythm disorder and / or hypersomnia disorder seen in circadian rhythm disorder. It was also suggested that the use of the circadian rhythm improving agent according to the present technology can contribute to the prevention or treatment of diseases caused by circadian rhythm disorders.

Claims (3)

A circadian rhythm improving agent comprising a casein hydrolyzate having the following physicochemical properties (a) to (c) as an active ingredient:
(A) The ratio of the fraction having a molecular weight of 1000 Daltons or less is 80% by mass or more,
(B) The ratio of the fraction having a molecular weight of 3500 daltons or more is less than 1% by mass,
(C) The ratio of the total mass of free amino acids to the total mass of all amino acids contained in the casein hydrolyzate is less than 10% by mass.   The circadian rhythm improving agent according to claim 1, wherein the circadian rhythm improvement is improvement of sleep / wake rhythm disorder and / or improvement of hypersomnia disorder.   A preventive or therapeutic agent for diseases caused by circadian rhythm disorders, comprising the circadian rhythm improving agent according to claim 1 or 2.

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