JP2010104364A - Food and drink containing neurogenesis promoter - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide food and drink containing a neurogenesis promoter, which has high safety, is obtained at a low cost, and is easy to continuously ingest. <P>SOLUTION: The food and drink as a decomposition product obtained by collagenase treating collagen or gelatin is obtained by mixing an aqueous solution containing the collagenase treated decomposition product and a synthetic absorbent SEPABEADS SP850 followed by recovering unabsorbed component, and mixed with a neurogenesis promoter containing peptide including amino acid arrays shown by Gly-Pro-Arg, Gly-Pro-Ser-Gly-Asn-Ala, Gly-Pro-Val-Gly-Ala-Arg, Gly-Pro-Ala-Gly-Pro-Ala, Gly-Pro-Hyp, Gly-Pro-Ile-Gly-Ser-Ala, Gly-Pro-Val-Gly-Pro-Ala, Gly-Pro-Ser-Gly-Glu-Arg-Gly-Pro-Hyp, and Gly-Leu-Ala-Gly-Pro-Hyp. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a food or drink containing a neurogenesis promoting agent that is highly safe and easy to continuously ingest. In more detail, it is related with the food-drinks containing the neurogenesis promoter containing the collagen-derived peptide.

  Neurodegenerative diseases are a major social problem. For example, an increase in the number of patients with dementia in an aging society, Parkinson's disease and amyotrophic lateral sclerosis, whose causes are still unknown, are typical examples. Moreover, even in healthy people, so-called forgetfulness frequently occurs in daily life or learning ability decreases, and it is considered that neurodegeneration in the brain is also a cause. These diseases and functional decline are certainly natural phenomena associated with aging, but it has also been pointed out that stress and anxiety are induced or promoted.

  Furthermore, it has been pointed out that neurodegeneration is also related to mood state and the resulting behavior. This is the case with crimes caused by withdrawal of extremely aggressiveness and increased aggression. In order to improve these mood states, it is essential to increase resistance to stress, but recent developments in brain science suggest that increasing neurogenesis is the fundamental solution. .

  On the other hand, in the modern society, there is an increasing demand for increasing learning ability, cognitive ability and memory ability due to intensifying global competition and intensifying competition for examinations. These demands may have existed universally in human history, but it seems to have increased especially in recent years. Promoting neurogenesis is also an effective means for enhancing these brain function-related abilities.

  Traditionally, neurogenesis was thought to occur only during embryonic and other developmental stages, but neural stem cells also exist in adult mammalian brain, including humans, and the subventricular zone around the lateral ventricle It was found that neurogenesis occurs in certain areas such as the dentate gyrus of the hippocampus and the hippocampus. (Non-Patent Document 1)

  There have been reports on the promotion of neurogenesis. Examples of substances that increase neurogenesis include various endogenous neurotrophic factors. These include epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). It has also been clarified that hepatocyte growth factor promotes neurogenesis (Patent Document 1). Insulin-like growth factor (IGF-1) is also a neurotrophic factor, a hormone that has a structure similar to insulin and also acts on the cranial nervous system. Although IGF-1 itself is known to promote neurogenesis, its N-terminal 3-residue peptide, Gly-Pro-Glu, has a neuroprotective action, but directly promotes neurogenesis. There are no reports. (Non-patent document 2) (Non-patent document 3) (Non-patent document 4) (Patent document 2). There is no doubt that these neurotrophic factors are important factors responsible for neurogenesis in vivo. However, treatment by intracerebroventricular administration of neurotrophic factor, which is a protein, is difficult from the technical, safety, and cost aspects, and of course, the effect of oral administration cannot be expected.

  On the other hand, sodium valproate and lithium carbonate are known as drugs other than neurotrophic factors that enhance hippocampal neurogenesis. (Non-patent document 5) (Non-patent document 6). These drugs have been used in clinical settings for many years as antiepileptic drugs or anti-manic drugs, but it has recently been clarified that these drugs have a neurogenesis-promoting action.

  Neogenesis is also promoted by administration of donepezil and menmatine, which are drugs for dementia. Although the relationship with neurogenesis is unclear, pharmaceuticals that improve anxiety and stress are known to act on brain monoamine metabolism and GABA receptors. Specifically, there are selective serotonin reuptake inhibitors and drugs called benzodiazepines. However, these pharmaceuticals have known side effects such as serotonin syndrome, and candidate compounds other than these monoamines are required. In addition, progesterone-like substances are also used in the United States and the like in addition to monoamine drugs to improve aggressiveness. In Japan, ethical issues regarding the feminization of men are pointed out and not approved. Even if a similar drug is approved or a food having a similar action is found, side effects such as sexual dysfunction are expected to appear as in the case of a monoamine drug.

  In any case, all of the above examples are used as pharmaceuticals, and there are many problems such as the risk of side effects and the cost of continuous intake. In particular, drugs that act on the nervous system have many side effects, and there are many social problems such as suicide and addiction. Moreover, it is impossible for a healthy person to continuously take for a long time.

  That is, in order to fundamentally solve the healing of neurodegenerative diseases due to neurogenesis or the improvement of QOL (Quality of Life) due to neurogenesis, neurogenesis promoting agents that can be continuously ingested as foods are required. .

  Only a few reports on food ingredients that have a nerve promoting effect have been disclosed. A composition that promotes neurogenesis in the mammalian hippocampus containing flavonoids as an active ingredient (Patent Document 3), a composition that promotes thermogenesis containing theanine (Patent Document 4), and is involved in the proliferation of neurons in the brain organ / hippocampus. Arachidonic acid (Non-patent Document 7), a neuroblast proliferating agent and a neurite extender (Patent Document 5), which contain an extract of a plant of the genus Amaranthaceae as active ingredients. However, none of them is a substance that exists in a large amount in the natural world, it is difficult to ingest in terms of flavor, it has defects such as lack of stability, and there is a problem in production cost. For example, catechin, which is a flavonoid, and theanine, which is an amino acid derivative, are mainly contained in tea and are often purified from tea industrially, but tea contains a relatively large amount of caffeine. Excluding the cost. Caffeine is highly irritating and often contraindicated. In addition, plants of the genus Amaranthaceae are limited as raw material resources. However, a peptide obtained by a relatively simple treatment from a protein present in a large amount in the natural world has high safety, high stability, and low cost. However, there has been no report of a peptide derived from such a property having neurogenesis activity in the brain.

There is a report on (Gly-XY) n (wherein n is a positive integer, X and Y are amino acid residues, and X and Y are the same or different arbitrary amino acid residues). However, a collagen tripeptide-containing external preparation for skin (Patent Document 6) is an action that reduces irritation and discomfort to the skin, and a biogenic collagen synthesis promoter (Patent Document 7) is an action that promotes collagen synthesis in the living body. The composition for promoting the production of type I collagen (Patent Document 8) has an action of promoting the production of type I collagen, and the hyaluronic acid production promoter (Patent Document 9) has an action of promoting the production of hyaluronic acid. In other words, it is completely unrelated to neurogenesis in the brain.

  From the above, there has been no report of a food-derived peptide having such a property having activity for promoting neurogenesis in the brain.

JP 2007-284410 A Special table 2007-509169 JP 2007-161606 A JP 2008-169143 A JP 2008-239505 A JP 2003-176219 A Japanese Patent No. 3802721 Japanese Patent No. 40338877 Japanese Patent Laid-Open No. 2004-123637

Nature Med. 4, 1313-1317, 1998. Ann NY Acad Sci. , 692: 183-191, 1993. Neuropharmacology, 47 (6): 892-903, 2004. Brain Res. 922 (1): 42-50,2001. J Neurosci. , 24: 6590-6599, 2004. Stem cells, 26 (7): 1758-1767, 2008. Suntory Corporation "Neuro2007" conference presentation

  The subject of this invention is providing the food-drinks containing the neurogenesis promoter which is safe and obtained at low cost, and is easy to continue ingestion.

  As a result of intensive studies on ingredients that can be used as foods having a neurogenesis-promoting action, the present inventors have an excellent neurogenesis-promoting action of a peptide derived from collagen, and further an anti-anxiety action, antidepressant action, It has been found that it has a brain function improving action such as a brain development promoting action, and has completed the present invention.

That is, the gist of the present invention is as follows.
[1] A degradation product obtained by treating collagen or gelatin with collagenase,
The aqueous solution containing the collagenase-treated degradation product and the synthetic adsorbent SEPABEADS SP850 are mixed, and the non-adsorbed components are collected.
Gly-Pro-Arg,
Gly-Pro-Ser-Gly-Asn-Ala (SEQ ID NO: 1),
Gly-Pro-Val-Gly-Ala-Arg (SEQ ID NO: 2),
Gly-Pro-Ala-Gly-Pro-Ala (SEQ ID NO: 3),
Gly-Pro-Hyp,
Gly-Pro-Ile-Gly-Ser-Ala (SEQ ID NO: 4),
Gly-Pro-Val-Gly-Pro-Ala (SEQ ID NO: 5),
Gly-Pro-Ser-Gly-Glu-Arg-Gly-Pro-Hyp (SEQ ID NO: 6) and Gly-Leu-Ala-Gly-Pro-Hyp (SEQ ID NO: 7)
A food and drink characterized by comprising a neurogenesis promoting agent containing a peptide consisting of the amino acid sequence represented by
[2] The food and beverage according to [1], wherein the neurogenesis promoting agent contains 50% by weight or more of a peptide having a molecular weight of 1,500 or less,
[3] The food or drink according to [1] or [2], wherein the neurogenesis promoting agent promotes neurogenesis in a hippocampus of a mammalian brain,
[4] The food or beverage according to any one of [1] to [3], wherein the neurogenesis promoting agent has a brain development promoting action,
[5] The food or beverage according to any one of [1] to [4], wherein the neurogenesis promoting agent has a brain function improving effect,
[6] The food / beverage product according to [5] above, wherein the brain function improving effect is an anxiolytic action or antidepressant action,
[7] Any of the above [1] to [6], wherein the synthetic adsorbent SEPABEADS SP850 from which the non-adsorbing component has been collected is mixed with a 20% (v / v) ethanol aqueous solution, and the non-adsorbing component is further recovered. It relates to the described food and drink.

The term “neurogenesis” as used in the present invention refers to differentiation of a young undifferentiated cell (for example, neural stem cell) in a mammal into a new nerve (neuron cell) via a neuroblast. Includes proliferation of differentiating neuroblasts and undifferentiated cells. Neurogenesis also includes regeneration of damaged nerves and nerve repair.
“Nerve” includes nerve centers (eg, brain, spinal cord, etc.) and peripheral nerves (eg, ulnar nerve, radial nerve, radial nerve). It is not limited to the neurite outgrowth action, the differentiation promotion action to the nerve cells, and the growth promotion action on the cell line (for example, PC12 etc.) in the experiment in vitro.

  According to the present invention, there is provided a food or drink containing a neurogenesis promoting agent that is highly safe, can be continuously ingested, can be produced at low cost, and has excellent processability. More specifically, an object of the present invention is to provide a food or drink containing an angiogenesis-promoting agent useful for an anxiolytic action, an antidepressant action and a brain development promoting action, which are brain function improving effects.

FIG. 1 is a graph showing the amount of food intake in each group of mice. FIG. 2 is a graph showing the amount of aqueous solution intake of each group of mice. FIG. 3 is a graph showing the weight of each group of mice. FIG. 4 is a graph showing the number of movements in the open field test. FIG. 5 is a graph showing the movement distance in the light / dark selection test. FIG. 6 is a graph showing the time spent on the closed arm in the elevated plus maze test. FIG. 7 is a graph showing the percentage of time spent in the closed arm in the elevated plus maze test. FIG. 8 is a graph showing the moving distance from the center in the tail suspension test. FIG. 9 is a graph showing brain weight measurement values. FIG. 10 shows immunostained images (black and white) of BrdU positive cells in the hippocampal dentate gyrus of each group of mice, and the figure inserted in the upper right of each image is a graph showing a nuclear stained image (green) of the same region. is there. FIG. 11 is a graph in which the average density of BrdU positive cells (number of BrdU positive cells / mmSGZ) is determined from each individual and compared between the two groups.

Embodiments of the present invention will be described below.
The present invention relates to a peptide having a specific rule among peptides obtained by degrading collagen or gelatin that is widely used as a food, and has an activity of promoting neurogenesis in the hippocampus of a mammalian brain. It has been found and completed for the first time that it has a brain development promoting effect, and has brain function improving effects such as anxiolytic action and antidepressant action.

  Examples of the peptide salts used in the present invention include salts with alkali metals such as sodium and potassium, alkaline earth metals such as magnesium and calcium, and organic bases such as lysine and ornithine, which can be used as food. . The peptide or salt thereof which is an active ingredient of the present invention may be isolated as a hydrate, various solvates, or a crystalline polymorphic substance, and the present invention includes those isolated and mixtures thereof. Are all included.

  The peptide or salt thereof used in the present invention can also be produced by a known method from the above-mentioned raw materials containing collagen or gelatin.

  For example, the collagen used as the raw material of the degradation product obtained by treating collagen or gelatin with collagenase is not particularly limited, and any type I to XIII type collagen can be used, and a mixed type collagen that is a mixture thereof. Can also be used. In reality, it is assumed that the collagen is a mixed type collagen obtained from various animals and fish. However, animals (for example, cattle, pigs, etc.) and fish (for example, cattle, pigs) from which this collagen is derived The types of flounder, salmon, sardines, tuna, etc.) and the collagen extraction site can also be bones, skins, tendons, duckweeds (fish), and the like.

  Extraction and purification of collagen from these components can be carried out using generally known methods. Specifically, for example, after pulverizing collagen-containing tissues such as bones, skin, tendons, and duckweed, washing with water, extraction with a dilute salt solution, extraction with an acid or alkaline solution, and an enzyme such as pepsin, trypsin or hyaluronidase Extraction is performed, and collagen is purified by applying known purification means such as salting out and dialysis. It can also be obtained as “regenerated collagen” by a generally known method. Commercially available collagen can also be used as a raw material.

  Gelatin is a water-soluble protein obtained by heating and extracting the aforementioned collagen with water. In the present invention, gelatin produced by a generally known method can be used as a raw material, and a commercially available product can also be used.

  The peptide used in the present invention can be produced by allowing collagenase to act on collagen or gelatin obtained as described above.

  Collagenase is not particularly limited, but it is derived from bacteria such as Clostridium histolyticum and Streptomyces parvulus, Streptomyces parvulus, and the like, and has a unique amino acid sequence [(Gly-A- B) n (wherein, A and B represent amino acid residues excluding glycine residues, which may be the same or different from each other, and n represents a positive integer): Is also referred to as a “specific amino acid sequence”)], by using a collagenase that specifically cleaves the amino group terminal side of the glycine residue, a collagenase degradation product rich in peptides of this specific amino acid sequence can be obtained. Possible and preferred. Further, the collagenase used here may be a collagenase obtained as a natural product, for example, a modified collagenase having the above-mentioned specificity obtained by modification by a protein engineering technique.

  The types of amino acid residues other than glycine residues that A and B can take are not particularly limited, and are usually amino acid residues of naturally occurring amino acids (excluding glycine), specifically, alanine. Residue, valine residue, leucine residue, isoleucine residue, proline residue, hydroxyproline residue, phenylalanine residue, tryptophan residue, methionine residue, serine residue, threonine residue, cysteine residue, glutamine residue It may be any amino acid residue of a group, an asparagine residue, a tyrosine residue, a lysine residue, an arginine residue, a histidine residue, an aspartic acid residue, or a glutamic acid residue.

  In addition, the peptide that can be used in the present invention is a free or immobilized chitobar or the like according to a generally known method, for example, the method described in JP-A-7-82299 or JP-A-9-176196. Collagenase immobilized on a carrier can be produced by a batch method, a column method or a combination of these methods, preferably by setting the reaction temperature to 40 to 45 ° C. and contacting with the collagen or gelatin. .

  The collagenase degradation product may be produced according to the above-described method and used as it is, or may be blended in various base materials. A compounding quantity and the kind of base material are not specifically limited, What is necessary is just to set suitably. As a base material, oral administration base materials, such as a tablet, a capsule, a bag, a gummi, or a drink, are preferable, for example. In addition, it is also possible to use a commercial item as a collagenase degradation product.

  Moreover, the collagenase degradation product manufactured according to the above-mentioned method or its commercially available product can be further purified using various resins to obtain a fraction with an increased concentration of low molecular peptides. Examples of the resin used include a cation exchange resin, an anion exchange resin, a porous resin, and a special resin (chelate resin, synthetic adsorbent, protein separating agent). Therefore, the synthetic adsorbent SEPABEADS SP850 (manufactured by Mitsubishi Chemical Corporation) is used. For elution of adsorbed organic substances, use acids, alkalis or various organic solvents such as lower alcohols such as methanol, ethanol, propanol, isopropanol and butanol, esters such as ethyl acetate and butyl acetate, and ketones such as acetone. However, it is not limited to these. These organic solvents may be used alone or in combination of two or more, and may be used as a mixed solvent of these with water, acid or alkali. From the viewpoint of economy and safety, it is preferable to elute using ethanol or an ethanol aqueous solution. Purification can be performed by a batch method or a column method. The collected fraction may be concentrated by reduced pressure or ultrafiltration, and further, if necessary, the solvent may be completely removed to dryness or lyophilization.

  Similarly, a fraction with an increased concentration of a low molecular weight peptide can also be obtained by purifying a collagenase degradation product or a commercially available product thereof by precipitation separation using an organic solvent or an inorganic salt.

  When collagen or gelatin is decomposed with collagenase and purified by various methods using the above methods, the absorption rate and absorption rate of peptides in the gastrointestinal tract are improved, the permeability of the blood-brain barrier is improved, and the stability to heat and protease is improved. From various viewpoints such as improvement, increase in activity of the peptide itself, and increase in the number of moles per unit weight, it is sufficient that the peptide having a molecular weight of 1,500 or less is contained by 50% by weight or more.

In particular, when purified by the above method, peptides include Gly-Pro-Arg,
Gly-Pro-Ser-Gly-Asn-Ala (SEQ ID NO: 1),
Gly-Pro-Val-Gly-Ala-Arg (SEQ ID NO: 2),
Gly-Pro-Ala-Gly-Pro-Ala (SEQ ID NO: 3),
Gly-Pro-Hyp,
Gly-Pro-Ile-Gly-Ser-Ala (SEQ ID NO: 4),
Gly-Pro-Val-Gly-Pro-Ala (SEQ ID NO: 5),
Gly-Pro-Ser-Gly-Glu-Arg-Gly-Pro-Hyp (SEQ ID NO: 6)
Gly-Leu-Ala-Gly-Pro-Hyp (SEQ ID NO: 7)
Etc.

  In the neurogenesis-promoting composition in which the above peptide is mixed, the peptide having the highest content in the obtained composition is Gly-Pro-Hyp, and Gly-Pro-Hyp is 5.0% by weight in the peptide. It is desirable to include the above.

  The peptide or salt thereof (hereinafter abbreviated as the peptide of the present invention) obtained as described above can be used as an active ingredient in a neurogenesis promoting agent.

  In the present invention, the neurogenesis promoting agent preferably promotes neurogenesis in the hippocampus among mammalian brains. Promote neurogenesis in the hippocampus to widely improve or improve the functions related to emotion, memory, learning and cognition that the hippocampus is responsible for, such as anxiolytic, antidepressant, anti-amnestic, memory-improving, and learning-enhancing functions. There is an advantage of making it. In addition, promotion of neurogenesis in the hippocampus of the brain can be confirmed using the method described in Examples described later.

  The neurogenesis promoting agent preferably has a brain development promoting action. The brain development promoting action refers to the action of increasing the weight of the whole brain, and is expressed by continuously administering the neurogenesis promoting agent. Specifically, it can confirm using the method as described in the below-mentioned Example.

  In addition, the neurogenesis promoting agent can be used for the purpose of improving brain function such as anxiolytic action and antidepressant action of mammals.

As drugs such as psychotropic drugs with anti-anxiety and antidepressant effects, various synthetic reagents have been commercially available, but they all act on the cranial nerves in the central nervous system and contribute to the mental activity of living organisms. It has some effect to alleviate symptoms such as anxiety and tension. However, it has been reported that these over-the-counter drugs are accompanied by various side effects such as sleep action and hallucinating action when administered incorrectly. In particular, symptoms such as anxiety and depression are difficult to distinguish objectively by diagnosis, and medication requires skill.
On the other hand, the neurogenesis promoting agent is a peptide constituting natural collagen, and is used as a food, and therefore has an advantage of excellent safety in terms of side effects.

  When the neurogenesis-promoting agent is used for improving the brain function of a mammal, an effective amount thereof can be administered to a human or non-human mammal according to conventional means. In this case, since it is a food, it is administered orally.

  In the case of oral administration as a food, various base materials can be selected depending on the purpose, and as described above, tablets, capsules, candy, gummi, beverages and the like can be mentioned. For example, when a small amount of an active ingredient is ingested continuously, a base material such as candy, gummi, or a beverage that is a base material that senses the taste is desirable. In this case, deliciousness and convenience can be imparted, and it is more effective for continuous intake. However, when a certain amount of the active ingredient is contained, the deliciousness of the active ingredient may be impaired, and in some cases, the physical properties of the base material itself may be changed. Therefore, when a certain amount of an active ingredient such as several grams is continuously ingested, a so-called supplement is desirable, and many of them are in the form of a solid composition that can be taken with water or hot water. Examples of the solid composition for oral administration according to the present invention include tablets, pills, capsules, fine granules, granules and the like. In such a solid composition, one or more active substances contain at least one inert diluent, for example, various sugars such as sucrose, lactose and glucose, various sugar alcohols such as mannitol and sorbitol, Furthermore, it is mixed with hydroxypropylcellulose, crystalline cellulose, various starches and the like. The composition is prepared according to conventional methods with additives other than inert diluents, such as lubricants such as magnesium stearate, disintegrants such as calcium calcium glycolate, stabilizers such as lactose, glutamic acid or A solubilizing or solubilizing aid such as aspartic acid may be contained. In some cases, additives such as fragrances and sweeteners can also be used. In addition, tablets, pills, granules, and capsule granules containing granules can be sugar-coated with sugars such as sucrose and sugar alcohols such as maltitol, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose as necessary. You can also apply a coating. Alternatively, it may be covered with a film of gastric or enteric material. Moreover, in order to improve the solubility of a formulation, a well-known solubilization process can also be performed.

  The dose of peptide or the like varies depending on the age, weight, symptom, therapeutic effect, route of administration, etc. of the subject, and is appropriately set in consideration of these. -30 g, preferably 100 mg to 10 g is suitable, and this is administered once a day or divided into 2 to several times. Since the dosage varies depending on the purpose of prevention and other various conditions, an amount smaller than the above dosage range may be sufficient. If the subject is a mammal other than a human, the dose may be adjusted according to the above human.

  Since the preparation thus obtained is safe and has low toxicity, for example, mammals (eg, humans, rats, mice, guinea pigs, rabbits, birds, sheep, pigs, cows, horses, cats, dogs, monkeys, For example, chimpanzees).

  In addition, because it has extremely excellent safety, absorbability, low allergenicity, stability of physical properties and excellent processing suitability, infants and infants such as formula milk and baby food are used for the purpose of promoting brain growth of infants and infants. It can also be used as a food for foods, and as a food for the prevention of aging and recovery of brain function such as brain function decline in the elderly, and in some cases, part of the raw materials for jelly-like nutritional foods for those who have difficulty chewing or swallowing Can also be used.

  Further, the neurogenesis promoting agent can be used in combination with other compounds and materials that are said to have an effect of improving brain function. In this case, it is conceivable that not only an additive effect but also a synergistic effect occurs. Examples of such compounds and materials include glucose, which is a nutrient source of the brain, purine derivatives such as caffeine that act on the nervous system, phosphatidylserine, phosphatidylcholine, arachidonic acid, docosahexaene, which are constituents of the brain. Examples thereof include lipids such as acids, compositions such as ginkgo biloba extract having an effect of improving blood flow, pyrroloquinoline quinone (PQQ) and coenzyme Q10 (CoQ10) which are coenzymes. The method of combination is not limited in any way, and one or more types that are suitable for the degree of expected effect, the taste surface such as taste, cost, processing suitability and stability can be used in combination.

  EXAMPLES Next, although an Example is given and this invention is demonstrated further in detail, this invention is not limited by this.

Example 1: Preparation of various samples Two types of test samples and control samples were prepared for various experiments. Both used common gelatin.
(Preparation and analysis of test sample)
20 L of water was added to 2.0 kg of pig skin gelatin (manufactured by Ruslo) and dissolved by heating. 20 g of collagenase type I (manufactured by Washington) was added and the enzyme reaction was carried out at 37 ° C. for 1 hour. After completion of the reaction, the reaction solution was heated at 100 ° C. for 5 minutes to deactivate the enzyme, and filtered through a 0.45 μm filter. This filtrate was fractionated using a synthetic adsorbent SEPABEADS SP850 (manufactured by Mitsubishi Chemical Corporation). 5 kg of the synthetic adsorbent and 5 L of the filtrate were mixed and stirred for 1 hour, and then allowed to stand to filter and collect the supernatant. Thereafter, 10 L of water was added and stirred, and then allowed to stand to filter and collect the supernatant. This operation was performed 3 times in total. Thereafter, a 20% (v / v) aqueous ethanol solution was added to the synthetic adsorbent and stirred, and then allowed to stand, and the supernatant was filtered and collected. This operation was performed 3 times in total. The collected filtrate was concentrated with a rotary evaporator under reduced pressure to remove ethanol, and then freeze-dried to obtain peptide powder. This was used as a test sample.
The molecular weight distribution of the obtained test sample was examined. Detection was performed by gel filtration chromatography using AKTA purifier and Superdex Peptide 10/300 GL (Amersham Biosciences). As a result, it was confirmed that a peptide having a molecular weight of 1500 or less was contained by 50% by weight or more.
Furthermore, when a part of the plurality of peptides contained in the test sample was isolated by high performance liquid chromatography (HPLC) and the structure was determined by a protein sequencer (Applied Biosystems),
Gly-Pro-Arg,
Gly-Pro-Ser-Gly-Asn-Ala (SEQ ID NO: 1),
Gly-Pro-Val-Gly-Ala-Arg (SEQ ID NO: 2),
Gly-Pro-Ala-Gly-Pro-Ala (SEQ ID NO: 3),
Gly-Pro-Hyp,
Gly-Pro-Ile-Gly-Ser-Ala (SEQ ID NO: 4),
Gly-Pro-Val-Gly-Pro-Ala (SEQ ID NO: 5),
Gly-Pro-Ser-Gly-Glu-Arg-Gly-Pro-Hyp (SEQ ID NO: 6)
Gly-Leu-Ala-Gly-Pro-Hyp (SEQ ID NO: 7)
And it certainly had a structure of (Gly-XY) n. Moreover, the peptide with the highest content in the test sample was Gly-Pro-Hyp, and contained in the test sample at 8.0% by weight.

(Preparation and analysis of control sample)
20 kg of water was added to 2.0 kg of pig skin gelatin and dissolved by heating. 20 g of protease N “Amano” G (manufactured by Amano Enzyme) was added, and the enzyme reaction was carried out at 55 ° C. for 1 hour. After completion of the reaction, the reaction solution was heated at 100 ° C. for 10 minutes to inactivate the enzyme and filtered through a 0.45 μm filter. This filtrate was fractionated by a precipitation method using ethanol. The filtrate: ethanol was mixed well at a volume ratio of 3: 2, and allowed to stand at 4 ° C. overnight, followed by centrifugation at 10,000 rpm and 4 ° C. for 15 minutes. The supernatant was collected and concentrated with a rotary evaporator under reduced pressure to remove ethanol and then lyophilized to obtain peptide powder. This was used as a control sample.
The molecular weight distribution of the obtained test sample was examined. Detection was performed by gel filtration chromatography (solvent: 1 × PBS) using AKTA purifier and Superdex Peptide 10/300 GL (manufactured by Amersham Bioscience). As a result, it was confirmed that there were few peptides having a molecular weight of 1500 or less, and the average molecular weight of this control sample was around 5000. Since the enzyme used is a mixture derived from microorganisms and has low substrate specificity, the N-terminal amino acid of the peptide is not specified in Gly.

In addition, the amino acid composition of the obtained test sample and the control sample was almost the same, and it could be said that there was no nutritional difference.

Example 2: Sample administration method and breeding method 32 5-week-old C57BL / 6J male mice were divided into two groups, each of which was mixed with a test sample and a control sample at 5% by weight in drinking water. And let them ingest freely. The test was started for 3 weeks and started at 8 weeks of age. Ingestion was continued after the start of the experiment. There were no significant differences between the two groups in food intake, water intake, and body weight. These results are shown in FIGS. 1 shows the amount of food intake of each group of mice, FIG. 2 shows the amount of aqueous solution intake of each group of mice, and FIG. 3 shows the weight of each group of mice.

Example 3: Examination of anti-anxiety effect 1
An open field test was conducted to investigate the effect of test samples on anxiety.
(Experimental design)
Measuring equipment: ImageJ OF2 for Open field test manufactured by Ohara Medical Industry Co., Ltd.
In the open field test, place the mouse in a wide open area (open field) of 50 x 50 cm surrounded by a 30 cm high wall, record the trajectory and stay time of the mouse, and observe its behavior. To do. The mouse repels large and bright places and exhibits the property of moving along the surrounding wall (contact mobility). Tactile mobility is perceived as anxiety-like behavior, and mice with reduced anxiety spend more time in the center of the open field. For the analysis, Image J XX (O'Hara & Co., Ltd.) made based on Image J was used. Image J is a freeware http://rsb.com product made at the National Institutes of Health. info. nih. It is available from gov / ij.

(Statistical analysis)
Each data was expressed as an average value ± standard deviation (means ± SD). SPSS 15.0J for Windows (registered trademark, the same applies hereinafter) was used for the data of each group, and a t-test that did not correspond to the comparison of the mean values between the two groups was performed. In addition, two-way analysis of variance was used for comparison of behavioral changes within the measurement time for the test sample and the control sample, and a comparison between the two groups was performed by an unmatched t-test at each time point. , Both were determined to be significant at less than 5%.

(result)
Eight-week-old male mice were placed in an open field and their behavior was observed for 10 minutes. Twelve mice were tested for each of the test sample-ingested mice and the control sample-ingested mice. There was no significant difference in the time spent in the central region, and there was no difference in the distance traveled between the two groups, but the number of movements within the test time (10 minutes) was significantly different as shown in FIG. There were many test sample ingestion mice. Test sample ingested mice are active and active, indicating reduced anxiety-like behavior. From this result, it was revealed that the test sample has an anxiolytic effect.

Example 4: Examination of anti-anxiety effect 2
In order to investigate the effect of the test sample on anxiety, a light / dark selection test was performed.
(Experimental design)
Measuring equipment: ImageJ LD1 for Light / dark transition test manufactured by Ohara Medical Industry Co., Ltd.
In the light / dark selection test, a mouse is placed in two connected white and black devices, and the number of times and the trajectory of the mouse moving between light and dark boxes are recorded, and the amount of activity and emotion are measured. Although the mouse has the property of favoring a dark place, anxiety is evaluated based on the amount of activity of the mouse and the number of times of traffic within the device that allows the user to freely move between the light box and the dark box. Mice with reduced anxiety will spend more time in the light box. For the analysis, Image J XX (O'Hara & Co., Ltd.) made based on Image J was used. Image J is a freeware produced at the National Institutes of Health, http: // rsb. info. nih. It is available from gov / ij.

(Statistical analysis)
Each data was expressed as an average value ± standard deviation (means ± SD). SPSS 15.0J for Windows was used for the data of each group, and a t-test that did not correspond to the comparison of the mean values between the two groups was performed. In addition, two-way analysis of variance was used for comparison of behavioral changes within the measurement time for the test sample and the control sample, and a comparison between the two groups was performed by an unmatched t-test at each time point. , Both were determined to be significant at less than 5%.
(result)
The light box was adjusted to 500 lux, an 8 week old male mouse was placed in the dark box, the experiment was started, and the behavior was observed for 10 minutes. Twelve mice were tested for each of the test sample-ingested mice and the control sample-ingested mice. As shown in FIG. 5, the test sample-ingested mice have a longer moving distance in the light box throughout the entire test time (10 minutes) than the control sample-ingested mice, and are significant between 6 and 7 minutes. There was a difference. Thus, similar to the open field test, the test sample-ingested mice are active and active, indicating a decrease in anxiety-like behavior. This result also revealed that the test sample had an anxiolytic effect.

Example 5: Examination of anti-anxiety effect 3
To investigate the effect of the test sample on anxiety, an elevated plus maze test was performed.
(Experimental design)
Measuring equipment: ImageJ EP1 for Evolved plus maze, manufactured by Ohara Medical Industry
In the elevated plus maze test, an experimental animal is placed on a cruciform stage placed at a high place and its behavior is observed. The two platforms in the symmetrical position of the cruciform stage are fitted with a wall (closed arm), and the other two contrasting platforms have no wall (open arm). Yes. The length of the arm is 45 cm on a side and 5 cm in width, and the height of the wall of the closed arm is 15 cm. Mice usually spend most of their time in a safer closed arm because of their fear of high altitude and their preference for narrow spaces, but mice with reduced anxiety have a longer stay in open arms. Show the trend. Record the number of times the mouse has been in each arm and the duration of the stay, and compare anxiety quantitatively. For the analysis, Image J XX (O'Hara & Co., Ltd.) made based on Image J was used. Image J is a freeware produced at the National Institutes of Health, http: // rsb. info. nih. It is available from gov / ij.

(Statistical analysis)
Each data was expressed as an average value ± standard deviation (means ± SD). SPSS 15.0J for Windows was used for the data of each group, and a t-test that did not correspond to the comparison of the mean values between the two groups was performed. In addition, two-way analysis of variance was used for comparison of behavioral changes within the measurement time for the test sample and the control sample, and a comparison between the two groups was performed by an unmatched t-test at each time point. , Both were determined to be significant at less than 5%.

(result)
Illumination was adjusted to 300 lux and an 8 week old male mouse was placed in the center of the elevated plus maze platform to begin the experiment and observed for 10 minutes. Twelve mice were tested for each of the test sample-ingested mice and the control sample-ingested mice. As shown in FIG. 6, the test sample ingested mice spent significantly less time in the closed arm than the control sample ingested mice. Further, as shown in FIG. 7, the proportion of the time spent in the closed arm was significantly less in the test sample ingested mice than in the control sample ingested mice. Thus, as in the open field test and the light / dark selection test, the test sample-ingested mice show that anxiety-like behavior is reduced. This result also revealed that the test sample had an anxiolytic effect.

Example 6: A tail suspension test was conducted to investigate the effect of a test sample on antidepressant depression-like behavior in mice fed with a test sample.
(Experimental design)
Measuring equipment: ImageJ TS1 for Tail suspension test manufactured by Ohara Medical Sangyo Co., Ltd.
In the tail suspension test, the mouse's tail is hooked on the ceiling hook, the mouse is hung upside down, the number of times that the mouse does not move (apathetic state) and the time are recorded, and the degree of depression is evaluated. Since the device is structured so that the mouse cannot escape, the mouse stops the escape behavior over time. This is thought to reflect the psychological background of giving up. Accordingly, the degree of depression can be quantitatively compared by measuring the immobility time. For the analysis, Image J XX (O'Hara & Co., Ltd.) made based on Image J was used. Image J is a freeware produced at the National Institutes of Health, http: // rsb. info. nih. It is available from gov / ij.

(Statistical analysis)
Each data was expressed as an average value ± standard deviation (means ± SD). SPSS 15.0J for Windows was used for the data of each group, and a t-test that did not correspond to the comparison of the mean values between the two groups was performed. In addition, two-way analysis of variance was used for comparison of behavioral changes within the measurement time for the test sample and the control sample, and a comparison between the two groups was performed by an unmatched t-test at each time point. , Both were determined to be significant at less than 5%.

(result)
The lighting was adjusted to 20 lux, and an 8 week old male mouse was hooked on the hook on the ceiling to start the experiment, and the behavior was observed for 5 minutes. Eleven test sample-ingested mice and 12 control sample-ingested mice were tested. Although there was no difference in the rate of immobility time, as shown in FIG. 8, the test sample-ingested mice had a significantly larger moving distance from the center point with time as compared to the control sample-ingested mice. That is, it can be said that the control sample-ingested mice gave up the escape behavior earlier, indicating that the test sample-ingested mice are more resistant to depression. This result revealed that the test sample had an antidepressant action.

Example 7: Brain Development Promoting Action In order to investigate the effect of the test sample on brain development, brain weight measurement was performed.
(Experimental settings)
The brains of test sample-ingested mice and control sample-ingested mice were removed and compared in weight. In addition, body weight was measured, and the brain weight ratio to body weight was compared.

(Statistical analysis)
Each data was expressed as an average value ± standard deviation (means ± SD). SPSS 15.0J for Windows was used for the data of each group, and a t-test that did not correspond to the comparison of the average value between the two groups was performed, and the result was judged to be significant with less than 5%.

(result)
Brain tissues were removed from 9-week-old test sample-ingested mice and control sample-ingested mice, and the brain weight and body weight were compared. Eight mice were tested for each of the test sample-ingested mice and the control sample-ingested mice. As shown in FIG. 9, the brain weight was significantly increased in the test sample-ingested mice compared to the control sample-ingested mice. Moreover, in the body weight, there was no difference between the two groups, and even when the ratio of the brain weight to the body weight was seen, the test sample-ingested mice showed a larger value. From this result, it can be said that the brain of the test sample-ingested mice was significantly larger than the control sample-ingested mice.
Therefore, it became clear that the test sample has a brain development promoting effect.

Example 8: Neurogenesis in the hippocampus of test sample-ingested mice The effect of the test sample on neurogenesis was examined. The hippocampus is a particularly active area of neurogenesis (neurogenesis), and the expansion of the granule cell layer (the neuronal cell layer of the hippocampal dentate gyrus) is thought to be due to increased neurogenesis. Therefore, using 5-bromo-2′-deoxyuridine (5-bromo-2′-deoxyuridine; BrdU), the dividing cells were specifically detected, and the cell densities were quantitatively compared.

(Experimental design)
In cell division, DNA replication is actively performed, but by administering a chemically labeled nucleic acid to a mouse, all dividing cells in the body can be labeled in vivo. . By using BrdU as a nucleic acid analog, it is mistaken for thymidine during DNA synthesis, and the chromosomal DNA of dividing cells is specifically labeled. BrdU was intraperitoneally administered at 100 g / kg per body weight, and after 2 hours, the mice were refluxed with PBS solution and fixed with paraformaldehyde solution. This procedure results in in vivo labeling of cells that have divided in 2 hours. For the hippocampal region, coronal sections having a thickness of 40 μm are prepared every 200 μm, and immunostaining is performed using an anti-BrdU antibody. Dividing cells that are BrdU positive cells are identified by fluorescence. At the same time, cell nuclei are stained using cyto13green to visualize the granular cell layer of the hippocampal dentate gyrus. Since neural progenitor cells are abundant in the subgranular zone (SGZ), BrdU positive cells are found in this region. Therefore, the density of dividing cells can be determined by measuring the number of BrdU positive cells along the granular cell layer. The density of dividing cells is the number of BrdU positive cells per unit length of the granular cell lower layer (cell number / mmSGZ), which is almost constant in any part of the hippocampus of the same solid. In addition, immunohistochemical staining of immature nerve cells was simultaneously performed using doublecortin (Dcx). The experiment was performed using 9-week-old male mice reared.

(Statistical analysis)
Each data was expressed as an average value ± standard deviation (means ± SD). SPSS 15.0J for Windows was used for the data of each group, and a t-test that did not correspond to the comparison of the average value between the two groups was performed, and the result was judged to be significant with less than 5%.

(result)
8 and 6 mice were used for the test sample-ingested mice and the control sample-ingested mice, respectively. Coronal sections were obtained every 200 μm in the area containing the mouse hippocampus (area: starting from the rostral end of the hippocampus at a position of 600 μm to a position of 1400 μm), and 4 sections per individual were used in the experiment. Immunohistochemical staining with anti-BrdU antibody, nuclear staining with cyto13green, and triple staining of Dcx immunostaining were performed, and photographs were taken of the left and right hippocampal dentate gyrus of each section (8 images per piece). Using LSM Image Browser (manufactured by Carl Zeiss MicroImaging), the distance of the subgranular cell layer (SGZ) is measured from the acquired image, then image analysis is performed using ImageJ software, and BrdU positive cells are visible Counted the number. FIG. 10 shows immunostained images (black and white) of BrdU positive cells in the hippocampal dentate gyrus of test sample-ingested mice and control sample-ingested mice. The figure inserted at the upper right of each image is a nuclear-stained image (green) of the same region. The cell layer below the granular cell layer shown in green is the granular cell lower layer (SGZ), and there are many neural progenitor cells. In both the test sample-ingested mice and the control sample-ingested mice, most of the BrdU-positive cells are present in the lower layer of the granule cell. However, in the test sample-ingested mice, BrdU-positive cells are more concentrated in a certain region than in the control sample-ingested mice. You can see that it exists. When counting the number of cells using image analysis software, the threshold and area of the light intensity are set and the number of cells exceeding the threshold and area is counted. When BrdU positive cells are concentrated in one place, even if there are multiple cells, the image analysis software count is “1”, which is inaccurate. Cells were counted. FIG. 11 shows the average of the density of BrdU positive cells (the number of BrdU positive cells / mmSGZ) from each individual based on these images. This analysis revealed that test sample-ingested mice had significantly higher dividing cell density in the granule cell lower layer of the hippocampal dentate gyrus compared to control sample-ingested mice. From this, it became clear that the test sample significantly increased the proliferation of nerve cells in the hippocampus. Moreover, since most of the dividing cells in the granular cell lower layer are neural progenitor cells, neurogenesis is considered to be active. That is, it became clear that the test sample promotes neurogenesis.

Claims (7)

A degradation product obtained by treating collagen or gelatin with collagenase,
The aqueous solution containing the collagenase-treated degradation product and the synthetic adsorbent SEPABEADS SP850 are mixed, and the non-adsorbed components are collected.
Gly-Pro-Arg,
Gly-Pro-Ser-Gly-Asn-Ala (SEQ ID NO: 1),
Gly-Pro-Val-Gly-Ala-Arg (SEQ ID NO: 2),
Gly-Pro-Ala-Gly-Pro-Ala (SEQ ID NO: 3),
Gly-Pro-Hyp,
Gly-Pro-Ile-Gly-Ser-Ala (SEQ ID NO: 4),
Gly-Pro-Val-Gly-Pro-Ala (SEQ ID NO: 5),
Gly-Pro-Ser-Gly-Glu-Arg-Gly-Pro-Hyp (SEQ ID NO: 6) and Gly-Leu-Ala-Gly-Pro-Hyp (SEQ ID NO: 7)
A food and drink comprising a neurogenesis promoting agent containing a peptide having the amino acid sequence represented by the formula:   The food or drink according to claim 1, wherein the neurogenesis-promoting agent contains 50% by weight or more of a peptide having a molecular weight of 1,500 or less.   The food or drink according to claim 1 or 2, wherein the neurogenesis promoting agent promotes neurogenesis in a hippocampus of a mammalian brain.   The food or drink according to any one of claims 1 to 3, wherein the neurogenesis-promoting agent has a brain development promoting action.   The food or beverage according to any one of claims 1 to 4, wherein the neurogenesis promoting agent has an effect of improving brain function.   The food / beverage product according to claim 5, wherein the brain function improving effect is an anxiolytic action or an antidepressant action.   The food / beverage product according to any one of claims 1 to 6, wherein the non-adsorbing component is further recovered by mixing the synthetic adsorbent SEPABEADS SP850 from which the non-adsorbing component has been recovered and a 20% (v / v) ethanol aqueous solution.