JP6863362B2 - Composition for food additive to promote brain endocrine of neurotrophic factors - Google Patents

Description

The present invention relates to a composition for adding food to promote the endocrine secretion of neurotrophic factors in the brain.

With the increase in the elderly population, the number of patients with senile dementia including Alzheimer's disease is increasing. According to the Ministry of Health, Labor and Welfare, the number of dementia patients in 2012 is estimated to increase from 4.62 million (1 in 7 people aged 65 and over) to 8.3 million in 2030. On the other hand, the dementia reserve army is called Mild Cognitive Impairment (MCI), and if MCI is diagnosed, it is necessary to prevent it by improving life. Since the number of dementia patients will increase 1.8 times from 2012 to 2030, the number of MCI patients in 2030 is estimated to be 7.2 million, assuming that the number will increase at the same rate. On the other hand, the number of patients suffering from brain problems such as depression due to various stresses such as work environment, family circumstances, and human relationships is increasing year by year. Recent studies have revealed that food ingredients affect brain function, and food ingredients that have effects on improving brain function, antidepressant, and dementia are drawing attention.

Neurotrophin is a general term for substances that promote the development, growth, maintenance, and regeneration of nerve cells, and various nutritional factors have been identified so far. Among them, brain-Derived Neurotrophic Factor (BDNF) exhibits actions such as maintaining the survival of nerve cells, promoting neurite outgrowth, and promoting the synthesis of neurotransmitters. The function of these BDNFs is mediated by the high affinity receptor (TrkB) (Non-Patent Documents 1 and 2).
BDNF has been reported to be associated with neuropsychiatric disorders such as Alzheimer's disease and depression. In the brains of patients with Alzheimer's disease, BDNF and TrkB levels are lower than in healthy individuals, especially in the cerebral cortex and hippocampus. In healthy individuals, TrkB mRNA levels decrease with age in the prefrontal cortex. It has been shown that age-related decline in spatial memory correlates with decreased BDNF mRNA expression in the hippocampus in rats. These facts suggest that a decrease in BDNF action is involved in memory loss in aging and Alzheimer's disease (Non-Patent Document 3).
Data showing the association between BDNF and depression have also been reported (Non-Patent Document 4). In the brains of depressed patients, decreased BDNF protein levels are observed in several areas, including the hippocampus. Hippocampal serotonergic nerve fiber shedding is observed in model animals of depression, but BDNF acts to maintain the survival of serotonergic neurons.

Therefore, it is strongly desired to develop a highly safe food material that prevents symptoms or diseases caused by decreased brain function by increasing the brain level of neurotrophic factors including BDNF, and further shows an improving effect. ing.

Huang EJ and Reichardt LF. Neurotrophins: roles in neuronal development and function. Annu. Rev. Neurosci. 2001, 24, 677. Schinder AF and Poo M. The neurotrophin hypothesis for synaptic plasticity. Trends Neurosci. 2000; 23: 639-645. Tapia-Arancibia L, Aliaga E, Silhol M, Arancibia S. New insights into brain BDNF function in normal aging and Alzheimer disease. Brain Res. Rev. 2008; 59: 201-220. Erickson KI, Miller DL, Roecklein KA. The aging hippocampus: interactions between exercise, depression, and BDNF. Neuroscientist. 2015; 18: 82-97.

An object of the present invention is to provide a food material capable of promoting the brain endocrine of neurotrophic factors including BDNF by adding it to a food, and adding a function of preventing neurological diseases and a function of improving brain function to the food. To do.

As a result of diligent research on highly safe food materials that efficiently increase the levels of neurotrophic factors in the brain, the present inventors have found oligopeptide mixtures containing dipeptides or tripeptides having lysine and / or arginine as constituent amino acids. , Have found that it efficiently increases neurotrophic factors in the brain, and have completed the present invention.

That is, the present invention
(1) A food-added composition for promoting brain endocrine secretion of neurotrophic factors, which comprises an oligopeptide mixture containing a dipeptide or tripeptide having lysine and / or arginine as constituent amino acids as an active ingredient.
(2) A food additive composition for promoting the brain endocrine secretion of the neurotrophic factor according to (1), wherein the ratio of the amount of lysine and / or arginine to the total amount of amino acids in the oligopeptide mixture is 5% by weight or more.
(3) To promote the endocrine secretion of the neurotrophic factor according to (1) or (2), wherein the content of the peptide having a molecular weight of less than 500 in the oligopeptide mixture is 50% by weight or more based on the total amount of the peptide and the free amino acid. Food additive composition,
(4) A neurotrophic factor brain endocrine promoter containing an oligopeptide mixture containing a dipeptide or tripeptide having lysine and / or arginine as constituent amino acids as an active ingredient.
(5) A method in which a dipeptide or tripeptide having lysine and / or arginine as constituent amino acids is added as an active ingredient to a food to produce a food used for promoting the brain endocrine secretion of neurotrophic factors.
Is.

Ingestion of the specific dipeptide or tripeptide of the present invention as an active ingredient can promote brain endocrine of neurotrophic factors such as BDNF. This can be useful for prevention of various cranial nerve diseases and improvement of brain function.

Hereinafter, embodiments of the present invention will be described in detail.

(Oligopeptide mixture)
An oligopeptide mixture is not a peptide having only a specific amino acid sequence, but a mixture of peptides having various amino acid sequences and molecular weights.
The oligopeptide mixture used as the food additive of the present invention is, in one embodiment, an acid hydrolyzate of a protein obtained by hydrolyzing a protein raw material with an acid or an enzymatic decomposition product of a protein enzymatically decomposed by a protein hydrolyzate (protease). Can be. In addition, it can be prepared by a conventional method by a chemical synthesis method or an enzymatic method.

Hereinafter, an embodiment of obtaining an oligopeptide mixture by an enzymatic decomposition method will be described.
As the protein raw material, various protein raw materials obtained by extracting, concentrating, or separating proteins from natural animal or vegetable raw materials can be used, and the suitable protein content in the protein raw materials is 50% by weight or more in terms of dry weight. It is preferably 70% by weight or more, more preferably 80% by weight or more, and further preferably 90% by weight or more.
The origins of animal protein raw materials include milk, eggs, livestock meat, seafood, and microorganisms, and the origins of vegetable protein raw materials include beans such as soybeans and almonds, rice, wheat, and barley. Examples include grains such as corn, and nuts such as almonds, cashew nuts, walnuts, pistachios, hazelnuts, and madacamian nuts.

Among these, it is preferable that the amino acid sequence of the protein contains a large amount of neutral amino acids such as lysine and arginine residues which are basic amino acids and glycine, and examples thereof include soybean, milk, livestock meat, and seafood. , Eggs, etc., and soybeans are more preferable. In the case of soybean, soymilk (regardless of total fat, degreasing, etc.), concentrated soybean protein, separated soybean protein, fractionated soybean protein, etc. can be used. In particular, when it is desired to ingest a larger amount of oligopeptide with a small amount of intake, it is preferable to use isolated soybean protein or fractionated soybean protein having a high protein content of 80% by weight or more in terms of dry weight.

The protein raw material is enzymatically decomposed by a protein hydrolase (protease), and the degree of enzymatic decomposition is appropriate that all molecules are not decomposed into free amino acids, and it is preferable that the degree of decomposition is higher. In particular, it is appropriate that the content of the peptide fraction having a molecular weight of less than 500 in the oligopeptide mixture is 50% by weight or more, preferably 60% by weight or more, based on the total amount of the peptide and free amino acids.

This peptide having a molecular weight of less than 500 is substantially composed of a dipeptide and a tripeptide in which 2 to 3 amino acids are bound.
If the molecular weight of the oligopeptide mixture is too large, the predominance of absorption rate will be reduced, and the effect of promoting neurotrophic factor secretion may be diminished.
The content of peptides having a molecular weight of less than 500 is determined in the protein hydrolyzate calculated by amino acid analysis after measuring the proportions of peptides having a molecular weight of less than 500 and free amino acid fractions in the oligopeptide mixture by gel filtration chromatography for peptides. It shall be calculated by subtracting the free amino acid content.

Further, the oligopeptide mixture specified as described above is preferably one in which the proportions of peptides other than peptides having a molecular weight of less than 500 and free amino acids are reduced as much as possible. That is, the content of free amino acids in the oligopeptide mixture is preferably 12% by weight or less, preferably 5% by weight or less, based on the total amount of peptides and free amino acids. This is because too much free amino acids can cause problems due to large intakes.

Furthermore, since it is desirable that the peptide form in the oligopeptide mixture is a smaller molecule, the proportion of fractions having a molecular weight of 500 or more in the oligopeptide mixture is 40% by weight or less based on the total amount of peptides and free amino acids. Is more preferable, 38% by weight or less is more preferable, and 35% by weight or less is further preferable.

Proteases used for enzymatic degradation to obtain oligopeptide mixtures, whether of animal, plant or microbial origin, are "metal proteases," "acidic proteases," "thiol proteases," and "serine proteases" in the protease classification. , Preferably, a protease classified into “metal protease”, “thiol protease”, and “serine protease” can be appropriately selected.

In particular, a decomposition method in which two or more or three or more enzymes belonging to different classifications are allowed to act sequentially or simultaneously is preferable because it can increase the proportion of peptides having a molecular weight of less than 500.
Furthermore, in order to reduce the content of free amino acids, it is preferable to use an enzyme having low exoprotease activity.

This protease classification is a method usually used in the field of enzyme science according to the type of amino acid in the active center.
As representatives of each, "metal protease" includes Bacillus-derived neutral protease, Streptomyces-derived neutral protease, Aspergillus-derived neutral protease, "samoase", etc., and "acidic protease" includes pepsin, Aspergillus-derived acidic protease, ""SumiteamFP" and the like, "thiol protease" includes bromeline, papain and the like, and "serine protease" includes trypsin, chymotrypsin, zubutyricin, alkaline protease derived from Streptomyces, "alcalase", "bioprase" and the like. ..

The classification of enzymes other than these can be confirmed by the action pH and the reactivity with the inhibitor.
Since the sites of action on the substrate are significantly different between enzymes having different active centers, it is possible to reduce "cut-offs" and efficiently obtain enzymatic degradation products.
Further, by using enzymes of different origins (origin organisms) in combination, an enzymatic decomposition product can be produced more efficiently.
Even in the same classification, if the origin is different, the site of action on the substrate protein is also different, and as a result, the proportion of peptides having a molecular weight of less than 500 can be increased.

The reaction pH and reaction temperature of the protease treatment may be set according to the characteristics of the protease to be used. Usually, the reaction pH may be set near the optimum pH, and the reaction temperature may be set near the optimum temperature.
The reaction temperature is generally 20 to 80 ° C, preferably 40 to 60 ° C. After the reaction, the enzyme is heated to a temperature sufficient to inactivate the enzyme (about 60 to 170 ° C.) to inactivate the residual enzyme activity.

The reaction solution after the protease treatment can be used as it is or concentrated, but it is usually sterilized, spray-dried, freeze-dried, etc. and used in the form of a dry powder.
The sterilization is preferably heat sterilization, the heating temperature is preferably 110 to 170 ° C., more preferably 130 to 170 ° C., and the heating time is preferably 3 to 20 seconds. Further, the reaction solution may be adjusted to an arbitrary pH.

Insoluble matter (precipitate or suspension) generated during protease treatment or pH adjustment may be removed by centrifugation, filtration, etc., and removal of this insoluble matter improves the titer of the active ingredient in the oligopeptide mixture. It is preferable because it can be caused. Further, it may be refined with activated carbon or an adsorption resin.

(Basic amino acid dipeptide or tripeptide containing lysine and / or arginine as constituent amino acids: NSPP)
The oligopeptide mixture used in the present invention may be abbreviated as "basic amino acid dipeptide or tripeptide containing lysine and / or arginine as constituent amino acids" [hereinafter, this may be abbreviated as "NSPP" (Neurotrophin Secretion Promoting Peptides). .. ] Is important to contain. That is, the essence of the present invention is to allow the NSPP to act as an effective component that promotes the secretion of neurotrophic factors from nerve cells in the brain.

In the case of a dipeptide, the NSPP contains one or two lysine residues or arginine residues, and in the case of only one residue, the remaining residues preferably contain neutral amino acids. In the case of a tripeptide, it preferably contains 1 to 3 residues of lysine or arginine, and in the case of only 1 to 2 residues, the remaining residues preferably contain neutral amino acids. Examples of the neutral amino acid include glycine.
The lysine residue or arginine residue may be present at either the N-terminal or the C-terminal of NSPP, and in the case of a tripeptide, it may be present in the middle of the amino acid sequence. In addition, since a peptide transporter independent of the amino acid transporter exists in the digestive tract, and it is known that not only dipeptides but also tripeptides are transported into cells in a peptide state, the substance is actually tripeptides. Is equivalent to ingesting dipeptides (Adibi SA. The oligopeptide transporter (Pept-1) in human intestine: biology and function. Gastroenterology. 1997; 113: 332-340.).

Further, the NSPP contained in the oligopeptide mixture may be a mixture of not only a single amino acid sequence containing lysine or arginine but also a mixture having two or more kinds of amino acid sequences containing lysine or arginine.
Among the NSPPs used in the present invention, those having higher expression and secretion of neurotrophic factors in the screening of active substances using astrocytes are preferable, and when measured by the method described in Examples, they are expressed or expressed. The enhancement of secretion is preferably 1.2 times or more, more preferably 1.3 times or more, still more preferably 1.5 times or more, as compared with the solvent-only control.
NSPPs that satisfy these indicators include, for example, Lys-Arg, Arg-Lys, Lys-Lys, Arg-Arg, Gly-Arg, Gly-Lys, Lsy-Gly, Pro-Arg, Arg-Pro, Ser for dipeptides. -Arg, Arg-Ser, Ala-Arg, Arg-Ala, Ile-Arg, Lys-Ile, Ala-Lys, Val-Arg, Arg-Gly, Val-Lys, Leu-Arg, Arg-Ile, Ser-Lys , Tyr-Arg, Lys-Tyr, Asn-Lys, Lys-Val and Lys-Ser. , Gly-Arg, Gly-Lys, Lsy-Gly, Pro-Arg, Arg-Pro, Ser-Arg, Arg-Ser, Ala-Arg, Arg-Ala, Ile-Arg, Lys-Ile, Ala-Lys, Val -Consists of Arg, Arg-Gly, Val-Lys, Leu-Arg and Arg-Ile, especially Lys-Arg, Arg-Lys, Lys-Lys, Arg-Arg, Gly-Arg, Gly-Lys, and Lys-Gly. Dipeptides selected from the group are preferred.
For tripeptides, Lys-Gly-Arg, Gly-Arg-Lys, Gly-Lys-Arg, Arg-Pro-Arg, Arg-Ser-Arg, Arg-Ala-Arg, Lys-Ile-Arg, Ser-Arg -Lys, Ser-Lys-Arg, Pro-Arg-Arg, Arg-Ala-Lys, Lys-Tyr-Arg, Lys-Val-Arg, Ala-Arg-Arg, Arg-Gly-Arg, Val-Lys-Arg , Val-Arg-Arg, Leu-Arg-Arg, Val-Arg-Lys, Pro-Arg-Lys, Asn-Lys-Arg, Arg-Ile-Arg and Lys-Ser-Arg Is preferable, and more preferably Lys-Gly-Arg, Gly-Arg-Lys, Gly-Lys-Arg, Arg-Pro-Arg, Arg-Ser-Arg, Arg-Ala-Arg and Lys-Ile-Arg, especially Lys. -Preferably, a tripeptide selected from the group consisting of Gly-Arg, Gly-Arg-Lys, and Gly-Lys-Arg.

It is considered that the higher the ratio of the amount of lysine and arginine to the total amount of amino acids in the oligopeptide mixture, the larger the amount of NSPP. Is preferable.

Although not essential, if the content of NSPP in the oligopeptide mixture is to be increased, the protein raw material can be enzymatically degraded by a protein hydrolase, and then the enzymatic degradation product of the protein can be further concentrated or purified.
The NSPP used in the present invention can be obtained by preparing an oligopeptide mixture by a plastic reaction, an enzymatic method using an amino acid ligase, or by chemically synthesizing it, but it is economical. In terms of efficiency, considering its use as a food material, it is preferable to concentrate or purify it from a protein hydrolyzate.

Concentration can be carried out by adsorbing a fraction containing a large amount of NSPP in the oligopeptide mixture using an adsorbent or the like.
For purification, a polar organic solvent such as ethanol is added to a solution of the oligopeptide mixture, and the precipitate is recovered to obtain a fraction containing a large amount of NSPP (International Publication No. WO 2008/123033).

(Physiological function of NSPP)
In the screening of active substances using astrosites, as oligopeptide mixtures containing dipeptides and / or tripeptides having basic amino acids containing lysine and arginine as constituent amino acids, for example, Lys-Gly-Arg, Gly-Arg- It has been found by the present invention that NSPPs with the Lys sequence have significantly higher expression and secretion of BDNF compared to controls.

From this empirical result, oligopeptides that promoted the expression or secretion of neurotrophic factors in the screening of active substances using NSPP, especially preferably astrocytes, were found to promote BDNF in the brain by ingesting them. It has been demonstrated by the present inventors that such a neurotrophic factor exhibits a high secretory-promoting effect.

(Composition for food additive to promote brain endocrine of neurotrophic factor containing oligopeptide mixture having NSPP)
A food additive composition for promoting the brain endocrine of a neurotrophic factor containing an oligopeptide mixture having NSPP can be incorporated into various foods. For example, it can be used in combination with products such as beverages, tablets, food bars, meat products, desserts, confectionery and dietary supplements.
These products may have the effect of preventing cranial nerve diseases or improving brain function clearly indicated on the package or advertising medium, or even without such indication, by using the food additive according to the present invention. The seller may substantially aim or expect the effect to be imparted.

(Cranial nerve disease)
Cerebral neurological disorders include higher brain dysfunctions such as memory disorders, attention disorders, executive dysfunctions, and social behavior disorders, and pathologically related symptoms of these disorders, such as cerebral infarction, head trauma, and brain. Examples include vascular dementia, Alzheimer's dementia, Parkinson's disease, schizophrenia, depression, and anxiety.

(Improvement of brain function)
Specific examples of the effect of improving brain function include improvement of memory, improvement of learning ability, improvement of attention, stress tolerance, antidepressant action, anxiolytic action, improvement of concentration, and improvement of sleep quality.

(Method for measuring free amino acid and peptide content)
The molecular weight distribution of the oligopeptide mixture shall be measured by the HPLC method using the following gel filtration column.
An HPLC system using a gel filtration column for peptides is assembled, a known peptide serving as a molecular weight marker is charged, and a calibration line is obtained in relation to the molecular weight and the retention time. The molecular weight markers are β-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe (SEQ ID NO: 1; molecular weight 1046) of [β-Asp] -Angiotensin II as an octapeptide, and Angiotensin IV as a hexapeptide. Val-Tyr-Ile-His-Pro-Phe (SEQ ID NO: 2; molecular weight 775), Leu-Enkephalin's Tyr-Gly-Gly-Phe-Leu (SEQ ID NO: 3; molecular weight 555) as a pentapeptide, Glu- as a tripeptide Glu-Glu (molecular weight 405) and Pro (molecular weight 115) are used as free amino acids.

The supernatant obtained by centrifuging the oligopeptide mixture (1%) at 10,000 rpm for 10 minutes is diluted 2-fold with a gel filtration solvent, and 5 μl thereof is applied to HPLC.
The ratio (%) of free amino acids and peptide fractions having a molecular weight of less than 500 in the protein hydrolyzate is determined by the ratio of the area having a molecular weight of less than 500 (time range) to the chart area of the total absorbance (column used:: Superdex Peptide 7.5 / 300GL (manufactured by GE Healthcare Japan Co., Ltd.), solvent: 1% SDS / 10 mM phosphate buffer, pH 8.0, column temperature 25 ° C, flow velocity 0.25 ml / min, detection wavelength: 220 nm).

Further, the ratio (%) of the peptide fraction having a molecular weight of 500 or more in the protein hydrolyzate is determined by the ratio of the area in the range of the molecular weight of 500 or more to the chart area of the total absorbance in the same manner as described above.

Next, the content of free amino acids in the protein hydrolyzate is measured by amino acid analysis. Add protein hydrolyzate (4 mg / ml) to an equal volume of 3% sulfosalicylic acid and shake at room temperature for 15 minutes. Centrifuge at 10,000 rpm for 10 minutes, filter the obtained supernatant with a 0.45 μm filter, and measure free amino acids with an amino acid analyzer “JLC500V” (manufactured by JEOL Ltd.).
The free amino acid content in the protein hydrolyzate is calculated as a ratio to the protein content obtained by the Kjeldahl method.
The value obtained by subtracting the "free amino acid content" from the "ratio of free amino acids and peptide fractions having a molecular weight of less than 500" is defined as the "content of peptides having a molecular weight of less than 500" in the proteolytic product.

Hereinafter, the present invention will be described in more detail with reference to composition examples, examples and experimental examples.

(peptide)
Peptides used in this study (Lys-Gly-Arg, Gly-Arg-Lys, Lys-Gly-Arg-Lys (SEQ ID NO: 4), Gly-Arg-Lys-Gly (SEQ ID NO: 5), Lys-Gly-Arg -Lys-Gly (SEQ ID NO: 6)) was synthesized by GenScript.

(Test method)
Female C57BL / 6J mice (8-10 weeks old, manufactured by Oriental Yeast Co., Ltd.) were pregnant, fetal brain cells 18 days old were isolated, and DMEM / F12 medium supplemented with G5-Supplement (manufactured by Invitrogen) (manufactured by Invitrogen). Invitrogen) induced differentiation into astrocytes. Seven days after the induction, the positive rate of GFAP, which is a differentiation marker, was examined, and it was confirmed that the positive rate was 95% or more.
Primary culture astrocytes derived from mouse fetal brain (final concentration 2 × 10 ⁵ cells / mL) were seeded in a 60 mm petri dish _{, cultured in a CO 2 incubator at 37 ° C and 5% CO 2} for 4 days, and then synthesized. Peptide (final concentration 1, 10 μM) was added. Medium was used as a control. After 6 hours of culturing, Total RNA was extracted using RNAiso Plus (manufactured by Takara Bio Inc.). Subsequently, real-time PCR was performed using the ReverTra Ace qPCR RT Kit (KAPA Biosystems) to quantify the mRNA expression level of brain-derived neurotrophic factor (BDNF). Standardized with the internal standard gene β-actin. The sequences of the primers used are shown in Table 1.

(Table 1) Primer sequence

(Examples 1 and 2) Examination of tripeptides As tripeptides, KGR (Lys-Gly-Arg) (Example 1) and GRK (Gly-Arg-Lys) (Example 2) were added to primary culture astrocytes. The mRNA expression level of brain-derived neurotrophic factor (BDNF) was examined.
The results are shown in Table 1. It was confirmed that the mRNA expression level of BDNF was significantly increased in all the tripeptides.

(Table 1)

(Comparative Examples 1 and 2) Examination of Tetrapeptides As tetrapeptides, KGRK (Lys-Gly-Arg-Lys) (SEQ ID NO: 4: Comparative Example 1), GRKG (Gly-Arg-Lys-Gly) (SEQ ID NO: 5: For Comparative Example 2), the mRNA expression level of BDNF was examined in the same manner as in Example 1.
The results are shown in Table 2. None of the tetrapeptides significantly increased BDNF mRNA expression.

(Table 2)

(Comparative Example 3) Examination of Pentapeptide As a pentapeptide, KGRKG (Lys-Gly-Arg-Lys-Gly) (SEQ ID NO: 6: Comparative Example 3) was examined for BDNF mRNA expression level in the same manner as in Example 1. ..
The results are shown in Table 3. Pentapeptide did not significantly increase BDNF mRNA expression.

(Table 3)

Claims (4)

Glycine as a constituent amino acid, lysine, and effective ingredient belt Ripepuchido which have a及beauty arginine, food additive compositions for promoting brain endocrine neurotrophic factors. The composition for food addition for promoting the endocrine secretion of the neurotrophic factor according to claim 1, wherein the tripeptide is KGR, GRK, or GKR . Glycine as a constituent amino acid, a belt Ripepuchido which have a lysine及beauty arginine and effective components, brain endocrine promoter of neurotrophic factors. To produce the food used for brain endocrine promoting neurotrophic factor, a method of adding to use glycine as a constituent amino acid, a belt Ripepuchido which have a lysine及beauty arginine, as the active ingredient in the food.