BDNF production promoting composition

Abstract

To provide a BDNF production promoting composition that has not been conventionally available, and BDNF production promoting food, pharmaceutical preparation and livestock feed which contain the composition.SOLUTION: Provided is a BDNF production promoting composition including a sialyl glyco peptide in which a sugar chain is bonded to a peptide having a threonine and / or serine residue, wherein a molecular weight of the sialyl glyco peptide is 500 or more and 3,000 or less, the sugar chain consists of sialic acid and galacto-N-biose, the galacto-N-biose bonds to threonine of the peptide or a hydroxyl group of serine residue, sialic acid bonds to galacto-N-biose, and a molar ratio of the sialic acid and the galacto-N-biose is 1:1 to 2:1. Also provided are BDNF production promoting food / drink, pharmaceutical preparation, and livestock feed which contain the composition.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to a composition for promoting BDNF production, which contains a sialylglycopeptide, and to a food, drink, pharmaceutical, or feed for promoting BDNF production, which contains the composition. [Background technology]

[0002] Brain-derived neurotrophic factor (BDNF) is one of the neurotrophic factors that plays the most important role in the expression of higher brain functions. BDNF has functions such as neurogenesis, maintaining the survival and growth of neurons, and enhancing synaptic function, and has been reported to be closely related to the maintenance and improvement of cognitive function and memory.

[0003] Furthermore, it has been reported that BDNF is involved in various biological functions, such as suppressing overeating and preventing weight gain, increasing energy expenditure, increasing expression of the uncoupling protein-1 (UCP1) gene in brown adipose tissue, promoting glucose utilization in peripheral tissues and contributing to improved glucose metabolism, maintaining erectile function, which declines with age, and maturing eggs. Therefore, increasing BDNF production is expected to exert beneficial functions in the body.

[0004] In light of the above, there is a need for the development of substances that promote BDNF production, not only as commonly used pharmaceuticals, but also as safe agents that can be added to foods and continuously ingested through daily eating habits. Examples include a BDNF production promoter containing milk-derived phospholipids as an active ingredient (Patent Document 1: JP 2017-171595 A), and a composition for increasing BDNF levels in the body containing whey protein hydrolysate containing Ile-Leu, Leu-Leu, Val-Leu, Ile-Val, Ile-Ile, Leu-Val, and Leu-Ile dipeptides (all with a molecular weight of approximately 200) as an active ingredient (Patent Document 2: Japanese Patent No. 6435079 A). However, it is not known that sialylglycopeptides (hereinafter abbreviated as SGP) promote BDNF production.

[0005] Oligosaccharides, glycoproteins, and glycolipids contained in human milk and cow's milk contain large amounts of acidic sugars (hereinafter sometimes referred to as "free sialic acid") and compounds containing sialic acid moieties (hereinafter sometimes referred to as "sialic acid moiety-containing compounds"), collectively known as "sialic acids (9-carbon modified forms of neuraminic acid with an amino group and a carboxylic acid (acidic) moiety)." Sialic acid moiety-containing compounds in milk are useful components that contribute to the establishment of intestinal bacteria in infants (puppies), the development of the nervous system and immune function, and infection defense. For example, it has been reported that feeding piglets a diet containing a sialic acid moiety-containing compound (casein glycomacropeptide) enhances their learning (Non-Patent Document 1: Wang B et al., Am J Clin Nutr, 85(2):561-9, 2007). In addition, it has been shown that milk fat globule membranes (MFGMs) containing sialic acid moiety-containing compounds promote BDNF production in the hippocampus, with an effect comparable to that of free sialic acid (Non-Patent Document 2: Brink L et al., J Nutr Biochem, 69:163-171, 2019). In addition to milk, there have also been reports on the relationship between compositions containing sialic acid moiety-containing compounds and BDNF production. It has been reported that BDNF production in the brain increases in offspring raised on the milk of mother mice fed bird's nest containing a sialic acid moiety-containing compound (a glycoprotein found in sparrow saliva) (Non-Patent Document 3: Mahaq O et al., Brain Behav, 10(11):e01817, 2020). Furthermore, it has been reported that a glycolipid containing a sialic acid moiety (ganglioside GQ1b) increases BDNF production in neuronal cell lines and primary cultured rat cortical neurons (Non-Patent Document 4: MK Shin et al., Neuropharmacology, 77:414-21, 2014). Thus, compositions containing compounds containing a sialic acid moiety are effective in promoting BDNF production, and are expected to improve brain and nervous system function through this effect.

[0006] Most sialic acids are bound to the non-reducing end of glycans, and the types and linkages of glycans to which sialic acids are bound are diverse. Sialic acids are bound to the 3- and 6-positions of galactose, N-acetylglucosamine, and N-acetylgalactosamine found in oligosaccharides, glycoproteins, and glycolipids, but they also exist as polysialic acids in which sialic acids are polymerized via α2-8 linkages. Furthermore, the types of proteins and lipids to which sialylglycans are bound are also diverse. Therefore, if the chemical form of sialic acid in a sialic acid moiety-containing compound differs, it is likely that the physiological functions of the compound will also differ. In fact, it has been reported that when mother mice were fed equal amounts of sea urchin ganglioside, bovine brain ganglioside GM1, and free sialic acid, the sea urchin ganglioside increased BDNF production in their offspring more effectively than GM1 or free sialic acid (Non-Patent Document 5: Wang X et al., Food Funct, 11:9912-23, 2020). Thus, the effectiveness of sialic acid moiety-containing compounds in promoting BDNF production depends not only on the amount of sialic acid contained in the composition, but also on its form. Given the same equivalent amount of sialic acid, it is more efficient to ingest sialic acid in a form that provides greater efficacy. Therefore, a sialic acid form that efficiently promotes BDNF production has been desired. Brink L et al. reported that the BDNF production-promoting effect of MFGM was observed even when the amount of free sialic acid was equivalent to the sialic acid moiety contained in MFGM. Therefore, if a compound containing a sialic acid moiety in a specific form that has a BDNF production-promoting effect greater than that of free sialic acid can be found in milk, it will become a novel, unprecedented milk-derived composition for promoting BDNF production. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2017-171595 [Patent Document 2] Patent No. 6435079 [Non-patent literature]

[0008] [Non-Patent Document 1] Wang B, Yu B, Karim M, Hu H, Sun Y, McGreevy P, Petocz P, Held S, Brand-Miller J. Dietary sialic acid supplementation improves learning and memory in piglets. Am J Clin Nutr. 2007 Feb;85(2):561-9. [Non-patent document 2] Brink LR, Gueniot JP, Lonnerdal B. Effects of milk fat globule membrane and its various components on neurologic development in a postnatal growth restriction rat model. J Nutr Biochem. 2019 Jul;69:163-171. [Non-patent document 3] Mahaq O, P Rameli MA, Jaoi Edward M, Mohd Hanafi N, Abdul Aziz S, Abu Hassim H, Mohd Noor MH, Ahmad H. The effects of dietary edible bird nest supplementation on learning and memory functions of multigenerational mice. Brain Behav. 2020 Nov;10(11):e01817. [Non-patent document 4] Shin MK, Jung WR, Kim HG, Roh SE, Kwak CH, Kim CH, Kim SJ, Kim KL. The ganglioside GQ1b regulates BDNF expression via the NMDA receptor signaling pathway. Neuropharmacology. 2014 Feb;77:414-21. [Non-patent document 5] Wang X, Cong P, Wang X, Liu Y, Wu L, Li H, Xue C, Xu J. Maternal diet with sea urchin gangliosides promotes neurodevelopment of young offspring via enhancing NGF and BDNF expression. Food Funct. 2020 Nov 18;11(11):9912-9923. Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a novel composition for promoting BDNF production, and a food, pharmaceutical, or feed for promoting BDNF production that contains the composition. [Means for solving the problem]

[0010] The present inventors have conducted extensive research into compounds containing a sialic acid moiety that promote BDNF production, and have found that sialylglycopeptide (SGP) has the effect of promoting BDNF production in neurons, and further that SGP increases BDNF levels in the blood and brain of mammals, leading to the completion of the present invention. [Aspect 1] A composition for promoting BDNF production, comprising a sialylglycopeptide in which a sugar chain is bound to a peptide having threonine and / or serine residues, the molecular weight of the sialylglycopeptide is 500 or more and 3,000 or less; the sugar chain is composed of sialic acid and galacto-N-biose, the galacto-N-biose is bound to a hydroxyl group of a threonine or serine residue of the peptide, and sialic acid is bound to the galacto-N-biose; A composition for promoting BDNF production, wherein the molar ratio of the sialic acid to the galacto-N-biose is 1:1 to 2:1. [Aspect 2] The composition for promoting BDNF production according to aspect 1, wherein the combined amount of the sialic acid and the galacto-N-biose is 20% by weight or more and 6090% by weight or less. [Aspect 3] 3. The composition for promoting BDNF production according to aspect 1 or 2, wherein the number of amino acid residues in the peptide chain of the sialylglycopeptide is 1 or more and 13 or less. [Aspect 4] 4. The composition for promoting BDNF production according to any one of aspects 1 to 3, wherein the sugar chain structure is one or more selected from the group consisting of the following (1) to (5): (1)Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAcα1 (2)Galβ1-3(Neu5Acα2-6)GalNAcα1 (3)Neu5Acα2-3Galβ1-3GalNAcα1 (4)Neu5Acα2-3Galβ1-3(O-Ac-Neu5Acα2-6)GalNAcα1 (5)Neu5Acα2-3Galβ1-3(O-diAc-Neu5Acα2-6)GalNAcα1 [Aspect 5] 5. The composition for promoting BDNF production according to any one of aspects 1 to 4, wherein the site where promotion of BDNF production is observed is the hippocampus of the subject. [Aspect 6] 5. The composition for promoting BDNF production according to any one of aspects 1 to 4, wherein the site where promotion of BDNF production is observed is in the blood of the subject. [Aspect 7] 7. The composition for promoting BDNF production according to any one of Aspects 1 to 6, wherein the sialylglycopeptide is derived from milk. [Aspect 8] A food or drink for promoting BDNF production, comprising the composition for promoting BDNF production according to any one of aspects 1 to 7. [Aspect 9] A pharmaceutical product for promoting BDNF production, comprising the composition for promoting BDNF production according to any one of aspects 1 to 7. [Aspect 10] A feed for promoting BDNF production, comprising the composition for promoting BDNF production according to any one of aspects 1 to 7. [Effects of the Invention]

[0011] The present invention provides a composition for promoting BDNF production containing SGP, as well as a food, drink, pharmaceutical, and feed for promoting BDNF production, each of which contains SGP as an active ingredient. [Brief explanation of the drawings]

[0012] [Figure 1] 1 shows the results of size exclusion chromatography (SEC) analysis of the SGP of the present invention (210 nm). [Figure 2] The results of LC / MS analysis of purified sialylglycopeptide purified from the SGP of the present invention are shown ((A) LC / MS chromatogram (TIC, m / z 400-2000) of the desalted sialylglycopeptide solution, (B) MS spectrum at 15.94 min). [Figure 3] 1 shows the structure of a purified sialylglycopeptide purified from SGP of the present invention. [Figure 4] 1 is a graph showing the amount of BDNF produced by a human neuronal cell line (SH-SY5Y) in a medium containing a sialic acid preparation or the SGP of the present invention. Tukey-Kramer's test was performed. Different letters indicate significant differences at the 5% level. [Figure 5] 1 is a graph showing the amount of BDNF produced by a human neuronal cell line (SH-SY5Y) in a medium containing SGP or a purified sialylglycopeptide purified from SGP of the present invention. Tukey-Kramer's test was performed. Different letters indicate significant differences at the 5% level. [Figure 6] 1 is a graph showing the amounts of BDNF in the plasma and brain of rats in the control group and the SGP-administered group. DETAILED DESCRIPTION OF THE INVENTION

[0013] The composition for promoting BDNF production containing the SGP of the present invention, and the food, drink, pharmaceutical and feed for promoting BDNF production containing the composition are described in detail below.

[0014] (Sialylglycopeptide: SGP) The SGP of the present invention has the following properties (a) to (e). (a) A sugar chain is bound to a peptide having threonine and / or serine residues. (b) The molecular weight is 500 or more and 3,000 or less. (c) The sugar chain consists of sialic acid and galacto-N-biose. (d) the galacto-N-biose of the sugar chain is bound to the hydroxyl group of a threonine or serine residue of the peptide, and sialic acid is bound to the galacto-N-biose. (e) The molar ratio of sialic acid to galacto-N-biose is in the range of 1:1 to 2:1. Furthermore, the combined amount of sialic acid and galacto-N-biose constituting the sugar chain of the SGP of the present invention is preferably 20% by weight or more and 90% by weight or less, more preferably 25% by weight or more and 70% by weight or less, and most preferably 30% by weight or more and 50% by weight or less. The SGP of the present invention preferably has a peptide chain consisting of 1 to 13 amino acid residues. Furthermore, the SGP of the present invention may contain acetylated sialic acid.

[0015] One embodiment of a composition for promoting BDNF production containing the SGP of the present invention is a composition containing a sialylglycopeptide having threonine and / or serine residues and a sugar chain bound to the hydroxyl group, wherein the sugar chain is one or more selected from the group consisting of (1) to (5) below. (1)Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAcα1 (2)Galβ1-3(Neu5Acα2-6)GalNAcα1 (3)Neu5Acα2-3Galβ1-3GalNAcα1 (4)Neu5Acα2-3Galβ1-3(O-Ac-Neu5Acα2-6)GalNAcα1 (5)Neu5Acα2-3Galβ1-3(O-diAc-Neu5Acα2-6)GalNAcα1 Since the peptide contains one or more of the above sugar chains, it also includes peptides containing two or more, such as those containing two or more of the same type of sugar chain or those containing two or more types. For example, it may be a peptide containing two of (1), or a peptide containing (1) and further containing one or more from the group consisting of (2) to (5).

[0016] The SGP of the present invention can typically be obtained by hydrolyzing a milk protein such as whey protein with an enzyme or the like and fractionating the hydrolyzed product. The whey protein used as the raw material for the SGP of the present invention can be any whey protein obtained from whey derived from cow's milk, as well as from whey derived from animal milk such as goat's or sheep's milk. The SGP of the present invention can also be prepared by enzymatically hydrolyzing extracts of microorganisms, plants, or animal organs and fractionating the hydrolyzed extracts. Furthermore, the SGP of the present invention can also be chemically synthesized or prepared by recombinant DNA technology.

[0017] (SGP manufacturing method) One embodiment of the method for producing the SGP of the present invention will be described below. The SGP of the present invention can be obtained, for example, by hydrolyzing whey protein and fractionating the hydrolyzed product. For example, dairy materials such as cheese whey, whey protein concentrate, and whey protein isolate can be used as raw materials. The SGP of the present invention can be produced by treating these dairy materials with an endo- or exo-protease and fractionating the resulting product.

[0018] The type of protease used to produce SGP is not particularly limited as long as it can hydrolyze peptide bonds to produce glycopeptides with the molecular weight and sugar chains described above (sialylglycopeptide: SGP). One or more types of endoproteases or exoproteases can be used. Enzymes that can be used in the production of foods and pharmaceuticals are preferred, and examples that can be used alone or in combination include actinase E, actinase AS (Kaken Pharma), nucleisin, orientase AY, orientase OP (HBI), Sumiteam FP, Sumiteam SPP-G (Shin Nippon Chemical Industry), protease A, peptidase R (Amano Enzyme), Alcalase, and Flavorzyme (Novozyme).

[0019] As described above, the SGP of the present invention is typically obtained by hydrolyzing whey and fractionating the hydrolyzed product, and therefore, depending on the degree of purification, it may be a purified single sialylglycopeptide, a mixture of multiple sialylglycopeptides, or a composition containing components other than the SGP of the present invention. In this specification, a composition containing the SGP of the present invention may be referred to as a composition containing SGP, and unless otherwise specified, this term is used to include all of the above cases.

[0020] (Evaluation of the BDNF production promoting effect of SGP) The BDNF production-promoting activity of SGP of the present invention can be evaluated by measuring the amount of BDNF produced by a human neuronal cell line (SH-SY5Y), or by administering SGP to animals such as rats or humans and measuring the amount of BDNF in the blood or brain, such as the hippocampus.

[0021] (Food, beverages, medicines, and feed containing SGP) The SGP or composition containing SGP of the present invention obtained by the above-mentioned production method can be used as a raw material for food and beverage products as is, and the food and beverage products can be produced according to the standard method for each food product, except for adding the composition containing SGP. Therefore, an effective amount of SGP of the present invention may be incorporated into any food or beverage, or may be added to raw materials during the manufacturing process of the food or beverage. Examples of foods and beverages include, but are not limited to, dairy products such as cheese, fermented milk, dairy lactic acid bacteria drinks, lactic acid bacteria drinks, butter, margarine, etc., milk drinks, fruit juice drinks, soft drinks, etc., egg products such as jelly, candy, pudding, mayonnaise, etc., sweets and breads such as butter cake, various types of milk powder, infant foods, nutritional compositions, etc. The food and drink containing SGP produced in this manner is provided as a food and drink for promoting BDNF production.

[0022] The SGP or composition containing SGP of the present invention obtained by the above-mentioned production method can be used as a raw material for pharmaceuticals as it is, and tablets, capsules, powders, syrups, etc. can be produced by conventional methods except for adding the composition containing SGP. Therefore, when formulating a pharmaceutical containing the SGP of the present invention as an active ingredient, it can be formulated by appropriately mixing it with approved excipients, stabilizers, flavoring agents, etc., or it can be dried directly and used as a powder or sachets. Furthermore, it can also be formulated by mixing it with excipients, binders, disintegrants, lubricants, flavoring agents, suspending agents, coating agents, and any other optional agents within the range that does not interfere with the BDNF production-promoting effect. Possible dosage forms include tablets, capsules, granules, powders, dusts, and syrups. Pharmaceuticals containing SGP produced in this manner are provided as pharmaceuticals for promoting BDNF production.

[0023] The SGP or SGP-containing composition of the present invention obtained by the above-mentioned production method can be used as a raw material for feed, which can be produced by standard methods for feed production except for the addition of the SGP-containing composition. Thus, an effective amount of SGP of the present invention can be incorporated into any feed, as in the case of the above-mentioned foods and beverages, or can be added to raw materials during the feed production process. The SGP-containing feed produced in this manner can be provided as a feed for promoting BDNF production.

[0024] (SGP intake) When the SGP of the present invention is incorporated into foods, pharmaceuticals, feeds, etc., the proportion of SGP to be incorporated is not particularly limited, and may be appropriately adjusted depending on the ease of production and the preferred daily dose. The daily dose of SGP of the present invention is determined individually for each subject taking into consideration the health condition, symptoms, age, etc. of the subject. Generally, for adults, SGP is administered in doses of 0.1 to 5,000 mg, 0.2 to 5,000 mg, 0.5 to 5,000 mg, 1 to 5,000 mg, 2 to 5,000 mg, 5 to 5,000 mg, 10 to 5,000 mg, 20 to 5,000 mg, 50 to 5,000 mg, 100 to 5 ,000mg, 200~5,000mg, 500~5,000mg, 1,000~5,000mg, 2,000~5,000mg, 0.2~2,000mg, 0.5~2,000mg, 1 ~2,000mg, 2~2,000mg, 5~2,000mg, 10~2,000mg, 20~2,000mg, 50~2,000mg, 100~2,000mg, 200~2,000m g, 500~2,000mg, 1,000~2,000mg, 0.5~1,000mg, 1~1,000mg, 2~1,000mg, 5~1,000mg, 10~1,000mg, 20~ 1,000mg, 50~1,000mg, 100~1,000mg, 200~1,000mg, 500~1,000mg, 1~500mg, 2~500mg, 5~500mg, 10~50 The amount of compounding may be adjusted so that the amount of 0 mg, 20 to 500 mg, 50 to 500 mg, 100 to 500 mg, 200 to 500 mg, 2 to 200 mg, 5 to 200 mg, 10 to 200 mg, 20 to 200 mg, 50 to 200 mg, 100 to 200 mg, 5 to 100 mg, 10 to 100 mg, 20 to 100 mg, 50 to 100 mg, 10 to 50 mg, or 20 to 50 mg can be ingested. By ingesting in this manner, the desired effect can be exerted.

[0025] The subject to which the composition containing SGP of the present invention is administered is not particularly limited, and may be humans or animals. [Example]

[0026] The present invention will be described in detail below with reference to examples and test examples, but these are merely illustrative and the present invention is not limited to them in any way. Example 1: Preparation method of SGP of the present invention 1. Manufacturing method of SGP 3 kg of whey protein isolate (WPI, Provon 190, Glanbia) was dissolved in 27 kg of water at 55°C to prepare 30 kg of a 10% WPI solution. Subsequently, 0.5% (v / v) endoprotease (Alcalase 2.4L FG, Novozyme, ≥2.4 U / g) and exoprotease (Flavorzyme 1000L, Novozyme, ≥1000 U / g) were added and incubated at 55°C for 8 hours. The enzymes were then inactivated by holding the mixture at 85°C for 30 minutes. The resulting 30 kg of WPI hydrolysate was subjected to diafiltration (DF) using an ultrafiltration membrane with a molecular weight cutoff of 1,000. The concentrate was continuously diluted with water and subjected to 10-fold DF. The retentate fraction was then dried to yield 120.0 g of SGP(1).

[0027] 2. Measurement of sialic acid content of SGP SGP was prepared in 50 mM potassium phosphate buffer (pH 5.0) at 250 μg / mL and sialidase (Neuraminidase isoenzyme S from Arthrobacter ureafaciens, #EC-32118-S, Cosmo Bio) at 0.2 U / mL. 50 μL of this solution was reacted for 1 hour at 37°C using a thermal cycler (Takara Bio). The reaction solution was diluted 4-fold with ultrapure water, and insoluble material was removed using a 0.45 μm filter (13A, Kurabo). Next, to measure the amount of free sialic acid, SGP material was dissolved in ultrapure water to a concentration of 1 mg / mL and centrifuged at 15,000 × g for 10 minutes (Tomy Seiko). The resulting supernatant was then passed through a 0.45 μm filter (13A, Kurabo). The sialic acid content was measured using a DIONEX ICS-5000DP system (Thermo Fisher Scientific) equipped with a CarboPac PA1 column (4 × 250 mm, Thermo Fisher Scientific). An electrochemical detector (pulsed amperometric mode) was used. The mobile phase consisted of 100 mM sodium hydroxide solution containing 60 mM sodium acetate, which was isocratically passed through the column for 7 min after sample introduction. The sodium acetate concentration was then linearly increased to 150 mM between 7 and 10 min and maintained at that concentration until 20 min. The column was then equilibrated by isocratically passing a 100 mM sodium hydroxide solution containing 60 mM sodium acetate for 5 min. The mobile phase flow rate was always 1 mL / min. N-Acetylneuraminic acid (#A2388, Sigma-Aldrich) was used as a sialic acid standard, and the sialic acid content was calculated from the calibration curve. The results are shown in Table 1.

[0028] 3. Measurement of galacto-N-biose (GNB) content in SGP A 50 mM potassium phosphate buffer (pH 5.0) solution containing 250 μg / mL SGP, 0.2 U / mL sialidase (Neuraminidase isoenzyme S from Arthrobacter ureafaciens, #EC-32118-S, Cosmo Bio), and 0.125 U / mL O-glycanase (#DG53 043a, Prozyme) was prepared. A 50 μL aliquot was incubated at 37°C for 24 hours in a thermal cycler (Takara Bio). The reaction mixture was diluted 2 to 10 times with ultrapure water, and insoluble material was removed using a 0.45 μm filter (13A, Kurabo). GNB was measured using a DIONEX ICS-5000DP system (Thermo Fisher Scientific) equipped with a CarboPac PA1 column. An electrochemical detector (pulsed amperometric mode) was used. The mobile phase consisted of 100 mM sodium hydroxide solution, which was isocratically passed through the column for 10 minutes after sample introduction. From 10 to 25 minutes, the sodium acetate concentration was linearly increased to 600 mM and maintained at that concentration until 30 minutes. From 30 to 45 minutes, 100 mM sodium hydroxide solution was isocratically passed through the column. The mobile phase was always passed through at 1 mL / min. The GNB content was calculated from a calibration curve prepared using a GNB standard (#A0167, Sigma). The results are shown in Table 1.

[0029] [Table 1]

[0030] [Example 2] Sialidase treatment of SGP SGP (1) prepared in Example 1 was diluted to 20 mg / mL in 100 mM acetate buffer (pH 5.0) containing 2 mM calcium chloride. Sialidase (Clostridium perfringens-derived: Sigma, N2876-25UN) was added to the solution at 10 U / mL and the mixture was incubated at 37°C for 20 hours. A blank without enzyme was prepared by reacting the mixture under the same conditions without enzyme addition. The reaction was terminated by boiling for 5 minutes. The resulting reaction mixture was subjected to size exclusion chromatography (SEC). SEC analysis was performed using an L-2000 (Hitachi) system equipped with two TSKgel G3000PW (Tosoh) columns, and UV absorption at 214 nm was detected. The mobile phase was 40% acetonitrile containing 0.1% trifluoroacetic acid, and the mixture was eluted isocratically at room temperature for 120 minutes at a flow rate of 0.3 mL / min. The results of the SEC analysis are shown in Figure 1. A large peak was observed around 70 minutes, which corresponds to the acetate buffer used in the enzyme reaction. The main peak of SGP was observed between 50 and 60 minutes, but this was shifted to lower molecular weights by sialidase treatment, indicating that the main component of the prepared SGP (1) was a compound containing sialic acid moieties.

[0031] [Example 3] Structural analysis of SGP using LC / MS LC / MS analysis was performed using an Orbitrap mass spectrometer Q-Exactive (Thermo Fisher Scientific) connected to a high-performance liquid chromatograph (HPLC) UltiMate 3000 (Thermo Fisher Scientific) (LC-ESI-IT MS). An InertSustain AQ-C18 (φ2.1 mm × 150 mm, GL Sciences) was used as the separation column. The mobile phase consisted of a 2% acetonitrile solution containing 0.1% formic acid (Solution A) and a 90% acetonitrile solution containing 0.1% formic acid (Solution B). SGP was dissolved in ultrapure water to a concentration of 100 μg / mL, and 10 μL was introduced into the HPLC. The mobile phase was run at 200 μL / min. The proportion of Solution B was 0% for 5 minutes after sample introduction, and then linearly increased from 0% to 10% over 25 minutes. The proportion of solution B was then linearly increased from 10% to 100% over a 20-minute period. The mass spectrometer performed MS (m / z 200–2000, positive mode) and MS / MS measurements. For MS / MS measurements, the five most intense ions were automatically selected as precursor ions, and the m / z values ​​of the resulting product ions were observed. The acquired MS / MS spectra were analyzed using Proteome Discoverer 2.2 (Thermo Fisher Scientific) with the Byonic (Protein Metrics) node. The protein database consisted of whey protein alone. The glycan database included 10 types of glycans, each consisting of one N-acetylhexosamine (HexNAc), 0–1 hexose (Hex), 0–2 N-acetylneuraminic acid (Neu5Ac), and 0–2 O-acetyl groups (O-Ac). The glycans identified as (HexNAc), (HexNAc)(Hex), or (HexNAc)(Hex)(Neu5Ac)2 by Byonic analysis were assigned to GalNAc, Galβ1-3GalNAc, or Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAc, respectively, according to previously reported O-linked glycan structures.Furthermore, for glycans assigned as (HexNAc)(Hex)(Neu5Ac), we manually confirmed the MS / MS spectra and assigned them to Neu5Acα2-3Galβ1-3GalNAc or Galβ1-3(Neu5Acα2-6)GalNAc. Similarly, for O-acetyl-modified Neu5Ac (O-Ac-Neu5Ac or O,O'-diAc-Neu5Ac), we manually confirmed the MS / MS spectra and assigned them to Neu5Acα2-3Galβ1-3(O-Ac-Neu5Acα2-6)GalNAc or Neu5Acα2-3Galβ1-3(O-diAc-Neu5Acα2-6)GalNAc. The glycan structures of glycopeptides contained in SGP (1) prepared in Example 1 were estimated from the MS and MS / MS spectra obtained by LC / MS analysis. Table 2 shows the glycan structures of glycopeptides detected by LC / MS analysis. As a result, all of the detected glycopeptides were peptides bound to glycans containing sialic acid. Many of the detected glycopeptides had the glycan structure Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAc, in which two molecules of sialic acid are bound (glycan types 3, 4, 5, 6, and 7). However, Neu5Acα2-3Galβ1-3GalNAc (glycan type 1) and Galβ1-3(Neu5Acα2-6)GalNAc (glycan type 2), in which one molecule of sialic acid is bound, were also detected. Furthermore, the O-acetyl forms of sialic acid, N,O-diacetylneuraminic acid (O-Ac-Neu5Ac, Glycan type 4) and N,O,O-triacetylneuraminic acid (O-diAc-Neu5Ac, Glycan type 5), were also detected. Glycopeptides with two glycans attached to a single peptide chain (Glycan types 6 and 7) were also detected. All detected glycopeptides contained one or more serine or threonine residues, and the peptide chain length of the glycopeptides ranged from one residue to 13 residues. The molecular weight of the sialylglycopeptides, including the glycans and sialic acid moieties, was between 500 and 3,000.

[0032] [Table 2]

[0033] Example 4: Purification of the major sialylglycopeptide in SGP 1. Purification method of sialylglycopeptides SGP prepared in Example 1 was dissolved in ultrapure water to a concentration of 10 mg / mL, and 5 mL of the solution was introduced into a preparative HPLC system PLC761 (GL Sciences, Inc.) connected to an NHP-90 column (20 × 300 mm, Shodex). The mobile phase consisted of ultrapure water (solution A) and 300 mM sodium dihydrogen phosphate aqueous solution (solution B). The column temperature was 40 °C and the flow rate was 10 mL / min. The detector used was a UV702 (GL Sciences, Inc.) set at 210 nm. After sample introduction, 10% solution B was passed through for 5 minutes, then the concentration of solution B was increased from 10% to 55% from 5 to 35 minutes, and then increased to 100% from 35 to 100 minutes. After 100% solution B was passed through for 10 minutes, 10% solution B was passed through for 35 minutes. Between 20 and 30 minutes, 6.7 mL of the eluate was collected, and fraction A containing the peak of the target sialylglycopeptide was collected (Figure 1). The same procedure was repeated 60 times. A graphite carbon (GC) solid-phase column, InertSep GC (1 g / 12 mL, GL Sciences Inc.), was washed with 10 mL of 80% acetonitrile containing 0.1% formic acid and then equilibrated with 10 mL of ultrapure water. The entire fraction A from 20 runs was loaded onto the GC column and washed with 20 mL of ultrapure water to remove salts. Sialylglycopeptides were recovered with 5 mL of 20% acetonitrile containing 0.1% formic acid. The sialylglycopeptides were eluted with 5 mL of 50% acetonitrile containing 0.1% formic acid and mixed with the previously recovered solution. 100 μL of concentrated aqueous ammonia was added and stirred. The acetonitrile was removed using a centrifugal evaporator (45°C), followed by lyophilization. This procedure was repeated three times, and the powders from each run were dissolved in 1 mL of ultrapure water. The resulting mixture was used as the sialylglycopeptide solution.

[0034] 2. Structural analysis method of purified sialylglycopeptides To determine the amino acid sequence and glycan structure of the purified sialylglycopeptide, 10 μL of the sialylglycopeptide solution was diluted 500-fold with ultrapure water, filtered through a 0.45 μm filter, and analyzed by LC / MS (Figure 2). An UltiMate 3000 (Thermo Fisher Scientific) was used for HPLC, and a Q Exactive (Thermo Fisher Scientific) was used for MS. The column used was an Inertsustain AQ-C18 (2.1 × 150 mm, Thermo Fisher Scientific), and the column temperature was set at 40 °C. The mobile phase used was a 2% acetonitrile solution containing 0.1% formic acid (Solution A) and a 90% acetonitrile solution containing 0.1% formic acid (Solution B), with a flow rate of 200 μL / min. After sample introduction, 100% solution A was allowed to flow for 5 minutes, then the proportion of solution B was linearly increased from 0% to 100% from 5 to 30 minutes, and 100% solution B was allowed to flow from 30 to 35 minutes. The ESI probe spray voltage was 3.5 kV, the capillary temperature was 275°C, and nitrogen gas was used for the sheath gas, auxiliary gas, and collision gas. The S-Lens RF Level was set to 90. MS spectra were acquired in positive mode using full scan measurements in the m / z range of 400–2000. The mass resolution was set to 70,000. MS2 spectra were acquired immediately after the MS spectrum in a data-dependent manner using the top five ions by signal intensity as precursor ions (top 5). The normalized collision energies were set to 15 and 27 (Figure 2). The acquired MS2 spectra were analyzed using Proteome Discoverer (Thermo Fisher Scientific Inc.) with the Byonic node, and the amino acid sequences and glycan structures of the purified sialylglycopeptides were determined (Figure 3).

[0035] 3. Quantitative analysis of purified sialylglycopeptides A 10 μL solution of sialylglycopeptide was diluted 500-fold with 50 mM phosphate buffer (pH 5.0), sialidase was added, and the mixture was incubated at 37°C for 6 hours. The released sialic acid was then measured. A CarboPac PA1 column (4 × 250 mm, Thermo Fisher Scientific) was used, and an HPAE-PAD (DIONEX ICS-5000DP system, Thermo Fisher Scientific) with an electrochemical detector (pulsed amperometric mode) was used. After sample introduction, a 60 mM aqueous solution of sodium acetate containing 100 mM sodium hydroxide was passed through the column for 7 minutes. The sodium acetate concentration was linearly increased from 60 mM to 150 mM between 7 and 10 minutes. A 150 mM aqueous solution of sodium acetate containing 100 mM sodium hydroxide was then passed through the column for 10 minutes. The column temperature was 25°C, and the flow rate was 1 mL / min. The amount of sialic acid was quantified using a calibration curve prepared using sialic acid standards of known concentrations. The concentration of sialylglycopeptide in the sialylglycopeptide solution was calculated using the quantitative value of sialic acid and the molecular weight calculated from the amino acid sequence and glycan structure of the sialylglycopeptide.

[0036] [Test Example 1] SH-SY5Y (human neuroblastoma) cell line (ECACC) was cultured in DMEM-high glucose media (Nakarai) + 10% FCS (Gibco) at 37°C under CO2. 1 × 10 cells were placed in each well of a 6-well plate. 4 Cells were seeded per well, and 72 hours later, 1 mg / ml of SGP prepared in Example 1 and 0.311 mg / ml of a sialic acid preparation (N-Acetylneuraminic Acid, Tokyo Chemical Industry Co., Ltd.) were added (the sialic acid content in the SGP and sialic acid preparation was the same), and the plate was left to stand at 37°C under 5% CO2 for 4 hours. 150 μl of RIPA buffer was added to each well, and the cells were left on ice for 10 minutes before being harvested with a cell scraper. The harvested cell lysate was centrifuged at 15,000 rpm for 10 minutes, and the supernatant was collected and used as a protein extract. The protein extract was prepared using PierceTM Protein concentration was measured using a BCA Protein Assay Kit (Thermo Scientific), followed by standard measurements using a Mature BDNF Rapid ELISA kit (Biosensis). The protein was diluted 5-fold with buffer according to the protocol and used as an ELISA sample. The chemiluminescence of the ELISA plate was measured using a VARIOSKAN FLASH (Thermo Scientific), and the sample concentration was calculated from a standard curve of mature BDNF standard. The values ​​obtained were then corrected for the total protein amount. The amount of BDNF (ng / mL) per total protein in the extract is shown in Figure 4. On the other hand, SGP significantly enhanced BDNF production compared to the control and the sialic acid-added sample.

[0037] [Test Example 2] SH-SY5Y (human neuroblastoma) cell line (ECACC) was cultured in DMEM-high glucose media (Nakarai) + 10% FCS (Gibco) at 37°C under CO2. 1 × 10 cells were placed in each well of a 6-well plate. 4 After 72 hours, purified sialylglycopeptide (prepared in Example 4) diluted with ultrapure water to the same sialylglycopeptide content as 10 μg / ml of SGP prepared in Example 1 was added to each well, and the cells were allowed to stand at 37°C under 5% CO2 for 4 hours. 150 μl of RIPA buffer was added to each well, and the cells were left on ice for 10 minutes before being harvested with a cell scraper. The harvested cell lysate was centrifuged at 15,000 rpm for 10 minutes, and the supernatant was collected and used as a protein extract. The protein extract was prepared using Pierce TM Protein concentration was measured using a BCA Protein Assay Kit (Thermo Scientific), followed by standard measurements using a Mature BDNF Rapid ELISA kit (Biosensis). The protein was diluted 5-fold with buffer according to the protocol and used as an ELISA sample. Chemiluminescence from the ELISA plate was measured using a VARIOSKAN FLASH (Thermo Scientific), and the sample concentration was calculated using a standard curve of mature BDNF standard. The values ​​obtained were then corrected for the total protein amount. The amount of BDNF (ng / ml) per total protein in the extract is shown in Figure 5. As in Test Example 1, SGP promoted BDNF production, and purified sialylglycopeptide also showed a similar increase in BDNF as SGP. While the purified sialylglycopeptide was effective, the BDNF production-promoting effect of SGP disappeared when SGP was treated with sialidase (data not shown), confirming that the active ingredient in SGP is sialylglycopeptide.

[0038] [Test Example 3] (Confirmation of BDNF production promoting effect in animal experiments) Male Wistar rats were divided into two groups (n = 10) with equal average body weights: a control group and an SGP group. The control group was fed a normal diet (AIN-93G), while the SGP group was fed a diet containing 10% SGP for 3 weeks. After that, plasma and brain protein extracts were analyzed by Pierce TM Protein concentration was measured using a BCA Protein Assay Kit (Thermo Scientific), followed by standard measurements using a Mature BDNF Rapid ELISA kit (Biosensis). As a result, the SGP intake group showed a significant increase in BDNF levels in the plasma (a) and hippocampus (b) compared to the control group (Figure 6).

[0039] [Example 5] Production of supplements 40 g of an equal mixture of vitamin C and citric acid, 100 g of granulated sugar, and 60 g of an equal mixture of cornstarch and lactose were added to 30 g of the SGP powder obtained in Example 1 and mixed. The mixture was packed into a stick-shaped bag to produce a supplement for promoting BDNF production of the present invention.

[0040] Example 6: Production of beverages The ingredients were mixed according to the composition shown in Table 3, filled into a container, and then heat sterilized to produce the beverage for promoting BDNF production of the present invention.

[0041] [Table 3]

[0042] [Example 7] Production of pharmaceuticals (capsules) The raw materials were mixed according to the formulation shown in Table 4, granulated to form granules, and then 10 mg of each was filled into empty capsules to produce capsules containing the pharmaceutical product for promoting BDNF production of the present invention.

[0043] [Table 4] [Industrial Applicability]

[0044] According to the present invention, it is possible to provide a composition for promoting BDNF production, which contains SGP as an active ingredient, and foods, beverages, pharmaceuticals, and feed for promoting BDNF production, which contain SGP as an active ingredient.

Claims

1. A composition for promoting BDNF production, comprising a sialylglycopeptide in which a sugar chain is bound to a peptide having a threonine and / or serine residue, the molecular weight of the sialylglycopeptide is 500 or more and 3,000 or less; the sugar chain is composed of sialic acid and galacto-N-biose, the galacto-N-biose is bound to a hydroxyl group of a threonine or serine residue of the peptide, and sialic acid is bound to the galacto-N-biose; A composition for promoting BDNF production, wherein the molar ratio of the sialic acid to the galacto-N-biose is 1:1 to 2:

1.

2. 2. The composition for promoting BDNF production according to claim 1, wherein the combined amount of said sialic acid and said galacto-N-biose is 20% by weight or more and 90% by weight or less.

3. The composition for promoting BDNF production according to claim 1, wherein the number of amino acid residues in the peptide chain of the sialylglycopeptide is 1 or more and 13 or less.

4. 2. The composition for promoting BDNF production according to claim 1, wherein the sugar chain structure is one or more selected from the group consisting of the following (1) to (5): (1) Neu5Acα2-3Galβ1-3 (Neu5Acα2-6)GalNAcα1 (2) Galβ1-3 (Neu5Acα2-6) GalNAcα1 (3) Neu5Acα2-3Galβ1-3GalNAcα1 (4) Neu5Acα2-3Galβ1-3(O-Ac-Neu5Acα2-6)GalNAcα1 (5) Neu5Acα2-3Galβ1-3(O-diAc-Neu5Acα2-6)GalNAcα1

5. 2. The composition for promoting BDNF production according to claim 1, wherein the site where promotion of BDNF production is observed is the hippocampus of the subject.

6. 2. The composition for promoting BDNF production according to claim 1, wherein the site where promotion of BDNF production is observed is in the blood of a subject.

7. The composition for promoting BDNF production according to claim 1, wherein the sialylglycopeptide is derived from milk.

8. A food or drink for promoting BDNF production, comprising the composition for promoting BDNF production according to any one of claims 1 to 7.

9. A pharmaceutical product for promoting BDNF production, comprising the composition for promoting BDNF production according to any one of claims 1 to 7.

10. A feed for promoting BDNF production, comprising the composition for promoting BDNF production according to any one of claims 1 to 7.

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