Composition for maintaining and / or improving memory / learning ability, and food, medicine, and feed each containing the composition

Abstract

To provide an unconventional, novel composition for maintaining and / or improving memory / learning ability; and a food or beverage, a medicine, and a feed, each of which is intended for maintaining and / or improving memory / learning ability and contains the composition.SOLUTION: Provided are: a composition for maintaining and / or improving memory / learning ability, the composition containing a sialyl glycopeptide as an active ingredient; and a food or beverage, a medicine, and a feed, each of which is intended for maintaining and / or improving memory / learning ability and contains the composition as an active ingredient.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present invention relates to a composition for maintaining and / or improving memory and learning ability, which contains a sialylglycopeptide, and to a food, drink, pharmaceutical, and feed for maintaining and / or improving memory and learning ability, which contains the composition. [Background technology]

[0002] As we enter a super-aging society, age-related cognitive decline and dementia have become major social issues. While progress is being made in the development of therapeutic drugs for dementia, a fundamental cure has yet to be realized. Furthermore, in modern society, we must process vast amounts of information each day, so forgetfulness and memory loss are no longer problems exclusive to the elderly. Memory loss can be caused by a variety of factors, including psychological stress, insomnia, and nutritional deficiencies, and it reduces worker productivity, leading to significant economic losses. Therefore, in addition to the development of pharmaceuticals that can be used regularly, there is a need for the development of highly safe agents that can be added to food and ingested continuously through daily dietary habits.

[0003] Oligosaccharides, glycoprotein sugar chains, and glycolipids contained in breast milk and cow's milk contain large amounts of acidic sugars called "sialic acids." Sialic acids in milk are components of brain gangliosides and polysialic acid-modified neural cell adhesion molecules and play an important role in the development of infants' brains and nerves. For example, it has been reported that rats fed milk from mothers who had ingested free sialic acid (N-acetylneuraminic acid) exhibit improved spatial working memory later in life (Non-Patent Document 1: J Nutr Sci Vitaminol (Tokyo). 2013;59(2):136-43). It has also been reported that feeding piglets milk replacer containing increased amounts of casein glycomacropeptide (GMP / CGMP)-binding sialic acid for 35 days promoted learning in the piglets and increased the expression of two learning-related genes (Non-Patent Document 2: Am J Clin Nutr. 2007 Feb;85(2):561-9). Thus, it is expected that ingesting free sialic acid or GMP containing sialic acid moieties will improve the function of the brain and nervous system.

[0004] Most sialic acids are bound to the non-reducing end of glycans, with diverse types and linkages of glycans. Sialic acids are bound to the 3- and 6-positions of galactose, N-acetylglucosamine, and N-acetylgalactosamine in oligosaccharides, glycoproteins, and glycolipids. However, 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 sialic acid chains are bound are diverse. Therefore, the physiological function of sialic acid may vary depending on the form of sialic acid present in a composition. In fact, one study reported that feeding piglets sialyllactose, in which sialic acid is bound to lactose, did not result in improved cognitive function (Non-Patent Document 3: Nutrients. 2018 Mar 23;10(4):395). Thus, the improvement of brain and nervous system function through the ingestion of free sialic acid or GMP containing a sialic acid moiety is likely dependent not only on the amount of sialic acid in the composition but also on its chemical bond form. Furthermore, if the same amount of sialic acid is to be ingested, it is thought that it would be more efficient to ingest a form of sialic acid moiety-containing compound that provides a greater effect in improving brain and nervous system function. Therefore, if a sialic acid moiety-containing compound can be found in milk that has a greater effect in improving brain and nervous system function than free sialic acid or GMP containing a sialic acid moiety, a composition containing such a compound would be a novel milk-derived composition for improving brain and nervous system function.

[0005] The nematode Caenorhabditis elegans (hereafter referred to as C. elegans) can sense various external stimuli and respond to diverse behaviors using a simple neural circuit consisting of 302 neurons. C. elegans is capable of associative learning, which involves associating food with chemicals, and is known to be an excellent model system for analyzing memory and cognitive functions, reflecting behavioral and neuroplasticity after conditioning with multiple stimuli. At the neural circuit level, changes in neuronal response properties and the degree of synaptic transmission over time can explain these changes.

[0006] Although nematodes and mammals are evolutionarily distant, they share many common molecular mechanisms at the cellular level. It has been reported that molecules known to be involved in mammalian memory are also involved in chemotaxis in nematodes. For example, it has been reported that the mammalian memory factor CREB (CRH-1 in nematodes) is involved in the acquisition and maintenance of memory through associative learning in nematodes (Non-Patent Document 4: EMBO Rep., 12, 855-862 (2011)), that serotonin, a neurotransmitter involved in mammalian memory, controls chemotaxis in nematodes (Non-Patent Document 5: PNAS 99, 19 pp. 12449-12454 (2002)), and that the mammalian memory factor calcineurin (RCAN-1 in nematodes) is involved in thermotaxis behavior in nematodes (Non-Patent Document 6: J. Mol. Biol., 427, 3457-3468 (2015)).

[0007] Furthermore, it has been reported that components that have been shown to be effective on the nervous system in nematodes also have effects on the brain and nervous system in mammals. For example, it has been reported that orally ingested alpha-lipoic acid improves age-related decline in associative learning behavior in nematodes and mammals (Neurobiol. Aging., 26, 899-905 (2005); J. Med. Food., 15, 713-7 (2012)), and that orally ingested curcumin improves age-related cognitive impairment in nematodes and mammals (Neurobiol. Aging., 39, 69-81 (2016); Geroscience., 40, 73-95 (2018)).

[0008] For these reasons, chemotaxis in C. elegans is considered to be a suitable model system for analyzing the molecular mechanisms of memory. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Seiichi Hiratsuka, Hiroyuki Honma, Yoichi Saitoh, Yuki Yasuda, Hidehiko Yokogoshi.: Effects of dietary sialic acid in n-3 fatty acid-deficient dams during pregnancy and lactation on the learning abilities of their pups after weaning. J Nutr Sci Vit. 59(2):136–43 (2013).

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[0010] The object of the present invention is to provide a novel, unprecedented composition for maintaining and / or improving memory and learning ability, and food, beverage, pharmaceutical and feed containing said composition for maintaining and / or improving memory and learning ability. [Means for solving the problem]

[0011] As a result of extensive research into materials that maintain and improve memory, the present inventors discovered sialylglycopeptide (SGP) that has a memory and learning enhancing ability superior to that of free sialic acid and GMP with the same amount of sialic acid, thereby completing the present invention. In other words, this invention is based on the discovery of a new, hitherto unreported use of SGP. Specifically, the present invention has the following features: [Aspect 1] A composition for maintaining and / or improving memory and learning ability, 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 maintaining and / or improving memory and learning ability, wherein the molar ratio of the sialic acid to the galacto-N-biose is 1:1 to 2:1. [Aspect 2] The composition for maintaining and / or improving memory and learning ability according to Aspect 1, wherein the combined amount of the sialic acid and the galacto-N-biose is 20% by weight or more and 90% by weight or less. [Aspect 3] 3. The composition for maintaining and / or improving memory and learning ability according to Aspect 1 or 2, wherein the peptide chain of the sialylglycopeptide has 1 to 13 amino acid residues. [Aspect 4] A composition for maintaining and / or improving memory and learning ability 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 maintaining and / or improving memory and learning ability according to any one of Aspects 1 to 4, wherein the sialylglycopeptide is derived from milk. [Aspect 6] A food or drink for maintaining and / or improving memory and learning ability, comprising the composition for maintaining and / or improving memory and learning ability described in any one of aspects 1 to 5. [Aspect 7] A pharmaceutical product for maintaining and / or improving memory and learning ability, comprising the composition for maintaining and / or improving memory and learning ability according to any one of aspects 1 to 5. [Aspect 8] A feed for maintaining and / or improving memory and learning ability, comprising the composition for maintaining and / or improving memory and learning ability according to any one of aspects 1 to 5. [Effects of the Invention]

[0012] The present invention provides a composition for maintaining and / or improving memory and learning ability, which contains sialylglycopeptide (SGP), and also provides foods, beverages, pharmaceuticals, and feeds for maintaining and / or improving memory and learning ability, which contain the SGP-containing composition as an active ingredient. [Brief explanation of the drawings]

[0013] [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] An outline of the chemotaxis test using C. elegans is shown. [Figure 5] 1 shows the memory index of young C. elegans that had been administered a sialic acid preparation or the SGP of the present invention. Tukey-Kramer's test: different letters indicate significant differences at the 5% level. [Figure 6] 1 shows the memory index of aged C. elegans that had been administered a sialic acid preparation or the SGP of the present invention. Tukey-Kramer's test: different letters indicate significant differences at the 5% level. [Figure 7] 1 shows the memory index of young C. elegans that ingested purified sialylglycopeptide purified from SGP of the present invention or SGP of the present invention. Tukey-Kramer's test: different letters indicate significant differences at the 5% level. [Figure 8] 1 shows the memory index of aged C. elegans ingesting GMP or SGP of the present invention. Tukey-Kramer's test. Different letters indicate significant differences at the 5% level. DETAILED DESCRIPTION OF THE INVENTION

[0014] The composition for maintaining and / or improving memory and learning ability, which contains the sialylglycopeptide (SGP) of the present invention as an active ingredient, and the food, beverage, pharmaceutical, and feed for maintaining and / or improving memory and learning ability, which contain the composition as an active ingredient, are described in detail below.

[0015] (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, preferably, the combined amount of sialic acid and galacto-N-biose constituting the sugar chain of the SGP of the present invention is 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 35% by weight or more and 50% by weight or less. The SGP of the present invention preferably has a peptide chain consisting of approximately 1 to 13 amino acid residues. Furthermore, the SGP of the present invention may also contain acetylated sialic acid.

[0016] One embodiment of a composition for maintaining and / or improving memory and learning ability containing the SGP of the present invention is a composition containing a sialylglycopeptide having a threonine and / or serine residue and a sugar chain attached to the hydroxyl group, wherein the structure of 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).

[0017] The SGP of the present invention can typically be obtained by hydrolyzing a milk protein such as whey protein with an enzyme, etc., followed by fractionation. 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 enzymatic hydrolysis and fractionation of extracts from microorganisms, plants, or animal organs, or by chemical synthesis or recombinant DNA technology.

[0018] (SGP manufacturing method) One embodiment of a method for producing the glycopeptide composition SGP of the present invention will be described below. The glycopeptide composition of the present invention can be obtained, for example, by hydrolyzing whey protein and fractionating it. For example, dairy ingredients such as cheese whey, whey protein concentrate, and whey protein isolate can be used as raw materials. The glycopeptide composition SGP of the present invention can be produced by treating these dairy materials with endoproteases or exoproteases and then subjecting the treated material obtained in the above steps to ultrafiltration with a molecular weight cutoff of 500 or more and 3,000 or less. The type of protease used in glycopeptide production is not particularly limited as long as it can hydrolyze peptide bonds to yield glycopeptides with the molecular weight and sugar chains described above (sialylglycopeptides: SGPs). One or more types of endoproteases or exoproteases can be used. Enzymes that can be used in food and pharmaceutical production are preferred, and examples include actinase E (Kaken Pharma), actinase AS (Kaken Pharma), nucleisin (HBI), orientase AY (HBI), Sumiteam FP (Shin-Nihon Chemical Industry), Sumiteam SPP-G (Shin-Nihon Chemical Industry), protease A (Amano Enzyme), and peptidase R (Amano Enzyme), which can be used alone or in combination.

[0019] As described above, the sialylglycopeptide of the present invention is typically obtained by hydrolyzing and fractionating whey. Therefore, depending on the degree of purification, it may be a purified single sialylglycopeptide or a mixture of multiple sialylglycopeptides.

[0020] As used herein, the phrase "maintaining and / or improving memory and learning ability" means that memory and learning ability is maintained and / or improved compared to when the composition containing the SGP of the present invention is not ingested.

[0021] In this invention, a chemotaxis test using C. elegans was conducted as a method for testing the maintenance and / or improvement of memory and learning abilities. Generally, when verifying the physiological functions of dietary components or drugs, higher animals such as mice, rats, dogs, and monkeys are used, but this requires a great deal of effort, time, and expense, and requires careful consideration of ethical issues. As an alternative to animal testing, tests using cultured cells isolated from animal tissues are also being conducted.

[0022] C. elegans is a non-parasitic, multicellular organism that lives in soil. It is widely used as a model organism in various research fields because it is easy to handle as an experimental animal, the lineages of the approximately 1,000 somatic cells that make up its body have been fully elucidated, and it possesses a nervous system. In particular, it has made significant contributions to research in various fields, including cell death, neuroscience, development, and aging. Furthermore, in recent years, C. elegans has been used as a model for various neurodegenerative diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, and Huntington's disease, to explore pharmaceuticals and dietary components that may be effective in preventing or ameliorating these diseases.

[0023] Although C. elegans has a simple neural circuit, it is known to associate the presence or absence of food with information about the salt concentration, temperature, and chemicals in the environment at that time, and thus can be used as a model system to evaluate cognitive functions such as associative learning and memory (Nature, 376, 344-348 (1995)).

[0024] (Food, beverages, medicines, and feed containing SGP) The SGP or a composition containing SGP obtained by the above-mentioned production method of the present invention can be used as a raw material or ingredient for food and beverages, and the food and beverages 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 an effective amount of SGP produced in this manner can be provided as a food and drink for maintaining and / or improving memory and learning ability.

[0025] The SGP or a composition containing SGP obtained by the above-mentioned production method of the present invention can be used as a raw material for pharmaceuticals as is, and can be produced in the form of tablets, capsules, powders, syrups, etc. by conventional methods, except for the addition of SGP. Therefore, when formulating pharmaceuticals containing the SGP of the present invention as an active ingredient, they can be formulated by appropriately mixing with approved excipients, stabilizers, flavoring agents, etc., or by drying the SGP as is and using it as a powder or sachets. They can also be formulated by mixing with excipients, binders, disintegrants, lubricants, flavoring agents, suspending agents, coating agents, and any other optional agents. Possible dosage forms include tablets, capsules, granules, powders, dusts, and syrups. A pharmaceutical containing an effective amount of SGP is provided as a pharmaceutical for maintaining and / or improving memory and learning ability.

[0026] The SGP or composition containing SGP obtained by the above-mentioned production method of the present invention can be used as a raw material for feed as is, and may be produced according to standard methods for feed, except for the addition of SGP. Therefore, the effective amount of SGP of the present invention may be blended into any feed, similar to the above-mentioned foods and beverages, or may be added to the raw materials during the production process of the feed. The feed containing an effective amount of SGP thus produced can be used as a feed for maintaining and / or improving memory and learning ability.

[0027] (SGP intake) When the SGP of the present invention is incorporated into ingredients such as food, beverages, medicines, and feed, or processed products of such ingredients, to produce a composition having the effect of maintaining and / or improving memory and learning ability, the proportion of incorporation is not particularly limited and may be adjusted appropriately depending on the ease of production and the preferred daily dosage. The daily dosage of the SGP of the present invention is determined individually taking into consideration the symptoms, age, etc. of the subject, but for adult humans, it is sufficient to take 0.1 g or more of the glycopeptide composition per day, preferably 1 g or more, and even more preferably 10 g or more.

[0028] 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]

[0029] Examples of the present invention will be described in detail below, but the present invention is not limited to these examples.

[0030] 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, Novozyme, Cat. #P4860, ≥2.4 U / g) and exoprotease (Flavorzyme, Novozyme) 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).

[0031] 2. Measurement of sialic acid content of SGP The SGP (1) prepared in Example 1 was diluted to 250 μg / mL and sialidase (Neuraminidase isoenzyme S from Arthrobacter ureafaciens, #EC-32118-S, Cosmo Bio) was diluted to 0.2 U / mL in 50 mM potassium phosphate buffer (pH 5.0). 50 μL of this solution was reacted at 37°C for 1 hour 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, the 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 filtered 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 free sialic acid standard, and the sialic acid content was calculated from the calibration curve. The results are shown in Table 1.

[0032] 3. Measurement of galacto-N-biose (GNB) content in SGP A 50 mM potassium phosphate buffer (pH 5.0) solution containing 250 μg / mL of SGP (1) prepared in Example 1, 0.2 U / mL of sialidase (Neuraminidase isoenzyme S from Arthrobacter ureafaciens, #EC-32118-S, Cosmo Bio), and 0.125 U / mL of O-glycanase (#DG53 043a, Prozyme) was prepared. A 50 μL aliquot was incubated at 37°C for 24 hours using a thermal cycler (Takara Bio). The reaction solution was diluted 2 to 10 times with ultrapure water, and insoluble material was removed using a 0.45 μm filter (13A, Kurabo). The amount of 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 as the detector. A 100 mM sodium hydroxide solution was isocratically passed through the mobile phase 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, a 100 mM sodium hydroxide solution was isocratically passed through the mobile phase. The mobile phase was always passed 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.

[0033] [Table 1]

[0034] [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.

[0035] [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 concentration of solution B was then increased linearly from 10% to 100% over a 20-minute period. Mass spectrometry was performed using MS (m / z 200-2000, positive mode) and MS / MS. For MS / MS, the five most intense ions were automatically selected as precursor ions, and the m / z values ​​of the resulting product ions were observed using collision energies of 15, 30, and 45. 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, and 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 glycopeptides detected contained one or more serine or threonine residues, and the peptide chain length ranged from one residue to 13 residues. The molecular weight of the sialylglycopeptides ranged from 500 to 3,000.

[0036] [Table 2]

[0037] [Example 4] Purification of sialylglycopeptides contained 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 10 minutes of 100% solution B, the solution was passed through for 35 minutes at 10% solution B. 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.

[0038] 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 x 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 sialylglycopeptides were determined (Figure 3).

[0039] 3. Quantitative analysis of purified sialylglycopeptides A 10 μL aliquot of sialylglycopeptide solution 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 a standard sample of free sialic acid with 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.

[0040] [Test Example 1] Evaluation of materials that maintain and / or improve memory and learning ability in young and aged models - Comparison of the effects of sialic acid (preparation) and SGP The wild-type N2 Bristol strain of C. elegans used in this study was reared according to the "Nematode Lab Manual." Specifically, NGM plates were used for rearing C. elegans, which were cultured with Escherichia coli OP50 strain.

[0041] 1. Test Method (1) Preparation of evaluation samples A sialic acid preparation (N-acetylneuraminic acid, manufactured by Nacalai Tesque) was dissolved in M9 buffer (0.04 M NaHPO, 0.02 M KHPO, 0.009 M NHCl, 0.02 M NaCl) to a concentration of 0.9 mg / ml, and 250 μl of each was plated on a C. elegans rearing plate to prepare a sample plate. The SGP used was that prepared in Example 1. SGP was dissolved in M9 buffer to a sialic acid content of 0.9 mg / ml, and 250 μl of each was plated on a C. elegans rearing plate to prepare a sample plate. As a control, 250 μl of M9 buffer was plated on a C. elegans rearing plate to prepare a control plate.

[0042] (2) Preparation of young and aged nematodes C. elegans eggs were collected by bleaching and placed on C. elegans rearing plates for 3 days to obtain juvenile nematodes. These adult nematodes were then collected in M9 buffer, transferred to sample or control plates, and reared for 4 days to obtain aged nematodes that had been fed the sample.

[0043] (3) Associative learning in young nematodes The method described by Kauffman et al. (PloS Biol, 8(5), e1000372(2010)) and Stein et al. (Neurobiol Learn Mem, 115, 86-94(2014)) was followed as follows. Synchronized cultures of young nematodes were prepared for testing. They were starved in M9 buffer for one hour. The nematodes were then transferred to a C. elegans rearing plate, and the inside of the plate lid was coated with 10% butanone dissolved in ethanol. An associative learning session was then conducted for one hour. After associative learning, some nematodes were subjected to a butanone-based chemotaxis test. The remaining nematodes were transferred to a sample or control plate and allowed to ingest the evaluation sample for up to four hours before the chemotaxis test.

[0044] (4) Associative learning in aging nematodes The method described by Kauffman et al. (PloS Biol, 8, e1000372 (2010)) and Stein et al. (Neurobiol Learn Mem, 115, 86-94 (2014)) was used as follows. Synchronized cultures of aged nematodes were prepared for testing. They were starved in M9 buffer for one hour. The nematodes were then transferred to C. elegans rearing plates, and 10% butanone dissolved in ethanol was applied to the inside of the plate lid. Associative learning was then performed for one hour. After associative learning, some nematodes were subjected to a butanone-based chemotaxis test. The remaining nematodes were transferred to C. elegans rearing plates, and a chemotaxis test was performed up to four hours later.

[0045] (5) Chemotaxis test The method described by Bargmann et al. (Cell, 74, 515-527 (1993)) and Stein et al. (Neurobiol Learn Mem, 115, 86-94 (2014)) was followed. 100-500 young or aged nematodes were placed at the start point on a 10-cm NGM plate. Equidistant from the start point were a butanone point (1 μl each of 10% butanone and sodium azide dissolved in ethanol) and a control ethanol point (1 μl each of ethanol and sodium azide). After 1 h, the number of nematodes in the butanone, ethanol, and start points was counted. Learning and memory were assessed by measuring the percentage of C. elegans that stayed at the butanone point (memory index) (Figure 4). Tukey's multiple-choice test was used to assess whether there were significant differences between the three groups (p<0.05 was considered significant).

[0046] 2. Test Results (1) Test results for young nematodes Figure 5 shows the test results for young nematodes. The memory index for naive on the X-axis indicates the value before learning. The memory index for 0 on the X-axis indicates the value immediately after learning. The memory index for 3 and 4 on the X-axis indicates the value 3 and 4 hours after learning. An increase in memory index due to learning was confirmed for all three test groups. The memory index for the control group decreased 3 and 4 hours after learning. In young nematodes that had been ingested with sialic acid preparations after learning, the memory index decreased 3 and 4 hours after learning, similar to the control group. In young nematodes that had been ingested with SGP after learning, the memory index remained higher than in the control group. Furthermore, young nematodes that had been ingested with SGP had a higher memory index than young nematodes that had been ingested with sialic acid preparations, and the memory index was significantly higher, especially 3 hours after learning.

[0047] (2) Test results on aging nematodes Figure 6 shows the test results for aged nematodes. The memory index for naive on the X-axis indicates the value before learning. The memory index for 0 on the X-axis indicates the value immediately after learning. The memory index for 2 and 4 on the X-axis indicates the value 2 and 4 hours after learning. An increase in memory index due to learning was confirmed for all three test groups. The memory index for the control decreased 2 and 4 hours after learning. In aged nematodes that had been fed a sialic acid preparation for 4 days, the memory index decreased 2 and 4 hours after learning, similar to the control. In aged nematodes that had been fed SGP for 4 days, the memory index remained higher than in the control. Furthermore, aged nematodes that had been fed SGP for 4 days had a higher memory index than aged nematodes that had been fed a sialic acid preparation, and the memory index was significantly higher, especially 4 hours after learning.

[0048] [Test Example 2] Evaluation of materials that maintain and / or improve memory and learning ability - Comparison of the effects of purified sialylglycopeptide and SGP 1. Test Method (1) Preparation of evaluation samples The SGP prepared in Example 1 and the purified sialylglycopeptide prepared in Example 5 were diluted with ultrapure water to the same sialylglycopeptide content, and 250 μl of each was inoculated onto a C. elegans rearing plate coated with the E. coli OP50 strain from Test Example 1 to prepare a sample plate. As a control, 250 μl of ultrapure water was inoculated onto a C. elegans rearing plate to prepare a control plate.

[0049] (2) Preparation of young nematodes The C. elegans eggs collected by bleaching were sown on a C. elegans rearing plate and left to stand for 3 days to obtain juvenile nematodes. Other conditions were the same as in Test Example 1.

[0050] 2. Test Results Figure 7 shows the results of the chemotaxis test when SGP or purified sialylglycopeptide was administered 3 and 4 hours after learning. In the control group, the chemotaxis index (CI) decreased over time after learning, confirming a decline in memory. In both the SGP and purified sialylglycopeptide groups, the CI was higher than that of the control group 3 and 4 hours after learning, confirming that memory was maintained. While the purified sialylglycopeptide was effective, the memory-maintaining effect disappeared when SGP was treated with sialidase (data not shown), confirming that the active ingredient in SGP is sialylglycopeptide.

[0051] [Test Example 3] Evaluation of materials that maintain and / or improve memory and learning ability - Comparison of the effects of GMP and SGP 1. Test Method (1) Preparation and intake method of evaluation samples 2.5 g of commercially available GMP material (CGMP-10, Arla Foods) and 1 g of SGP prepared in Example 1 were dissolved in 1 ml of M9 buffer, and 250 μl of each solution was seeded onto a C. elegans rearing plate coated with the E. coli OP50 strain from Test Example 1. As a control, 250 μl of M9 buffer was seeded onto a C. elegans rearing plate to serve as a control plate. The nematodes that had stopped laying eggs were allowed to feed for 5 days, with plates replaced every two days to prevent food shortage.

[0052] (2) Associative learning in aging nematodes C. elegans associative learning and chemotaxis assays were performed with reference to the methods described by Kauffman et al. (PloS Biol, 8, e1000372 (2010)) and Stein et al. (Neurobiol Learn Mem, 115, 86-94 (2014)). C. elegans were starved in M9 buffer for 1 hour, after which butanone was smeared on the inside of the plate lid. C. elegans were then transferred to an OP50 NGM plate and allowed to ingest OP50 for 1 hour to perform associative learning.

[0053] (3) Chemotaxis test The conditions were the same as in Test Example 1.

[0054] 2. Test Results Figure 8 shows the results of a chemotaxis test in which subjects ingested SGP or commercially available GMP material for 5 days and then ingested E. coli OP50 strain in the absence of butanone odor 1 and 2 hours after learning. In the control group, CI decreased over time after learning, confirming a decline in memory. GMP and SGP were tested with the same amount of glycan content. Both GMP and SGP had higher CI than the control group 2 hours after learning, indicating that memory was maintained, but the CI of SGP was significantly higher than that of GMP.

[0055] [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 glycopeptide composition powder obtained in Example 1 and mixed. The mixture was packed into a stick-shaped bag to produce a supplement for maintaining and / or improving memory and learning ability according to the present invention.

[0056] 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 a beverage for maintaining and / or improving memory and learning ability of the present invention.

[0057] [Table 3]

[0058] [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 maintaining and / or improving memory and learning ability of the present invention.

[0059] [Table 4] [Industrial Applicability]

[0060] According to the present invention, it is now possible to provide a composition for maintaining and / or improving memory and learning ability, which contains sialylglycopeptide as an active ingredient, as well as food, beverages, medicines, and feed for maintaining and / or improving memory and learning ability, which contain said composition as an active ingredient.

Claims

1. A composition for maintaining and / or improving memory and learning ability, 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 maintaining and / or improving memory and learning ability, wherein the molar ratio of said sialic acid to said galacto-N-biose is 1:1 to 2:

1.

2. 2. The composition for maintaining and / or improving memory and learning ability 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 maintaining and / or improving memory and learning ability 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 maintaining and / or improving memory and learning ability 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 maintaining and / or improving memory and learning ability according to claim 1, wherein the sialylglycopeptide is derived from milk.

6. A food or drink for maintaining and / or improving memory and learning ability, comprising the composition for maintaining and / or improving memory and learning ability according to any one of claims 1 to 5.

7. A pharmaceutical for maintaining and / or improving memory and learning ability, comprising the composition for maintaining and / or improving memory and learning ability according to any one of claims 1 to 5.

8. A feed for maintaining and / or improving memory and learning ability, comprising the composition for maintaining and / or improving memory and learning ability according to any one of claims 1 to 5.

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