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

Abstract

The present invention addresses the problem of providing: an unconventional and novel composition for maintaining and / or improving memory / learning ability; and a food or beverage, a medicine, and a feed, each of which is for maintaining and / or improving memory / learning ability and contains the composition.　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 for maintaining and / or improving memory / learning ability and contains the composition as an active ingredient.

Description

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

[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.

[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 in human 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 the infant's brain and nervous system. For example, it has been reported that pups fed milk from dams that had been given 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). Furthermore, it has 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 the ingestion of free sialic acid or GMP containing a sialic acid moiety will improve the function of the brain and nervous system.

[0004] Most sialic acids are bound to the non-reducing end of glycans, with a wide variety of glycan types and binding modes. 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 also diverse. Therefore, the strength of the physiological function of sialic acid may vary depending on the form of sialic acid present in a composition. In fact, there is a report 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, not only the amount of sialic acid in a composition but also its chemical binding mode is likely important for improving brain and nervous system function through the ingestion of free sialic acid or GMP containing a sialic acid moiety. Furthermore, if the same amount of sialic acid is to be ingested, it is considered more efficient to ingest a sialic acid moiety-containing compound in a form 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 brain and nervous system function-improving effect superior to that of 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) uses a simple neural circuit consisting of 302 neurons to sense various external stimuli and exhibit diverse response behaviors. C. elegans are capable of associative learning, which involves memorizing associations between food and chemicals. They are known to be an excellent model system for analyzing memory and cognitive functions that reflect behavior after conditioning with multiple stimuli and the plasticity of neural functions. At the neural circuit level, these changes can be explained by the response properties of neurons and the degree of synaptic transmission over time.

[0006] Although nematodes and mammals are evolutionarily distant, they share a large degree of commonality in molecular mechanisms at the cellular level. It has been reported that molecules known to be involved in memory in mammals 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 brain neurotransmitter involved in memory in mammals, controls chemotaxis in nematodes (Non-Patent Document 5: PNAS 99, 19 p12449-12454 (2002)), and that the mammalian memory factor calcineurin (RCAN-1 in nematodes) is involved in the thermotaxis behavior of 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), Non-Patent Document 8: 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), Non-Patent Document 10: 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.

[0009] 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 Vitaminol. 59(2):136-43 (2013).Bing Wang, Bing Yu, Muhsin Karim, Honghua Hu, Yun Sun, Paul McGreevy, Peter Petocz, Suzanne Held, Jennie Brand-Miller.: Dietary sialic acid supplementation improves learning and memory in piglets. Am J Clin Nutr Feb;85(2):561-9 (2007)Stephen A. Fleming, Maciej Chichlowski, Brian M. Berg, Sharon M. Donovan and Ryan N. Dilger.: Dietary Sialyllactose Does Not Influence Measures of Recognition Memory or Diurnal Activity in the Young Pig. Nutrients Volume 10 Issue 4 (2018)Nishida, Y., Sugi, T., Nonomura, M. et al.: Identification of the AFD neuron as the site of action of the CREB protein in Caenorhabditis elegans thermotaxis. EMBO Rep., 12, 855-862 (2011)William M. Nuttley, Karen P.Atkinson-Leadbeater, and Derek van der Kooy., Serotonin mediates food-odor associative learning in the nematode Caenorhabditis elegans. PNAS vol. 99 no. 19 12449-12454, September 17, 2002Li, W., Bell, H. W., Ahnn, J., Lee, S. K.: Regulator of Calcineurin (RCAN-1) Regulates Thermotaxis Behavior in Caenorhabditis elegans. J. Mol. Biol., 427, 3457-3468 (2015)Murakami, S., Murakami, H.: The effects of aging and oxidative stress on learning behavior in C. elegans. Neurobiol. Aging., 26, 899-905 (2005)Cui, Y., Shu, Y., Zhu, Y., Shi, Y., Le, G.: High-fat diets impair spatial learning of mice in the Y-maze paradigm: ameliorative potential of α-lipoic acid. J. Med. Food. 15, 713-7 (2012)Miyasaka, T., Xie, C., Yoshimura, S., et al. Curcumin improves tau-induced neuronal dysfunction of nematodes. Neurobiol. Aging., 39, 69-81 (2016)Sarker, M. R., Franks, S. F. Efficacy of curcumin for age-associated cognitive decline: a narrative review of preclinical and clinical studies. Geroscience.40, 73-95 (2018).

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

[0011] As a result of extensive research into materials for maintaining and improving memory, the present inventors discovered a sialylglycopeptide (SGP) that has a memory and learning enhancement ability superior to that of free sialic acid and GMP with the same sialic acid content, leading to the completion of the present invention. In other words, this invention is based on the discovery of a new, previously unreported use of SGP. Specifically, the present invention has the following configurations: [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 a threonine and / or serine residue, wherein the molecular weight of the sialylglycopeptide is 500 to 3,000, the sugar chain is composed of sialic acid and galacto-N-biose, the galacto-N-biose is bound to a hydroxyl group of the threonine or serine residue of the peptide, and the sialic acid is bound to the galacto-N-biose, and 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, which is for oral administration. [Aspect 3] The composition for maintaining and / or improving memory and learning ability according to Aspect 1 or 2, 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 4] The composition for maintaining and / or improving memory and learning ability according to any of Aspects 1 to 3, wherein the peptide chain of the sialylglycopeptide has 1 to 13 amino acid residues. [Aspect 5] The composition for maintaining and / or improving memory and learning ability according to any of Aspects 1 to 4, wherein the structure of the sugar chain 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 [Embodiment 6] The composition for maintaining and / or improving memory and learning ability according to any one of embodiments 1 to 5, wherein the sialylglycopeptide is derived from milk.[Aspect 7] The composition for maintaining and / or improving memory and learning ability according to any one of Aspects 1 to 6, wherein the memory and learning ability maintenance and / or improvement is that of a healthy subject. [Aspect 8] The composition for maintaining and / or improving memory and learning ability according to any one of Aspects 1 to 7, wherein the memory and learning ability maintenance and / or improvement is that of a subject whose memory function has declined due to aging, psychological stress, insomnia, or nutritional deficiency. [Aspect 9] The composition for maintaining and / or improving memory and learning ability according to any one of Aspects 1 to 8, for non-therapeutic use. [Aspect 10] The composition for maintaining and / or improving memory and learning ability according to any one of Aspects 1 to 9, wherein the memory and learning ability maintenance and / or improvement is not that of an Alzheimer's disease patient. [Aspect 11] The composition for maintaining and / or improving memory and learning ability according to any one of Aspects 1 to 6, for therapeutic use. [Aspect 12] 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 Aspects 1 to 10. [Aspect 13] 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 Aspects 1 to 6 and 11. [Aspect 14] 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 10. Aspect 15: A method for maintaining and / or improving the memory and learning ability of a subject, comprising the step of administering to the subject a composition comprising a sialylglycopeptide in which a sugar chain is bound to a peptide having a threonine and / or serine residue, wherein 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 the sialic acid is bound to the galacto-N-biose; and the molar ratio of the sialic acid to the galacto-N-biose is 1:1 to 2:1. Aspect 16: The method for maintaining and / or improving the memory and learning ability of a subject according to Aspect 15, wherein the subject is a healthy individual.[Aspect 17] The method for maintaining and / or improving memory and learning ability according to Aspect 15 or 16, wherein the subject has a decline in memory function due to aging, psychological stress, insomnia, or nutritional deficiency. [Aspect 18] The method for maintaining and / or improving memory and learning ability according to any one of Aspects 15 to 17, wherein the subject is not an Alzheimer's disease patient. [Aspect 19] The method for maintaining and / or improving memory and learning ability according to any one of Aspects 15 to 18, wherein the administration is oral administration.

[0012] The present invention provides a composition for maintaining and / or improving memory and learning ability, which contains a sialylglycopeptide (SGP), and the composition. It also provides a food, drink, pharmaceutical, and feed for maintaining and / or improving memory and learning ability, which contain the composition containing the SGP as an active ingredient.

[0013] 1 shows the results of size exclusion chromatography (SEC) analysis of the SGP of the present invention (210 nm). 2 shows the results of LC / MS analysis of purified sialylglycopeptide purified from the SGP of the present invention ((A) LC / MS chromatogram (TIC, m / z 400-2000) of the sialylglycopeptide solution after desalting, (B) MS spectrum at 15.94 min). 3 shows the structure of purified sialylglycopeptide purified from the SGP of the present invention. 4 shows an outline of a chemotaxis test using C. elegans. 5 shows the memory index of young C. elegans that ingested a sialic acid preparation or the SGP of the present invention. 6 shows the results of Tukey-Kramer's test; different letters indicate significant differences at the 5% level. 7 shows the memory index of aged C. elegans that ingested a sialic acid preparation or the SGP of the present invention. 8 shows the results of Tukey-Kramer's test; different letters indicate significant differences at the 5% level. 1 shows the memory index of young C. elegans that ingested purified sialylglycopeptide purified from the SGP of the present invention or the SGP of the present invention. Tukey-Kramer's test: different letters indicate significant differences at the 5% level.

[0039] FIG. 1 shows the memory index of aged C. elegans that ingested GMP or the SGP of the present invention. Tukey-Kramer's test: different letters indicate significant differences at the 5% level.

[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 a threonine and / or serine residue; (b) the molecular weight is 500 or more and 3,000 or less; (c) the sugar chain is composed of sialic acid and galacto-N-biose; (d) the galacto-N-biose of the sugar chain is bound to the hydroxyl group of the threonine or serine residue of the peptide, and the sialic acid is bound to the galacto-N-biose; and (e) the molar ratio of sialic acid to galacto-N-biose is in the range of 1:1 to 2:1. Preferably, the SGP of the present invention has a total content of sialic acid and galacto-N-biose constituting the sugar chain of 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. Furthermore, the SGP of the present invention preferably has a peptide chain 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 bound to the hydroxyl group, wherein the sugar chain structure 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 peptides contain one or more of the above sugar chains, peptides containing two or more of them are also included, and examples thereof include those containing two or more of the same type of sugar chain or those containing two or more types. For example, the peptide may be a peptide containing two of (1), or a peptide containing (1) and further containing one or more of 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 or the like and fractionating it. 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 or from animal milk such as goat or sheep. The SGP of the present invention can also be prepared by hydrolyzing an extract of a microorganism, plant, or animal organ with an enzyme or the like and fractionating it. Furthermore, the SGP may be chemically synthesized or prepared using a genetic recombinant.

[0018] (Method for Producing SGP) 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 and fractionating whey protein. For example, dairy materials 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 an endoprotease or exoprotease and then subjecting the treated material obtained in the above step to ultrafiltration with a molecular weight cutoff of 500 to 3,000. The type of protease used for glycopeptide production is not particularly limited, as long as it can hydrolyze peptide bonds to produce glycopeptides having the molecular weight and sugar chains described above (sialylglycopeptide: SGP). One or more types of endoproteases or exoproteases can be used. Preferably, enzymes that can be used in the production of foods and pharmaceuticals are used, and examples that can be used alone or in combination include actinase E (Kaken Pharma), actinase AS (Kaken Pharma), nucleisin (HBI), orientase AY (HBI), Sumiteam FP (Shin Nippon Chemical Industry), Sumiteam SPP-G (Shin Nippon Chemical Industry), protease A (Amano Enzyme), and peptidase R (Amano Enzyme).

[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 a composition containing the SGP of the present invention is not ingested.

[0021] In the present invention, a chemotaxis test using C. elegans was conducted as a method for testing the maintenance and / or improvement of memory and learning ability. 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 aspects. As an alternative to animal testing, tests using cultured cells isolated from animal tissues have also been 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 various research fields, including cell death, neuroscience, development, and aging. Furthermore, in recent years, C. elegans models of various neurodegenerative diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, and Huntington's disease, have been used to explore pharmaceuticals and dietary components effective in preventing and ameliorating these neurodegenerative diseases.

[0023] Although C. elegans has a simple neural circuit, it is known to associate the presence or absence of food with information on 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] (Foods, beverages, pharmaceuticals, and feeds containing SGP) The SGP or a composition containing SGP obtained by the above-described production method of the present invention can be used directly as a material or ingredient for foods and beverages, which can be produced according to standard methods for each food and beverage, except for the addition of the SGP-containing composition. Therefore, an effective amount of SGP of the present invention can be incorporated into any food and beverage, or added to the raw materials during the production process of the food and 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, and margarine; beverages such as milk drinks, fruit juice drinks, and soft drinks; egg products such as jelly, candy, pudding, and mayonnaise; confectioneries and breads such as butter cake; various types of powdered milk; infant foods; and nutritional compositions. The foods and beverages thus produced, containing an effective amount of SGP, can be used as foods and beverages for maintaining and / or improving memory and learning ability.

[0025] The SGP or a composition containing SGP obtained by the above-described production method of the present invention can be used directly as a raw material for pharmaceuticals, and can be produced by standard methods such as tablets, capsules, powders, and syrups, except for the addition of SGP. Therefore, when formulating pharmaceuticals containing the SGP of the present invention as an active ingredient, the SGP can be appropriately mixed with approved excipients, stabilizers, flavoring agents, etc., or the SGP can be dried and used as a powder or syrup. It can also be formulated by mixing excipients, binders, disintegrants, lubricants, flavoring agents, suspending agents, coating agents, and other optional agents. Possible dosage forms include tablets, capsules, granules, powders, dusting agents, and syrups. Pharmaceuticals containing an effective amount of SGP can be used as pharmaceuticals for maintaining and / or improving memory and learning ability.

[0026] The SGP or composition containing SGP obtained by the above-described production method of the present invention can be used as a raw material for feed, and can be produced by standard methods for feed production except for the addition of SGP. Therefore, an effective amount of SGP of the present invention can be incorporated into any feed, as in the case of the above-described foods and beverages, or can be added to raw materials during the feed production process. The feed containing an effective amount of SGP produced in this manner can be provided as a feed for maintaining and / or improving memory and learning ability.

[0027] (Intake of SGP) When the SGP of the present invention is incorporated into materials such as foods, beverages, pharmaceuticals, and feeds, or processed products of these materials 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 appropriately adjusted depending on the ease of production and the preferred daily dose. The daily dose of the SGP of the present invention is determined individually taking into account the symptoms, age, etc. of the subject, but for adult humans, it is sufficient to ingest 0.1 g or more of the glycopeptide composition per day, preferably 1 g or more, and more preferably 10 g or more.

[0028] There are no particular limitations on the subjects to which the composition containing the SGP of the present invention can be administered, and it can be humans or animals.

[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: Method for Preparing SGP of the Present Invention 1. Method for Producing 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 reacted at 55°C for 8 hours. After the reaction, the enzymes were inactivated by holding at 85°C for 30 minutes. Diafiltration (DF) was performed on this 30 kg WPI hydrolysate using an ultrafiltration membrane with a molecular weight cutoff of 1,000. Water was added to the concentrate, and the retentate fraction was obtained after 10-fold DF. The retentate fraction was dried to give 120.0 g of SGP (1).

[0031] 2. Measurement of Sialic Acid Content of SGP 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), and 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 matter 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 (Tomy Seiko) at 15,000 × g for 10 minutes. The resulting supernatant was filtered through a 0.45 μm filter (13A, Kurabo). The amount of sialic acid 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. A 100 mM sodium hydroxide solution containing 60 mM sodium acetate was isocratically passed through the mobile phase for 7 minutes after sample introduction. The sodium acetate concentration was linearly increased to 150 mM between 7 and 10 minutes and maintained at that concentration until 20 minutes. The column was then equilibrated by isocratically passing a 100 mM sodium hydroxide solution containing 60 mM sodium acetate for 5 minutes. The mobile phase was always passed at 1 mL / min. N-acetylneuraminic acid (#A2388, Sigma-Aldrich) was used as a free sialic acid standard, and the amount of sialic acid was calculated from the prepared calibration curve. The results are shown in Table 1.

[0032] 3. Measurement of Galacto-N-biose (GNB) Content in SGP SGP (1) prepared in Example 1 was diluted to 250 μg / mL, sialidase (Neuraminidase isoenzyme S from Arthrobacter ureafaciens, #EC-32118-S, Cosmo Bio) was diluted to 0.2 U / mL, and O-glycanase (#DG53 043a, Prozyme) was diluted to 0.125 U / mL in 50 mM potassium phosphate buffer (pH 5.0). A 50 μL aliquot was reacted 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 matter was removed using a 0.45 μm filter (13A, Kurabo). A DIONEX ICS-5000DP system (Thermo Fisher Scientific) equipped with a CarboPac PA1 column was used to measure the GNB content. 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]

[0034] Example 2: Sialidase treatment of SGP SGP (1) prepared in Example 1 was adjusted to 20 mg / mL in 100 mM acetate buffer (pH 5.0) containing 2 mM calcium chloride. Sialidase (derived from Clostridium perfringens: Sigma, N2876-25UN) was added to the solution at 10 U / mL, and the mixture was allowed to react at 37°C for 20 hours. A blank sample without enzyme addition was prepared by reacting the mixture under the same conditions without adding any enzyme. After the reaction, the mixture was boiled for 5 minutes to terminate the reaction. The resulting reaction solution was subjected to size exclusion chromatography (SEC). For SEC analysis, an L-2000 (Hitachi) system equipped with two TSKgel G3000PW (Tosoh) columns was used to detect UV absorption at 214 nm. The mobile phase was a 40% acetonitrile solution 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 enzymatic reaction. The main peak of SGP was observed between 50 and 60 minutes, but this was shifted overall to lower molecular weights by sialidase treatment. This demonstrated that the majority 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 installed as the separation column. A 2% acetonitrile solution containing 0.1% formic acid (Solution A) and a 90% acetonitrile solution containing 0.1% formic acid (Solution B) were used as the mobile phase. 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 passed at 200 μL / min. After sample introduction, the proportion of solution B was 0% for 5 minutes, after which the proportion of solution B was linearly increased from 0% to 10% over 25 minutes. The proportion of solution B was then linearly increased from 10% to 100% over 20 minutes. 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 after destruction at 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 used was a database consisting only of whey protein, and the glycan database used included 10 types of glycans consisting of one molecule of N-acetylhexosamine (HexNAc), 0 to 1 molecule of hexose (Hex), 0 to 2 molecules of N-acetylneuraminic acid (Neu5Ac), and 0 to 2 O-acetyl groups (O-Ac). Analysis using Byonic revealed that the glycans were classified as (HexNAc), (HexNAc)(Hex), or (HexNAc)(Hex)(Neu5Ac). ２The glycans assigned as "(HexNAc)(Hex)(Neu5Ac)" were assigned to GalNAc, Galβ1-3GalNAc, or Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAc, respectively, according to previously reported O-linked glycan structures. Furthermore, the glycans assigned as "(HexNAc)(Hex)(Neu5Ac)" were manually confirmed by MS / MS spectrum and assigned to Neu5Acα2-3Galβ1-3GalNAc or Galβ1-3(Neu5Acα2-6)GalNAc. Similarly, the MS / MS spectrum of O-acetyl-modified Neu5Ac (O-Ac-Neu5Ac or O,O'-diAc-Neu5Ac) was manually confirmed and assigned 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 sialic acid-containing glycans. Many of the glycopeptides detected had the glycan structure Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAc, which has two sialic acid molecules bound to it (Glycan types 3, 4, 5, 6, and 7). However, Neu5Acα2-3Galβ1-3GalNAc (Glycan type 1) and Galβ1-3(Neu5Acα2-6)GalNAc (Glycan type 2), which have one sialic acid molecule, were also detected. Furthermore, the O-acetylated 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 sugar chains bound to one peptide chain (Glycan types 6 and 7) were also detected. All of the detected glycopeptides contained one or more serine or threonine residues, and the peptide chain length ranged from one residue for the shortest to 13 residues for the longest.The molecular weight of the sialylglycopeptide was 500 or more and 3,000 or less.

[0036]

[0037] Example 4 Purification of Sialylglycopeptides Contained in SGP 1. Method for Purifying 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 NH2P-90 column (20 x 300 mm, Shodex). The mobile phase consisted of ultrapure water (Solution A) and 300 mM aqueous sodium dihydrogen phosphate (Solution B). The column temperature was 40°C, and the flow rate was 10 mL / min. A UV702 (GL Sciences, Inc.) detector was used, set at 210 nm. After sample introduction, 10% Solution B was passed through for 5 minutes, and then 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 the column for 10 minutes, 10% solution B was passed through the column for 35 minutes. Between 20 and 30 minutes, 6.7 mL of eluate was collected, and fraction A containing the peak of the target sialylglycopeptide was recovered (Figure 1). This 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 solution containing 0.1% formic acid and then equilibrated with 10 mL of ultrapure water. The entire amount of fraction A from 20 runs was loaded onto the GC column and washed with 20 mL of ultrapure water to remove salts. The sialylglycopeptide was recovered with 5 mL of 20% acetonitrile solution containing 0.1% formic acid. The sialylglycopeptide was then eluted with 5 mL of 50% acetonitrile solution containing 0.1% formic acid and mixed with the previously recovered solution. 100 μL of concentrated aqueous ammonia was added and stirred, and the acetonitrile was removed using a centrifugal evaporator (45°C), followed by freeze-drying. The same procedure was repeated three times, and each of the three freeze-dried powders was dissolved in 1 mL of ultrapure water. The resulting mixture was used as a sialylglycopeptide solution.

[0038] 2. Structural Analysis of Purified Sialylglycopeptides To determine the amino acid sequence and glycan structure of the purified sialylglycopeptides, 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 phases used were 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 passed through the column 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 passed through 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 as 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 data-dependently immediately after the MS spectrum, using the top five ions in signal intensity as precursor ions (top 5). The normalized collision energy was set to 15 and 27 (Figure 2). The acquired MS2 spectra were analyzed using Proteome Discoverer (Thermo Fisher Scientific) with a Byonic node, and the amino acid sequences and glycan structures of the sialylglycopeptides were determined (Figure 3).

[0039] 3. Quantitative Method for Purified Sialylglycopeptide: 10 μL 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 amount of liberated sialic acid was then measured. A CarboPac PA1 column (4 × 250 mm, Thermo Fisher Scientific Inc.) was used for the measurement, and an HPAE-PAD (DIONEX ICS-5000DP system, Thermo Fisher Scientific Inc.) equipped with an electrochemical detector (pulsed amperometric mode) was used as the detector. After sample introduction, a 60 mM aqueous sodium acetate solution containing 100 mM sodium hydroxide was passed through the column for 7 minutes, and the sodium acetate concentration was linearly increased from 60 mM to 150 mM from 7 to 10 minutes. A 150 mM aqueous sodium acetate solution 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 a known concentration. 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 sugar chain 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 specification was reared according to the "Nematode Lab Manual." Specifically, an NGM plate coated with Escherichia coli OP50 strain and cultured on it was used as the plate for rearing C. elegans.

[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 Na2HPO4, 0.02 M KH2PO4, 0.009 M NH4Cl, 0.02 M NaCl) to a concentration of 0.9 mg / ml, and 250 μl of the solution was seeded on a C. elegans rearing plate to prepare a sample plate. The SGP prepared in Example 1 was used. SGP was dissolved in M9 buffer to a sialic acid content of 0.9 mg / ml, and 250 μl of the solution was seeded on a C. elegans rearing plate to prepare a sample plate. For the control, 250 μl of M9 buffer was seeded on a C. elegans rearing plate to prepare a control plate.

[0042] (2) Preparation of young and aged nematodes: C. elegans eggs collected by bleaching were plated on C. elegans rearing plates and left to stand for 3 days to obtain young nematodes. These adult nematodes were then recovered in M9 buffer, transferred to a sample plate or control plate, and reared for 4 days to obtain sample-fed aged nematodes.

[0043] (3) Associative learning in young nematodes. This study was performed with reference to the methods described by Kauffman et al. (PloS Biol, 8(5), e1000372(2010)) and Stein et al. (Neurobiol Learn Mem, 115, 86-94(2014)). Synchronized young nematodes were prepared for testing. They were starved in M9 buffer for 1 hour. The nematodes were transferred to a C. elegans rearing plate, and the inside of the plate lid was coated with 10% butanone dissolved in ethanol. Associative learning was then performed for 1 hour. After associative learning, some nematodes were subjected to a chemotaxis test using butanone. The remaining nematodes were transferred to a sample or control plate and allowed to ingest the evaluation sample for up to 4 hours before undergoing a chemotaxis test.

[0044] (4) Associative learning in aging nematodes This study was 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)). Synchronized cultures of aging nematodes were prepared as follows. They were starved in M9 buffer for 1 hour. The nematodes were transferred to C. elegans rearing plates, and the inside of the plate lid was coated with 10% butanone dissolved in ethanol. Associative learning was then performed for 1 hour. After associative learning, some nematodes were subjected to a chemotaxis test using butanone. The remaining nematodes were transferred to C. elegans rearing plates, and a chemotaxis test was performed after a maximum of 4 hours.

[0045] (5) Chemotaxis Test. Chemotaxis tests were performed using the methods described by Bergmann et al. (Cell, 74, 515-527 (1993)) and Stein et al. (Neurobiol Learn Mem, 115, 86-94 (2014)). 100-500 young or aged nematodes were placed at the start point of 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 hour, the number of nematodes at the butanone point, ethanol point, and start point was counted. Learning and memory were assessed by measuring the percentage of C. elegans located at the butanone point, which was defined as the memory index (Figure 4). Furthermore, the presence or absence of significant differences when comparing the three groups was evaluated by Tukey's multiple test (p<0.05 was determined to be significant).

[0046] 2. Test Results (1) Test Results for Young Nematodes Figure 5 shows the test results for young nematodes. The memory index at "naive" on the X-axis indicates the value before learning. The memory index at 0 on the X-axis indicates the value immediately after learning. The memory index at 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 the three test groups. The memory index of the control decreased 3 and 4 hours after learning. In young nematodes that had been ingested with a sialic acid preparation after learning, the memory index decreased 3 and 4 hours after learning, similar to the control. In young nematodes that had been ingested with SGP after learning, the memory index remained high compared to the control. Furthermore, young nematodes that had been ingested with SGP had a higher memory index than young nematodes that had been ingested with a sialic acid preparation, and the memory index was significantly higher, especially 3 hours after learning.

[0047] (2) Test Results for Aged Nematodes Figure 6 shows the test results for aged nematodes. The memory index at "naive" on the X-axis indicates the value before learning. The memory index at "0" on the X-axis indicates the value immediately after learning. The memory index at "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 the three test groups. The memory index of 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 high compared to 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 4 hours after learning was significantly higher.

[0048] Test Example 2 Evaluation of Materials Maintaining and / or Improving Memory and Learning Ability - Comparison of Effects of Purified Sialylglycopeptide and SGP 1. Test Method (1) Preparation of Evaluation Samples 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 Escherichia 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 C. elegans eggs collected by bleaching were sown on a C. elegans rearing plate and left to stand for 3 days to obtain young nematodes. Other conditions were the same as in Test Example 1.

[0050] 2. Test Results The results of the chemotaxis test when SGP or purified sialylglycopeptide was administered 3 and 4 hours after learning are shown in Figure 7. In the control group, the chemotaxis index (CI) decreased over time after learning, confirming a decline in memory. The CI of both the SGP and purified sialylglycopeptide groups 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 Maintaining and / or Improving Memory and Learning Ability - Comparison of the Effects of GMP and SGP 1. Test Method (1) Preparation and Intake Method of Evaluation Sample 2.5 g of a 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 of 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. For nematodes that had stopped laying eggs, the plates were changed every two days to prevent food starvation, and the nematodes were allowed to feed for five days.

[0052] (2) Associative learning in aging nematodes Associative learning and chemotaxis assays in nematodes 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)) as follows. C. elegans were starved in M9 buffer for 1 hour, after which butanone was smeared on the inside of the plate lid, and the nematodes were 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 mice were given SGP or commercially available GMP material for 5 days and then given E. coli OP50 strain in the absence of butanone odor 1 and 2 hours after learning. The control group's CI decreased over time after learning, confirming a decline in memory. GMP and SGP were administered with the same amount of sugar chains. Both GMP and SGP had higher CIs 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 a supplement 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 of the present invention.

[0056] [Example 6] Production of beverage The ingredients were mixed according to the formulation 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]

[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 pharmaceuticals of the present invention for maintaining and / or improving memory and learning ability.

[0059]

[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, pharmaceuticals, and feed for maintaining and / or improving memory and learning ability, which contain the 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, wherein the molecular weight of the sialylglycopeptide is between 500 and 3,000, the sugar chain is composed of sialic acid and galacto-N-biose, the galacto-N-biose is bound to a hydroxyl group of the threonine or serine residue of the peptide, the sialic acid is bound to the galacto-N-biose, and the molar ratio of the sialic acid to the galacto-N-biose is 1:1 to 2:

1.

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. A 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 to 13.

4. 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. A 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 described in 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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