Composition for inhibiting mumps virus infection

Abstract

To provide a novel composition for inhibiting mumps virus infection.SOLUTION: The present invention provides a composition for inhibiting mumps virus infection comprising GMP or a sialyl sugar peptide shown below as an active ingredient. The sialyl sugar peptide has a molecular weight of 500 or more and 3,000 or less, and comprises a sugar chain composed of sialic acid and galacto-N-biose, wherein the galacto-N-biose is bound to a hydroxyl group of a 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 1:1-2:1.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a composition for inhibiting mumps virus infection, and in particular to a composition for inhibiting mumps virus infection, which comprises, as an active ingredient, a sialic acid-binding peptide such as GMP or a sialylglycopeptide. [Background technology]

[0002] The mumps virus belongs to the Paramyxoviridae family and is the causative agent of epidemic parotitis (mumps). Mumps is an infectious disease characterized by swelling of the salivary glands, which develops after an incubation period of 2-3 weeks, but usually subsides within 1-2 weeks. Its main symptoms are swelling of the salivary glands, fever, and pain when swallowing, and complications can include aseptic meningitis, encephalitis, orchitis, oophoritis, sensorineural hearing loss (mumps hearing loss), and pancreatitis, among other serious diseases. The most frequent complication, aseptic meningitis, is particularly important, occurring in 1-10% of patients and often requiring hospitalization. Mumps hearing loss is seen in 0.1-1% of patients, and in the aforementioned survey conducted by the Oto-Rhino-Laryngological Society of Japan during the mumps epidemic of 2015-2016, at least 348 cases of mumps hearing loss were reported, most of which were ineffective for treatment, and 90% of them had severe or severe hearing loss (Non-Patent Document 1). There is no specific treatment for mumps and its complications, and treatment is basically symptomatic.

[0003] The only way to prevent mumps is through vaccination. However, in Japan, since the discontinuation of the MMR vaccine, the vaccination rate for the mumps vaccine, which has become optional, has been low at around 30-40%. As a result, mumps epidemics still occur nationwide every 4-5 years. In neighboring South Korea, the vaccination rate has remained at over 95% since two doses of the MMR vaccine were made mandatory in 2001, but the number of reported cases has been steadily increasing since 2003. Many infected people have received MMR two or more times and develop the disease despite having immunity to mumps, which is secondary vaccine failure, and it has been pointed out that immunity weakens over time (Non-Patent Document 1).

[0004] For these reasons, there is a need for a method for preventing mumps other than vaccination or a specific treatment for mumps. The main routes of infection for the mumps virus are droplet infection caused by coughing or sneezing, or contact infection. After binding to receptors on the cell surface, the mumps virus penetrates the cell and infects it by fusing the cell membrane with the viral membrane. The infected mumps virus multiplies within the cell and is released from the cell, causing further infection. It is expected that mumps virus infection can be suppressed by inhibiting either of these mumps virus infection mechanisms.

[0005] Regarding the suppression of mumps virus infection by food components, it has been reported that the catechins contained in tea exhibit an inhibitory effect on mumps virus infection by suppressing the membrane fusion activity of the virus (Non-Patent Document 2). If other food ingredients that are easily available and that inhibit the adhesion of the mumps virus to cells can be found, they may be expected to suppress mumps virus infection.

[0006] κ-casein glycomacropeptide (hereinafter sometimes simply referred to as GMP) is a glycopeptide produced by the action of rennet or pepsin on κ-casein from cow's milk, and is contained in cheese whey. GMP is composed of Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAc, Galβ1-3(Neu5Acα2-6)GalNAc, Neu5Acα2-3Galβ1-3GalNAc, Galβ1-3GalNAc, GalNac, Neu5Acα2-3Galβ1-3(O-Ac-Neu5Acα2-6)GalNAc, and Neu5Acα2-3Galβ1-3(O-diAc-Neu5Acα2-6)GalNAc bound to a peptide chain consisting of 64 amino acids on the C-terminal side of κ-casein (Non-Patent Documents 3 and 4).

[0007] Non-Patent Document 5 describes that GMP inhibits infection with multiple pathogenic bacteria by binding to the bacteria. Non-Patent Document 6 reports that GMP inhibits hemagglutination caused by influenza virus, and Non-Patent Document 7 reports that κ-casein acts to inhibit human rotavirus infection. However, none of the above documents discloses or suggests the infection-suppressing effect of GMP on mumps virus.

[0008] Patent Document 1 also discloses that κ-casein or GMP is found to have an effect of preventing adhesion of enteric Escherichia coli to cells, hemagglutination activity (HI activity) against influenza virus, or an inhibitory effect on lymphocyte transformation caused by Epstein-Barr virus (EBV), and therefore is believed to be effective in preventing the onset of diarrhea due to infection, mass prevention against influenza epidemics, or cancerization of lymphocytes caused by EBV. However, Patent Document 1 does not discuss the protective effect against infection with the mumps virus.

[0009] Patent Document 2 discloses an antiviral agent that exhibits influenza virus infection inhibitory activity and contains as an active ingredient a sialo-glycan-containing polymer in which sialo-glycan is bound to γ-polyglutamic acid. However, as in the above patent documents, only the effect on influenza virus is disclosed, and the protective effect against mumps virus infection is not examined. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Japanese Patent Application Publication No. 63-284133 [Patent Document 2] JP 2008-031156 A [Non-patent literature]

[0011] [Non-Patent Document 1] Kidokoro, M. 2018.2. Prospects for mumps vaccine. Virus. 68:125-136. [Non-Patent Document 2] T.Takahashi,Y.Kurebayashi,K.Tani,M.Yamazaki,A.Minami,H.Takeuchi.2021.The antiviral effect of catechins on mumps virus infection.J Finc Foods.87:104817. [Non-Patent Document 3] T. Saito, T. Itoh. 1992. Variations and distributions of O-Glycosidically linked sugar chains in bovine k-casein. J Dairy Sci.75:1768-1774. [Non-Patent Document 4] H.Fukudome,T.Yamaguchi,J.Higuchi,A.Ogawa,Y.Taguchi,J.Li,T.Kabuki,K.Ito,F.Sakai.2021.Large-scale preparation and glycan characterization of sialylglycopeptide from bovine milk glycomacro peptide and its bifidogenic properties.J Dairy Sci.104:1433-1444.

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Summary of the invention

[0012] An object of the present invention is to provide a novel composition for inhibiting mumps virus infection. [Means for solving the problem]

[0013] The present inventors have conducted extensive research to solve the above problems and have found that sialic acid-binding peptides have a strong inhibitory effect on mumps virus infection, leading to the completion of the present invention. <1> A composition for inhibiting mumps virus infection, comprising a sialic acid-binding peptide as an active ingredient. <2> Sialic acid-binding peptides are derived from milk <1> A composition for inhibiting mumps virus infection described in claim 1. <3> The sialic acid-bound peptide is a sialylglycopeptide as shown below: <1> A composition for inhibiting mumps virus infection described in claim 1. Sialylglycopeptides; The molecular weight 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; The molar ratio of the sialic acid to the galacto-N-biose is 1:1 to 2:1. <4> The combined amount of the sialic acid and the galacto-N-biose is 20% by weight or more. <3> A composition for inhibiting mumps virus infection described in claim 1. <5> The number of amino acid residues in the peptide chain of the sialylglycopeptide is 1 to 13. <3> A composition for inhibiting mumps virus infection described in claim 1. <6> The sugar chain structure is one or more selected from the group consisting of the following (1) to (5): <3> A composition for inhibiting mumps virus infection described in claim 1. (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 <7> The sugar chain structure is one or more selected from the group consisting of (1) and (2) to (5). <6> A composition for inhibiting mumps virus infection described in claim 1. <8> The sialic acid-binding peptide is GMP. <1> A composition for inhibiting mumps virus infection described in claim 1. <9> <1> ~ <8> 2. A food or drink for inhibiting mumps virus infection, comprising the composition for inhibiting mumps virus infection described in any one of claims 1 to 11. <10> <1> ~ <8> 2. A pharmaceutical for suppressing mumps virus infection, comprising the composition for suppressing mumps virus infection according to any one of claims 1 to 11. Effect of the Invention

[0014] According to the present invention, it is possible to provide a composition for suppressing mumps virus infection, which contains a sialic acid-binding peptide as an active ingredient, and also to provide a food or drink for suppressing mumps virus infection, and a pharmaceutical for suppressing mumps virus infection, which contain the sialic acid-binding peptide as an active ingredient. [Brief description of the drawings]

[0015] [Figure 1] 1 is a chart showing the results of size exclusion chromatography (SEC) analysis of a sialylglycopeptide composition of the present invention. [Diagram 2] 1 is a graph showing the mumps virus infection-inhibiting effect when the GMP or sialylglycopeptide composition of the present invention and mumps virus (13V165E2 strain) were mixed and added to African green monkey kidney-derived Vero cells. [Diagram 3] 1 is an image showing the hemagglutination inhibition (HI) effect of the GMP or sialylglycopeptide composition of the present invention. The thick frame indicates a sample determined to exhibit HI. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention relates to a composition for inhibiting mumps virus infection, which contains, as an active ingredient, a sialic acid-binding peptide such as GMP or a sialylglycopeptide, and a food, drink, or medicine for inhibiting mumps virus infection, which contains the composition. Each of these will be described in detail below.

[0017] (GMP) GMP, an active ingredient of the composition for suppressing mumps virus infection of the present invention, is a sialic acid-binding peptide produced when rennet or pepsin is allowed to act on κ-casein, and has been known to be contained in cheese whey and rennet casein whey. As described above, GMP is a C-terminal peptide containing the sugar chain of κ-casein, and in the case of cow's milk, it corresponds to residues 106-169 of κ-casein, and has a molecular weight of about 7,000. In the present invention, any GMP obtained from whey derived from animal milk such as goat or sheep, other than cow's milk, can be used.

[0018] (GMP manufacturing method) The GMP used in the present invention can be prepared by the method described in Japanese Patent No. 2673828 or Japanese Patent Publication No. 25297 / 1994. That is, a method of preparing GMP by adjusting a milk raw material containing GMP, such as cheese whey, whey protein concentrate, deproteinized cheese whey, etc., to a pH of less than 4, and then ultrafiltrating the filtrate using a membrane with a molecular weight cutoff of 10,000 to 50,000, and preferably adjusting the filtrate to a pH of 4 or more again, and then desalting and concentrating the filtrate using a membrane with a molecular weight cutoff of 50,000 or less. In addition, a method of preparing a composition with a high GMP content by adjusting milk whey to a pH of 6.0 or more and heating it at 40 to 79°C to remove whey protein, recovering GMP in the concentrate, reducing the pH of the concentrate to 5.5 or less, and then subjecting it to membrane treatment again to recover GMP in the permeate can be mentioned. The amount of GMP in the obtained composition can be analyzed by, for example, urea-SDS electrophoresis as shown in the examples of Japanese Patent No. 3372499.

[0019] (Sialylglycopeptide) The sialylglycopeptide, which is the active ingredient of the composition for suppressing mumps virus infection of the present invention, has the following properties (a) to (d). (a) The molecular weight is 500 or more and 3,000 or less. (b) A sugar chain consisting of sialic acid and galacto-N-biose is attached to the peptide chain. (c) 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. (d) the molar ratio of the sialic acid to the galacto-N-biose is 1:1 to 2:1 Here, the combined amount of sialic acid and galacto-N-biose is preferably 20% by weight or more (based on solid weight) in the composition. Furthermore, the lower limit of the molecular weight of the sialylglycopeptide is more preferably not less than 1,000. The molecular weight of GMP is 7,000, which is significantly different from the sialylglycopeptide of the present invention, which has a molecular weight of 500 or more and not more than 3,000, and the physical properties are expected to be significantly different as well. In addition, the sialylglycopeptide of the present invention preferably has a peptide chain consisting of 1 to 13 amino acid residues. Furthermore, the sialylglycopeptide of the present invention may also contain acetylated sialic acid.

[0020] A more specific embodiment of the sialylglycopeptide of the present invention is a peptide having a threonine and / or serine residue and a sugar chain bound to the hydroxyl group of the residue, wherein the sugar chain is one or more selected from the group consisting of (1) to (5) below. (1)Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAcα1 (2)Galβ1-3(Neu5Acα2-6)GalNAcα1 (3)Neu5Acα2-3Galβ1-3GalNAcα1 (4)Neu5Acα2-3Galβ1-3(O-Ac-Neu5Acα2-6)GalNAcα1 (5)Neu5Acα2-3Galβ1-3(O-diAc-Neu5Acα2-6)GalNAcα1 The peptides include those containing one or more of the above sugar chains, and include peptides containing two or more of the same type of sugar chain, or peptides containing two or more types of sugar chains. For example, the peptides may be peptides containing two of (1), or peptides containing (1) and further containing one or more of the group consisting of (2) to (5).

[0021] The sialylglycopeptide of the present invention can typically be prepared by hydrolyzing a casein material, a κ-casein material, a whey material, a GMP material, or the like with an enzyme or the like. The GMP can be any GMP that can be obtained from whey derived from cow's milk, goat's milk, sheep's milk, or other animal milk. The sialylglycopeptide of the present invention can also be prepared by hydrolyzing an extract of a microorganism, a plant, or an animal organ with an enzyme, etc. Furthermore, it may be chemically synthesized or prepared by a genetic recombinant.

[0022] (Method of Producing Sialylglycopeptide) One embodiment of the method for producing the sialylglycopeptide of the present invention will be described below. The sialylglycopeptide of the present invention can be obtained, for example, by hydrolyzing GMP. The sialylglycopeptide of the present invention can be produced by treating the GMP obtained by the above method with an endoprotease or an exoprotease, and subjecting the treated product obtained in the above step to ultrafiltration with a molecular weight cutoff of 500 to 3,000. In addition, a similar sialylglycopeptide can be prepared by directly enzymatically treating a milk raw material containing GMP without separating GMP from the milk raw material. The sialylglycopeptide of the present invention can be obtained, for example, by hydrolyzing whey protein without the step of separating GMP as described above. For example, dairy materials such as cheese whey, whey protein concentrate, and whey protein isolate can be used as raw materials. The sialylglycopeptide of the present invention can be produced by a step of treating these dairy materials with endo- or exo-proteases and a step of subjecting the treated product obtained in the above step to ultrafiltration with a molecular weight cutoff of 500 to 3,000.

[0023] The type of protease used in the production of the sialylglycopeptide of the present invention is not particularly limited as long as it can hydrolyze peptide bonds to obtain the sialylglycopeptide of the present invention having the above-mentioned molecular weight and sugar chain, and one or more types of endo- or exo-type proteases can be used. Preferably, enzymes that can be used in the production of foods and pharmaceuticals are used, and 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), Peptidase R (Amano Enzyme), Alcalase (Novozyme), Flavorzyme (Novozyme), etc. can be used alone or in combination.

[0024] As described above, the sialylglycopeptide of the present invention is typically obtained by hydrolysis of GMP, and therefore, depending on the degree of purification, it may be a purified single sialylglycopeptide, a mixture of multiple sialylglycopeptides, or a mixture containing components other than the sialylglycopeptide of the present invention. In the present specification, a substance containing the sialylglycopeptide of the present invention may be referred to as a sialylglycopeptide composition, and unless otherwise specified, the term is used in the sense that it includes all of the above cases.

[0025] (Evaluation of mumps virus infection inhibitory effect) The mumps virus infection-inhibiting effect of a composition containing the sialic acid-binding peptide of the present invention can be evaluated by comparing the presence or absence of mumps virus infection when the sialic acid-binding peptide composition is administered and when it is not administered, and if the infection rate is lower when the sialic acid-binding peptide composition is administered than when it is not administered, the mumps virus infection-inhibiting effect of the present invention can be evaluated. Furthermore, in an in vitro mumps virus infection evaluation test, the amount of mumps virus in the medium or the number of mumps virus-infected cells when a sialic acid-binding peptide composition is added to the medium at the same time as the mumps virus is compared with when it is not added, and if the amount of mumps virus or the number of mumps virus-infected cells is lower when a sialic acid-binding peptide composition is added than when it is not added, it can be evaluated that the mups virus infection inhibitory effect of the present invention is present.

[0026] (Food, beverages, and medicines containing sialic acid-binding peptides) The sialic acid-binding peptide composition obtained by the above-mentioned production method of the present invention can be used as it is as a material or ingredient for food and beverages, and the food and beverages can be produced according to standard methods for each food product, except for adding the sialic acid-binding peptide composition. Therefore, an effective amount of the sialic acid-binding peptide composition of the present invention may be incorporated into any food or drink, or may be added to the raw materials during the manufacturing process of the food or drink. Examples of the food or drink 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., beverages such as milk drinks, fruit juice drinks, and soft drinks, egg products such as jelly, candy, pudding, and mayonnaise, confectionery and bread such as butter cake, and further various types of milk powder, foods for infants, and nutritional compositions. The food or drink containing an effective amount of the sialic acid-binding peptide thus produced is provided as a food or drink having an inhibitory effect on mumps virus infection.

[0027] The sialic acid-binding peptide composition obtained by the above-mentioned production method of the present invention can be used as a raw material for pharmaceuticals as is, and apart from adding the sialic acid-binding peptide composition, it can be produced in the usual manner into tablets, capsules, powders, syrups, etc. Therefore, when preparing a pharmaceutical product containing the sialic acid-binding peptide of the present invention as an active ingredient, it can be formulated by appropriately mixing excipients, stabilizers, flavoring agents, etc. that are approved for pharmaceutical use, or the sialic acid-binding peptide composition can be dried as is and used as a powder or dust. It can also be formulated by mixing excipients, binders, disintegrants, lubricants, flavoring agents, suspending agents, coating agents, and other optional drugs within a range that does not interfere with the mumps virus infection inhibitory effect. Possible dosage forms include tablets, capsules, granules, powders, dusts, syrups, etc. A pharmaceutical containing an effective amount of the sialic acid-binding peptide thus produced can be provided as a pharmaceutical having an inhibitory effect on mumps virus infection.

[0028] (Intake of sialic acid-binding peptides) When the sialic acid-binding peptide composition of the present invention is blended with ingredients such as food, beverages, and pharmaceuticals, or processed products of such ingredients to produce a composition having an effect of inhibiting mumps virus infection, the blending ratio is not particularly limited and may be appropriately adjusted according to the ease of production and the preferred daily dosage. The daily dosage of the sialic acid-binding peptide composition of the present invention is determined individually taking into consideration the symptoms, age, etc. of the subject to be administered, but for adult humans, it is sufficient to ingest 0.1 g or more of the sialic acid-binding peptide composition per day, preferably 1 g or more, and even more preferably 10 g or more.

[0029] The mumps virus infection suppression effect of the sialic acid-binding peptide of the present invention as an active ingredient is intended for all humans, including healthy individuals, and is particularly preferably used for humans at high risk of mumps virus infection. For example, the effect can be confirmed by taking the peptide for children at high risk of infection or unvaccinated individuals. EXAMPLES

[0030] Examples of the present invention will be described in detail below, but the present invention is not limited to these examples.

[0031] [Example 1] Method for preparing the sialylglycopeptide composition of the present invention 1. GMP manufacturing method 1 kg of whey protein concentrate (Sunlact N-2, Taiyo Kagaku) ​​was dissolved in 50 L of water at 50°C, and the pH was adjusted to 3.5 with concentrated hydrochloric acid. This was filtered using an ultrafiltration membrane (GR61PP, DDS) with a molecular weight cutoff of 20,000 at 50°C, pressure of 0.4 MPa, and average permeate flow rate of 52.4 L / M. 2 When the amount of permeate reached 40 L, 40 L of water at 50°C was added to the concentrated liquid, and ultrafiltration was continued. Continuous operation was carried out in this manner, and 160 L of permeate was obtained. The resulting permeate was added with 25% caustic soda to adjust the pH to 7.0, and ultrafiltration was performed again under the same conditions with the same ultrafiltration membrane until the concentrate reached 5 L, for desalting and concentration. Next, water at 50°C was added, and diafiltration was performed under the same conditions with the same ultrafiltration membrane while keeping the concentrate volume at 10 L, for further desalting. When the permeate volume reached 80 L through this diafiltration, the addition of water to the concentrate was stopped, and the concentrate was concentrated by ultrafiltration until the concentrate volume reached 2 L, and this concentrate was dried to obtain 54 g of GMP.

[0032] 2. Method for producing sialylglycopeptide composition Using the GMP obtained in 1 above, 25 kg of 5% GMP solution was prepared. After adjusting the pH to 7 with 8N potassium hydroxide aqueous solution (Fujifilm Wako Pure Chemical Industries, Ltd.), endo-type protease (Alcalase, Novozyme) and exo-type protease (Flavorzyme, Novozyme) were added and reacted at 50°C for 4 hours. This was concentrated 1.25 times using an ultrafiltration membrane with a molecular weight cutoff of 1,000 (Membralox EP19-40, Pall). Subsequently, 20 kg of the concentrate was subjected to 5 times diafiltration to obtain a concentrate fraction. This concentrate was dried to obtain 125 g of sialylglycopeptide composition (1).

[0033] 3. Method for producing a sialylglycopeptide composition from whey protein isolate 3 kg of whey protein isolate (WPI, Provon190, Glanbia) was dissolved in 27 kg of water at 55°C to prepare 30 kg of 10% WPI solution. Then, endo-type protease (Alcalase, Novozyme) and exo-type protease (Flavorzyme, Novozyme) were added and reacted at 55°C for 8 hours. After the reaction, the mixture was kept at 85°C for 30 minutes to inactivate the enzymes. Diafiltration was performed on this 30 kg of WPI hydrolyzate using an ultrafiltration membrane with a molecular weight cutoff of 1,000. Water was added to the concentrate and the mixture was continuously diafiltered, and a retentate fraction was obtained after 10-fold diafiltration. This retentate fraction was dried to obtain 120.0 g of sialylglycopeptide composition (2).

[0034] 4. Measurement of sialic acid content in sialylglycopeptide compositions The sialylglycopeptide composition was prepared in 50 mM potassium phosphate buffer (pH 5.0) at 250 μg / mL and sialidase (Neuraminidase isoenzyme S from Arthrobacter ureafaciens, #EC-32118-S, Cosmo Bio) at 0.2 U / mL, and 50 μL of the 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, in order to measure the amount of free sialic acid, the sialylglycopeptide composition was dissolved in ultrapure water to 1 mg / mL, and then centrifuged at 15,000×g for 10 minutes (Tomy Seiko), and the resulting supernatant was passed through a 0.45 μm filter (13A, Kurabo) to remove insoluble matter. 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 as the detector. As the mobile phase, a 100 mM sodium hydroxide solution containing 60 mM sodium acetate was isocratically passed through the column until 7 minutes after the sample was introduced, and the sodium acetate concentration was linearly increased to 150 mM between 7 and 10 minutes and maintained at that concentration until 20 minutes. Then, a 100 mM sodium hydroxide solution containing 60 mM sodium acetate was isocratically passed through the column for 5 minutes to equilibrate the column. The mobile phase was always passed at 1 mL / min. N-Acetylneuraminic acid (#A2388, Sigma-Aldrich) was used as the sialic acid standard, and the amount of sialic acid was calculated from the calibration curve created.

[0035] 5. Measurement of Galacto-N-biose (GNB) Content in Sialylglycopeptide Compositions The sialylglycopeptide composition was prepared at 250 μg / mL, sialidase (Neuraminidase isoenzyme S from Arthrobacter ureafaciens, #EC-32118-S, Cosmo Bio) at 0.2 U / mL, and O-glycanase (#DG53 043a, Prozyme) at 0.125 U / mL in 50 mM potassium phosphate buffer (pH 5.0), and 50 μL of the solution 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 amount of GNB. An electrochemical detector (pulsed amperometric mode) was used as the detector. As the mobile phase, 100 mM sodium hydroxide solution was isocratically passed through the column for 10 minutes after sample introduction, and the sodium acetate concentration was linearly increased to 600 mM from 10 to 25 minutes and maintained at that concentration until 30 minutes. From 30 to 45 minutes, 100 mM sodium hydroxide solution was isocratically passed through the column. The mobile phase was always passed through at 1 mL / min. The GNB content was calculated from a calibration curve prepared using a GNB standard (#A0167, Sigma-Aldrich).

[0036] [Table 1]

[0037] [Example 2] Sialidase treatment of sialylglycopeptide composition The sialylglycopeptide composition (1) prepared in Example 1 was adjusted to 20 mg / mL with 100 mM acetate buffer (pH 5.0) containing 2 mM calcium chloride, and sialidase (derived from Clostridium perfringens, Sigma-Aldrich, N2876-25UN) was added to the mixture at 10 U / mL, followed by reaction at 37°C for 20 hours. A blank without enzyme addition was reacted under the same conditions without adding the enzyme. After the reaction, the mixture was boiled for 5 minutes to stop the reaction. The reaction solution obtained was subjected to size exclusion chromatography (SEC). For SEC analysis, an L-2000 (HITACHI) system equipped with two TSKgel G3000PW (Tosoh) was used to detect UV214 nm absorption. A 40% acetonitrile solution containing 0.1% trifluoroacetic acid was used as the mobile phase, and the mixture was eluted isocratically at room temperature for 120 minutes at a flow rate of 0.3 ml / min.

[0038] 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 the sialylglycopeptide composition was observed between 50 and 60 min, but these were generally shifted to lower molecular weights by sialidase treatment, indicating that the main component of the prepared sialylglycopeptide composition (1) was a compound containing sialic acid.

[0039] [Example 3] Structural analysis of sialylglycopeptide composition using LC / MS For LC / MS analysis, an Orbitrap mass spectrometer Q-Exactive (Thermo Fisher Scientific) connected to a high-performance liquid chromatograph (HPLC) UltiMate 3000 (Thermo Fisher Scientific) was used (LC-ESI-IT MS), and an InertSustain AQ-C18 (φ2.1 mm × 150 mm, GL Sciences) was installed as the separation column. The mobile phases used were a 2% acetonitrile solution containing 0.1% formic acid (liquid A) and a 90% acetonitrile solution containing 0.1% formic acid (liquid B). The sialylglycopeptide composition was dissolved in ultrapure water to prepare a 100 μg / mL solution, and 10 μL was introduced into the HPLC. The mobile phase was passed at 200 μL / min, and the proportion of liquid B was 0% for 5 minutes after sample introduction, after which the proportion of liquid B was increased linearly from 0% to 10% over 25 minutes. The proportion of liquid B was then increased linearly from 10% to 100% over 20 minutes. The mass spectrometer performed MS measurements (m / z 200-2000, positive mode) and MS / MS measurements. For MS / MS measurements, the five most intense ions were automatically selected as precursor ions, and the m / z values ​​of the product ions generated by destruction with collision energies of 15, 30, and 45 were observed. The acquired MS / MS spectra were analyzed using Proteome Discoverer 2.2 (Thermo Fisher Scientific) including the Byonic (protein metrics) node. The protein database used was a database consisting only of GMP, 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). The glycans assigned as (HexNAc), (HexNAc)(Hex), or (HexNAc)(Hex)(Neu5Ac)2 by the Byonic analysis were assigned to GalNAc, Galβ1-3GalNAc, or Neu5Acα2―3Galβ1-3(Neu5Acα2―6)GalNAc, respectively, according to the previously reported glycan structure of GMP. In addition, the glycans assigned as (HexNAc)(Hex)(Neu5Ac) were assigned to Neu5Acα2―3Galβ1-3GalNAc or Galβ1-3(Neu5Acα2―6)GalNAc by manually checking the MS / MS spectrum. 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.

[0040] The glycan structures of the glycopeptides contained in the sialylglycopeptide composition (1) prepared in Example 1 were estimated from the MS spectrum and MS / MS spectrum obtained by LC / MS analysis. Table 2 shows the glycan structures of the 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 a glycan structure (Glycan type 3, 4, 5, 6, 7) of Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAc with two molecules of sialic acid bound, but Neu5Acα2-3Galβ1-3GalNAc (Glycan type 1) and Galβ1-3(Neu5Acα2-6)GalNAc (Glycan type 2) with one molecule of sialic acid were also detected. Furthermore, O-acetylated 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 bound to one 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 for the shortest to 13 residues for the longest. The molecular weights of the detected peptide chains ranged from 105 to 2928, and the molecular weights of the sialylglycopeptides ranged from 762 to 2987.

[0041] [Table 2]

[0042] [Test Example 1] Study on the effect of GMP and sialylglycopeptide composition in suppressing mumps virus infection 1. Test Method (1) Preparation of cells African green monkey kidney-derived Vero cells were cultured in a 24-well culture plate (CORNING Falcon, 353047) at 1.0 × 10 5 The cells were seeded at 500 μL / well and cultured overnight at 37°C in the presence of 5% CO2 until they became confluent.

[0043] (2) Preparation of test levels 150 μL of mumps virus (13V165E2 strain)-serum-free MEM suspension with an infectivity of 400 focus forming units (FFU) / mL was mixed with 150 μL of sialylglycopeptide composition (1) or (2) diluted with serum-free MEM or GMP in equal amounts, and allowed to stand on ice for 1 hour. As a control (negative control), a mixture of 150 μL of virus-serum-free MEM suspension and 150 μL of MEM in equal amounts was prepared. The final concentrations of the sialylglycopeptide composition and GMP in the medium were 0.00977, 0.0391, 0.156, 0.625, 2.5, and 10 mg / mL.

[0044] (3) Infection inhibition test Confluent cells in a 24-well culture plate were washed with 250 μL / well of PBS, and the mixture of virus and each sample was added at 250 μL / well and infected on ice for 1.5 hours. The cells were washed with 250 μL / well of PBS, and 2 mL / well of agar medium containing 0.8% Bacto Agar (BD, 214010) and SFM (Thermo Fisher Scientific, 12300067, Hybridoma-SFM) containing acetylated trypsin (Sigma-Aldrich, T6763, final concentration 2 μg / mL) was added, and the cells were cultured at 37°C in the presence of 5% CO2 for 72 hours. 50μL / well of 300μM sialidase fluorescent imaging agent 2-(Benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (Tokyo Chemical Industry, B6235, BTP3-Neu5Ac) was added to the agar medium and left to stand at 37℃ for 3 hours. UV light with a wavelength of 365nm was irradiated using a UV transilluminator (Natural Immunity GUV-720), and the clusters (foci) of virus-infected cells that were fluorescently imaged were counted. Each sample was measured three times independently, and the average value was calculated and used. The relative value of the number of foci was calculated when the number of foci in the control was set to 100%. The 50% inhibitory concentration (IC 50 ) values ​​and 95% confidence interval (95% CI) ranges were calculated using GraphPad Prism version 5 (GraphPad Prism Software).

[0045] 2. Test Results As a result of the evaluation, the number of foci was reduced in a concentration-dependent manner at the level where the sialylglycopeptide composition or GMP was added, and the mumps virus infection-inhibiting effect was observed (Figure 2). The IC 50 The IC value of the infection-suppressing effect of sialylglycopeptide composition (2) was 5.23 mg / mL, with a 95% CI of 3.89-7.03 mg / mL. 50 The IC50 value for GMP infection suppression was 4.18 mg / mL, with a 95% CI of 3.53-4.96 mg / mL. 50 was 11.1 mg / mL, with a 95% CI of 7.51-16.40 mg / mL.

[0046] [Test Example 2] Examination of the hemagglutination inhibition (HI) effect of GMP and sialylglycopeptide composition 1. Test Method Sialylglycopeptide composition (1), (2) or GMP diluted with PBS was added at 50 μL / well to a 96-well U-bottom plate (WATOSON, 195-96U). The concentrations added were 0.00977, 0.0195, 0.0391, 0.0781, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10, and 20 mg / mL. Virus-PBS suspension (25 μL / well) of mumps virus (13V165E2 strain) diluted to 4 hemagglutination units (HAU) was added, stirred for 30 seconds with a microplate mixer (Touyo, PM-1), and then allowed to stand on ice for 1 hour. 0.5% guinea pig red blood cells (Kohjin Bio, 12072702)-PBS suspension was added at 50 μL / well and stirred for 30 seconds with a microplate mixer. The mixture was allowed to stand on ice for 4 hours, and the maximum dilution concentration of the sample showing HI was measured.

[0047] 2. Test Results As a result of the evaluation, sialylglycopeptide composition (1) and GMP showed HI at concentrations of 0.625 mg / mL or more (Figure 3). In addition, sialylglycopeptide composition (2) showed HI at concentrations of 0.313 mg / mL or more. These results suggested that the mumps virus infection-suppressing effect of the sialylglycopeptide composition or GMP of the present invention is mediated by inhibition of virus receptor binding.

[0048] [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 and mixed with 30 g of the powder of sialylglycopeptide composition (1) obtained in Example 1. The mixture was packed into a stick bag to produce a supplement for suppressing mumps virus infection of the present invention.

[0049] [Example 6] Production of beverage 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 inhibiting mumps virus infection of the present invention.

[0050] [Table 3]

[0051] [Example 7] Production of pharmaceuticals (capsules) The raw materials were mixed according to the ratio shown in Table 4, granulated into granules, and then 10 mg of each was filled into empty capsules to produce capsules containing the pharmaceutical agent for inhibiting mumps virus infection of the present invention.

[0052] [Table 4] [Industrial Applicability]

[0053] According to the present invention, it is possible to provide a novel composition for suppressing mumps virus infection, which contains a sialic acid-binding peptide as an active ingredient, and foods, beverages, and pharmaceuticals for suppressing mumps virus infection, which contain a sialic acid-binding peptide as an active ingredient.

Claims

1. A composition for inhibiting mumps virus infection, comprising a sialic acid-binding peptide as an active ingredient.

2. The composition for inhibiting mumps virus infection according to claim 1, wherein the sialic acid-binding peptide is derived from milk.

3. A composition for inhibiting mumps virus infection according to claim 1, wherein the sialic acid-binding peptide is a sialylglycopeptide having the following characteristics: Sialylglycopeptides; (a) The molecular weight is 500 or more and 3,000 or less. (b) A sugar chain consisting of sialic acid and galacto-N-biose is bound to the peptide chain. (c) 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; (d) the molar ratio of said sialic acid to said galacto-N-biose is 1:1 to 2:1

4. The composition for inhibiting mumps virus infection according to claim 3, wherein the combined amount of said sialic acid and said galacto-N-biose is 20% by weight or more.

5. A composition for inhibiting mumps virus infection according to claim 3, wherein the number of amino acid residues in the peptide chain of the sialylglycopeptide is 1 or more and 13 or less.

6. The composition for inhibiting mumps virus infection according to claim 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

7. The composition for inhibiting mumps virus infection according to claim 6, wherein the sugar chain structure is one or more selected from the group consisting of (1) and (2) to (5).

8. 2. The composition for inhibiting mumps virus infection according to claim 1, wherein the sialic acid-binding peptide is GMP.

9. A food or drink for inhibiting mumps virus infection, comprising the composition for inhibiting mumps virus infection according to any one of claims 1 to 8.

10. A pharmaceutical for suppressing mumps virus infection, comprising the composition for suppressing mumps virus infection according to any one of claims 1 to 8.

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