Nutritional composition for preventing bronchitis

Abstract

The present invention addresses the problem of exploring a component for preventing the onset of bronchitis from the components of breast milk to provide a novel nutritional composition. The present invention relates to a nutritional composition for preventing the onset of bronchitis, said nutritional composition being characterized by containing N-acetylneuraminic acid, 6 '-sialyllactose, alpha-2, 6-disialic acid N-linked glycans, and disialic acid N-linked glycans of a specific structure as active ingredients. The nutritional composition is also an active ingredient not only for infants, but also for the elderly who are at high risk of bronchitis and those who have basic diseases, and therefore, the nutritional composition can be used for treating bronchitis. The composition is effective as a nutritional composition for preventing the onset of bronchitis when it is added to food and nutritional compositions for adults and elderly people.

Description

Technical Field

[0001] The present invention relates to a nutritional composition for preventing bronchitis. More specifically, the present invention relates to a nutritional composition for preventing bronchitis containing N-acetylneuraminic acid as an active ingredient. Background Art

[0002] Bronchitis is a disease in which a virus or bacteria infects the airways (bronchi) that branch off the trachea and causes inflammation. The causes of bronchitis are mostly viral infections such as respiratory syncytial (RS) virus, rhinovirus, adenovirus, coronavirus and influenza virus. Bacterial pathogens such as mycoplasma and Bordetella pertussis can also cause bronchitis. After the initial infection with these pathogens, bacteria such as Streptococcus pneumococcus and Haemophilus influenzae can cause secondary infections, leading to severe illness.

[0003] Infants and young children are at high risk of developing bronchitis because their immune function is not yet mature. In particular, infants up to about 3 months old have thin bronchi, which makes their ability to expel sputum weak, so they are at risk of becoming seriously ill due to the expansion of inflammation.

[0004] In addition, older people with weakened immune systems are at high risk for bronchitis, which, if it worsens, can develop into pneumonia, leading to severe, life-threatening symptoms.

[0005] Therefore, in order to live a healthy social life, it is important for everyone from infants to the elderly to prevent bronchitis. So far, various studies have been conducted on methods for treating and preventing bronchitis. Regarding treatment methods after suffering from bronchitis, the administration of anti-influenza virus drugs, treatment with various antibiotics, treatment with cough suppressants and expectorants, etc. have been developed. Patent document 1 discloses an anti-ulcer agent or a bifidobacterium growth promoter containing a specific sialic acid derivative as an active ingredient. It is also proven that sialic acid derivatives have a therapeutic effect on infectious diseases caused by viruses such as influenza virus, rotavirus and herpes virus. However, the document does not disclose the preventive effect of sialic acid derivatives on bronchitis. The patent document discloses the preventive effect of sialic acid derivatives on diarrhea caused by rotavirus. However, rotavirus is not the causative virus of bronchitis, so the preventive effect of sialic acid derivatives on bronchitis is not disclosed. Therefore, there is no effective method for preventing bronchitis, and the only thing to do is to generally encourage hand washing and gargling.

[0006] In addition, breast milk is known to contain various components that can reduce the risk of infectious diseases (Patent Document 2). In particular, it is reported that carbohydrates represented by oligosaccharides contained in breast milk inhibit the stage in which specific viruses attach to host cells (Patent Document 3).

[0007] In order to explore active ingredients that prevent the onset of bronchitis, the present inventors have focused on ingredients contained in breast milk. However, it is not clear whether these can prevent the onset of bronchitis itself, and it is not possible to infer which ingredients in breast milk can prevent the onset of bronchitis.

[0008] Patent Literature

[0009] Patent Document 1: JP 3,930,559 B

[0010] Patent Document 2: JP 2,514,375 B

[0011] Patent Document 3: JP 3,789,146 B Summary of the invention

[0012] Problem that the invention aims to solve

[0013] The present inventors have conceived that if a component capable of preventing the onset of bronchitis can be found among the components of breast milk, it would be possible to supply a nutritional composition for infants that prevents the onset of bronchitis by incorporating the component into a nutritional composition for infants. The component is an effective component not only for infants but also for the elderly who are at high risk of bronchitis and people with underlying diseases, so the present inventors believe that it would be possible to incorporate the component into foods and nutritional compositions for adults and the elderly to provide them as nutritional compositions for preventing the onset of bronchitis.

[0014] The present invention solves the problem of discovering a component that prevents the onset of bronchitis from the components of breast milk to provide a novel nutritional composition.

[0015] Solutions for solving problems

[0016] In order to explore the components in breast milk that prevent the onset of bronchitis, the inventors conducted a statistical analysis on the relationship between the amount of carbohydrate components in breast milk and the history of bronchitis in infants. As a result, the inventors found that N-acetylneuraminic acid, 6'-sialyllactose, α-2,6-disialo N-linked glycan, and disialo N-linked glycan with a specific structure in breast milk have a preventive effect on bronchitis, and completed the present invention.

[0017] That is, the present invention relates to a nutritional composition for preventing the onset of bronchitis, which contains one or more active ingredients selected from the group consisting of N-acetylneuraminic acid, 6'-sialyllactose, α-2,6-disialyl N-linked glycans, and disialyl N-linked glycans having a specific structure. Specifically, the present invention has the following configuration.

[0018] <1> A nutritional composition for preventing bronchitis, comprising N-acetylneuraminic acid as an active ingredient.

[0019] <2> according to <1> The nutritional composition comprises N-acetylneuraminic acid bound to one or more of protein, peptide, oligosaccharide or lipid.

[0020] <3> according to <1> or <2> A nutritional composition wherein the linkage pattern of N-acetylneuraminic acid is α-2,6 linkage.

[0021] <4> according to <3> A nutritional composition wherein N-acetylneuraminic acid is present as 6'-sialyllactose.

[0022] <5> according to <3> A nutritional composition wherein N-acetylneuraminic acid is present as an N-linked glycan.

[0023] <6> according to <5> The nutritional composition of claim 1, wherein the N-linked glycan is α-2,6-disialyl N-linked glycan.

[0024] <7> according to <6> A nutritional composition, wherein the α-2,6-disialyl N-linked glycan has the structure shown in (1) or (2) below:

[0025] (1)Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAc

[0026] (2) Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc.

[0027] <8> according to <1> to <7> The nutritional composition of any one of the above, wherein it is for infants and young children.

[0028] <9> A food and drink for preventing bronchitis, comprising <1> to <8> A nutritional composition for preventing bronchitis according to any one of the claims.

[0029] <10> A bronchitis prevention drug containing <1> to <8> A nutritional composition for preventing bronchitis according to any one of the claims.

[0030] The present invention also has the following configurations.

[0031] <11> A method for preventing bronchitis, comprising the step of administering to a human a nutritional composition containing an effective amount of N-acetylneuraminic acid.

[0032] <12> according to <11> The method for preventing bronchitis, wherein N-acetylneuraminic acid is bound to any one or more of a protein, a peptide, an oligosaccharide or a lipid.

[0033] <13> according to <11> or <12> A method for preventing bronchitis, wherein the linkage pattern of N-acetylneuraminic acid is α-2,6 linkage.

[0034] <14> according to <13> A method for preventing bronchitis wherein N-acetylneuraminic acid is present as 6'-sialyllactose.

[0035] <15> according to <13> The method for preventing bronchitis, wherein N-acetylneuraminic acid is an N-linked glycan.

[0036] <16> according to <15> The method for preventing bronchitis, wherein the N-linked glycan is α-2,6-disialyl N-linked glycan.

[0037] <17> according to <16> A method for preventing bronchitis, wherein the α-2,6-disialyl N-linked glycan has the structure shown in (1) or (2) below:

[0038] (1)Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAc

[0039] (2) Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc.

[0040] <18> according to <11> to <17> The method for preventing bronchitis of any one of claims , wherein the human is an infant.

[0041] <19> according to <11> to <18> The method for preventing bronchitis according to any one of claims 1 to 5, wherein the nutritional composition is a food or beverage.

[0042] <20> according to <11> to <18> The method for preventing bronchitis of any one of claims , wherein the nutritional composition is a medicament.

[0043] Effects of the Invention

[0044] According to the present invention, the onset of bronchitis can be prevented by a nutritional composition containing N-acetylneuraminic acid as an active ingredient. This ingredient can also be an effective ingredient not only for infants, but also for the elderly who are at high risk of bronchitis and those with underlying diseases. Therefore, the present invention can prevent the onset of bronchitis in adults and the elderly. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] [ Figure 1 ] Figure 1 The effect of N-acetylneuraminic acid concentration in breast milk on the onset of bronchitis in infants is shown.

[0046] [ Figure 2 ] Figure 2 The effect of 6'-SL concentration in breast milk on the onset of bronchitis in infants is shown.

[0047] [ Figure 3 ] Figure 3 The effect of disialyl N-linked glycan A concentration in breast milk on the onset of bronchitis in infants is shown.

[0048] [ Figure 4 ] Figure 4 The effect of disialyl N-linked glycan B concentration in breast milk on the onset of bronchitis in infants is shown.

[0049] [ Figure 5 ] Figure 5 The effect of the concentration of α-2,6-disialyl N-linked glycans in breast milk on the onset of bronchitis in infants is shown. DETAILED DESCRIPTION

[0050] (N-acetylneuraminic acid)

[0051] The N-acetylneuraminic acid of the present invention can be in a free state or in a state of being bound to other sugars as an oligosaccharide. N-acetylneuraminic acid can be in a state of being bound to a peptide and / or a protein or a lipid. N-acetylneuraminic acid can be derived from a fermentation method of milk, eggs, bird's nests or genetically modified bacteria, and can be any synthetic product, semi-synthetic product or natural product. The natural material containing the natural product can be added to the nutritional composition of the present invention as a supply source as it is. As used herein, natural product means a product extracted from a natural material containing N-acetylneuraminic acid by a well-known method, a product obtained by crude purification of the extract, or a product obtained by further subjecting it to high purification. For example, include glycomacropeptide derived from cow's milk and sialic acid glycopeptide obtained by condensing the polysaccharide part of glycomacropeptide.

[0052] In the present invention, the linkage mode of N-acetylneuraminic acid is desirably an α-2,6 bond. α-2,6-linked N-acetylneuraminic acid refers to N-acetylneuraminic acid that is bound to a galactose residue in an oligosaccharide or glycoprotein glycan via an α-2,6 bond, or to an N-acetylgalactosamine residue. More specifically, α-2,6-linked N-acetylneuraminic acid refers to a compound having a Neu5Acα2-6Gal structure or a Neu5Acα2-6GalNAc structure.

[0053] (6'-sialyllactose)

[0054] In the present invention, the polysaccharide containing N-acetylneuraminic acid is desirably present as 6'-sialyllactose (Neu5Acα2-6Galβ1-4Glc). 6'-sialyllactose (hereinafter sometimes referred to as 6'-SL) can be derived from a fermentation method of milk or genetically modified bacteria, and can be any synthetic product, semi-synthetic product or natural product. The natural material containing the natural product can be added to the nutritional composition of the present invention as a supply source as it is. As used herein, a natural product means a product extracted from a natural material containing 6'-sialyllactose by a well-known method, a product obtained by crudely purifying the extract, or a product obtained by further highly purifying it.

[0055] (N-linked glycans)

[0056] In the present invention, the polysaccharide containing N-acetylneuraminic acid is desirably present as an N-linked polysaccharide (N-LinkedGlycan / N-Linked Sugar Chain). The N-linked polysaccharide containing N-acetylneuraminic acid of the present invention can be in a free state or in a state of being bound to a peptide and / or protein. It can be derived from a fermentation method of milk, eggs, meat, microorganisms or genetically modified bacteria, and can be any synthetic product, semi-synthetic product or natural product. The natural material containing the natural product can be added to the nutritional composition of the present invention as a supply source as it is. As used herein, a natural product means a product extracted from a natural material containing an N-linked polysaccharide by a well-known method, a product obtained by crude purification thereof, or a product obtained by further highly purifying it.

[0057] (α-2,6-disialyl N-linked glycan)

[0058] The α-2,6-disialyl N-linked glycans in the present invention refer to N-linked glycans that reduce the onset of bronchitis. The α-2,6-disialyl N-linked glycans of the present invention may be in a free state or in a state bound to a peptide and / or protein. They may be derived from a fermentation method of milk, eggs, meat or genetically modified bacteria, and may be any synthetic product, semi-synthetic product or natural product. The natural material containing the natural product may be added to the nutritional composition of the present invention as a supply source as it is. Here, the natural product means a product extracted from a natural material containing α-2,6-disialyl N-linked glycans by a well-known method, a product obtained by crudely purifying it, or a product obtained by further highly purifying it.

[0059] Specific examples of the α-2,6-disialyl N-linked glycan in the present invention include Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAc (hereinafter sometimes referred to as disialyl N-linked glycan A) and Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc (hereinafter sometimes referred to as disialyl N-linked glycan B). In addition, for example, N-linked polysaccharides having two molecules of Neu5Acα-2,6 bonds at the non-reducing end, such as Neu5Acα2-6Galβ1-4(Fucα1-3)GlcNAcβ1-2Manα1-3(Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcN Acβ1-4GlcNAc, Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2-6Galβ1-4(Fucα1-3)GlcNAcβ1-2Manα1-6)Man β1-4GlcNAcβ1-4GlcNAc and Neu5Acα2-6Galβ1-4GlcNAcβ1-2(Neu5Acα2-6Galβ1-4GlcNAcβ1-4)Manα1-3(Galβ1-4GlcN Acβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAc.

[0060] The nutritional composition of the present invention needs to contain an effective amount of N-acetylneuraminic acid for preventing bronchitis. A preferred range of the required amount per 100 g of solid matter of the nutritional composition of the present invention is shown below.

[0061] The content of N-acetylneuraminic acid is preferably 170 mg / 100 g solid or more, and more preferably 189 mg / 100 g solid or more. When the nutritional composition is milk powder, the content of N-acetylneuraminic acid in the milk after preparation is preferably 221 mg / L or more, and more preferably 246 mg / L or more.

[0062] The nutritional composition of the present invention preferably contains 46.9 mg / 100 g solid or more of 6'-sialyllactose, and more preferably 104 mg / 100 g solid or more.

[0063] The adjusted milk preferably contains 61.0 mg / L or more of 6'-SL, and more preferably 135 mg / L or more.

[0064] The nutritional composition of the present invention preferably contains 0.198 μmol / 100 g solid or more of α-2,6-disialyl N-linked glycans, and more preferably 0.338 μmol / 100 g solid or more. The adjusted milk preferably contains 0.257 μmol / L or more of α-2,6-disialyl N-linked glycans, and more preferably 0.440 μmol / L or more.

[0065] The nutritional composition of the present invention preferably contains 0.0728 μmol / 100 g solid or more, and more preferably 0.241 μmol / 100 g solid or more of disialyl N-linked glycan A. The adjusted milk preferably contains 0.0947 μmol / L or more, and more preferably 0.313 μmol / L or more of disialyl N-linked glycan A.

[0066] The nutritional composition of the present invention preferably contains 0.0935 μmol / 100 g solid or more, and more preferably 0.188 μmol / 100 g solid or more of disialyl N-linked glycan B. The adjusted milk preferably contains 0.122 μmol / L or more, and more preferably 0.245 μmol / L or more of disialyl N-linked glycan B.

[0067] (Nutritional compositions, food and beverages, and medicines)

[0068] The nutritional composition for preventing bronchitis of the present invention may be a composition containing an effective amount of N-acetylneuraminic acid, etc. The nutritional composition of the present invention may contain proteins, lipids, sugars, vitamins, minerals, etc. in addition to the active ingredient. The nutritional composition of the present invention is used as a nutritional composition for infants, adults, or the elderly.

[0069] Nutritional compositions for infants and young children include powder and liquid infant formulas, powder toddler formulas (follow-up milk and growing-up milk), powder formulas for low-birth-weight infants, allergen-free foods for milk allergies, and lactose-free foods for lactose intolerance, etc.

[0070] Nutritional compositions for adults and the elderly include formula milk powder and formula liquid milk for adults, allergen-free foods for cow's milk allergy, lactose-free foods and supplements for lactose intolerance, and the like.

[0071] As the food and drink for preventing bronchitis containing N-acetylneuraminic acid etc. of the present invention, any food and drink with effective amount of N-acetylneuraminic acid is preferred. N-acetylneuraminic acid etc. can be added to raw materials during the manufacturing process of food and drink. Examples of food and drink include but are not limited to dairy products such as cheese, fermented milk, lactic acid bacteria beverage, butter and margarine, beverages such as milk beverage, fruit juice beverage and soft drink, jelly, candy, egg processed products such as pudding and mayonnaise, and confectionery and bread such as butter cake.

[0072] The nutritional composition for preventing bronchitis of the present invention can be used as a raw material of a medicine as it is. The medicine of the present invention can be manufactured by conventional methods such as tablets, capsules, powders, granules, powders and syrups.

[0073] The drug of the present invention can be prepared by mixing N-acetylneuraminic acid and the like with pharmaceutically acceptable excipients, stabilizers and flavorings, etc. The composition containing N-acetylneuraminic acid and the like can be dried as it is and used as a powder or a powder. Any excipient, binder, disintegrant, lubricant, flavoring corrective, suspending agent, coating agent and other optional agents can be mixed for preparation within the range that does not interfere with the effect of preventing bronchitis.

[0074] Example

[0075] [Test Example 1]

[0076] Various carbohydrate components were measured in 153 breast milk samples and analyzed for their association with the presence or absence of episodes of bronchitis in infants during the first year of life using logistic regression. The methods and results are presented below.

[0077] 1. Methods

[0078] (1) Investigation of breast milk samples and infant infection history

[0079] Snow Brand BeanStalk Co., Ltd. (Bean Stalk Snow Co., Ltd.) conducted a prospective cohort study (UMINID 000015494) from 2014 to 2019 to investigate the relationship between the composition of Japanese breast milk and the health status of mothers and children in Japan. In this study, breast milk samples collected from 1,210 mothers and questionnaires investigating the health status of mothers and children were used for the study. In addition, 200 samples were randomly selected from 1,210 samples. Breast milk collected between 1 month and 2 months after delivery was used as breast milk samples for analysis. Parents filled out a questionnaire surveying the health status of their babies for a total of 6 times, once every 2 months after birth for 1 year, and collected their results. From the questionnaire results, the results gathered whether the baby was diagnosed as suffering from bronchitis by a doctor within one year after delivery. If the description of the presence or absence of an attack of bronchitis was unclear, they were excluded from the study.

[0080] As a result, in the questionnaire survey conducted 6 times in total within one year, 153 samples correctly answered whether the infant was diagnosed with bronchitis, and the samples became the subject to be analyzed in the study. Among the 153 samples, 18 samples were from infants diagnosed with bronchitis within one year after delivery.

[0081] (2) Measurement of various sugar components in breast milk

[0082] (2-1) Measurement of total N-acetylneuraminic acid

[0083] The ultrapure water of 800 μL and the 1N sulfuric acid of 100 μL are added to the breast milk of 100 μL from 153 samples to be analyzed, then heated at 80 ℃ for 45 minutes.It is cooled to room temperature, adds the sodium hydroxide aqueous solution of 0.1N of 1mL, then carries out the ultrafiltration membrane process that molecular weight cut-off is 10,000, analyzes permeate solution (permeate solution) by HPLC.For HPLC, use the DIONEX ICS-5000DP system (Thermo Fisher Scientific Co., Ltd.) connected to electrochemical detector, and CarboPac PA1 post (Thermo Fisher Scientific Co., Ltd.) is used for post.As mobile phase, will be ultrapure water (solution A), 200mM sodium hydroxide solution (solution B) and contain 600mM sodium acetate 100mM sodium hydroxide solution (solution C) three kinds of solution pass through with the flow of 1mL / minute.First 7 minutes, 45% solution B and 10% solution C pass through. Thereafter, by 10 minutes, solution B was linearly decreased to 37.5%, and solution C was linearly increased to 25%. From 10 minutes to 20 minutes, 37.5% solution B and 25% solution C were passed. From 20.1 minutes to 25 minutes, 45% solution B and 10% solution C were passed. The amount of total N-acetylneuraminic acid in each breast milk sample was calculated from a calibration curve created using standards with known concentrations of N-acetylneuraminic acid.

[0084] (2-2) Measurement of oligosaccharides

[0085] Five hundred and eighty (580) μ L of ultrapure water are added to the breast milk of 20 μ L from 153 samples to be analyzed.After this, the ultrafiltration membrane process of molecular weight cut-off is 10,000, and the permeate solution is analyzed by HPLC.For HPLC, the DIONEX ICS-5000DP system (Thermo Fisher Scientific Co., Ltd.) connected to an electrochemical detector is used, and CarboPac PA1 post (Thermo Fisher Scientific Co., Ltd.) is used for post.As mobile phase, three kinds of solutions of ultrapure water (solution A), 200mM sodium hydroxide solution (solution B) and 100mM sodium hydroxide solution (solution C) containing 600mM sodium acetate are passed through with a flow rate of 1mL / minute.For the first 10 minutes, 50% solution B is passed through.After this, by 18 minutes, solution B is linearly decreased to 47.5%, and solution C is linearly increased to 5%. From 18 minutes to 28 minutes, 47.5% of solution B and 5% of solution C passed. From 28 minutes to 32 minutes, solution B was linearly decreased to 42%, and solution C was linearly increased to 16%. From 32 minutes to 39 minutes, 42% of solution B and 16% of solution C passed. Thereafter, from 39 minutes to 43 minutes, 30% of solution B and 40% of solution C passed. The content of each oligosaccharide in each breast milk sample was calculated from a calibration curve created using standards having known concentrations of 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose (hereinafter sometimes referred to as 3'-SL), 6'-sialyllactose (hereinafter sometimes referred to as 6'-SL), lactodifucotetraose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-difucohexaose I, and lacto-N-difucohexaose II. The total value of these 11 oligosaccharides was defined as "total oligosaccharides".

[0086] (2-3) Measurement of N-linked glycans

[0087] Ten (10) μL of 3M sodium borohydride aqueous solution is added to 20 μL of breast milk from 153 samples to be analyzed, and they are left standing at room temperature for 30 minutes. Then, 7.5 μL of acetic acid is added, and they are left standing at room temperature for 30 minutes. 15 μL of 1M ammonium bicarbonate aqueous solution and 7.5 μL of ultrapure water are added. Then, 5 μL of 400mM dithiothreitol aqueous solution containing 5% sodium dodecyl sulfate is added, heated at 100 ° C for 10 minutes to denature the protein. It is cooled to room temperature, 10 μL of 123mM iodoacetamide aqueous solution is added, and they are left standing at room temperature for 1 hour. 7 μL of 500mM sodium acetate aqueous solution (pH 6.0) and 9 μL of 10% NP-40 solution are added. Thereafter, 10 μL of 500 units / μL peptide-N-glycanase F (Peptide-N-glycanase F) solution is added. Then, the mixture was left to stand at 37 ° C for 24 hours to release N-linked polysaccharides. 109 μL of ultrapure water was added, and the entire amount was then introduced into a Vivaspin 500 (Sartorius Co., Ltd.) with a molecular weight cutoff of 10,000. The mixture was centrifuged at 15,000 × g and 25 ° C for 10 minutes to recycle the permeate solution. 50 μL of acetonitrile was added to 50 μL of the permeate solution to be analyzed by LC / MS. LC / MS analysis was performed as follows. For HPLC, UltiMate 3000 (Thermo Fisher Scientific Co., Ltd.) was used, and Q-Exactive (Thermo Fisher Scientific Co., Ltd.) was used for MS. For the column, Glycan Pac AXH-1 (Thermo Fisher Scientific Co., Ltd.) was used, and the column temperature was set to 40 ° C. As mobile phase, acetonitrile (solution A) and pH are adjusted to 4.4 50mM ammonium formate aqueous solution (solution B) for passing through with the flow rate of 0.4mL / minute.After introducing sample, by 55 minutes, the ratio of solution B is increased linearly from 10% to 35%.Thereafter, from 55 minutes to 65 minutes, 35% solution B passes through.The spray voltage (spray voltage) of ESI probe is 3.5kV, and capillary temperature is 275 ℃, and nitrogen is used for sheath gas (sheathgas), auxiliary gas (auxiliary gas) and collision gas (collision gas).S-Lens RF Level is set to 90.MS mass spectrum is obtained by full scan measurement in the range of m / z 400-2000 in positive mode.The MS mass spectrum obtained uses Compound Discover 2.1 (Thermo Fisher Scientific Co., Ltd.) to analyze.They were compared with a glycan database constructed from previously reported N-linked glycans of milk proteins to detect glycans. Compound Discover created an extracted chromatogram corresponding to the molecular ion peak of each glycan and calculated the signal area value of each glycan detected in each breast milk sample. The calculated signal area value of the glycan was used as a relative quantitative value for each glycan. There were 16 types of N-linked glycans detected. The total value of their area values ​​was used as a relative value for "total N-linked glycans".

[0088] (2-4) Measurement of O-linked glycans

[0089] The glycan release reagent contained in the O-linked glycan analysis kit EZGlyco Prep kit (Sumitomo Bakelite Co., Ltd.) was added to 153 breast milk samples to be analyzed, which were heated at 50°C for 20 minutes. After dilution with ultrapure water, it was introduced into Vivaspin 500 with a molecular weight cutoff of 10,000. They were centrifuged at 15,000 × g and 25°C for 15 minutes, and the permeate solution was collected. 50 μL of acetonitrile was added to 50 μL of the permeate solution, and LC / MS analysis was performed on it. UltiMate3000 was used for HPLC, and Q-Exactive was used for MS. GlycanPac AXH-1 column was used for the column, and the column temperature was set to 40°C. As mobile phase, acetonitrile (solution A) and a 50 mM ammonium formate aqueous solution (solution B) adjusted to pH 4.4 were used to pass through at a flow rate of 0.4 mL / min. After the sample was introduced, the proportion of solution B was linearly increased from 10% to 35% at 55 minutes. Thereafter, 35% solution B was passed from 55 minutes to 65 minutes. The spray voltage of the ESI probe was 3.5 kV, the capillary temperature was 275 ° C, and nitrogen was used for sheath gas, auxiliary gas and collision gas. The S-Lens RF Level was set to 90. The MS mass spectrum was obtained by full scan measurement in the range of m / z 300-2000 in positive mode. The acquired MS mass spectrum was analyzed using Compound Discover 2.1 (Thermo Fisher Scientific Co., Ltd.). They were compared with the O-linked glycan database to detect glycans. Compound Discover creates an extracted chromatogram corresponding to the molecular ion peak of each glycan and calculates the signal area value of each glycan detected in each breast milk sample. The calculated signal area value of the glycan is used as the relative quantitative value of each glycan. There are 12 types of O-linked glycans detected. The total value of these area values ​​was used as a relative value of "total O-linked glycans".

[0090] (3) Determination of the structures of disialyl N-linked glycans A and B

[0091] (3-1) Structural analysis

[0092] One of the 153 samples prepared in the above-mentioned "Measurement of N-linked glycans" was subjected to LC / MS / MS measurement to perform structural analysis of disialyl N-linked glycans A and B. LC / MS / MS analysis was performed as follows. UltiMate 3000 was used for HPLC, and Q-Exactive MS was used for MS. GlycanPac AXH-1 column was used for the column, and the column temperature was set to 40°C. As mobile phases, acetonitrile (solution A) and a 50mM ammonium formate aqueous solution (solution B) adjusted to pH 4.4 were used to pass at a flow rate of 0.4mL / min. After the sample was introduced, the proportion of solution B was linearly increased from 10% to 35% at 55 minutes. Thereafter, from 55 minutes to 65 minutes, 35% solution B passed. The spray voltage of the ESI probe was 3.5kV, the capillary temperature was 275°C, and nitrogen was used for sheath gas, auxiliary gas, and collision gas. The S-Lens RF Level was set to 90. MS / MS spectra were acquired in positive mode and data-dependent Top 10 mode. The acquired MS / MS spectra were analyzed using SimGlycan software (PREMIER Biosoft International) to analyze the structures of disialyl N-linked glycans A and B.

[0093] (3-2) Determination of connection mode

[0094] In order to determine the connection pattern of N-acetylneuraminic acid in disialyl N-linked polysaccharides A and B, LC / MS analysis was performed after treatment with sialidase. Sialidase (α-2,3-sialidase (α-2,3-Sialidase)) that specifically decomposes α-2,3 bonds or any of sialidase that decomposes both α-2,3 and α-2,6 bonds was used as sialidase. In a sample identical to the sample in (3-1) above, 37 μL of ultrapure water and 5 μL of enzyme-attached buffer were added to 5 μL of N-linked polysaccharide solution. 3 μL of α-2,3-sialidase or neuraminidase diluted 10 times with enzyme-attached buffer was added and incubated at 37 ° C for 15 minutes. After heating it in boiling water for 5 minutes for inactivating the enzyme, 50 μL of acetonitrile was added and LC / MS analysis was performed. LC / MS analysis was performed as follows. UltiMate 3000 was used for HPLC, and Q-Exactive was used for MS. GlycanPac AXH-1 was used for the column, and the column temperature was set to 40°C. As mobile phase, acetonitrile (solution A) and a 50mM ammonium formate aqueous solution (solution B) adjusted to pH 4.4 were used to pass through at a flow rate of 0.4mL / min. After the sample was introduced, the proportion of solution B was linearly increased from 10% to 35% at 55 minutes. Thereafter, from 55 minutes to 65 minutes, 35% solution B passed. The spray voltage of the ESI probe was 3.5kV, the capillary temperature was 275°C, and nitrogen was used for sheath gas, auxiliary gas, and collision gas. S-Lens RF Level was set to 90. The MS mass spectrum obtained was analyzed using Xcalibur software (Thermo Fisher Scientific Co., Ltd.).

[0095] (4) Absolute quantification of disialyl N-linked glycans A and B

[0096] The absolute amounts of disialyl N-linked glycans A and B in breast milk, which have been found to be associated with the onset of bronchitis, were measured. The content of disialyl N-linked glycan A in breast milk was measured by a standard addition method using a standard substance of disialyl N-linked glycopeptide. The absolute amount of disialyl N-linked glycan B in breast milk was calculated by multiplying the absolute amount of disialyl N-linked glycan A by the relative ratio of the area value of disialyl N-linked glycan B to the area value of disialyl N-linked glycan A obtained from the LC / MS results.

[0097] The method for measuring disialyl N-linked polysaccharide A by standard addition method is described below. Ten (10) μL of 3M sodium borohydride aqueous solution is added to 20 μL of breast milk of 1 sample randomly selected from 153 breast milk samples to be analyzed, and it is left to stand at room temperature for 30 minutes. Then, 7.5 μL of acetic acid is added and it is left to stand at room temperature for 30 minutes. Fifteen (15) μL of 1M ammonium bicarbonate aqueous solution is added, and then 7.5, 5.5, 3.5 or 1.5 μL of ultrapure water are added. In addition, 0, 2, 4 or 6 μL of sialic acid glycopeptide (sialylglycopeptide) (Tokyo Chemical Industry Co., Ltd.) is prepared into 20 μg / mL as an aqueous solution of a standard substance for glycopeptide for binding to disialyl N-linked polysaccharide A. Then, 5 μL of 120mM dithiothreitol aqueous solution is added and heated at 60 ° C for 30 minutes. Then, 10 μL of 123mM iodoacetamide aqueous solution was added and allowed to stand at room temperature for 1 hour. 20 μL of 40 units / μL trypsin solution was added and incubated at 37°C for 1 hour. The mixture was heated at 100°C for 5 minutes to inactivate trypsin. Then, 500 units / μL peptide-N-glycanase F solution was added and allowed to stand at 37°C for 18 hours to release N-linked glycans. Using the N-linked glycan preparation kit BlotGlyco (Sumitomo Bakelite), the released N-linked glycans were labeled and purified with 2-aminobenzamide and analyzed by LC / MS. UltiMate 3000 was used for HPLC, and Q-Exactive was used for MS. GlycanPac AXH-1 was used for the column, and the column temperature was set to 40°C. As mobile phases, acetonitrile (solution A) and a 50mM ammonium formate aqueous solution (solution B) adjusted to pH 4.4 were used to pass through at a flow rate of 0.4mL / min. After the sample was introduced, the proportion of solution B was linearly increased from 10% to 35% by 55 minutes. Thereafter, from 55 minutes to 65 minutes, 35% solution B was passed. The spray voltage of the ESI probe was 3.5 kV, the capillary temperature was 275° C., and nitrogen was used for sheath gas, auxiliary gas, and collision gas.

[0098] The S-Lens RF level was set to 90. In the MS measurement, selective ion monitoring of m / z 1172.43 was performed in positive mode. The acquired MS chromatogram was analyzed using Xcalibur software. The area value of the MS chromatogram was plotted relative to the amount of disialylglycopeptide added, thereby using the standard addition method to absolutely quantify the amount of sialic acid N-linked polysaccharide A in breast milk. The absolute quantitative value and the relative quantitative value of 153 breast milk samples obtained in "2-3. Measurement of N-linked polysaccharides" described above are used to calculate the absolute quantitative value of disialyl N-linked polysaccharide A in each breast milk sample of the 153 samples. For each breast milk sample, the absolute quantitative value of disialyl N-linked polysaccharide B is calculated by the ratio of the area value of the MS chromatogram of disialyl N-linked polysaccharide B to the area value of the MS chromatogram of disialyl N-linked polysaccharide A.

[0099] (5) Analysis of the correlation between the content of various sugar components in breast milk and bronchitis

[0100] For 153 breast milk samples, logistic regression was used to analyze the relationship between the concentrations of N-acetylneuraminic acid, oligosaccharides, N-linked glycans, O-linked glycans, disialyl N-linked glycan A, and disialyl N-linked glycan B and the onset of infantile bronchitis. The sex of the infant, the presence or absence of siblings, the method of delivery (natural delivery, cesarean section), and birth weight were used as adjustment factors.

[0101] 2. Results

[0102] (1) The relationship between the content of various sugar components in breast milk and the onset of infant bronchitis

[0103] For 153 breast milk samples, the relationship between the content of various carbohydrate components (total N-acetylneuraminic acid, total oligosaccharides, total N-linked polysaccharides and total O-linked polysaccharides) and the onset of infant bronchitis was analyzed using logistic regression. As a result, a statistically significant relationship was found between the total N-acetylneuraminic acid concentration and the onset of bronchitis (Table 1). On the other hand, such a relationship was not observed in total oligosaccharides, total N-linked polysaccharides or total O-linked polysaccharides. The odds ratio and regression coefficient between the total N-acetylneuraminic acid concentration and the onset of bronchitis were 0.098 and -2.32, respectively, indicating that infants who had drunk breast milk with a high concentration of total N-acetylneuraminic acid had a lower risk of suffering from bronchitis.

[0104] [Table 1]

[0105] Table 1. Relationship between the onset of bronchitis and the content of various sugar components in breast milk

[0106]

[0107] The breast milk samples were divided into two groups based on whether the infants had ever suffered from bronchitis, and the total N-acetylneuraminic acid concentration in the breast milk was compared. As a result, the breast milk drunk by infants who had never suffered from bronchitis contained a significantly higher concentration of total N-acetylneuraminic acid ( Figure 1 ). This indicates that the nutritional composition for infants containing the same level of N-acetylneuraminic acid as breast milk drunk by infants who have never suffered from bronchitis is effective in reducing the risk of suffering from bronchitis. The distribution range of total N-acetylneuraminic acid concentration in breast milk drunk by infants who have never suffered from bronchitis is 181-977 mg / L, with a median of 395 mg / L (Table 2). The distribution of total N-acetylneuraminic acid concentration, unlike the normal distribution, is a distribution in which its tail extends to the side with higher concentration. Such a distribution is seen in the distribution of height and weight of infants, and when creating a growth curve for infants, the 3rd to 97th percentiles of the overall distribution are used as standards (Infant Physical Development Survey Report 2000). Based on this, the 3rd to 97th percentiles are used as the standard range of total N-acetylneuraminic acid concentration in breast milk. The minimum value of the total N-acetylneuraminic acid concentration in breast milk drunk by infants who have never suffered from bronchitis is 221 mg / L corresponding to the 3rd percentile of the distribution. The total N-acetylneuraminic acid content in breast milk was divided into two groups: samples with a content of 246 mg / L or more and samples with a content of 246 mg / L or less, and the relationship between the total N-acetylneuraminic acid content and whether the infant suffers from bronchitis was tested by Fisher's exact test, reaching p < 0.05 or less. Therefore, when the total N-acetylneuraminic acid content in breast milk is 246 mg / L or more, the risk of suffering from bronchitis has been shown to be reduced.

[0108] Therefore, as a nutritional composition for infants to prevent the onset of bronchitis, the prepared milk needs to contain 221 mg / L or more of N-acetylneuraminic acid. Preferably, it is expected to contain 246 mg / L or more. Assuming that the prepared milk concentration is 13%, the nutritional composition for infants needs to contain at least 170 mg of N-acetylneuraminic acid per 100 g of solid matter. Preferably, it is expected to contain 189 mg or more of N-acetylneuraminic acid.

[0109] The mechanism of the onset of bronchitis is the same in infants and adults, so it is believed that the nutritional composition for infants is effective for adults and the elderly who are at high risk of bronchitis. Therefore, the composition is also effective as a nutritional composition for adults containing the same N-acetylneuraminic acid content as above.

[0110] [Table 2]

[0111] Table 2. Distribution of N-acetylneuraminic acid concentrations in breast milk of mothers whose infants never developed bronchitis

[0112]

[0113] (2) Exploration of N-acetylneuraminic acid compounds for preventing infant bronchitis

[0114] These results suggest that infants who drink breast milk with a high total N-acetylneuraminic acid content have a lower risk of developing bronchitis. Most N-acetylneuraminic acid in breast milk exists as oligosaccharides or glycans of glycoproteins, so we explored which molecular form of N-acetylneuraminic acid helps prevent bronchitis.

[0115] The association between the concentration of oligosaccharides (2 types), N-linked glycans (6 types), and O-linked glycans (5 types) detected in breast milk and the onset of bronchitis in infants was analyzed using logistic regression. Table 3 shows part of the analysis results.

[0116] As a result of the analysis, 6'-SL (p=0.035), disialyl N-linked glycan A (p=0.038), and disialyl N-linked glycan B (p=0.046) were statistically significantly associated with the onset of bronchitis. On the other hand, no such association was observed with the 3'-SL content.

[0117] The regression coefficient and odds ratio between 6'-SL and disialyl N-linked glycans A and B and the onset of bronchitis were negative and less than 1, respectively, indicating that infants drinking breast milk with high levels of 6'-SL and disialyl N-linked glycans A and B had a lower risk of developing bronchitis.

[0118] [Table 3]

[0119] Table 3. Relationship between the onset of bronchitis in infants and N-acetylneuraminic acid-containing glycans in breast milk

[0120]

[0121] (3) Confirmation of the preventive effect of 6'-SL on infantile bronchitis

[0122] The breast milk samples were divided into two groups based on whether the infants had bronchitis or not, and the concentration of 6'-SL in breast milk was compared. As a result, the amount of 6'-SL was significantly higher in breast milk drunk by infants who had never had bronchitis than in breast milk drunk by infants who had had bronchitis ( Figure 2 ). This suggests that a nutritional composition for infants containing the same level of 6'-SL concentration as breast milk drunk by infants who have never had bronchitis is effective for reducing the risk of bronchitis using the nutritional composition for infants. The distribution range of 6'-SL concentration in breast milk drunk by infants who have never had bronchitis was 25.7 to 701 mg / L, with a median of 203 mg / L (Table 4).

[0123] Similar to the distribution of N-acetylneuraminic acid, the distribution of 6'-SL concentration in breast milk is not a normal distribution, but a distribution whose tail extends toward the side with higher concentration. Therefore, the 3rd to 97th percentiles are set as the range of 6'-SL concentration in breast milk. It is believed that the 6'-SL concentration in breast milk drunk by infants who have never suffered from bronchitis is 61.0 mg / L or more corresponding to the 3rd percentile or more. The samples were divided into two groups, namely, samples with a 6'-SL concentration of 135 mg / L or more in breast milk and those with a 6'-SL concentration of 135 mg / L or less in breast milk, and their relationship with the presence or absence of an attack of bronchitis was evaluated using Fisher's exact test. The test result was p<0.05, indicating that a 6'-SL concentration of 135 mg / L or more in breast milk reduces the risk of suffering from bronchitis.

[0124] [Table 4]

[0125] Table 4. Distribution of concentrations of N-acetylneuraminic acid-containing glycans in breast milk of mothers whose infants never developed bronchitis

[0126]

[0127] Therefore, for a nutritional composition for infants that prevents the onset of bronchitis, the adjusted milk needs to contain 61.0 mg / L or more of 6'-SL. Preferably, it is expected to contain 135 mg / L or more. Assuming that the adjusted milk concentration is 13%, the nutritional composition for infants needs to contain at least 46.9 mg or more of 6'-SL per 100 g of solid matter. Preferably, it is expected to contain 104 mg or more of 6'-SL. The mechanism of the onset of bronchitis is the same in infants and adults, so it is believed that the nutritional composition for infants is also effective for adults and the elderly who are at high risk of bronchitis. Therefore, the composition is effective as a nutritional composition for adults having the same 6'-SL content as above.

[0128] (4) Confirmation of the preventive effect of disialyl N-linked glycan A on infantile bronchitis

[0129] The breast milk samples were divided into two groups based on whether the infants had bronchitis or not, and the concentration of disialyl N-linked glycan A in the breast milk was compared. As a result, the amount of disialyl N-linked glycan A was significantly higher in the breast milk drunk by infants who had never had bronchitis than in the breast milk drunk by infants who had had bronchitis ( Figure 3 ). This indicates that a nutritional composition for infants containing a concentration of disialyl N-linked glycan A at the same level as the concentration of disialyl N-linked glycan A in breast milk drunk by infants who have never suffered from bronchitis is effective for reducing the risk of suffering from bronchitis using the nutritional composition for infants. The distribution range of the concentration of disialyl N-linked glycan A in breast milk drunk by infants who have never suffered from bronchitis is 0.0740 to 2.88 μmol / L, and the median is 0.302 μmol / L (Table 4). Similar to the distribution of N-acetylneuraminic acid, the distribution of the concentration of disialyl N-linked glycan A in breast milk is not a normal distribution, but a distribution whose tail extends toward the higher concentration side. Therefore, the 3rd to 97th percentiles are set as the range of the concentration of disialyl N-linked glycan A in breast milk. It is believed that the concentration of disialyl N-linked glycan A in breast milk drunk by infants who have never suffered from bronchitis is 0.0947 μmol / L or more corresponding to the 3rd percentile or more. The breast milk samples were divided into two groups, which were samples with a disialyl N-linked glycan A concentration of 0.313 μmol / L or more in breast milk and samples with a disialyl N-linked glycan A concentration of 0.313 μmol / L or less, and the relationship between the presence or absence of an attack of bronchitis was evaluated using Fisher's exact test. The test result was p<0.05, indicating that a disialyl N-linked glycan A concentration of 0.313 μmol / L or more in breast milk reduces the risk of suffering from bronchitis.

[0130] Therefore, for a nutritional composition for infants that prevents the onset of bronchitis, the adjusted milk needs to contain 0.0947 μmol / L or more of disialyl N-linked glycan A. Preferably, it is expected to contain 0.313 μmol / L or more. Assuming that the adjusted milk concentration is 13%, the nutritional composition for infants needs to contain at least 0.0728 μmol or more of disialyl N-linked glycan A per 100 g of solid matter. Preferably, it is expected to contain 0.241 μmol or more of disialyl N-linked glycan A. Since the mechanism of the onset of bronchitis is the same in infants and adults, it is also believed that containing the nutritional composition for infants is effective for adults and the elderly who are at high risk of bronchitis. Therefore, the composition is also effective as a nutritional composition for adults containing the same content of disialyl N-linked glycan A as above.

[0131] (5) Confirmation of the preventive effect of disialyl N-linked glycan B on infantile bronchitis

[0132] The breast milk samples were divided into two groups based on whether the infants had ever had bronchitis, and the concentration of disialyl N-linked glycan B in breast milk was compared. As a result, the amount of disialyl N-linked glycan B was significantly higher in breast milk drunk by infants who had never had bronchitis than in breast milk drunk by infants who had ever had bronchitis ( Figure 4 ). This indicates that a nutritional composition for infants containing a disialyl N-linked glycan B concentration at the same level as breast milk drunk by infants who have never suffered from bronchitis is effective for reducing the risk of suffering from bronchitis using the nutritional composition for infants. The concentration of disialyl N-linked glycan B in breast milk drunk by infants who have never suffered from bronchitis ranges from 0.0853 to 5.06 μmol / L, with a median of 0.599 μmol / L (Table 4). Similar to the distribution of N-acetylneuraminic acid, the distribution of the concentration of disialyl N-linked glycan B in breast milk is not a normal distribution, but a distribution in which its tail extends toward the higher concentration side. Therefore, the 3rd to 97th percentiles are set as the range of the concentration of disialyl N-linked glycan B in breast milk. It is believed that the concentration of disialyl N-linked glycan B in breast milk drunk by infants who have never suffered from bronchitis is 0.122 μmol / L or more corresponding to the 3rd percentile or more. The samples were divided into two groups, i.e., samples with a disialyl N-linked glycan B concentration of 0.245 μmol / L or more in breast milk and those with a disialyl N-linked glycan B concentration of 0.245 μmol / L or less in breast milk, and their relationship with the presence or absence of an attack of bronchitis was evaluated using Fisher's exact test. The test result was p<0.05 or less, indicating that a disialyl N-linked glycan B concentration of 0.245 μmol / L or more in breast milk reduces the risk of developing bronchitis.

[0133] Therefore, for a nutritional composition for infants that prevents the onset of bronchitis, the adjusted milk needs to contain 0.122 μmol / L or more of disialyl N-linked glycan B. Preferably, it is expected to contain 0.245 μmol / L or more. Assuming that the adjusted milk concentration is 13%, the nutritional composition for infants needs to contain at least 0.0935 μmol or more of disialyl N-linked glycan B per 100 g of solid matter. Preferably, it is expected to contain 0.188 μmol or more of disialyl N-linked glycan B. Since the mechanism of developing bronchitis is the same in infants and adults, containing the nutritional composition for infants is also effective for adults and the elderly who are at high risk of bronchitis. Therefore, the composition is also effective as a nutritional composition for adults having the same disialyl N-linked glycan B content as above.

[0134] (6) Determination of the structures of disialyl N-linked glycans A and B

[0135] In order to study the structures of disialyl N-linked glycans A and B in breast milk that have been shown to be associated with the onset of infantile bronchitis, the retention time of the LC / MS chromatogram was compared with the retention time of α-2,3 or α-2,6-disialyl N-linked glycan standards. As a result, the retention times of both disialyl N-linked glycans A and B were consistent with the retention time of the α-2,6-disialyl N-linked glycan standards.

[0136] When disialyl N-linked glycans A and B were treated with nonspecific sialidase, decomposition of glycans was observed. On the other hand, when they were treated with sialidase that specifically cleaves α-2,3-linked N-acetylneuraminic acid, glycans were not decomposed. From the above, disialyl N-linked glycans A and B were confirmed to be α-2,6-disialyl N-linked glycans represented by the following structure.

[0137] <Disialyl N-linked glycan A>

[0138] Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAc

[0139] <Disialyl N-linked glycan B>

[0140] Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc

[0141] (7) Confirmation of the preventive effect of α-2,6-disialyl N-linked glycans on infantile bronchitis

[0142] The difference in the glycan structure of disialyl N-linked glycans A and B is only whether fucose (fucose) forms a molecule bond. What they both have in common is that there are two molecules of N-acetylneuraminic acid α-2,6-bound to galactose at the non-reducing end. Therefore, it is believed that N-linked glycans with this α-2,6-disialyl bond have the possibility of being associated with infantile bronchitis. Therefore, the correlation between the amount (molar concentration) of α-2,6-disialyl N-linked glycans in breast milk, which is the total amount (molar concentration) of disialyl N-linked glycans A and B in breast milk, and bronchitis was analyzed by logistic regression. The result was p = 0.034, revealing a statistically significant correlation between the amount (molar concentration) of α-2,6-disialyl N-linked glycans in breast milk and the onset of bronchitis (Table 3). This association had a negative regression coefficient and an odds ratio of less than 1, indicating that infants drinking breast milk with a high molar concentration of α-2,6-disialyl N-linked glycans in breast milk had a lower risk of developing bronchitis.

[0143] The breast milk samples were divided into two groups based on whether the infants had ever suffered from bronchitis, and the amount (molar concentration) of the substance of α-2,6-disialyl N-linked glycan in the breast milk was compared. As a result, the amount (molar concentration) of the substance of α-2,6-disialyl N-linked glycan in the breast milk drunk by infants who had never suffered from bronchitis was significantly higher than that in the breast milk drunk by infants who had ever suffered from bronchitis ( Figure 5). This indicates that a nutritional composition for infants containing α-2,6-disialyl N-linked glycans at the same level as breast milk drunk by infants who have never suffered from bronchitis is effective for reducing the risk of suffering from bronchitis using the nutritional composition for infants. The distribution range of the amount of α-2,6-disialyl N-linked glycans in breast milk drunk by infants who have never suffered from bronchitis was 0.179 to 6.76 μmol / L, with a median of 0.933 μmol / L (Table 4). Similar to the distribution of N-acetylneuraminic acid, the distribution of α-2,6-disialyl N-linked glycans in breast milk is not a normal distribution, but a distribution in which its tail extends toward the higher concentration side. Therefore, the concentration range of α-2,6-disialyl N-linked glycans in breast milk is set to the 3rd to 97th percentiles. The α-2,6-disialyl N-linked glycans in breast milk drunk by infants who have never suffered from bronchitis are considered to be 0.257 μmol / L or more corresponding to the 3rd percentile or more. The samples were divided into two groups, namely, samples with α-2,6-disialyl N-linked glycans in breast milk of 0.440 μmol / L or more and those with α-2,6-disialyl N-linked glycans in breast milk of 0.440 μmol / L or less, and their relationship with the presence or absence of an attack of bronchitis was evaluated using Fisher's exact test. The result was p<0.05, indicating that α-2,6-disialyl N-linked glycans above 0.440 μmol / L in breast milk reduce the risk of an attack of bronchitis.

[0144] Therefore, for a nutritional composition for infants that prevents the onset of bronchitis, the adjusted milk needs to contain 0.257 μmol / L or more of α-2,6-disialo N-linked glycans. Preferably, it is expected to contain 0.440 μmol / L or more. Assuming that the adjusted milk concentration is 13%, the nutritional composition for infants needs to contain at least 0.198 μmol or more of α-2,6-disialo N-linked glycans per 100 g of solid matter. Preferably, it is necessary to contain 0.338 μmol or more of α-2,6-disialo N-linked glycans. Since the mechanism of bronchitis is the same in infants and adults, the nutritional composition for infants is also considered to be effective for adults and the elderly who are at high risk of bronchitis. Therefore, the composition is also effective as a nutritional composition for adults containing the same α-2,6-disialo N-linked glycans as above.

[0145] [Example 1] Preparation of a formula milk powder containing N-acetylneuraminic acid

[0146] Fifty-two point seven (52.7) kg of whey powder, 239 kg of skim milk and 28 g of N-acetylneuraminic acid were dissolved in 500 kg of water, mixed with 23.9 kg of vegetable oil, and then the solution was homogenized. The resulting solution was sterilized, concentrated by standard methods, and dried to obtain 99 kg of milk powder. The milk powder was mixed with 1 kg of vitamin and mineral components to finally obtain 100 kg of milk powder. The amount of N-acetylneuraminic acid in the resulting milk powder was 221 mg / 100 g solid.

[0147] [Example 2] Preparation of a formula milk powder containing 6'-sialyllactose

[0148] Fifty-two point seven (52.7) kg of whey powder, 239 kg of skim milk and 50 g of 6'-sialyllactose were dissolved in 500 kg of water, mixed with 23.9 kg of vegetable oil, and then the solution was homogenized. The resulting solution was sterilized, concentrated by standard methods, and dried to obtain 99 kg of milk powder. The milk powder was mixed with 1 kg of vitamin and mineral components to finally obtain 100 kg of milk powder. The amount of 6'-sialyllactose in the resulting milk powder was 50 mg / 100 g of solid.

[0149] [Example 3] Preparation of milk powder containing glycopeptide

[0150] Fifty-two point seven (52.7) kg of whey powder, 239 kg of skim milk and 567 mg of sialic acid glycopeptide were dissolved in 500 kg of water, and the solution was mixed with 23.9 kg of vegetable oil and homogenized. The resulting solution was sterilized, concentrated by standard methods, and dried to obtain 99 kg of milk powder. The milk powder was mixed with 1 kg of vitamins and mineral components to obtain 100 kg of modulated milk powder. The amount of α-2,6-disialyl N-linked polysaccharide in the resulting milk powder was 0.198 μmol / 100 g solid.

[0151] [Example 4] Preparation of milk powder containing glycopeptide

[0152] Fifty-two point seven (52.7) kg of whey powder, 239 kg of skim milk and 421 g of egg yolk powder were dissolved in 500 kg of water, and the solution was mixed with 23.9 kg of vegetable oil and homogenized. The resulting solution was sterilized, concentrated by standard methods, and dried to obtain 99 kg of milk powder. The milk powder was mixed with 1 kg of vitamins and mineral components to finally obtain 100 kg of modulated milk powder. The α-2,6-disialyl N-linked polysaccharide of the obtained milk powder was 0.198 μmol / 100 g solid.

[0153] [Example 5] Production of supplement

[0154] One (1) g of N-acetylneuraminic acid powder was mixed with 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 corn starch and lactose. The mixture was filled into a stick-shaped bag to prepare a supplement for preventing bronchitis of the present invention.

[0155] [Example 6] Production of supplement

[0156] Ten (10) mg of α-2,6-disialyl N-linked glycans were mixed with 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 corn starch and lactose. The mixture was filled into a stick bag to prepare a supplement for preventing bronchitis of the present invention.

[0157] [Example 7] Production of beverages

[0158] The raw materials were mixed according to the composition shown in Table 5, and then filled into a container and heat-sterilized to produce the beverage for preventing bronchitis of the present invention.

[0159] [Table 5]

[0160] Content (weight %) N-Acetylneuraminic acid 2.5 granulated sugar 7.5 Citric Acid 0.6 Apple Juice 10.0 water 79.4

[0161] [Example 8] Production of beverages

[0162] The raw materials were mixed according to the composition shown in Table 6, and filled into a container and then heat-sterilized to manufacture the beverage for preventing bronchitis of the present invention.

[0163] [Table 6]

[0164] Content (weight %) α-2,6-disialyl N-linked glycans 0.1 granulated sugar 7.5 Citric Acid 0.6 Apple Juice 10.0 water 81.8

[0165] Industrial Applicability

[0166] According to the present invention, a nutritional composition containing N-acetylneuraminic acid as an active ingredient can prevent the onset of bronchitis. This ingredient is also an effective ingredient not only for infants but also for the elderly who are at high risk of bronchitis and people with underlying diseases. Therefore, the nutritional composition of the present invention can also prevent the onset of bronchitis in adults and the elderly.

Claims

1. A nutritional composition for preventing bronchitis, comprising N-acetylneuraminic acid as an active ingredient. 2 . The nutritional composition according to claim 1 , wherein N-acetylneuraminic acid is bound to any one or more of protein, peptide, oligosaccharide or lipid.

3. The nutritional composition according to claim 1 or 2, wherein the linkage pattern of N-acetylneuraminic acid is α-2,6 linkage.

4. The nutritional composition according to claim 3, wherein N-acetylneuraminic acid is present as 6'-sialyllactose. The nutritional composition according to claim 3 , wherein N-acetylneuraminic acid is present as an N-linked glycan. The nutritional composition according to claim 5 , wherein the N-linked glycan is an α-2,6-disialyl N-linked glycan.

7. The nutritional composition according to claim 6, wherein the α-2,6-disialyl N-linked glycan has the structure shown in (1) or (2) below: (1)Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAc (2) Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc.

8. The nutritional composition according to any one of claims 1 to 7, wherein the nutritional composition is for infants and young children. 9 . A food or beverage for preventing bronchitis, comprising the nutritional composition for preventing bronchitis according to claim 1 .

10. A medicament for preventing bronchitis, comprising the nutritional composition for preventing bronchitis according to any one of claims 1 to 8.

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