Nutritional composition for preventing food allergy

Abstract

To discover a component for preventing the onset of a food allergy among components of breast milk, and provide a novel nutritional composition.SOLUTION: The present invention relates to a nutritional composition for preventing the onset of a food allergy, the nutritional composition containing, as active ingredients, 3-fucosyllactose, lacto-N-fucopentaose II, and a sugar chain having a Lewis X structure. The nutritional composition is an effective component not only for infants but also for adults having a high risk of food allergy. Therefore, the nutritional composition is also effective as a nutritional composition for preventing the onset of a food allergy when being blended into an adult food or a nutritional composition.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a nutritional composition for preventing food allergies. More specifically, the present invention relates to a nutritional composition for preventing food allergies, which contains one or more active ingredients selected from the group consisting of 3-fucosyllactose, lacto-N-fucopentaose II, and sugar chains having Lewis X structure. [Background technology]

[0002] Food allergy is defined as a phenomenon in which adverse symptoms are induced in the body through antigen-specific immunological mechanisms induced by food. It is a disease in which an allergic reaction occurs upon ingestion or contact with a specific food, and depending on the food, it can cause anaphylactic shock, which can be life-threatening. In some groups, it can later progress to atopic dermatitis or asthma.

[0003] Food allergies often develop during infancy, with chicken eggs and milk being the main causes. From adulthood onwards, the main foods that cause food allergies are shellfish, wheat, fruit, and seafood. Although the prevalence of food allergies peaks at birth and decreases with age, in many cases, management is still necessary even in adulthood. Food allergies can also develop suddenly in adulthood.

[0004] Therefore, preventing food allergies is important for everyone to live a healthy social life, and various methods of prevention have been studied. For example, it has been shown that food allergies may be prevented by restricting the diet of pregnant and breastfeeding mothers and by starting solid food at a certain time. However, there is still not enough evidence, and there is no effective method of preventing food allergies.

[0005] Incidentally, breast milk is known to contain various components that are useful for infants. In searching for an active ingredient that prevents the onset of food allergies, the present inventors focused on the components contained in breast milk. Breast milk contains various useful ingredients, and it has been reported that carbohydrates, particularly oligosaccharides, contained in breast milk may alleviate or reduce allergic reactions (Patent Document 1, Patent Document 2). However, it is unclear whether breast milk components can specifically prevent the onset of food allergies themselves, and it has not been possible to infer what components in breast milk can prevent the onset of food allergies. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special table 2000-504019 publication [Patent Document 2] JP 2021-130665 A Summary of the Invention [Problem to be solved by the invention]

[0007] The present inventors thought that if a component that prevents the onset of food allergies could be found in breast milk, then it would be possible to provide a nutritional composition for infants that prevents the onset of food allergies by incorporating that component into a nutritional composition for infants. Furthermore, because the component is effective not only for infants but also for adults, it would also be possible to incorporate the component into foods and nutritional compositions for adults and the elderly and provide them as nutritional compositions that prevent the onset of food allergies. An object of the present invention is to find components in breast milk that can prevent the onset of food allergies, and to provide a new nutritional composition. [Means for solving the problem]

[0008] In order to search for a component in breast milk that prevents the onset of food allergies, the present inventors statistically analyzed the relationship between the amount of carbohydrate components in breast milk and the history of food allergy onset in infants, and as a result of intensively studying new components that prevent the onset of food allergies, they found that 3-fucosyllactose (hereinafter sometimes simply referred to as 3-FL), lacto-N-fucopentaose-II (hereinafter sometimes simply referred to as LNFP-II), and two types of O-linked glycans (O-linked glycan A and O-linked glycan B) in breast milk are effective in preventing food allergies. Furthermore, as a result of analyzing the structures of O-linked glycan A and O-linked glycan B, they found that both are O-linked glycans with a common Lewis X structure, and thus completed the present invention. That is, the present invention relates to a nutritional composition for infants and a nutritional composition which prevent the onset of food allergy, characterized by containing 3-FL, LNFP-II, or a sugar chain having a Lewis X structure as an active ingredient. Specifically, the present invention has the following configuration.

[0009] <1> A nutritional composition for preventing food allergies that uses a glycan as an active ingredient, the nutritional composition containing one or more of the group consisting of 3-fucosyllactose, a glycan having a Lewis X structure, and lacto-N-fucopentaose II as an active ingredient. <2> The glycan having a Lewis X structure is an O-linked glycan having the structure shown in (1) or (2) below: <1> The nutritional composition described in (1)Neu5Acα2-3Galβ1-3[Galβ1-4(Fucα1-3)GlcNAcβ1-6]GalNAc (2)Galβ1-4(Fucα1-3)GlcNAcβ1-3GalNAc <3> For infants and toddlers, <1> or <2> The nutritional composition described in <4> <1> or <2> A food or drink for preventing food allergies, comprising the nutritional composition for preventing food allergies described in the above. <5> <1> or <2> A pharmaceutical for preventing food allergies, comprising the nutritional composition for preventing food allergies described in claim 1. <6> A nutritional composition for infants and young children containing 3.1 mg / 100 g solid or more of Neu5Acα2-3Galβ1-3[Galβ1-4(Fucα1-3)GlcNAcβ1-6]GalNAc. <7> A nutritional composition for infants and young children containing 0.14 mg / 100 g solid or more of Galβ1-4(Fucα1-3)GlcNAcβ1-3GalNAc. Effect of the Invention

[0010] According to the present invention, a novel nutritional composition for preventing food allergies is provided. The nutritional composition contains one or more active ingredients selected from the group consisting of 3-FL, LNFP-II, and sugar chains having Lewis X structures, and the onset of food allergies can be prevented by ingesting the composition. Moreover, the ingredient can prevent the onset of food allergies not only in infants, but also in adults and the elderly. [Brief description of the drawings]

[0011] [Figure 1] This shows the effect of 3-FL concentration in breast milk on the development of food allergy in infants. [Diagram 2] This shows the effect of LNFP-II concentration in breast milk on the development of food allergy in infants. [Diagram 3] The effect of O-linked glycan A concentration in breast milk on the development of food allergy in infants is shown. [Figure 4] This shows the effect of the concentration of O-linked glycan B in breast milk on the development of food allergy in infants. [Diagram 5] This shows the effect of Lewis X glycan concentration in breast milk on the development of food allergies in infants. [Figure 6] The figure shows the expression level of the TFF3 gene (intestinal barrier-related gene) 48 hours after adding 10 mg / ml of Lewis X sugar chain to LS174T cells, a human colonic goblet cell model. In the figure, * indicates P<0.05. [Figure 7] The expression level of HLA-DR (dendritic cell activation marker) 3 days after adding LPS and various sugar chains to TDDCs, which were differentiated into dendritic cells from the human monocyte-derived cell line THP-1. In the figure, lowercase letters indicate significant differences between different letters. [Figure 8] The expression level of CD86 (dendritic cell activation marker) 3 days after adding LPS and various sugar chains to TDDCs, which were differentiated into dendritic cells from the human monocyte-derived cell line THP-1. In the figure, lowercase letters indicate significant differences between different letters. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] (Active ingredient) The nutritional composition for preventing food allergies of the present invention is characterized by containing at least one of the group consisting of 3-fucosyllactose, lacto-N-fucopentaose II, and a sugar chain having a Lewis X structure as an active ingredient. The nutritional composition of the present invention also contains an effective amount of the sugar chain for preventing food allergies. The following describes the preferred range of each active ingredient and the required amount per 100g of solid matter of the nutritional composition.

[0013] (3-Fucosyl lactose (3-FL)) The 3-fucosyllactose used in the present invention is an oligosaccharide in which fucose and lactose are linked (3-O-(α-L-fucopyranosyl)-4-O-(β-D-galactopyranosyl)-D-glucopyranose). In the present invention, 3-FL may be derived from milk or from fermentation of recombinant bacteria, and may be any of synthetic, semi-synthetic, or natural products. In addition, natural materials containing natural products may be used as a supply source and added to the nutritional composition of the present invention. Here, the natural product means a product extracted from a natural material containing 3-FL by a known method, a crude product, or a product further purified by these. The nutritional composition of the present invention containing 3-FL as an active ingredient preferably contains 70.1 mg / 100 g solid of 3-FL or more, more preferably 348 mg / 100 g solid or more. When the nutritional composition is a powdered milk, the milk after preparation preferably contains 91.1 mg / L or more of 3-FL, more preferably 452 mg / L or more.

[0014] (Lacto-N-fucopentaose-II (LNFP-II)) In the present invention, LNFP-II may be derived from milk or from the fermentation method of genetically modified bacteria, and may be any of synthetic products, semi-synthetic products, or natural products.In addition, natural materials containing natural products may be used as a supply source and incorporated into the nutritional composition of the present invention.Here, natural products refer to those extracted from natural materials containing LNFP-II by known methods, those roughly purified, or those further highly purified. The nutritional composition of the present invention containing LNFP-II as an active ingredient preferably contains LNFP-II at 0.68 mg / 100 g solid or more, more preferably 243 mg / 100 g solid or more. The content of LNFP-II in the prepared milk is preferably 0.88 mg / L or more, more preferably 315 mg / L or more.

[0015] (Sugar chains with Lewis X structure) In the present invention, the sugar chain having a Lewis X structure refers to a sugar chain that has a Galβ1-4(Fucα1-3)GlcNAc structure in the sugar chain structure and reduces the onset of food allergy, specifically, Neu5Acα2-3Galβ1-3[Galβ1-4(Fucα1-3)GlcNAcβ1-6]GalNAc (hereinafter, sometimes simply referred to as O-linked sugar chain A) and Galβ1-4(Fucα1-3)GlcNAcβ1-3GalNAc (hereinafter, sometimes simply referred to as O-linked sugar chain B). Other examples include Galβ1-4(Fucα1-3)GlcNAcβ1-3Gal, Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc, Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc, Galβ1-4(Fucα1-3)GlcNAcβ1-2Manα1-3(Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc, etc. Hereinafter, glycans having Lewis X structures are sometimes simply referred to as "Lewis X glycans".

[0016] The Lewis X sugar chain of the present invention may be in a free state or bound to a peptide and / or protein. It may also be derived from milk, eggs, meat, microorganisms, or fermentation of genetically modified bacteria, and may be any of synthetic, semi-synthetic, or natural products. Natural materials containing natural products may be used as a supply source and added to the nutritional composition of the present invention. Here, the term "natural product" refers to a product extracted from a natural material containing Lewis X sugar chain by a known method, a crude product, or a product further purified by these methods.

[0017] The nutritional composition of the present invention containing Lewis X sugar chain as an active ingredient preferably contains Lewis X sugar chain at 3.3 μmol / 100 g solid or more, more preferably 7.8 μmol / 100 g solid or more. In addition, the content of Lewis X sugar chain in the milk after preparation is preferably 4.3 μmol / L or more, more preferably 10.1 μmol / L or more.

[0018] (O-linked glycan A) In the present invention, the nutritional composition containing O-linked glycan A Neu5Acα2-3Galβ1-3[Galβ1-4(Fucα1-3)GlcNAcβ1-6]GalNAc as an active ingredient preferably contains 3.1 mg / 100 g solid or more of O-linked glycan A, more preferably 8.0 mg / 100 g solid or more. In addition, the O-linked glycan A in the prepared milk is preferably 4.0 mg / L or more, more preferably 10.4 mg / L or more.

[0019] (O-linked glycan B) In the present invention, the nutritional composition containing Galβ1-4(Fucα1-3)GlcNAcβ1-3GalNAc as an active ingredient preferably contains 0.14 mg / 100 g solid or more of O-linked glycan B, more preferably 0.25 mg / 100 g solid or more of O-linked glycan B. The O-linked glycan B in the prepared milk is preferably 0.18 mg / L or more, more preferably 0.33 mg / L or more.

[0020] (Preventive effect against food allergies) The nutritional composition for preventing food allergies of the present invention has the effect of preventing the onset of food allergies by ingesting effective amounts of 3-FL, LNFP-II, and Lewis X sugar chain, and the mechanism of action includes strengthening the intestinal barrier function and inhibiting the activation of dendritic cells, as shown in the examples described below. It is believed that the above action provides a preventive effect against food allergies regardless of the type of food.

[0021] (Nutritional compositions, food and beverages, pharmaceuticals) The nutritional composition for preventing food allergies containing the 3-FL, LNFP-II, and Lewis X glycan of the present invention (hereinafter, these may be collectively referred to as simply 3-FL, etc.) as active ingredients may be any composition containing an effective amount of 3-FL, etc., and may contain proteins, fats, carbohydrates, vitamins, minerals, etc. in addition to the active ingredients, and may be used as a nutritional composition for infants, as well as adults and the elderly. Nutritional compositions for infants and young children include infant formulas and liquid formulas, toddler formulas (follow-up milk and growing-up milk), formulas for low birth weight infants, allergen-free foods for those with cow's milk allergies, and lactose-free foods for those with lactose intolerance. Nutritional compositions for adults and the elderly include powdered milk formulas and liquid milk formulas for adults, allergen-free foods for those with milk allergies, lactose-free foods for those with lactose intolerance, and supplements. Furthermore, the food and drink of the present invention containing 3-FL etc. for preventing food allergies may be blended into any food and drink as long as it contains an effective amount of 3-FL etc., or may be added to the raw materials during the manufacturing process of the food and drink. Examples of foods and drinks include, but are not limited to, dairy products such as cheese, fermented milk, 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, and sweets and breads such as butter cake, etc. The nutritional composition for preventing food allergies of the present invention can be used as a raw material for pharmaceuticals as it is, and may be manufactured into tablets, capsules, powders, syrups, etc. by conventional methods. Therefore, when preparing a pharmaceutical product containing the food allergy prevention composition of the present invention containing 3-FL or the like as an active ingredient, it can be formulated by appropriately mixing it with excipients, stabilizers, flavorings, etc. that are approved for formulation, or the composition containing 3-FL or the like can be dried as it is and used as a powder or dust. In addition, it can be formulated by mixing excipients, binders, disintegrants, lubricants, flavorings, suspending agents, coating agents, and other optional drugs within a range that does not interfere with the food allergy prevention effect. The dosage form can be tablets, capsules, granules, powders, dusts, syrups, etc.

[0022] (Nutritional composition for infants and young children) A further aspect of the present invention relates to a nutritional composition for infants containing a certain amount or more of a specific Lewis X sugar chain, i.e., a nutritional composition for infants containing 3.1 mg / 100 g solid or more of Neu5Acα2-3Galβ1-3[Galβ1-4(Fucα1-3)GlcNAcβ1-6]GalNAc, or a nutritional composition for infants containing 0.14 mg / 100 g solid or more of Galβ1-4(Fucα1-3)GlcNAcβ1-3GalNAc. EXAMPLES

[0023] [Test Example 1] The carbohydrate components of 155 breast milk samples were measured, and the relationship with the onset of food allergies in the infants' first year of life was analyzed using logistic regression. The method and results are shown below.

[0024] 1. Method (1) Investigation of breast milk samples and infant infection histories In a prospective cohort study (UMIN ID 000015494) conducted by Snow Brand Bean-Stalk Co., Ltd. between 2014 and 2019 to investigate the relationship between breast milk components and the health status of mothers and children in Japan, breast milk samples collected from 1,210 mothers and questionnaire results investigating the health status of mothers and children were used in this study. In addition, 200 samples were randomly selected from the 1,210 samples. Breast milk collected between 1 and 2 months after birth was used as the breast milk samples for analysis. The questionnaire investigating the health status of the infant was filled out by the parents a total of six times every two months over the course of one year after birth, and the results were collected. From the questionnaire results, the results of whether or not the infant had been diagnosed with a food allergy by a doctor within one year after birth were tallied. If the statement regarding the presence or absence of a diagnosis was unclear, the infant was excluded from the survey. As a result, 155 samples were properly answered regarding the presence or absence of a food allergy in a total of six questionnaires over the course of one year, and these were the subjects of analysis in this survey. Of the 155 samples, 18 were from infants who were diagnosed with food allergies within one year of birth.

[0025] (2) Measurement of various carbohydrate components in breast milk (2-1) Measurement of oligosaccharides 580μL of ultrapure water was added to 20μL of breast milk from 155 samples to be analyzed, and then ultrafiltration with a molecular weight cutoff of 10,000 was performed, and the permeate was analyzed by HPLC. For the HPLC, a DIONEX ICS-5000DP system (Thermo Fisher Scientific Co., Ltd.) connected to an electrochemical detector and a CarboPac PA1 column (Thermo Fisher Scientific Co., Ltd.) were used. The mobile phase consisted of three solutions: ultrapure water (solution A), 200mM sodium hydroxide solution (solution B), and 100mM sodium hydroxide solution containing 600mM sodium acetate (solution C) at a flow rate of 1mL / min. Solution B was passed at 50% for 10 minutes from the start, and then solution B was linearly decreased to 47.5% by 18 minutes, and solution C was linearly increased to 5%. From 18 minutes to 28 minutes, solution B was passed at 47.5% and solution C was passed at 5%. From 28 to 32 minutes, solution B was linearly decreased to 42%, and solution C was linearly increased to 16%. From 32 to 39 minutes, 42% solution B and 16% solution C were passed through, and then from 39 to 43 minutes, 30% solution B and 40% solution C were passed through. The content of each oligosaccharide in each breast milk sample was calculated from a calibration curve prepared using the standard products of known concentrations of 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3'-sialyllactose, 6'-sialyllactose, lactodifucotetraose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II (LNFP-II), lacto-N-difucohexaose I, and lacto-N-difucohexaose II.

[0026] (2-2) Measurement of N-linked glycans 10 μL of 3M sodium borohydride aqueous solution was added to 20 μL of breast milk from 155 samples to be analyzed, and the mixture was left to stand at room temperature for 30 minutes. Then, 7.5 μL of acetic acid was added and the mixture was left to stand at room temperature for 30 minutes. 15 μL of 1M ammonium bicarbonate aqueous solution and 7.5 μL of ultrapure water were added. Then, 5 μL of 400 mM dithiothreitol aqueous solution containing 5% sodium lauryl sulfate was added, and the mixture was heated at 100 °C for 10 minutes to denature the protein. The mixture was allowed to cool to room temperature, and 10 μL of 123 mM iodoacetamide aqueous solution was added and the mixture was left to stand at room temperature for 1 hour. After adding 7 μL of 500 mM sodium acetate aqueous solution (pH 6.0) and 9 μL of 10% NP-40 solution, 10 μL of 500 units / μL Peptide-N-glycanase F solution was added, and the mixture was left to stand at 37 °C for 24 hours to release N-linked glycans. After adding 109 μL of ultrapure water, the entire amount was introduced into Vivaspin 500 (Sartorius Co., Ltd.) with a molecular weight cutoff of 10,000, and centrifuged at 15,000 × g and 25 ° C for 10 minutes to recover the permeate. 50 μL of acetonitrile was added to 50 μL of the permeate and subjected to LC / MS analysis. LC / MS analysis was performed as follows. HPLC was performed using UltiMate 3000 (Thermo Fisher Scientific Co., Ltd.), and MS was performed using Q-Exactive (Thermo Fisher Scientific Co., Ltd.). The column used was GlycanPac AXH-1 (Thermo Fisher Scientific Co., Ltd.), and the column temperature was set to 40 ° C. The mobile phase used was acetonitrile (liquid A) and a 50 mM ammonium formate aqueous solution (liquid B) with a pH adjusted to 4.4, and the flow rate was 0.4 mL / min. After sample introduction, the ratio of solution B was increased linearly from 10% to 35% by 55 min, and then solution B was passed at 35% from 55 min to 65 min. The spray voltage of the ESI probe was 3.5 kV, the capillary temperature was 275°C, and nitrogen gas was used as the sheath gas, auxiliary gas, and collision gas. The S-Lens RF Level was set to 90. MS spectra were acquired by full scan measurement in the positive mode in the range of m / z 400-2000.The acquired MS spectra were analyzed using Compound Discover 2.1 (Thermo Fisher Scientific). Glycans were detected by comparing them with a glycan database constructed from previously reported N-linked glycans of milk proteins. In addition, an extraction chromatogram corresponding to the molecular ion peak of each glycan was created using Compound Discover, and the signal area value of each glycan detected in each breast milk sample was calculated. The calculated signal area value of the glycan was used as the relative quantitative value of each glycan.

[0027] (2-3) Measurement of O-linked glycans The 155 breast milk samples to be analyzed were added with the glycan release reagent included in the O-linked glycan analysis kit EZGlyco Prep kit (Sumitomo Bakelite Co., Ltd.) and heated at 50°C for 20 minutes. After dilution with ultrapure water, the mixture was introduced into a Vivaspin 500 with a molecular weight cutoff of 10,000, centrifuged at 15,000×g and 25°C for 15 minutes, and the permeate was collected. 50 μL of the permeate was added with 50 μL of acetonitrile and subjected to LC / MS analysis. The HPLC was performed using an UltiMate 3000, and the MS was performed using a Q-Exactive. The column used was a GlycanPac AXH-1, and the column temperature was set to 40°C. The mobile phase used was acetonitrile (liquid A) and a 50 mM ammonium formate aqueous solution (liquid B) with a pH adjusted to 4.4, and the flow rate was 0.4 mL / min. After sample introduction, the proportion of solution B was increased linearly from 10% to 35% by 55 minutes, and then solution B was passed at 35% 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 gas was used for the sheath gas, auxiliary gas, and collision gas. The S-Lens RF Level was set to 90. MS spectra were acquired by full scan measurement in the range of m / z 300-2000 in positive mode. The acquired MS spectra were analyzed using Compound Discover 2.1 (Thermo Fisher Scientific Co., Ltd.). Glycans were detected by matching with the O-linked glycan database. In addition, an extraction chromatogram corresponding to the molecular ion peak of each glycan was created by Compound Discover, and the signal area value of each glycan detected in each breast milk sample was calculated. The calculated signal area value of the glycan was used as the relative quantitative value of each glycan.

[0028] (3) Structural determination of O-linked glycan A and O-linked glycan B (3-1) Structural analysis One of the 155 samples prepared in the above "O-linked glycan measurement" was subjected to LC / MS / MS measurement, and the structures of O-linked glycan A and O-linked glycan B were analyzed. LC / MS / MS analysis was performed as follows. An UltiMate 3000 was used for HPLC, and a Q-Exactive was used for MS. A GlycanPac AXH-1 was used as the column, and the column temperature was set to 40°C. Acetonitrile (liquid A) and a 50 mM ammonium formate aqueous solution (liquid B) adjusted to pH 4.4 were used as the mobile phase, and the flow rate was 0.4 mL / min. After sample introduction, the proportion of liquid B was increased linearly from 10% to 35% by 55 minutes, and then 35% of liquid B was used 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 gas was used as the 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-dependently in Top10 mode. The acquired MS / MS spectra were analyzed using SimGlycan Software (PREMIER Biosoft International), and the structures of O-linked glycan A and O-linked glycan B were analyzed.

[0029] (3-2) Determination of binding mode To determine the linkage mode of N-acetylneuraminic acid in O-linked glycan A, the samples were treated with either a sialidase that specifically degrades α2,3 linkages or a sialidase that degrades both α2,3 and α2,6 linkages, and then analyzed by LC / MS. To determine the linkage mode of fucose in O-linked glycan A and O-linked glycan B, the samples were treated with either a fucosidase that specifically degrades α1,2 linkages, a fucosidase that specifically degrades α1,3 and α1,4 linkages, or a fucosidase that specifically degrades α1,2, α1,4 and α1,6 linkages, and then analyzed by LC / MS. Furthermore, to determine the binding mode of galactose, the samples were treated with either a fucosidase that specifically degrades α1,3 and α1,4 bonds and a galactosidase that specifically degrades β1,3 bonds simultaneously, or a fucosidase that specifically degrades α1,3 and α1,4 bonds and a galactosidase that specifically degrades β1,4 bonds simultaneously, and then analyzed by LC / MS. For one sample identical to that in (3-1) above, 27 μL of ultrapure water, 5 μL of the buffer solution provided with the enzyme, and 5 μL of the bovine serum albumin solution provided with the enzyme were added to 10 μL of O-linked glycan solution, followed by addition of 3 μL of each enzyme solution and incubation at 37°C for 1 hour. After heating in boiling water for 5 minutes to inactivate the enzyme, the samples were treated in the same manner as in (2-4) above and subjected to LC / MS analysis. LC / MS analysis was performed as follows. An UltiMate 3000 was used for HPLC, and a Q-Exactive was used for MS. The column used was a GlycanPac AXH-1, and the column temperature was set to 40°C. The mobile phase was acetonitrile (liquid A) and a 50 mM ammonium formate aqueous solution (liquid B) adjusted to pH 4.4, and the flow rate was 0.4 mL / min. After sample introduction, the proportion of liquid B was linearly increased from 10% to 35% by 55 minutes, and then 35% of liquid 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 gas was used as the sheath gas, auxiliary gas, and collision gas. The S-Lens RF Level was set to 90. The acquired MS spectra were analyzed using FreeStyle software (Thermo Fisher Scientific Co., Ltd.).

[0030] (4) Absolute quantification of O-linked glycan A and O-linked glycan B The absolute amounts in breast milk of O-linked glycan A and O-linked glycan B, which have been found to be associated with the onset of food allergies, were measured. The contents of O-linked glycan A and O-linked glycan B were measured by HPLC connected to a mass spectrometer (MS) and a fluorescence detector (FL) after fluorescent labeling of O-linked glycan A and O-linked glycan B. The contents of O-linked glycan A and O-linked glycan B in breast milk were calculated by dividing the results by the recovery rate of the O-linked glycan analysis kit, which was calculated separately.

[0031] The details of the measurement of O-linked glycan A and O-linked glycan B are described below. 10 μL of breast milk from one sample randomly selected from the 155 breast milk samples analyzed was subjected to 2-aminobenzamide (AB) labeling and purification using the O-linked glycan analysis kit EzGlyco O-glycan Prep kit (Sumitomo Bakelite), and then subjected to LC / MS analysis. The HPLC was performed on an UltiMate 3000 and the MS on a Q-Exactive. The column used was a GlycanPac AXH-1, and the column temperature was set at 40°C. The mobile phase used was acetonitrile (liquid A) and a 50 mM ammonium formate aqueous solution (liquid B) adjusted to pH 4.4, and the flow rate was 0.4 mL / min. After sample introduction, the proportion of solution B was increased linearly from 10% to 35% by 55 min, and then solution B was passed at 35% from 55 min to 65 min. The spray voltage of the ESI probe was 3.5 kV, the capillary temperature was 275°C, and nitrogen gas was used for the sheath gas, auxiliary gas, and collision gas. The S-Lens RF level was set to 90. MS measurements were performed in positive mode with selected ion monitoring of m / z 853.3566 and m / z 1306.5049. Fluorescence measurements were performed at an excitation wavelength of 330 nm and an emission wavelength of 420 nm. The acquired MS chromatograms and fluorescence chromatograms were analyzed using FreeStyle software to obtain the fluorescence area values ​​of O-linked glycan A and O-linked glycan B. O-linked glycan A and O-linked glycan B were quantified from a calibration curve prepared using a fluorescently labeled O-linked glycan standard (Neu5Acα2,3Galβ1,3GalNAc-AB) with a known concentration. Furthermore, the contents of O-linked glycan A and O-linked glycan B in breast milk were calculated by dividing the results by the recovery rate of the kit calculated separately. The absolute quantitative values ​​of O-linked glycan A and O-linked glycan B in each of the 155 breast milk samples were obtained using the relative quantitative values ​​of the 155 breast milk samples obtained by the above-mentioned "O-linked glycan measurement."

[0032] (5) Analysis of the relationship between the content of various carbohydrate components in breast milk and food allergies The 155 breast milk samples were divided into two groups, secretory breast milk (2'-FL>100 mg / L) and nonsecretory breast milk (2'-FL<100 mg / L), and the relationship to food allergy was evaluated using Fisher's exact test. In addition, the relationship between the concentrations of oligosaccharides, N-linked glycans, and O-linked glycans and the onset of food allergy in infants was analyzed using logistic regression. The sex of the infant, the presence or absence of siblings, the method of delivery (natural birth, Caesarean section), and birth weight were used as adjustment factors.

[0033] (6) Evaluation of the relationship between Lewis X glycans and intestinal barrier function (6-1) Cells and reagents Human colonic goblet cell model LS174T cells (EC87060401-F0) were purchased from KAC Co., Ltd. The medium used was MEM containing Earle's salts, NEAA, and L-glutamine (21443-15, Nacalai) supplemented with 10% FBS (Gibco) and 1% P / S (15140-122, Gibco). Culture was performed at 37°C under 5% CO2 conditions. Lewis X (OL06490) was purchased from Biosynth.

[0034] (6-2) Sample addition test to LS174T cells 1 x 10 LS174T cells 5 The cells were seeded in a 48-well plate (3830-048, AGC Technoglass Co., Ltd.) at 16.5 cells / well. After overnight incubation, the plate was washed once with PBS, and the medium was replaced with medium containing 5 or 10 mg / ml of each sample, followed by incubation for 48 hours.

[0035] (6-3) RNA extraction, cDNA synthesis, and gene expression measurement The cells were washed once with PBS, and total RNA was extracted using a nucleic acid extraction reagent (Maxwell® RSC simplyRNA Tissue, AS1340, Promega) and an instrument (Maxwell® RSC Instrument, Promega). The extracted total RNA was reverse transcribed using ReverTra Ace qPCR RT Master Mix (FSQ-201, Toyobo) to obtain cDNA. The expression levels of the TFF3 gene (TFF3) and the housekeeping gene 18S rRNA gene (18S rRNA) were measured for the obtained cDNA using THUNDERBIRD® NextSYBR® qPCR Mix (QPX-201, Toyobo) and QuantStudio 6 Pro (Therno Fisher Scientific). Standard curves were created for each gene to calculate the relative expression levels, which were then corrected by the 18S rRNA expression level. The primers used are shown in the table below.

[0036] [Table 1]

[0037] (6-4) Statistical analysis Data are shown as arithmetic mean + standard deviation. Comparison between two groups was performed using Student's t-test. Multiple comparison with the control group was performed using Dunnett's test. The significance level was set at P = 0.05.

[0038] (7)Assessment of the association between 3-FL and dendritic cells (7-1) Cells and reagents Human monocyte-derived cell line THP-1 was obtained from ATCC. The medium used was RPMI 1640 medium (30264-85, Nacalai) supplemented with 10% FBS (Gibco), 1% P / S (15140-122, Gibco) and 0.05 mM 2-mercaptoethanol (133-14571, Fujifilm Wako Pure Chemical Industries, Ltd.). The cells were cultured at 37°C and 5% CO2. Lactose Monohydrate (128-00095) was purchased from Fujifilm Wako Pure Chemical Industries, Ltd. 2'-FL (OF137553) and 3-FL (OF05673) were purchased from Carbosynth. Lewis A Trisaccharide (OL04541) and Lewis X Trisaccharide (OL06490) were purchased from Biosynth. Human Recombinant GM-CSF (17365-88) and Human Recombinant IL-4 (19268-08) were purchased from PeproTech. Cell Staining buffer (420201, Biolegend), FITC anti-human CD86 Antibody(374203), PE anti-human HLA-DR Antibody(307605), FITC Mouse IgG1, κ Isotype Ctrl Antibody(400107), PE Mouse IgG2a,κ isotype Ctrl Antibody(400211), Zombie Aqua Fixable Viability Kit (423101) and Human TruStain FcX TM (422302) was purchased from Biolegend.

[0039] (7-2) THP-1 derived dendritic cells ( T HP-1 D erived D endritic C ell, TDDC) 5 × 10 THP-1 cells 5 The cells were seeded in a 6-well plate (3810-006, AGC Technoglass Co., Ltd.) at 2 × 10 cells / ml and cultured in a medium containing 1500 U / ml GM-CSF and 1500 U / ml IL-4 for 6 days (medium was changed on day 3) to differentiate into TDDCs. TDDCs were harvested on day 6 and cultured at 2 × 10 5The cells were seeded into a 96-well plate (3860-096, AGC Technoglass Co., Ltd.) at 16 cells / well and cultured for 3 days in medium containing 100 ng / ml LPS and 10 mg / ml of various glycans (2'-FL, 3-FL, Lewis A glycan, and Lewis X glycan).

[0040] (7-3) Surface antigen staining and FACS analysis The harvested TDDCs were washed with PBS and the viable cell count was determined to be 1 × 10 5 The cells were placed in a FACS tube so that the number of cells / tube was 1 or more. The cells were centrifuged to remove the supernatant, and Zombie Dye / PBS was added and incubated at room temperature in the dark for 15 minutes. After 15 minutes, the cells were washed with Cell Staining buffer. The cells were centrifuged to remove the supernatant, and 0.1% PFA / PBS was added and incubated at room temperature in the dark for 20 minutes. After 20 minutes, the cells were washed with Cell Staining buffer. The cells were centrifuged to remove the supernatant, and Human TruStain FcX was added. TM After adding Cell Staining buffer, the cells were incubated at 4°C for 10 minutes in the dark. After 10 minutes, the cells were washed with Cell Staining buffer. The supernatant was removed by centrifugation, and various cell surface staining solutions diluted with Cell Staining buffer were added and incubated for 30 minutes in the dark. After 30 minutes, the cells were washed with Cell Staining buffer and incubated with BDFACSCanto TM The results were analyzed using a BD Biosciences II flow cytometer. Median fluorescence intensity (MFI) of HLA-DR and CD86 was calculated from the flow cytometry analysis results.

[0041] (7-4) Statistical analysis Data are shown as arithmetic mean ± standard deviation. Intergroup comparisons were performed using Tukey-Kramer's test. The significance level was set at P = 0.05.

[0042] 2.Results (1) The relationship between the content of various carbohydrate components in secretory and non-secretory breast milk and the onset of food allergies in infants When 155 breast milk samples were divided into two groups, secretory breast milk (2'-FL>100 mg / L) and non-secretory breast milk (2'-FL<100 mg / L), no food allergies were observed in infants who drank non-secretory breast milk (Table 2). In addition, Fisher's exact test was used to examine the relationship between the incidence of food allergies in infants who drank both groups, with p<0.05, indicating that the incidence of food allergies was significantly lower in infants who drank non-secretory breast milk.

[0043] [Table 2]

[0044] The above results clarified that infants who drank non-secretory breast milk had a lower risk of developing food allergies. Therefore, we investigated which components in non-secretory breast milk are related to the prevention of the onset of food allergies. The relationship between the concentrations of oligosaccharides (4 types), N-linked glycans (2 types), and O-linked glycans (5 types) that were higher in non-secretory breast milk compared to secretory breast milk and the onset of food allergies in infants was analyzed using logistic regression. Some of the analysis results are shown in Table 3. The analysis showed a significant association between 3-FL (p=0.034), LNFP-II (p=0.035), and O-linked glycan A (p=0.022) and food allergy, and a tendency for an association between O-linked glycan B (p=0.089) and food allergy. The regression coefficients between these carbohydrate components and the onset of food allergy were negative, and the odds ratios were less than 1, indicating that infants who drank breast milk with a high content of 3-FL, LNFP-II, O-linked glycan A, or O-linked glycan B have a lower risk of developing food allergy.

[0045] [Table 3]

[0046] (2) Confirmation of the effectiveness of 3-FL in preventing the onset of food allergies in infants Breast milk samples were divided into two groups based on whether the infants had developed food allergies, and the 3-FL concentrations in breast milk were compared. The results showed that the 3-FL concentrations in breast milk from infants who did not develop food allergies tended to be higher than those from infants who did develop food allergies (Figure 1). This indicates that infant nutritional compositions containing the same level of 3-FL as breast milk from infants who did not develop food allergies are effective in reducing the risk of developing food allergies. The distribution range of 3-FL concentrations in breast milk from infants who did not develop food allergies was 38 to 2341 mg / L, with a median of 557 mg / L (Table 4). The distribution of 3-FL concentrations was different from a normal distribution, with the tail spreading toward the higher concentration side. This type of distribution is also observed in the distribution of height and weight of infants, and when creating growth curves for infants, the 3rd to 97th percentile of the overall distribution is used as the standard (Infant Physical Development Survey Report 2000). Based on this, the range of 3-FL concentrations in breast milk was set as the standard, from the 3rd to the 97th percentile. The lowest 3-FL concentration in breast milk consumed by infants who did not develop food allergies was 91.1 mg / L, which corresponds to the 3rd percentile of the distribution. In addition, breast milk 3-FL concentrations were divided into two groups, samples with 452 mg / L or higher and samples with a lower concentration, and the relationship with the presence or absence of food allergies was subjected to Fisher's exact test, with p<0.05 or lower, indicating that breast milk 3-FL concentrations of 452 mg / L or higher are associated with a lower risk of developing food allergies. Therefore, in order to prevent the onset of food allergies, it is necessary for the infant nutritional composition to contain 91.1 mg / L or more of 3-FL in the prepared milk, and preferably 452 mg / L or more. If the concentration of the prepared milk is 13%, it is necessary to incorporate at least 70.1 mg of 3-FL per 100 g of solid matter in the infant nutritional composition, and preferably 348 mg or more of 3-FL. In addition, because the mechanism of food allergy onset is the same in infants and adults, the formulation of this nutritional composition for infants is also considered to be effective for adults and the elderly who are at high risk of food allergies. Therefore, the same 3-FL content as above is also effective as a nutritional composition for adults.

[0047] [Table 4]

[0048] (3) Confirmation of the effectiveness of LNFP-II in preventing food allergy in infants Breast milk samples were divided into two groups based on the presence or absence of food allergy in the infants, and the LNFP-II concentrations in breast milk were compared. As a result, the breast milk of infants who did not develop food allergies tended to have higher LNFP-II concentrations than the breast milk of infants who did develop food allergies (Figure 2). This indicates that an infant nutritional composition containing the same level of LNFP-II concentration as that of breast milk of infants who did not develop food allergies is effective in reducing the risk of developing food allergies in infant nutritional compositions. The distribution range of LNFP-II concentrations in breast milk of infants who did not develop food allergies was 0 to 4123 mg / L, with a median of 388 mg / L (Table 4). As with the distribution of 3-FL, the distribution of LNFP-II concentrations in breast milk was not a normal distribution, but was a distribution with a wider tail toward the higher concentration side, so the 3rd to 97th percentile was set as the concentration range of LNFP-II in breast milk. The LNFP-II concentration in breast milk consumed by infants who did not develop food allergies is considered to be 0.88 mg / L or higher, which corresponds to the 3rd percentile or higher. In addition, samples were divided into two groups: those with LNFP-II concentrations of 315 mg / L or higher, and those with concentrations below that level. The relationship between the presence or absence of food allergies was evaluated using Fisher's exact test, with a p<0.05 result, indicating that those with LNFP-II concentrations of 315 mg / L or higher in breast milk have a lower risk of developing food allergies. Therefore, as an infant nutritional composition that prevents the onset of food allergy, it is necessary that the milk after preparation contains 0.88 mg / L or more of LNFP-II, and preferably 315 mg / L or more. If the concentration of the milk preparation is 13%, it is necessary to incorporate at least 0.68 mg or more of LNFP-II per 100 g of solid matter in the infant nutritional composition, and preferably 243 mg or more of LNFP-II is incorporated. Since the mechanism of onset of food allergy is the same in infants and adults, it is considered that the incorporation of this infant nutritional composition is also effective for adults and elderly people who are at high risk of food allergy. Therefore, it is also effective as an adult nutritional composition containing the same LNFP-II content as above.

[0049] (4) Confirmation of the preventive effect of O-linked glycan A on the onset of food allergies in infants Breast milk samples were divided into two groups based on the presence or absence of food allergy in infants, and the concentration of O-linked glycan A in breast milk was compared. As a result, the amount of O-linked glycan A in breast milk drank by infants who did not develop food allergies was significantly higher than that in breast milk drank by infants who developed food allergies (Figure 3). This indicates that an infant nutritional composition containing a concentration of O-linked glycan A equivalent to that of breast milk drank by infants who did not develop food allergies is effective in reducing the risk of developing food allergies in infant nutritional compositions. The distribution range of O-linked glycan A concentrations in breast milk drank by infants who did not develop food allergies was 0.33 to 321 mg / L, with a median of 15.2 mg / L (Table 4). As with the distribution of 3-FL, the concentration distribution of O-linked glycan A in breast milk was not a normal distribution, but was a distribution with a wider tail toward the higher concentration, so the concentration range of O-linked glycan A in breast milk was set to the 3rd to 97th percentile. The O-linked glycan A concentration in breast milk consumed by infants who did not develop food allergies is considered to be 4.0 mg / L or higher, which corresponds to the 3rd percentile or higher. In addition, samples were divided into two groups, those with O-linked glycan A concentrations of 10.4 mg / L or higher, and those with concentrations below 10.4 mg / L. The relationship between the presence or absence of food allergy was evaluated using Fisher's exact test, with p<0.05, indicating that a breast milk O-linked glycan A concentration of 10.4 mg / L or higher reduces the risk of developing food allergies. Therefore, as an infant nutritional composition that prevents the onset of food allergy, it is necessary that the milk after preparation contains 4.0 mg / L or more of O-linked glycan A, and preferably 10.4 mg / L or more. If the concentration of the prepared milk is 13%, it is necessary to incorporate at least 3.1 mg or more of O-linked glycan A per 100 g of solid matter in the infant nutritional composition, and preferably 8.0 mg or more of O-linked glycan A is incorporated. Since the mechanism of onset of food allergy is the same in infants and adults, it is considered that the formulation of this infant nutritional composition is also effective for adults and elderly people who are at high risk of food allergy. Therefore, it is also effective as a nutritional composition for adults that contains the same O-linked glycan A content as above.

[0050] (5) Confirmation of the preventive effect of O-linked glycan B on food allergy in infants Breast milk samples were divided into two groups based on the presence or absence of food allergy in infants, and the concentration of O-linked glycan B in breast milk was compared. As a result, the amount of O-linked glycan B in breast milk drank by infants who did not develop food allergies was significantly higher than that in breast milk drank by infants who developed food allergies (Figure 4). This indicates that an infant nutritional composition containing a concentration of O-linked glycan B at the same level as that of breast milk drank by infants who did not develop food allergies is effective in reducing the risk of developing food allergies in infant nutritional compositions. The distribution range of O-linked glycan B concentrations in breast milk drank by infants who did not develop food allergies was 0.07 to 22.1 mg / L, with a median of 1.26 mg / L (Table 4). As with the distribution of 3-FL, the distribution of O-linked glycan B concentrations in breast milk was not a normal distribution, but was a distribution with a wider tail toward the higher concentration side, so the concentration range of O-linked glycan B in breast milk was set to the 3rd to 97th percentile. The O-linked glycan B concentration in breast milk consumed by infants who did not develop food allergies is considered to be 0.18 mg / L or higher, which corresponds to the 3rd percentile or higher. In addition, samples were divided into two groups, samples with di-O-linked glycan B concentrations of 0.33 mg / L or higher, and samples with concentrations below 0.33 mg / L, and the relationship with the onset of food allergies was evaluated using Fisher's exact test, with p<0.05 or lower, indicating that a breast milk O-linked glycan B concentration of 0.33 mg / L or higher reduces the risk of developing food allergies. Therefore, as an infant nutritional composition that prevents the onset of food allergy, it is necessary that the milk after preparation contains 0.18 mg / L or more of O-linked glycan B, and preferably 0.33 mg / L or more. If the concentration of the prepared milk is 13%, it is necessary to incorporate at least 0.14 mg or more of O-linked glycan B per 100 g of solid matter in the infant nutritional composition, and preferably 0.25 mg or more of O-linked glycan B is incorporated. Since the mechanism of onset of food allergy is the same in infants and adults, it is considered that the formulation of this infant nutritional composition is also effective for adults and elderly people who are at high risk of food allergy. Therefore, it is also effective as a nutritional composition for adults that contains the same O-linked glycan B content as above.

[0051] (6) Determination of the structures of O-linked oligosaccharide A and O-linked oligosaccharide B To investigate the structures of O-linked oligosaccharide A and O-linked oligosaccharide B in breast milk, which have been found to be related to the onset of food allergies in infants, LC / MS measurement and LC / MS / MS measurement were carried out. As a result, O-linked oligosaccharide A was estimated to be Neu5Ac-Galβ1-3[Gal-(Fuc-)GlcNAcβ1-6]GalNAc, and O-linked oligosaccharide B was estimated to be Gal-(Fuc-)GlcNAcβ1-3GalNAc. To determine the binding mode of fucose in O-linked oligosaccharide A and O-linked oligosaccharide B, LC / MS measurement was performed after treatment with any one of fucosidases that specifically cleave α1,2 linkages, fucosidases that specifically cleave α1,3 and α1,4 linkages, or fucosidases that specifically cleave α1,2, α1,4, and α1,6 linkages. As a result, degradation of O-linked oligosaccharide A and O-linked oligosaccharide B was observed only when fucosidase that specifically cleaves α1,3 and α1,4 linkages was used. Therefore, it was revealed that the fucose in O-linked oligosaccharide A and O-linked oligosaccharide B is an α1,3 linkage. Similarly, when galactosidase that specifically cleaves β1,4 linkages was allowed to act simultaneously with fucosidase that specifically cleaves α1,3 and α1,4 linkages, degradation of the oligosaccharide was observed. Therefore, it was revealed that the galactose bound to GlcNAc in O-linked oligosaccharide A and O-linked oligosaccharide B is a β1,4 linkage. Furthermore, since O-linked oligosaccharide A was degraded by sialidase that specifically cleaves α2,3 linkages, it was revealed that the N-acetylneuraminic acid in O-linked oligosaccharide A is an α2,3 linkage. From the above, it was shown that O-linked oligosaccharide A and O-linked oligosaccharide B are O-linked oligosaccharides represented by the following structures.

[0052] <O-linked oligosaccharide A> Neu5Acα2-3Galβ1-3[Galβ1-4(Fucα1-3)GlcNAcβ1-6]GalNAc <O-linked oligosaccharide B> Galβ1-4(Fucα1-3)GlcNAcβ1-3GalNAc

[0053] Since O-linked glycan A and O-linked glycan B both had a Lewis X structure (Galβ1-4(Fucα1-3)GlcNAc), it is believed that Lewis X glycan has a preventive effect against food allergies.

[0054] (7) Confirmation of the preventive effect of Lewis X sugar chains on food allergies in infants Since both O-linked glycan A and O-linked glycan B have a common Lewis X structure at the non-reducing end, it was thought that Lewis X glycans would have a preventive effect against food allergies. Therefore, the association between the amount (molar concentration) of Lewis X glycans in breast milk, which is the sum of the amounts (molar concentration) of O-linked glycan A and O-linked glycan B in breast milk, and the history of onset of food allergy was analyzed by logistic regression. As a result, p=0.016, it was found that there is a statistically significant association between the amount (molar concentration) of Lewis X glycans in breast milk and the history of onset of food allergy (Table 5). These associations were shown to have negative regression coefficients and odds ratios of less than 1, indicating that infants who drank breast milk with a high amount (molar concentration) of Lewis X glycans in breast milk have a lower risk of developing food allergies.

[0055] [Table 5]

[0056] Breast milk samples were divided into two groups based on the presence or absence of a history of food allergy in infants, and the amount of Lewis X glycan in breast milk (molar concentration) was compared. As a result, the amount of Lewis X glycan in breast milk of infants who had no history of food allergy was significantly higher than that of breast milk of infants who had developed food allergy (Figure 5). This indicates that infant nutritional compositions containing Lewis X glycan at the same level as breast milk of infants who had not developed food allergy are effective in reducing the risk of developing food allergy in infant nutritional compositions. The distribution range of the amount of Lewis X glycan in breast milk of infants who had not developed food allergy was 0.66 to 301.4 μmol / L, and the median was 16.9 μmol / L (Table 6). As with the distribution of 3-FL, the distribution of Lewis X glycans in breast milk was not normal, but had a wider tail toward higher concentrations, so the concentration range of Lewis X glycans in breast milk was set to 3-97 percentile. The Lewis X glycans in breast milk consumed by infants who did not develop food allergies are considered to be 4.3 μmol / L or higher, which corresponds to the 3rd percentile or higher. In addition, samples were divided into two groups, samples with Lewis X glycans of 10.1 μmol / L or higher and samples with Lewis X glycans below 10.1 μmol / L, and the relationship with the presence or absence of food allergies was evaluated using Fisher's exact test, with p<0.05, indicating that Lewis X glycans in breast milk of 10.1 μmol / L or higher are associated with a lower risk of developing food allergies.

[0057] [Table 6]

[0058] Therefore, as an infant nutritional composition that prevents the onset of food allergy, it is necessary that the milk after preparation contains 4.3 μmol / L or more of Lewis X sugar chain, and preferably 10.1 μmol / L or more. If the concentration of the milk preparation is 13%, it is necessary to incorporate at least 3.3 μmol or more of Lewis X sugar chain per 100 g of solid matter of the infant nutritional composition, and preferably 7.8 μmol or more of Lewis X sugar chain is incorporated. Since the mechanism of onset of food allergy is the same in infants and adults, it is considered that the incorporation of this infant nutritional composition is also effective for adults and elderly people who are at high risk of developing food allergy. Therefore, it is also effective as a nutritional composition for adults that contains the same Lewis X sugar chain as above.

[0059] (8) Evaluation of the effect of glycans with Lewis X structure on intestinal barrier function Based on the above results, we investigated whether Lewis X glycans affect functions related to food allergy. The onset and progression of food allergy is related to the influx of allergens due to the breakdown of the intestinal barrier. Therefore, we evaluated the effect of Lewis X glycans on the expression of intestinal barrier-related genes.

[0060] LS174T cells, a model of human colonic goblet cells, were cultured for 48 hours after adding 10mg / ml Lewis X glycan (administered in the form of trisaccharide), and the expression level of TFF3 (a protein that strengthens barrier function) was evaluated. As a result, a significant increase in TFF3 expression was observed with the addition of Lewis X glycan (Figure 6). In other words, it was shown that Lewis X glycan has the effect of enhancing TFF3 expression.

[0061] TFF3 is known to contribute to comprehensive strengthening of the intestinal barrier function through various actions such as strengthening mucin binding, mucosal repair, inflammation suppression, and induction of antimicrobial peptide production. Therefore, it was suggested that Lewis X glycans strengthen the intestinal barrier through enhancing TFF3 expression and have a preventive effect against food allergies.

[0062] (9)Evaluation of the effects of 3-FL, Lewis A glycan, and Lewis X glycan on dendritic cells It is known that the onset and progression of food allergy involves not only the intestinal barrier function but also the responsiveness of dendritic cells, a type of immune cell. Therefore, we evaluated the effects of various glycans on the activation of dendritic cells. Since LNFP-II is a glycan with a Lewis A structure (also called Lewis A glycan), we evaluated Lewis A glycans as an alternative to LNFP-II.

[0063] Dendritic cells were activated by culturing THP-1-derived dendritic cells (TDDCs) in a medium containing LPS for 3 days. LPS stimulation increased the expression of HLA-DR and CD86, which are activation markers of dendritic cells, indicating that the dendritic cells were activated (Figs. 7 and 8). When various glycans (lactose, 2'-FL, 3-FL, Lewis A glycan (administered in the form of trisaccharide), and Lewis X glycan (administered in the form of trisaccharide)) were added in addition to LPS and cultured, lactose did not affect the expression of either HLA-DR or CD86 (Figs. 7 and 8). 2'-FL and Lewis A glycan did not affect the expression of HLA-DR (Fig. 7), but significantly suppressed the increase in CD86 expression caused by LPS (Fig. 8). It was shown that 3-FL and Lewis X strongly suppressed the expression of both HLA-DR and CD86 (Fig. 7). Therefore, it was shown that 2'-FL, 3-FL, Lewis A sugar chain, and Lewis X sugar chain all have an inhibitory effect on dendritic cell activation. HLA-DR is an MHC class II molecule that presents antigens to T cells in adaptive immunity, and a decrease in HLA-DR expression means a decrease in the antigen-presenting ability of dendritic cells, which acts as an inhibitor against autoimmune diseases and allergies. In addition, CD86 is one of the costimulatory molecules that transmits auxiliary signals necessary for antigen presentation, and it has been reported that allergic symptoms are alleviated in mice in which the CD86 gene is knocked down. Therefore, it was suggested that Lewis A sugar chains and Lewis X sugar chains such as 3-FL and LNFP-II have a preventive effect against food allergies by suppressing the activation of dendritic cells.

[0064] [Example 1] Preparation of 3-FL-containing infant formula 52.7 kg of whey powder, 239 kg of skim milk, and 348 g of 3-FL were dissolved in 500 kg of water, and this solution was mixed with 23.9 kg of vegetable oil and homogenized. The resulting solution was sterilized, concentrated and dried by a conventional method to obtain 99 kg of milk powder. The milk powder was mixed with 1 kg of vitamin and mineral ingredients to obtain 100 kg of milk powder. The amount of 3-FL in the resulting milk powder was 348 mg / 100 g solids.

[0065] [Example 2] Preparation of LNFP-II-containing infant formula 52.7 kg of whey powder, 239 kg of skim milk, and 242 g of LNFP-II were dissolved in 500 kg of water, and this solution was mixed with 23.9 kg of vegetable oil and homogenized. The resulting solution was sterilized, concentrated and dried by a conventional method to obtain 99 kg of milk powder. The milk powder was mixed with 1 kg of vitamin and mineral ingredients to obtain 100 kg of milk powder. The amount of LNFP-II in the resulting milk powder was 242 mg / 100 g solids.

[0066] [Example 6] Preparation of mucin-containing milk powder 52.7 kg of whey powder, 239 kg of skim milk, and 32 g of mucin material derived from porcine stomach as an O-linked glycan A-containing material were dissolved in 500 kg of water, and this solution was mixed with 23.9 kg of vegetable oil and homogenized. The resulting solution was sterilized, concentrated and dried by a standard method 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 modified milk powder. The amount of O-linked glycan A in the obtained milk powder was 8.0 mg / 100 g solid.

[0067] [Example 4] Preparation of powdered milk containing glycopeptides 52.7 kg of whey powder, 239 kg of skim milk, and 250 mg of O-linked glycan B powder were dissolved in 500 kg of water, and this solution was mixed with 23.9 kg of vegetable oil and homogenized. The resulting solution was sterilized, concentrated and dried by a standard method 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 modified milk powder. The amount of O-linked glycan B in the obtained milk powder was 0.25 mg / 100 g solid.

[0068] [Example 5] Production of supplements 1 g of 3-FL 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 cornstarch and lactose. The mixture was packed into a stick bag to produce the food allergy prevention supplement of the present invention.

[0069] [Example 6] Production of beverage The ingredients were mixed according to the ratio shown in the table below, filled into a container, and then heat sterilized to produce the beverage for preventing food allergies of the present invention.

[0070] [Table 7]

[0071] [Example 7] Production of beverage The ingredients were mixed according to the ratio shown in the table below, filled into a container, and then heat sterilized to produce the beverage for preventing food allergies of the present invention.

[0072] [Table 8] [Industrial Applicability]

[0073] According to the present invention, the onset of food allergies can be prevented by a nutritional composition containing as an active ingredient 3-FL, LNFP-II, or a sugar chain having a Lewis X structure. Moreover, since the ingredient is effective not only for infants but also for adults at high risk of food allergies, it is possible to prevent the onset of food allergies in adults.

Claims

1. A nutritional composition for preventing food allergies, which contains a sugar chain as an active ingredient, and which contains at least one of the group consisting of 3-fucosyllactose, a sugar chain having a Lewis X structure, and lacto-N-fucopentaose II as an active ingredient.

2. The nutritional composition according to claim 1, wherein the glycan having a Lewis X structure is an O-linked glycan having the structure shown in (1) or (2) below. (1) Neu5Acα2-3Galβ1-3[Galβ1-4(Fucα1-3)GlcNAcβ1-6]GalNAc (2) Galβ1-4 (Fucα1-3)GlcNAcβ1-3GalNAc

3. 3. The nutritional composition according to claim 1 or 2, which is for infants.

4. A food or drink for preventing food allergies, comprising the nutritional composition for preventing food allergies according to claim 1 or 2.

5. A pharmaceutical for preventing food allergies, comprising the nutritional composition for preventing food allergies according to claim 1 or 2.

6. A nutritional composition for infants and young children containing 3.1 mg / 100 g solids or more of Neu5Acα2-3Galβ1-3[Galβ1-4(Fucα1-3)GlcNAcβ1-6]GalNAc.

7. A nutritional composition for infants and young children containing 0.14 mg / 100 g solid or more of Galβ1-4(Fucα1-3)GlcNAcβ1-3GalNAc.

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