Nutritional composition for preventing food allergy

Abstract

The present invention addresses the problem of discovering a component for preventing the onset of food allergy from among components of breast milk, and providing a novel nutritional composition.　The present invention relates to a nutritional composition for preventing the onset of food allergy, the nutritional composition being characterized by containing, as an active ingredient, 3-fucosyllactose, lacto-N-fucopentaose II, and a sugar chain having a Lewis X structure. The nutritional composition is effective 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 food allergy when being blended into an adult food or a nutritional composition.

Description

Nutritional composition for preventing food allergies

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

[0002] Food allergy is defined as a phenomenon in which adverse symptoms are induced in the body through antigen-specific immunological mechanisms triggered 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 lead to anaphylactic shock, which can be life-threatening. In some groups, this 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 age 0 and decreases with age, in many cases management is required even in adulthood. Food allergies can also suddenly develop in adulthood.

[0004] Therefore, preventing food allergies is important for everyone to lead a healthy social life, and various methods for prevention have been studied. For example, it has been shown that food allergies can be prevented by restricting the diet of mothers during pregnancy and breastfeeding, or by starting solid foods at an appropriate time. However, there is still insufficient evidence, and no effective method for preventing food allergies exists.

[0005] Breast milk is known to contain various components that are beneficial to infants. In searching for active ingredients that prevent the onset of food allergies, the present inventors focused on the components contained in breast milk. Breast milk contains a variety of useful components, 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 which components in breast milk are capable of preventing the onset of food allergies.

[0006] JP 2000-504019 A JP 2021-130665 A

[0007] The present inventors believed that if a component that prevents the onset of food allergies could be found among the components of breast milk, then incorporating that component into a nutritional composition for infants could provide a nutritional composition for infants that prevents the onset of food allergies. Furthermore, because the component is effective not only for infants but also for adults, it could be incorporated into foods and nutritional compositions for adults and the elderly, and provided as a nutritional composition that prevents the onset of food allergies. The objective of the present invention is to identify a component that prevents the onset of food allergies from the components of breast milk and to provide a new nutritional composition.

[0008] In order to search for components in breast milk that prevent 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 allergies in infants, and conducted extensive research to identify new components that prevent the onset of food allergies. As a result, they discovered 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, leading to the completion of the present invention. Specifically, the present invention relates to a nutritional composition for infants and a nutritional composition that prevent the onset of food allergies, characterized by containing 3-FL, LNFP-II, or a glycan with a Lewis X structure as an active ingredient. Specifically, the present invention has the following configurations.

[0009] <1> A nutritional composition for preventing food allergies, which contains a sugar chain as an active ingredient, the nutritional composition comprising, as an active ingredient, any one or more of the group consisting of 3-fucosyllactose, a sugar chain having a Lewis X structure, and lacto-N-fucopentaose II. <2> The nutritional composition according to <1>, wherein the sugar chain having a Lewis X structure is an O-linked sugar chain 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> The nutritional composition according to <1> or <2>, which is for infants. <4> A food or drink for preventing food allergies, which comprises the nutritional composition for preventing food allergies according to <1> or <2>. <5> A pharmaceutical product for preventing food allergies, comprising the nutritional composition for preventing food allergies according to <1> or <2>. <6> 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. <7> A nutritional composition for infants containing 0.14 mg / 100 g solid or more of Galβ1-4(Fucα1-3)GlcNAcβ1-3GalNAc. <8> A method for preventing food allergies, comprising the step of administering to a subject any one or more of the group consisting of 3-fucosyllactose, a sugar chain having a Lewis X structure, and lacto-N-fucopentaose II. <9> The method for preventing food allergies according to <8>, wherein the sugar chain having a Lewis X structure is an O-linked sugar chain 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 <10> The method for preventing food allergies according to <8> or <9>, wherein the subject is an infant. <11> Use of any one or more of the group consisting of 3-fucosyllactose, a sugar chain having a Lewis X structure, and lacto-N-fucopentaose II for producing a composition for preventing food allergies.<12> The use according to <11>, wherein the sugar chain having a Lewis X structure is an O-linked sugar chain 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 <13> The use according to <11> or <12>, wherein the food allergy is a food allergy in infants.

[0010] The present invention provides a novel nutritional composition for preventing food allergies. The nutritional composition contains, as an active ingredient, one or more selected from the group consisting of 3-FL, LNFP-II, and sugar chains having a Lewis X structure, and ingesting the composition can prevent the onset of food allergies. Furthermore, the ingredient can prevent the onset of food allergies not only in infants, but also in adults and the elderly.

[0011] This shows the effect of 3-FL concentration in breast milk on the onset of food allergies in infants. This shows the effect of LNFP-II concentration in breast milk on the onset of food allergies in infants. This shows the effect of O-linked glycan A concentration in breast milk on the onset of food allergies in infants. This shows the effect of O-linked glycan B concentration in breast milk on the onset of food allergies in infants. This shows the effect of Lewis X glycan concentration in breast milk on the onset of food allergies in infants. This shows the expression level of TFF3 gene (intestinal barrier-related gene) 48 hours after adding 10 mg / ml Lewis X glycan to LS174T cells, a human colon goblet cell model. In the figure, ＊ P<0.05 is indicated. The figures show the expression levels of HLA-DR (dendritic cell activation marker) 3 days after the addition of LPS and various sugar chains to TDDCs, which were differentiated from the human monocyte-derived cell line THP-1 into dendritic cells. In the figures, lowercase letters indicate significant differences between different letters. The figures show the expression levels of CD86 (dendritic cell activation marker) 3 days after the addition of LPS and various sugar chains to TDDCs, which were differentiated from the human monocyte-derived cell line THP-1 into dendritic cells. In the figures, lowercase letters indicate significant differences between different letters.

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

[0013] (3-Fucosyllactose (3-FL)) The 3-fucosyllactose used in the present invention is an oligosaccharide (3-O-(α-L-fucopyranosyl)-4-O-(β-D-galactopyranosyl)-D-glucopyranose) in which fucose and lactose are linked together. In the present invention, 3-FL may be derived from milk or from fermentation using genetically modified bacteria, and may be a synthetic product, semi-synthetic product, or natural product. 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 products extracted from natural materials containing 3-FL by known methods, crudely purified products, or products further purified from 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 powdered milk, the prepared milk 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 fermentation using genetically modified bacteria, and may be any of synthetic, semi-synthetic, or natural products. Furthermore, natural materials containing natural products may be used as a supply source and incorporated directly into the nutritional composition of the present invention. Here, "natural products" refers to products extracted from natural materials containing LNFP-II by known methods, crudely purified products, or products further purified from these. The nutritional compositions of the present invention containing LNFP-II as an active ingredient preferably contain 0.68 mg / 100 g solid content or more of LNFP-II, more preferably 243 mg / 100 g solid content or more. Furthermore, the milk after preparation preferably contains 0.88 mg / L or more of LNFP-II, more preferably 315 mg / L or more.

[0015] (Sugar chains having Lewis X structure) In the present invention, a 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, it refers to 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)GlcNAcβ1-3Galβ1-4Glc, Galβ1-4(Fucα1-3)GlcNAcβ1-2Manα1-3(Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc, etc. Hereinafter, sugar chains having a Lewis X structure may be simply referred to as "Lewis X sugar chains."

[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 methods using genetically modified bacteria, and may be a synthetic, semi-synthetic, or natural product. Natural materials containing natural products may also be used as a supply source and incorporated directly into 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 from such a natural material.

[0017] The nutritional composition of the present invention containing Lewis X sugar chains as an active ingredient preferably contains Lewis X sugar chains at a concentration of 3.3 μmol / 100 g solid or more, more preferably 7.8 μmol / 100 g solid or more. The content of Lewis X sugar chains in the prepared milk 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. Furthermore, the O-linked glycan A content 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 O-linked glycan B, 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. Furthermore, the O-linked glycan B content in the prepared milk is preferably 0.18 mg / L or more, more preferably 0.33 mg / L or more.

[0020] (Preventive effect on 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 chains, and its mechanism of action includes strengthening the intestinal barrier function and inhibiting the activation of dendritic cells, as shown in the examples below. It is believed that these actions result in a preventive effect on food allergies regardless of the type of food.

[0021] (Nutritional Compositions, Foods and Drinks, Pharmaceuticals) The nutritional compositions of the present invention for preventing food allergies containing 3-FL, LNFP-II, or Lewis X sugar chain (hereinafter, these may be collectively referred to as 3-FL, etc.) as active ingredients may be any compositions containing an effective amount of 3-FL, etc., and may contain proteins, fats, carbohydrates, vitamins, minerals, etc. in addition to the active ingredient. They are used as nutritional compositions for infants, as well as for adults and the elderly. Nutritional compositions for infants include infant formulas and liquid formulas, infant formulas (follow-up milk and growing-up milk), formulas for low birth weight infants, allergen-free foods for cow's milk allergies, and lactose-free foods for lactose intolerance. Nutritional compositions for adults and the elderly include adult formulas and liquid formulas, allergen-free foods for cow's milk allergies, lactose-free foods for lactose intolerance, and supplements. Furthermore, the food and beverage product of the present invention for preventing food allergies containing 3-FL or the like may be incorporated into any food and beverage product containing an effective amount of 3-FL or the like, or may be added to raw materials during the manufacturing process of the food and beverage product. Examples of food and beverage products include, but are not limited to, dairy products such as cheese, fermented milk, lactic acid bacteria drinks, butter, and margarine; beverages such as milk drinks, fruit juice drinks, and soft drinks; egg products such as jelly, candy, pudding, and mayonnaise; and confectioneries and breads such as butter cake. The nutritional composition for preventing food allergies of the present invention can be used directly as a raw material for pharmaceuticals and can be manufactured by conventional methods into tablets, capsules, powders, syrups, and the like. Therefore, when formulating 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, flavoring agents, etc. that are approved for formulation. In addition, the composition containing 3-FL or the like can be dried directly and used as a powder or dusting agent. In addition, the product can be formulated by mixing excipients, binders, disintegrants, lubricants, flavorings, suspending agents, coating agents, and other optional agents within the scope that does not impair the food allergy prevention effect.Dosage forms that can be used include tablets, capsules, granules, powders, dusts, syrups, etc.

[0022] (Nutritional Composition for Infants) A further aspect of the present invention relates to a nutritional composition for infants containing a specific Lewis X sugar chain at a certain level or higher, i.e., a nutritional composition for infants containing 3.1 mg / 100 g solid or higher 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 higher of Galβ1-4(Fucα1-3)GlcNAcβ1-3GalNAc.

[0023] [Test Example 1] Various carbohydrate components were measured for 155 breast milk samples, and the relationship between the presence or absence of food allergies in the infants during the first year of life was analyzed using logistic regression. The method and results are shown below.

[0024] 1. Methods (1) Investigation of breast milk samples and infant infection history. This study used breast milk samples collected from 1,210 mothers and questionnaire results investigating maternal and infant health status from a prospective cohort study (UMIN ID 000015494) conducted by Snow Brand Beanstalk Co., Ltd. between 2014 and 2019 to investigate the relationship between breast milk components and maternal and infant health status in Japanese women. Furthermore, 200 samples were randomly selected from the 1,210 samples. Breast milk samples collected between 1 and 2 months after birth were used for analysis. Parents completed questionnaires investigating their infants' health status six times, every two months for one year after birth, and the results were collected. From these questionnaire results, we collated whether or not the infants had been diagnosed with food allergies by a doctor within one year after birth. Subjects whose diagnosis was unclear were excluded from the study. As a result, 155 subjects correctly answered whether they had been diagnosed with food allergies in six questionnaires over the course of one year, and these were the subjects of this study. Of the 155 subjects, 18 were infants diagnosed with food allergies within one year of birth.

[0025] (2) Measurement of Various Carbohydrate Components in Breast Milk (2-1) Measurement of Oligosaccharides 20 μL of breast milk from 155 samples was added to 580 μL of ultrapure water, then subjected to ultrafiltration with a molecular weight cutoff of 10,000. The permeate was analyzed by HPLC. The HPLC was performed using a DIONEX ICS-5000DP system (Thermo Fisher Scientific Inc.) connected to an electrochemical detector, and a CarboPac PA1 column (Thermo Fisher Scientific Inc.). The mobile phase consisted of three solutions: ultrapure water (Solution A), 200 mM sodium hydroxide solution (Solution B), and 100 mM sodium hydroxide solution containing 600 mM sodium acetate (Solution C), which were passed through the system at a flow rate of 1 mL / min. The first 10 minutes were spent at 50% solution B, after which solution B was linearly reduced to 47.5% by 18 minutes and solution C was linearly increased to 5%. From 18 to 28 minutes, solution B was 47.5% and solution C was 5%. From 28 to 32 minutes, solution B was linearly reduced to 42% and solution C was linearly increased to 16%. From 32 to 39 minutes, solution B was 42% and solution C was 16%, and then from 39 to 43 minutes, solution B was 30% and solution C was 40%. The content of each oligosaccharide in each breast milk sample was calculated from a calibration curve prepared using known concentrations of standard samples 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 To 20 μL of breast milk from 155 samples analyzed, 10 μL of 3 M sodium borohydride aqueous solution was added and allowed to stand at room temperature for 30 minutes. Subsequently, 7.5 μL of acetic acid was added and allowed to stand at room temperature for 30 minutes. 15 μL of 1 M ammonium bicarbonate aqueous solution and 7.5 μL of ultrapure water were added. Subsequently, 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. After cooling to room temperature, 10 μL of 123 mM iodoacetamide aqueous solution was added and allowed to stand at room temperature for 1 hour. After adding 7 μL of 500 mM aqueous sodium acetate 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 volume was introduced into a Vivaspin 500 (Sartorius) with a molecular weight cutoff of 10,000, and centrifuged at 15,000 x g and 25°C for 10 minutes to collect 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. The HPLC was performed using an UltiMate 3000 (Thermo Fisher Scientific Inc.), and the MS was performed using a Q-Exactive (Thermo Fisher Scientific Inc.). The column used was a GlycanPac AXH-1 (Thermo Fisher Scientific Inc.), 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) adjusted to pH 4.4, and the flow rate was 0.4 mL / min. After sample introduction, the proportion of solution B was linearly increased from 10% to 35% by 55 minutes, and then 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 gas was used as the sheath gas, auxiliary gas, and collision gas. The S-Lens RF Level was set to 90. MS spectra were acquired in the positive mode by full scan measurement in the m / z range of 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, Compound Discover was used to create an extraction chromatogram corresponding to the molecular ion peak of each glycan, and the signal area value of each glycan detected in each breast milk sample was calculated. The calculated glycan signal area value was used as the relative quantitative value of each glycan.

[0027] (2-3) Measurement of O-linked Glycans The 155 breast milk samples analyzed were treated with 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 and centrifuged at 15,000 x g and 25°C for 15 minutes, and the permeate was collected. 50 μL of acetonitrile was added to 50 μL of the permeate and subjected to LC / MS analysis. An UltiMate 3000 HPLC and a Q-Exactive MS were used. A GlycanPac AXH-1 column was used, and the column temperature was set to 40°C. The mobile phase consisted of acetonitrile (liquid A) and 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 linearly increased from 10% to 35% by 55 minutes, and then 35% solution B was passed from 55 minutes to 65 minutes. The ESI probe spray voltage was 3.5 kV, the capillary temperature was 275°C, and nitrogen gas was used as the sheath gas, auxiliary gas, and collision gas. The S-Lens RF Level was set to 90. MS spectra were acquired in the positive mode using full scan measurements in the m / z range of 300-2000. The acquired MS spectra were analyzed using Compound Discover 2.1 (Thermo Fisher Scientific). Glycans were detected by comparing them with an O-linked glycan database. Furthermore, 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 each 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 LC / MS / MS measurements were performed on one of the 155 samples prepared in the "O-Linked Glycan Measurement" section above, and structural analysis of O-linked glycan A and O-linked glycan B was performed. LC / MS / MS analysis was performed as follows. An UltiMate 3000 HPLC and a Q-Exactive MS were used. A GlycanPac AXH-1 column was used, and the column temperature was set to 40°C. The mobile phase consisted of acetonitrile (Solution A) and a 50 mM aqueous ammonium formate solution (Solution B) adjusted to pH 4.4, and the flow rate was 0.4 mL / min. After sample introduction, the proportion of Solution B was linearly increased from 10% to 35% by 55 minutes, and then 35% Solution B was passed from 55 to 65 minutes. The ESI probe spray voltage was 3.5 kV, the capillary temperature was 275°C, and nitrogen gas was used as the sheath gas, auxiliary gas, and collision gas. The S-Lens RF Level was set to 90. MS / MS spectra were acquired in positive mode and data-dependently in Top 10 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 determined.

[0029] (3-2) Determination of Linkage Mode To determine the linkage mode of N-acetylneuraminic acid contained in O-linked glycan A, the glycans were treated with either a sialidase that specifically degrades α2,3 linkages or a sialidase that degrades both α2,3 and α2,6 linkages, followed by LC / MS analysis. To determine the linkage mode of fucose contained in O-linked glycan A and O-linked glycan B, the glycans 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, followed by LC / MS analysis. Furthermore, to determine the galactose linkage, the samples were treated with either a fucosidase specifically degrading α1,3 and α1,4 linkages and a galactosidase specifically degrading β1,3 linkages simultaneously, or a fucosidase specifically degrading α1,3 and α1,4 linkages and a galactosidase specifically degrading β1,4 linkages simultaneously, followed by LC / MS analysis. For the same sample as 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 3 μL of each enzyme solution and incubation at 37°C for 1 hour. The enzyme was inactivated by heating in boiling water for 5 minutes, and then the sample was treated in the same manner as in (2-4) above and subjected to LC / MS analysis. LC / MS analysis was performed as follows. The HPLC was performed using an UltiMate 3000 and the MS was Q-Exactive. The column was a GlycanPac AXH-1, and the column temperature was set to 40°C. The mobile phase consisted of acetonitrile (liquid A) and a 50 mM ammonium formate 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 linearly increased from 10% to 35% by 55 minutes, and then 35% solution B was passed from 55 minutes to 65 minutes. The ESI probe spray voltage was 3.5 kV, the capillary temperature was 275°C, and nitrogen gas was used as the sheath gas, auxiliary gas, and collision gas. The S-Lens RF Level was set to 90.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 of O-linked glycan A and O-linked glycan B in breast milk, which have been found to be associated with the onset of food allergy, were measured. The contents of O-linked glycan A and O-linked glycan B were measured by fluorescently labeling the O-linked glycan A and O-linked glycan B, followed by HPLC connected to a mass spectrometer (MS) and a fluorescence detector (FL). The contents of O-linked glycan A and O-linked glycan B in breast milk were calculated by dividing by the recovery rate of the O-linked glycan analysis kit, which was calculated separately.

[0031] Details of the measurement of O-linked glycan A and O-linked glycan B are described below. 10 μL of breast milk from one randomly selected sample from the 155 breast milk samples analyzed was subjected to 2-aminobenzamide (AB) labeling and purification using the EzGlyco O-glycan Prep kit (Sumitomo Bakelite), an O-linked glycan analysis kit, followed by LC / MS analysis. An UltiMate 3000 HPLC and a Q-Exactive MS were used. A GlycanPac AXH-1 column was used, and the column temperature was set at 40°C. The mobile phase consisted of acetonitrile (Solution A) and a 50 mM aqueous ammonium formate solution (Solution B) adjusted to pH 4.4, and the flow rate was 0.4 mL / min. After sample introduction, the proportion of solution B was linearly increased from 10% to 35% by 55 minutes, and then solution B was passed at 35% from 55 to 65 minutes. The ESI probe spray voltage was 3.5 kV, the capillary temperature was 275°C, and nitrogen gas was used as the sheath gas, auxiliary gas, and collision gas. The S-Lens RF Level was set to 90. MS measurements were performed in positive mode using 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 a fluorescence wavelength of 420 nm. The acquired MS chromatograms and fluorescence chromatograms were analyzed using FreeStyle software to obtain fluorescence area values ​​for 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) of known concentration. Furthermore, the contents of O-linked glycan A and O-linked glycan B in breast milk were calculated by dividing by the recovery rate of the kit, which was calculated separately. 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 association between the content of various carbohydrate components in breast milk and food allergy. 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), and the association with food allergy was evaluated using Fisher's exact test. Furthermore, the association 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. Adjustment factors included the infant's gender, presence or absence of siblings, method of delivery (natural birth, cesarean section), and birth weight.

[0033] (6) Evaluation of the relationship between Lewis X sugar chains 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 (21443-15, Nacalai) containing Earle's salts, NEAA, and L-glutamine, supplemented with 10% FBS (Gibco) and 1% P / S (15140-122, Gibco). Culture was performed at 37°C and 5% CO ２ Lewis X (OL06490) was purchased from Biosynth.

[0034] (6-2) Sample addition test to LS174T cells LS174T cells 1 × 10 ５ The cells were seeded onto a 48-well plate (3830-048, AGC Technoglass Co., Ltd.) at 100 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. 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 resulting cDNA was used to measure the expression levels of the TFF3 gene (TFF3) and the housekeeping gene 18S rRNA gene (18S rRNA) using THUNDERBIRD® NextSYBR® qPCR Mix (QPX-201, Toyobo) and QuantStudio 6 Pro (Therno Fisher Scientific). A calibration curve was created for each gene to calculate the relative expression level, which was then corrected for by the 18S rRNA expression level. The primers used are shown in the table below.

[0036]

[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) Evaluation of the Relationship between 3-FL and Dendritic Cells (7-1) Cells and Reagents The 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% CO ２The experiments were carried out under the following conditions. 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) Sample addition test to THP-1 derived dendritic cells (TDDC) THP-1 cells were added at 5 × 10 ５ The cells were seeded onto a 6-well plate (3810-006, AGC Technoglass Co., Ltd.) at 2 × 10 cells / ml and cultured for 6 days (medium change on day 3) in a medium containing 1500 U / ml GM-CSF and 1500 U / ml IL-4 to differentiate into TDDCs. TDDCs were collected on day 6 and cultured at 2 × 10 cells / ml. ５ The cells were seeded onto a 96-well plate (3860-096, AGC Technoglass Co., Ltd.) at 100 ng / ml LPS and 10 mg / ml of various sugar chains (2'-FL, 3-FL, Lewis A sugar chain, Lewis X sugar chain) for 3 days.

[0040] (7-3) Surface Antigen Staining and FACS Analysis The collected 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 was equal to or greater than 100. After centrifugation, the supernatant was removed, 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. After centrifugation, the supernatant was removed, 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. After centrifugation, the supernatant was removed, and the cells were incubated with Human TruStain FcX. ＴＭ 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 in the dark for 30 minutes. After 30 minutes, the cells were washed with cell staining buffer and analyzed by BD FACSCanto. ＴＭ The cells 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 comparison was performed using the Tukey-Kramer's test. The significance level was set at P = 0.05.

[0042] 2. Results (1) Relationship between the content of various carbohydrate components in secretory and non-secretory breast milk and the onset of food allergies in infants. 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 consumed non-secretory breast milk (Table 2). Furthermore, a Fisher's exact test was performed to examine the relationship between the two groups and the onset of food allergies in infants, with a p<0.05 result, indicating that infants who consumed non-secretory breast milk had a significantly lower incidence of food allergies.

[0043]

[0044] These results clearly demonstrate that infants who drink non-secretory breast milk have a reduced risk of developing food allergies. Therefore, we sought to determine which components in non-secretory breast milk are associated with the prevention of food allergies. The concentrations of four oligosaccharides, two N-linked glycans, and five O-linked glycans, which were found to be higher in non-secretory breast milk than in secretory breast milk, were analyzed using logistic regression to examine the relationship between their concentrations in breast milk and the onset of food allergies in infants. A portion of the analysis results is shown in Table 3. As a result of the analysis, significant associations were observed between 3-FL (p = 0.034), LNFP-II (p = 0.035), and O-linked glycan A (p = 0.022) and food allergies, and a tendency for an association between O-linked glycan B (p = 0.089) and food allergies was observed. 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 had a reduced risk of developing food allergy.

[0045]

[0046] (2) Confirmation of the preventive effect of 3-FL on the development of food allergies in infants. Breast milk samples were divided into two groups based on whether or not the infants had developed food allergies, and the 3-FL concentrations in the breast milk were compared. Results showed that breast milk from infants without food allergies tended to have higher 3-FL concentrations than breast milk from infants with food allergies (Figure 1). This indicates that infant nutritional compositions containing 3-FL at levels equivalent to those in breast milk from infants without food allergies are effective in reducing the risk of developing food allergies. The 3-FL concentrations in breast milk from infants without food allergies ranged from 38 to 2341 mg / L, with a median of 557 mg / L (Table 4). The distribution of 3-FL concentrations differed from a normal distribution, exhibiting a broader tail toward higher concentrations. This type of distribution is also observed in the height and weight distributions of infants. When creating growth curves for infants, the 3rd to 97th percentile of the overall distribution is used as the standard (2000 Infant Physical Development Survey Report). Based on this, the 3rd to 97th percentile was used as the standard range for the 3-FL concentration in breast milk. 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. Furthermore, breast milk samples were divided into two groups: those with 3-FL concentrations of 452 mg / L or higher and those with concentrations below 452 mg / L. Fisher's exact test was performed to examine the relationship between the presence or absence of food allergies and the risk of developing food allergies. Results showed a p<0.05, indicating that breast milk 3-FL concentrations of 452 mg / L or higher are associated with a lower risk of developing food allergies. Therefore, infant nutritional compositions that prevent the onset of food allergies must contain 91.1 mg / L or more of 3-FL in the formula after preparation, preferably 452 mg / L or more. Assuming a formula concentration of 13%, the infant nutritional composition must contain at least 70.1 mg or more of 3-FL per 100 g of solids, preferably 348 mg or more. Furthermore, because the mechanism of food allergy onset is the same in infants and adults, this infant nutritional composition is believed to be effective for adults and elderly people who are at high risk of food allergies.Therefore, it is also effective as a nutritional composition for adults containing the same 3-FL content as above.

[0047]

[0048] (3) Confirmation of the Effect of LNFP-II on the Prevention of Food Allergies in Infants Breast milk samples were divided into two groups based on whether or not the infants had developed food allergies, and the LNFP-II concentrations in the breast milk were compared. Results showed that breast milk from infants who did not develop food allergies tended to have higher LNFP-II concentrations than breast milk from infants who did develop food allergies ( Figure 2 ). This indicates that infant nutritional compositions containing LNFFP-II concentrations equivalent to those of breast milk from infants who did not develop food allergies are effective in reducing the risk of developing food allergies. The range of LNFP-II concentrations in breast milk from infants who did not develop food allergies was 0 to 4123 mg / L, with a median of 388 mg / L ( Table 4 ). Similar to the distribution of 3-FL, the distribution of LNFP-II concentrations in breast milk was not normal, but rather broadened toward higher concentrations. Therefore, the 3rd to 97th percentiles were used as the range for LNFP-II concentrations in breast milk. The LNFFP-II concentrations in breast milk consumed by infants who did not develop food allergies are considered to be 0.88 mg / L or higher, which corresponds to the 3rd percentile or higher. Furthermore, breast milk samples were divided into two groups: those with LNFP-II concentrations of 315 mg / L or higher and those with lower concentrations. The correlation between these concentrations and the presence or absence of food allergies was evaluated using Fisher's exact test. The results showed a p<0.05, indicating that breast milk LNFP-II concentrations of 315 mg / L or higher are associated with a lower risk of developing food allergies. Therefore, for infant nutritional compositions that prevent the onset of food allergies, it is necessary for the prepared milk to contain 0.88 mg / L or higher of LNFP-II, with 315 mg / L or higher being preferred. Assuming a formula concentration of 13%, it is necessary to incorporate at least 0.68 mg of LNFP-II per 100 g of solids in the infant nutritional composition, and it is preferable to incorporate at least 243 mg of LNFP-II. Because the mechanism of food allergy onset is the same in infants and adults, this infant nutritional composition is also thought to be effective for adults and elderly people who are at high risk of food allergies. Therefore, a nutritional composition for adults containing the same LNFP-II content as above is also effective.

[0049] (4) Confirmation of the preventive effect of O-linked glycan A on the development of food allergies in infants. Breast milk samples were divided into two groups based on whether or not the infants had developed food allergies, and the concentrations of O-linked glycan A in the breast milk were compared. The results showed that the breast milk from infants who did not develop food allergies had significantly higher levels of O-linked glycan A than the breast milk from infants who did develop food allergies (Figure 3). This indicates that infant nutritional compositions containing O-linked glycan A at a concentration equivalent to that of breast milk from infants who did not develop food allergies are effective in reducing the risk of developing food allergies. The distribution of O-linked glycan A concentrations in breast milk from infants who did not develop food allergies ranged from 0.33 to 321 mg / L, with a median of 15.2 mg / L (Table 4). Similar to the distribution of 3-FL, the concentration distribution of O-linked glycan A in breast milk was not normal but broadened toward higher concentrations. Therefore, the 3rd to 97th percentile was defined as the range of O-linked glycan A concentrations in breast milk. 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, corresponding to the 3rd percentile or higher. Furthermore, breast milk samples were divided into two groups: those with O-linked glycan A concentrations of 10.4 mg / L or higher and those with O-linked glycan A concentrations of 10.4 mg / L or lower. The correlation with the presence or absence of food allergies was evaluated using Fisher's exact test. The results showed a p<0.05, indicating that an O-linked glycan A concentration of 10.4 mg / L or higher in breast milk reduces the risk of developing food allergies. Therefore, for infant nutritional compositions that prevent the onset of food allergies, it is necessary for the prepared milk to contain 4.0 mg / L or higher of O-linked glycan A, preferably 10.4 mg / L or higher. When the formula concentration is 13%, it is necessary to incorporate at least 3.1 mg of O-linked glycan A per 100 g of solids in the infant nutritional composition, and preferably 8.0 mg or more of O-linked glycan A. Since the mechanism of food allergy onset is the same in infants and adults, this infant nutritional composition is thought to be effective for adults and elderly people who are at high risk of food allergies. Therefore, it is also effective as a nutritional composition for adults containing the same O-linked glycan A content as above.

[0050] (5) Confirmation of the preventive effect of O-linked glycan B on the development of food allergies in infants. Breast milk samples were divided into two groups based on whether or not the infants had developed food allergies, and the concentrations of O-linked glycan B in the breast milk were compared. The results showed that the breast milk from infants who did not develop food allergies had significantly higher levels of O-linked glycan B than the breast milk from infants who did develop food allergies (Figure 4). This indicates that infant nutritional compositions containing O-linked glycan B at a concentration equivalent to that of breast milk from infants who did not develop food allergies are effective in reducing the risk of developing food allergies. The distribution of O-linked glycan B concentrations in breast milk from infants who did not develop food allergies ranged from 0.07 to 22.1 mg / L, with a median of 1.26 mg / L (Table 4). Similar to the distribution of 3-FL, the distribution of O-linked glycan B concentrations in breast milk was not a normal distribution, but rather a distribution with a wider tail at higher concentrations. Therefore, the 3rd to 97th percentile was defined as the range of O-linked glycan B concentrations in breast milk. The O-linked glycan B concentrations in breast milk consumed by infants who did not develop food allergies are considered to be 0.18 mg / L or higher, which corresponds to the 3rd percentile or higher. Furthermore, breast milk samples were divided into two groups: those with di-O-linked glycan B concentrations of 0.33 mg / L or higher and those with concentrations below 0.33 mg / L. The relationship with the presence or absence of food allergies was evaluated using Fisher's exact test, with p<0.05, indicating that O-linked glycan B concentrations in breast milk of 0.33 mg / L or higher are associated with a lower risk of developing food allergies. Therefore, for an infant nutritional composition that prevents the onset of food allergies, it is necessary for the formula to contain 0.18 mg / L or more of O-linked glycan B, preferably 0.33 mg / L or more. If the formula concentration is 13%, it is necessary to incorporate at least 0.14 mg or more of O-linked glycan B per 100 g of solids in the infant nutritional composition, preferably 0.25 mg or more of O-linked glycan B. Because the mechanism of food allergy onset is the same in infants and adults, this infant nutritional composition is also thought to be effective for adults and elderly people who are at high risk of food allergies. Therefore, a nutritional composition for adults containing the same O-linked glycan B content as above is also effective.

[0051] (6) Determination of the Structures of O-linked Glycan A and O-linked Glycan B To investigate the structures of O-linked glycan A and O-linked glycan B in breast milk, which have been shown to be associated with the onset of food allergies in infants, LC / MS and LC / MS / MS measurements were performed. As a result, O-linked glycan A was estimated to be Neu5Ac-Galβ1-3[Gal-(Fuc-)GlcNAcβ1-6]GalNAc, and O-linked glycan B was estimated to be Gal-(Fuc-)GlcNAcβ1-3GalNAc. To determine the fucose linkage in O-linked glycan A and O-linked glycan B, LC / MS measurements were performed after treatment with a fucosidase that specifically degrades α1,2 bonds, a fucosidase that specifically degrades α1,3 and α1,4 bonds, or a fucosidase that specifically degrades α1,2, α1,4, and α1,6 bonds. Degradation of O-linked glycan A and O-linked glycan B was observed only when a fucosidase that specifically degrades α1,3 and α1,4 bonds was used, revealing that the fucose in O-linked glycan A and O-linked glycan B is α1,3-linked. Similarly, when galactosidase, which specifically degrades β1,4 linkages, was used simultaneously with fucosidase, which specifically degrades α1,3 and α1,4 linkages, degradation of the glycans was observed, demonstrating that the galactose linked to GlcNAc in O-linked glycan A and O-linked glycan B is a β1,4 linkage. Furthermore, since O-linked glycan A was degraded by sialidase, which specifically degrades α2,3 linkages, it was revealed that the N-acetylneuraminic acid in O-linked glycan A is an α2,3 linkage. These results demonstrate that O-linked glycan A and O-linked glycan B are O-linked glycans represented by the following structures:

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

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

[0054] (7) Confirmation of the preventive effect of Lewis X sugar chains on food allergies in infants. Both O-linked sugar chain A and O-linked sugar chain B shared a Lewis X structure at their non-reducing ends, suggesting that Lewis X sugar chains may have a preventive effect on food allergies. Therefore, the correlation between the amount (molar concentration) of Lewis X sugar chains in breast milk, which is the sum of the amounts (molar concentrations) of O-linked sugar chain A and O-linked sugar chain B in breast milk, and a history of food allergy was analyzed using logistic regression. The results showed a p = 0.016, indicating a statistically significant correlation between the amount (molar concentration) of Lewis X sugar chains in breast milk and a history of food allergy (Table 5). These correlations were confirmed by the 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 sugar chains in breast milk had a reduced risk of developing food allergies.

[0055]

[0056] Breast milk samples were divided into two groups based on whether the infants had a history of food allergy, and the amount of Lewis X sugar chains (molar concentration) in the breast milk was compared. The breast milk from infants without a history of food allergy had significantly higher levels of Lewis X sugar chains (molar concentration) than the breast milk from infants with a history of food allergy (Figure 5). This suggests that infant nutritional compositions containing Lewis X sugar chains at levels equivalent to those found in breast milk from infants without food allergies are effective in reducing the risk of developing food allergies. The distribution of Lewis X sugar chains in the breast milk from infants without food allergies ranged from 0.66 to 301.4 μmol / L, with a median of 16.9 μmol / L (Table 6). Similar to the distribution of 3-FL, the distribution of Lewis X glycans in breast milk was not normal, but rather broadened toward higher concentrations. Therefore, the 3rd to 97th percentile was defined as the concentration range for Lewis X glycans in breast milk. 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. Furthermore, breast milk samples were divided into two groups: those with Lewis X glycans of 10.1 μmol / L or higher and those with lower concentrations. 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 breast milk Lewis X glycans of 10.1 μmol / L or higher are associated with a lower risk of developing food allergies.

[0057]

[0058] Therefore, infant nutritional compositions that prevent the onset of food allergies must contain 4.3 μmol / L or more of Lewis X sugar chains in the prepared milk, preferably 10.1 μmol / L or more. If the concentration of the prepared milk is 13%, the infant nutritional composition must contain at least 3.3 μmol or more of Lewis X sugar chains per 100 g of solids, preferably 7.8 μmol or more. Since the mechanism of food allergy onset is the same in infants and adults, this infant nutritional composition is also considered to be effective for adults and elderly people who are at high risk of developing food allergies. Therefore, this infant nutritional composition containing the same Lewis X sugar chain as above is also effective as a nutritional composition for adults.

[0059] (8) Evaluation of the effect of glycans with Lewis X structures on intestinal barrier function Based on the above results, we investigated whether Lewis X glycans affect functions related to food allergies. The onset and progression of food allergies 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 with 10 mg / ml Lewis X sugar chains (administered in the form of trisaccharide), and the expression level of TFF3 (a protein that strengthens the barrier function) was evaluated. As a result, a significant increase in TFF3 expression was observed with the addition of Lewis X sugar chains (Figure 6). This indicates that Lewis X sugar chains have 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 sugar chains 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 sugar chains, and Lewis X sugar chains on dendritic cells. The onset and progression of food allergies are known to involve 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 sugar chains on dendritic cell activation. Since LNFP-II is a sugar chain with a Lewis A structure (also known as a Lewis A sugar chain), we evaluated Lewis A sugar chains as an alternative to LNFP-II.

[0063] THP-1-derived dendritic cells (TDDCs) were activated by culturing them in LPS-containing medium for 3 days. LPS stimulation increased the expression of HLA-DR and CD86, markers of dendritic cell activation, indicating dendritic cell activation (Figures 7 and 8). When cultured with various sugar chains (lactose, 2'-FL, 3-FL, Lewis A sugar chain (administered in the form of a trisaccharide), and Lewis X sugar chain (administered in the form of a trisaccharide)) in addition to LPS, lactose did not affect the expression of either HLA-DR or CD86 (Figures 7 and 8). 2'-FL and Lewis A sugar chains did not affect HLA-DR expression (Figure 7), but significantly suppressed the LPS-induced increase in CD86 expression (Figure 8). 3-FL and Lewis X were shown to strongly suppress both HLA-DR and CD86 expression (Figure 7). Therefore, it was shown that 2'-FL, 3-FL, Lewis A sugar chains, and Lewis X sugar chains all have the effect of suppressing 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 a suppressor against autoimmune diseases and allergies. Furthermore, CD86 is a costimulatory molecule 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 dendritic cell activation.

[0064] Example 1: Preparation of 3-FL Formulated Formula Milk Powder 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 using standard methods to obtain 99 kg of milk powder. The milk powder and 1 kg of vitamin and mineral components were powder-mixed to finally 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 Formulated Formulated Milk Powder 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 standard method to obtain 99 kg of milk powder. The milk powder and 1 kg of vitamin and mineral components were powder-mixed to finally obtain 100 kg of milk powder. The amount of LNFP-II in the resulting milk powder was 242 mg / 100 g solids.

[0066] Example 3: Preparation of mucin-blended milk powder. 52.7 kg of whey powder, 239 kg of skim milk, and 32 g of porcine stomach-derived mucin material (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 standard methods 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 resulting milk powder was 8.0 mg / 100 g solids.

[0067] Example 4: Preparation of glycopeptide-containing milk powder. 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 23.9 kg of vegetable oil was mixed with this solution and homogenized. The resulting solution was sterilized, concentrated, and dried by standard methods 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 resulting milk powder was 0.25 mg / 100 g solids.

[0068] Example 5: Production of a supplement 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-shaped bag to produce a supplement for preventing food allergies of the present invention.

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

[0070]

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

[0072]

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

Claims

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

2. The nutritional composition according to claim 1, wherein the sugar chain having the Lewis X structure is an O-linked sugar chain 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. A nutritional composition according to claim 1 or 2, for use by infants and young children.

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

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

6. A nutritional composition for infants 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 containing 0.14 mg / 100 g solids or more of Galβ1-4(Fucα1-3)GlcNAcβ1-3GalNAc.

8. A method for preventing food allergies, comprising the step of administering to a subject one or more of the group consisting of 3-fucosyllactose, a sugar chain having a Lewis X structure, and lacto-N-fucopentaose II.

9. The method for preventing food allergy according to claim 8, wherein the sugar chain having a Lewis X structure is an O-linked sugar chain 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 10. The method for preventing food allergies described in claim 8 or 9, wherein the subject is an infant or young child.

11. Use of any one or more of the group consisting of 3-fucosyllactose, a sugar chain having a Lewis X structure, and lacto-N-fucopentaose II for producing a composition for preventing food allergies.

12. The use according to claim 11, 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 13. The use according to claim 11 or 12, wherein the food allergy is a food allergy in infants and young children.

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