Dendritic cell activation-suppressing composition

Abstract

To provide a novel dendritic cell activation-suppressing composition.SOLUTION: A dendritic cell activation-suppressing composition comprises at least one sugar chain selected from the group consisting of 3-fucosyllactose, a sugar chain having a Lewis A structure, and a sugar chain having a Lewis X structure as an active ingredient. The present invention also provides a dendritic cell activation-suppressing pharmaceutical composition, a dendritic cell activation-suppressing food composition or a dendritic cell activation-suppressing feed composition, each comprising the dendritic cell activation-suppressing composition. The present invention further provides an allergy disease preventing and / or improving composition comprising the dendritic cell activation-suppressing composition.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a composition for inhibiting dendritic cell activation, which contains a sugar chain as an active ingredient, and also to a pharmaceutical composition, food composition, or feed composition for inhibiting dendritic cell activation, which contains the composition. [Background technology]

[0002] The immune system is an important mechanism for maintaining the functions of the living body. An immune response must be induced against foreign antigens and tumor antigens, but the response must eventually converge. In addition, immune tolerance must be induced against allergens and self-antigens, and failure of this mechanism leads to the development of allergies and autoimmune diseases. Therefore, not only immune responses but also induction of immune tolerance are extremely important for maintaining the living body.

[0003] Dendritic cells are important immune cells in immune regulation. Dendritic cells are one of the antigen-presenting cells that take in foreign substances and present antigens to immune cells such as T cells, inducing antigen-specific immune responses. Dendritic cells are important immune cells that are the starting point of immune responses, but when they are overactivated by inflammatory inducers, they can cause various inflammatory diseases such as autoimmune diseases and allergies. Therefore, suppressing the increase of overactivated dendritic cells is thought to contribute to the prevention and treatment of these diseases. Therefore, the importance of functional food materials that can regulate the action of dendritic cells to maintain the immune system and prevent or reduce the risk of diseases is immeasurable.

[0004] HLA-DR and CD86 are known as surface antigens important for antigen presentation by dendritic cells. HLA-DR is an MHC class II molecule that presents antigens to T cells in acquired immunity. A decrease in HLA-DR expression means a decrease in the antigen presentation ability of dendritic cells, and acts suppressively against autoimmune diseases and allergies. On the other hand, CD86 is one of the costimulatory molecules that transmits auxiliary signals necessary for antigen presentation by MHC class II molecules. It has been reported that the symptoms of allergic asthma are alleviated in mice with the CD86 gene knocked down (Non-Patent Document 1). Therefore, it is considered that a decrease in HLA-DR and CD86 expression contributes to the alleviation of symptoms of autoimmune diseases and allergies. In fact, it has been reported that triptolide, one of the components with immunosuppressive effects, suppresses HLA-DR and CD86 expression in dendritic cells (Non-Patent Document 2).

[0005] Various food ingredients that suppress the activity of dendritic cells have been reported. For example, vitamin D is known to inhibit the activation of dendritic cells through binding to vitamin D receptors expressed on dendritic cells (Non-Patent Document 3). It has also been reported that the increase in HLA-DR and CD86 expression in dendritic cells stimulated with lipopolysaccharide (LPS) is suppressed by the addition of lactoferrin (Non-Patent Document 4). Furthermore, it has been reported that apple polyphenols reduce the expression of CD86 in dendritic cells (Non-Patent Document 5). Therefore, the intake of these food ingredients is expected to have an effect of alleviating the symptoms of autoimmune diseases and allergies.

[0006] Incidentally, various sugar chains having physiological activity have been reported, and examples of such sugar chains include Lewis X sugar chains and Lewis A sugar chains. Lewis X sugar chains and Lewis A sugar chains are the general names for oligosaccharides in which fucose is bound at α1-3 or α1-4 to type II and type I lactosamine. Glycolipids and glycoproteins having such sugar chain structures play an important role in intercellular information transmission and recognition mechanisms as blood group antigens and cancer-related antigens. Patent Document 1 reports that Lewis X sugar chains and Lewis A sugar chains have the effect of suppressing inflammatory response reactions (passive Arthus reaction) to intravenous administration of anti-ovalbumin rabbit antiserum and intradermal administration of ovalbumin antigen to guinea pigs. However, this patent document does not examine whether Lewis X sugar chains and Lewis A sugar chains directly suppress the activation of dendritic cells. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO94 / 20514 Brochure [Non-patent literature]

[0008] [Non-Patent Document 1] Y. Asai-Tajiri et al., “Small interfering RNA against CD86 during allergen challenge blocks experimental allergic asthma,” Respir. Res., vol. 15, no. 1, pp. 1-11, 2014. [Non-Patent Document 2] KJ Zhu, QY Shen, H. Cheng, XH Mao, LM Lao, and GL Hao, “Triptolide affects the differentiation, maturation and function of human dendritic cells,” Int. Immunopharmacol., vol. 5, no. 9, pp. 1415-1426, 2005. [Non-Patent Document 3] M. Bscheider and EC Butcher, “Vitamin D immunoregulation through dendritic cells,” Immunology, vol. 148, no. 3, pp. 227-236, 2016. [Non-Patent Document 4] HW Park et al., “Lactoferrin induces tolerogenic bone marrow-derived dendritic cells,” Immune Netw., vol. 20, no. 5, pp. 1-12, 2020. [Non-Patent Document 5] S. Katayama et al., “Apple polyphenols suppress antigen presentation of ovalbumin by THP-1-derived dendritic cells,” Food Chem., vol. 138, no. 2-3, pp. 757-761, Jun. 2013. Summary of the Invention [Problem to be solved by the invention]

[0009] An objective of the present invention is to provide a novel composition for inhibiting dendritic cell activation. [Means for solving the problem]

[0010] As a result of intensive research to solve the above problems, the present inventors have found that a sugar chain selected from the group consisting of 3-fucosyllactose (hereinafter referred to as 3-FL), a sugar chain having a Lewis A structure, and a sugar chain having a Lewis X structure has an inhibitory effect on dendritic cell activation, and have completed the present invention. That is, the present invention is as follows. <1> A composition for inhibiting dendritic cell activation comprising as an active ingredient one or more glycans from the group consisting of 3-fucosyllactose, glycans having a Lewis A structure, and glycans having a Lewis X structure. <2> The glycan having the Lewis A structure is a Lewis A trisaccharide. <1> A composition for inhibiting dendritic cell activation described in claim 1. <3> The glycan having a Lewis X structure is a Lewis X trisaccharide. <1> A composition for inhibiting dendritic cell activation described in claim 1. <4> <1> ~ <3> 2. A pharmaceutical composition, a food composition, or a feed composition for inhibiting dendritic cell activation, comprising the composition for inhibiting dendritic cell activation according to any one of claims 1 to 11. <5> <1> from <3> 2. A composition for preventing and / or ameliorating an allergic disease, comprising the composition for inhibiting dendritic cell activation described in any one of claims 1 to 11. Effect of the Invention

[0011] According to the present invention, it is possible to provide a composition for inhibiting dendritic cell activation, which contains as an active ingredient at least one type of sugar chain selected from the group consisting of 3-FL, a sugar chain having a Lewis A structure, and a sugar chain having a Lewis X structure. It is also possible to provide a pharmaceutical composition, food composition, or feed composition for inhibiting dendritic cell activation, which contains the composition. [Brief description of the drawings]

[0012] [Figure 1]This is a graph showing the expression level of HLA-DR 3 days after adding LPS and various glycans to TDDCs, which are dendritic cell-like cells differentiated from the human monocyte-derived cell line THP-1. In the figure, lowercase letters indicate significant differences between different letters. [Diagram 2] This is a graph showing the expression level of CD86 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

[0013] The composition for inhibiting dendritic cell activation of the present invention is characterized by containing 3-FL, a sugar chain having a Lewis A structure, and a sugar chain having a Lewis X structure as active ingredients. The present invention also relates to a pharmaceutical composition, food composition, and feed composition for inhibiting dendritic cell activation, which contain the composition. The present invention further relates to a composition for preventing or ameliorating allergic diseases, which contains the composition for inhibiting dendritic cell activation. Each active ingredient and the composition for each use will be described below.

[0014] (3-FL) The active ingredient of the present invention, 3-fucosyllactose, is an oligosaccharide in which fucose and lactose are linked together (3-O-(α-L-fucopyranosyl)-4-O-(β-D-galactopyranosyl)-D-glucopyranose). In this specification, the term "glycan" refers to a group of compounds in which various sugars are linked together like a chain through glycosidic bonds, and the number of linked sugars is two or more. Therefore, oligosaccharides such as 3-FL in which three sugars are linked are also included in the glycochain of the present invention. In addition, the glycochain may be in a free state or linked to a peptide and / or protein.

[0015] The 3-FL in the present invention 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 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 highly purified from the above.

[0016] The composition for inhibiting dendritic cell activation of the present invention, which contains 3-FL as an active ingredient, preferably contains 70.1 mg / 100 g or more of 3-FL per 100 g solid, more preferably 348 mg / 100 g or more of 3-FL. When the composition for inhibiting dendritic cell activation is in the form of powdered milk, the milk after preparation preferably contains 91.1 mg / L or more of 3-FL, more preferably 452 mg / L or more.

[0017] (Lewis A sugar chain) The sugar chain having a Lewis A structure, which is an active ingredient of the present invention, is a sugar chain having a Galβ1-3(Fucα1-4)GlcNAc structure in the sugar chain structure, and refers to a sugar chain having an inhibitory effect on dendritic cell activation. Specific examples include Galβ1-3(Fucα1-4)GlcNAc and lacto-N-fucopentaose-II (LNFP-II). Hereinafter, a sugar chain having a Lewis A structure may be simply referred to as a "Lewis A sugar chain".

[0018] The Lewis A 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 or from a fermentation method of a genetically modified bacterium, and may be any of a synthetic product, a semi-synthetic product, or a natural product. In addition, a natural material containing a natural product may be used as a supply source as it is and added to the nutritional composition of the present invention. Here, the natural product means a product extracted from a natural material containing a Lewis A sugar chain by a known method, a crudely purified product, or a product further purified from the above.

[0019] The composition for inhibiting dendritic cell activation of the present invention, which contains LNFP-II, a Lewis A sugar chain, as an active ingredient, preferably contains 0.68 mg / 100 g solid or more of LNFP-II, more preferably 243 mg / 100 g solid or more. When the composition for inhibiting dendritic cell activation is in the form of powdered milk, the milk after preparation preferably contains 0.88 mg / L or more of LNFP-II, more preferably 315 mg / L or more.

[0020] (Lewis X sugar chain) The sugar chain having a Lewis X structure, which is an active ingredient of the present invention, is a sugar chain having a Galβ1-4(Fucα1-3)GlcNAc structure in the sugar chain structure, and refers to a sugar chain having an inhibitory effect on dendritic cell activation. Specifically, examples include Neu5Acα2-3Galβ1-3[Galβ1-4(Fucα1-3)GlcNAcβ1-6]GalNAc, Galβ1-4(Fucα1-3)GlcNAcβ1-3GalNAc, Galβ1-4(Fucα1-3)GlcNAcβ1-3Gal, Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc, Galβ1-4(Fucα1-3)GlcNAcβ1-2Manα1-3(Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc, etc. Hereinafter, glycans having Lewis X structures are sometimes simply referred to as "Lewis X glycans".

[0021] 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.

[0022] The composition for inhibiting dendritic cell activation of the present invention, which contains 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. When the composition for inhibiting dendritic cell activation is in the form of powdered milk, the prepared milk preferably contains Lewis X sugar chain at 4.3 μmol / L or more, more preferably 10.1 μmol / L or more.

[0023] (How to use) As described above, the composition of the present invention can contain as active ingredients various sugar chains derived from milk, from fermentation of genetically modified bacteria, synthetic products, semi-synthetic products, or natural products, and therefore can be widely used as raw materials for pharmaceuticals, foods, beverages, and feeds.

[0024] (Pharmaceutical composition for inhibiting dendritic cell activation) When the composition for inhibiting dendritic cell activation of the present invention is formulated as a pharmaceutical product, it can be appropriately mixed with excipients, stabilizers, flavorings, etc. that are approved for pharmaceutical use, and then concentrated and lyophilized. These dried products, concentrates, and paste-like products are also included. 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 inhibitory action of various sugar chains on dendritic cell activation. Possible dosage forms include tablets, capsules, granules, powders, dusts, syrups, and the like, and it is preferable to administer these orally.

[0025] (Food composition for inhibiting dendritic cell activation) The composition for inhibiting dendritic cell activation of the present invention may be incorporated into any food or drink, or may be added to the raw materials during the manufacturing process of the food or drink. Examples of the food or drink include, but are not limited to, dairy products such as cheese, fermented milk, dairy lactic acid bacteria drinks, lactic acid bacteria drinks, butter, margarine, etc., beverages such as milk drinks, fruit juice drinks, and soft drinks, egg products such as jelly, candy, pudding, and mayonnaise, confectioneries and breads such as butter cake, and further various types of milk powder, foods for infants and children, and nutritional compositions.

[0026] The classification of the above-mentioned food and drink is not particularly limited, and the food and drink can be general food, health functional food, special purpose food, etc. In addition, the health functional food can be a specific health food, a nutrient functional food, a functional food, etc.

[0027] When indicating the use of the composition for inhibiting dendritic cell activation of the present invention, examples of the indication include indications stipulated by various laws, enforcement regulations, guidelines, etc. Indications of use include indications on packages such as wrapping and containers, as well as indications on advertising media such as pamphlets.

[0028] (Feed composition for inhibiting dendritic cell activation) The composition for inhibiting dendritic cell activation of the present invention can be added to feed. It may be added to any feed, similar to the above-mentioned foods and beverages, or may be added to the raw materials during the production process of the feed.

[0029] The subject to which the composition for inhibiting dendritic cell activation of the present invention is administered is not particularly limited, and may be a human or a non-human animal (e.g., a dog, a cat, a horse, or a rabbit). When the subject is a human, the composition may be administered to a minor under 20 years of age, an adult, or an elderly person over 65 years of age. Subjects to which the composition for inhibiting dendritic cell activation of the present invention is administered include healthy subjects and subjects diagnosed with an autoimmune disease or allergy. Further examples include subjects who have not yet developed allergic symptoms and are hoping for allergy prevention.

[0030] (Method for evaluating the ability to inhibit dendritic cell activation) The inhibitory effect of the present invention on dendritic cell activation can be evaluated by confirming whether the component to be evaluated inhibits dendritic cell activation, for example, by the following method shown in the Examples using THP-1 cells differentiated into dendritic cells. (1) THP-1 cells are cultured in a specific medium to differentiate into THP-1 derived dendritic cells (TDDCs). TDDCs are harvested and cultured in media containing various glycans. (2) After culture, the TDDCs recovered are stained with various cell surface antigen staining solutions and analyzed by flow cytometry. (3) From the analysis results, the median fluorescence intensity (MFI) of HLA-DR and CD86 is calculated. If the MFI calculated in this manner is found to be reduced when the component to be evaluated is contained compared to when it is not contained, then the component to be evaluated can be evaluated as having an inhibitory effect on dendritic cell activation.

[0031] (Allergy prevention effect) The composition for preventing or ameliorating allergic diseases of the present invention is a composition containing the composition for inhibiting dendritic cell activation, which has the effect of improving allergic symptoms or preventing the onset of allergic diseases by ingesting an effective amount of one or more active ingredients selected from the group consisting of 3-FL, Lewis A sugar chain, and Lewis X sugar chain through the inhibitory effect on dendritic cell activation. EXAMPLES

[0032] The present invention will be described in more detail below with reference to test examples and examples, but the present invention should not be construed as being limited to these examples, etc. Unless otherwise specified, % indicates % by mass.

[0033] [Test Example 1] Evaluation of the relationship between dendritic cells and various sugar chains To verify the inhibitory effect of various glycans on dendritic cell activation, the following evaluation was carried out.

[0034] 1. Method (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.

[0035] 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.

[0036] (2) Sample addition test to THP-1 derived dendritic cells (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 changed on day 3) to differentiate into TDDCs. TDDCs were harvested on day 6 and cultured at 2 × 10 5 The cells were seeded at 100 ng / ml LPS and 10 mg / ml of each sugar chain in a 96-well plate (3860-096, AGC Technoglass Co., Ltd.) at 10 cells / well and cultured for 3 days in a medium containing 100 ng / ml LPS and 10 mg / ml of each sugar chain.

[0037] (3) Surface antigen staining and FACS analysis After incubation, TDDCs were collected and 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℃ 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 antigen 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 BD FACSCanto TM The results were analyzed using a BD Biosciences II flow cytometer. The MFI of HLA-DR and CD86 was calculated from the results of the flow cytometry analysis.

[0038] (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.

[0039] 2.Results Evaluation of the effects of 3-FL, Lewis A glycan, and Lewis X glycan on dendritic cells Dendritic cells were activated by culturing 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 dendritic cells were activated (Figs. 1 and 2). When various sugar chains (lactose, 2'-FL, 3-FL, Lewis A (administered in the form of trisaccharide), and Lewis X (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. 1 and 2). 2'-FL and Lewis A did not affect the expression of HLA-DR (Fig. 1), but significantly suppressed the increase in CD86 expression (Fig. 2). 3-FL and Lewis X were shown to strongly suppress the expression of both HLA-DR and CD86 (Figs. 1 and 2). Thus, it was shown that 2'-FL, 3-FL, Lewis A, and Lewis X all have an inhibitory effect on dendritic cell activation, and that the effect is particularly strong for 3-FL and Lewis X.

[0040] [Test Example 2] Analysis of 3-FL, LNFP-II, and glycoprotein glycans in breast milk, and glycoprotein glycans in porcine stomach mucin We analyzed 3-FL and LNFP-II in human milk, glycoprotein glycans, and glycoprotein glycans with Lewis X structures in mucin derived from porcine stomach.

[0041] 1. Method (1) Analysis of 3-FL and LNFP-II oligosaccharides in breast milk 580 μL of ultrapure water was added to 20 μL of breast milk (200 samples), and then ultrafiltration with a molecular weight cutoff of 10,000 was performed, and the permeate was analyzed by HPLC. For 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. Three solutions were passed through the mobile phase at a flow rate of 1 mL / min: ultrapure water (solution A), 200 mM sodium hydroxide solution (solution B), and 100 mM sodium hydroxide solution containing 600 mM sodium acetate (solution C). 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 oligosaccharide content in each breast milk sample was calculated from a calibration curve created using 3-FL and LNFP-II standards of known concentrations.

[0042] (2) Measurement of O-linked glycans 2-AB-labeled O-linked glycans were prepared from breast milk and porcine stomach mucins using the EZGlyco Prep kit (Sumitomo Bakelite Co., Ltd.), an O-linked glycan analysis kit, and subjected to LC / MS and LC / MS / 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 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 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 range of m / z 300-2000 in positive mode. The acquired MS spectra were analyzed using Compound Discover 2.1 (Thermo Fisher Scientific). Glycans were detected by matching with an O-linked glycan database. MS / MS spectra were acquired in a positive mode, data-dependently in Top10 mode, and analyzed using SimGlycan Software (PREMIER Biosoft International).

[0043] (3) Determination of the binding mode of O-linked glycans contained in human milk and pig stomach mucin materials To determine the linkage of N-acetylneuraminic acid in O-linked glycans in human milk or porcine stomach mucin materials, the samples were treated with either sialidase that specifically degrades α2,3 linkages or sialidase that degrades both α2,3 and α2,6 linkages, and then analyzed by LC / MS. To determine the linkage of fucose, the samples were treated with either fucosidase that specifically degrades α1,2 linkages, fucosidase that specifically degrades α1,3 and α1,4 linkages, or fucosidase that specifically degrades α1,2 linkages, α1,4 linkages, and α1,6 linkages, and then analyzed by LC / MS. Furthermore, to determine the binding mode of galactose, the samples were treated with either galactosidase that specifically degrades β1,3 bonds and fucosidase that specifically degrades α1,3 bonds and α1,4 bonds at the same time, or galactosidase that specifically degrades β1,4 bonds at the same time as fucosidase that specifically degrades α1,3 bonds and α1,4 bonds at the same time, and then analyzed by LC / MS. 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 2-AB-labeled O-linked glycan solution prepared from breast milk or pig stomach mucin material, and then 3 μL of each enzyme solution was added and incubated at 37°C for 1 hour. The enzyme was inactivated by heating in boiling water for 5 minutes, and then 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 GlycanPac AXH-1, and the column temperature was set to 40°C. Acetonitrile (liquid A) and 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 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 spectrum was analyzed using FreeStyle software (Thermo Fisher Scientific Co., Ltd.).

[0044] 2.Results (1) Analysis of 3-FL and LNFP-II in breast milk Analysis of 200 breast milk samples revealed that LNFP-II, which has 3-FL and Lewis A structures, was detected in all breast milk samples, suggesting that these two types of oligosaccharides in breast milk may suppress the activation of dendritic cells in infants.

[0045] (2) Analysis of glycans of glycoproteins with Lewis X structures in breast milk Analysis of glycoprotein glycans with Lewis X structures in breast milk revealed that Neu5Acα2-3Galβ1-3[Galβ1-4(Fucα1-3)GlcNAcβ1-6]GalNAc (Lewis X glycan A) and Galβ1-4(Fucα1-3)GlcNAcβ1-3GalNAc (Lewis X glycan B). Therefore, it is possible that these two types of glycans with Lewis X structures in breast milk suppress the activation of infant dendritic cells.

[0046] (3) Analysis of glycoprotein glycans with Lewis X structures contained in mucin material derived from porcine stomach Analysis of glycoprotein glycans with Lewis X structures contained in the food material, porcine stomach-derived mucin, detected Lewis X glycan A. Therefore, it is expected that the food material, porcine stomach-derived mucin, inhibits the activation of dendritic cells through the glycans with Lewis X structures contained therein.

[0047] [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 components to obtain 100 kg of milk powder. The amount of 3-FL in the resulting milk powder was 348 mg / 100 g solid.

[0048] [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 components to obtain 100 kg of milk powder. The amount of LNFP-II in the resulting milk powder was 242 mg / 100 g solid.

[0049] [Example 3] 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 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 Lewis X sugar chain A in the obtained milk powder was 8.0 mg / 100 g solid.

[0050] [Example 4] Preparation of powdered milk containing glycopeptides 52.7 kg of whey powder, 239 kg of skim milk, and 250 mg of Lewis X sugar chain 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 Lewis X sugar chain B in the obtained milk powder was 0.25 mg / 100 g solid.

[0051] [Example 5] Preparation of Lewis A sugar chain blended milk powder 52.7 kg of whey powder, 239 kg of skim milk, and 250 mg of Lewis A trisaccharide 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 finally obtain 100 kg of modified milk powder.

[0052] [Example 6] Preparation of Lewis X sugar chain blended milk powder 52.7 kg of whey powder, 239 kg of skim milk, and 250 mg of Lewis X trisaccharide 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 finally obtain 100 kg of modified milk powder.

[0053] [Example 7] 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 supplement for preventing and / or improving food allergies of the present invention.

[0054] [Example 8] Production of beverages The ingredients were mixed according to the composition shown in Table 1, filled into a container, and then heat sterilized to produce a beverage for preventing and / or improving allergies and autoimmune diseases of the present invention.

[0055] [Table 1]

[0056] [Example 9] Production of beverages The ingredients were mixed according to the composition shown in Table 2, filled into containers, and then heat sterilized to produce the beverage for preventing allergies and autoimmune diseases of the present invention.

[0057] [Table 2] [Industrial Applicability]

[0058] According to the present invention, it is possible to provide a composition for inhibiting dendritic cell activation, which contains as an active ingredient at least one type of sugar chain selected from the group consisting of 3-FL, a sugar chain having a Lewis A structure, and a sugar chain having a Lewis X structure.

Claims

1. A composition for inhibiting dendritic cell activation, comprising as an active ingredient one or more sugar chains from the group consisting of 3-fucosyllactose, sugar chains having a Lewis A structure, and sugar chains having a Lewis X structure.

2. 2. The composition for suppressing dendritic cell activation according to claim 1, wherein the sugar chain having a Lewis A structure is Lewis A trisaccharide.

3. 2. The composition for suppressing dendritic cell activation according to claim 1, wherein the sugar chain having a Lewis X structure is Lewis X trisaccharide.

4. A pharmaceutical composition, a food composition, or a feed composition for inhibiting dendritic cell activation, comprising the composition for inhibiting dendritic cell activation according to any one of claims 1 to 3.

5. A composition for preventing and / or ameliorating an allergic disease, comprising the composition for inhibiting dendritic cell activation according to any one of claims 1 to 3.

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