Application of breast milk oligosaccharide in regulating brain immunity

Abstract

The invention belongs to the technical field of food, and relates to application of breast milk oligosaccharide in regulating brain immunity. Specifically, the invention relates to the non-therapeutic purpose of adjusting brain immunity by adjusting polarization of microglial cells M1 / M2, and the breast milk oligosaccharide is any one of 3-fucosyllactose, lactose-N-tetraose, lactose-N-neotetraose, 3 '-sialic acid lactose and 6'-sialic acid lactose. Research results show that a specific variety of breast milk oligosaccharides (especially 3-FL) can obviously inhibit the generation of neurotoxic factors NO, inflammatory factors TNF-alpha and IL-1beta in microglial cells, and promote the generation of anti-inflammatory cytokines IL-10 in the microglial cells, so that the state of the microglial cells is improved, damage repair is promoted, and the microglial cells are prevented from being damaged. Therefore, the effect of regulating brain immunity is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of food technology and relates to the use of human milk oligosaccharides in regulating brain immunity, and specifically relates to the use of human milk oligosaccharides to achieve the non-therapeutic purpose of regulating brain immunity by regulating microglial M1 / M2 polarization. Background Art

[0002] Glial cells, also known as glial cells for short, are another major type of cells in the nervous tissue besides neurons. They have protrusions, but no distinction between dendrites and axons. They are widely distributed in the central and peripheral nervous systems. The main types of glial cells in the central nervous system (CNS) are: astrocytes, oligodendrocytes, microglia, ependymal cells, and radial glia.

[0003] Microglia are the only nerve cells that originate from the mesoderm. As a specialized macrophage, they perform immune functions in the nervous system. The brain establishes many connections between neurons, allowing them to transmit information back and forth. In fact, under normal circumstances, far more synapses are produced than are needed, and only the strongest and most important synapses survive. Microglia are directly involved in the "pruning" process of synapses by engulfing synapses marked as unnecessary, removing harmful substances, repairing damage, and protecting various types of neurons in the central nervous system.

[0004] Microglia, as resident macrophages in the CNS, account for 5% to 12% of all CNS-specific cells. They are the most abundant immune cells in the CNS and play a major role in maintaining or promoting the health of the central nervous system. When subjected to adverse stimuli, microglia are rapidly activated and eventually polarize into two cell populations with opposite functions, M1 or M2. M1 phenotype microglia mainly release pro-inflammatory cytokines, such as tumor necrosis factor (TNF-α), interleukin 1β (IL-1β), and chemical signal nitric oxide (nitric oxid, NO), which may cause an inflammatory response in the CNS, leading to neuronal damage and ultimately death; M2 phenotype microglia mainly promote damage repair and exert a neuroprotective effect by secreting anti-inflammatory cytokines, such as interleukin 10 (IL-10).

[0005] Breast milk contains about 7% carbohydrates, of which 80% is lactose, and the remaining 20% ​​is composed of human milk oligosaccharides (HMOs). The content of HMOs is highest in colostrum, about 20-25g / L, and in mature milk it is about 5-20g / L. It is the third largest solid component in breast milk, second only to lactose and fat.

[0006] Studies have shown that HMOs play an important role in improving intestinal flora, maintaining intestinal barrier, regulating immunity, resisting pathogen infection and promoting neural development. HMOs are mainly composed of five core monomer structures: glucose (Glc), sialic acid (SA), fucose (Fuc), N-acetylglucosamine (GlcNAc) and galactose (Gal). Different HMOs have different fucosylation and sialylation in their structures, so HMOs in breast milk can be divided into neutral fucosylated HMOs (such as 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL)), neutral non-fucosylated HMOs (such as lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT)) and acidic sialylated HMOs (such as 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL)).

[0007] Reference 1 discloses the use of an oligosaccharide 2′-FL in the preparation of a product for improving neural development and functional disorders. Specifically, the use of the oligosaccharide 2′-FL in the preparation of a protein inhibitor, which can inhibit the accumulation of Aβ protein, which can lead to neuronal synaptic disorders, neuronal apoptosis, brain damage and functional disorders. 2'-FL improves neurological diseases caused by the accumulation of Aβ protein; in addition, the use of 2'-FL in the preparation of a cholinergic modulator is also provided, which can inhibit the activity of acetylcholinesterase, improve and protect the normal development of cholinergic neurons, and increase the content of neurotransmitters.

[0008] Reference 2 discloses an oligosaccharide composition for improving neural development and dysfunction, wherein the oligosaccharide composition includes 6′-sialyllactose and lactose-N-trisaccharide. The oligosaccharide composition can effectively inhibit the accumulation of Aβ protein, thereby protecting nerves. In addition, it can also inhibit the activity of acetylcholinesterase, protect the normal development of cholinergic neurons and increase the content of neurotransmitters.

[0009] Reference 3 discloses a nutritional composition for promoting neurodevelopment, which includes 2'-fucosyllactose and osteopontin. Studies have found that the nutritional composition has a significant synergistic effect in promoting neurodevelopment such as neuronal maturation, synaptogenesis and myelination, especially in promoting the proliferation, maturation and differentiation of oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes (OLs) and / or the myelination characteristics of OLs.

[0010] Reference 4 discloses a nutritional composition comprising human milk oligosaccharides, milk phospholipids and choline and / or edible choline derivatives. Studies have found that the nutritional composition can promote neural development such as neuronal maturation, synaptogenesis and myelination.

[0011] The study cited in reference 5 found that rats received intracerebroventricular injection of 2'-FL or saline before middle cerebral artery occlusion (MCAo) surgery, and the IBA1 immunoreactivity (IBA1-ir) of the rats was detected 2 days after treatment. The results showed that the administration of 2'-FL reduced IBA1-ir and the morphological activation of microglia in the perilesional area.

[0012] Reference 6 discloses a nutritional composition containing a therapeutically effective amount of 2'-fucosyllactose, which is used for treating, preventing or alleviating neuroinflammation, and is also used for treating cognitive dysfunction, preventing cognitive impairment, or reducing cognitive damage.

[0013] References:

[0014] Reference 1: CN118105395A

[0015] Reference 2: CN118104826A

[0016] Reference 3: CN114586983A

[0017] Reference 4: CN114223723A

[0018] Cited literature 5: Wu KJ, Chen YH, Bae EK, et al. Human milk oligosaccharide2′-fucosyllactose reduces neurodegeneration in stroke brain[J]. TranslationalStroke Research, 2020,11:1001-1011.

[0019] Reference 6: WO2015100091A1 Summary of the invention

[0020] Problem that the invention aims to solve

[0021] Although existing technologies have been used to study the functions of human milk oligosaccharides and their compositions, on the one hand, current research directions of human milk oligosaccharides are mostly aimed at promoting neural development, while less attention is paid to regulating brain immunity, which is equally important as promoting neural development in the brain; on the other hand, current research on human milk oligosaccharides is more focused on 2'-fucosyllactose, while research on other types of human milk oligosaccharides is relatively rare; therefore, there is still room for further exploration of nutrients that regulate brain immunity, and there is still development prospect for functional research and application of other types of human milk oligosaccharides.

[0022] Therefore, the primary purpose of the present invention is to provide a new use of human milk oligosaccharides, specifically, to provide a new use of human milk oligosaccharides for the non-therapeutic purpose of regulating brain immunity by regulating microglial M1 / M2 polarization.

[0023] Further research of the present invention also shows that some specific types of human milk oligosaccharides (especially 3-FL) have verifiable effects in regulating brain immunity. The present invention uses microglial cell lines (BV2 cells) stimulated by LPS as the research object, analyzes and measures the amount of pro-inflammatory cytokines and anti-inflammatory cytokines produced by them, and verifies and demonstrates the effect of human milk oligosaccharides on regulating brain immunity from the effects of human milk oligosaccharides on reducing the content of NO, TNF-α, IL-1β and increasing the content of IL-10 in the cell supernatant within a certain concentration range of human milk oligosaccharides.

[0024] In addition, the present invention also provides a non-therapeutic use of an edible human milk oligosaccharide composition in regulating brain immunity.

[0025] Solutions for solving problems

[0026] The present invention finds that the above technical problems can be solved by the following technical solutions:

[0027] [1] The non-therapeutic use of human milk oligosaccharides in regulating brain immunity, characterized in that the human milk oligosaccharides are any one of 3-fucosyllactose, lactose-N-tetraose, lactose-N-neotetraose, 3'-sialyllactose and 6'-sialyllactose.

[0028] [2]. The use according to [1], wherein the regulation of brain immunity is achieved by regulating microglial M1 / M2 polarization, and the regulation of microglial M1 / M2 polarization includes at least one of inhibiting microglial polarization to M1 phenotype, promoting microglial polarization to M2 phenotype, and inducing microglial transformation from M1 phenotype to M2 phenotype.

[0029] [3] The use according to [2], wherein the regulation of microglia M1 / M2 polarization includes inhibiting microglia from producing pro-inflammatory cytokines and / or promoting microglia from producing anti-inflammatory cytokines.

[0030] [4] The use according to [3], wherein the pro-inflammatory cytokines include one or more of TNF-α, IL-1β and NO; and the anti-inflammatory cytokines include IL-10.

[0031] [5] A non-therapeutic use of a human milk oligosaccharide composition in regulating brain immunity, wherein the human milk oligosaccharide composition comprises at least two of 3-fucosyllactose, lactose-N-tetraose, lactose-N-neotetraose, 3'-sialyllactose and 6'-sialyllactose, and optionally further comprises a solvent;

[0032] Wherein, the regulation of brain immunity is achieved by regulating microglial M1 / M2 polarization, and the regulation of microglial M1 / M2 polarization includes at least one of inhibiting microglial polarization to M1 phenotype, promoting microglial polarization to M2 phenotype and inducing microglial transformation from M1 phenotype to M2 phenotype.

[0033] [6]. The use according to [5], wherein the solvent comprises any one of water, physiological saline, and glucose water.

[0034] [7]. The use according to [5] or [6], wherein the regulation of microglia M1 / M2 polarization includes inhibiting microglia from producing pro-inflammatory cytokines and / or promoting microglia from producing anti-inflammatory cytokines.

[0035] [8] The use according to [7], wherein the pro-inflammatory cytokines include one or more of TNF-α, IL-1β and NO; and the anti-inflammatory cytokines include IL-10.

[0036] [9]. The use according to any one of [5] to [8], wherein the human milk oligosaccharide composition is an edible composition.

[0037]

[10] . The use according to [9], wherein the edible composition is a dairy product.

[0038] Effects of the Invention

[0039] Based on the implementation of the above technical solution, the present invention can achieve the following technical effects:

[0040] 1) The experimental results of the present invention show that certain types of human milk oligosaccharides have the effect of regulating brain immunity, especially by regulating microglial M1 / M2 polarization (especially regulating the state of microglial cells in the central nervous system);

[0041] 2) Further research in the present invention shows that specific types of human milk oligosaccharides (especially 3-FL) can significantly inhibit the production of neurotoxic factors NO, inflammatory factors TNF-α and IL-1β in microglia, and promote the production of anti-inflammatory cytokine IL-10 in microglia, thereby improving the state of microglia, promoting damage repair, and regulating brain immunity;

[0042] 3) The technical solution provided by the present invention has no special requirements for the application mode of human milk oligosaccharides and the form of the edible human milk oligosaccharide composition, and is convenient in implementation;

[0043] 4) The present invention provides a new application method for a specific type of human milk oligosaccharides (especially 3-FL), and provides a new idea for the development of nutritional supplementary foods that have the function of regulating brain immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 : Schematic diagram of the experimental process of studying the regulatory effect of functional nutrients on microglia in the present invention. DETAILED DESCRIPTION

[0045] The following describes the embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various structures described below, and various changes can be made within the scope of the invention claim, and the embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention.

[0046] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints numerical values ​​A and B.

[0047] In this specification, unless otherwise specified, the word "multiple" in "multiple", "multiple", "plurality", etc. means a numerical value of 2 or more.

[0048] In this specification, "comprising", "having", "including" or "containing" may mean inclusive or open-ended, not excluding additional, uncited elements or method steps. At the same time, "comprising", "having", "including" or "containing" may also mean closed, excluding additional, uncited elements or method steps.

[0049] In this specification, the numerical range expressed using "above" or "below" means a numerical range including the number.

[0050] In this specification, the word "may" includes both performing a certain process and not performing a certain process.

[0051] In this specification, the use of "optional" or "optional" indicates that certain substances, components, execution steps, application conditions and other factors are used or not used.

[0052] In this specification, unless otherwise specified, the "normal temperature" used generally refers to the temperature of 23±2°C.

[0053] In this specification, the unit names used are all international standard unit names, and unless otherwise stated, the "%" used indicates weight or mass percentage.

[0054] In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0055] Unless otherwise defined, other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0056] The present invention mainly provides a new use of human milk oligosaccharides, that is, a new effect of human milk oligosaccharides is discovered through in vitro cell experiments, and based on this research, the existing application of human milk oligosaccharides is expanded. The present invention is mainly based on the following insights:

[0057] Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria. Studies have shown that LPS can enter the brain through the blood-brain barrier to activate microglia and induce neuroinflammatory reactions, induce the release of proinflammatory cytokines, and cause memory impairment. The present invention uses microglia stimulated by LPS as a microglia activation model to study the regulatory effects of nutrients of different types and concentrations on microglia, and explore functional nutrients (such as LPS) that are safe (non-cytotoxic) and have obvious effects on regulating brain immunity. Figure 1 The present invention has found through research that the use of specific types of breast milk oligosaccharides (especially 3-FL, LNnT, etc.) to intervene in microglia stimulated by LPS can increase the amount of IL-10 released by microglia and reduce the amount of TNF-α, IL-1β and NO released by microglia.

[0058] (Human milk oligosaccharides)

[0059] Human milk oligosaccharides (HMOs) are substances unique to breast milk, and their content is second only to lactose and fat. Within 3 days after giving birth, the HMOs content in breast milk can exceed 20g / L, and the content in mature milk after half a month is about 5-20g / L.

[0060] HMOs are mainly composed of five core monomer structures: glucose, sialic acid, fucose, N-acetylglucosamine and galactose. Different HMOs have different fucosylation and sialylation in their structures, so HMOs in breast milk can be divided into neutral fucosylated HMOs, acidic sialylated HMOs, and neutral non-fucosylated HMOs. Among them, neutral fucosylated HMOs are considered to be the HMOs with the highest content in mature breast milk, including 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), etc.

[0061] Related studies predict that there are more than a thousand types of HMOs in human breast milk, mainly because the molecular structure and spatial configuration of the constituent monomers are different. Currently, more than 200 HMOs have been reported, and the most studied ones are 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lactose-N-neotetraose (LNnT), lactose-N-tetraose (LNT), 6'-sialyllactose (6'-SL) and 3'-sialyllactose (3'-SL). At present, some regions use 2'-FL, LNnT, LNT, 3'-sialyllactose sodium salt and 6'-sialyllactose sodium salt as new food raw materials for infant formula and food supplements.

[0062] At present, the production methods of human milk oligosaccharides mainly include chemical synthesis, enzymatic synthesis and bioengineering synthesis. At the same time, these components can also be indirectly introduced from various existing milk components.

[0063] It should be noted that the use of the various human milk oligosaccharides mentioned above in the present invention should comply with the requirements of local laws and regulations. In some cases, where permitted by laws and regulations, these ingredients can be directly introduced into the food composition in the form of separate raw materials; in other cases, where permitted by laws and regulations, they can be indirectly introduced into the composition or food by adding milk raw materials that meet the requirements.

[0064] The present invention does not particularly limit the source of human milk oligosaccharides, and typically, they can be brought in from different milk-containing raw materials, such as animal milk raw materials (such as cow's milk, milk powder, etc.). In the present invention, "animal milk" is used to refer to the liquid obtained from the mammary gland of a mammal in the lactation period. The term "animal milk" should be interpreted broadly and covers both raw milk (i.e., liquid obtained directly from the mammary gland) and standardized dairy products. In some embodiments, the human milk oligosaccharides described in the present invention are any one of 3-fucosyllactose, lactose-N-tetraose, lactose-N-neotetraose, 3'-sialyllactose and 6'-sialyllactose, preferably 3-fucosyllactose (3-FL).

[0065] (Edible human milk oligosaccharide composition)

[0066] The present invention further provides an edible human milk oligosaccharide composition, wherein the human milk oligosaccharide composition comprises at least two of 3-fucosyllactose, lactose-N-tetraose, lactose-N-neotetraose, 3'-sialyllactose and 6'-sialyllactose, and optionally also comprises a solvent.

[0067] The present invention does not specifically limit the use form of the human milk oligosaccharide composition, as long as it complies with the requirements of relevant laws and regulations. Within the permitted range, it can be used in the form of a combination of two or more components, or other solvents that can be added to the composition can be used to give the desired human milk oligosaccharide in the use form. In some specific embodiments, the solvent includes any one of water, saline, and glucose water. In the present invention, there is no particular limitation on the final form of the human milk oligosaccharide composition, which can be, for example, a liquid (with the help of a desired solvent), a solid (such as a powder, granules or a block (pressed)) at room temperature, etc.

[0068] (food)

[0069] Furthermore, the present invention provides a food based on the above human milk oligosaccharide, especially based on the above edible human milk oligosaccharide composition. The food has the effect of regulating brain immunity due to the use of the above specific type of human milk oligosaccharide.

[0070] The food of the present invention may include, in addition to the above-mentioned human milk oligosaccharides or human milk oligosaccharide composition, other optional ingredients according to the needs of the final product, which may include:

[0071] Plants or plant extracts, including fruits such as figs, pomegranates, kiwis, tangerines, oranges, pineapples, strawberries, apples, bananas, grapes, pears, cherries, blueberries, blackberries, raspberries and bilberries or their extracts; fruits and vegetables such as onions, cucumbers, tomatoes, cauliflower, carrots, spinach, kale, Brussels sprouts, garlic, basil, oregano or their extracts; grains such as rice (indica rice, japonica rice, glutinous rice), wheat (wheat, barley, oats, rye), corn, sorghum, millet, millet, yellow rice, buckwheat, soybeans, broad beans, peas, mung beans, red beans, kidney beans or their extracts; nuts such as walnuts, cashews, hazelnuts, almonds, apricots, pine nuts, pistachios, peanuts, melon seeds, chestnuts, macadamia nuts, ginkgo nuts or their extracts; coffee or its extracts.

[0072] Contains milk ingredients, including fresh milk from raw cow (sheep) milk, milk powder, whey protein or cheese.

[0073] Animal ingredients include meat products from beef, lamb, fish or poultry.

[0074] The fat component may include at least one of saturated fatty acids, polyunsaturated fatty acids, monounsaturated fatty acids, OPO structured lipids, DHA, EPA, ARA and phospholipids, and more specifically, the fat includes safflower seed oil, walnut oil, peanut oil, soybean oil, argan oil, olive oil, tea oil, sacha inchi oil, olive oil, coconut oil, perilla oil, deep-sea fish oil, cocoa butter, palm oil, butter, cream, lard, medium-chain triglycerides, lecithin, etc.

[0075] Functional added ingredients, including vitamins (vitamin A, β-carotene, vitamin D3, vitamin E, vitamin K1, vitamin B1, vitamin B2, vitamin B6, vitamin B 12 , vitamin C, pantothenic acid, folic acid, niacin, choline, inositol, biotin, etc.), starch, modified starch, amino acids (L-lysine-L-glutamic acid, L-glutamic acid, L-arginine, L-tryptophan, L-glutamine, taurine, L-valine, L-isoleucine, L-leucine, etc.), traditional Chinese medicine or traditional Chinese medicine extracts, dietary fiber (inulin, konjac flour, galacto-oligosaccharides, fructo-oligosaccharides, isomaltooligosaccharides, soybean polysaccharides, cyclodextrin, resistant dextrin, soybean fiber, etc.).

[0076] Trace element supplements, including calcium citrate, L-calcium lactate, calcium hydrogen phosphate, potassium gluconate, sodium citrate, ferrous gluconate, zinc gluconate, sodium selenite, copper gluconate, manganese gluconate, magnesium gluconate, etc.

[0077] Any food-acceptable excipients, including solvents, antioxidants, antibacterial agents, thickeners, diluents, cosolvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, edible flavors, edible colorings, etc.

[0078] In some preferred embodiments, the food comprises human milk oligosaccharides, antioxidant vitamins (vitamin A, vitamin C, vitamin E, vitamin B6, etc.) and protein components.

[0079] In principle, there is no particular limitation on the content of the human milk oligosaccharide in the food. In some specific embodiments of the present invention, the content of the human milk oligosaccharide in the food is 0.001% by mass or more, preferably 0.005% by mass or more, and more preferably 0.01% by mass or more, based on dry weight, from the perspective of having the desired effect of regulating brain immunity, and there is no particular limitation on the upper limit of the content.

[0080] The present invention does not specifically limit the specific form of the food. Exemplarily, at room temperature, the food may be in solid form, semi-solid form or liquid form. In some specific embodiments, the food may be powdered reconstituted food (instant coffee, cereal powder, nut powder or lotus root powder, etc.), baked food (bread, cake or biscuit baked food, etc.), beverage (carbonated beverage, fruit and vegetable juice beverage, functional beverage, tea beverage or milk beverage, etc.), milk and dairy products (fresh milk, milk powder, whey powder, fermented milk, cheese or condensed milk derived from fresh cow (sheep) milk, etc.), pasta products (dried noodles, instant noodles, steamed buns, dumplings or wontons, etc.), etc.

[0081] (Use in regulating brain immunity)

[0082] The present invention proposes the use of the above-mentioned human milk oligosaccharide in regulating brain immunity, so the above-mentioned edible human milk oligosaccharide composition and food can also be used for the above-mentioned purpose. Therefore, the present invention believes that eating these foods can have the effect of regulating brain immunity.

[0083] The "regulating brain immunity" mentioned in the present invention means that the immune activity in the brain is maintained at the most appropriate level in the most appropriate form by regulating the interactions between immune cells and immune molecules and other systems such as the neuroendocrine system. At the same time, the regulating brain immunity mentioned in the present invention is not intended to prevent and / or treat diseases.

[0084] In some specific embodiments, the present invention relates to regulating brain immunity by regulating the state of microglia, wherein the state of microglia includes their tendency to polarize to M1 or M2, and the amount or ratio of various cytokines secreted by them.

[0085] In some more specific embodiments, the regulating brain immunity is achieved by regulating microglial M1 / M2 polarization, and the regulating microglial M1 / M2 polarization includes at least one of inhibiting microglial polarization to M1 phenotype, promoting microglial polarization to M2 phenotype, and inducing microglial transformation from M1 phenotype to M2 phenotype.

[0086] In some more specific embodiments, the regulation of microglia M1 / M2 polarization described in the present invention includes inhibiting microglia from producing proinflammatory cytokines and / or promoting microglia from producing anti-inflammatory cytokines. In some specific embodiments, the regulation of microglia M1 / M2 polarization described in the present invention includes inhibiting microglia from producing proinflammatory cytokines and promoting microglia from producing anti-inflammatory cytokines.

[0087] In some specific embodiments, the proinflammatory cytokines include one or more of TNF-α, IL-1β, and NO.

[0088] In some specific embodiments, the anti-inflammatory cytokine comprises IL-10.

[0089] Example

[0090] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0091] 1. Materials and Methods

[0092] 1.1 Raw materials

[0093] Table 1. Main raw materials for the experiment

[0094] serial number raw material Product Name company batch number 1 2'-FL 2'-fucosyllactose DSM DK22296001 2 LqCy GlyCare LNnT 9000 DSM 20454001 3 LNT GlyCare LNT 8001 DSM 19242101 4 3'-SL 3SL 9001 DSM DK21294101 5 6'-SL GlyCare 6SL 9001 DSM 19487101 6 3-FL GlyCare 3FL 9001 DSM DK22381101

[0095] 1.2 Cells

[0096] Mouse microglial cell line BV2 cells were purchased from Shanghai Enzyme Research Biotechnology (CC-Y2022).

[0097] 1.3 Reagents

[0098] Table 2. Main reagents for the experiment

[0099] serial number Reagent name factory model 1 DMEM high glucose SkyJet CA0004-500ML 2 0.25% pancreatin solution SkyJet CN0004-100ML 3 Penicillin-Streptomycin Mixture SkyJet CM0001-100ML 4 Special grade fetal bovine serum Vicente 085-150 5 PBS Meilun Biotechnology MA0020 6 Cell cryopreservation medium Meilun Biotechnology MA0405 7 LPS Sigma L2880 8 CCK-8 Kit White Shark BS350A 9 Nitric Oxide Test Kit Blue Sky S0021S 10 IL-10 ELISA Kit Chenglin Biotechnology AD2837Mo 11 IL-β ELISA Kit Chenglin Biotechnology AD3364Mo 12 TNF-α ELISA Kit Chenglin Biotechnology AD3051Mo

[0100] 1.4 Instruments

[0101] Table 3. Main instruments and equipment for the experiment

[0102] serial number Instrument Name factory 1 VD-1320 clean workbench Beijing Donglian Har Instrument Manufacturing Company 2 Carbon dioxide incubator Thermo Fisher Scientific China Co., Ltd. 3 Motic Microscope Harbin Wayne Trading Co., Ltd. 4 Cell Imaging Microplate Reader Meigu Molecular Instruments Co., Ltd. 5 Constant temperature water bath Quanzhou Yiguan Technology Co., Ltd. 6 Multifunctional ELISA reader Dynatech Corporation 7 Centrifuge Thermo Fisher Scientific China Co., Ltd.

[0103] 1.5 Cell culture

[0104] Mouse microglial cell line (BV2) was cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37° C. in a 5% CO 2 incubator.

[0105] 1.6 Nutrient cytotoxicity assay

[0106] CCK-8 kit was used to detect the cytotoxicity of nutrients.

[0107] The working principle of the CCK-8 kit is: using the dehydrogenase in the cell mitochondria, in the presence of the electron carrier 1-methoxy-5-methylphenazine dimethyl sulfate, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt is reduced to yellow formazan. Because only mitochondria in living cells can produce dehydrogenase, the production of formazan is directly proportional to the number of living cells, and the OD value is detected at 450nm on a microplate reader.

[0108] BV2 cells were seeded into 96-well plates at 4 × 10 4 cells, 200 μL / well, 3 replicates per group. Place in an incubator and culture for 12 hours. After the cells are completely attached, add the prepared culture medium of each nutrient to the 96-well plate. After 12 hours of culture, add 20 μL CCK-8 solution (protected from light) to each well and continue incubating in the cell culture incubator for 0.5 to 1 hour. Use an enzyme marker to measure the absorbance at 450nm, and then calculate the cell survival rate according to the formula: cell survival rate = [(absorbance of experimental well-absorbance of blank well) / (absorbance of control well-absorbance of blank well)] × 100% (wherein the experimental well contains cells, culture medium, nutrients and CCK-8 solution; the blank well contains culture medium and CCK-8 solution, but does not contain cells and nutrients; the control well contains cells, culture medium and CCK-8 solution, but does not contain nutrients). The CCK8 method was used to detect the viability of BV2 microglia.

[0109] 1.7 Establishment of microglial activation model and experimental grouping

[0110] Mouse microglial cell line (BV2) was cultured in a 5% CO2 incubator (37°C, saturated humidity) using high-glucose DMEM medium (containing 10% fetal bovine serum). BV2 cells in logarithmic growth phase were selected, and cells in logarithmic growth phase and reaching 90% confluence were digested with trypsin. The cell concentration was adjusted, and the cells were inoculated in a suitable cell culture dish, and then cultured for another 24 hours before the cells were grouped. 4 The number of cells / mL was inoculated in a 96-well plate, 200 μL / well, and 3 replicates were added to each group. The experiment was divided into 20 groups, namely blank control group, LPS model group, and 18 experimental groups (6 types of HMO, 2'-FL, LNnT, LNT, 3'-SL, 6'-SL, 3-FL, each with three concentration gradients of low, medium and high). (1) Control group: conventional cell culture; (2) LPS model group: LPS (1 μg / mL) was used to intervene BV2 cells for 24 hours to establish a microglial activation model; (3) Experimental group: LPS stimulated BV2 cells for 24 hours, and each nutrient was added for intervention for 12 hours.

[0111] Table 4 Experimental groups and doses

[0112] Serial number Group LPS (μg / mL) HMO (mg / mL) 1 Blank control group - - 2 LPS model group 1 - 3 2'-FL low dose 1 0.5 4 2'-FL medium dose 1 1 5 2'-FL high dose 1 2 6 LNnT low dose 1 0.05 7 LNnT medium dose 1 0.1 8 LNnT High Dose 1 0.2 9 LNT low dose 1 0.05 10 LNT medium dose 1 0.1 11 LNT High Dose 1 0.2 12 3'-SL low dose 1 1.25 13 3'-SL medium dose 1 5.0 14 3'-SL high dose 1 10 15 6'-SL low dose 1 1.25 16 6'-SL medium dose 1 5.0 17 6'-SL High Dose 1 10 18 3-FL low dose 1 0.5 19 3-FL medium dose 1 1 20 3-FL high dose 1 2

[0113] 1.8 NO kit to detect the NO content in the supernatant of BV2 cells

[0114] The BV2 cell experimental grouping and nutrient intervention methods were the same as above. After the culture was completed, the cells were placed in a 4°C temperature environment and centrifuged at 1000 rpm for 5 min. The supernatant was collected and the NO content in the cell supernatant was determined using the NO kit according to the operating procedures.

[0115] The principle of NO kit to detect NO content is: NO is easily oxidized into nitrite ions (NO2 - ), under acidic conditions, NO2 - It can react with sulfonamide to produce diazo compounds, which can then react with naphthylethylenediamine to produce colored compounds. The concentration of the product of this reaction is similar to that of NO2 - There is a linear relationship between the concentration and the absorption peak at 520-560nm. - The NO content in the liquid was calculated using the standard curve.

[0116] 1.9 Detection of TNF-α, IL-1β and IL-10 in the supernatant of BV2 cell culture by enzyme-linked immunosorbent assay (ELISA)

[0117] The BV2 cell experimental grouping and nutrient intervention methods were the same as above. After the culture was completed, the cells were placed in a 4°C temperature environment and centrifuged at 1000 rpm for 5 min. The supernatant was collected and the amounts of TNF-α, IL-1β and IL-10 in the cell supernatant were detected according to the operating steps in the kit instructions.

[0118] 1.10 Statistical analysis

[0119] All experiments were repeated three times independently, and SPSS22.0 statistical software was used to analyze the experimental data. The experimental results were tested for homogeneity of variance, and the statistical differences between the groups were analyzed using one-way ANOVA, expressed as mean ± standard deviation (x ± SD). p>0.05 indicated no statistical significance, and p<0.05 indicated statistically significant differences.

[0120] 2. Experimental results

[0121] 2.1 Nutrient cytotoxicity assay

[0122] Table 5 shows the effects of nutrients at different concentrations on the cell viability of BV2 cells. The results show that nutrients have no significant effect on cell viability.

[0123] Table 5 Effects of different concentrations of HMO on cell viability of BV2 cells

[0124] Nutrients Concentration (mg / mL) Cell survival rate (%) 2'-FL low dose 0.5 105.12±4.63 2'-FL medium dose 1 100.47±6.18 2'-FL high dose 2 101.00±9.78 LNnT low dose 0.05 100.84±1.50 LNnT medium dose 0.1 99.06±1.69 LNnT High Dose 0.2 98.97±0.75 LNT low dose 0.05 101.28±0.14 LNT medium dose 0.1 99.59±1.26 LNT High Dose 0.2 98.38±6.32 3'-SL low dose 1.25 101.28±0.27 3'-SL medium dose 5.0 100.94±1.69 3'-SL high dose 10 98.78±0.19 6'-SL low dose 1.25 104.12±5.81 6'-SL medium dose 5.0 101.09±11.28 6'-SL High Dose 10 99.97±0.61 3-FL low dose 0.5 101.22±0.66 3-FL medium dose 1 99.22±9.69 3-FL high dose 2 97.16±4.17

[0125] 2.2 Effects of nutrients on NO production in LPS-stimulated BV2 cells

[0126] Table 6 Effects of different HMOs on NO production in BV2 cells stimulated by LPS

[0127] Serial number Group HMO (mg / mL) NO(μmol / L) Reduction of NO compared with the model group 1 Blank control group - 0.16±0.09 2 LPS model group - <![CDATA[11.85±0.46 *** ]]> 3 2'-FL low dose 0.5 <![CDATA[8.51±0.15 ∧∧∧ ]]> 3.34 4 2'-FL medium dose 1 <![CDATA[8.45±0.18 ∧∧∧ ]]> 3.40 5 2'-FL high dose 2 <![CDATA[8.49±0.16 ∧∧∧ ]]> 3.36 6 LNnT low dose 0.05 11.28±0.27 0.57 7 LNnT medium dose 0.1 <![CDATA[9.73±0.36 ∧∧∧ ]]> 2.12 8 LNnT High Dose 0.2 <![CDATA[9.64±0.09 ∧∧∧ ]]> 2.21 9 LNT low dose 0.05 12.64±0.55 -0.79 10 LNT medium dose 0.1 11.73±0.18 0.12 11 LNT High Dose 0.2 11.28±0.27 0.57 12 3'-SL low dose 1.25 11.28±0.27 0.57 13 3'-SL medium dose 5.0 11.64±0.27 0.21 14 3'-SL high dose 10 11.91±0 -0.06 15 6'-SL low dose 1.25 10.91±0.46 0.94 16 6'-SL medium dose 5.0 11.37±1.82 0.48 17 6'-SL High Dose 10 11.82±0.09 0.03 18 3-FL low dose 0.5 <![CDATA[7.08±0.27 ∧∧∧ ]]> 4.77 19 3-FL medium dose 1 <![CDATA[7.36±0.18 ∧∧∧ ]]> 4.49 20 3-FL high dose 2 <![CDATA[7.72±0.36 ∧∧∧ ]]> 4.13

[0128] Note: *** Compared with the blank control group, p < 0.001; ∧∧∧ Compared with the LPS model group, p<0.001.

[0129] The results of NO research showed that compared with the blank control group, the NO in the cell supernatant of the LPS model group was significantly increased (p<0.001). After different concentrations of 2'-FL, LNnT and 3-FL were added to the culture system, except for the low-dose LNnT group, the NO in the cell supernatant was significantly reduced compared with the LPS model group (p<0.001), and the effect of reducing NO content was 3-FL>2'-FL>LNnT. However, after different concentrations of LNT, 3'-SL and 6'-SL were added to the culture system, no significant changes in NO in the cell supernatant were observed compared with the LPS model group.

[0130] 2.3 Effects of nutrients on pro-inflammatory factors in LPS-stimulated BV2 cells

[0131] Table 7 Effects of different HMOs on the production of proinflammatory factors in BV2 cells stimulated by LPS

[0132]

[0133] Note: *** Compared with the blank control group, p < 0.001; ∧∧ Compared with the LPS model group, p < 0.01; ∧∧∧ Compared with the LPS model group, p<0.001.

[0134] The results of TNF-α research showed that compared with the blank control group, the TNF-α in the cell supernatant of the LPS model group was significantly increased (p<0.001). After different concentrations of 2'-FL, LNnT and 3-FL were added to the culture system, except for the low-dose LNnT group, the TNF-α in the cell supernatant was significantly reduced compared with the LPS model group (p<0.001), and the effect of reducing TNF-α content was 3-FL>2'-FL>LNnT. However, after different concentrations of LNT, 3'-SL and 6'-SL were added to the culture system, no significant changes in TNF-α in the cell supernatant were observed compared with the LPS model group.

[0135] The results of IL-1β research showed that compared with the blank control group, the IL-1β in the cell supernatant of the LPS model group was significantly increased (p<0.001). After different concentrations of 2'-FL, LNnT, LNT, 3'-SL, 6'-SL and 3-FL were added to the culture system, except for the high-dose 6'-SL group (p<0.01), the IL-1β in the cell supernatant was significantly reduced compared with the LPS model group (p<0.001).

[0136] 2.4 Effects of nutrients on anti-inflammatory factors in LPS-stimulated BV2 cells

[0137] Table 8 Effects of different HMOs on the production of anti-inflammatory factors in BV2 cells stimulated by LPS

[0138] Serial number Group HMO (mg / mL) IL-10 (pg / mL) Compared with the model group, the increase in IL-10 1 Blank control group - 229.48±1 2 LPS model group - 213.96±2.63 3 2'-FL low dose 0.5 <![CDATA[267.42±7.78 ∧∧∧ ]]> 53.46 4 2'-FL medium dose 1 <![CDATA[274.32±7.78 ∧∧∧ ]]> 60.36 5 2'-FL high dose 2 <![CDATA[282.94±31.91 ∧∧∧ ]]> 68.98 6 LNnT low dose 0.05 <![CDATA[249.02±5.17 ∧∧ ]]> 35.06 7 LNnT medium dose 0.1 <![CDATA[271.44±3.45 ∧∧∧ ]]> 57.48 8 LNnT High Dose 0.2 <![CDATA[341.57±12.94 ∧∧∧ ]]> 127.61 9 LNT low dose 0.05 214.53±8.62 0.57 10 LNT medium dose 0.1 <![CDATA[236.38±9.5 ∧ ]]> 22.42 11 LNT High Dose 0.2 <![CDATA[241.55±4.34 ∧ ]]> 27.59 12 3'-SL low dose 1.25 205.34±6.06 -8.62 13 3'-SL medium dose 5.0 <![CDATA[257.07±4.34 ∧∧∧ ]]> 43.11 14 3'-SL high dose 10 <![CDATA[235.23±5.17 ∧ ]]> 21.27 15 6'-SL low dose 1.25 <![CDATA[251.9±6.06 ∧∧∧ ]]> 37.94 16 6'-SL medium dose 5.0 <![CDATA[247.3±6.9 ∧∧ ]]> 33.34 17 6'-SL High Dose 10 232.93±4.34 18.97 18 3-FL low dose 0.5 <![CDATA[343.29±26.73 ∧∧∧ ]]> 129.33 19 3-FL medium dose 1 <![CDATA[326.05±11.22 ∧∧∧ ]]> 112.09 20 3-FL high dose 2 <![CDATA[300.76±3.45 ∧∧∧ ]]> 86.8

[0139] Note: ∧ Compared with the LPS model group, p < 0.05; ∧∧ Compared with the LPS model group, p < 0.01; ∧∧∧ Compared with the LPS model group, p<0.001.

[0140] The results of IL-10 research showed that after different concentrations of 2'-FL, LNnT, LNT, 3'-SL, 6'-SL and 3-FL were added to the culture system respectively, except for the LNT low-dose group, 3'-SL low-dose group and 6'-SL high-dose group, the IL-10 in the cell supernatant was significantly increased compared with the LPS model group. The effect of increasing IL-10 content was that 3-FL, 2'-FL and LNnT were higher than LNT, 3'-SL and 6'-SL.

[0141] Industrial Applicability

[0142] The use of the human milk oligosaccharide provided by the present invention can be widely applied in industry.

Claims

1. The non-therapeutic use of human milk oligosaccharides in regulating brain immunity, characterized in that: The human milk oligosaccharide is any one of 3-fucosyllactose, lactose-N-tetraose, lactose-N-neotetraose, 3'-sialyllactose and 6'-sialyllactose.

2. The use according to claim 1, characterized in that The regulation of brain immunity is achieved by regulating microglial M1 / M2 polarization, and the regulation of microglial M1 / M2 polarization includes at least one of inhibiting microglial polarization to M1 phenotype, promoting microglial polarization to M2 phenotype, and inducing microglial transformation from M1 phenotype to M2 phenotype.

3. The use according to claim 2, characterized in that: The regulation of microglia M1 / M2 polarization includes inhibiting microglia from producing pro-inflammatory cytokines and / or promoting microglia from producing anti-inflammatory cytokines.

4. The use according to claim 3, characterized in that The pro-inflammatory cytokines include one or more of TNF-α, IL-1β and NO; the anti-inflammatory cytokines include IL-10.

5. A non-therapeutic use of a human milk oligosaccharide composition in regulating brain immunity, characterized in that: The human milk oligosaccharide composition comprises at least two of 3-fucosyllactose, lactose-N-tetraose, lactose-N-neotetraose, 3'-sialyllactose and 6'-sialyllactose, and optionally further comprises a solvent; Wherein, the regulation of brain immunity is achieved by regulating microglial M1 / M2 polarization, and the regulation of microglial M1 / M2 polarization includes at least one of inhibiting microglial polarization to M1 phenotype, promoting microglial polarization to M2 phenotype and inducing microglial transformation from M1 phenotype to M2 phenotype.

6. The use according to claim 5, characterized in that The solvent includes any one of water, physiological saline and glucose water.

7. The use according to claim 5 or 6, characterized in that The regulation of microglia M1 / M2 polarization includes inhibiting microglia from producing pro-inflammatory cytokines and / or promoting microglia from producing anti-inflammatory cytokines.

8. The use according to claim 7, characterized in that The pro-inflammatory cytokines include one or more of TNF-α, IL-1β and NO; the anti-inflammatory cytokines include IL-10.

9. The use according to any one of claims 5 to 8, characterized in that: The human milk oligosaccharide composition is an edible composition.

10. The use according to claim 9, characterized in that The edible composition is a dairy product.

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