Use of human milk oligosaccharides in regulation of brain immunity

Abstract

The use of human milk oligosaccharides for a non-therapeutic purpose of regulating brain immunity by means of regulating the M1 / M2 polarization of microglia, wherein the human milk oligosaccharides are any one of 3-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, 3'-sialyllactose and 6'-sialyllactose.

Description

Use of human milk oligosaccharides in modulating brain immunity TECHNICAL FIELD

[0001] The present invention belongs to the field of food technology, and relates to the use of human milk oligosaccharides in modulating brain immunity, in particular to the use of human milk oligosaccharides in modulating brain immunity by regulating M1 / M2 polarization of microglia for non-therapeutic purposes. BACKGROUND

[0002] Glial cells, simply referred to as glia, are another major class of cells in neural tissue in addition to neurons, have processes, but no distinction between dendrites and axons, and are widely distributed in the central and peripheral nervous system. The glial cells in the central nervous system (CNS) mainly include astrocytes, oligodendrocytes, microglia, ependymal cells, and radial glia.

[0003] Microglia are the only neural cells derived from mesoderm, and as a specialized macrophage, they perform immune functions in the nervous system. The brain establishes many connections between neurons, enabling them to transmit information back and forth. In fact, under normal circumstances, the number of synapses produced is much larger than needed, and only the strongest and most important synapses survive. Microglia, by phagocytosis, directly participate in the "pruning" process of synapses, clear harmful substances, repair damage, and protect various neurons in the central nervous system.

[0004] As the resident macrophages of the CNS, microglia account for 5% to 12% of all CNS-specific cells, and are the most abundant immune cells in the CNS, playing 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 functionally opposite cell populations, M1 and M2. M1 phenotype microglia mainly release pro-inflammatory cytokines such as tumor necrosis factor (TNF-α), interleukin 1β (IL-1β), and the chemical signal nitric oxide (NO), which can cause inflammation of the CNS, leading to damage and eventually death of neurons; M2 phenotype microglia mainly promote damage repair by secreting anti-inflammatory cytokines such as interleukin 10 (IL-10), and exert neuroprotective effects.

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

[0006] Studies have shown that HMOs play an important role in improving intestinal flora microecology, maintaining intestinal barrier, regulating immunity, resisting pathogenic bacterial infection and promoting neural development. HMOs are mainly composed of five core monomer structures of glucose (Glc), sialic acid (SA), fucose (Fuc), N-acetylglucosamine (GlcNAc) and galactose (Gal). Different HMOs will appear different fucosylation and sialylation on the structure, so HMOs in human 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 Document 1 discloses the use of oligosaccharide 2'-FL in the preparation of a product for improving neural development and improving dysfunction. Specifically, the use of oligosaccharide 2'-FL in the preparation of a protein inhibitor, which can inhibit the accumulation of Aβ protein, which can cause synaptic dysfunction of neurons, apoptosis of neurons, brain damage and dysfunction, 2'-FL improves the accumulation of Aβ protein leading to neurological diseases; 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 Document 2 discloses an oligosaccharide composition for improving neurodevelopment and dysfunction, wherein 6'-sialyllactose and lacto-N-triose are included in the oligosaccharide composition. The oligosaccharide composition can effectively inhibit the accumulation of Aβ protein, thereby playing a role in protecting nerves, and in addition, can inhibit the activity of acetylcholinesterase, protect the normal development of cholinergic neurons, and increase the content of neurotransmitters.

[0009] Reference Document 3 discloses a neurodevelopment-promoting nutritional composition comprising 2'-fucosyllactose and osteopontin. It is found that the nutritional composition has a synergistic effect on promoting neurodevelopment, in particular, the proliferation, maturation, and differentiation of oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes (OLs) and / or myelination of OLs.

[0010] Reference Document 4 discloses a nutritional composition comprising human milk oligosaccharides, milk phospholipids, and choline and / or edible choline derivatives. It is found that the nutritional composition can promote neurodevelopment, such as neuronal maturation, synapse formation, and myelination.

[0011] Reference Document 5 finds that rats received intracerebroventricular injection of 2'-FL or normal saline before middle cerebral artery occlusion (MCAo) surgery, and the rats were tested for IBA1 immunoreactivity (IBA1-ir) 2 days after treatment. The results show that administration of 2'-FL reduces IBA1-ir and morphological activation of microglia in the perilesional area.

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

[0013] Reference Document:

[0014] Reference Document 1: CN118105395A

[0015] Reference Document 2: CN118104826A

[0016] Reference Document 3: CN114586983A

[0017] Reference Document 4: CN114223723A

[0018] Reference 5: Wu KJ, Chen YH, Bae EK, et al. Human milk oligosaccharide 2'-fucosyllactose reduces neurodegeneration in stroke brain [J]. Translational Stroke Research, 2020, 11: 1001-1011.

[0019] Reference 6: WO2015100091A1 SUMMARY

[0020] Problems to be solved by the invention

[0021] Although the prior art has studied the functions of human milk oligosaccharides and their compositions, on the one hand, the current research direction of human milk oligosaccharides is more focused on promoting neural development, and less attention is paid to regulating brain immunity, which is equally important for promoting neural development in the brain; on the other hand, the current research on human milk oligosaccharides is more focused on 2'-fucosyllactose, and less on other types of human milk oligosaccharides; therefore, there is still room for further exploration of nutritional substances for regulating brain immunity, and there is also development prospects for the functional research and application of other types of human milk oligosaccharides.

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

[0023] Further research of the present application also shows that some specific types of human milk oligosaccharides (especially 3-FL) have verifiable effects on regulating brain immunity. The present application takes the LPS-stimulated microglial cell line (BV2 cells) as the research object, analyzes and determines the amount of pro-inflammatory cytokines and anti-inflammatory cytokines generated by the cells, and verifies and demonstrates the function of human milk oligosaccharides in regulating brain immunity in terms of the effect of human milk oligosaccharides on reducing the content of NO, TNF-α and 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 application also provides a non-therapeutic use of an edible human milk oligosaccharide composition for regulating brain immunity.

[0025] Solution to the problem

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

[0027] [1]. Use of human milk oligosaccharides for non-therapeutic purposes in modulating brain immunity, characterized in that the human milk oligosaccharides are any one of 3-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, 3'-sialyllactose and 6'-sialyllactose.

[0028] [2]. Use according to [1], wherein the modulating brain immunity is achieved via modulating microglia M1 / M2 polarization, including at least one of inhibiting microglia polarization to M1 phenotype, promoting microglia polarization to M2 phenotype and inducing microglia conversion from M1 phenotype to M2 phenotype.

[0029] [3]. Use according to [2], wherein the modulating microglia M1 / M2 polarization includes inhibiting microglia production of pro-inflammatory cytokines and / or promoting microglia production of anti-inflammatory cytokines.

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

[0031] [5]. Use of a human milk oligosaccharide composition for non-therapeutic purposes in modulating brain immunity, wherein the human milk oligosaccharide composition includes at least two of 3-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, 3'-sialyllactose and 6'-sialyllactose, and optionally further includes a solvent.

[0032] wherein the modulating brain immunity is achieved via modulating microglia M1 / M2 polarization, including at least one of inhibiting microglia polarization to M1 phenotype, promoting microglia polarization to M2 phenotype and inducing microglia conversion from M1 phenotype to M2 phenotype.

[0033] [6]. Use according to [5], wherein the solvent includes any one of water, physiological saline, glucose water.

[0034] [7]. Use according to [5] or [6], wherein the modulating microglia M1 / M2 polarization includes inhibiting microglia production of pro-inflammatory cytokines and / or promoting microglia production of anti-inflammatory cytokines.

[0035] [8]. Use according to [7], wherein the pro-inflammatory cytokines include one or more of TNF-a, IL-1b and NO; 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]

[0010] . 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 solutions, the present application can obtain the following technical effects:

[0040] 1) The experimental results of the present application show that specific types of human milk oligosaccharides have the effect of regulating brain immunity, and in particular can regulate brain immunity by regulating the M1 / M2 polarization of microglia, especially the state of immune cells in the central nervous system, i.e. microglia;

[0041] 2) Further research of the present application 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, so that the state of microglia is improved, the damage is repaired, and the effect of regulating brain immunity is played;

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

[0043] 4) The present application provides a new application mode for specific types of human milk oligosaccharides, especially 3-FL, which provides a new idea for the development of nutritional supplement foods with the effect of regulating brain immunity. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1: Schematic diagram of the experimental process of the present application for studying the regulating effect of functional nutrients on microglia. DETAILED DESCRIPTION

[0045] The embodiments of the present application are described below, but the present application is not limited thereto. The present application is not limited to each of the configurations described below, and various modifications can be made within the scope of the present application, and embodiments obtained by appropriately combining the technical means disclosed in each of the different embodiments are also included in the technical scope of the present application.

[0046] In the present specification, the numerical range represented by "numerical value A to numerical value B" means a range including the end point values A and B.

[0047] In the present specification, "a number of" or "numbers of" or "a plurality of" means 2 or more, unless specifically stated otherwise.

[0048] In the present specification, "comprising" or "including" or "containing" or "having" can mean that the elements included are included or open, and does not exclude additional, unmentioned elements or method steps. At the same time, "comprising" or "including" or "containing" or "having" can also mean closed, excluding additional, unmentioned elements or method steps.

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

[0050] In the present specification, the meaning indicated using "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0051] In the present specification, "optional" or "optionally" means that a certain substance, component, execution step, applied condition, etc. is used or not used.

[0052] In the present specification, "room temperature" used, unless specifically stated, generally means a temperature at 23±2℃.

[0053] In the present specification, the unit name used is the international standard unit name, and if not specifically stated, "%" used means a weight or mass percentage content.

[0054] In the present specification, "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc. mentioned refer to the specific elements (e.g. features, structures, properties, and / or characteristics) described in relation to the embodiments being included in at least one embodiment described herein, and can be present in other embodiments or can not be present in other embodiments. In addition, it should be understood that the elements can be combined in various embodiments in any suitable manner.

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

[0056] The present application mainly provides a new use of human milk oligosaccharides, i.e. a new function of human milk oligosaccharides is found through in vitro cell experiment research, and based on this research, the existing application mode of human milk oligosaccharides is expanded. The present application 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, activate microglia cells and induce neuroinflammatory responses, induce the release of pro-inflammatory cytokines, and cause memory deficits. In the present application, LPS-stimulated microglial cells are used as a model of microglial cell activation to study the regulatory effects of different types and concentrations of nutrients on microglial cells, and to explore functional nutrients that are safe (non-cytotoxic) and have obvious regulatory effects on brain immunity (as shown in Figure 1). The present application has found that the use of specific types of human milk oligosaccharides (especially 3-FL, LNnT, etc.) to intervene in LPS-stimulated microglial cells can increase the amount of IL-10 released by microglial cells and reduce the amount of TNF-α, IL-1β and NO released by microglial cells.

[0058] (Human milk oligosaccharides)

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

[0060] HMOs are mainly composed of five core monomer structures: glucose, sialic acid, fucose, N-acetylglucosamine and galactose. Different HMOs will have different fucosylation and sialylation on their structures, so HMOs in human 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 highest content of HMOs in mature human milk, including 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL) and the like.

[0061] Related studies predict that there are more than a thousand types of HMOs in human milk, mainly due to differences in molecular structure and spatial configuration of the constituent monomers. More than 200 types of HMOs have been reported, and the most studied 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). Currently, 2'-FL, LNnT, LNT, 3'-sialyllactose sodium salt and 6'-sialyllactose sodium salt are used as new food raw materials in some regions and are applied to infant formula and food supplements.

[0062] Currently, the main methods for producing human milk oligosaccharides are chemical synthesis, enzymatic synthesis and biological engineering synthesis, and these components can also be indirectly introduced from existing various milk components.

[0063] It should be noted that the use of the various human milk oligosaccharides described above in the present application should comply with the requirements of the local laws and regulations. In some cases, these ingredients can be directly introduced into the food composition in the form of a single raw material as allowed by the laws and regulations; in other cases, they can be indirectly introduced into the composition or food through the addition of a qualified milk raw material as allowed by the laws and regulations.

[0064] The present application does not make any particular limitation on the source of the human milk oligosaccharides, which can typically be brought in from different milk-containing raw materials, such as animal milk raw materials (e.g. cow milk, cow milk powder, etc.). In the present application, "animal milk" refers to the liquid obtained from the mammary glands of a mammal during lactation. The term "animal milk" should be interpreted broadly and covers both raw milk (i.e. the liquid obtained directly from the mammary glands) and standardized dairy products. In some embodiments, the human milk oligosaccharides described in the present application are any one of 3-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, 3'-sialyllactose and 6'-sialyllactose, preferably 3-fucosyllactose (3-FL).

[0065] (Eatable human milk oligosaccharide composition)

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

[0067] The present application does not make any particular limitation on the form of use of the human milk oligosaccharide composition, which should comply with the requirements of the relevant laws and regulations. Within the allowed range, it can be used in the form of a combination of two or more ingredients, or other solvents containing the composition can be added to impart the desired human milk oligosaccharides in the form of use. In some specific embodiments, the solvent comprises any one of water, physiological saline, glucose water. In the present application, the final form of the human milk oligosaccharide composition is not particularly limited, which can be, for example, a liquid (with the desired solvent), a solid (such as a powder, a granule or a block (compressed)) at room temperature, etc.

[0068] (Food)

[0069] Further, the present application provides a food based on the above-mentioned human milk oligosaccharides, in particular based on the above-mentioned eatable human milk oligosaccharide composition. The food has the effect of regulating brain immunity due to the use of the above-mentioned specific types of human milk oligosaccharides.

[0070] For the food of the present application, in addition to the above-mentioned human milk oligosaccharide or human milk oligosaccharide composition, other optional ingredients can be included as needed for the final product, which can include, for example:

[0071] Fruits or extracts thereof, including figs, pomegranates, kiwis, oranges, oranges, pineapples, strawberries, apples, bananas, grapes, pears, cherries, blueberries, blackberries, strawberries, and mulberries, etc.; fruit and vegetable materials or extracts thereof, including onions, cucumbers, tomatoes, cauliflowers, red radishes, spinach, kohlrabi, Brussels sprouts, garlic, basil, Oregon grass, etc.; cereals or extracts thereof, including rice (indica rice, japonica rice, waxy rice), wheat (wheat, barley, oat, rye), corn, sorghum, millet, foxtail millet, japonica, buckwheat, soybeans, beans, peas, mung beans, adzuki beans, lentils, etc.; nut materials or extracts thereof, including walnuts, cashews, hazelnuts, almonds, pine nuts, pistachios, peanuts, melon seeds, chestnuts, macadamia nuts, ginkgo nuts, etc.; coffee or extracts thereof.

[0072] Milk-containing ingredients, including fresh milk, milk powder, whey protein, or cheese derived from raw fresh cow (sheep) milk, etc.

[0073] Animal-derived ingredients, including meat products derived from cows, sheep, fish, or poultry.

[0074] Fat ingredients, which can include at least one of saturated fatty acids, polyunsaturated fatty acids, monounsaturated fatty acids, OPO structured fats, DHA, EPA, ARA, and phospholipids, and more specifically, include 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 oil, palm oil, beef tallow, butter, lard, medium-chain triglycerides, lecithin, etc.

[0075] Functional additive components, including vitamins (vitamin A, beta-carotene, vitamin D3, vitamin E, vitamin K1, vitamin B1, vitamin B2, vitamin B6, vitamin B 12 12, 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 medicines or extracts thereof, dietary fibers (inulin, konjac powder, galactooligosaccharides, fructooligosaccharides, isomaltooligosaccharides, soybean polysaccharides, cyclodextrins, resistant dextrins, 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 additives that are acceptable to use include solvents, antioxidants, antibacterial agents, thickeners, diluents, solubilizers, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, food flavorings, food colorings, etc.

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

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

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

[0081] (Uses in regulating brain immunity)

[0082] This invention discloses the use of the aforementioned human milk oligosaccharides in regulating brain immunity; therefore, the aforementioned edible human milk oligosaccharide compositions and foods can also be used for the aforementioned purposes. Thus, this invention suggests that consuming these foods can have a modulating effect on brain immunity.

[0083] The "regulation of brain immunity" described in this invention refers to maintaining the brain's immune activity at an optimal level by regulating the interactions between immune cells and molecules, as well as with other systems such as the neuroendocrine system. Furthermore, the brain immune regulation described in this invention is not intended to prevent or treat diseases.

[0084] In some specific embodiments, the modulating brain immunity according to the present application is modulating the state of microglia, including the tendency of the microglia to polarize into M1 or M2 type, and the amount or ratio of various cytokines secreted by the microglia.

[0085] In some more specific embodiments, the modulating brain immunity according to the present application is achieved via modulating microglia M1 / M2 polarization, including at least one of inhibiting microglia polarization into M1 phenotype, promoting microglia polarization into M2 phenotype, and inducing microglia conversion from M1 phenotype to M2 phenotype.

[0086] In some more specific embodiments, the modulating microglia M1 / M2 polarization according to the present application includes inhibiting microglia production of pro-inflammatory cytokines and / or promoting microglia production of anti-inflammatory cytokines. In some specific embodiments, the modulating microglia M1 / M2 polarization according to the present application includes inhibiting microglia production of pro-inflammatory cytokines and promoting microglia production of anti-inflammatory cytokines.

[0087] In some specific embodiments, the pro-inflammatory cytokines include one or more of TNF-a, IL-1b and NO.

[0088] In some specific embodiments, the anti-inflammatory cytokines include IL-10.

[0089] Examples

[0090] Embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will understand that the following examples are for illustrative purposes only and should not be construed as limiting the scope of the present application. The specific conditions not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be obtained by purchase.

[0091] 1. Materials and methods

[0092] 1.1 Raw materials

[0093] Table 1. Main raw materials for experiments

[0094] 1.2 Cells

[0095] Mouse microglial cell line BV2 cells were purchased from Shanghai Zety Yang Biological Technology Co., Ltd. (CC-Y2022).

[0096] 1.3 Reagents

[0097] Table 2. Main reagents for experiments

[0098] 1.4 Instruments

[0099] Table 3. Main experimental instruments and equipment

[0100] 1.5 Cell culture

[0101] The mouse microglial cell line (BV2) was cultured in DMEM high-sugar medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and was placed in a temperature environment of 37°C in a 5% CO2 incubator.

[0102] 1.6 Nutrient cytotoxicity detection

[0103] The CCK-8 kit was used to detect the cytotoxicity of the nutrients.

[0104] The working principle of the CCK-8 kit is as follows: in the presence of an electron carrier 1-methoxy-5-methyl phenazine dimethyl sulfate, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid phenyl)-2H-tetrazole monosodium salt is reduced to yellow methylene by dehydrogenase in the mitochondria of cells. Because only mitochondria in living cells can produce dehydrogenase, the production of methylene and the number of living cells are positively related, and the OD value is detected at 450 nm by an enzyme marker.

[0105] The BV2 cells were inoculated into a 96-well plate at 4x10 4 cells per well, 200 μL / well, and 3 replicates per group. After 12 hours of adhesion culture in the incubator, the prepared nutrient medium was added to the 96-well plate, and after 12 hours of culture, 20 μL of CCK-8 solution (avoiding light) was added to each well, and the cells were incubated in the cell incubator for 0.5-1 hour. The absorbance at 450 nm was measured by an enzyme marker, and then the cell survival rate was calculated according to the formula: cell survival rate = [(experimental well absorbance-blank well absorbance) / (control well absorbance-blank well absorbance)]x100% (wherein the experimental well contains cells, medium, nutrients and CCK-8 solution; the blank well contains medium and CCK-8 solution, but does not contain cells and nutrients; the control well contains cells, medium and CCK-8 solution, but does not contain nutrients). Finally, the cell survival rate was calculated. The CCK8 method was used to detect the activity of BV2 microglial cells.

[0106] 1.7 Establishment of microglial cell activation model and experimental grouping

[0107] The mouse microglial cell line (BV2) was cultured with high-sugar DMEM medium containing 10% fetal bovine serum in a 5% CO2 incubator (37°C, saturated humidity). The logarithmic phase BV2 cells were selected, and the cells in the logarithmic growth phase and reaching 90% confluence were digested with trypsin. The cell concentration was adjusted, and the cells were inoculated into appropriate cell culture dishes and then cultured for 24 h. The cells were then divided into groups. The cells were inoculated into 96-well plates at a cell number of 4 x 10 4 4 / mL per well, 200 μL / well, and 3 replicate wells per group. The experiment was divided into 20 groups, including a blank control group, an LPS model group, and 18 experimental groups (2'-FL, LNnT, LNT, 3'-SL, 6'-SL, 3-FL, a total of 6 HMOs, each set at low, medium, and high concentration gradients). (1) The control group was cultured with cells as usual. (2) The LPS model group was intervened with LPS (1 μg / mL) for 24 h to establish a microglial cell activation model. (3) The experimental group was intervened with LPS for 24 h, and then each nutrient was added for 12 h.

[0108] Table 4 Experimental grouping and dose

[0109] 1.8 Detection of NO content in BV2 cell supernatant by NO kit

[0110] The BV2 cells were grouped and intervened with nutrients as described above. After the culture, the cells were centrifuged at 1000 rpm for 5 min at 4°C. The supernatant was collected, and the NO content in the cell supernatant was determined using the NO kit according to the operation steps.

[0111] The principle of the NO kit for detecting the NO content is as follows: After the generation of NO, it is very easy to be oxidized into nitrite ion (NO2 - ). Under acidic conditions, NO2 - can react with sulfanilamide to produce diazonium compounds, which can be coupled with naphthalene ethylene diamine to generate colored compounds. The product concentration of this reaction is linearly related to the NO2 - concentration, and there is an absorption peak at 520-560 nm. The NO content in the liquid can be calculated according to the OD value and the NO2 - standard curve.

[0112] 1.9 Detection of TNF-α, IL-1β, and IL-10 contents in the BV2 cell culture supernatant by enzyme-linked immunosorbent assay (ELISA)

[0113] The BV2 cell experiment grouping and nutrient intervention method are the same as above. After the culture is completed, centrifugation is performed at a temperature of 4°C and a speed of 1000 rpm for 5 min. The supernatant is collected, and the amount of TNF-α, IL-1β and IL-10 in the cell supernatant is detected according to the operation steps in the kit instructions.

[0114] 1.10 Statistical analysis

[0115] All experiments were independently repeated three times. The experimental data were statistically analyzed by SPSS22.0 statistical software. The experimental results were subjected to homogeneity of variance test, and the statistical differences between groups were analyzed by one-way analysis of variance. The mean ± standard deviation (x ± SD) was used to represent the statistical difference. p>0.05 indicates no statistical significance, and p<0.05 indicates that the difference is statistically significant.

[0116] 2. Experimental results

[0117] 2.1 Nutrient cytotoxicity detection

[0118] Table 5 shows the effect of different concentrations of nutrients on the cell viability of BV2 cells. The results show that the nutrients have no significant effect on cell viability.

[0119] Table 5 Effect of different concentrations of HMOs on the cell viability of BV2 cells

[0120] 2.2 Effect of nutrients on NO production of LPS-stimulated BV2 cells

[0121] Table 6 Effect of different HMOs on NO production of LPS-stimulated BV2 cells Note: *** p<0.001 compared with the blank control group; ∧∧∧ p<0.001 compared with the LPS model group.

[0122] The results of the NO study 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 adding different concentrations of 2'-FL, LNnT and 3-FL 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 adding different concentrations of LNT, 3'-SL and 6'-SL to the culture system, no significant change in NO in the cell supernatant was observed compared with the LPS model group.

[0123] 2.3 Effect of nutrients on pro-inflammatory factors of LPS-stimulated BV2 cells

[0124] Table 7 Effect of different HMOs on pro-inflammatory cytokine production in LPS-stimulated BV2 cells Notes: *** p<0.001 compared with the blank control group; ∧∧ p<0.01 compared with the LPS model group; ∧∧∧ p<0.001 compared with the LPS model group.

[0125] The results of the TNF-a study showed that the TNF-a in the cell supernatant of the LPS model group was significantly higher than that of the blank control group (p<0.001). After different concentrations of 2'-FL, LNnT and 3-FL were added to the culture system, the TNF-a in the cell supernatant was significantly lower than that of the LPS model group (p<0.001) except for the low-dose LNnT group, and the effect of reducing the TNF-a 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 change in the TNF-a in the cell supernatant was observed compared with the LPS model group.

[0126] The results of the IL-1 b study showed that the IL-1 b in the cell supernatant of the LPS model group was significantly higher than that of the blank control group (p<0.001). After different concentrations of 2'-FL, LNnT, LNT, 3'-SL, 6'-SL and 3-FL were added to the culture system, the IL-1 b in the cell supernatant was significantly lower than that of the LPS model group (p<0.001) except for the high-dose 6'-SL group (p<0.01).

[0127] 2.4 Effect of nutrients on anti-inflammatory cytokines in LPS-stimulated BV2 cells

[0128] Table 8 Effect of different HMOs on anti-inflammatory cytokine production in LPS-stimulated BV2 cells Notes: ∧ p<0.05 compared with the LPS model group; ∧∧ p<0.01 compared with the LPS model group; ∧∧∧ p<0.001 compared with the LPS model group.

[0129] The results of the IL-10 study showed that after different concentrations of 2'-FL, LNnT, LNT, 3'-SL, 6'-SL and 3-FL were added to the culture system, the IL-10 in the cell supernatant was significantly higher than that of the LPS model group except for the low-dose LNT group, the low-dose 3'-SL group and the high-dose 6'-SL group, and the effect of increasing the IL-10 content was 3-FL, 2'-FL and LNnT higher than LNT, 3'-SL and 6'-SL.

[0130] Industrial applicability

[0131] The use of the breast milk oligosaccharides provided by the present application can be widely applied in industry.

Claims

1. Use of a human milk oligosaccharide for non-therapeutic purposes in modulating brain immunity, characterized in that, The human milk oligosaccharide is any one of 3-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, 3'-sialyllactose, and 6'-sialyllactose.

2. Use according to claim 1, characterized in that, The modulating brain immunity is achieved via modulating microglia M1 / M2 polarization, which includes at least one of inhibiting microglia polarization to M1 phenotype, promoting microglia polarization to M2 phenotype, and inducing microglia transformation from M1 phenotype to M2 phenotype.

3. Use according to claim 2, characterized in that, The modulating microglia M1 / M2 polarization includes at least one of inhibiting microglia from producing pro-inflammatory cytokines and / or promoting microglia from producing anti-inflammatory cytokines.

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

5. Use of a human milk oligosaccharide composition for non-therapeutic purposes in modulating brain immunity, characterized in that, The human milk oligosaccharide composition includes at least two of 3-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, 3'-sialyllactose, and 6'-sialyllactose, and optionally further includes a solvent. The modulating brain immunity is achieved via modulating microglia M1 / M2 polarization, which includes at least one of inhibiting microglia polarization to M1 phenotype, promoting microglia polarization to M2 phenotype, and inducing microglia transformation from M1 phenotype to M2 phenotype.

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

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

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

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

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

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