Use of nutritional compositions to modulate brain immunity

Abstract

The invention belongs to the field of nutrient substance research, and particularly relates to application of a nutrient composition to regulation of brain immunity. The invention provides application of a nutritional composition in preparation of food for regulating brain immunity. Wherein the nutritional composition comprises the following necessary components: lactoferrin and fucosyllactose, and in the nutritional composition, the mass ratio of the lactoferrin to the fucosyllactose is 1: (0.1-30). Research finds that after lactoferrin and fucosyllactose are combined according to a certain proportion, the lactoferrin and fucosyllactose have a synergistic effect of regulating brain immunity. Experimental data show that after the combination of lactoferrin and fucosyllactose is used for treating the BV2 cells, the trend that the BV2 cells are differentiated into M2 phenotype microglial cells is higher than the trend that the BV2 cells are differentiated into M1 phenotype microglial cells, and the amount of secreted inflammatory factors is obviously reduced.

Description

Technical Field

[0001] The present invention belongs to the field of nutrient research, and specifically relates to the use of a nutrient composition for regulating brain immunity, and more specifically relates to the use of a nutrient composition containing lactoferrin and fucosyllactose in regulating brain immunity and assisting in improving the health of the central nervous system. Background Art

[0002] In recent years, studies have shown that the brain is not an organ with immune privileges, and the brain and the immune system are closely connected. In addition, it is now known that the brain has its own immune cells, such as microglia, which are resident macrophages in the central nervous system (CNS), accounting for 5% to 12% of all CNS-specific cells and are the most abundant immune cells in the CNS. During brain development, microglia are involved in controlling neurogenesis, the fate of oligodendrocyte progenitor cells, and the formation of myelin, and can indirectly affect synaptic and neuronal function by regulating the activation of microglia and the activity of other non-neuronal cells.

[0003] After activation, microglia can polarize into two cell groups with opposite functions, M1 or M2. M1 phenotype microglia mainly release pro-inflammatory cytokines, such as tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) and chemical signal nitric oxide (NO), which can easily cause excessive inflammatory response in the CNS, leading to neuronal damage and eventually death; M2 phenotype microglia mainly promote damage repair and play a neuroprotective role by secreting anti-inflammatory cytokines, such as interleukin 10 (IL-10).

[0004] Microglia play an important role in neuroinflammation, which is a complex immune response in which nerve tissue clears pathogens, damaged cells, and harmful substances. Excessive neuroinflammatory response can affect the health of the CNS.

[0005] Lactoferrin (LF) belongs to the transferrin superfamily. It is a mammalian non-heme cationic protein with a relative molecular mass of about 80,000 Da and composed of about 700 amino acids. It has a symmetrical bilobed structure, and its unique structure is mainly used to control the binding and release of iron ions. LF is widely distributed in the body and is expressed in mammalian body fluids such as milk, tears, saliva, and vaginal secretions. The content is most abundant in milk, and the concentration of LF in human colostrum is 1-8 g / L. LF is not only involved in iron transport, but also has powerful biological functions such as broad-spectrum antibacterial, antioxidant, anti-cancer, and immune system regulation. In recent years, studies have found that lactoferrin can enter the blood-brain barrier through receptor-mediated endocytosis, activate microglia / macrophage coupling to induce proinflammatory response, and play an important role in regulating iron homeostasis, inflammatory response, oxidative stress, cell apoptosis, and angiogenesis.

[0006] Reference 1 discloses a composite liposome for improving brain cognition memory and neuroinflammation and a preparation method thereof, which belongs to the technical field of liposome preparation. Liposomes capable of simultaneously loading the hydrophobic substance curcumin and the hydrophilic substance epigallocatechin gallate were prepared, and on this basis, the surface was modified with lactoferrin, and hyaluronic acid was used to further stabilize the system through electrostatic interaction. The composite liposome has good stability and free radical scavenging efficiency, can significantly inhibit lipopolysaccharide-induced cognitive dysfunction, and is used to improve brain cognition memory and neuroinflammation.

[0007] Cited document 2 discloses the use of lactoferrin combined with choline in the preparation of drugs for preventing and / or treating Alzheimer's disease. Lactoferrin and choline are combined in a specific ratio as pharmacological substances, which can increase the level of anti-inflammatory factors in mice with neurodegenerative diseases, reduce the level of pro-inflammatory factors, enhance the brain's ability to resist inflammatory response, and reduce tau protein phosphorylation and serum Aβ levels, improve learning ability and cognitive function, and improve autonomous exploration ability, providing a new option for the clinical prevention or treatment of Alzheimer's disease.

[0008] 3-fucosyllactose (3-FL) is a trisaccharide composed of D-galactose (Gal), D-glucose (Glc) and L-fucose (Fuc). 3-FL is one of the most abundant human milk oligosaccharides (HMOs) in breast milk. Studies have shown that the concentration of 3-FL in breast milk increases during lactation. 3-FL plays a key role in the healthy development of infants, including regulating the immune system, antibacterial and anti-inflammatory effects, regulating the proliferation of intestinal microbiota, and promoting brain maturation.

[0009] Reference 3 investigated the correlation of HMOs with MRI indices of tissue microstructure and regional cerebral blood flow (rCBF) in infants. In mother-infant pairs (N=20) at 1 month postpartum, 2'-FL, 3-FL, 3'-SL, and 6'-SL concentrations in breast milk were analyzed. The data showed that fucosylated and sialylated HMOs had different associations with tissue microstructure and rCBF indices, suggesting that 2'-FL, 3-FL, and 3'-SL may play a special role in early brain maturation.

[0010] In addition, there are also studies on nutritional compositions containing both human milk oligosaccharides and lactoferrin, such as:

[0011] Reference 4 discloses a nutritional composition, food and use containing human milk oligosaccharides and lactoferrin, wherein the nutritional composition comprises or consists of: human milk oligosaccharides and lactoferrin. Human milk oligosaccharides are preferably neutral fucosylated human milk oligosaccharides, which are preferably selected from one or more of 2'-fucosyllactose, 3'-fucosyllactose, lactose-N-fucopentaose I, lactose-N-difucohexaose I, and lactose-N-difucohexaose II. The nutritional composition has the effect of promoting the development of the immune system of offspring and improving the intestinal flora of offspring through consumption by human or animal mothers.

[0012] Reference 5 discloses a nutritional composition for promoting physical development, wherein the raw materials for making the effective ingredients thereof include, by weight, 50-60 parts of galacto-oligosaccharide, 35-40 parts of human milk oligosaccharide and 5-10 parts of lactoferrin, wherein the human milk oligosaccharide includes 2'-fucosyllactose and / or lactose-N-neotetraose. The combination of galacto-oligosaccharide, human milk oligosaccharide and lactoferrin can synergistically enhance the utilization rate of calcium by the body and effectively promote physical development.

[0013] References:

[0014] Reference 1: CN114470236B;

[0015] Reference 2: CN115737787B;

[0016] Cited literature 3: Berger PK, Bansal R, Sawardekar S, Yonemitsu C, Furst A, HampsonHE, Schmidt KA, Alderete TL, Bode L, Goran MI, Peterson BS. Associations of HumanMilk Oligosaccharides with Infant Brain Tissue Organization and RegionalBlood Flow at 1Month of Age.Nutrients.2022Sep16;14(18):3820.

[0017] Reference 4: CN115918916A;

[0018] Reference 5: CN115644458A. Summary of the invention

[0019] Problem that the invention aims to solve

[0020] At present, existing technologies such as the above-mentioned references 1 to 5 have studied the physiological activity functions of lactoferrin and fucosyllactose, but such research is still not sufficient. There is still room for development in the functional research of the combination of lactoferrin and fucosyllactose.

[0021] During the research process, the present invention unexpectedly discovered that the combination of lactoferrin and fucosyllactose has a synergistic effect in regulating brain immunity, which can regulate the M1 / M2 polarization trend of microglia, and can improve neuroinflammation to assist in improving the health of the central nervous system, especially when lactoferrin and fucosyllactose are combined in a certain ratio, the effect is better.

[0022] Therefore, the primary object of the present invention is to provide a non-therapeutic effect of a nutritional composition comprising lactoferrin and fucosyllactose in modulating brain immunity and assisting in improving the health of the central nervous system.

[0023] Solutions for solving problems

[0024] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0025] [1]. Use of a nutritional composition in the preparation of a food for regulating brain immunity; wherein the nutritional composition comprises the following essential components: lactoferrin and fucosyllactose, and in the nutritional composition, the mass ratio of lactoferrin to fucosyllactose is 1:(0.1-30).

[0026] [2]. The use according to [1], wherein the fucosyllactose includes 3-fucosyllactose.

[0027] [3] The use according to [1] or [2], wherein the regulation of brain immunity includes at least one of inhibiting the polarization of microglia to the M1 phenotype, promoting the polarization of microglia to the M2 phenotype, and inducing the transformation of microglia from the M1 phenotype to the M2 phenotype.

[0028] [4] The use according to any one of [1] to [3], wherein the regulating brain immunity comprises at least one of increasing the amount of IL-10 released by microglia, reducing the amount of TNF-α released by microglia, reducing the amount of IL-1β released by microglia, and reducing the amount of NO released by microglia.

[0029] [5]. The use according to any one of [1] to [4], wherein the food is candy, a beverage, a dairy product or a baked product.

[0030] [6]. The use according to [5], wherein the candy comprises at least one of hard candy, jelly candy, crisp candy, compressed candy and aerated candy; the beverage comprises at least one of carbonated beverage, tea beverage, coffee beverage, fruit and vegetable juice beverage and lactic acid bacteria beverage; the dairy product comprises at least one of fermented milk, cheese and milk powder; and the baked food comprises at least one of bread, cake and biscuits.

[0031] [7]. The use according to any one of [1] to [4], wherein the food is an oral preparation, and the oral preparation includes any one or more of tablets, pills, granules, powders, capsules and oral liquids.

[0032] [8]. Use of a nutritional composition in the preparation of a food for assisting in improving the health of the central nervous system; wherein the nutritional composition comprises the following essential components: lactoferrin and fucosyllactose, and, in the nutritional composition, the mass ratio of lactoferrin to fucosyllactose is 1:(0.1-30).

[0033] [9]. The use according to [8], wherein the fucosyllactose includes 3-fucosyllactose.

[0034]

[10] . The use according to [8] or [9], wherein the auxiliary improvement of central nervous system health includes improving neuroinflammation for non-therapeutic purposes.

[0035] Effects of the Invention

[0036] The present invention has found that the combination of lactoferrin and fucosyllactose, especially in a certain ratio, has a synergistic effect of regulating brain immunity and assisting in improving the health of the central nervous system. Experimental data show that in an in vitro model, when BV2 cells are treated with a combination of lactoferrin and fucosyllactose, the tendency of differentiation into M2 phenotype microglia is higher than that of differentiation into M1 phenotype microglia, and the amount of secreted inflammatory factors is significantly reduced. In addition, lactoferrin and fucosyllactose have a good synergistic effect, which can effectively regulate brain immunity and assist in improving the health of the central nervous system. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 : Schematic diagram of the experimental process of studying the regulatory effects of nutrients on microglia in the present invention.

[0038] Figure 2A : Western blot results of the effects of nutrients on the TLR4 / NF-κB signaling pathway in BV2 cells stimulated by LPS; among them, group 1 was the LPS model group, group 2 was the blank control group, group 3 was the LF high-dose group, group 4 was the 3-FL high-dose group, group 5 was the LF high-dose + 3-FL low-dose group (LF:3-FL was 1:0.5), and group 6 was the LF low-dose + 3-FL high-dose group (LF:3-FL was 1:25).

[0039] Figure 2B : Western blot analysis results of the effects of nutrients on TLR4 protein expression in the TLR4 / NF-κB signaling pathway of BV2 cells stimulated by LPS; Group 1 was the LPS model group, Group 2 was the blank control group, Group 3 was the LF high-dose group, Group 4 was the 3-FL high-dose group, Group 5 was the LF high-dose + 3-FL low-dose group (LF:3-FL was 1:0.5), Group 6 was the LF low-dose + 3-FL high-dose group (LF:3-FL was 1:25), ns means no significant difference, *P<0.05, ***P<0.001, ****P<0.0001.

[0040] Figure 2C: Western blot analysis results of the effects of nutrients on the expression of NF-κBp65 protein in the TLR4 / NF-κB signaling pathway of BV2 cells stimulated by LPS; Group 1 was the LPS model group, Group 2 was the blank control group, Group 3 was the LF high-dose group, Group 4 was the 3-FL high-dose group, Group 5 was the LF high-dose + 3-FL low-dose group (LF:3-FL was 1:0.5), Group 6 was the LF low-dose + 3-FL high-dose group (LF:3-FL was 1:25), ***P<0.001, ****P<0.0001.

[0041] Figure 2D : Western blot analysis results of the effects of nutrients on the expression of p-NF-κBp65 protein in the TLR4 / NF-κB signaling pathway of BV2 cells stimulated by LPS; Group 1 was the LPS model group, Group 2 was the blank control group, Group 3 was the LF high-dose group, Group 4 was the 3-FL high-dose group, Group 5 was the LF high-dose + 3-FL low-dose group (LF:3-FL was 1:0.5), Group 6 was the LF low-dose + 3-FL high-dose group (LF:3-FL was 1:25), ***P<0.001, ****P<0.0001.

[0042] Figure 2E : Western blot analysis results of the effects of nutrients on the expression of IKB-α protein in the TLR4 / NF-κB signaling pathway of BV2 cells stimulated by LPS; Group 1 was the LPS model group, Group 2 was the blank control group, Group 3 was the LF high-dose group, Group 4 was the 3-FL high-dose group, Group 5 was the LF high-dose + 3-FL low-dose group (LF:3-FL was 1:0.5), Group 6 was the LF low-dose + 3-FL high-dose group (LF:3-FL was 1:25), ****P<0.0001.

[0043] Figure 2F : Western blot analysis results of the effects of nutrients on the expression of p-IKB-α protein in the TLR4 / NF-κB signaling pathway of BV2 cells stimulated by LPS; Group 1 was the LPS model group, Group 2 was the blank control group, Group 3 was the LF high-dose group, Group 4 was the 3-FL high-dose group, Group 5 was the LF high-dose + 3-FL low-dose group (LF:3-FL was 1:0.5), Group 6 was the LF low-dose + 3-FL high-dose group (LF:3-FL was 1:25), *P<0.05, **P<0.01, ****P<0.0001. DETAILED DESCRIPTION

[0044] The following describes the embodiments of the present invention, but the present invention is not limited thereto. Various changes can be made within the scope of the present invention, 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.

[0045] In the present invention, a numerical range expressed using “value A to value B”, “value A - value B”, “value A or more” or “value A or less” means a range including the endpoints A and B.

[0046] In the present invention, the word "may" includes both performing a certain process and not performing a certain process.

[0047] In the present invention, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes instances where the event occurs and instances where it does not occur.

[0048] In the present invention, the term "a" or "an" or "the" may mean "one", and may also mean "one or more", "at least one" and "one or more than one".

[0049] In the present invention, the terms "comprising", "having", "including" or "containing" may mean inclusive or open-ended, and do not exclude 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.

[0050] In the present invention, the term "about" is used to define the numerical range and parameters of the present invention are approximate values, and the specific relevant values ​​are presented as accurately as possible. Unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in the present invention are modified by "about". Here, "about" usually means that the actual value is within ±5%, ±3%, ±1% or ±0.5% of a particular value or range.

[0051] 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.

[0052] The present invention is mainly completed based on the following insights:

[0053] Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria. It can enter the brain through the blood-brain barrier to activate microglia and induce neuroinflammatory response, 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 nutrients that are safe (non-cytotoxic) and have obvious effects on regulating brain immunity and maintaining the health of the nervous system (such as Figure 1 The present invention has found through research that the combination of lactoferrin and 3-fucosyllactose can be used to intervene in microglia stimulated by LPS, thereby inhibiting the polarization of microglia to M1 phenotype, promoting the polarization of microglia to M2 phenotype, inducing the transformation of microglia from M1 phenotype to M2 phenotype, and increasing the amount of IL-10 released by microglia, and reducing the amount of TNF-α, IL-1β and NO released by microglia.

[0054] I. Nutritional Composition

[0055] The nutritional composition of the present invention comprises the following essential components: lactoferrin and fucosyllactose. In some embodiments, lactoferrin and fucosyllactose are the main effective ingredients in the nutritional composition of the present invention. The "main effective ingredient" of the present invention refers to a component that exerts a specific physiologically active function in the nutritional composition, such as regulating brain immunity. In some embodiments, based on the total dry weight of the nutritional composition, the total content of lactoferrin and fucosyllactose in the nutritional composition is 60% or more, for example, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95%, etc., preferably 85% or more, more preferably 90% or more. In some embodiments, the nutritional composition may contain a small amount of other types of carbohydrate substances, fat substances and / or protein substances, such as lactose, in addition to lactoferrin and fucosyllactose.

[0056] In some embodiments, the nutritional composition consists of lactoferrin and fucosyllactose.

[0057] The present invention does not specifically limit the source of lactoferrin. For example, lactoferrin from mammals such as cows, sheep, and camels can be used, and bovine lactoferrin is preferred. The present invention does not specifically limit the method for obtaining lactoferrin. For example, fresh milk or dairy products can be used as raw materials, and lactoferrin can be separated and extracted by methods such as protein flocculation, or commercial lactoferrin products can be directly used.

[0058] At present, the production methods of fucosyllactose mainly include chemical synthesis, enzymatic synthesis and bioengineering synthesis. At the same time, it can also be indirectly introduced from various existing milk ingredients.

[0059] It should be noted that the use of fucosyllactose should comply with the requirements of local laws and regulations. In some cases, where permitted by laws and regulations, fucosyllactose can be directly introduced into food compositions as a separate raw material; in other cases, where permitted by laws and regulations, it can be indirectly introduced into compositions or foods by adding milk raw materials that meet the requirements.

[0060] The present invention does not particularly limit the source of fucosyllactose, for example, it can be brought in from different animal milk raw materials (such as cow's milk, milk powder, etc.), or it can be fucosyllactose prepared by microbial fermentation, or commercial fucosyllactose products can be directly used. 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.

[0061] In some embodiments, the fucosyllactose in the nutritional composition comprises 3-fucosyllactose. In some preferred embodiments, the fucosyllactose in the nutritional composition is 3-fucosyllactose.

[0062] In some embodiments, in the nutritional composition, the mass ratio of lactoferrin to fucosyllactose is 1:(0.1-30), for example, 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12. 2, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29 or 1:30, etc.; preferably 1:(0.1-25); more preferably 1:(0.1-20); further preferably 1:(0.1-10); even more preferably 1:(0.2-10).

[0063] II. Food

[0064] The food of the present invention contains or uses the above-mentioned nutritional composition, so that the food of the present invention also contains lactoferrin and fucosyllactose. By adding or using the nutritional composition, in some embodiments, in the food, the mass ratio of lactoferrin to fucosyllactose is 1: (0.1-30).

[0065] The present invention does not specifically limit the absolute content of lactoferrin and fucosyllactose in food, as long as it complies with the requirements of relevant laws and regulations. In some embodiments, the content of lactoferrin in food is 0.001% to 1.5%, preferably 0.01% to 1%; the content of fucosyllactose in food is 0.001% to 4%, preferably 0.01% to 2%.

[0066] The present invention does not particularly limit the types of food, and examples thereof include candies, beverages, dairy products or baked goods. For candies, examples include hard candies, crisp candies, gel candies, compressed candies and aerated candies. For beverages, examples include carbonated beverages, tea beverages, coffee beverages, fruit and vegetable juice beverages and lactic acid bacteria beverages. For dairy products, examples include fermented milk, cheese and milk powder. For baked goods, examples include bread, cakes and biscuits. In addition, the food of the present invention can also be a health food, such as various types of oral preparations, including tablets, pills, granules, powders, capsules and oral liquids.

[0067] In addition to the nutritional composition and the lactoferrin and fucosyllactose therein, depending on the type of food and the final needs of the applicable objects, in some embodiments, the food of the present invention also contains any one or more of the following ingredients: plant product ingredients, animal dairy product ingredients, animal meat product ingredients, functional added ingredients and any acceptable excipients.

[0068] As for the ingredients of plant products, examples include fruits such as figs, pomegranates, kiwis, tangerines, oranges, pineapples, strawberries, apples, bananas, grapes, pears, cherries, blueberries, blackberries, black currants, cranberries, raspberries, melons, emblica chinensis and bilberry or their extracts; vegetables such as onions, cucumbers, tomatoes, cauliflowers, 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, sorghum, yellow rice, buckwheat, soybeans, broad beans, peas, mung beans, red beans, kidney beans or their extracts; nuts such as walnuts, pistachios, cashews, hazelnuts, almonds, apricots, pine nuts, peanuts, melon seeds, chestnuts, macadamia nuts, ginkgo nuts or their extracts; coffee or its extracts; and some botanical Chinese medicinal materials or their extracts that are both medicine and food.

[0069] As for animal dairy ingredients, examples include fresh milk from mammals such as cows, sheep, and camels, as well as reprocessed dairy products such as whole milk powder, skim milk powder, concentrated whey protein powder, desalted whey powder, whey protein powder, and hydrolyzed whey protein powder.

[0070] Examples of animal meat ingredients include meat ingredients from pigs, cattle, sheep, aquatic animals or poultry.

[0071] Functional added ingredients include vitamin supplements, mineral supplements, nucleotide supplements, dietary fiber, functional polyunsaturated fatty acid supplements, and the like.

[0072] As for any acceptable excipients, examples include 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.

[0073] III. Uses of regulating brain immunity

[0074] The present invention unexpectedly discovered that the combination of lactoferrin and fucosyllactose can play a role in regulating brain immunity. Moreover, when lactoferrin and fucosyllactose are combined in a certain ratio, there is a synergistic effect between the two, which can further enhance the effect of regulating brain immunity. Therefore, the nutritional composition containing lactoferrin and fucosyllactose described in the present invention and the food added or used with the nutritional composition have the effect of regulating brain immunity.

[0075] 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 regulation of brain immunity mentioned in the present invention is not intended to prevent and treat diseases.

[0076] Furthermore, the present invention provides use of a nutritional composition containing lactoferrin and fucosyllactose in preparing food for regulating brain immunity.

[0077] In some embodiments, the effect of regulating brain immunity includes regulating microglia polarization. In some specific embodiments, the regulating brain immunity includes inhibiting microglia polarization to M1 phenotype, promoting microglia polarization to M2 phenotype, and inducing microglia to transform from M1 phenotype to M2 phenotype. In some specific embodiments, the regulating brain immunity includes inhibiting microglia polarization to M1 phenotype, promoting microglia polarization to M2 phenotype, and inducing microglia to transform from M1 phenotype to M2 phenotype. In some specific embodiments, the regulating brain immunity includes increasing the amount of IL-10 released by microglia and reducing the amount of TNF-α, IL-1β and / or NO released by microglia. In some specific embodiments, the regulating brain immunity includes increasing the amount of IL-10 released by microglia and reducing the amount of TNF-α, IL-1β and / or NO released by microglia. In some specific embodiments, the regulating brain immunity comprises increasing the amount of IL-10 released by microglia and reducing the amount of TNF-α, IL-1β and NO released by microglia.

[0078] IV. Uses to assist in improving the health of the central nervous system

[0079] The present invention unexpectedly found that the combination of lactoferrin and fucosyllactose can play a role in assisting the improvement of the health of the central nervous system. Moreover, when lactoferrin and fucosyllactose are combined in a certain ratio, there is a synergistic effect between the two, which can further enhance the role of assisting the improvement of the health of the central nervous system. Therefore, the nutritional composition containing lactoferrin and fucosyllactose described in the present invention and the food added or used with the nutritional composition also have the role of assisting the improvement of the health of the central nervous system, and this role is non-therapeutic purpose.

[0080] Furthermore, the present invention provides use of a nutritional composition containing lactoferrin and fucosyllactose in preparing a food for assisting in improving the health of the central nervous system.

[0081] In some embodiments, the aiding improvement of central nervous system health includes improving neuroinflammation, such neuroinflammation does not reach disease levels. In some embodiments, the central nervous system health is affected by neuroinflammation, and the food aids in improving central nervous system health by improving neuroinflammation.

[0082] In some embodiments, the improvement of neuroinflammation is achieved by affecting the TLR4 / NF-κB signaling pathway in microglia. In some specific embodiments, the effect on the TLR4 / NF-κB signaling pathway in microglia includes inhibiting the expression of TLR4, inhibiting the expression of NF-κB p65, inhibiting the phosphorylation of NF-κB p65, increasing the expression of IKB-α and inhibiting the phosphorylation of IKB-α.

[0083] Example

[0084] The embodiments of the present invention will be described in detail below in conjunction with 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 under normal conditions or conditions recommended by the manufacturer. Unless otherwise specified, the materials or instruments used are all commercially available conventional products.

[0085] 1. Materials and Methods

[0086] 1.1 Raw materials

[0087] Lactoferrin (LF): Hilmar, Hilmar 1000 lactoferrin.

[0088] 3-Fucosyllactose (3-FL): DSM, GlyCare 3-FL 9001.

[0089] 1.2 Cells

[0090] Mouse microglial cell line BV2 cells were purchased from Shanghai Enzyme Research Biotechnology (CC-Y2022).

[0091] 1.3 Reagents

[0092] Table 1 Main reagents for the experiment

[0093] serial number Drug 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 13 EASYspinPlus Tissue / Cell RNA Rapid Extraction Kit Adele Biology RN2802 14 Reverse transcription kit Adele Biology PC7002 15 SGExcel FastSYBR Master Mix Bioengineering B532955-0005 16 Primers Bioengineering H520001-0001 / H510001-0002 17 NF-κB p65 Antibody Wuhan Tri-Eagle Biotech 10745-1-AP 18 Phospho-NF-κB p65 Antibody Wuhan Tri-Eagle Biotech 80379-2-RR 19 IKB-α Antibody Wuhan Tri-Eagle Biotech 10268-1-AP 20 Phospho-IKB-α Antibody Wuhan Tri-Eagle Biotech 82349-1-RR 21 GAPDH Antibody Wuhan Tri-Eagle Biotech 10494-1-AP 22 TLR4 antibodies Wuhan Tri-Eagle Biotech 19811-1-AP 23 HRP-conjugated Goat Anti-Rabbit IgG Wuhan Tri-Eagle Biotech SA00001-2

[0094] 1.4 Instruments

[0095] Table 2 Main instruments and equipment for the experiment

[0096] 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. 8 Fluorescence quantitative PCR instrument Thermo Fisher Scientific China Co., Ltd. 9 Water bath Quanzhou Yiguan Technology Co., Ltd. 10 Nucleic acid protein analyzer Thermo Fisher Scientific China Co., Ltd. 11 Ordinary PCR instrument Harbin Wayne Trading Co., Ltd. 12 Multifunctional imaging system Wuhan Sewell Biotechnology Co., Ltd. 13 Transfer electrophoresis instrument Wuhan Sewell Biotechnology Co., Ltd.

[0097] 1.5 Cell culture

[0098] 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.

[0099] 1.6 Nutrient cytotoxicity assay

[0100] The CCK-8 kit detects the toxicity of nutrients to cells. The working principle of the CCK-8 kit is to use the dehydrogenase in the cell mitochondria to reduce 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonate benzene)-2H-tetrazole monosodium salt to yellow formazan in the presence of the electron carrier 1-methoxy-5-methylphenazine dimethyl sulfate. 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 the microplate reader.

[0101] 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 0.5-1 hour. Use an enzyme marker to measure the absorbance at 450nm, and then use 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), and finally calculate the cell survival rate. The CCK8 method was used to detect the viability of BV2 microglia.

[0102] 1.7 Establishment of microglial activation model and experimental grouping

[0103] 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 14 groups, namely blank control group, LPS model group, and 12 experimental groups. (1) Control group: conventional cell culture was used; (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.

[0104] Table 3 Experimental groups and doses

[0105] Corresponding experiment Dose Group Description LPSμg / mL LF mg / mL 3-FL mg / mL LF:3-FL Blank control Blank control group - - - Positive Control LPS model group 1 - - Experiment 1 LF high dose group 1 1 - Experiment 2 LF medium dose group 1 0.5 - Experiment 3 LF low dose group 1 0.2 - Experiment 4 3-FL high dose group 1 - 5 Experiment 5 3-FL medium dose group 1 - 2 Experiment 6 3-FL low dose group 1 - 0.5 Experiment 7 LF high + 3-FL high 1 1 5 1:5 Experiment 8 LF high + 3-FL medium 1 1 2 1:2 Experiment 9 LF high + 3-FL low 1 1 0.5 1:0.5 Experiment 10 LF medium + 3-FL high 1 0.5 5 1:10 Experiment 11 LF medium + 3-FL medium 1 0.5 2 1:4 Experiment 12 LF low + 3-FL high 1 0.2 5 1:25

[0106] 1.8 NO kit to detect the content of NO in the supernatant of BV2 cells

[0107] 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 minutes. The supernatant was collected and the NO content in the cell supernatant was determined using the NO kit according to the operating procedures. The principle of the NO kit to detect NO content is that NO is easily oxidized to 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 520nm-560nm. - The NO content in the liquid was calculated using the standard curve.

[0108] 1.9 Detection of TNF-α, IL-1β and IL-10 in the supernatant of BV2 cell culture by enzyme-linked immunosorbent assay (ELISA)

[0109] 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.

[0110] 1.10 Detection of iNOS and CD206, markers of M1 and M2 phenotypes of BV2 cells

[0111] qRT-PCR was used to detect the mRNA expression levels of iNOS and CD206-related genes and then determine the changes in the content of M1 cell population and M2 cell population. The third-generation BV2 microglial cell suspension was inoculated in a 24-well culture plate with a cell density of 4×10 5Each well was divided into groups according to the experimental design. After treatment, the cells were washed twice with PBS, using 200 μL per well. Digested with trypsin for 2.5 min, and digested with 400 μL of culture medium. The cells were blown up with a pipette and collected in enzyme-free centrifuge tubes. Total RNA of each group of cells was extracted. Reverse transcription was performed using a reverse transcription kit according to the operating procedures to obtain cDNA, and the amplified cDNA was mixed with primers, fluorescent probes and qPCR Master Mix. The corresponding PCR cycle program and parameters were set according to the requirements of qPCR, and the corresponding mRNA expression level was analyzed in the real-time PCR detection system. The specific primer information is as follows:

[0112] iNOS:

[0113] Upstream: 5'-GGCTTGCCCCTGGAAGTTT-3' (SEQ ID NO. 1),

[0114] Downstream: 5′-TGCAAGTGAAATCCGATGTGG-3′ (SEQ ID NO. 2);

[0115] CD206:

[0116] Upstream: 5'-TTCAGCTATTGGACGCGAGG-3' (SEQ ID NO. 3),

[0117] Downstream: 5'-GAATCTGACACCCAGCGGAA-3' (SEQ ID NO. 4);

[0118] GAPDH:

[0119] Upstream: 5'-GGTTGTCTCCTGCGACTTCA-3' (SEQ ID NO. 5),

[0120] Downstream: 5'-TGGTCCAGGGTTTCTTACTCC-3' (SEQ ID NO. 6).

[0121] 1.11 Western blot detection of relative expression of proteins in BV2 cells

[0122] Western blot was used to detect the expression of TLR4, NF-κB, p-NF-κB, IKB-α and p-IKB-α, and the metabolic pathways of TLR4, NF-κB, p-NF-κB, IKB-α and p-IKB-α were studied. The third generation BV2 cell suspension was inoculated in a 6-well culture plate with a cell density of 1×10 5 / well, and treated in groups according to the experimental design. After treatment, wash the cells 3 times with 4℃ precooled PBS solution, lyse the cells on ice with RIPA lysis buffer containing protease inhibitors, centrifuge at 4℃ for 15min, take the supernatant, use BCA quantitative kit to detect protein concentration, and quantitatively divide the samples. Add 5 times the concentration of SDS-PAGE protein loading buffer and boil in a 100℃ metal water bath for 5min to fully denature the protein. After equal amounts of each group of proteins are loaded, electrophoresis and transfer are performed, and the membrane is blocked with protein-free rapid blocking solution (1×) for 1h. Take out the PVDF membrane and add the primary antibody (1:1000) and incubate at 4℃ overnight. Recover the primary antibody, wash 3 times with TBST solution, add the corresponding secondary antibody (dilution ratio 1:5000) and incubate at room temperature for 1h. Wash again with TBST solution 3 times, prepare ECL developer (ratio 1:1), expose and develop with chemiluminescence imaging analysis system, and collect images. Image J software was used to perform grayscale analysis on protein bands, and the final results were expressed as the grayscale value of the target protein / grayscale value of the internal reference protein, with GAPDH as the internal reference protein.

[0123] 1.12 Statistical analysis

[0124] All experiments were repeated three times independently, and SPSS22.0 statistical software was used to analyze the experimental data. The experimental results were analyzed by variance homogeneity test, and the statistical differences between the groups were analyzed by one-way ANOVA, and expressed as mean ± standard deviation (x ± SD). P>0.05 indicated no statistical significance, and P<0.05 indicated statistically significant differences.

[0125] 2. Experimental results

[0126] 2.1 Nutrient cytotoxicity assay

[0127] Table 4 shows the effects 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.

[0128] Table 4 Effects of different nutrients on cell viability of BV2 cells

[0129] Corresponding experiment Dose Group Description LF mg / mL 3-FL mg / mL LF:3-FL Cell survival rate% Blank control Blank control group - - 100.46±0.22 Positive Control LPS model group - - 94.87±0.37 Experiment 1 LF high dose group 1 - 99.04±0.88 Experiment 2 LF medium dose group 0.5 - 97.94±0.64 Experiment 3 LF low dose group 0.2 - 96.39±0.68 Experiment 4 3-FL high dose group - 5 96.28±0.85 Experiment 5 3-FL medium dose group - 2 96.15±0.36 Experiment 6 3-FL low dose group - 0.5 96.25±0.47 Experiment 7 LF high + 3-FL high 1 5 1:5 99.28±0.21 Experiment 8 LF high + 3-FL medium 1 2 1:2 97.28±1.91 Experiment 9 LF high + 3-FL low 1 0.5 1:0.5 97.80±0.47 Experiment 10 LF medium + 3-FL high 0.5 5 1:10 98.24±0.21 Experiment 11 LF medium + 3-FL medium 0.5 2 1:4 97.69±0.47 Experiment 12 LF low + 3-FL high 0.2 5 1:25 97.87±0.57

[0130] 2.2 Effects of nutrients on NO production in LPS-stimulated BV2 cells

[0131] To measure the effects of mixed or single nutrient treatments on LPS-induced BV2 cells, the present invention evaluated the secretion of the pro-inflammatory chemical signal NO to estimate the effect of regulating microglial polarization.

[0132] Table 5 shows the effects of different nutrient composition samples containing LF and 3-FL on NO production in BV2 cells stimulated by LPS. The results of NO research showed that compared with the LPS model group, LF or 3-FL, when used alone, showed the effect of reducing NO production. When the two were used in combination, especially when the stoichiometric ratio of LF:3-FL was controlled at 1:0.5 to 1:25, a significant effect of reducing LPS-induced NO production could be obtained.

[0133] Specifically, by comparing Experimental Example 2, Experimental Example 4 and Experimental Example 10, it is shown that relative to the LPS model group, cell experiment Examples 2 and 4 reduced the NO content by 0.54 and 1.39, respectively, while cell experiment Example 10 reduced NO by 4.24, which is greater than the sum of the previous two (0.54+1.39), indicating that there is a synergistic effect between the two components, which can synergistically reduce the NO production of BV2 cells stimulated by LPS.

[0134] Table 5 Effects of different nutrient compositions on NO production in BV2 cells stimulated by LPS

[0135]

[0136] 2.3 Effects of nutrients on the production of pro-inflammatory factors in LPS-stimulated BV2 cells

[0137] In order to measure the effects of mixed nutrition or single nutrition treatment on LPS-induced BV2 cells, we evaluated the secretion of proinflammatory factors TNF-α and IL-1β to estimate the effect of regulating microglial polarization. Table 6 shows the effects of different nutritional composition samples containing LF and 3-FL on the production of proinflammatory factors in LPS-stimulated BV2 cells. The results showed that compared with the LPS model group, when LF or 3-FL was used alone, the effect of reducing proinflammatory factors was not obvious. When the two were used in combination, the LF:3-FL ratio was 1:0.5 to 1:25, and a significant effect of reducing the production of LPS-induced proinflammatory factors could be obtained.

[0138] Specifically, by comparing Experimental Example 2, Experimental Example 4 and Experimental Example 10, it is illustrated that relative to the LPS model group, cell experiment Experimental Examples 2 and 4 reduced the TNF-α content by -2.78 and 4.86, and the IL-1β content by 0.57 and 1.32, respectively, while cell experiment Experimental Example 10 reduced TNF-α by 47.57, which is greater than the sum of the first two (-2.78+4.86), and reduced IL-1β by 3.67, which is greater than the sum of the first two (0.57+1.32), indicating that there is a synergistic effect between the two components, which can synergistically reduce the production of proinflammatory factors in BV2 cells stimulated by LPS.

[0139] Table 6 Effects of different nutritional compositions on the production of pro-inflammatory factors in BV2 cells stimulated by LPS

[0140]

[0141] 2.4 Effects of nutrients on anti-inflammatory factors in LPS-stimulated BV2 cells

[0142] In order to measure the effects of mixed nutrition or single nutrition treatment on LPS-induced BV2 cells, we evaluated the secretion of the anti-inflammatory factor IL-10 to estimate the effect of regulating microglial polarization. Table 7 shows the effects of different nutritional composition samples containing LF and 3-FL on the production of anti-inflammatory factors in BV2 cells stimulated by LPS. The results showed that compared with the LPS model group, when LF or 3-FL was used alone, both showed the effect of increasing the production of anti-inflammatory factors. At the same time, when the two are used in combination, a significant effect of increasing IL-10 production can be obtained.

[0143] Specifically, by comparing Experimental Example 2, Experimental Example 4 and Experimental Example 10, it is shown that compared with the single nutritional treatment of Experimental Example 2 and Experimental Example 4, the IL-10 content of the mixed nutritional treatment of the cell experiment Experimental Example 10 was significantly increased (P<0.001), indicating that the two can synergistically increase the production of anti-inflammatory factors.

[0144] Table 7 Effects of different nutritional compositions on the production of anti-inflammatory factors in BV2 cells stimulated by LPS

[0145]

[0146] 2.5 Effects of nutrients on iNOS and CD206, markers of M1 and M2 phenotypes of BV2 cells stimulated by LPS

[0147] After activation, microglia eventually polarize into two cell groups 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 (NO), which may cause inflammatory response in the CNS, leading to neuronal damage and eventually death; M2 phenotype microglia mainly promote damage repair and exert neuroprotective effects by secreting anti-inflammatory cytokines, such as interleukin 10 (IL-10). The main phenotypic marker of M1 microglia is iNOS, and the main phenotypic marker of M2 microglia is CD206.

[0148] In order to measure the effect of mixed nutrition or single nutrition treatment on LPS-induced BV2 cells, we used qRT-PCR to detect the mRNA expression levels of iNOS and CD206-related genes and then measured the changes in the content of M1 cell populations and M2 cell populations to estimate the effect of regulating microglial polarization. Table 8 shows the effects of different nutrient composition samples containing LF and 3-FL on the mRNA expression levels of BV2 cell markers stimulated by LPS.

[0149] The results showed that compared with the LPS model group, when LF or 3-FL was used alone, it increased the relative expression of CD206 and decreased the relative expression of iNOS. At the same time, when the two were used in combination, the dosage ratio of LF:3-FL was 1:0.5 to 1:10, and a more obvious effect could be obtained.

[0150] Specifically, by comparing Experimental Example 2, Experimental Example 4 and Experimental Example 10, relative to the LPS model group, cell experiment Experimental Examples 2 and 4 reduced the relative expression of iNOS by 2.21 and 2.51, respectively, and increased the relative expression of CD206 by 2.54 and 2.76, while cell experiment Experimental Example 10 reduced the relative expression of iNOS by 5.27, which was greater than the sum of the first two (2.21+2.51), and increased the relative expression of CD206 by 6.82, which was greater than the sum of the first two (2.54+2.76), indicating that there is a synergistic effect between the two components, which can synergistically reduce the polarization of LPS-stimulated BV2 cells to the M1 phenotype.

[0151] Table 8 Effects of different nutritional compositions on the expression levels of BV2 cell marker mRNAs stimulated by LPS

[0152]

[0153] 2.6 Effects of nutrients on the TLR4 / NF-κB signaling pathway in LPS-stimulated BV2 cells

[0154] LPS induces the activation of Toll-like receptors (TLR4), which in essence activates the NF-κB signaling pathway and accelerates the transcription of inflammatory genes. Under normal conditions, NF-κB is in an inactive form and combines with the inhibitor IKB-α in the cytoplasm to form a dimer. After being stimulated by inflammation, the IKB-α protein is phosphorylated and degraded, releasing NF-κB to transfer to the cell nucleus and participate in the expression of several inflammatory factors such as IL-1β and TNF-α. At the same time, these inflammatory factors are involved in regulating the TLR-4 / NF-κB signaling pathway, thus forming a feedback loop and jointly participating in the process of neuroinflammation.

[0155] In order to explore the pathways that affect mixed nutrients or single nutrient treatment on LPS-induced BV2 cells, we extracted the whole protein of each group of cells and used Western blot to detect the expression of TLR4, NF-κB, p-NF-κB, IKB-α and p-IKB-α. Figures 2A to 2F The effects of LF, 3-FL and their combination on the expression of TLR4 / NF-κB signaling pathway proteins in LPS-stimulated BV2 cells were demonstrated.

[0156] The results showed that compared with the control group, the protein expressions of TLR4, NF-κB p65 and p-NF-κBp65 (phosphorylated NF-κB p65) in the LPS model group were significantly increased (P < 0.0001, Figures 2B to 2D ); Compared with the LPS model group, there was no significant difference in TLR4 expression in the high-dose 3-FL group, but the TLR4 expression in the high-dose LF group was significantly decreased, and the inhibitory effect of the combination was more significant (P < 0.0001, Figure 2B The expressions of NF-κBp65 and p-NF-κB p65 in the high-dose LF group, high-dose 3-FL group and combination group were inhibited, and the inhibitory effect of the combination was more significant (P<0.0001, Figure 2C , 2D At the same time, the phosphorylation degree of IKB-α protein was determined. The results showed that compared with the control group, the expression of IKB-α protein in the LPS model group was significantly reduced, and the expression of p-IKB-α (phosphorylated IKB-α) protein was significantly increased, and the difference was statistically significant (P < 0.0001, Figure 2E , 2F ); Compared with the LPS model group, the high-dose LF group, high-dose 3-FL group and the combination group inhibited the phosphorylation of IKB-α in BV2 cells, and the inhibitory effect of the combination was more significant (P < 0.0001, Figure 2F ). Therefore, the combination of LF and 3-FL alleviates neuroinflammatory response, which may be achieved through the TLR4 / NF-κB signaling pathway.

Claims

1. Use of the nutritional composition in preparing food for regulating brain immunity; wherein, The nutritional composition comprises the following essential components: lactoferrin and fucosyllactose, and in the nutritional composition, the mass ratio of the lactoferrin to the fucosyllactose is 1:(0.1-30).

2. The use according to claim 1, characterized in that The fucosyllactose includes 3-fucosyllactose.

3. The use according to claim 1 or 2, characterized in that The regulating brain immunity includes at least one of inhibiting the polarization of microglia to M1 phenotype, promoting the polarization of microglia to M2 phenotype, and inducing the transformation of microglia from M1 phenotype to M2 phenotype.

4. The use according to any one of claims 1 to 3, characterized in that The regulating brain immunity includes at least one of increasing the amount of IL-10 released by microglia, reducing the amount of TNF-α released by microglia, reducing the amount of IL-1β released by microglia, and reducing the amount of NO released by microglia.

5. The use according to any one of claims 1 to 4, characterized in that: The food is candy, beverage, dairy product or baked food.

6. The use according to claim 5, characterized in that The candies include at least one of hard candies, jelly candies, crisp candies, compressed candies and aerated candies; the beverages include at least one of carbonated beverages, tea beverages, coffee beverages, fruit and vegetable juice beverages and lactic acid bacteria beverages; the dairy products include at least one of fermented milk, cheese and milk powder; the baked goods include at least one of bread, cakes and biscuits.

7. The use according to any one of claims 1 to 4, characterized in that The food is an oral preparation, which includes any one or more of tablets, pills, granules, powders, capsules and oral liquids.

8. Use of the nutritional composition in preparing food for assisting in improving the health of the central nervous system; wherein: The nutritional composition comprises the following essential components: lactoferrin and fucosyllactose, and in the nutritional composition, the mass ratio of the lactoferrin to the fucosyllactose is 1:(0.1-30).

9. The use according to claim 8, characterized in that The fucosyllactose includes 3-fucosyllactose.

10. The use according to claim 8 or 9, characterized in that The auxiliary improvement of central nervous system health includes the improvement of neuroinflammation for non-therapeutic purposes.

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