Use of nutritional composition for regulating brain immunity

Abstract

The use of a nutritional composition in the preparation of a food product for regulating brain immunity, and in the preparation of a food product for assisting with improving the health of the central nervous system, wherein the nutritional composition contains 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).

Description

Use of nutritional composition to modulate brain immunity TECHNICAL FIELD

[0001] The present application belongs to the field of nutritional substances, and particularly relates to the use of nutritional composition to modulate brain immunity, and more particularly to the use of nutritional composition containing lactoferrin and fucosyllactose in modulating brain immunity and assisting in improving the health of central nervous system. BACKGROUND

[0002] In recent years, studies have shown that the brain is not an immune-privileged organ, and the brain and the immune system are closely connected. Moreover, it is known that the brain has its own immune cells, such as microglia, which are macrophages that reside in the central nervous system (CNS) and account 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 myelin formation, and can indirectly affect synapse and neuron function by regulating the activation of microglia and the activity of other non-neuronal cells.

[0003] After activation, microglia can polarize into two functionally opposite cell populations, M1 and M2. M1 phenotype microglia mainly release pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), and the chemical signal nitric oxide (NO), which can easily cause excessive inflammatory response in the CNS, leading to damage to neurons and ultimately causing their death; M2 phenotype microglia mainly promote damage repair by secreting anti-inflammatory cytokines such as interleukin 10 (IL-10), etc., and play a neuroprotective role.

[0004] Microglia play an important role in neuroinflammation. Neuroinflammation is a complex immune response of neural tissue to eliminate pathogens, damaged cells, and harmful substances, and excessive neuroinflammatory response can affect the health of the CNS.

[0005] Lactoferrin (LF) belongs to the transferrin superfamily, is a non-hematin cationic protein with a relative molecular mass of about 80,000 Da, composed of about 700 amino acids, and has a symmetrical bilobed structure. 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, with the highest content in milk. The concentration of LF in human colostrum is 1-8 g / L. LF not only participates in iron transport, but also has strong biological functions such as broad-spectrum antibacterial, antioxidant, anticancer, 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 pro-inflammatory responses, and play an important role in regulating iron homeostasis, inflammatory response, oxidative stress, apoptosis, and angiogenesis.

[0006] Reference document 1 discloses a complex liposome for improving brain cognitive memory and neuroinflammation and a preparation method thereof, and belongs to the technical field of liposome preparation. Liposomes capable of simultaneously loading hydrophobic substance curcumin and hydrophilic substance epigallocatechin gallate are prepared, and on this basis, lactoferrin is used for surface modification, and hyaluronic acid is further used for stabilization by electrostatic interaction. The complex liposome has good stability and free radical scavenging efficiency, can significantly inhibit lipopolysaccharide-induced cognitive dysfunction, and is used for improving brain cognitive memory and neuroinflammation.

[0007] Reference document 2 discloses the use of lactoferrin combined with choline in the preparation of a drug for preventing and / or treating Alzheimer's disease. Lactoferrin and choline combined in a specific ratio as a drug efficacy substance can increase the level of anti-inflammatory factors in mice with neurodegenerative diseases, reduce the level of pro-inflammatory factors, enhance the ability of the brain to resist inflammatory response, reduce tau protein phosphorylation and serum Aβ level, improve learning ability and cognitive function, and improve autonomous exploration ability, providing a new choice for 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, and 3-FL plays a key role in the healthy development of infants, including regulating the immune system, antibacterial and anti-inflammatory, regulating the proliferation of intestinal microbiota, and promoting brain maturation.

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

[0010] In addition, there are also studies on nutritional compositions comprising both human milk oligosaccharides and lactoferrin, for example:

[0011] Reference 4 discloses nutritional compositions, foods and uses comprising or consisting of human milk oligosaccharides and lactoferrin. The human milk oligosaccharides are preferably neutral fucosylated human milk oligosaccharides, which are preferably selected from one or more of 2’-fucosyllactose, 3’-fucosyllactose, lacto-N-fucopen-taose I, lacto-N-difucohexaose I, lacto-N-difucohexaose II. The nutritional compositions have the effect of promoting the development of the immune system and improving the intestinal flora of the offspring of the human or animal mother after being ingested by the human or animal mother.

[0012] Reference 5 discloses a nutritional composition for promoting physical development, the effective ingredient raw material for making it includes, in weight parts: 50-60 parts of galacto-oligosaccharides, 35-40 parts of human milk oligosaccharides and 5-10 parts of lactoferrin, wherein the human milk oligosaccharides include 2’-fucosyllactose and / or lacto-N-neotetraose. The combination of galacto-oligosaccharides, human milk oligosaccharides and lactoferrin can synergistically improve the utilization rate of calcium in the body and effectively promote physical development.

[0013] Reference:

[0014] Reference 1: CN114470236B;

[0015] Reference 2: CN115737787B;

[0016] Reference 3: Berger PK, Bansal R, Sawardekar S, Yonemitsu C, Furst A, Hampson HE, Schmidt KA, Alderete TL, Bode L, Goran MI, Peterson BS. Associations of Human Milk Oligosaccharides with Infant Brain Tissue Organization and Regional Blood Flow at 1 Month of Age. Nutrients. 2022 Sep 16; 14(18): 3820.

[0017] Reference 4: CN115918916A;

[0018] Reference 5: CN115644458A. SUMMARY

[0019] PROBLEMS TO BE SOLVED BY THE INVENTION

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

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

[0022] Therefore, the primary purpose of the present application is to provide a non-therapeutic purpose of the nutritional composition containing lactoferrin and fucosyllactose in regulating brain immunity and assisting in improving central nervous system health.

[0023] SOLUTIONS TO PROBLEMS

[0024] In order to solve the above technical problems, the present application 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 comprises 3-fucosyllactose.

[0027] [3]. The use according to [1] or [2], wherein the modulating brain immunity comprises at least one of suppressing polarization of microglia to Ml phenotype, promoting polarization of microglia to M2 phenotype, and inducing conversion of microglia from Ml phenotype to M2 phenotype.

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

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

[0030] [6]. The use according to [5], wherein the confectionery comprises at least one of a hard candy, a gummy candy, a crisp candy, a pressed candy, and an aerated candy; the beverage comprises at least one of a carbonated beverage, a tea-based beverage, a coffee-based beverage, a fruit / vegetable juice beverage, and a lactic acid bacteria beverage; the dairy product comprises at least one of a fermented milk, a cheese, and a milk powder; and the bakery product comprises at least one of a bread, a cake, and a cookie.

[0031] [7]. The use according to any one of [1] to [4], wherein the food is an oral preparation comprising any one or more of a tablet, a pill, a granule, a powder, a capsule, and an oral liquid.

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

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

[0034]

[0010] . The use according to [8] or [9], wherein the assisting improvement in central nervous system health comprises improvement in neuroinflammation for non-therapeutic purposes.

[0035] Effects of the Invention

[0036] The present application researches and finds that the combination of lactoferrin and fucosyllactose, especially in a certain proportion, has a synergistic effect of regulating brain immunity and assisting in improving the health of central nervous system. Experimental data shows that in an in vitro model, the combination of lactoferrin and fucosyllactose is used to treat BV2 cells, the trend of differentiating into M2 phenotype microglial cells is higher than that of differentiating into M1 phenotype microglial cells, the amount of secreted inflammatory factors is obviously reduced, and there is a good synergistic effect between lactoferrin and fucosyllactose, which can play an effective role in regulating brain immunity and assisting in improving the health of central nervous system. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is a schematic diagram of the experimental process of the present application for studying the regulating effect of nutrients on microglial cells.

[0038] Figure 2A is the Western blot detection result of the effect of nutrients on the TLR4 / NF-κB signaling pathway of LPS-stimulated BV2 cells; wherein group 1 is the LPS model group, group 2 is the blank control group, group 3 is the LF high dose group, group 4 is the 3-FL high dose group, group 5 is the LF high dose + 3-FL low dose group (LF:3-FL is 1:0.5), and group 6 is the LF low dose + 3-FL high dose group (LF:3-FL is 1:25).

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

[0040] Figure 2C is the Western blot detection analysis result of the effect of nutrients on the expression of NF-κB p65 protein in the TLR4 / NF-κB signaling pathway of LPS-stimulated BV2 cells; wherein group 1 is the LPS model group, group 2 is the blank control group, group 3 is the LF high dose group, group 4 is the 3-FL high dose group, group 5 is the LF high dose + 3-FL low dose group (LF:3-FL is 1:0.5), and group 6 is the LF low dose + 3-FL high dose group (LF:3-FL is 1:25), ***P<0.001, ****P<0.0001.

[0041] Figure 2D: Western blot detection analysis results of the effect of nutrients on the expression of p-NF-κB p65 protein in the TLR4 / NF-κB signaling pathway of LPS-stimulated BV2 cells; wherein group 1 is the LPS model group, group 2 is the blank control group, group 3 is the LF high-dose group, group 4 is the 3-FL high-dose group, group 5 is the LF high-dose + 3-FL low-dose group (LF:3-FL is 1:0.5), group 6 is the LF low-dose + 3-FL high-dose group (LF:3-FL is 1:25), ***P<0.001, ****P<0.0001.

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

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

[0044] The embodiments of the present application are described below, but the present application is not limited thereto. 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 and examples are also included in the technical scope of the present application.

[0045] In the present application, the numerical range indicated by "numerical value A to numerical value B", "numerical value A-numerical value B", "numerical value A or more" or "numerical value A or less" means a range including the end point values A and B.

[0046] In the present application, the meaning indicated by "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0047] In the present application, "optional" or "optionally" means that the subsequently described event or circumstance can or can not occur, and that the description includes the situation where the event occurs and the situation where the event does not occur.

[0048] In the present application, the term "a" or "an" or "the" can refer to "one", but also to "one or more", "at least one" and "one or more than one".

[0049] In the present application, the term "comprising", "having", "including" or "containing" can refer to inclusive or open-ended, and does not exclude additional, unrecited elements or method steps. At the same time, "comprising", "having", "including" or "containing" can also mean closed, excluding additional, unrecited elements or method steps.

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

[0051] Unless otherwise defined, other technical and scientific terms used in the present application have the same meaning as commonly understood by a person of ordinary skill in the art to which the present application belongs.

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

[0053] Lipopolysaccharide (LPS) is the main component of the outer membrane of gram-negative bacteria, which can enter the brain to activate microglia and induce neuroinflammatory responses, induce pro-inflammatory cytokine release, and cause memory deficits, etc. In the present application, the LPS-stimulated microglia is used as a model of microglial activation, and the regulatory effects of different types and concentrations of nutrients on microglia are studied to explore nutrients that are safe (non-cytotoxic) and have obvious effects on regulating brain immunity and maintaining nervous system health (as shown in Figure 1). The present application found that, by using a combination of lactoferrin and 3-fucosyllactose to intervene in LPS-stimulated microglia, the polarization of microglia to M1 phenotype can be inhibited, the polarization of microglia to M2 phenotype can be promoted, the transformation of microglia from M1 phenotype to M2 phenotype can be induced, and the amount of IL-10 released by microglia can be increased, and the amount of TNF-α, IL-1β and NO released by microglia can be reduced.

[0054] I. Nutritional composition

[0055] The nutritional composition described in the present application comprises the following essential components: lactoferrin and fucosyl lactose. In some embodiments, lactoferrin and fucosyl lactose are the main effective components in the nutritional composition described in the present application. "Main effective component" described in the present application refers to a component that exerts a specific physiological activity function, such as regulating brain immunity, in the nutritional composition. In some embodiments, the total content of lactoferrin and fucosyl lactose in the nutritional composition is 60% or more, for example, can be 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, based on the total dry weight of the nutritional composition. In some embodiments, in addition to lactoferrin and fucosyl lactose, the nutritional composition can also contain a small amount of other types of carbohydrate substances, fat substances, and / or protein substances, such as lactose, etc.

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

[0057] The present application does not make special limitations on the source of lactoferrin, for example, lactoferrin from mammals such as cows, sheep, camels, etc. can be used, and preferably bovine lactoferrin is used. The present application does not make special limitations on the method of obtaining lactoferrin, for example, lactoferrin can be isolated and extracted by protein flocculation, etc. from fresh milk or dairy products, or commercial lactoferrin products can be directly used.

[0058] Currently, the production methods of fucosyl lactose mainly include chemical synthesis, enzymatic synthesis, and biological engineering synthesis, etc. Meanwhile, fucosyl lactose can also be indirectly introduced from existing various milk components.

[0059] It should be noted that the use of fucosyl lactose should comply with the requirements of local laws and regulations. In some cases, fucosyl lactose can be directly introduced into food compositions in the form of a single raw material under the permission of laws and regulations; in other cases, fucosyl lactose can be indirectly introduced into compositions or foods by adding required milk raw materials under the permission of laws and regulations.

[0060] The present application does not particularly limit the source of fucosyllactose, which can be, for example, fucosyllactose brought in from different animal milk raw materials (e.g., cow milk, cow milk powder, etc.), fucosyllactose prepared by microbial fermentation, or a commercial fucosyllactose product. In the present application, "animal milk" refers to a liquid obtained from mammary glands of mammals during lactation. The term "animal milk" should be interpreted broadly and encompasses both raw milk (i.e., a liquid obtained directly from mammary glands) 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, the mass ratio of lactoferrin to fucosyllactose in the nutritional composition 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, 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 application contains or uses the above-mentioned nutritional composition, and thus also contains lactoferrin and fucosyllactose. By adding or using the nutritional composition, in some embodiments, the mass ratio of lactoferrin to fucosyllactose in the food is 1:(0.1-30).

[0065] The present application does not particularly limit the absolute content of lactoferrin and fucosyllactose in the food, which should meet the requirements of relevant laws and regulations. In some embodiments, the content of lactoferrin in the food is 0.001%-1.5%, preferably 0.01%-1%; and the content of fucosyllactose in the food is 0.001%-4%, preferably 0.01%-2%.

[0066] The food of the present application is not particularly limited in kind, and can include, for example, confectionery, beverages, dairy products, or baked goods. As for the confectionery, examples can include hard candy, crisp candy, gummy candy, pressed candy, and aerated candy. As for the beverages, examples can include carbonated beverages, tea-based beverages, coffee-based beverages, fruit and vegetable juice beverages, and lactic acid bacteria beverages. As for the dairy products, examples can include fermented milk, cheese, and milk powder. As for the baked goods, examples can include bread, cakes, and cookies. In addition, the food of the present application 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 fucosyl lactose contained therein, the food of the present application can contain, in some embodiments, any one or more of the following ingredients, depending on the kind of food and the final needs of the target subject: a plant product ingredient, an animal dairy product ingredient, an animal meat product ingredient, a functional additive ingredient, and any acceptable adjuvant.

[0068] As for the plant product ingredient, examples can include fruits such as figs, pomegranates, kiwis, oranges, tangerines, pineapples, strawberries, apples, bananas, grapes, pears, cherries, blueberries, blackberries, blackcurrants, cranberries, raspberries, melons, emblics, and mulberries, or extracts thereof; vegetable materials such as onions, cucumbers, tomatoes, cauliflowers, red beets, spinach, kohlrabi, Brussels sprouts, garlic, basil, Oregon grass, or extracts thereof; cereals such as rice (indica rice, japonica rice, waxy rice), wheat (wheat, barley, oat, rye), corn, sorghum, millet, foxtail millet, broomcorn millet, buckwheat, soybeans, fava beans, peas, mung beans, adzuki beans, kidney beans, or extracts thereof; nut materials such as walnuts, pistachios, cashews, hazelnuts, almonds, apricot kernels, pine nuts, peanuts, melon seeds, chestnuts, macadamia nuts, ginkgo nuts, or extracts thereof; coffee or extracts thereof; and some medicinal plants or extracts thereof.

[0069] As for the animal dairy product ingredient, examples can include fresh milk derived 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] As for the animal meat product ingredient, examples can include meat product ingredients derived from pigs, cows, sheep, aquatic animals, or birds.

[0071] As for the functional additive ingredient, examples can include vitamin supplements, mineral supplements, nucleotide supplements, dietary fibers, functional polyunsaturated fatty acid supplements, and the like.

[0072] For any acceptable adjuvant, examples can include solvents, antioxidants, antibacterial agents, thickening agents, diluents, co-solvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweetening agents, food flavors, food colors, and the like.

[0073] III. Use for modulating brain immunity

[0074] The present application surprisingly found that the combination of lactoferrin and fucosyl lactose can play a role in modulating brain immunity. Moreover, when lactoferrin and fucosyl lactose are combined in a certain ratio, there is a synergistic effect between the two, which can further enhance the efficacy of modulating brain immunity. Therefore, the nutritional composition containing lactoferrin and fucosyl lactose and the food to which the nutritional composition is added or used according to the present application have the effect of modulating brain immunity.

[0075] The "modulating brain immunity" according to the present application means that by modulating the interaction between immune cells and immune molecules and with other systems such as the neuroendocrine system, the immune activity in the brain allows the brain to be maintained at the most appropriate level in the most appropriate form. At the same time, the modulating brain immunity according to the present application is not intended for the purpose of preventing and treating diseases.

[0076] Further, the present application provides the use of the nutritional composition containing lactoferrin and fucosyl lactose in the preparation of a food for modulating brain immunity.

[0077] In some embodiments, the effect of modulating brain immunity includes modulating microglial cell polarization. In some specific embodiments, the modulating brain immunity includes at least one of inhibiting microglial cell polarization to an Ml phenotype, promoting microglial cell polarization to an M2 phenotype, and inducing microglial cell conversion from an Ml phenotype to an M2 phenotype. In some specific embodiments, the modulating brain immunity includes inhibiting microglial cell polarization to an Ml phenotype, promoting microglial cell polarization to an M2 phenotype, and inducing microglial cell conversion from an Ml phenotype to an M2 phenotype. In some specific embodiments, the modulating brain immunity includes at least one of increasing the amount of IL-10 released by microglial cells and decreasing the amount of TNF-a, IL-1β, and / or NO released by microglial cells. In some specific embodiments, the modulating brain immunity includes increasing the amount of IL-10 released by microglial cells and decreasing the amount of TNF-a, IL-1β, and / or NO released by microglial cells. In some specific embodiments, the modulating brain immunity includes increasing the amount of IL-10 released by microglial cells and decreasing the amount of TNF-a, IL-1β, and NO released by microglial cells.

[0078] IV. Use for assisting in improving central nervous system health

[0079] The present application surprisingly found that lactoferrin and fucosyllactose in combination can play a role in assisting to improve the health of central nervous system. And 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 to improve the health of central nervous system. Therefore, the nutritional composition containing lactoferrin and fucosyllactose and the food added with or using the nutritional composition also have the role of assisting to improve the health of central nervous system, and this role is not for therapeutic purposes.

[0080] Further, the present application provides the use of the nutritional composition containing lactoferrin and fucosyllactose in the preparation of food for assisting to improve the health of central nervous system.

[0081] In some embodiments, the assisting to improve the health of central nervous system includes improving neuroinflammation, which does not reach the level of disease. In some embodiments, the health of central nervous system is affected by neuroinflammation, and the food assists to improve the health of central nervous system by improving neuroinflammation.

[0082] In some embodiments, the improving neuroinflammation is achieved by affecting the TLR4 / NF-κB signaling pathway in microglial cells. In some specific embodiments, the affecting the TLR4 / NF-κB signaling pathway in microglial cells includes at least one of 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] Examples

[0084] The embodiments of the present application will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. The specific conditions not indicated in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the materials or instruments used are conventional products that can be used by purchasing.

[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 purchased from Shanghai Zymo Biological (CC-Y2022).

[0091] 1.3 Reagents

[0092] Table 1 Main reagents for experiment

[0093] 1.4 Instruments

[0094] Table 2 Main instruments and equipment for experiment

[0095] 1.5 Cell culture

[0096] Mouse microglial cell line (BV2) was cultured in DMEM high glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and placed in a temperature environment of 37°C, and cultured in a 5% CO2 incubator.

[0097] 1.6 Nutrient cytotoxicity detection

[0098] CCK-8 kit was used to detect the toxicity of nutrients to cells. The working principle of CCK-8 kit is that in the presence of electron carrier 1-methoxy-5-methyl phenazine sulfate dimethyl, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid phenyl)-2H-tetrazole monosodium salt is reduced to yellow formazan by dehydrogenase in cell mitochondria. Because only mitochondria in living cells can produce dehydrogenase, the production of formazan and the number of living cells show a positive correlation, and the OD value is detected at 450 nm by a microplate reader.

[0099] The BV2 cells were inoculated into a 96-well plate at 4x10 4 cells per well, 200 μL / well, and 3 replicate wells per group. After 12 hours of adhesion culture in the incubator, the prepared nutrient medium was added to the 96-well plate after the cells were completely adhered, and incubated for 12 hours. Then 20 μL of CCK-8 solution was added to each well (avoiding light), and incubated in the cell incubator for 0.5-1 hour. The absorbance at 450 nm was measured by a microplate reader, 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)

[0100] 1.7 Establishment of microglial cell activation model and experimental grouping

[0101] The mouse microglial cell line (BV2) was cultured in high-glucose 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, followed by 24 h of culture. The cells were then grouped. 4 x 10 4 cells / mL were inoculated into 96-well plates, 200 μL / well, and 3 replicate wells per group. The experiment was divided into 14 groups, including a blank control group, an LPS model group, and 12 experimental groups. (1) Control group: cells were cultured routinely; (2) LPS model group: BV2 cells were treated with LPS (1 μg / mL) for 24 h to establish a microglial cell activation model; (3) Experimental group: BV2 cells were stimulated with LPS for 24 h, and then each nutrient was added for 12 h.

[0102] Table 3 Experimental grouping and dosage

[0103] 1.8 Detection of NO content in BV2 cell supernatant by NO kit

[0104] The BV2 cell experimental grouping and nutrient intervention method were the same as above. After culture, the cells were centrifuged at 1000 rpm at 4°C for 5 min, and the supernatant was collected. The NO content in the cell supernatant was determined using the NO kit according to the operation steps. The principle of the NO kit for detecting 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.

[0105] 1.9 Detection of TNF-α, IL-1β, and IL-10 contents in BV2 cell culture supernatant by enzyme-linked immunosorbent assay (ELISA)

[0106] 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.

[0107] 1.10 Detection of M1 phenotype and M2 phenotype markers iNOS and CD206 of BV2 cells

[0108] The mRNA expression levels of iNOS and CD206 related genes are detected by qRT-PCR to determine the content changes of M1 cell population and M2 cell population. The 3rd passage of BV2 microglial cell suspension is inoculated in a 24-well culture plate, the cell density is 4x10 5 cells / well, and the cells are treated according to the experimental design. After the treatment, the cells are washed with PBS for 2 times, 200 μL is used per well. Trypsin digestion is performed for 2.5 min, and 400 μL of culture solution is used to terminate the digestion. The cells are blown with a pipette gun and collected into an enzyme-free centrifuge tube. The total RNA of each group of cells is extracted. Reverse transcription is performed using a reverse transcription kit according to the operation steps to obtain cDNA. The amplified cDNA is mixed with primers, fluorescent probes and qPCR Master Mix. The corresponding PCR cycle program and parameters are set according to the requirements of qPCR, and the corresponding mRNA expression level is analyzed by a real-time PCR detection system. The specific primer information is as follows:

[0109] iNOS:

[0110] Upstream: 5'-GGCTTGCCCCTGGAAGTTT-3'(SEQ ID NO. 1),

[0111] Downstream: 5'-TGCAAGTGAAATCCGATGTGG-3'(SEQ ID NO. 2);

[0112] CD206:

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

[0114] Downstream: 5'-GAATCTGACACCCAGCGGAA-3'(SEQ ID NO. 4);

[0115] GAPDH:

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

[0117] Downstream: 5'-TGGTCCAGGGTTTCTTACTCC-3' (SEQ ID NO. 6).

[0118] 1.11 Western blot detection of the relative expression of each protein in BV2 cells

[0119] The expression levels of TLR4, NF-κB, p-NF-κB, IKB-α and p-IKB-α were detected by Western blot to study the metabolic pathways of TLR4, NF-κB, p-NF-κB, IKB-α and p-IKB-α. The 3rd generation of BV2 cell suspension was inoculated in a 6-well culture plate, with a cell density of 1 x 10 5 / well, and was treated according to the experimental design. After treatment, the cells were washed with 4°C pre-cooled PBS solution for 3 times, and the cells were lysed with RIPA lysis buffer containing protease inhibitors on ice, centrifuged at 15000 r / min for 15 min at 4°C, and the supernatant was taken. The protein concentration was detected using the BCA quantitative kit, and the sample was quantitatively packaged. 5 times the concentration of SDS-PAGE protein loading buffer was added and boiled in a 100°C metal water bath for 5 min to denature the protein. Equal amounts of protein from each group were loaded for electrophoresis and membrane transfer, and the protein-free quick blocking solution (1x) was used for blocking for 1 h. The PVDF membrane was taken out and incubated with the primary antibody (1:1000) at 4°C overnight. The primary antibody was recovered and washed with TBST solution for 3 times, and the corresponding secondary antibody (dilution ratio 1:5000) was added and incubated at room temperature for 1 h. The TBST solution was used for washing for 3 times again, and the ECL developing solution (ratio 1:1) was prepared. The chemiluminescence imaging analysis system was exposed and developed, and the image was collected. The gray scale of the protein band was analyzed using Image J software, and the final result was expressed as the gray value of the target protein / GAPDH.

[0120] 1.12 Statistical analysis

[0121] All experiments were repeated three times independently, and the experimental data were statistically analyzed using SPSS22.0 statistical software. The experimental results were tested by variance homogeneity test, and the statistical differences between groups were analyzed by one-way ANOVA. The mean ± standard deviation (x ± SD) was used. P>0.05 indicates no statistical significance, and P<0.05 indicates that the difference is statistically significant.

[0122] 2. Experimental results

[0123] 2.1 Nutrient cytotoxicity detection

[0124] Table 4 shows the effect of different concentrations of nutrients on the cell viability of BV2 cells, and the results show that nutrients have no significant effect on cell viability.

[0125] Table 4 Effect of different nutrients on cell viability of BV2 cells

[0126] 2.2 Effect of nutrients on NO production of LPS-stimulated BV2 cells

[0127] To measure the effect of mixed nutrients or individual nutrient treatment on LPS-induced BV2 cells, the inventors evaluated the amount of pro-inflammatory chemical signal NO secretion to estimate the effect of modulating microglial polarization. Table 5 shows the effect of different nutrient composition samples containing LF, 3-FL on NO production of LPS-stimulated BV2 cells. The results of NO study show that, compared with the LPS model group, LF or 3-FL alone shows a role in reducing NO production. When the two are used in combination, especially when the LF:3-FL ratio is controlled at 1:0.5 to 1:25, a significant effect of reducing LPS-induced NO production can be achieved.

[0128] Specifically, as compared with experimental example 2, experimental example 4 and experimental example 10, relative to the LPS model group, the cell experiment experimental example 2, 4 respectively reduced the NO content by 0.54 and 1.39, while the cell experiment experimental example 10 reduced the NO by 4.24, which is greater than the sum of the former two (0.54+1.39), indicating that there is a synergistic effect between the two components, which can synergistically reduce the NO production of LPS-stimulated BV2 cells.

[0129] Table 5 Effect of different nutrient compositions on NO production of LPS-stimulated BV2 cells

[0130] 2.3 Effect of nutrients on pro-inflammatory factor production of LPS-stimulated BV2 cells

[0131] To measure the effect of mixed nutrients or individual nutrient treatment on LPS-induced BV2 cells, the inventors evaluated the amount of pro-inflammatory factors TNF-a and IL-1 b secretion to estimate the effect of modulating microglial polarization. Table 6 shows the effect of different nutrient composition samples containing LF, 3-FL on pro-inflammatory factor production of LPS-stimulated BV2 cells. The results of the study show that, compared with the LPS model group, LF or 3-FL alone shows no obvious effect in reducing pro-inflammatory factors. When the two are used in combination, especially when the LF:3-FL ratio is controlled at 1:0.5 to 1:25, a significant effect of reducing LPS-induced pro-inflammatory factor production can be achieved.

[0132] Specifically, comparing the experimental example 2, experimental example 4 and experimental example 10, compared with the LPS model group, the cell experiment experimental example 2, 4 respectively makes the TNF-α content reduced by-2.78 and 4.86, the IL-1β content reduced by 0.57 and 1.32, and the cell experiment experimental example 10 makes the TNF-α reduced by 47.57, which is greater than the sum of the former two(-2.78+4.86), and makes the IL-1β reduced by 3.67, which is greater than the sum of the former two(0.57+1.32), indicating that there is a synergistic effect between the two components, which can synergistically reduce the production of pro-inflammatory factors of BV2 cells stimulated by LPS.

[0133] Table 6 Effects of different nutritional compositions on the production of pro-inflammatory factors of BV2 cells stimulated by LPS

[0134] 2.4 Effects of nutritional substances on anti-inflammatory factors of BV2 cells stimulated by LPS

[0135] In order to measure the effect of mixed nutrition or single nutrition treatment on LPS-induced BV2 cells, we evaluated the secretion amount of anti-inflammatory factor IL-10 to estimate the effect of regulating microglia polarization. Table 7 shows the effects of different nutritional composition samples containing LF, 3-FL on the production of anti-inflammatory factors of BV2 cells stimulated by LPS. The research results show that compared with the LPS model group, LF or 3-FL alone can increase the production of anti-inflammatory factors. At the same time, when the two are compounded, the effect of increasing IL-10 production can be obtained.

[0136] Specifically, comparing the experimental example 2, experimental example 4 and experimental example 10, compared with the LPS model group, the cell experiment experimental example 2, 4 respectively makes the TNF-α content reduced by-2.78 and 4.86, the IL-1β content reduced by 0.57 and 1.32, and the cell experiment experimental example 10 makes the TNF-α reduced by 47.57, which is greater than the sum of the former two(-2.78+4.86), and makes the IL-1β reduced by 3.67, which is greater than the sum of the former two(0.57+1.32), indicating that there is a synergistic effect between the two components, which can synergistically reduce the production of pro-inflammatory factors of BV2 cells stimulated by LPS.

[0137] Table 7 Effects of different nutritional compositions on the production of anti-inflammatory factors of BV2 cells stimulated by LPS

[0138] 2.5 Effects of nutritional substances on M1 phenotype and M2 phenotype markers iNOS and CD206 of BV2 cells stimulated by LPS

[0139] After activation, microglia 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 chemical signal nitric oxide (NO), which may cause inflammation in 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 play a neuroprotective role. The main phenotype marker of M1 microglia is iNOS, and the main phenotype marker of M2 microglia is CD206.

[0140] 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 to determine the content changes of M1 and M2 cell populations, and to estimate the effect of regulating microglia polarization. Table 8 shows the effect of different nutritional composition samples containing LF and 3-FL on the mRNA expression levels of LPS-stimulated BV2 cell markers.

[0141] The results show that, compared with the LPS model group, LF or 3-FL alone can increase the relative expression of CD206 and reduce the relative expression of iNOS. At the same time, when the two are used in combination, the ratio of LF:3-FL is 1:0.5 to 1:10, and more obvious effects can be obtained.

[0142] Specifically, as compared with experimental example 2, experimental example 4 and experimental example 10, compared with the LPS model group, the cell experiment experimental example 2, 4 respectively makes the relative expression of iNOS decrease by 2.21 and 2.51, and the relative expression of CD206 increases by 2.54 and 2.76, while the cell experiment experimental example 10 makes the relative expression of iNOS decrease by 5.27, which is greater than the sum of the former two (2.21+2.51), and the relative expression of CD206 increases by 6.82, which is greater than the sum of the former 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 M1 phenotype.

[0143] Table 8 Effect of different nutritional compositions on mRNA expression levels of LPS-stimulated BV2 cell markers

[0144] 2.6 Effect of nutritional substances on TLR4 / NF-κB signaling pathway of LPS-stimulated BV2 cells

[0145] LPS induces activation of Toll-like receptor (TLR4), which essentially activates the NF-κB signaling pathway, and accelerates the transcription of inflammatory genes. Under normal conditions, NF-κB is in an inactive form, combined 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 the nucleus, participating in the expression of several inflammatory factors such as IL-1β and TNF-α. At the same time, these inflammatory factors also participate in the regulation of the TLR-4 / NF-κB signaling pathway, thereby forming a feedback loop and participating in the process of neuroinflammation.

[0146] In order to explore the effect of mixed nutrients or single nutrient treatment on the LPS-induced BV2 cell pathway, the whole protein of each group of cells was extracted, and Western blot was used to detect the expression of TLR4, NF-κB, p-NF-κB, IKB-α and p-IKB-α. Figures 2A-2F show the effect of LF, 3-FL and their combination on the expression of TLR4 / NF-κB signaling pathway proteins in LPS-stimulated BV2 cells.

[0147] The results showed that compared with the control group, the protein expression of TLR4, NF-κB p65 and p-NF-κB p65 (phosphorylated NF-κB p65) in the LPS model group was significantly increased, and the difference was statistically significant (P<0.0001, Figures 2B-2D); compared with the LPS model group, the expression of TLR4 in the high-dose 3-FL group had no significant difference, the expression of TLR4 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 expression of NF-κB p65 and p-NF-κB p65 in the high-dose LF group, the high-dose 3-FL group and the combination group was inhibited, and the inhibitory effect of the combination was more significant (P<0.0001, Figures 2C, 2D). The phosphorylation degree of IKB-α protein was also measured, and the results showed that compared with the control group, the expression of IKB-α protein in the LPS model group was significantly decreased, and the expression of p-IKB-α (phosphorylated IKB-α) protein was significantly increased, and the difference was statistically significant (P<0.0001, Figures 2E, 2F); compared with the LPS model group, the phosphorylation of IKB-α in the BV2 cells was inhibited in the high-dose LF group, the high-dose 3-FL group and the combination group, and the inhibitory effect of the combination was more significant (P<0.0001, Figure 2F). Therefore, the LF and 3-FL combination may alleviate the neuroinflammatory response through the TLR4 / NF-κB signaling pathway.

Claims

1. Use of a nutritional composition in the manufacture of a foodstuff for modulating brain immunity; wherein, The nutritional composition contains the following essential components: lactoferrin and fucosyl lactose, and in the nutritional composition, the mass ratio of the lactoferrin and fucosyl lactose is 1:(0.1-30).

2. Use according to claim 1, characterized in that, The fucosyl lactose includes 3-fucosyl lactose.

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

4. Use according to any one of claims 1 to 3, characterized in that, The modulating 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. Use according to any one of claims 1 to 4, characterized in that, The food is a confectionery, a beverage, a dairy product, or a baked food.

6. Use according to claim 5, characterized in that, The confectionery includes at least one of hard candy, gummy candy, crisp candy, pressed candy, and aerated candy; the beverage includes at least one of carbonated beverage, tea-based beverage, coffee-based beverage, fruit and vegetable juice beverage, and lactic acid bacteria beverage; the dairy product includes at least one of fermented milk, cheese, and milk powder; and the baked food includes at least one of bread, cake, and cookie.

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

8. Use of a nutritional composition in the manufacture of a foodstuff to assist in improving central nervous system health; wherein, The nutritional composition contains the following essential components: lactoferrin and fucosyl lactose, and in the nutritional composition, the mass ratio of the lactoferrin and fucosyl lactose is 1:(0.1-30).

9. Use according to claim 8, characterized in that, The fucosyl lactose includes 3-fucosyl lactose.

10. Use according to claim 8 or 9, characterized in that, The assisting in improving central nervous system health includes improving neuroinflammation for non-therapeutic purposes.

Images