Use of nutritional compositions for improving anxiety and memory ability

Abstract

The invention discloses application of a nutritional composition to improvement of anxiety and memory ability, and belongs to the field of nutrient substance research. According to the nutritional composition containing the lactose-N-neotetraose and the 2 '-fucosyllactose provided by the invention, through the synergistic effect of the lactose-N-neotetraose and the 2'-fucosyllactose, the mood disorder and the reduction of memory and recognition ability can be improved, and the mood disorder and the reduction of memory and recognition ability are caused by maternal immune abnormal activation. Furthermore, the composition can improve the activity of neurons by inhibiting the transition activation of brain microglial cells, and help is provided for improving mood disorder and memory and recognition ability decline. A new thought is provided for development of products for improving offspring emotional disorder and memory and recognition ability decline caused by maternal immune activation.

Description

TECHNICAL FIELD

[0001] The present application relates to the application of nutritional compositions in helping to improve anxiety and memory capacity, and belongs to the field of nutritional substances. BACKGROUND

[0002] Social anxiety (SA) is a common emotional experience of people, which is characterized by the negative emotions caused by the fear of negative evaluation of others in social situations. When the degree of social anxiety is more serious and leads to functional impairment, it becomes social anxiety disorder.

[0003] The cognitive theory of social anxiety believes that social anxiety is due to the cognitive bias of social anxiety, which deploys attention to the surrounding threatening information, strengthens the individual's negative self-perception, and later the individual needs to concentrate on the current goal, and needs to suppress the threatening stimuli of interference, which gradually develops into avoidance of threatening stimuli. As an executive function, this inhibitory control ability also plays an important role in prospective memory processing. Prospective memory refers to the ability to remember to perform planned intentions or behaviors at a future time or situation. Studies have found that in natural situations, patients with social anxiety have significantly lower performance in event-based prospective memory tasks than the control group, and there is a defect in event-based prospective memory ability.

[0004] In recent years, studies have shown that there is a close relationship between the gut microbiota and the central nervous system (i.e. the "gut-brain axis"), and prebiotics, as a substance that can promote the growth of beneficial bacteria, have a significant effect on regulating the intestinal flora and improving metabolic function. Human milk oligosaccharides, as a prebiotic, play an important role in the establishment of intestinal microecology, the strengthening of the immune system, the promotion of brain and cognitive development, and other related aspects. For example, reference document 1 discloses a human milk oligosaccharide composition containing 2'-FL and LNnT and other human milk oligosaccharides, and further discloses that the human milk oligosaccharide composition as a prebiotic can reduce the risk of intestinal inflammation, protect the intestinal barrier, and have a potential anti-colon cancer effect. Reference document 2 discloses a human milk oligosaccharide composition containing 2'-FL and LNnT, which can specifically increase the abundance of Akkermansia muciniphila in the intestine, and is used for preventing or treating various metabolic, inflammatory and nervous system diseases.

[0005] And there are also documents speculate that the mechanism of HMOs to promote brain and neural development mainly includes three aspects: 1) gut-brain axis; 2) effects in the gut that are independent of the microbiome; 3) support brain development as a direct or indirect source of sialic acid. However, there is no clear research that breast milk oligosaccharides can directly improve social anxiety and memory decline caused by maternal immune activation.

[0006] CITATIONS:

[0007] CITATION 1: CN110650635A

[0008] CITATION 2: CN111683665A SUMMARY

[0009] Problems to be solved by the invention

[0010] The adverse effects of maternal immune activation on offspring can be traced back to the fetal development stage. This early-occurring damage often shows stronger persistence and severity compared to acquired damage after birth. There is no clear improvement or intervention program for the current offspring anxiety, memory and recognition ability decline caused by maternal immune activation. In the process of exploration, it is accidentally found that the breast milk oligosaccharides containing specific types have obvious improvement effect on the offspring memory and recognition ability decline caused by maternal immune activation. Therefore, on this basis, the present application provides a new use of a nutritional composition for improving the offspring anxiety, memory and recognition ability decline caused by maternal immune activation.

[0011] The nutritional composition in the present application can be provided to infants and young children as edible products, thereby giving infants and young children the brain and nervous system of the golden period of 1000 days of early life, which can help to improve abnormal brain development as early as possible, especially the offspring brain development abnormalities caused by maternal immune activation.

[0012] It should be noted that the anxiety, memory and recognition ability decline and brain development abnormalities described in the present application refer to the non-disease state of the offspring health insufficiency caused by maternal immune activation.

[0013] Solutions to the problems

[0014] [1]. Use of a nutritional composition in the preparation of a product for improving mood disorders, memory and recognition ability decline, wherein the mood disorders, memory and recognition ability decline are caused by maternal immune abnormal activation;

[0015] The nutritional composition comprises the following essential active ingredients: lacto-N-neotetraose and 2'-fucosyllactose.

[0016] [2]. The use according to [1], wherein the mass ratio of the lacto-N-neotetraose and 2'-fucosyllactose in the nutritional composition is 1 : (1-10), preferably 1 : (1-6), more preferably 1 : (3-6).

[0017] [3]. The use according to [1] or [2], wherein the mood disorder comprises anxiety.

[0018] The improvement of the mood disorder comprises improvement of anxiety behavior.

[0019] [4]. The use according to any one of [1] to [3], wherein the improvement of the mood disorder, the decline of memory and recognition ability comprises inhibition of activation and proliferation of microglia and / or modulation of neuronal activity.

[0020] [5]. The use according to [4], wherein the modulation of neuronal activity comprises increasing the expression of BDNF in the hippocampus of the brain, preferably increasing the expression of BDNF in the DG region and the CA3 region of the hippocampus of the brain, and / or

[0021] increasing the expression of Snap 25 gene in the cortex of the brain.

[0022] [6]. Use of a nutritional composition for the manufacture of a product for improving abnormal brain development, wherein the abnormal brain development comprises over-activation of microglia, decreased expression of BDNF and / or decreased expression of Snap 25 gene, which is caused by abnormal maternal immune activation.

[0023] The nutritional composition comprises the essential active ingredients: lacto-N-neotetraose and 2'-fucosyllactose.

[0024] [7]. The use according to [6], wherein the mass ratio of the lacto-N-neotetraose and 2'-fucosyllactose in the nutritional composition is 1 : (1-10), preferably 1 : (1-6), more preferably 1 : (3-6), more preferably 1 : (3.4-6).

[0025] [8]. The use according to [6] or [7], wherein the over-activation of microglia comprises increased expression of IBA-1 in the hippocampus of the brain, in particular increased expression of IBA-1 in the DG region and the CA3 region of the hippocampus of the brain.

[0026] The decreased expression of BDNF comprises decreased expression of BDNF in the hippocampus of the brain, in particular decreased expression of BDNF in the DG region and the CA3 region of the hippocampus of the brain.

[0027] The Snap 25 gene expression amount reduction includes a cerebral cortex Snap 25 gene expression amount reduction.

[0028] [9]. The use according to any one of [1] to [8], wherein the product comprises a food product, which is a beverage, a dairy product, a bakery product, or a confectionery.

[0029]

[10] . The use according to any one of [1] to [8], wherein the product is an oral preparation; the oral preparation comprises at least one form of a tablet, a pill, a granule, a powder, a capsule, a beverage, a jelly, a gummy, and an oral liquid.

[0030] Effects of the invention

[0031] The nutritional composition provided by the present application contains 2'-fucosyllactose and lacto-N-neotetraose, and the improvement of the anxiety, memory and recognition ability caused by maternal immune activation can be significantly amplified by the synergistic effect of 2'-fucosyllactose and lacto-N-neotetraose, especially lacto-N-neotetraose and 2'-fucosyllactose. The improvement is mainly reflected in the improvement of the total movement distance, the central area movement distance and time, the number of times of entering the center, the recognition time of new objects, the recognition time of old objects, the recognition index, and the total recognition time. At the same time, the present application finds that the composition can also inhibit the excessive activation of brain microglia cells and improve the activity of neurons, thereby providing help for further improving the anxiety, memory and recognition ability. DETAILED DESCRIPTION

[0032] Various exemplary embodiments, features, and aspects of the present application will be described in detail below. The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any implementation described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other implementations.

[0033] In addition, in order to better illustrate the present application, numerous specific details are given in the following detailed description. Those skilled in the art will understand that the present application can be implemented without certain specific details. In some other examples, methods, means, apparatus and steps that are well known to those skilled in the art are not described in detail in order to highlight the main idea of the present application.

[0034] Unless otherwise specified, the units used in the specification are international standard units, and the numerical values and numerical ranges appearing in the present application should be understood to include the systematic errors that are inevitable in industrial production.

[0035] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0036] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0037] In this specification, "optional" and "optionally" mean that the events or circumstances described below may or may not occur, and the description includes both cases where the events or circumstances occur and cases where the events or circumstances do not occur.

[0038] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0039] As used herein, the term “and / or” covers all combinations of items connected by the term and should be regarded as if each combination had been listed separately herein. For example, “A and / or B” covers “A,” “A and B,” and “B.” For example, “A, B, and / or C” covers “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.”

[0040] This invention is mainly based on the following insights:

[0041] Human milk oligosaccharides (HMOs) have been shown to improve inflammation and neurological disorders, but these conditions are acquired due to lifestyle habits and environmental factors. The adverse effects of maternal immune activation on offspring can be traced back to the fetal development stage. This early damage occurring in the womb often exhibits greater persistence and severity compared to acquired damage. Currently, there are no reports on anxiety, memory, and cognitive decline in offspring caused by maternal immune activation, and it is not readily known whether or how these effects can be improved. This invention established a maternal immune homeostasis dysregulation model in mice. Using offspring mice born to this model as research subjects, we unexpectedly discovered that HMOs can improve anxiety, memory, and cognitive decline in offspring caused by maternal immune activation.

[0042] I. Nutritional Composition

[0043] The nutritional composition provided by this invention is a human milk oligosaccharide composition. Human milk oligosaccharides are a class of structurally complex non-digestible sugars, mainly composed of 3 to 10 monosaccharides (such as glucose, galactose, N-acetylglucosamine, fucose, and sialic acid). In some specific embodiments of this invention, the human milk oligosaccharide is one or two of 2'-fucosylated lactose and lactose-N-neotetrasaccharide.

[0044] In some specific embodiments, the nutritional composition comprises the following essential active ingredients: lactose-N-neotetrasaccharide and 2'-fucosylated lactose.

[0045] The 2'-fucosyllactose (2'-FL) described in this invention is a neutral trisaccharide composed of L-fucose, D-galactose, and D-glucose units, wherein the monosaccharide L-fucose is linked to the disaccharide D-lactose via an α (1→2) bond. Its molecular formula is C2. 18 H 32 O 15 Its molecular weight is 488.439 g / mol.

[0046] The lactose-N-neotetrasaccharide (LNnT) described in this invention is a tetrasaccharide composed of β-D-galactose-(1→4)-β-D-glucose NAc-(1→3)-β-D-galactose-(1→4)-D-glucose units linked by glycosidic bonds. Its molecular formula is C2. 26 H 45 NO 21 Its molecular weight is 707.63 g / mol.

[0047] This invention does not particularly limit the source of the 2'-fucosyllactose and lactose-N-neotetrasaccharide, which can be, for example, from natural sources, synthetic sources, or microbial fermentation sources. Typically, 2'-fucosyllactose can be synthesized through steps such as glycosylation reaction between lactose acceptor and fucose donor; alternatively, it can be synthesized using exogenously added lactose as a substrate and 5'-guanine diphosphate nucleoside-fucose disodium salt formed through the microbial metabolic pathway as a precursor, under the action of fucosyltransferase. For lactose-N-neotetrasaccharide, it can be synthesized stepwise from lactose through a protecting group and a deprotection reaction via chemical reaction; it can also be synthesized from lactose and UDP-N-acetylglucosamine as substrates via β-1,3-N-acetylglucosamine transferase (LgtA); or it can be synthesized from LNT and UDP-galactose as substrates via β-1,4-galactosyltransferase (GalT).

[0048] In some embodiments, the mass content of 2'-fucosylated lactose and lactose-N-neotetraose may be 60% or more, preferably 80% or more, more preferably 90% or more, or any other content, relative to the total mass of the products from each source of 2'-fucosylated lactose and lactose-N-neotetraose.

[0049] In some embodiments, the nutritional composition comprises an active ingredient (a component that performs a specific physiological function, such as a component that improves mood disorders, memory, and cognitive impairment) and an inactive ingredient (a component that does not perform a specific physiological function, such as a substance that does not improve mood disorders, memory, and cognitive impairment). Exemplarily, the inactive ingredient may be other nutrients, any food-acceptable excipient, and / or substances that are generated during the production or acquisition of the active ingredient and cannot be effectively separated from the active ingredient or do not require separation. In some embodiments, the nutritional composition consists of the active ingredient and the inactive ingredient.

[0050] In some alternative embodiments, the mass ratio of 2'-fucosylated lactose to lactose-N-neotetrasaccharide in the human milk oligosaccharide composition is 1:(1~10), preferably 1:(1~6), more preferably 1:(3~6), even more preferably 1:(3.4~6), for example, it can be 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1: 2.0, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3.0, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4.0, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1 1:4.7, 1:4.8, 1:4.9, 1:5.0, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6.0, 1:6.1, 1:6.2, 1:6.3, 1:6.4, 1:6.5, 1:6.6, 1:6.7, 1:6.8, 1:6.9, 1:7.0, 1:7.1, 1:7.2, 1:7.3 1:7.4, 1:7.5, 1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8, 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:9.0, 1:9.1, 1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9, 1:10.0, etc.

[0051] II. Products

[0052] The product of this invention contains or uses the aforementioned human milk oligosaccharides, especially 2'-fucosylated lactose and lactose-N-neotetrasaccharide. The product of this invention can be a food product.

[0053] This invention does not particularly limit the specific form of the food. At room temperature, the edible nutrient can be solid, semi-solid, or liquid. Exemplarily, it may include a drinkable composition, a powder or granule composition, a gel, or a frozen or partially frozen composition. The food may optionally be part of a capsule filler, or may optionally be part of a beverage, dairy product, non-dairy cream, sauce, or baked goods.

[0054] In some specific implementations, the food may be powdered reconstituted food (solid beverages, instant coffee, cereal powder, nut powder or lotus root powder, etc.), baked goods (bread, cake or biscuit baked goods, etc.), beverages (carbonated beverages, fruit and vegetable juice beverages, functional beverages, tea beverages, milk beverages or alcoholic beverages, etc.), candy (gel candy, hard candy, compressed candy, etc.), milk and dairy products (fresh milk derived from raw cow (sheep) milk, milk powder, whey powder, fermented milk, cheese or condensed milk, etc.), etc.

[0055] In other specific embodiments, the food described in this invention can also be a health food, such as various types of oral preparations, including but not limited to tablets, pills, granules, powders, capsules, beverages, jellies, gummies, and oral liquids.

[0056] This invention does not specifically limit the absolute content of 2'-fucosyllactose and lactose-N-neotetrasaccharide in food, as long as it meets the requirements of local food-related laws and regulations. In some embodiments, the mass content of 2'-fucosyllactose and lactose-N-neotetrasaccharide relative to the total mass of the food is at least 0.05%, preferably at least 0.1%, more preferably at least 1%, and even more preferably at most 12%.

[0057] In addition to the 2'-fucosylated lactose and lactose-N-neotetrasaccharide mentioned above, other ingredients may be included, such as proteins / amino acids, carbohydrates, fats, vitamins, minerals, and other components commonly found in foods. Furthermore, depending on the type of food and the end-user's needs, in some embodiments, the food described in this invention may also contain any one or more of the following ingredients: plant-based ingredients, animal-based ingredients, animal meat product ingredients, functional additives, and any acceptable excipients.

[0058] Examples of plant-based ingredients include fruits such as fig, pomegranate, kiwi, orange, tangerine, pineapple, strawberry, apple, banana, grape, pear, cherry, blueberry, blackberry, blackcurrant, cranberry, raspberry, melon, amla, and bilberry, or their extracts; vegetables such as onion, cucumber, tomato, cauliflower, carrot, spinach, kale, Brussels sprouts, garlic, basil, and oregano, or their extracts; and rice (indica rice). Grains or their extracts, including japonica rice, glutinous rice, cereals (wheat, barley, oats, rye), corn, sorghum, millet, sorghum, yellow millet, buckwheat, soybeans, broad beans, peas, mung beans, red beans, kidney beans, etc.; nuts or their extracts, including walnuts, pistachios, cashews, hazelnuts, almonds, apricot kernels, pine nuts, peanuts, sunflower seeds, chestnuts, macadamia nuts, ginkgo nuts, etc.; coffee or its extracts; and some medicinal and edible plant-based Chinese medicinal materials or their extracts.

[0059] Animal product ingredients include animal dairy products, animal meat products, and animal by-products. Examples of animal dairy products include fresh milk from mammals such as cows, sheep, and camels, as well as processed dairy products such as whole milk powder, skim milk powder, whey protein concentrate, demineralized whey powder, whey protein powder, and hydrolyzed whey protein powder. Examples of animal meat products include meat products from pigs, cows, sheep, aquatic animals, or poultry. Examples of animal by-products include (collagen) proteins and corresponding hydrolyzed protein peptides extracted from pigs, cows, sheep, horses, donkeys, aquatic animals, or poultry.

[0060] Examples of functional additives include vitamin supplements, mineral supplements, nucleotide supplements, dietary fiber, and functional polyunsaturated fatty acid supplements.

[0061] Any acceptable excipients may include solvents, antioxidants, antibacterial agents, thickeners, diluents, cosolvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, food flavorings, and food colorings.

[0062] III. Uses to improve mood disorders, memory, and cognitive decline.

[0063] This invention discovers that combining lactose-N-neotetrasaccharide and 2'-fucosylated lactose allows them to mutually enhance each other's beneficial effects. Specifically, lactose-N-neotetrasaccharide amplifies the benefits of 2'-fucosylated lactose for improving mood disorders, memory, and cognitive impairment in offspring caused by maternal immune abnormality activation, while 2'-fucosylated lactose also amplifies the benefits of lactose-N-neotetrasaccharide for these same conditions. This ensures that the composition containing both ingredients significantly improves mood disorders, memory, and cognitive impairment caused by maternal immune abnormality activation.

[0064] In some implementations, the mood disorder includes anxiety; the improvement of the mood disorder includes improvement of anxious behaviors, particularly socially anxious behaviors.

[0065] In some implementations, the improvement in social anxiety behavior includes: increasing total distance traveled, increasing central area distance and time traveled, and / or increasing the number of visits to the center.

[0066] In some specific implementations, the improvement of social anxiety behavior includes: increasing the total distance traveled.

[0067] In some specific implementations, the improvement of social anxiety behavior includes: increasing the distance and time spent moving around the central area.

[0068] In some specific implementations, the improvement of social anxiety behavior includes increasing the frequency of visits to the center.

[0069] In some specific implementation schemes, the improvement of social anxiety behavior includes: increasing total distance traveled and increasing distance and time spent traveling in the central area.

[0070] In some specific implementations, the improvement of social anxiety behavior includes: increasing central area movement distance and time, and increasing the frequency of visits to the center.

[0071] In some specific implementation plans, the improvement of social anxiety behavior includes: increasing the total distance traveled and increasing the number of visits to the center.

[0072] In some specific implementation schemes, the improvement of social anxiety behavior includes: increasing total distance traveled, increasing distance and time spent in the central area, and increasing the number of visits to the center.

[0073] In some implementations, the improvement in memory and recognition ability includes increasing the recognition time for new objects, reducing the recognition time for old objects, reducing the recognition index, and / or increasing the total recognition time.

[0074] In some specific implementations, the improvement in memory and recognition ability includes increasing the time to recognize new objects.

[0075] In some specific implementations, the improvement in memory and recognition ability includes reducing the recognition time for old objects.

[0076] In some specific implementations, the improvement in memory and recognition ability includes reducing the recognition index.

[0077] In some specific implementations, the improvement in memory and recognition ability includes increasing total recognition time.

[0078] In some specific implementations, the improvement in memory and recognition ability includes increasing the recognition time for new objects and decreasing the recognition time for old objects.

[0079] In some specific implementations, the improvement in memory and recognition ability includes increasing the recognition time for new objects and reducing the recognition index.

[0080] In some specific implementations, the improvement in memory and recognition ability includes increasing the time to recognize new objects and increasing the total recognition time.

[0081] In some specific implementations, the improvement in memory and recognition ability includes reducing the recognition time for old objects and lowering the recognition index.

[0082] In some specific implementations, the improvement in memory and recognition ability includes reducing the recognition time for old objects and increasing the total recognition time.

[0083] In some specific implementations, the improvement in memory and recognition ability includes reducing the recognition index and increasing the total recognition time.

[0084] In some specific implementations, the improvement in memory and recognition ability includes increasing the recognition time for new objects, reducing the recognition time for old objects, and reducing the recognition index.

[0085] In some specific implementations, the improvement in memory and recognition ability includes increasing the recognition time for new objects, reducing the recognition time for old objects, and increasing the total recognition time.

[0086] In some specific implementations, the improvement in memory and recognition ability includes increasing the recognition time for new objects, reducing the recognition index, and increasing the total recognition time.

[0087] In some specific implementations, the improvement in memory and recognition ability includes reducing the recognition time for old objects, reducing the recognition index, and increasing the total recognition time.

[0088] In some specific implementations, the improvement in memory and recognition ability includes increasing the recognition time for new objects, reducing the recognition time for old objects, reducing the recognition index, and increasing the total recognition time.

[0089] In some specific implementations, the improvement of mood disorders, memory and recognition impairment includes: inhibiting the overactivation and proliferation of microglia and / or regulating neuronal activity.

[0090] In some specific implementations, the improvement of mood disorders, memory and recognition impairment includes: inhibiting the overactivation and proliferation of microglia.

[0091] In some specific implementations, the improvement of mood disorders, memory and recognition decline includes: regulating neuronal activity.

[0092] In some specific implementations, the improvement of mood disorders, memory and recognition impairment includes: inhibiting the overactivation and proliferation of microglia and regulating neuronal activity.

[0093] In some implementations, microglia overactivation is characterized by a significant increase in microglia expression in the brain, particularly in the hippocampus, compared to a healthy human brain. Inhibition of microglia overactivation and proliferation is manifested as a decrease in IBA-1 expression in the DG and CA3 regions of the hippocampus.

[0094] In this invention, the "hippocampus" is an important component of the limbic system of the brain, located in the medial temporal lobe, and named for its resemblance to a seahorse. It plays a central role in memory formation (especially episodic and spatial memory), learning, emotion regulation, and spatial navigation. The hippocampus comprises: (1) the hippocampus; (2) the dentate gyrus (DG); (3) the subiculum; (4) the entorhinal cortex; and (5) the hippocampal accessory structures surrounding the corpus callosum. The hippocampus is often divided into four regions: CA1, CA2, CA3, and CA4.

[0095] In addition, the regulation of neuronal activity includes increasing the expression of BDNF in the DG and CA3 regions of the hippocampus and / or increasing the expression of the Snap 25 gene in the cerebral cortex.

[0096] In some specific implementations, the regulation of neuronal activity includes increasing BDNF expression in the DG and CA3 regions of the hippocampus.

[0097] In some specific implementations, the regulation of neuronal activity includes increasing the expression of the Snap 25 gene in the cerebral cortex.

[0098] In some specific implementations, the regulation of neuronal activity includes increasing the expression of BDNF in the DG and CA3 regions of the hippocampus and increasing the expression of the Snap 25 gene in the cerebral cortex.

[0099] Therefore, the nutritional composition containing lactose-N-neotetrasaccharide and 2'-fucosylated lactose provided by this invention helps improve mood disorders, memory and cognitive impairment caused by maternal immune activation. This invention's improvement of mood disorders, memory and cognitive impairment is not intended to prevent or treat disease. Furthermore, the mood disorders, memory and cognitive impairment described in this invention do not reach a level that can be considered a disease.

[0100] IV. Uses to improve abnormal brain development

[0101] The present invention unexpectedly discovered that when the human milk oligosaccharide composition is applied to offspring mice with mood disorders, memory and recognition impairment caused by maternal immune activation, it can significantly improve, for example, the overactivation and proliferation of microglia in the cerebral cortex of the offspring mice, the reduction in BDNF expression in the cerebral cortex, and the reduction in Snap 25 gene expression. Furthermore, when lactose-N-neotetrasaccharide and 2'-fucosylated lactose in the human milk oligosaccharide composition are combined in a mass ratio of 1:(1~10), preferably 1:(1~6), more preferably 1:(3~6), and even more preferably 1:(3.4~6), they have a synergistic effect. Lactose-N-neotetrasaccharide can amplify the effect of 2'-fucosylated lactose on improving the abnormal brain development in offspring caused by abnormal maternal immune activation. The abnormal brain development includes microglia overactivation, decreased BDNF expression, and decreased Snap 25 gene expression. 2'-fucosylated lactose can also amplify the effect of lactose-N-neotetrasaccharide on improving the abnormal brain development in offspring caused by abnormal maternal immune activation. Thus, the mutual amplification effect between the two helps to further improve the effect of improving abnormal brain development.

[0102] In some implementations, microglia overactivation is a state in which microglia in the brain, especially in the cerebral cortex, undergo morphological changes and oxidative stress compared to a healthy state.

[0103] Therefore, the present invention provides the use of a human milk oligosaccharide composition comprising lactose-N-neotetrasaccharide and 2'-fucosylated lactose in the preparation of a product for improving abnormal brain development, said microglia overactivation, decreased BDNF expression and / or decreased Snap 25 gene expression.

[0104] In some specific embodiments, the present invention provides the use of a human milk oligosaccharide composition comprising lactose-N-neotetrasaccharide and 2'-fucosylated lactose in the preparation of a product for improving microglia overactivation in the hippocampus of the brain.

[0105] In some specific embodiments, the present invention provides the use of a human milk oligosaccharide composition comprising lactose-N-neotetrasaccharide and 2'-fucosylated lactose in the preparation of a product for improving reduced BDNF expression in the DG and CA3 regions of the hippocampus.

[0106] In some specific embodiments, the present invention provides the use of a human milk oligosaccharide composition comprising lactose-N-neotetrasaccharide and 2'-fucosylated lactose in the preparation of a product for improving reduced expression of the Snap 25 gene in the cerebral cortex.

[0107] In some specific embodiments, the present invention provides the use of a human milk oligosaccharide composition comprising lactose-N-neotetrasaccharide and 2'-fucosylated lactose in the preparation of a product for improving microglia overactivation in the hippocampus and reducing BDNF expression in the DG and CA3 regions of the hippocampus.

[0108] In some specific embodiments, the present invention provides the use of a human milk oligosaccharide composition comprising lactose-N-neotetrasaccharide and 2'-fucosylated lactose in the preparation of a product for improving microglia overactivation in the hippocampus and reducing expression of the Snap25 gene in the cerebral cortex.

[0109] In some specific embodiments, the present invention provides the use of a human milk oligosaccharide composition comprising lactose-N-neotetrasaccharide and 2'-fucosylated lactose in the preparation of a product for improving reduced BDNF expression in the DG and CA3 regions of the hippocampus and reduced Snap 25 gene expression in the cerebral cortex.

[0110] In some specific embodiments, the present invention provides the use of a human milk oligosaccharide composition comprising lactose-N-neotetrasaccharide and 2'-fucosylated lactose in the preparation of a product for improving microglia overactivation in the hippocampus, reduced BDNF expression in the DG and CA3 regions of the hippocampus, and reduced Snap 25 gene expression in the cerebral cortex.

[0111] In some more specific embodiments, the hyperactivation of microglia in the hippocampus includes increased expression of IBA-1 in the DG and CA3 regions of the hippocampus.

[0112] In some implementations, the improvement of brain developmental abnormalities is not intended to prevent or treat diseases; similarly, the improvement of microglia overactivation, the improvement of BDNF expression reduction, and the improvement of Snap 25 gene expression reduction described in this invention are also not intended to prevent or treat diseases. Furthermore, the brain developmental abnormalities, microglia overactivation, BDNF expression reduction, and Snap 25 gene expression reduction described in this invention do not reach a level that can be identified as a disease.

[0113] Example

[0114] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0115] Embodiment one, the model construction and human milk oligosaccharide intervention experiment involved in the present application

[0116] 1. Construction of a model of maternal immune homeostasis disorder

[0117] 150 female mice and 30 male mice were selected, primarily for offspring breeding. When grouping them together, healthy mice with shiny fur were chosen, and a female-to-male ratio of 2:1 was maintained. The weight of the female mice was measured regularly over the following week. Once the female mice showed significant weight gain, they were separated and injected intraperitoneally with 20 mg / kg poly(I:C) solution to establish a maternal immune homeostasis dysregulation model.

[0118] 2. Offspring Intervention

[0119] In this experiment, male offspring rats were used as experimental subjects. Three weeks after the female offspring gave birth, the male offspring were weaned and fed separately. When the male offspring were four weeks old, they were administered either PBS solution or HMOs solutions of different proportions via gavage for six weeks. One week before the end of the gavage period, when the offspring were nine weeks old, animal behavior tests were conducted to analyze the effect of the HMOs combination on the social behavior of offspring activated by maternal immunity.

[0120] 3. Intervention grouping of offspring animals

[0121] The experiment consisted of 11 groups, with 12 mice in each group: a control group, a model group, and 9 HMO intervention groups. "PBS mice" referred to control mice born after their mothers were injected with PBS solution during pregnancy, while "MIA mice" referred to offspring born after their mothers were injected with Poly(I:C) solution during pregnancy. During the experiment, each mouse was housed in a standard cage under a 12-hour light / dark cycle, with humidity of 50 ± 15% and temperature of 22 ± 2℃. Mice had free access to food and water.

[0122] 4. Monitoring of basic growth indicators

[0123] During the 6-week intervention, the mice's body weight, food intake, and water intake were recorded weekly.

[0124] 5. Experimental intervention design

[0125] In this invention, two human milk oligosaccharides, 2'-FL and LNnT, were selected and combined at different single doses or in different ratios to investigate the effect of different dosage combinations on repetitive stereotyped behaviors. The experimental design is shown in Table 1. According to the literature, the feeding dose of LNnT was fixed at 250 mg / kg body weight / day, while the dose of 2'-FL was selected at three gradients: 250 mg / kg body weight / day, 500 mg / kg body weight / day, 850 mg / kg body weight / day, and 1500 mg / kg body weight / day. These five single-factor intervention doses of the two substances served as comparative examples. Four different ratios of LNnT:2'-FL (1:1, 1:2, 1:3.4, and 1:6) were then used as four examples.

[0126] Table 1: Experimental Intervention Design

[0127]

[0128] Example 2: Evaluation of Basal Growth Indicators in Mice

[0129] Table 2 shows the weight changes of mice in the control group, model group, and 9 intervention groups over 6 weeks of intervention. As can be seen from the table, the weight of mice in each group was not significantly different on day 0 of the intervention, with the model group having the lowest average weight of 11.85g. However, after significance analysis, no significant difference was found in the baseline average weight of mice in the control group, model group, 5 comparative groups, and 4 example groups (p > 0.05). With the extension of the intervention time, the average weight of mice in each group continuously increased. Weight monitoring and recording were conducted on days 7, 14, 21, 28, 35, and 42. The final analysis showed no significant difference in the average weight of mice in the 11 groups at each time point (p > 0.05). Furthermore, the weight gain of mice in each group after 42 days of intervention was approximately 9g. However, after significance analysis, it was found that the intervention of different HMOs (monomeric or monomeric) groups did not affect the normal weight gain of mice in all 11 groups, including the model group and different HMOs, and there was no significant difference between groups (p > 0.05).

[0130] Table 2: Weight changes (g) of 9 groups of mice during 6 weeks of intervention.

[0131]

[0132] Meanwhile, the food intake and water consumption of the 11 groups of mice were recorded during the intervention period, and the results are shown in Table 3. The table shows that the food intake of the 11 groups of mice during the entire intervention period varied by approximately 3 g / day. However, after significance analysis, it was found that, except for Comparative Examples 1, 4, and 5, where the average daily food intake was significantly higher than that of the control group (p < 0.05), the food intake of the other groups was not significantly different from that of the control group (p > 0.05). Water consumption was also recorded and compared among the groups, and the table shows that there was no significant difference in water consumption among the 11 groups (p > 0.05).

[0133] Table 3: Comparison of average daily food intake and water consumption of mice in each group during the intervention period

[0134]

[0135] Example 3: Evaluation of Anxiety Behavior

[0136] Analysis of basal growth indicators revealed that the basal growth and development of mice in the model group and the various intervention groups of HMOs were not affected, and there were no significant differences in food and water intake.

[0137] With no differences in baseline growth indicators and development, the inventors first evaluated the effects of modeling and HMO intervention on anxiety behavior in mice.

[0138] The open field test (OFT) was used to assess anxiety behavior. The OFT is a commonly used behavioral test primarily used to evaluate the exploratory behavior, anxiety levels, and motor activity of laboratory animals (such as mice or rats) in new environments. Recording the mouse's activity in an open space reflects its anxiety level, while the total distance traveled reflects its overall motor activity level. The principle is based on the animal's natural aversion to open spaces, exhibiting an approach-avoidance behavior; on the other hand, it also shows curiosity and explores new environments. A square open area was selected, surrounded by walls to prevent escape. The area was divided into a central area and peripheral areas, with the central area considered more likely to elicit an anxiety response. During the experiment, the mouse was placed individually in the center of a box (50 cm × 50 cm × 40 cm), with the central area measuring 25 cm × 25 cm. Before the experiment, the mouse was gently placed in the box for 5 minutes to acclimatize. During the experiment, the mouse was gently placed back in the center of the box while data was collected, and video recording was conducted for 5 minutes. Throughout the process, the experimenter remained quiet and away from the test mouse. After the experiment, the time the mice spent in the central and peripheral areas, and the distance they traveled were analyzed. The enclosure was cleaned with 75% alcohol after each mouse was replaced.

[0139] The activity level of mice was assessed by the total movement distance; the anxiety level of animals was assessed by the time spent in the central area and the number of times they entered the central area, with animals with high anxiety levels tending to move in the peripheral areas.

[0140] The experimental results are shown in Table 4. The table shows that the overall motor activity level of the mice decreased after modeling, but after significant difference analysis, there was no significant difference between the model group and the control group (p > 0.05), indicating that maternal immune activation had little impact on the overall motor activity level of the offspring. After intervention with LNnT or 2'-FL monomers and different doses, the total locomotor distance increased to varying degrees. However, after One Way Anova significant difference analysis, except for Example 4, where the total locomotor distance was significantly higher than the model group (p < 0.05), Comparative Example 1 (p < 0.05), and Comparative Example 2 (p < 0.05), there were no significant differences among the other groups (p > 0.05). These results indicate that modeling did not have a significant negative impact on the overall motor activity level of the mice.

[0141] Furthermore, the inventors compared the anxiety behaviors of the different groups of mice. Since mice tend to avoid the central area once they become anxious, spending more time near the perimeter of the square enclosure, the anxiety level was reflected by the distance the mice traveled in the central area, the time spent there, and the number of times they entered the center. The results are shown in Tables 4 and 4-1. The tables show that after modeling, the distance the mice traveled in the center significantly decreased to an average of 6.19 meters (p < 0.0001), indicating that modeling induced a certain degree of anxiety in the mice, preventing them from spending more time in the center. However, intervention with LNnT and 2'-FL monomers or different ratios significantly increased the time spent in the center compared to the model group (p < 0.0001 or p < 0.001), indicating that intervention with human milk oligosaccharides improved the anxiety behavior of the mice. Furthermore, the inventors discovered that after intervention with the two monomers (Comparative Examples 1-5), the distance traveled in the central region by mice was between 7 and 9 meters. However, when the two oligosaccharides were compounded in four different ratios (1:1, 1:2, 1:3.4, and 1:6) (Examples 1-4), the distance traveled in the central region by mice was between 10 and 14 meters. Difference analysis revealed that the distance traveled in the central region of the four examples was significantly higher than that of the five comparative examples (p < 0.0001). Further comparison of the four examples showed that Example 4 was significantly higher than Examples 1, 2, and 3 (p < 0.0001 or p < 0.001), while there was no significant difference among Examples 1-3 (p > 0.05).

[0142] Subsequently, the inventors analyzed the activity time of mice in the center. Tables 4 and 4-2 show that the average activity time in the center for untreated mice was approximately 34.43 s, while after modeling, the average activity time in the center decreased significantly to approximately 14.36 s, with a significant difference between the two groups (p < 0.0001). Intervention with different doses of LNnT and 2'-FL monomers significantly increased the activity time in the center, averaging approximately 23-26 s, showing a significant difference from the model group (p < 0.001). Furthermore, intervention with mice using a mixture of the two oligosaccharides LNnT and 2'-FL at ratios of 1:1, 1:2, 1:3.4, and 1:6 significantly increased the activity time in the center to between 33-37 s, showing a highly significant difference from the model group (p < 0.0001). Further comparison revealed that the central activity time of the four compounded examples was significantly higher than that of comparative examples 1-5 (p<0.0001), and the improvement in central activity time of examples 1-4 compared to the model group was greater than the sum of the improvement values ​​of the corresponding comparative examples, indicating that the two nutrients have a synergistic effect.

[0143] The above two results indicate that maternal immune activation in offspring induces anxiety behavior, significantly reducing the distance and time spent moving in the central region, resulting in severe approach-avoidance behavior. This anxiety behavior was improved after intervention with two types of human milk oligosaccharides, and the distance and time spent moving in the central region after combining the two oligosaccharides were significantly better than those after single-saccharide intervention.

[0144] Next, the number of times mice entered the center was analyzed, as this number reflects their social avoidance behavior under anxiety. The results are shown in Tables 4 and 4-3. The tables show that mice without any treatment entered the center an average of 30 times during the experiment, while the number of entries significantly decreased to an average of 17.2 times after modeling (p < 0.0001). However, intervention with LNnT and 2'-FL, either as monomers or in combination, significantly increased the number of entries (p < 0.0001, p < 0.001, or p < 0.05). Furthermore, the number of entries after combining the two oligosaccharides (Examples 1-4) was higher than that after oligosaccharide monomer intervention (Comparative Examples 1-5), and the difference was highly significant (p < 0.0001) after significance analysis. In the four examples, Example 4 had the highest number of entries, significantly higher than Example 1 (p < 0.0001), Example 2 (p < 0.01), and Example 3 (p < 0.0001). This indicates that the combination of the two oligosaccharides can effectively and significantly improve social avoidance under anxious conditions.

[0145] Table 4: Anxiety Behavior of Mice in Each Group

[0146]

[0147] Table 4-1: Analysis of significant differences in central distance among groups of mice

[0148]

[0149] Table 4-2: Analysis of significant differences in central activity time among groups of mice

[0150]

[0151] Table 4-3: Significant differences in the number of times each group of mice entered the center.

[0152]

[0153] Example 4: Evaluation of Memory and Recognition Abilities

[0154] This invention employs a novel object recognition experiment to assess memory and recognition abilities. Memory ability is evaluated using the novel object recognition experiment, a commonly used behavioral experiment in laboratories to assess the memory and recognition abilities of laboratory animals. This experiment is based on animals' innate tendency to explore new objects, assessing their memory ability by comparing the exploration time for new and old objects. Specifically, the total exploration time is used to assess the memory ability of each group of mice, while the recognition index (the ratio of the mouse's exploration time for the new object to the total exploration time) is used to assess the mouse's recognition ability. The novel object recognition experiment is conducted over three days.

[0155] (1) Adapting to the weather

[0156] On the first day, remove the mice from their cages and place them in an empty rectangular box (40 cm × 40 cm × 40 cm) for free exploration for 5 minutes. After exploration, temporarily place the mice in an empty cage. Repeat this process until all mice in that cage have completed the acclimatization training. Thoroughly clean the rectangular box with 75% ethanol each time.

[0157] (2) Training days

[0158] The next day, two identical objects (i.e., the northeast corner and the southwest corner) were placed inside the box. The test mice were removed from their cages and placed in the center of the box, equidistant from the two identical objects, allowing them to explore freely for 10 minutes. At the end of the experiment, the mice were temporarily placed in an empty cage until all mice in that cage had completed the adaptation training. The box and objects were thoroughly cleaned with 75% ethanol each time.

[0159] (3) Test day

[0160] On the third day, place one object used on the second day (i.e., the old object) and one new object in the same relative positions as on the second day. Remove the mouse from the cage and place it in the center of the box, equidistant from the old and new objects. Allow the mouse to explore freely for 5 minutes. Experimenters unfamiliar with the grouping process should record the time taken for the mouse to explore the new / old objects. Record the entire exploration process using a camera and store the data in a computer. Exploration is defined as the mouse pointing its nose at an object from a distance of 2 cm or less (i.e., touching or sniffing the object). Climbing onto an object without sniffing or chewing it does not qualify as exploration.

[0161] The total time a mouse spends exploring old and new objects is used to determine its memory ability. The new object recognition index is an indicator of memory ability, calculated as the time spent exploring new objects minus the time spent exploring old objects, divided by the total exploration time.

[0162] The experimental results are shown in Tables 5 and 5-1. The tables show that after maternal modeling, the total time for offspring to recognize both new and old objects was significantly reduced (p < 0.0001), and the recognition times for new and old objects were not significantly different. This indicates that the memory and recognition abilities of the offspring mice were impaired to some extent. However, after intervention with the two oligosaccharides, the recognition time for new objects significantly increased, while the recognition time for old objects remained relatively consistent. The corresponding total recognition time also improved to varying degrees. Furthermore, a significant difference analysis of the total recognition time revealed that Comparative Examples 1-5 and Examples 1-4 all showed significantly higher total recognition times than the model group (p < 0.0001 or p < 0.001). At the same time, the total recognition time of Examples 1-4 was significantly higher than that of Comparative Examples 1-5 (p < 0.0001 or p < 0.001 or p < 0.05), and the improvement in recognition ability of Examples 1-4 compared to the model group was greater than the sum of the improvement values ​​of the corresponding comparative examples. This indicates that the combination of two types of LNnT and 2'-FL in ratios of 1:1, 1:2, 1:3.4, and 1:4 can significantly improve the memory impairment in offspring caused by maternal immune activation, and that the two have a synergistic effect. Further comparison of the four examples revealed that the total recognition time in Example 4 was significantly higher than that in Example 1 (p < 0.01), Example 3 (p < 0.0001), and Example 4 (p < 0.01).

[0163] Subsequently, the inventors used the recognition index of the offspring mice for new and old objects to evaluate the mice's recognition ability. The results are shown in Table 5 and Table 5-2 (p < 0.001 or p < 0.01), and Examples 1-4 were significantly higher than Comparative Examples 1-5 (p < 0.0001). This indicates that the combination of LNnT and 2'-FL in ratios of 1:1, 1:2, 1:3.4, and 1:4 significantly improved the recognition ability of the offspring mice compared to single-component intervention. In contrast, the recognition index of Example 4 was 0.48, which was higher than that of Examples 1, 2, and 3.

[0164] Table 5: Evaluation of memory and recognition abilities of mice in each group

[0165]

[0166] Table 5-1: Significant differences in total time for recognizing new and old objects among different groups of mice

[0167]

[0168] Table 5-2: Significant differences in the recognition index of new and old objects among different groups of mice

[0169]

[0170] The above experiments used a mouse model to simulate social anxiety disorder and memory decline in offspring of human maternal immune activation. The experiments showed that after modeling, mice with normal basic growth and development and normal food and water intake exhibited significant social anxiety and memory decline. However, after intervention for 42 days with two human milk oligosaccharides, LNnT and 2'-FL, in different ratios (1:1, 1:2, 1:3.4, 1:6), their social behavior was improved.

[0171] Example 5: Research on Behavior Improvement Mechanisms

[0172] The behavioral experiments described above demonstrate that the combination of two human milk oligosaccharides, 2'-FL and LNnT, significantly improves anxiety behavior and memory in offspring mice with maternal immune activation. Previous studies have also shown that maternal inflammation during pregnancy may harm fetal neurodevelopment and increase the incidence of neurological diseases. Fetal microglia are particularly sensitive to maternally derived inflammatory signals, and they may undergo phenotypic changes in the maternal immune activation (MIA) model, further leading to misconnections in neuronal circuits and resulting in abnormal behavioral manifestations. Based on this theory, this invention examines the changes in brain neural tissue at the cellular and gene levels behind offspring-related behavioral manifestations caused by maternal immune homeostasis dysregulation, and the effects of human milk oligosaccharide intervention on these indicators, thereby clarifying the underlying mechanisms of these behavioral improvements.

[0173] First, the inventors used immunofluorescence to analyze the effects of maternal immune activation on microglia and neurons in the brain tissue of offspring. Ionized calcium-binding adapter molecule 1 (IBA-1) is a calcium-binding protein commonly used as a marker for microglia. This invention analyzed the expression levels of IBA-1 and BDNF in the DG and CA3 regions of the hippocampus of offspring mice in the control, model, and intervention groups to examine the activation of microglia and neuronal activity in the brain tissue of each group of mice.

[0174] The specific experimental steps are as follows:

[0175] 1. Brain tissue collection

[0176] Mice were fasted for 12 hours before sacrifice. They were anesthetized with an intraperitoneal injection of 1.25% tribromoethanol solution at a dose of 0.2 mL / 10 g bw, and euthanized by cervical dislocation. Brain tissue samples were then collected by dissection on ice, wrapped in aluminum foil, rapidly frozen in liquid nitrogen, and transferred to a -80°C freezer. Tissue samples for pathological sections were placed in 4% (v / v) paraformaldehyde solution for subsequent experiments.

[0177] 2. Immunofluorescence staining to measure IBA-1 and BDNF levels in the hippocampus of the brain.

[0178] (1) Preparation of paraffin sections

[0179] The hemibrain was fixed in 4% (v / v) paraformaldehyde / PBS fixative for 24 h. After dehydration, it was embedded in paraffin and cut into 5 μm thin slices using a microtome. The slices were then flattened on a 42℃ water surface, retrieved using APES-coated slides, inserted into a slide holder, and dried in a 37℃ oven.

[0180] (2) Immunofluorescence staining

[0181] After drying overnight in a 37°C oven, the tissue sections were dewaxed and rehydrated using xylene I for 10 min, xylene II for 10 min, 100% ethanol I for 5 min, 100% ethanol II for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, and 70% ethanol for 5 min. They were then washed three times with PBS for 5 min each time. The permeabilization buffer was poured into a beaker, the tissue sections were placed inside, and permeabilized at room temperature for 15 min. They were then washed three times with PBS for 5 min each time. Antigen retrieval was performed using the boiling method: the retrieval buffer was poured into a container, and the sections were boiled in a microwave oven on medium heat for 8 min, or in an induction cooker for 10 min. They were then washed three times with PBS for 5 min each time. The tissue was dried with absorbent filter paper, and one drop of 3% H2O2 deionized water was added to the tissue. The tissue was then placed in a humidified chamber and incubated at room temperature for 10 min to block endogenous peroxidase. Finally, the tissue was washed three times with PBS for 5 min each time. Add one drop of 10% goat serum to the tissue, place it in a humidified chamber, and incubate at room temperature for 60 min. After incubation, discard the goat serum, add the corresponding primary antibody (rabbit anti-IBA-1 antibody for IBA-1; mouse anti-BDNF antibody for BDNF), and incubate overnight at 4°C.

[0182] Tissue sections were removed and allowed to warm to room temperature for 30 min. They were then washed 10 times with PBS, 3 min each time. The tissue was dried with absorbent filter paper, and secondary antibody solutions corresponding to the primary antibody species were added (IBA-1 secondary antibody: anti-rabbit IgG - Alexa Fluor 488 green fluorescence; BDNF secondary antibody: anti-mouse IgG - Alexa Fluor 594 red fluorescence). The sections were placed in a humidified chamber and incubated at room temperature for 2 h. Afterward, the sections were washed 10 times with PBS, 3 min each time. The tissue was dried, mounted with a DAPI-containing anti-fluorescence attenuation mounting medium, and observed and photographed under a fluorescence microscope (Olympus, Tokyo, Japan). Fluorescence microscope channel selection: Alexa Fluor 488: excitation 488 nm, emission 525 nm (green); Alexa Fluor 594: excitation 594 nm, emission 617 nm (red); DAPI: excitation 358 nm, emission 461 nm (blue).

[0183] result:

[0184] 1. As shown in Tables 6 and 6-1, it can be seen from the tables that after the mothers in the model group underwent immune homeostasis activation during pregnancy, the microglia in the hippocampus of the offspring mice were significantly activated, and the fluorescence intensity of the cell marker IBA-1 increased significantly (p < 0.0001). However, after 42 days of intervention with breast milk oligosaccharides, the offspring showed a decreasing trend in IBA-1 fluorescence intensity. Furthermore, One-Way Anova differential analysis revealed that the expression levels of IBA-1 in the DG region of the hippocampus of mice in all nine groups (Comparative Examples 1-5 and Examples 1-4) were significantly lower than those in the model group (p < 0.0001), and the expression levels of IBA-1 in the CA3 region of the hippocampus were also significantly lower than those in the model group (p < 0.0001). This indicates that intervention with breast milk oligosaccharides can significantly inhibit excessive activation of microglia. Furthermore, the inventors discovered that the fluorescence intensity of IBA-1 in the two regions of the hippocampus of the brain was lower than that of LNnT and 2'-FL monotherapy after being combined in ratios of 1:1, 1:2, 1:3.4, and 1:6. After significant difference analysis, it was found that the fluorescence intensity of IBA-1 in the DG region of the hippocampus in Examples 1-4 was significantly lower than that in Comparative Examples 1-5 (p < 0.0001 or p < 0.001 or p < 0.01 or p < 0.05); the fluorescence intensity of IBA-1 in the CA3 region of the hippocampus in Examples 1-4 was significantly lower than that in Comparative Examples 1-5 (p < 0.0001 or p < 0.001 or p < 0.01 or p < 0.05), and the reduction in fluorescence intensity of IBA-1 in the CA3 region of the hippocampus in Examples 1-4 compared to the model group was greater than the sum of the reduction values ​​of the corresponding comparative examples. This indicates that LNnT and 2'-FL, when combined in ratios of 1:1, 1:2, 1:3.4, and 1:6, exhibit a synergistic effect, demonstrating better efficacy in inhibiting the activation of microglia in the cerebral cortex. Comparison of the four examples revealed that the composition of Example 4 showed the lowest fluorescence intensity for IBA-1 in the CA3 region, approximately 0.06, but after significance analysis, no significant difference was found among the four groups (p > 0.05). However, for IBA-1 in the DG region, the fluorescence intensity in Example 4 was significantly lower than that in Example 2 (p < 0.01), but there was no significant difference compared to Examples 1 and 3 (p > 0.05).

[0185] Table 6: Comparative Analysis of Quantitative Expression of IBA-1 in Brain Tissue of Different Groups of Mice

[0186]

[0187] Table 6-1: Analysis of statistical significance of differences in IBA-1 quantitative expression in the brain tissue of offspring mice (adjusted p-values)

[0188]

[0189] 2. In this invention, it was found that the activity of offspring neurons was affected after intraperitoneal injection of poly(I:C) solution into the maternal immune homeostasis model during pregnancy. The fluorescence intensity of the neurotrophic factor BDNF in the DG and CA3 regions of the hippocampus was significantly reduced (p < 0.0001) (as shown in Tables 7 and 7-1). After intervention with two oligosaccharides, it was found that the fluorescence intensity in the DG region of the hippocampus in Comparative Examples 1-5 and Examples 1-4 was significantly higher than that in the model group (p < 0.001 or p < 0.01 or p < 0.05), and the fluorescence intensity in the CA3 region of the hippocampus in Comparative Examples 1-5 and Examples 1-4 was significantly higher than that in the model group (p < 0.001 or p < 0.01 or p < 0.05). This indicates that intervention with the two oligosaccharides can improve the reduced activity of offspring neurons caused by pregnancy stress. Further comparison revealed that the BDNF production after combining LNnT and 2'-FL was significantly higher than that of monotherapy in both regions. After significance analysis, it was found that in the DG region, the BDNF fluorescence intensity in Examples 1-4 was significantly higher than that in Comparative Examples 1-5 (p < 0.001, p < 0.01, p < 0.05). In the CA3 region, the BDNF fluorescence intensity in Examples 1-4 was significantly higher than that in Comparative Examples 1-5 (p < 0.001, p < 0.01, p < 0.05). This indicates that the combination of LNnT and 2'-FL has a synergistic effect, and the beneficial effect on neuronal activity after combined intervention is significantly higher than that of monotherapy. Further analysis of the four examples revealed that Example 4 showed the highest BDNF fluorescence intensity in both regions. However, after significant difference analysis, Example 4 was significantly higher than Example 1 in the CA3 region (p < 0.001), while there was no significant difference compared to Examples 2 and 3. In the DG region, Example 4 was significantly higher than Example 1 (p < 0.001), Example 2 (p < 0.01), and Example 3 (p < 0.05).

[0190] Table 7: BDNF fluorescence intensity in the hippocampus of each group of mice

[0191]

[0192] Table 7-1: Differential p-values ​​of quantitative BDNF expression in the hippocampus of rats in each group

[0193]

[0194] Second, the inventors further analyzed the expression of specific alleles related to anxiety behavior and memory ability in offspring mice with maternal immune activation at the genetic level. For example, the allele Snap25 (synaptosome-associated protein 25) is a gene that plays a key role in neuronal synapses. Its encoded protein is a core component of the SNARE complex and participates in neurotransmitter release. Its basic functions in the human brain include: 1) Snap 25 protein mediates the fusion of synaptic vesicles with the cell membrane and regulates neurotransmitter release by forming the SNARE complex with proteins such as Syntaxin and VAMP; 2) In terms of neuroplasticity, it plays an important role in the development of learning, memory, and social behavior by influencing synapse formation and signal transduction between neurons. The results showed that the expression level of the allele Snap 25 varied in different groups of mice.

[0195] This invention used PCR technology to study the expression level of the Snap 25 gene in the cerebral cortex of mice in each group, specifically:

[0196] Quantitative real-time polymerase chain reaction (qRT-PCR) technology is mainly used to analyze changes in the gene transcription levels of specific molecules in the cortex and colon. The specific operation is as follows:

[0197] (1) Tissue RNA extraction

[0198] TRIzol was used to extract mRNA from the tissue, following the instructions. A certain amount of mouse cerebral cortex tissue was taken, added to grinding beads, and a tissue homogenate was prepared. Then, 1 mL of TRIzol was added, and the homogenate was lysed. Subsequently, the RNA was obtained by layering (chloroform extraction), precipitation (isopropanol), and washing (75% ethanol) according to the instructions. The RNA was then dissolved in DEPC water and stored at -80°C.

[0199] (2) Reverse transcription (cDNA synthesis)

[0200] The extracted mRNA was reverse transcribed using the PrimeScript™ RTMaster Mix Reverse Transcription Kit (TaKaRa PrimeScript RTMasterMix, Dalian), following the instructions in the product manual.

[0201] (3) Indicator Measurement

[0202] According to the instructions for the Takara Premix Ex Taq™ II RR036A kit, the PCR amplification system was prepared as follows: 1 μL upstream primer, 1 μL downstream primer, 6 μL ddH2O, 10 μL Premix Ex Taq II, and 2 μL cDNA template. Primers for each detection index are shown in Table 8.

[0203] The standard two-step PCR amplification procedure is as follows: 95°C for 30 s, one cycle; 95°C for 3 s, 60°C for 30 s, 40 cycles; then proceed to the melting curve stage. Finally, GAPDH mRNA is used as an internal control, based on 2... -△△Ct The relative expression level of genes is calculated.

[0204] Table 8: Gene Primer Sequences

[0205]

[0206] The results are shown in Tables 9 and 9-1. Tables 9 and 9-1 show that the mRNA expression level of the Snap 25 allele in the cerebral cortex of mice in the model group was significantly reduced after maternal immune activation (p < 0.0001). However, after timely intervention with two human milk oligosaccharides, LNnT and 2'-FL, in early life, the mRNA expression level of Snap 25 was enhanced, possibly through epigenetic regulation (such as DNA methylation or histone modification). Specifically, the difference analysis between Comparative Examples 1-5 and Examples 1-4 and the model group shows that the mRNA expression level of Snap 25 in the intervention group was significantly higher than that in the model group (p < 0.0001). Further comparison revealed that when LNnT and 2'-FL were combined in ratios of 1:2, 1:3.4, and 1:6, the mRNA expression level of the Snap 25 gene was significantly higher than that in Comparative Examples 1-5 (p < 0.0001 or p < 0.001 or p < 0.05). Further comparison of the four examples revealed that the expression level of Snap 25 gene mRNA was the highest in Example 4.

[0207] Table 9: Expression levels of the Snap 25 mRNA in the cerebral cortex of mice in each group

[0208]

[0209] Table 9-1: Significant differences in the expression levels of the Snap 25 mRNA in the cerebral cortex of mice from different groups

[0210]

[0211] Through the above analysis, the inventors discovered that maternal immune activation leads to excessive activation of microglia, the immune cells in the offspring's brain, while simultaneously affecting neuronal activity, thus damaging the entire neural network structure and biological environment of the brain. Furthermore, at the gene level, the mRNA expression level of the Snap25 allele, which is related to synapse formation, neural signal transduction, and learning and memory abilities, was significantly reduced. This indicates that maternal immune activation may have a serious impact on the formation of neural networks in offspring's brains. However, when offspring were immediately treated with a combination of human milk oligosaccharides (LNnT and 2'-FL in different ratios of 1:1, 1:2, 1:3.4, and 1:4) after birth, the activation level of microglia in the offspring's brain was significantly reduced, neuronal activity was improved, and the mRNA expression level of the synapse formation-related Snap25 allele was significantly increased, resulting in a significant improvement in the offspring's anxiety behavior and memory recognition abilities.

Claims

1. The use of a nutritional composition in the preparation of products that improve mood disorders, memory, and cognitive impairment, characterized in that, The aforementioned mood disorders, memory loss, and cognitive impairment are caused by abnormal activation of the maternal immune system; The nutritional composition contains the following essential active ingredients: lactose-N-neotetrasaccharide and 2'-fucosylated lactose.

2. The use according to claim 1, characterized in that, In the nutritional composition, the mass ratio of lactose-N-neotetrasaccharide and 2'-fucosylated lactose is 1:(1~10).

3. The use according to claim 1 or 2, characterized in that, The mood disorders mentioned include anxiety; The improvement of mood disorders includes the improvement of anxiety behavior.

4. The use according to any one of claims 1 to 3, characterized in that, The improvement of mood disorders, memory and recognition impairment includes: inhibiting the activation and proliferation of microglia and / or regulating neuronal activity.

5. The use according to claim 4, characterized in that, The regulation of neuronal activity includes increasing the expression level of BDNF in the hippocampus and / or increasing the expression level of the Snap 25 gene in the cerebral cortex.

6. The use of the nutritional composition in the preparation of products that improve abnormal brain development, characterized in that, The brain developmental abnormalities include microglia overactivation, decreased BDNF expression, and / or decreased Snap 25 gene expression, which are caused by abnormal activation of the maternal immune system. The nutritional composition contains the following essential active ingredients: lactose-N-neotetrasaccharide and 2'-fucosylated lactose.

7. The use according to claim 6, characterized in that, In the nutritional composition, the mass ratio of lactose-N-neotetrasaccharide and 2'-fucosylated lactose is 1:(1~10).

8. The use according to claim 6 or 7, characterized in that, The microglia overactivation includes increased IBA-1 expression in the hippocampus of the brain; and / or The decrease in BDNF expression includes a decrease in BDNF expression in the hippocampus of the brain; and / or The reduced expression of the Snap 25 gene includes a reduced expression of the Snap 25 gene in the cerebral cortex.

9. The use according to any one of claims 1 to 8, characterized in that, The products include food, which may be beverages, milk and dairy products, baked goods, or confectionery.

10. The use according to any one of claims 1 to 8, characterized in that, The product is an oral preparation; the oral preparation includes at least one of the following forms: tablets, pills, granules, powders, capsules, beverages, jellies, gummies, and oral liquids.

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