Use of nutritional compositions containing breast milk oligosaccharides to help improve repeated platelet behavior

Abstract

The invention discloses application of a nutritional composition containing breast milk oligosaccharide to improvement of repeated engraving behaviors, and belongs to the field of nutrient substance research. The present invention provides a composition comprising lactose-N-neotetraose and 2 '-fucosyllactose, both of which have a synergistic effect and can improve a repeated engraving behavior, which is caused by maternal immune abnormal activation. Furthermore, the composition can inhibit the transition activation of brain microglial cells and improve the activity of neurons, so that the brain neural network damage is improved, and help is provided for improving repeated engraving behaviors. And a new thought is provided for the development of products for improving repeated engraving behaviors caused by maternal immune activation.

Description

Technical Field

[0001] This invention relates to the use of a nutritional composition containing human milk oligosaccharides to help improve repetitive and stereotyped behaviors, and belongs to the field of nutrient research. Background Technology

[0002] Repetitive and stereotyped behaviors refer to a series of frequent, purposeless, and meaningless behaviors exhibited by children or adults. Currently, treatment methods for repetitive and stereotyped behaviors mainly include behavioral interventions and pharmacological therapy. However, these methods have limitations in efficacy and significant side effects, especially for children. Therefore, finding safe and effective interventions is crucial. How to effectively improve repetitive and stereotyped behaviors in children has become a common concern in the academic community.

[0003] Recent studies have shown a close connection between the gut microbiota and the central nervous system (i.e., the "gut-brain axis"), and prebiotics, as substances that promote the growth of beneficial bacteria, play a significant role in regulating the gut microbiota and improving metabolic function. Human milk oligosaccharides (HMOs), as a prebiotic, possess various biological activities, including immunomodulation, gut microbiota regulation, and promotion of neural development. For example, cited reference 1 discloses a HMO composition containing 2'-FL and LNnT, which can be used to prevent or treat various metabolic, inflammatory, and neurological diseases by specifically increasing the abundance of Akkermansia muciniphila in the gut. Cited reference 2 discloses a HMO composition containing 2'-FL, LNnT, and other HMOs, and further discloses that the HMO composition, as a prebiotic, can reduce the risk of intestinal inflammation, protect the intestinal barrier, and has potential anti-colon cancer effects.

[0004] However, there are currently no clear studies or methods to improve repetitive and stereotyped behaviors in offspring caused by maternal immune activation.

[0005] References:

[0006] Reference 1: CN111683665A

[0007] Reference 2: CN110650635A Summary of the Invention

[0008] The problem the invention aims to solve

[0009] Maternal immune activation has a particularly profound impact on offspring. Because this effect begins in the fetal stage, the damage occurs earlier and often has more lasting and severe consequences compared to other external injuries suffered after birth. Currently, there are no clear solutions to address repetitive and stereotyped behaviors in offspring caused by maternal immune activation. During the research process, this invention unexpectedly discovered that compositions containing specific types of human milk oligosaccharides have a significant ameliorative effect on repetitive and stereotyped behaviors caused by maternal immune activation. Based on this, this invention provides a new use for compositions that improve repetitive and stereotyped behaviors.

[0010] The composition of this invention can be provided to infants and young children as an edible product, thereby providing them with early-stage brain development and a healthy nervous system during their golden period of development. It can help improve abnormal brain development as early as possible, especially repetitive stereotyped behaviors and damage to the brain's neural networks caused by maternal immune activation.

[0011] It should be noted that the repetitive stereotyped behaviors and brain neural network damage mentioned in this invention refer to non-disease states in offspring that are not fully healthy due to maternal immune activation.

[0012] Solution for solving the problem

[0013] [1]. Use of the composition in the preparation of a product that improves repetitive stereotyped behaviors, wherein the repetitive stereotyped behaviors are caused by damage to the brain's neural network; the damage to the brain's neural network is caused by abnormal activation of the maternal immune system;

[0014] The composition contains the following essential active ingredients: lactose-N-neotetrasaccharide and 2'-fucosylated lactose.

[0015] [2]. According to the use described in [1], wherein, in the composition, the mass ratio of lactose-N-neotetrasaccharide and 2'-fucosylated lactose is 1:(1~10), preferably 1:(2~6), more preferably 1:(2~3.5), and even more preferably 1:(2~3.4).

[0016] [3]. According to the use described in [1] or [2], wherein the improvement of repetitive stereotyped behavior includes: inhibiting the overactivation of microglia in the hippocampus, especially in the CA1 region of the hippocampus, and / or regulating neuronal activity.

[0017] [4]. According to the use described in [3], wherein the regulation of neuronal activity includes increasing the expression level of BDNF in the hippocampus and / or increasing the expression level of the PTEN gene in the cerebral cortex.

[0018] [5]. Use of the composition in the preparation of a product that improves brain neural network damage, wherein the brain neural network damage includes microglia overactivation, decreased BDNF expression and / or decreased PTEN gene expression, wherein the brain neural network damage is caused by abnormal activation of the maternal immune system;

[0019] The composition contains the following essential active ingredients: lactose-N-neotetrasaccharide and 2'-fucosylated lactose.

[0020] [6]. According to the use described in [5], wherein in the composition, the mass ratio of lactose-N-neotetrasaccharide and 2'-fucosylated lactose is 1:(1~10), preferably 1:(1~6), and more preferably 1:(2~3.5).

[0021] [7]. According to the use described in [5] or [6], wherein the microglia hyperactivation includes an increase in IBA-1 expression in the hippocampus, and further, the microglia hyperactivation includes an increase in IBA-1 expression in the CA1 region of the hippocampus; and / or

[0022] The decreased BDNF expression includes decreased BDNF expression in the hippocampus, particularly in the CA1 region of the hippocampus; and / or

[0023] The decreased PTEN gene expression level includes a decrease in PTEN gene expression level in the cerebral cortex.

[0024] [8]. The use according to any one of [1] to [7], wherein the product includes food, and the food is a beverage, milk and dairy products, baked goods or confectionery.

[0025] [9]. The use according to any one of [1] to [7], wherein the product is an oral preparation; the oral preparation includes at least one form of tablets, pills, granules, powders, capsules, beverages, jellies, gummies and oral liquids.

[0026]

[10] . According to any one of [1] to [9], wherein the product contains at least 0.05% by mass of lactose-N-neotetrasaccharide and 2'-fucosylated lactose.

[0027] The effects of the invention

[0028] The present invention provides a composition comprising lactose-N-neotetrasaccharide and 2'-fucosylated lactose. The lactose-N-neotetrasaccharide and 2'-fucosylated lactose in this composition have a synergistic effect. In this composition, lactose-N-neotetrasaccharide and 2'-fucosylated lactose can significantly amplify each other's effect on improving repetitive stereotyped behaviors induced by maternal immune activation. Especially when combined in a specific ratio, they can achieve a better synergistic effect, thereby improving repetitive stereotyped behaviors, primarily by reducing the frequency and duration of these behaviors. Furthermore, the present invention has found that the composition can improve damage to the brain's neural networks by inhibiting the overactivation of microglia and improving neuronal activity, thus further contributing to the improvement of repetitive stereotyped behaviors. Attached Figure Description

[0029] Figure 1 The study presents a comparative analysis of quantitative expression of IBA-1 in the CA1 region of the hippocampus.

[0030] Figure 2 The quantitative comparative analysis of BDNF in the CA1 region of the hippocampus is shown.

[0031] Figure 3 The expression levels of the PTEN mRNA in the cerebral cortex are shown. Detailed Implementation

[0032] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0033] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0034] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable 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 play a role in regulating immunity and gut microbiota, as well as improving neurological disorders. However, these symptoms or diseases are mostly caused by environmental influences, and the impact of maternal immune activation on offspring is particularly profound. Because this effect begins in the fetal stage, the damage occurs earlier and often has more lasting and severe consequences compared to other external injuries suffered after birth. Currently, there are no reports on repetitive stereotyped behaviors in offspring caused by maternal immune activation, and it is not easy to know whether or how these behaviors can be improved. This invention uses a mouse model of maternal immune homeostasis dysregulation and, by using offspring mice born from this model as research subjects, unexpectedly discovered that HMOs can improve repetitive stereotyped behaviors in offspring caused by maternal immune activation.

[0042] I. Composition

[0043] The 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] 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.

[0045] 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.

[0046] 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).

[0047] 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.

[0048] In some embodiments, the composition comprises an active ingredient (a component that performs a specific physiological function, such as a component that improves repetitive stereotyped behaviors) and an inactive ingredient (a component that does not perform a specific physiological function, such as a substance that does not improve repetitive stereotyped behaviors). 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 composition consists of the active ingredient and the inactive ingredient.

[0049] In some optional embodiments, the mass ratio of 2'-fucosylated lactose to lactose-N-neotetrasaccharide in the human milk oligosaccharide composition is 1:(1~10), preferably 1:(2~6), more preferably 1:(2~3.5), and even more preferably 1:(2~3.4). 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, etc. 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 1:4.7, 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.

[0050] II. Products

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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%.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] Examples of functional additives include vitamin supplements, mineral supplements, nucleotide supplements, dietary fiber, and functional polyunsaturated fatty acid supplements.

[0060] 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.

[0061] III. Uses to improve repetitive and stereotyped behaviors

[0062] 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 effect of 2'-fucosylated lactose on improving repetitive stereotyped behaviors in offspring caused by maternal immune abnormal activation, and 2'-fucosylated lactose also amplifies the effect of lactose-N-neotetrasaccharide on improving repetitive stereotyped behaviors in offspring caused by maternal immune abnormal activation. This ensures that the composition containing both ingredients has a significant effect on improving repetitive stereotyped behaviors caused by maternal immune abnormal activation.

[0063] In some implementations, the improvement of repetitive stereotyped behavior includes reducing the frequency and duration of repetitive stereotyped behavior.

[0064] In some implementations, the improvement of repetitive stereotyped behaviors includes: inhibiting excessive activation of microglia in the offspring brain and / or regulating neuronal activity.

[0065] In some specific implementations, the improvement of repetitive stereotyped behaviors includes: inhibiting the overactivation of microglia in the offspring brain.

[0066] In some specific implementations, the improvement of repetitive and stereotyped behaviors includes: modulating the activity of neurons in the offspring brain.

[0067] In some specific implementations, the improvement of repetitive stereotyped behaviors includes: inhibiting the overactivation of microglia in the offspring brain and regulating the activity of neurons in the offspring brain.

[0068] Furthermore, the inhibition of microglia overactivation manifests as a reduction in the expression of IBA-1 in the CA1 region of the hippocampus.

[0069] 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.

[0070] In some implementations, microglia overactivation is a state in which microglia in the brain, especially in the hippocampus, undergo morphological changes and oxidative stress compared to a healthy state.

[0071] In addition, the regulation of neuronal activity includes increasing the expression level of BDNF in the CA1 region of the hippocampus and / or increasing the expression level of the PTEN gene in the cerebral cortex.

[0072] Therefore, in some specific implementations, the improvement of repetitive stereotyped behaviors includes: reducing the expression level of IBA-1 in the CA1 region of the hippocampus.

[0073] In some specific implementations, the improvement of repetitive stereotyped behaviors includes: increasing BDNF expression in the CA1 region of the hippocampus.

[0074] In some specific implementations, the improvement of repetitive and stereotyped behaviors includes increasing the expression level of the PTEN gene in the cerebral cortex.

[0075] In some specific implementations, the improvement of repetitive stereotyped behaviors includes: reducing the expression level of IBA-1 in the CA1 region of the hippocampus and increasing the expression level of BDNF in the CA1 region of the hippocampus.

[0076] In some specific implementations, the improvement of repetitive and stereotyped behaviors includes: reducing the expression level of IBA-1 in the CA1 region of the hippocampus and increasing the expression level of the PTEN gene in the cerebral cortex.

[0077] In some specific implementations, the improvement of repetitive and stereotyped behaviors includes: increasing the expression level of BDNF in the CA1 region of the hippocampus and increasing the expression level of the PTEN gene in the cerebral cortex.

[0078] In some specific implementations, the improvement of repetitive stereotyped behaviors includes: reducing the expression level of IBA-1 in the CA1 region of the hippocampus, increasing the expression level of BDNF in the CA1 region of the hippocampus, and increasing the expression level of the PTEN gene in the cerebral cortex.

[0079] Therefore, the composition containing lactose-N-neotetrasaccharide and 2'-fucosylated lactose provided by this invention, or the food containing said composition, helps to improve repetitive stereotyped behaviors in offspring caused by abnormal maternal immune activation. This invention does not aim to prevent or treat diseases by improving repetitive stereotyped behaviors. Furthermore, the repetitive stereotyped behaviors described in this invention do not reach a level that can be considered a disease.

[0080] IV. Uses to improve brain neural network damage

[0081] The present invention unexpectedly discovered that when the human milk oligosaccharide composition is applied to offspring with brain neural network damage caused by maternal immune activation, it can significantly improve the excessive activation of microglia in the offspring's cerebral cortex caused by brain neural network damage, such as the hippocampus, especially the CA1 region of the hippocampus, the decrease in BDNF expression in the cerebral cortex, and the decrease in PTEN 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:(2~6), more preferably 1:(2~3.5), and especially 1:(2~3.4), they exhibit a synergistic effect. Lactose-N-neotetrasaccharide can amplify the effect of 2'-fucosylated lactose on improving the damage to the brain neural network of offspring caused by abnormal maternal immune activation. This brain neural network damage includes the improvement effect on microglia overactivation, decreased BDNF expression, and decreased PTEN gene expression. Similarly, 2'-fucosylated lactose can also amplify the effect of lactose-N-neotetrasaccharide on improving the damage to the brain neural network of offspring caused by abnormal maternal immune activation. Thus, the mutual amplification effect between the two helps to further enhance the effect of improving the damage to the brain neural network.

[0082] In some implementations, microglia overactivation is characterized by a significant increase in the expression of microglia in the brain, particularly in the hippocampus and especially in the CA1 region of the hippocampus, compared to the level in healthy individuals.

[0083] 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 brain neural network damage, said brain neural network damage including microglia overactivation, decreased BDNF expression and / or decreased PTEN gene expression.

[0084] In some implementations, the microglia overactivation includes increased expression of IBA-1 in the hippocampus, particularly in the CA1 region of the hippocampus.

[0085] In some implementations, the reduced BDNF expression includes a decrease in BDNF expression in the hippocampus, particularly in the CA1 region of the hippocampus.

[0086] In some implementations, the reduction in PTEN gene expression includes a reduction in PTEN gene expression in the cerebral cortex.

[0087] 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 the overactivation of microglia in the CA1 region of the hippocampus of offspring brains, said overactivation of microglia including increased expression of IBA-1 in the CA1 region of the hippocampus.

[0088] 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 CA1 region of the hippocampus of offspring brains.

[0089] 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 PTEN gene in the cerebral cortex of offspring.

[0090] 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 and reduced BDNF expression in the CA1 region of the hippocampus of offspring brains; wherein the microglia overactivation includes increased expression of IBA-1 in the CA1 region of the hippocampus.

[0091] 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 CA1 region of the hippocampus of offspring and reduced PTEN gene expression in the cerebral cortex of offspring.

[0092] 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 the overactivation of microglia in the CA1 region of the hippocampus of offspring brains and the reduced expression of the PTEN gene in the cerebral cortex of offspring brains; said overactivation of microglia includes increased expression of IBA-1 in the CA1 region of the hippocampus of the brain.

[0093] 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 CA1 region of the hippocampus of offspring brain, reduced expression of the PTEN gene in the cerebral cortex of offspring brain, and reduced expression of BDNF in the CA1 region of the hippocampus of offspring brain; wherein the microglia overactivation includes increased expression of IBA-1 in the CA1 region of the hippocampus.

[0094] In some implementations, the improvement of brain neural network damage is not intended to prevent or treat disease. Similarly, the improvements in microglia overactivation, BDNF expression reduction, and PTEN gene expression reduction described in this invention are also not intended to prevent or treat disease. Furthermore, the brain neural network damage, microglia overactivation, BDNF expression reduction, and PTEN gene expression reduction described in this invention do not reach a level that can be considered a disease.

[0095] Example

[0096] 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.

[0097] Example 1: Construction of the model and human milk oligosaccharide intervention experiment involved in this invention

[0098] 1. Construction of a model of maternal immune homeostasis disorder

[0099] 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.

[0100] 2. Offspring Intervention

[0101] 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. At four weeks of age, the male offspring 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, behavioral tests were conducted to analyze the effect of the HMOs combination on stereotyped and repetitive behaviors in offspring induced by maternal immune activation. The offspring were then sacrificed, and brain tissue was collected for biochemical analysis.

[0102] 3. Intervention grouping of offspring animals

[0103] 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.

[0104] 4. Monitoring of basic growth indicators

[0105] During the 6-week intervention, the mice's body weight, food intake, and water intake were recorded weekly.

[0106] 5. Experimental intervention design

[0107] 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 effects and mechanisms 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.

[0108] Table 1: Experimental Intervention Design

[0109]

[0110] Example 2: Evaluation of Basal Growth Indicators in Mice

[0111] 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).

[0112] 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).

[0113] Table 2: Changes in body weight (g) of mice in 9 groups during 6 weeks of intervention.

[0114]

[0115] Table 3: Comparison of average daily food intake and water consumption of mice in each group during the intervention period

[0116]

[0117] Example 3: Evaluation of Repetitive Stereotyped Behaviors

[0118] 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. Furthermore, the inventors evaluated the effect of HMO intervention on improving repetitive and stereotyped behaviors in mice.

[0119] The repetitive stereotyped behaviors in this invention are mainly evaluated in terms of self-grooming and bead embedding.

[0120] Mice are known to have a natural instinct to groom themselves. This grooming behavior reveals complex, fixed patterns of grooming actions, which are ritualized into a stereotyped display. Common self-grooming actions include grooming the forelimbs, rubbing and washing the face, ventral side of the body, tail, and genitals. Furthermore, the inventors used another repetitive stereotyped assessment experiment—the bead burial test—to examine the ameliorative effect of HMO on this symptom. Digging and burying are typical behaviors in mice. While initially used to assess anxiety behavior in rodents, this test is more often used to measure repetitive or compulsive digging behaviors and can quantitatively detect repetitive stereotyped behaviors in the MIA mouse model.

[0121] 3.1 Self-reflection experiment

[0122] The self-grooming test is often used to test repetitive stereotyped behaviors in MIA mice. Mice have a natural instinct to groom their fur, and common grooming behaviors include grooming with the forelimbs, rubbing and cleaning the face, ventral side of the body, tail, and genitals.

[0123] In the experiment, mice were placed in a rectangular box (40 cm × 34 cm × 40 cm) and allowed to acclimatize for 10 minutes. Then, video analysis software was used to record the mice's self-grooming behaviors over the next 10 minutes, including licking their bodies and fur, rubbing their faces with their front paws, and scratching their torsos. The box was cleaned with 75% alcohol after each mouse was replaced.

[0124] 3.2 Bead embedding test

[0125] The marble-burying tests were originally used to assess anxiety behavior in rodents, but they are more commonly used to measure repetitive or compulsive digging behaviors and can quantitatively detect repetitive and stereotyped behaviors in rat models. In the experiment, fresh, unscented bedding was added to a brand-new cage (30 cm × 17 cm × 16 cm) to a depth of 5 cm and compacted. Standard glass marbles (approximately 2 cm in diameter and weighing approximately 5 g) were gently placed on the bedding in five rows of five at equal intervals. A camera was used to record the mice's movements over 30 minutes. The marble burying standard was defined as more than two-thirds of the marble volume being buried in the bedding. Between each experiment, the cage and bedding were changed, and the marbles were washed with a mild detergent, rinsed thoroughly with distilled water, and dried before use.

[0126] 3.3 Results

[0127] Table 4 shows the self-grooming time of mice in the control group, model group, and different HMO intervention groups within 10 minutes. The table shows that the self-grooming time of mice in the model group was significantly higher than that in the control group (p=0.0008). After intervention with two low-concentration doses of LNnT and 2'-FL (Comparative Example 1, Comparative Example 2, and Comparative Example 3), self-grooming time decreased, but statistical analysis showed no significant difference between Comparative Example 1-3 and the model group (p>0.05). In Comparative Example 4, the self-grooming time was higher than that in the model group after intervention with a higher concentration of 2'-FL, while in Comparative Example 5, the self-grooming time was comparable to the mean of the model group after intervention with the highest dose of 2'-FL. Statistical analysis for these two groups also showed no significant difference from the model group (p>0.05), indicating that single-component intervention with LNnT and 2'-FL did not significantly improve self-grooming. However, when the two HMOs were combined in different proportions (Examples 1-4), a significant reduction in self-grooming time was observed. This indicates that intervention with LNnT and 2'-FL in four ratios of 1:1, 1:2, 1:3.4, and 1:6 can significantly reduce the self-grooming time of repetitive stereotyped behaviors in MIA mice. The self-grooming time of mice in the four example groups was approximately equal to that of Example 1 < Example 2 < Example 3 ≈ Example 4, but after significance analysis, no significant difference was found among the four groups (p > 0.05).

[0128] Table 4 shows that the mice developed severe repetitive stereotyped behaviors after the model was established, with a significant increase in the number of implanted beads (p < 0.0001), approximately three times that of the normal control group. However, the same model mice treated with different doses and ratios of HMOs showed a significant decrease in the number of implanted beads. One-way ANOVA analysis of the number of implanted beads in the 11 groups of mice yielded the results shown in Table 4-1. The table shows that the number of implanted beads in mice treated with LNnT monomer, 2'-FL monomer, and different doses of both was significantly lower than that in the model group (p < 0.05), reaching the level of the control group, and showing no difference from the untreated healthy control mice (p > 0.05). This indicates that intervention with the two HMOs can effectively improve the repetitive stereotyped behaviors in the model mice. Furthermore, the inventors compared the effects of LNnT and 2'-FL compounded in four ratios of 1:1, 1:2, 1:3.4 and 1:6. As can be seen from the table, Examples 1-4 were significantly lower than Comparative Examples 1-5 (as shown in Table 4), and LNnT and 2'-FL had a synergistic effect.

[0129] Table 4: Repetitive stereotyped behaviors of mice in each group

[0130]

[0131] Table 4-1: Analysis of differences in repetitive stereotyped behaviors and the number of embedded beads among different groups of mice

[0132]

[0133] The above experiments used a mouse model to simulate repetitive stereotyped behaviors in humans. The experiments showed that after modeling, mice with normal basic growth and development and normal food and water intake exhibited significant repetitive stereotyped behaviors. 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), the repetitive stereotyped behaviors were significantly reduced. In particular, the effects of the combination of the two oligosaccharides in the ratios of 1:2, 1:3.4, and 1:6 were significantly higher than the effects of single-agent intervention.

[0134] Example 4: Effects of human milk oligosaccharides on brain neural tissue at the cellular and genetic levels

[0135] Through the above behavioral experiments, the inventors observed that the combination of two human milk oligosaccharides, 2'-FL and LNnT, could significantly restore repetitive and stereotyped behaviors in mice. 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 alter their phenotype in a maternal immune activation MIA model, further leading to misconnections in neuronal circuits and resulting in abnormal behavioral performance. Based on this theory, this invention examines the changes in brain neural tissue at the cellular and gene levels behind the behavioral performance of offspring caused by maternal immune homeostasis dysregulation, and the effects of human milk oligosaccharide intervention on these indicators, thereby clarifying the deep-seated mechanisms of behavioral improvement.

[0136] First, the inventors used immunofluorescence to analyze the effects of maternal immune activation on microglia and neurons in offspring brain tissue. Ionized calcium-binding adapter molecule 1 (IBA-1) is a calcium-binding protein commonly used as a marker for microglia. BDNF is a neurotrophic protein that plays an important role in the survival, differentiation, growth, and development of neurons.

[0137] The specific experimental steps are as follows:

[0138] 1. Brain tissue collection

[0139] 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.

[0140] 2. Immunofluorescence staining to determine IBA-1 and BDNF

[0141] (1) Preparation of paraffin sections

[0142] 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.

[0143] (2) Immunofluorescence staining

[0144] 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.

[0145] 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).

[0146] result:

[0147] This invention analyzed the expression level of IBA-1 in the CA1 region of the hippocampus of progeny mice from the control group, model group, and intervention group to examine the activation of microglia in the brain tissue of each group of mice. The experimental results are as follows: Figure 1 As shown in Table 5, the model group showed significant activation of microglia in the CA1 region of the hippocampus in offspring after maternal intervention during pregnancy, with a significant increase in the fluorescence intensity of the cell marker IBA-1 (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, after significant difference analysis, it was found that the expression level of IBA-1 in the CA1 region of the hippocampus in all nine groups (Comparative Examples 1-5 and Examples 1-4) was significantly lower than that in the model group (as shown in Table 5). This indicates that intervention with breast milk oligosaccharides can significantly inhibit excessive microglia activation. The inventors further discovered that the fluorescence intensity of IBA-1 in the CA1 region of the hippocampus was lower than that of LNnT and 2'-FL monoclonal intervention when combined in ratios of 1:1, 1:2, 1:3.4, and 1:6. Significant difference analysis showed that the fluorescence intensity in Examples 1-4 was significantly lower than that in Comparative Examples 1-5. This indicates that LNnT and 2'-FL, when combined in ratios of 1:2, 1:3.4, and 1:6, can synergistically reduce microglia activation. Compared to the four examples, Example 2 showed significantly lower results than Example 1 (p < 0.0001), Example 3 (p < 0.001), and Example 4 (p < 0.01).

[0148] Table 5: Comparative analysis of quantitative expression of IBA-1 in the CA1 region of the hippocampus.

[0149]

[0150] The above results indicate that maternal immune imbalance during pregnancy leads to a large-scale activation of microglia in the offspring's brain.

[0151] Furthermore, this invention found that intraperitoneal injection of poly(I:C) solution into pregnant mice to construct a maternal immune homeostasis dysregulation model affected the activity of offspring neurons, with a significant decrease in the fluorescence intensity of the neurotrophic factor BDNF in the CA1 region of the hippocampus (p < 0.0001) (as shown in Tables 6 and 6-1). However, intervention with human milk oligosaccharides (Comparative Examples 1-5, Examples 1-4) resulted in a significant increase in fluorescence intensity (p < 0.001), indicating that human milk oligosaccharides can improve the activity of offspring neurons caused by pregnancy stress. Further comparison revealed that the intervention effect of LNnT and 2'-FL monomers was less than that of their complex, and after significant difference analysis, Examples 1-5 showed significantly higher efficacy than Comparative Examples 1-4 (p < 0.05), indicating a synergistic effect. This suggests that LNnT and 2'-FL, when combined in ratios of 1:1, 1:2, 1:3.4, and 1:6, can synergistically enhance neuronal cell activity. Among them, Example 2 was significantly higher than Example 1 (p < 0.01), Example 3 (p < 0.01), and Example 4 (p < 0.01).

[0152] Table 6: Quantitative expression of BDNF in the CA1 region of the hippocampus of mice in each group

[0153]

[0154] Table 6-1: Comparative Analysis of Quantitative Expression of BDNF in CA1 Region of the Hippocampus

[0155]

[0156] Second, the inventors further analyzed the expression of specific alleles associated with repetitive and stereotyped behaviors at the gene level, such as the PTEN gene, which is crucial in brain development and affects neuronal proliferation, migration, synapse formation, and neural network stability.

[0157] This invention used PCR technology to study the expression level of the PTEN gene in the cerebral cortex of mice in each group, specifically:

[0158] 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:

[0159] (1) Tissue RNA extraction

[0160] 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.

[0161] (2) Reverse transcription (cDNA synthesis)

[0162] 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.

[0163] (3) Indicator Measurement

[0164] 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 7.

[0165] 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.

[0166] Table 7: Gene Primer Sequences

[0167]

[0168] The results are shown in Table 8. The table shows that the expression level of the PTEN gene mRNA in mice after maternal immune activation was significantly reduced in the model group. However, timely intervention with two human milk oligosaccharides, LNnT and lactose-N-neotetrasaccharide, in early life enhanced PTEN mRNA expression through epigenetic regulation (such as DNA methylation or histone modification) (Comparative Examples 1-5 and Examples 1-4). However, after significant difference analysis, it was found that low-dose intervention with the two oligosaccharides (Comparative Examples 1 and 2) showed no significant difference from the model group, while Comparative Examples 3-5 showed significant differences from the model group. Further analysis of LNnT and 2'-FL at ratios of 1:1, 1:2, 1:3.4, and 1:6 revealed that PTEN expression levels were significantly higher than in the model group in all four examples, and significantly higher in the five comparative examples, indicating a synergistic effect. In particular, Example 2 showed significantly higher expression levels than Examples 1, 3, and 4.

[0169] Table 8: Expression levels of PTEN mRNA in the cerebral cortex of mice in each group

[0170]

[0171] Table 8-1: Comparative Analysis of PTEN mRNA Expression Levels in the Cerebral Cortex

[0172]

[0173] Through the above analysis, the inventors discovered that maternal immune activation leads to excessive activation of microglia, the immune cells in the offspring brain. Simultaneously, the stress response caused by immune activation affects the neuronal activity of the offspring, with a significant decrease in BDNF. Furthermore, at the gene level, the mRNA expression level of the PTEN allele, which is related to neuronal proliferation, migration, synapse formation, and neural network stability, was significantly reduced. This indicates that maternal immune activation severely impacts the formation of neural networks in the offspring brain. 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, a significant decrease in microglia activation, improved neuronal activity, a significant increase in BDNF expression, and a significant increase in PTEN mRNA expression were observed. Behavioral studies also showed a significant improvement in repetitive and stereotyped behaviors.

Claims

1. The use of the composition in the preparation of products that improve repetitive and stereotyped behavior, characterized in that, The repetitive stereotyped behavior is caused by abnormal activation of the maternal immune system; The 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 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 improvement of repetitive and stereotyped behaviors includes: inhibiting the overactivation of microglia in the hippocampus and / or regulating neuronal activity.

4. The use according to claim 3, 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 PTEN gene in the cerebral cortex.

5. The use of the composition in the preparation of products that improve damage to the brain's neural networks, characterized in that, The brain neural network damage includes microglia overactivation, decreased BDNF expression, and / or decreased PTEN gene expression, which is caused by abnormal activation of the maternal immune system. The composition contains the following essential active ingredients: lactose-N-neotetrasaccharide and 2'-fucosylated lactose.

6. The use according to claim 5, characterized in that, In the composition, the mass ratio of lactose-N-neotetrasaccharide and 2'-fucosylated lactose is 1:(1~10).

7. The use according to claim 5 or 6, 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 decreased expression of the PTEN gene includes a decrease in the expression of the PTEN gene in the cerebral cortex.

8. The use according to any one of claims 1 to 7, characterized in that, The products include food, which may be beverages, milk and dairy products, baked goods, or confectionery.

9. The use according to any one of claims 1 to 7, 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.

10. The use according to any one of claims 1 to 9, characterized in that, The product contains at least 0.05% by mass of lactose-N-neotetrasaccharide and 2'-fucosylated lactose.

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