Nutritional composition for synergistically regulating neural stem cells to differentiate into specific nerve cells

Abstract

The invention discloses a nutritional composition for synergistically regulating neural stem cells to differentiate into specific nerve cells. The invention provides a nutritional composition. The nutritional composition comprises the following necessary components: a grease component and a sialic acid oligosaccharide component, moreover, in the nutritional composition, the molar ratio of the grease component to the sialic acid oligosaccharide component is (1-5): (0.01-5). The nutritional composition provided by the invention can effectively promote the neural stem cells to differentiate into specific nerve cells and achieve function maturation, especially significantly promote the neural stem cells to differentiate into neurons, and shows a synergistic enhancement effect on the structure and function aspects, and the components have a synergistic effect.

Description

Technical Field

[0001] This invention belongs to the food field and relates to a nutritional composition that synergistically regulates the differentiation of neural stem cells into specific neural cells. More specifically, it relates to a nutritional composition that promotes the differentiation of neural stem cells into neurons, thereby benefiting the development and functional improvement of the nervous system. Background Technology

[0002] In recent years, research on neural development and nutritional regulation has gradually expanded from "morphological development" to the "fate determination" stage. Neural stem cells, as the basic cell population for the development and regeneration of the nervous system, possess self-renewal and multi-directional differentiation potential, capable of differentiating into neurons, oligodendrocytes, and astrocytes. In the early stages of central nervous system development, key factors influencing the differentiation of neural stem cells into specific types of neural cells mainly include endogenous signaling pathways (such as Notch, Wnt, and BDNF-TrkB) and exogenous nutritional and metabolic signals.

[0003] Existing research indicates that docosahexaenoic acid (DHA) and arachidonic acid (ARA) are the most abundant long-chain polyunsaturated fatty acids (LC-PUFAs) in brain tissue, playing crucial roles in neural stem cell membrane structure formation, signal transduction, and gene transcription regulation. Studies have found that DHA significantly promotes the differentiation and neurite growth of neural stem cells or primary neurons, while the addition of ARA increases the proportion of astrocytes in glial lineage-prone precursor cells, suggesting a link between ARA and glial formation. Furthermore, both DHA and ARA have a synergistic effect on neurogenesis at certain ratios, and are important structural lipid components of the nervous system development.

[0004] On the other hand, sialic acid-human milk oligosaccharides such as 3′-sialic acid lactose (3′-SL) and 6′-sialic acid lactose (6′-SL) are abundant in breast milk, and multiple animal and breast milk studies support their association with cognitive development and neuronal maturation.

[0005] Currently, publicly available technologies for early differentiation research of neural stem cells mainly focus on the following categories:

[0006] (1) Methods to promote differentiation with a single type of nutrient: For example, synthetic nutrient combinations of fatty acids such as DHA and ARA are used to promote myelin formation and infant cognitive development (reference 1).

[0007] (2) Research on the improvement of neurological function by human milk oligosaccharides or sialic acid: Some patents suggest that human milk oligosaccharides, especially 2'-fucosylated lactose (2'-FL), can promote the enhancement of neuronal synaptic signals, thereby improving learning and memory functions (referencing reference 2), but do not involve the synergistic effect with lipid signals; Reference 3 patent points out that administering an effective amount of sialic acid (3'-SL and 6'-SL are important exogenous sources of sialic acid in the neonatal brain) to infants or premature infants has an enhancing effect on learning and memory.

[0008] (3) Directional study of cell differentiation regulation: For example, the differentiation of neural stem cells into neural cells is induced by exogenous small molecule chemical reagents (refer to reference 4), but this method depends on specific chemical structures and lacks nutritional convertibility.

[0009] In summary, while existing research has confirmed that both lipids and carbohydrates promote neural development, there is still a lack of understanding regarding the "synergistic regulatory mechanism of lipid and carbohydrate signaling" and its application in the "differentiation of neural stem cells into specific neuronal lineages".

[0010] References

[0011] Reference 1: US11191293B2

[0012] Reference 2 HK1195713A1

[0013] Reference 3 WO2015085077A1

[0014] Reference 4 WO2009076529A1 Summary of the Invention

[0015] The problem the invention aims to solve

[0016] Although existing technologies have been used to study the early differentiation of neural stem cells, there are still shortcomings. For example, the following shortcomings exist in cited references 1-4:

[0017] ① Focus on single components and neglect synergy between components: Focus on the promoting effect of single nutrients (such as DHA, ARA or human milk oligosaccharides) on neural development, but fail to reveal the interaction and synergistic relationship between multiple factors, especially lacking systematic research on the "lipid-glucose signaling axis" at the cell differentiation level.

[0018] ② The differentiation direction is not specific: Traditional nutritional interventions or growth factor induction programs can only improve the overall differentiation level, lacking clear quantitative evidence and directional control methods, and lacking directionality and precision.

[0019] ③ The evaluation indicators are too simplistic and lack a multi-layered, multi-dimensional chain of evidence.

[0020] Many studies rely on a single biochemical indicator or behavioral endpoint to support their effects (e.g., detecting only the expression of a certain protein or animal behavioral scores), and rarely establish a multi-level, closed-loop evidence system from "molecular drive → morphology → function / maturation", which reduces the interpretability of the mechanism and the credibility of the review.

[0021] Therefore, this invention aims to provide a synergistic regulatory method through the combined action of docosahexaenoic acid (DHA), eicosapentaenoic acid (ARA), and sialic acid-human milk oligosaccharides (3′-SL, 6′-SL). It explores the directional regulation of neural stem cell differentiation into specific neuronal lineages (such as neuronal types) under different combination ratios, revealing the synergistic mechanism of lipid-glyconutrient compositions in the early stages of cell differentiation. This invention aims to clarify the synergistic effect of DHA, ARA, and SLs in regulating the differentiation direction of neural stem cells, while optimizing the ratio and concentration of these two types of nutrients to achieve integrated controllable differentiation and nutritional intervention, providing new scientific and technological basis for infant brain development formulas or functional nutritional compositions.

[0022] Solution for solving the problem

[0023] [1]. A nutritional composition, wherein the nutritional composition comprises the following essential components:

[0024] Oil components and sialic acid oligosaccharide components;

[0025] Furthermore, in the nutritional composition, the molar ratio of the oil component to the sialic acid oligosaccharide component is (1-5):(0.01-5);

[0026] Preferably, the molar ratio of the oil component to the sialic acid oligosaccharide is (1-3):(0.5-5).

[0027] [2]. According to the nutritional composition described in [1], wherein the oil component includes one or more of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids;

[0028] Preferably, the oil component includes polyunsaturated fatty acids;

[0029] More preferably, the polyunsaturated fatty acid comprises any one or more of α-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), linoleic acid (LA), arachidonic acid (ARA), and conjugated linoleic acid (CLA).

[0030] [3]. The nutritional composition according to [1] or [2], wherein the oil component comprises docosahexaenoic acid and arachidonic acid;

[0031] Preferably, the molar ratio of docosahexaenoic acid to arachidonic acid in the oil component is 1:(1~5).

[0032] [4]. The nutritional composition according to any one of [1]-[3], wherein the sialic acid oligosaccharide comprises any one or more of 3'-sialylated lactose, 6'-sialylated lactose, sialylated-lactose-N-tetrasaccharide a, sialylated-lactose-N-tetrasaccharide b, sialylated-lactose-N-tetrasaccharide c, and disialialic acid-lactose-N-tetrasaccharide.

[0033] Preferably, the sialic acid oligosaccharides include 3'-sialylated lactose and 6'-sialylated lactose;

[0034] More preferably, the mass ratio of 3'-sialylated lactose to 6'-sialylated lactose in the sialic acid oligosaccharide is (1~20): (15~1).

[0035] [5]. A food product comprising or using any of the nutritional compositions described in [1]-[3] as ingredients.

[0036] [6]. The food according to [5], wherein the food includes any one or more of infant food, children's food, adolescent food, maternal food, adult food and middle-aged and elderly food.

[0037] [7]. The food according to [5] or [6], wherein the food further contains any one or more of the following ingredients: plant product ingredients, animal dairy product ingredients, animal meat product ingredients, functional additives and any acceptable excipients.

[0038] [8]. Application of a composition containing two essential components, an oil component and a sialic acid oligosaccharide component, in the preparation of a nutritional composition for regulating the differentiation of neural stem cells into neurons and / or promoting the functional maturation of neurons.

[0039] [9]. Application of a composition containing two essential components, an oil component and a sialic acid oligosaccharide component, in the preparation of foods that regulate the differentiation of neural stem cells into neurons and / or promote the functional maturation of neurons.

[0040]

[10] . According to the application described in [8] or [9], wherein the regulation of neural stem cell differentiation into neurons and / or promotion of neuronal functional maturation includes at least one of promoting neural stem cell differentiation into neurons, promoting myelin development, promoting axonal extension, enhancing cytoskeleton stability, and / or enhancing neuronal morphological development.

[0041] The effects of the invention

[0042] 1) The present invention provides a composition for promoting neural development, comprising an oil component and a sialylated substance component, which can effectively promote the differentiation and functional maturation of neural stem cells into specific neural cells, especially significantly promote the differentiation of neural stem cells into neurons, and exhibit a synergistic enhancement effect at the structural and functional levels, and the components synergistically enhance each other.

[0043] 2) The composition provided by the present invention has broad prospects in promoting brain development and plays an important role in various cellular mechanisms such as promoting neural stem cell proliferation, differentiation into specific nerve cells, promoting synapse construction, promoting myelin development, and promoting neuronal maturation.

[0044] 3) The composition of the present invention has been fully validated through a complete structure-function dual verification system covering key aspects such as neural stem cell differentiation, synaptic connection, and myelin development.

[0045] 4) By employing multiple techniques such as protein quantification, fluorescence imaging, and morphological measurement, experiments were conducted to verify the scientific combination of sialic acidified substances and lipid components, which showed synergistic effects at multiple levels and promoted the efficient differentiation of neural stem cells into neurons.

[0046] 5) The composition of the present invention has a wide range of applications and is suitable for various groups of people, including infants, children, adolescents, pregnant women, adults and the elderly. It is also convenient to add to or prepare various foods or health products. Attached Figure Description

[0047] Figure 1 This diagram illustrates the cell viability results of neural stem cells co-cultured with samples of different concentrations. The figure shows… " indicates P < 0.05, " " indicates P < 0.001.

[0048] Figure 2 This is a schematic diagram showing the statistical results of relative fluorescence intensity in confocal fluorescence imaging of neuron-specific β-III tubulin (Tuj-1 antibody labeling) in cells co-cultured with neural stem cells in different proportions of nutrient compositions.

[0049] Figure 3 This is a schematic diagram showing the statistical results of relative fluorescence intensity in confocal fluorescence imaging of synaptophysin in cells co-cultured with neural stem cells in different proportions of nutrient compositions.

[0050] Figure 4 This is a schematic diagram of fluorescence confocal imaging results of neuron-specific β-III tubulin (labeled with Tuj-1 antibody) and synaptophysin in cells co-cultured with neural stem cells in different proportions of nutrient compositions.

[0051] Figure 5 This is a schematic diagram showing the expression results of the brain-derived neurotrophic factor BDNF in each group.

[0052] Figure 6 This is a schematic diagram showing the expression results of the myelin-associated glycoprotein MAG in each group. Detailed Implementation

[0053] 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.

[0054] 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.

[0055] 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.

[0056] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0057] 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.

[0058] 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.

[0059] In this invention, the term "about" is used to define that the numerical ranges and parameters of this invention are approximate values, while specific related values ​​have been presented as precisely as possible. Unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this invention are modified by "about". Here, "about" generally means that the actual value is within ±3%, ±2%, ±1%, or ±0.5% of a specific value or range.

[0060] In this invention, the term "infant" is used to refer to the human group aged 0-6 months.

[0061] In this invention, the term "older infant" refers to the human group aged 6 to 12 months.

[0062] In this invention, the term "infant" refers to the human group aged 12 to 36 months.

[0063] In this invention, the term "infant" refers to the human group under the age of 3 years.

[0064] In this invention, the term "children" refers to a group of human beings who are older than 3 years and younger than 12 years and are in the growth and development stage.

[0065] In this manual, the term "adult" refers to a person who is 18 years of age or older.

[0066] In this manual, the term "teenager" refers to people aged 7-40.

[0067] In this specification, the terms "optional" or "optional" are used to indicate the use or omission of certain substances, components, procedures, application conditions, etc.

[0068] <First Aspect>

[0069] The first aspect of the present invention provides a nutritional composition comprising the following essential components: an oil component, including one or more of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids; and a sialic acid oligosaccharide component, wherein the molar ratio of the oil component to the sialic acid oligosaccharide component in the nutritional composition is (1-5):(0.01-5).

[0070] In some preferred embodiments, the molar ratio of the oil component to the sialic acid oligosaccharide is (1-3):(0.5-5).

[0071] The nutritional composition described in this invention can promote the development of the nervous system and regulate the differentiation and functional maturation of neural stem cells into specific nerve cells. Specifically, the nutritional composition of this invention can promote the proliferation of neural stem cells, the differentiation into specific nerve cells, the construction of synapses, the development of myelin sheaths, the maturation of neurons, and the formation of key stages in the formation of neural network connections, among other aspects of nervous system development.

[0072] The nutritional composition described in this invention can regulate the differentiation and functional maturation of neural stem cells into specific neural cells, and has a significant synergistic effect in promoting the differentiation of neural stem cells into neurons and the maturation of neurons.

[0073] (Oil and fat components)

[0074] In some embodiments, the oil component includes one or more of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids.

[0075] In some embodiments, the oil component includes polyunsaturated fatty acids, which include any one or more of alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), linoleic acid (LA), arachidonic acid (ARA), and conjugated linoleic acid (CLA).

[0076] In some preferred embodiments, the oil components include docosahexaenoic acid and arachidonic acid.

[0077] docosahexaenoic acid

[0078] Docosahexaenoic acid (DHA) refers to cis-4,7,10,13,16,19-docosahexaenoic acid, commonly abbreviated as DHA in this field. It is an essential polyunsaturated fatty acid for the human body. It is a straight-chain fatty acid containing 22 carbon atoms and 6 carbon-carbon double bonds, with the molecular formula C2. 22 H 32 O2.

[0079] In this specification, the terms "docosahexaenoic acid" and "DHA" are used interchangeably.

[0080] Arachidonic acid

[0081] Arachidonic acid refers to all-cis-5,8,11,14-eicosatetraenoic acid, commonly abbreviated as ARA in this field. It is a higher unsaturated fatty acid containing 20 carbon atoms and 4 carbon-carbon double bonds, with the molecular formula CH3(CH2)4(CH=CH-CH2)4(CH2)2COOH.

[0082] In this specification, the terms "arachidonic acid" and "ARA" are used interchangeably.

[0083] This invention does not specifically limit the source of the oil components; typically, they can be derived from various milk-containing raw materials, such as animal milk raw materials (e.g., cow's milk, milk powder, etc.). In this invention, "animal milk" is used to refer to the liquid obtained from the mammary glands of mammals in lactation.

[0084] Those skilled in the art can commercially obtain DHA and ARA, such as, but not limited to, food-grade DHA and food-grade ARA sold by JABY Biotech Co., Ltd.

[0085] The term “animal milk” should be interpreted broadly and encompass both raw milk (i.e., the liquid obtained directly from the mammary glands) and standardized dairy products.

[0086] In some embodiments, the molar ratio of docosahexaenoic acid (DHA) to arachidonic acid (ARA) in the oil component is 1:(1~5).

[0087] In some preferred embodiments, the molar ratio of docosahexaenoic acid (DHA) to arachidonic acid (ARA) in the oil component is 1:(1~3).

[0088] In some more preferred embodiments, the molar ratio of docosahexaenoic acid (DHA) to arachidonic acid (ARA) in the oil component is 1:(1~2.5).

[0089] In some further preferred embodiments, the molar ratio of docosahexaenoic acid (DHA) to arachidonic acid (ARA) in the oil component is 1:(1~2). For example, the molar ratio of docosahexaenoic acid (DHA) to arachidonic acid (ARA) can be 1:(1.5~2), 1:(1.5~1.8), for example, 1:1.5, 1:1.6, 1:1.7, 1:1.8, etc.

[0090] In some exemplary embodiments, the mass ratio of docosahexaenoic acid (DHA) to arachidonic acid (ARA) in the oil component may be 1:1.7.

[0091] The oil components of the present invention can exist in a liquid state, or in a semi-solid or solid state.

[0092] When the oil component of the present invention exists in a liquid state, in some preferred embodiments, the concentration of the oil component in the nutritional composition is 1-300 nM.

[0093] In some preferred embodiments, the concentration of the oil component in the nutritional composition is 1-200 nM, and more preferably, the concentration of the oil component is 50-120 nM, for example: 53 nM, 54 nM, 55 nM, 80 nM, 81 nM, 82 nM, 107 nM, 108 nM, 109 nM, etc.

[0094] In some exemplary embodiments, the DHA concentration range is 20 nM-400 nM. More specifically, the suitable DHA concentration range of the nutritional composition is 20 nM-80 nM, such as 20 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 60 nM, etc. For example, the DHA concentration is 40 nM.

[0095] In some embodiments, the concentration of arachidonic acid (ARA) is between 30 and 120 nM, for example: 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, etc.

[0096] In some preferred embodiments, the concentration of arachidonic acid (ARA) is between 30 and 80 nM, for example: 40 nM, 50 nM, 60 nM, 65 nM, 70 nM, etc.

[0097] In some preferred embodiments, the concentration of arachidonic acid (ARA) is between 50 and 70 nM, for example: 60 nM, 65 nM, 68 nM, 70 nM, etc.

[0098] In some exemplary embodiments, the concentration of arachidonic acid (ARA) in the nutritional composition is 68 nM.

[0099] When the content of docosahexaenoic acid and arachidonic acid in the nutritional composition of the present invention is within the above range, the nutritional composition can regulate the differentiation of neural stem cells into specific neural cells (especially into neurons) and their functional maturation.

[0100] (Sialic acid oligosaccharide component)

[0101] Sialylation is a type of glycosylation involving the covalent addition of sialic acid to the terminal glycans of glycoproteins and glycolipids. This enzymatic process is tightly regulated by sialyltransferases (STs) and sialylases / neuraminidases (NEUs). Based on the type of glycosidic bond, sialylation can be classified into three types: α2-3-, α2-6-, and α2-8-sialylation. Sialylated oligosaccharides (SOS) are an important subclass of oligosaccharides, containing sialic acid residues in their molecular structure. Sialic acid is a nine-carbon monosaccharide, usually located at the end of the sugar chain, giving SOS a negative charge and important biological functions.

[0102] In some embodiments, the sialic acid oligosaccharides of the present invention include sialyl lactose.

[0103] Sialyllactose (SL) is the most basic and core member of sialic acid oligosaccharides, and one of the most important components of human milk oligosaccharides (HMOs). Its structure consists of sialic acid molecules and lactose molecules linked by glycosidic bonds.

[0104] In some embodiments, the sialylated lactose of the present invention includes any one or more of 3'-sialylated lactose, 6'-sialylated lactose, sialylated-lactose-N-tetrasaccharide a, sialylated-lactose-N-tetrasaccharide b, sialylated-lactose-N-tetrasaccharide c, and disialialylated-lactose-N-tetrasaccharide.

[0105] In some preferred embodiments, the sialic acid oligosaccharides of the present invention are 3'-sialylated lactose (3'-SL) and 6'-sialylated lactose (6'-SL).

[0106] This invention does not specifically limit the source of sialic acid oligosaccharides; typically, they can be derived from various milk-containing raw materials, such as animal milk raw materials (e.g., cow's milk, milk powder, etc.). In this invention, "animal milk" is used to refer to the liquid obtained from the mammary glands of mammals in lactation.

[0107] Those skilled in the art can commercially obtain 3'-SL and 6'-SL, such as food-grade 3'-SL (GlyCare™ 3SL 9001) and food-grade 6'-sialic acid lactose (GlyCare™ 6SL 9001) sold by Royal DSM, but are not limited thereto.

[0108] In some embodiments, the mass ratio of 3'-sialylated lactose (3'-SL) to 6'-sialylated lactose (6'-SL) in the sialic acid oligosaccharide component is (1~20): (15~1).

[0109] In some preferred embodiments, the mass ratio of 3'-sialylated lactose (3'-SL) to 6'-sialylated lactose (6'-SL) in the sialic acid oligosaccharide component is (1~15): (10~1).

[0110] In some more preferred embodiments, the mass ratio of 3'-sialylated lactose (3'-SL) to 6'-sialylated lactose (6'-SL) in the sialic acid oligosaccharide component is (8~13):(13~8).

[0111] In some further preferred embodiments, the mass ratio of 3'-sialylated lactose (3'-SL) to 6'-sialylated lactose (6'-SL) in the sialic acid oligosaccharide component is (8~12):(12~8). For example, the mass ratio of 3'-sialylated lactose (3'-SL) to 6'-sialylated lactose (6'-SL) can be 12:8, 11:9, 10:10, etc.

[0112] In some exemplary embodiments, the mass ratio of 3'-sialylated lactose (3'-SL) to 6'-sialylated lactose (6'-SL) can be 11:9.

[0113] The sialylated component of the present invention can exist in a liquid state, or in a semi-solid or solid state.

[0114] When the sialylated component of the present invention is present in a liquid state, in some preferred embodiments, the concentration of the sialyl oligosaccharide in the nutritional composition is 10-200 nM.

[0115] In some preferred embodiments, the concentration of the sialylated component is in the range of 10-100 nM, for example: 23 nM, 24 nM, 25 nM, 59 nM, 60 nM, 61 nM, 75 nM, 76 nM, 77 nM, etc.

[0116] In some further preferred embodiments, the concentration of 3'-sialylated lactose (3'-SL) in the sialylated component is between 5-55 nM, and the concentration of 6'-sialylated lactose (6'-SL) is between 5-45 nM.

[0117] In some more specific embodiments, the concentration of the 3'-sialylated lactose (3'-SL) is between 10 and 45 nM, for example: 10 nM, 13 nM, 13.2 nM, 14 nM, 31 nM, 32 nM, 33 nM, 40 nM, 41.8 nM, 42 nM, 43 nM, etc.

[0118] In some more specific embodiments, the concentration of the 6'-sialylated lactose (6'-SL) is between 5 nM and 40 nM, for example: 9 nM, 10 nM, 10.8 nM, 11.9 nM, 26 nM, 27 nM, 28 nM, 33 nM, 34 nM, 34.2 nM, 35 nM, etc.

[0119] In some exemplary embodiments, the concentration of 3'-sialylated lactose (3'-SL) in the nutritional composition is 13.2 nM and the concentration of 6'-sialylated lactose (6'-SL) is 10.8 nM; the concentration of 3'-sialylated lactose (3'-SL) in the nutritional composition is 33 nM and the concentration of 6'-sialylated lactose (6'-SL) is 27 nM; the concentration of 3'-sialylated lactose (3'-SL) in the nutritional composition is 41.8 nM and the concentration of 6'-sialylated lactose (6'-SL) is 34.2 nM.

[0120] When the 3'-sialylated lactose and 6'-sialylated lactose in the sialylated component are used in the above ratio and amount, they can play a synergistic role. When used in combination with other components of the nutritional composition, the nutritional composition can achieve better synergistic effect in regulating the differentiation of neural stem cells into specific neural cells (especially into neurons) and functional maturation.

[0121] When sialic acid oligosaccharide components and lipid components are used in combination, the nutritional composition achieves better synergistic effects in regulating the differentiation and functional maturation of neural stem cells into specific neural cells.

[0122] (Synergistic effect)

[0123] To obtain a more synergistic composition for promoting the maturation of nerve cell function, in some embodiments, the molar ratio of the lipid component to the sialic acid oligosaccharide component in the nutritional composition is (1-5):(0.01-5).

[0124] In some embodiments, the molar ratio of the oil component to the sialic acid oligosaccharide component in the nutritional composition is (1-4): (0.1-5).

[0125] In some preferred embodiments, the molar ratio of the oil component to the sialic acid oligosaccharide component in the nutritional composition is (1-3):(0.1-4).

[0126] To achieve a further and better synergistic effect in promoting neurodevelopment, in some more preferred embodiments, the molar ratio of the oil component to the sialic acid oligosaccharide in the nutritional composition is (2-3):(0.5-2).

[0127] In some exemplary embodiments, the molar ratio of the oil component and the sialic acid oligosaccharide component in the nutritional composition may be 2.7:0.6; 2.7:1.5; 2.7:1.9.

[0128] In some preferred embodiments, the molar ratio of the oil component to the sialic acid component in the nutritional composition is 2.7:(0.5-1.8).

[0129] In some more preferred embodiments, the molar ratio of the oil component and the sialic acidification component in the nutritional composition is 2.7:0.6; 2.7:1.5.

[0130] In some further preferred embodiments, the molar ratio of the oil component and the sialic acidification component in the nutritional composition is 2.7:(0.7-1.8), 2.7:(0.8-1.7), 2.7:(0.9-1.6), or 2.7:(1.2-1.6), for example, 2.7:1.5.

[0131] In this invention, when the lipid component and the sialic acid oligosaccharide component are within the above-mentioned range, the composition further achieves better synergistic effects in regulating the differentiation and functional maturation of neural stem cells into specific neural cells, such as enhancing neural stem cell activity, promoting the differentiation of neural stem cells into specific neuronal lineages (e.g., neuronal type), promoting myelin development, promoting synapse formation, improving cytoskeleton stability, and / or promoting neuronal structure.

[0132] (Composition)

[0133] The nutritional composition of the present invention includes at least the lipid component and sialic acid oligosaccharide component described above. There are no particular limitations on the formation method of the composition; it can be formed by dissolving and mixing the substances containing these components as described above in a solvent, or by mixing various high-purity extracts.

[0134] Furthermore, there are no particular limitations on other components that can be used in the compositions of the present invention. Other edible ingredients, food additives, or solvent components commonly used in the art can be used without impairing the effects of the present invention.

[0135] The nutritional composition of the present invention may exist in a liquid state, or in a semi-solid or solid state.

[0136] When the nutritional composition of the present invention is present in a liquid form, in some embodiments, the concentration of the oil component in the nutritional composition is below 300 nM.

[0137] In some specific embodiments, the concentration of the oil component in the nutritional composition is between 50 nM and 200 nM. In some exemplary embodiments, the concentration of the oil component in the nutritional composition is 108 nM.

[0138] In some embodiments, the concentration of the sialic acid oligosaccharide component in the nutritional composition is below 200 nM.

[0139] In some preferred embodiments, the concentration of the sialic acid oligosaccharide component in the nutritional composition is below 100 nM. For example, the concentration of the sialic acid oligosaccharide in the nutritional composition is between 20 and 80 nM, such as 23 nM, 24 nM, 25 nM, 59 nM, 60 nM, 61 nM, 75 nM, 76 nM, 77 nM, etc.

[0140] In some exemplary embodiments, the concentration of the sialic acid oligosaccharide component in the nutritional composition is 24 nM, 60 nM, or 76 nM.

[0141] In the nutritional composition, when the concentrations of the oil component and the sialic acid oligosaccharide component are within the above-mentioned range, they can synergistically enhance each other and have the effect of regulating the differentiation of neural stem cells into specific neural cells and their functional maturation.

[0142] (use)

[0143] Furthermore, the above-mentioned composition of the present invention has been experimentally verified to have the effect of regulating the differentiation and functional maturation of neural stem cells into specific neural cells. The regulation of neural stem cell differentiation and functional maturation into specific neural cells includes at least one of the following effects: enhancing neural stem cell activity, promoting the differentiation of neural stem cells into specific neuronal lineages (such as neuronal type), promoting myelin development, promoting synapse formation, improving cytoskeleton stability, and / or promoting the structural and functional maturation of neurons.

[0144] Specifically, the composition can promote enhanced expression of neuron-specific β-III tubulin (labeled with Tuj-1 antibody) and synaptophysin, thereby improving cytoskeleton stability and / or enhancing neuronal morphological development; the composition can increase brain-derived neurotrophic factor (BDNF) expression to promote neural stem cell differentiation; the composition can promote the expression of myelin-associated glycoprotein MAG, further promoting myelination; the composition can promote an increase in the average length of neurons, further promoting axonal extension after neuronal differentiation.

[0145] Therefore, it can be used as a food, functional health product, or functional health additive.

[0146] <Second aspect>

[0147] In a second aspect of the present invention, a food product is provided, the food product comprising or using the nutritional composition described in the first aspect above.

[0148] The food products of this invention can be suitable for infants, adolescents, adults and / or middle-aged and elderly people to regulate the differentiation of neural stem cells into specific neural cells and achieve functional maturity.

[0149] The food products described in this invention include any one or more of the following: infant food, children's food, adolescent food, pregnant and postpartum food, adult food, and food for the middle-aged and elderly.

[0150] This invention does not impose any particular limit on the absolute content of oil components and sialic acidified substances (and their specific components) in food, as long as the requirements of local food-related laws and regulations are met.

[0151] In some specific embodiments, the food can be obtained by processing the nutritional composition according to the first aspect described above.

[0152] There are no particular limitations on the food products of this invention; they can generally be pasta, beverages, instant foods, baked goods, sauces, or functional nutritional supplements.

[0153] For pasta-based foods, this can include staple foods made from flour-based raw materials, as well as whole grain staple foods, such as steamed buns, pancakes, noodles, and filled staple foods.

[0154] For baked goods, this can be cakes or cookies that are mainly based on butter, eggs, and baking powder.

[0155] There are no particular restrictions on beverages; they can include fruit drinks, vegetable drinks, milk tea drinks, tea drinks, milk, yogurt, vitamin drinks, etc. For fruit or vegetable drinks, in addition to including the functional components of this invention, they may also include fruit or vegetable juices or their solid components. For vitamin drinks, in addition to including the two functional components of this invention, they mainly contain various functional vitamins and other functional ingredients, specifically including, for example, white sugar, cyclamate, acesulfame potassium, taurine, potassium sorbate, lysine, inositol, niacin (vitamin B3), vitamin B6, vitamin B12, citric acid, etc.

[0156] For reconstituteable foods, typical examples are reconstituteable milk powder products, such as infant formula, adult milk powder, and milk powder for middle-aged and elderly people.

[0157] In addition, there are no particular restrictions on functional nutritional supplements, which can be used as nutritional supplements or meal replacements. Such foods may include one or more of the following components in addition to the two components of this invention: plant product ingredients, animal dairy product ingredients, animal meat product ingredients, protein ingredients, vitamin supplements, mineral supplements, nucleotide supplements, polyunsaturated fatty acid supplements, and any food-acceptable excipients.

[0158] Plants or plant extracts, including fruits such as fig, pomegranate, kiwi, orange, tangerine, pineapple, strawberry, apple, rubber, grape, pear, cherry, blueberry, blackberry, blackcurrant, cranberry, raspberry, melon, amla, and bilberry, or their extracts; fruits and vegetables such as onion, cucumber, tomato, cauliflower, carrot, spinach, kale, Brussels sprouts, garlic, basil, and oregano, or their extracts; grains such as rice (indica, japonica, glutinous rice), cereals (wheat, barley, oats, rye), corn, sorghum, millet, foxtail millet, yellow millet, buckwheat, soybean, broad bean, pea, mung bean, red bean, and kidney bean, or their extracts; nuts such as walnut, pistachio, cashew, hazelnut, almond, apricot kernel, pine nut, peanut, sunflower seed, chestnut, macadamia nut, and ginkgo, or their extracts; and coffee or its extracts.

[0159] Animal-derived ingredients, including meat products from cattle, sheep, fish, or poultry.

[0160] The fat component may include at least one of saturated fatty acids, polyunsaturated fatty acids, monounsaturated fatty acids, OPO structured lipids, DHA, EPA, ARA, and phospholipids. More specifically, the fat includes safflower seed oil, walnut oil, peanut oil, corn oil, soybean oil, argan oil, olive oil, tea oil, sacha inchi oil, coconut oil, perilla oil, deep-sea fish oil, cocoa butter, palm oil, tallow, cream, lard, medium-chain triglycerides, or lecithin, etc.

[0161] Functional additives include vitamins (one or more of vitamin A, beta-carotene, vitamin D3, vitamin E, vitamin K1, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, pantothenic acid, folic acid, niacin, choline, inositol, and biotin), starch, modified starch, amino acids (L-lysine-L-glutamic acid, L-glutamic acid, L-arginine, L-tryptophan, L-glutamine, taurine, L-valine, L-isoleucine, or L-leucine, etc.), traditional Chinese medicine or its extracts, and dietary fiber (inulin, konjac flour, galactooligosaccharides, fructooligosaccharides, isomaltooligosaccharides, soybean polysaccharides, cyclodextrin, resistant dextrin, or soybean fiber, etc.).

[0162] The protein component is selected from at least one of whey protein powder, soy protein isolate, whole milk powder, whole egg powder, lactoferrin, bovine colostrum, amino acids, and protein peptides; and the amino acid is selected from at least one of L-lysine, L-glutamic acid, L-arginine, L-tryptophan, L-glutamine, taurine, L-valine, L-isoleucine, and L-leucine; the protein peptide is selected from one or more of soy oligopeptides, wheat protein peptides, silkworm pupa protein peptides, marine fish oligopeptide powder, cola peptides, amino peptides, and ovalbumin peptides.

[0163] Micronutrient supplements may include metal ion salts of organic acids, such as one or more of the following: calcium citrate, L-calcium lactate, calcium hydrogen phosphate, potassium gluconate, sodium citrate, ferrous gluconate, potassium iodide, zinc gluconate, sodium selenite, copper gluconate, chromium sulfate, manganese gluconate, and magnesium gluconate.

[0164] Any food additives that are acceptable, including but not limited to solvents, antioxidants, antibacterial agents, thickeners, diluents, solubilizers, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, food flavorings, and food colorings.

[0165] There is no particular limitation on the specific form of the nutritional / health food of the present invention, which can be solid (powder or block, etc.), semi-solid (soft, paste or thick, etc.) or liquid.

[0166] This invention does not impose any particular limitations on specific foods that can be prepared using the above-mentioned nutritional composition and have the effect of promoting the functional maturation of nerve cells, such as promoting synaptic connections, promoting the differentiation of neural stem cells, improving the stability of the cytoskeleton, and / or promoting the maturation of neurons.

[0167] In some implementations, the food is a solid dairy product or a liquid dairy product; for example, milk powder, cheese, yogurt, liquid milk, etc.

[0168] In some specific implementations, the nutritional or health food can be a powdered reconstituteable food (solid beverage, milk powder, instant coffee, cereal powder, nut powder or lotus root powder, etc.) or a liquid beverage (carbonated beverage, fruit and vegetable juice beverage, functional beverage, tea beverage, milk beverage or alcoholic beverage, etc.).

[0169] Typically, the aforementioned foods include: infant formula, maternal formula, children's formula, or nutritional or dietary supplements.

[0170] <Third aspect>

[0171] This invention also provides the use of a composition containing the aforementioned lipid component and the aforementioned sialic acid oligosaccharide component, both essential components, in the preparation of a nutritional composition for non-therapeutic purposes that regulates the differentiation of neural stem cells into specific nerve cells (neurons) and / or promotes the functional maturation of neurons. This invention further provides the use of a composition containing the aforementioned lipid component and the aforementioned sialic acid oligosaccharide component, both essential components, in the preparation of a food product that regulates the differentiation of neural stem cells into specific nerve cells (neurons) and / or promotes the functional maturation of neurons.

[0172] In some specific embodiments, the nutritional composition or food can regulate the differentiation of neural stem cells into specific nerve cells (neurons) and / or promote the functional maturation of neurons.

[0173] In some specific implementations, the regulation of neural stem cell differentiation into specific neural cells (neurons) and the promotion of neuronal functional maturation include at least one of the following: promoting neural stem cell differentiation into specific neuronal lineages (such as neuronal type), promoting myelin development, promoting axonal extension, enhancing cytoskeleton stability, and / or enhancing neuronal morphological development.

[0174] In some specific embodiments, the nutritional composition can enhance cytoskeleton stability and neuronal morphological development, and promote synaptic connection formation efficiency and neural network construction level.

[0175] In some embodiments, the nutritional composition can promote the expression of neuron-specific β-III tubulin, which, as a neuron-specific cytoskeletal protein, plays a key role in the differentiation of neural stem cells into neurons and the formation of neurites, further promoting early structural development of neurons.

[0176] In some embodiments, the nutritional composition can also promote the expression of synaptophysin, a presynaptic vesicle marker. Synaptophysin is a presynaptic vesicle membrane protein that reflects the number and distribution of synaptic vesicles. Promoting its expression indicates that it promotes neuronal synapsis and increases the number of synapses.

[0177] In some specific embodiments, the nutritional composition can promote neural stem cell differentiation, for example, by regulating neuronal dendritic differentiation, synapse formation, and synaptic plasticity.

[0178] In some embodiments, the composition can increase the expression of brain-derived neurotrophic factor (BDNF), demonstrating that the nutritional composition can enhance the differentiation drive of nerve cells and maintain the stability of neural development by regulating neurotrophic signaling pathways.

[0179] In some specific implementations, the nutritional composition can promote myelination and neuronal functional maturation.

[0180] In some embodiments, the nutritional composition can promote the expression of myelin-associated glycoprotein (MAG), demonstrating that the nutritional composition not only promotes early neuronal differentiation but also contributes to the early maturation of neurons and the formation of myelin, thereby supporting the development of more complete neural network functions.

[0181] In some specific embodiments, the nutrient composition can promote axonal elongation, which contributes to neuronal morphological development and the establishment of neural network structures and supports neural development.

[0182] In some implementations, the specific selection and ratio of the oil component and the sialic acid oligosaccharide component are as described in the preceding <First Aspect>.

[0183] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0184] (1) From exogenous induction to synergistic transformation of nutrient signals

[0185] Existing technologies often use growth factors (such as epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2)) or small molecule chemical signals to simulate in vivo signaling pathways to induce differentiation, which have problems such as poor safety and difficulty in transformation. This invention is the first to achieve differentiation induction at the nutritional level through the synergistic effect of DHA, ARA and SLs, which has a higher safety and edibility basis.

[0186] (2) Establish neuron-specific differentiation pathways

[0187] By enhancing indicators such as Tuj-1, Synaptophysin, and BDNF, it was clarified that the differentiation direction mainly leans towards the neuronal lineage, which is different from the traditional "non-specific differentiation" method that does not distinguish cell types.

[0188] (3) Evaluation based on both morphology and function

[0189] Traditional methods often use single protein expression as an indicator. This invention combines fluorescence imaging, synapse length quantification, and myelin-associated protein MAG expression to form a complete structure-function dual verification system.

[0190] (4) The synergistic mechanism is clear

[0191] This study proposes for the first time a synergistic model involving DHA (membrane fluidity regulation), ARA (signal transduction stabilization), and SL (glycosylation environment optimization), explaining the complementary role of multiple nutrient signals in cell fate decision-making.

[0192] Example

[0193] 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.

[0194] Test samples

[0195] In the following examples, the DHA to ARA concentration ratio is fixed at 1:1.7, and the concentrations of other components are set proportionally to form multiple nutritional compositions with different ratios. The internal proportions of sialic acid oligosaccharides (SL) are set as follows:

[0196] Table 1. Ratio of sialic acid oligosaccharides (SL) (by mass)

[0197]

[0198] Various nutritional compositions were prepared by mixing DHA, ARA, and SL in the proportions shown in the following examples, as shown in Table 2.

[0199] Table 2 Composition of the nutritional composition

[0200]

[0201] Note: "-" indicates that it has not been added.

[0202] The experimental materials and cell culture procedures used in the following implementation sections are as follows.

[0203] 1. Experimental Materials

[0204] ① Experimental sample

[0205] Docosahexaenoic acid (DHA) lipids (CABIO, content (in C...) 22 H 32 (calculated as O2 triglycerides), w / %≥35), arachidonic acid oil (ARA, content (as C) 20 H 32 O2 triglycerides (w / %≥38.0) (CABIO); 3'-sialyl lactose (GlyCare™ 3SL 9001, DSM), 6'-sialyl lactose (GlyCare™ 6SL 9001, DSM).

[0206] ② Experimental cells and their sources

[0207] Neural stem cells were derived from mice within 24 hours of birth obtained by excision and delivery of C57bL / 6 wild-type pregnant mice. The pregnant mice were purchased from Shanghai Slack Animal Experiment Co., Ltd.

[0208] ③ Consumables and reagents

[0209] Immunostaining permeabilization solution (Triton X-100, P0096, Beyotime), TBSTw (10X, ST673, Beyotime), QuickBlock™ immunostaining blocking solution (P0260, Beyotime), primary immunostaining antibody dilution solution (P0262, Beyotime), secondary immunofluorescence staining antibody dilution solution (P0265, Beyotime), DAPI staining solution (C1006, Beyotime), PBS (BL310A, Bioshop), CoraLite488-conjugated Goat Anti-Mouse IgG (H+L) (SA00013-1, Proteintech), CoraLite594-conjugated Goat Anti-Rabbit IgG (H+L) (SA00013-4, Proteintech), Anti-MAP2 Rabbit pAb (GB115559, Saive Biotech), TUBB3-specific / TUJ1 Monoclonal antibody (66375-1-Ig, Proteintech), Poly-L-lysine (ST509, Beyotime), and complete culture medium for mouse neural stem cells (CM-M139, Pronosai).

[0210] The ELISA kits for brain-derived neurotrophic factor BDNF and myelin-associated glycoprotein (MAG) were purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd.

[0211] (2) Cell culture

[0212] C57bL / 6 wild-type suckling mice within 24 hours were provided by the Animal Experiment Ethics Committee of East China Normal University. First, Ca-free... 2+ and Mg 2+ Hanks' balanced salt solution was prepared and kept in an ice bath. Mice were then anesthetized in a sterile cell culture room, and their brains were quickly removed and placed in the prepared solution. After dissecting the brain and removing the cerebral cortex tissue, it was placed in papain solution and incubated for 15 minutes to allow neurons to detach from the tissue. The cortical tissue was then pipetted several times, placed in an ice bath for 1 minute to settle, and then centrifuged (1200 rpm, 5 minutes) to obtain neural stem cells. An appropriate amount of plating medium was added, and the cells were cultured in a 37°C, 5% CO2 cell culture incubator, with the culture medium changed every 2-3 days. The cells were then placed in cell culture dishes containing poly-L-lysine (PDL) solution, with the cell density controlled at 1×10⁻⁶ cells / mL. 6Approximately 100 cells were placed in an incubator for 4 hours, after which the plating medium was removed. Then, 2 mL of growth medium was added, and the cells were cultured in a 37°C cell culture incubator. The growth status of neural stem cells was observed daily, and the growth medium was changed as needed. The growth medium was changed or partially changed every 3 days. After 3 days, the neurons could be used for related cell experiments.

[0213] Test Example 1: Screening for the optimal concentration of DHA

[0214] After neural stem cell culture, a pre-screening experiment was conducted to determine the effective concentration of the nutrient composition, using DHA concentration as the benchmark, with a concentration range of 20 nM–400 nM. The results showed that cells survived within the concentration range of 20 nM–400 nM, and cell viability was assessed using the CCK-8 assay within this concentration range.

[0215] All cell experiments were repeated at least three times (independent biological replicates), with each replicate containing ≥3 technical replicates. Results are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used for comparisons among multiple groups, followed by pairwise comparisons after Tukey or Dunnett correction; a two-sided p-value < 0.05 was considered statistically significant. Significance indicators are shown in the figures. The results indicate that... Figure 1 The nutrient composition had no effect on cell viability in the range of 20 nM-80 nM and exhibited a dose-dependent promoting effect, with the highest cell viability observed at 80 nM. Therefore, the suitable concentration range for the nutrient composition in subsequent experiments was determined to be 20 nM-80 nM (based on DHA concentration), and 40 nM was used as a representative concentration. Thus, the optimal concentration for DHA intervention in subsequent experiments was set at 40 nM, with other nutrients added in corresponding proportions.

[0216] Test Example 2: Neuron-specific β-III tubulin expression promotes the expression of neuronal characteristic proteins; Synaptophysin promotes synapse formation and neural network construction.

[0217] Neuron-specific β-III tubulin (labeled with Tuj-1 antibody), as a neuron-specific cytoskeletal protein, plays a crucial role in the differentiation of neural stem cells into neurons and the formation of neurites, and is an important indicator for evaluating early neuronal structural development. Synaptophysin is a presynaptic vesicle membrane protein that reflects the number and distribution of synaptic vesicles; higher expression levels indicate more active neuronal synapsis and a greater number of synapses. Dual-labeled fluorescence imaging with Tuj-1 and Synaptophysin showed that synaptic colocalization signals were more concentrated in a certain ratio, and synaptic length and branch number were higher than in the control group, indicating a significant improvement in synaptic connection formation efficiency and neural network construction level.

[0218] (1) Experimental steps

[0219] ① Cell Culture and Treatment: Neuronal cells were seeded in pretreated culture dishes and cultured until they reached the differentiation and maturation stage. After differentiation, the cells were gently washed three times with cold PBS buffer to remove residual culture medium.

[0220] ② Fixation and permeabilization: Cells were fixed with 4% paraformaldehyde (PFA) aqueous solution and incubated at room temperature for 15–20 minutes, followed by washing three times with PBS buffer for 5 minutes each time. The fixed cells were then permeabilized with 0.3% Triton X-100 aqueous solution and incubated at room temperature for 10 minutes, followed by washing three times again with PBS buffer.

[0221] ③ Blocking and antibody incubation: Cells were blocked with 5% bovine serum albumin (BSA) at room temperature for 1 hour to reduce non-specific binding. Then, rabbit primary antibodies of Tuj-1 and Synaptophysin were added and incubated overnight at 4°C.

[0222] ④ Incubation with fluorescently labeled secondary antibody: The next day, wash cells three times with PBS buffer for 5 minutes each time, add fluorescently labeled secondary antibody, and incubate at room temperature for 1 hour in the dark. After incubation, wash three more times with PBS buffer, then stain the nuclei with DAPI staining solution (1 μg / mL), incubate at room temperature in the dark for 5 minutes, and finally wash three times with PBS buffer.

[0223] ⑤ Sample mounting and imaging: Mount the samples with anti-fluorescence quenching mounting medium and observe them under a confocal fluorescence microscope. Select appropriate excitation and detection wavelengths for each dye and adjust the microscope parameters to obtain the best imaging results. Use multi-channel imaging to simultaneously observe the fluorescence signals of β-III tubulin (Tuj-1 antibody labeled) and synaptophysin, as well as the nuclear fluorescence signals of DAPI-stained cells.

[0224] ⑥ Data Acquisition and Analysis: High-resolution images were acquired using confocal fluorescence microscopy to record the expression distribution and cell morphology characteristics of β-III tubulin (labeled with Tuj-1 antibody), synaptophysin, and DAPI. Fluorescence intensity was quantitatively analyzed using image processing software, and the expression levels of Tuj-1 and synaptophysin were assessed by comparing experimental groups.

[0225] ⑦ Data Processing and Graphing: Relevant graphs of the experimental results were arranged into a series of graphs using software.

[0226] (2) Experimental results

[0227] Depend on Figure 2 , 3It was found that the fluorescence intensity of Tuj-1 and Synaptophysin in the mixture of DHA, ARA, and SLs (Examples 1-3) was higher than that in the DHA, ARA, and single-factor groups (Comparative Example 1) and the SLs single-factor groups (Comparative Examples 2-4), indicating that the mixture promoted cytoskeleton stability and simultaneously enhanced neuronal morphological development through the synergistic effect of nutrient signals. Furthermore, when both nutrients were present, with the amount of fixed DHA and ARA remaining constant, the expression of Tuj-1 and Synaptophysin showed a trend of "first increasing and then decreasing" with the increase of SLs population content, reaching its maximum value when the SLs population ratio was 1.5 (Example 2).

[0228] Figure 4 Fluorescence confocal imaging showed that neural stem cells (NSCs) under uninterrupted conditions (control group) exhibited a low natural differentiation rate and extremely weak expression of Tuj-1 and Synaptophysin. However, after intervention with the nutrient composition, the morphology of neurons was more complete and their density higher than that of the single-factor group, with overall enhanced fluorescence intensity and significantly improved differentiation efficiency (Examples 1-3). When the proportion of SLs was 1.5 (Example 2), neurons showed significant process elongation and increased branching, with Synaptophysin fluorescent spots regularly distributed along the processes and clustering at synaptic sites. Further increases in SL concentration resulted in a significant decrease in fluorescence intensity, suggesting that there is an optimal concentration range for the nutrients in the nutrient composition.

[0229] Test Example 3: Increased expression of neurotrophic factors drives differentiation process

[0230] Brain-derived neurotrophic factor (BDNF) plays a crucial role in regulating neuronal dendritic differentiation, synapse formation, and synaptic plasticity. As an important driver of neural differentiation and synaptic plasticity, its upregulated expression indicates that this nutrient composition can enhance the differentiation-driving force of nerve cells and maintain the stability of neural development by regulating neurotrophic signaling pathways.

[0231] (1) Experimental steps

[0232] The expression level of brain-derived neurotrophic factor BDNF in neuronal cells was detected using enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows:

[0233] ① Sample preparation: After culturing neuronal cells and differentiating to the predetermined time point, wash the cells three times with cold PBS buffer to remove residual culture medium. Add an appropriate amount of cell lysis buffer (containing protease inhibitor), lyse the cells at 4°C for 30 minutes, then centrifuge at 12000 rpm for 10 minutes, collect the supernatant as the protein sample, and store it at -80°C for later use.

[0234] ② ELISA kit preparation: Using BDNF's specific ELISA kit, bring all reagents to room temperature and prepare a gradient concentration of standards.

[0235] ③ Testing steps:

[0236] 1) Add the standard and sample to the 96-well plate, 100 μL per well, and set up the standard curve gradient and blank control.

[0237] 2) Add the capture antibody and incubate at room temperature for 2 hours. Then wash the plate 3 times, using 300 μL of washing buffer each time.

[0238] 3) Add biotin-labeled BDNF detection antibody, incubate for 1 hour, and then repeat the washing step.

[0239] 4) Add streptavidin-horseradish peroxidase (HRP), incubate at room temperature for 30 minutes, and then wash the well plate.

[0240] 5) Add substrate solution (TMB), incubate in the dark for 15 minutes, and then add stop solution to stop the reaction.

[0241] ④ Data acquisition and analysis: Use an ELISA reader to read the absorbance value (OD value) of each well at a wavelength of 450nm. According to the kit instructions, use a series of standard concentrations to plot a standard curve, and calculate the concentration of BDNF in the sample accordingly.

[0242] (2) Experimental results

[0243] ① Effects of the ratio of various nutritional compositions on the expression of brain-derived neurotrophic factor BDNF

[0244] To assess the effects of multiple nutrient compositions relative to single nutrients, and the effects of different nutrient composition ratios on the proliferation and differentiation of neural stem cells, a BDNF kit was used for quantitative analysis. Results are shown below. Figure 5 .

[0245] As shown in the figures, the expression level of BDNF in the composition (Examples 1-3) was significantly better than that of any single factor (Comparative Examples 1-4). With the DHA and ARA ratio fixed, BDNF expression showed a trend of "first increasing and then decreasing" with the concentration of SLs (Solar Roots), reaching its maximum at an SLs ratio of 1.5, suggesting a dose-dependent optimal ratio. Furthermore, in the composition, DHA+ARA played a dominant role in neural stem cell differentiation (Comparative Example 1), suggesting that DHA and ARA provide the main driving force for neuronal structural maturation.

[0246] Table 3 also shows the increase in BDNF expression levels in each group compared to the blank control. Taking Example 1 ((DHA, ARA) + low SLs) as an example, the increase in BDNF expression compared to the blank control was 69, which is 7.6 (69-47.2-14.2=7.6) compared to the sum of single factors. Similarly, the BDNF expression levels in each combination group of Examples 2 and 3 were greater than the sum of single factors, indicating that DHA, ARA, and SLs can synergistically increase BDNF expression, better promote neural stem cell proliferation and differentiation, and provide a good driving force for neuronal structural maturation. Among them, the BDNF combination in Example 2 showed the largest increase compared to the sum of single factors, at 7.9.

[0247] Table 3. Comparison of expression of brain-derived neurotrophic factor BDNF in each group

[0248]

[0249] Note: "-" indicates that it has not been added.

[0250] Test Example 4: Promotes the expression of myelination-related proteins and supports neural maturation.

[0251] Myelin-associated glycoproteins (MAGs) are involved in axonal myelination and are one of the important markers of neuronal functional maturation. These results indicate that the present invention not only promotes early neuronal differentiation but also contributes to earlier neuronal maturation and myelination, thereby supporting more complete neural network functional development.

[0252] (1) Experimental steps

[0253] The expression level of myelin-associated glycoprotein MAG in neuronal cells was detected using enzyme-linked immunosorbent assay (ELISA). The specific steps were the same as those in test example 3 (1).

[0254] (2) Experimental results

[0255] ① Effects of the ratio of various nutrient compositions on the expression of myelin-associated glycoprotein MAG

[0256] To assess the effects of multiple nutrient compositions relative to single nutrients, and the effects of different nutrient composition ratios on the proliferation and differentiation of neural stem cells, a MAG reagent kit was used for quantitative analysis. Results are shown below. Figure 6 .

[0257] As shown in the figure, the MAG expression levels of the compositions (Examples 1-3) were higher than those of the single factors (Comparative Examples 1-4). With a fixed SLs ratio, MAG expression exhibited a "slow increase followed by a decrease" trend with SLs concentration, reaching a high value at an SLs ratio of 1.5, suggesting a possible dose-dependent optimal ratio. Furthermore, the comparative results showed that DHA+ARA played a dominant role in neural stem cell differentiation (Comparative Example 1), while the SLs (Comparative Examples 2-4) provided a synergistic enhancing effect.

[0258] Table 4 also shows the increase in MAG expression levels in each group compared to the blank control. Taking Example 1 ((DHA, ARA) + low SLs) as an example, the increase in MAG compared to the blank control was 56.4, which is 20.2 higher than the sum of single factors (56.4 - 34.6 - 1.6 = 20.2). Similarly, the MAG expression levels of each nutritional combination in Examples 1-3 were greater than the sum of single factors, indicating a synergistic effect between DHA, ARA, and SLs, which can synergistically increase MAG expression levels. This suggests that while structural development is enhanced, neurons may be developing towards myelination and functional maturation.

[0259] Table 4. Comparison of expression of myelin-associated glycoprotein MAG in each group

[0260]

[0261] Note: "-" indicates that it has not been added.

[0262] Test Example 5: Statistics on the Mean Length of Neurons Based on Fluorescence Microscopy

[0263] Based on the confocal images from the previous test case 2, image analysis software was used to quantitatively measure the average protrusion length of neurons, objectively reflecting the morphological maturation of neurons.

[0264] (1) Experimental steps

[0265] Representative fluorescence confocal images with intact neuronal morphology and complete Tuj-1 staining were selected. Five representative images from each group were randomly selected for analysis. The images were then imported into the software, and one to two neurons with clear, unobstructed axons were randomly selected from each field of view. The measurement line segment was then manually drawn from the center of the neuron cell body to the end of its corresponding axon using the "line segment tool," and the length of each neuron was recorded. A total of 5-10 neurons were measured in each group of images. The measurement results were exported, the mean length was calculated, and one-way ANOVA was used for inter-group comparisons. All data are expressed as mean ± standard deviation.

[0266] (2) Experimental results

[0267] The lengths of neuronal processes were calculated based on the proportions of nutrients used, as shown in Table 5. Statistical analysis revealed that the neuronal length in Example 2 ((DHA, ARA) + SLs) was significantly higher than in other groups (44.0% and 105.9% higher than in Comparative Examples 1 and 3, respectively). This indicates that the nutrient composition can more effectively promote the axonal extension of neural stem cells after differentiation into neurons, contributing to neuronal morphological development and the establishment of neural network structures, and demonstrating a significant supportive effect on neural development.

[0268] Table 5. Statistics on the average length of neurons based on fluorescence microscopy.

[0269]

[0270] Note: "-" indicates that it has not been added.

[0271] Technical effect

[0272] The implementation of the "synergistic regulation of neural stem cell differentiation into specific neural cell types by DHA, ARA and sialic acid-human milk oligosaccharides" described in this invention can significantly promote the differentiation of neural stem cells into neurons under in vitro culture conditions, and exhibit a synergistic enhancement effect at both structural and functional levels.

[0273] ① Promotes the expression of neuronal characteristic proteins: Tuj-1, as a marker of neuronal differentiation, showed significantly upregulated expression levels in the DHA+ARA+SL combined group, suggesting accelerated differentiation of cells into neuronal lineages. Fluorescence imaging showed that the cell morphology was more extended, with longer and thinner processes, exhibiting typical neuronal morphology.

[0274] ② Promotes synapse formation and neural network construction: The Synaptophysin signal intensity and the Tuj-1 / Synaptophysin co-localization area are significantly increased, indicating that this combination can enhance synapse generation and networked connections, laying the foundation for neuronal functionalization.

[0275] ③ Upregulation of neurotrophic factor BDNF levels: Increased BDNF expression is an upstream driver of differentiation signals, demonstrating that this combination can promote the differentiation decisions of neural stem cells and the determination of neuronal fate through nutrient signals.

[0276] ④ Promotes the upregulation of maturation and myelin-related proteins: Increased MAG expression suggests that the tendency of cells to mature and form myelin in the later stages of differentiation is enhanced, reflecting the advancement of the neuronal maturation process.

[0277] ⑤ Significantly prolonged neuronal process length: Morphological quantification results showed that the average process length of neurons in the combined group was 44.0% and 105.9% higher than that in the control group, respectively, further verifying the synergistic promoting effect of structural development and neural network expansion.

[0278] In summary, DHA, ARA, and SL work synergistically at multiple levels to form a continuous mechanism chain from structural feature formation (Tuj-1) to synaptic connection (Synaptophysin) to differentiation signal drive (BDNF) to maturation stabilization (MAG), thereby achieving efficient differentiation of neural stem cells into neurons.

[0279] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0280] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A nutritional composition comprising, The nutritional composition comprises the following essential components: a lipid component and a sialyl-oligosaccharide component; and, in the nutritional composition, the molar ratio of the lipid component and the sialyl-oligosaccharide component is (1-5):(0.01-5); Preferably, the molar ratio of the lipid component and the sialyl-oligosaccharide is (1-3):(0.5-5).

2. The nutritional composition of claim 1, wherein, The lipid component comprises one or more of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids; Preferably, the lipid component comprises polyunsaturated fatty acids; More preferably, the polyunsaturated fatty acids comprise any one or more of alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), linoleic acid (LA), arachidonic acid (ARA), and conjugated linoleic acid (CLA).

3. The nutritional composition according to claim 1 or 2, wherein, The lipid component comprises docosahexaenoic acid and arachidonic acid; Preferably, the molar ratio of docosahexaenoic acid and arachidonic acid in the lipid component is 1:(1-5).

4. The nutritional composition of any one of claims 1-3, wherein, The sialyl-oligosaccharide comprises any one or more of 3'-sialyllactose, 6'-sialyllactose, sialyl-lactose-N-tetraose a, sialyl-lactose-N-tetraose b, sialyl-lactose-N-tetraose c, and disialyl-lactose-N-tetraose, Preferably, the sialyl-oligosaccharide comprises 3'-sialyllactose and 6'-sialyllactose; More preferably, the mass ratio of 3'-sialyllactose and 6'-sialyllactose in the sialyl-oligosaccharide is (1-20):(15-1).

5. A food product, wherein, The food product comprises or uses, as a raw material, the nutritional composition of any one of claims 1-4.

6. The food product of claim 5, wherein, The food product comprises any one or more of infant food, children's food, adolescent food, pregnant and postpartum women's food, adult food, and elderly food.

7. The food product of claim 5 or 6, wherein, The food product further comprises any one or more of the following components: a plant product component, an animal milk product component, an animal meat product component, a functional additive component, and any acceptable auxiliary material.

8. Use of a composition comprising two essential components of a lipid component and a sialyl-oligosaccharide component in the preparation of a nutritional composition for regulating the differentiation of neural stem cells into neurons and / or promoting the functional maturation of neurons.

9. Use of a composition comprising two essential components of a lipid component and a sialyl-oligosaccharide component in the preparation of a food product for regulating the differentiation of neural stem cells into neurons and / or promoting the functional maturation of neurons.

10. Use according to claim 8 or 9, wherein, The regulation of the differentiation of neural stem cells into neurons and / or the promotion of the functional maturation of neurons comprises at least one of promoting the differentiation of neural stem cells into neurons, promoting myelin development, promoting axon extension, enhancing cytoskeletal stability, and / or enhancing the morphological development of neurons.

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