Nutritional composition for promoting neurodevelopment

Abstract

The invention discloses a nutritional composition for promoting neurodevelopment. The invention provides a nutritional composition, the nutritional composition comprises the following necessary components: a grease component, a phospholipid component and a sialylated substance component, and in the nutritional composition, the molar ratio of the grease component to the phospholipid component to the sialylated substance component is (1-5): (1-10): (0.01-5). The composition provided by the invention has the advantages that the components are synergistic, the nerve development is effectively promoted, the application range is wide, and various foods or health-care products can be conveniently added or prepared.

Description

Technical Field

[0001] This invention relates to a nutritional composition, belonging to the food field, and more specifically, to a nutritional composition that promotes nerve development. Background Technology

[0002] Brain development is a highly complex and dynamic biological process involving finely regulated multiple stages, including the proliferation and differentiation of neural stem cells (NSCs), neuronal migration, synapsis, the construction and maturation of neural circuits, synaptic pruning, and the formation of myelin and glial cells. As the core cell population in brain development, the self-renewal capacity and potential for differentiation into neurons and glial cells determine the developmental level of the central nervous system. Increasing research indicates that specific trophic factors play a crucial regulatory role in the proliferation, directed differentiation, and maturation of NSCs by influencing the neural microenvironment and modulating signaling pathways.

[0003] Docosahexaenoic acid (DHA) and arachidonic acid (ARA) among polyunsaturated fatty acids are considered key components of nerve membranes, which can promote neuronal differentiation by regulating cell membrane fluidity, neurotransmitter release, and signaling pathways (cited in references 1-3). Phospholipid nutrients such as phosphatidylcholine (PC) and phosphatidylserine (PS) play important roles in neuronal membrane structure building, synaptic plasticity, and nerve signal transduction (cited in references 4 and 5). Sialic acid oligosaccharides such as 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) are naturally occurring active sugars in breast milk, which can participate in neural development by influencing cell glycosylation modification and regulating synapse formation (cited in references 6 and 7).

[0004] However, the above studies have mostly focused on the mechanism of action of single nutrient factors, and there is a lack of systematic research to explore the synergistic effects among multiple key nutrients and their impact on the entire process of neural stem cell development.

[0005] In response, existing technologies have explored complex nutritional compositions, for example:

[0006] Reference 8 discloses a nutritional composition that can enhance and / or improve an individual's social behavior, wherein by combining DHA, ARA, and phospholipids (sphingomyelin and / or lecithin), the composition and its food products help improve an individual's social behavior. Reference 9 provides a nutritional composition containing phospholipids, their metabolic precursors, and / or their metabolites, for use in infants and young children, designed to promote or support optimal myelination trajectory in the brain, approximating the trajectory observed in infants exclusively breastfed in human milk. Reference 10 provides a mixture containing lipids that promotes brain-derived neurotrophic factor (BDNF) protein expression, with effects of promoting or improving neural development and enhancing learning and memory. Reference 11 discloses a nutritional composition containing phospholipids, milk fat globule membrane protein, and d-α-tocopherol or d-α-tocopherol derivatives, further comprising docosahexaenoic acid and / or arachidonic acid, with effects of promoting brain development and enhancing cognition.

[0007] However, the aforementioned research cannot be considered complete or sufficient. For example, existing nutritional compositions do not target multiple time-series indicators with interconnected effects (e.g., covering key stages of neural stem cell development from proliferation and differentiation, synapse formation, myelin development, neuronal maturation, and network connection formation). Furthermore, considering the construction of scientifically formulated composite nutritional compositions and systematically validating their effects on promoting neural stem cell differentiation and neural network maturation at the cellular level has significant scientific value and application prospects. New nutritional compositions for promoting neural development still need to be developed and researched in this field.

[0008] References

[0009] Cited literature 1 Ma D, Zhang M, Larsen CP, et al. DHA promotes the neuronaldifferentiation of rat neural stem cells transfected with GPR40 gene. BrainRes. 2010;1330:1-8.

[0010] Cited literature 2 Calder PC. n-3 polyunsaturated fatty acids, inflammation, and inflammatory diseases. Am J Clin Nutr. 2006;83(6 Suppl):1505S-1519S.

[0011] Cited literature 3 Yaqoob P, Calder PC. Fatty acids and immune function: new insights into mechanisms. Br J Nutr. 2007;98 Suppl 1:S41-S45.

[0012] Cited literature 4 Kim HY, Huang BX, Spector AA. Phosphatidylserine in thebrain: metabolism and function. Prog Lipid Res. 2014;56:1-18.

[0013] Cited literature 5 Chaung HC, Chang CD, Chen PH, Chang CJ, Liu SH, Chen CC. Docosahexaenoic acid and phosphatidylserine improves the antioxidantactivities in vitro and in vivo and cognitive functions of the developing brain. Food Chem. 2013;138(1):342-347.

[0014] Cited literature 6 Wang B, Brand-Miller J, McVeagh P, Petocz P. Concentration and distribution of sialic acid in human milk and infant formulas. Am J ClinNutr. 2001;74(4):510-515.

[0015] Reference 7: Wang B, Brand-Miller J. The role and potential of sialicacid in human nutrition. Eur J Clin Nutr. 2003;57(11):1351-1369.

[0016] Reference 8 CN119366561A

[0017] Reference 9 CN120203237A

[0018] Reference 10 CN117678641A

[0019] Reference 11 CN115191604B Summary of the Invention

[0020] The problem the invention aims to solve

[0021] Current research on nutritional factors crucial for neural stem cell differentiation and development, which are essential for brain development, largely focuses on the effects of single nutrients (such as DHA or PS alone), neglecting their potential "synergistic network effects" during neural development. Furthermore, systematic, multi-indicator cell experiments are lacking to validate the different sites, characteristics, and stages of neural cell differentiation and development, particularly regarding the roles of key neural factors such as BDNF, MAG, and MAP-2 at different stages of neuronal development. In addition, in actual product development, the proportions of various functional components are often not precisely defined, potentially leading to inefficient formulation design and unstable efficacy.

[0022] Therefore, this invention aims to provide a nutritional composition based on lipid components, phospholipid components, and sialylated substances. Cell experiments have demonstrated that it has a significant synergistic effect under specific ratios, which can significantly improve the differentiation efficiency and maturity of neural stem cells. In particular, it is far superior to the effect of using each component alone in terms of the expression of neurotrophic factors such as BDNF and NGF. This achieves synergistic effects among multiple nutrient factors, thereby significantly promoting the development and differentiation of neural cells and providing a better nutritional support solution for the brain development of infants and young children.

[0023] Solution for solving the problem

[0024] [1]. A nutritional composition, wherein the nutritional composition comprises the following essential components:

[0025] Oil components include one or more of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids;

[0026] Phospholipid components, including one or more of phosphatidylcholine (PC) and / or its metabolic precursors, phosphatidylethanolamine (PE) and / or its metabolic precursors, phosphatidylserine (PS) and / or its metabolic precursors, phosphatidylinositol (PI) and / or its metabolic precursors, phosphatidylglycerol (PG) and / or its metabolic precursors, diphosphatidylglycerol (DPG) and / or its metabolic precursors, and sphingomyelin (SM) and / or its metabolic precursors;

[0027] Sialidized components include one or more of N-acetylneuraminic acid, sialic acid oligosaccharides, sialylated proteins, and sialylated lipids;

[0028] Furthermore, in the nutritional composition, the molar ratio of the oil component, the phospholipid component, and the sialic acidified substance component is (1-5): (1-10):(0.01-5);

[0029] Preferably, the molar ratio of the oil component, the phospholipid component, and the sialic acidification component is (1-3): (1-5): (0.5-2);

[0030] More preferably, the molar ratio of the oil component, the phospholipid component, and the sialic acidification component is (1-3): (1-3):(0.5-2).

[0031] [2]. According to the nutritional composition described in [1], wherein the oil component comprises polyunsaturated fatty acids, the polyunsaturated fatty acids comprising 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).

[0032] Preferably, the oil components include docosahexaenoic acid and arachidonic acid;

[0033] More preferably, the molar ratio of docosahexaenoic acid to arachidonic acid in the oil component is 1:(1~5).

[0034] [3]. The nutritional composition according to [1] or [2], wherein the sialylated component comprises sialic acid oligosaccharides, wherein the sialic acid oligosaccharides comprise 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.

[0035] Preferably, the sialylated substance component includes 3'-sialylated lactose and 6'-sialylated lactose;

[0036] More preferably, the mass ratio of 3'-sialylated lactose to 6'-sialylated lactose in the sialylated substance component is (1~20): (15~1).

[0037] [4]. The nutritional composition according to any one of [1] to [3], wherein the phospholipid component comprises phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) and sphingomyelin (SM).

[0038] Preferably, the phospholipid components, based on the total mass of the phospholipids, include 15-50% by mass of phosphatidylethanolamine (PE), 15-40% by mass of phosphatidylcholine (PC), 15-40% by mass of sphingomyelin (SM), 1-20% by mass of serine phospholipid (PS), and 3-15% by mass of phosphatidylinositol (PI).

[0039] [5]. A food product, wherein the food product comprises or uses the nutritional composition described in any one of [1] to [4] as a raw material.

[0040] [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.

[0041] [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.

[0042] [8]. Application of a composition containing three essential components—lipid component, phospholipid component and sialic acidified substance component—in the preparation of a nutritional composition that promotes nerve development.

[0043] [9]. Application of a composition containing three essential components—lipid component, phospholipid component and sialic acid component—in the preparation of foods that promote neurodevelopment.

[0044]

[10] . According to the use described in [8] or [9], wherein the promotion of neural development includes at least one of promoting neural cell proliferation and differentiation, promoting synaptic connections, promoting myelin development and / or promoting neuronal maturation.

[0045] The effects of the invention

[0046] This invention constructs a systematic functional evaluation index system focusing on the entire process of neural stem cell differentiation into neurons and the gradual formation of mature neural networks. Through six categories of experimental indicators, arranged chronologically and in a coordinated manner, it covers the key stages of neural stem cell development from proliferation and differentiation → synapse construction → myelin sheath development → neuronal maturation → network connection formation, systematically verifying the effects and mechanisms of action of different proportions of nutrient compositions on each stage of development. Unlike existing technologies that focus on the evaluation of a single component or single indicator, this invention:

[0047] 1) The present invention provides a composition for promoting nerve development, comprising an oil component, a phospholipid component and a sialic acidified substance component, which can effectively promote nerve development, and the components synergistically promote nerve development.

[0048] 2) The compositions provided by this invention can promote myelin development and / or myelin maturation. Specifically, the compositions provided by this invention show great promise in promoting brain development, and play an important role in various cellular mechanisms during phylogenetic development, such as promoting the proliferation and differentiation of neural stem cells, promoting synapse construction, promoting myelin development, promoting neuronal maturation, and promoting the formation of neural network connections.

[0049] 3) In in vitro models, cell cultures treated with the composition provided by the present invention promote the proliferation and differentiation of neural stem cells, promote synapse construction, promote myelin development, and promote neuronal maturation.

[0050] 4) The composition of the present invention was fully verified by a cross-stage, multi-dimensional evaluation system covering five key links: neural stem cell proliferation, differentiation, synaptic connection, myelin development and neural network maturation.

[0051] 5) By employing multiple techniques such as protein quantification, fluorescence imaging, and morphological measurement, experiments were conducted to verify that the scientific combination of sialic acidified substances, phospholipids, and lipids exhibited synergistic effects at multiple developmental stages, improving the quality of nerve cell development and providing experimental evidence for the composition ratio and functional verification of early intervention in infant brain development.

[0052] 6) 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

[0053] Figure 1 The results show the cell viability of neural stem cells after co-culturing with samples of different concentrations.

[0054] The image shows " " indicates P < 0.05, " " indicates P < 0.001.

[0055] Figure 2 The expression of brain-derived neurotrophic factor BDNF in each group.

[0056] Figure 3 The expression of nerve growth factor (NGF) in each group.

[0057] Figure 4 The expression of microtubule-associated protein 2 (MAP-2) in each group.

[0058] Figure 5 The expression of the myelin-associated glycoprotein MAG in each group.

[0059] Figures 6A-6CFluorescence confocal imaging of neuron-specific β-III tubulin (labeled with Tuj-1 antibody) and synaptophysin in cells after co-culturing with various nutrient compositions and neural stem cells. Figure 6A Fluorescence confocal imaging of neuron-specific β-III tubulin (labeled with Tuj-1 antibody) and synaptophysin in cells after co-culturing the nutrient composition with neural stem cells in this example. Figure 6B Fluorescence confocal imaging of neuron-specific β-III tubulin (labeled with Tuj-1 antibody) and synaptophysin in neural stem cells after co-culture with comparative nutritional compositions; Figure 6C for Figure 6A and Figure 6B Fluorescence confocal imaging of neuron-specific β-III tubulin (labeled with Tuj-1 antibody) and synaptophysin in cells after co-culturing with nutrient compositions from representative groups in the middle part of the study. Detailed Implementation

[0060] 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.

[0061] 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.

[0062] 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.

[0063] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] In this invention, the term "infant" is used to refer to the human group aged 0 to 6 months.

[0068] In this invention, the term "older infant" refers to the human group aged 6 to 12 months.

[0069] In this invention, the term "infant" is used to refer to the human group aged 12 to 36 months.

[0070] In this invention, the term "infant" refers to the human group under the age of 3 years.

[0071] 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.

[0072] In this manual, the term "adult" refers to a person who is 18 years of age or older.

[0073] In this manual, the term "teenager" refers to people aged 7-40.

[0074] In this specification, the terms "optional" or "optional" are used to indicate the use or omission of certain substances, components, procedures, application conditions, etc.

[0075] <First Aspect>

[0076] 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; a phospholipid component, including one or more of phosphatidylcholine (PC) and / or its metabolic precursor, phosphatidylethanolamine (PE) and / or its metabolic precursor, phosphatidylserine (PS) and / or its metabolic precursor, phosphatidylinositol (PI) and / or its metabolic precursor, phosphatidylglycerol (PG) and / or its metabolic precursor, diphosphatidylglycerol (DPG) and / or its metabolic precursor, and sphingomyelin (SM) and / or its metabolic precursor; and a sialylated substance component, including one or more of N-acetylneuraminic acid, sialic acid oligosaccharides, sialylated proteins, and sialylated lipids; and wherein, in the nutritional composition, the molar ratio of the oil component, the phospholipid component, and the sialylated substance component is (1-5):(1-10):(0.01-5).

[0077] In some preferred embodiments, the molar ratio of the oil component, the phospholipid component, and the sialic acidification component is (1-3): (1-5): (0.5-2).

[0078] In some more preferred embodiments, the molar ratio of the oil component, the phospholipid component, and the sialic acidification component is (1-3): (1-3):(0.5-2).

[0079] The nutritional composition described in this invention can promote the development of the nervous system. Specifically, the nutritional composition of this invention can promote the development of the nervous system in many aspects, including the proliferation and differentiation of neural stem cells, the formation of synapses, the development of myelin sheaths, the maturation of neurons, and the formation of neural network connections.

[0080] (Oil and fat components)

[0081] In some embodiments, the oil component includes one or more of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids.

[0082] 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).

[0083] In some preferred embodiments, the oil components include docosahexaenoic acid and arachidonic acid.

[0084] docosahexaenoic acid

[0085] 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.

[0086] In this specification, the terms "docosahexaenoic acid" and "DHA" are used interchangeably.

[0087] Arachidonic acid

[0088] 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: .

[0089] In this specification, the terms "arachidonic acid" and "ARA" are used interchangeably.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] In some embodiments, the molar ratio of docosahexaenoic acid (DHA) to arachidonic acid (ARA) in the oil component is 1:(1~5).

[0094] In some preferred embodiments, the molar ratio of docosahexaenoic acid (DHA) to arachidonic acid (ARA) in the oil component is 1:(1~3).

[0095] 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).

[0096] 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.

[0097] In some exemplary embodiments, the mass ratio of docosahexaenoic acid (DHA) to arachidonic acid (ARA) in the oil component may be 1:1.7.

[0098] The oil components of the present invention can exist in a liquid state, or in a semi-solid or solid state.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] In some exemplary embodiments, the concentration of arachidonic acid (ARA) in the nutritional composition is 68 nM.

[0106] 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 promote nerve cell development.

[0107] (Phospholipid components)

[0108] Phospholipids are a collective term for the various components of phospholipids.

[0109] Phospholipids, also known as phospholipid derivatives or phospholipid lipids, are lipids containing phosphoric acid and belong to the category of complex lipids. Phospholipids are composed of five elements: C, H, O, N, and P. They are major components of biological membranes, characterized by producing a mixture containing fatty acids and phosphoric acid upon hydrolysis. Phospholipids are amphoteric molecules, with one end being a hydrophilic nitrogen- or phosphorus-containing head and the other end being a hydrophobic (lipophilic) long hydrocarbon chain. For this reason, the hydrophilic ends of phospholipid molecules are close to each other, and the hydrophobic ends are close to each other. They often form the phospholipid bilayer, i.e., the structure of the cell membrane, together with other molecules such as proteins, glycolipids, and cholesterol.

[0110] In some embodiments, the phospholipids include glycerophospholipids and / or sphingomyelin.

[0111] In some preferred embodiments, the phospholipids include glycerophospholipids and sphingomyelins.

[0112] In some embodiments, the glycerophospholipids include one or more selected from: phosphatidylcholine and / or its metabolic precursor (also known as lecithin) (PC), phosphatidylethanolamine and / or its metabolic precursor (also known as cephalin) (PE), serine phospholipids and / or their metabolic precursors (PS), phosphatidylglycerol and / or its metabolic precursors (PG), diphosphatidylglycerol and / or its metabolic precursors (DPG), and phosphatidylinositol and / or its metabolic precursors (PI).

[0113] In some embodiments, the present invention does not particularly limit the metabolic precursors of the phosphate component. Examples of such precursors include at least one of glycerol-3-phosphate, phosphatidic acid, diglyceride, CDP-choline, CDP-ethanolamine, and CDP-diglyceride. In some preferred embodiments, the glycerophospholipids include phosphatidylcholine (PC), phosphatidylethanolamine (also known as cephalin) (PE), serine phospholipid (PS), and phosphatidylinositol (PI).

[0114] In some preferred embodiments, the phospholipids include phosphatidylcholine (PC), phosphatidylethanolamine (PE), serine phospholipids (PS), phosphatidylinositol (PI), and sphingomyelin (SM).

[0115] In some embodiments, the phospholipids may optionally further include other edible phospholipids, such as one or more of phosphatidylglycerol, diphosphatidylglycerol (also known as cardiolipin), etc.

[0116] In some embodiments, the phospholipid is a plant-derived phospholipid and / or an animal-derived phospholipid.

[0117] In some embodiments, the phospholipid may be a plant-derived phospholipid.

[0118] In some embodiments, the phospholipid may be an animal-derived phospholipid.

[0119] In some preferred embodiments, the phospholipids may be plant-derived phospholipids or animal-derived phospholipids.

[0120] In some embodiments, the plant-derived phospholipids are one or more plant phospholipids derived from soybean, and / or coconut, and / or flaxseed, and / or walnut, and / or sunflower seeds.

[0121] In some embodiments, the plant-derived phospholipids may be provided from one or more of soybean oil, and / or coconut oil, and / or flaxseed oil, and / or walnut oil, and / or soybean phospholipids, and / or sunflower phospholipids, and / or sunflower seed oil, or food ingredients made therefrom.

[0122] This invention does not impose any particular limitation on the source of phospholipids.

[0123] In some embodiments, the phospholipid or plant-derived phospholipid may be soybean phospholipid.

[0124] In some preferred embodiments, the phospholipid or plant-derived phospholipid may be soybean-derived phosphatidylcholine (or soybean lecithin).

[0125] In some preferred embodiments, the animal-derived phospholipids are one or more phospholipids derived from cow's milk, and / or sheep's milk, and / or egg yolk.

[0126] Animal-derived phospholipids and plant-derived phospholipids contain glycerophospholipids with similar or identical components and compositions.

[0127] In some exemplary embodiments, phospholipids can be commercially obtained by those skilled in the art, such as food-grade soybean phosphatidylcholine, L-α-phosphatidylethanolamine, L-α-phosphatidylinositol, sphingomyelin, and L-α-phosphatidylserine sold by Shanghai Zhenzhun Biotechnology, but are not limited thereto.

[0128] In some embodiments, the phospholipids comprise, by weight, 15-50% phosphatidylethanolamine (PE), 15-40% phosphatidylcholine (PC), 15-40% sphingomyelin (SM), 1-20% serine phospholipid (PS), and 3-15% phosphatidylinositol (PI).

[0129] In some preferred embodiments, the phospholipids comprise, by weight of total mass, 20-45% phosphatidylethanolamine (PE); 20-35% phosphatidylcholine (PC); 20-30% sphingomyelin (SM); 5-15% serine phospholipid (PS); and 5-10% phosphatidylinositol (PI).

[0130] In some preferred embodiments, the phospholipids comprise, by weight, 25-35% phosphatidylethanolamine (PE); 20-30% phosphatidylcholine (PC); 23-28% sphingomyelin (SM); 8-12% serine phospholipids (PS); and 5-10% phosphatidylinositol (PI).

[0131] In some specific embodiments, the phospholipids comprise 25%, 27%, 30%, 33%, or 35% by mass of phosphatidylethanolamine (PE) based on the total mass of the phospholipids.

[0132] In some specific embodiments, the phospholipids comprise 20%, 23%, 25%, 27%, or 30% by mass of phosphatidylcholine (PC) based on the total mass of the phospholipids.

[0133] In some specific embodiments, the phospholipids comprise 23%, 24%, 25%, 26%, 27%, or 28% by mass of sphingomyelin (SM) based on the total mass of the phospholipids.

[0134] In some specific embodiments, the phospholipids comprise 8%, 9%, 10%, 11%, or 12% by mass of serine phospholipids (PS) based on the total mass of the phospholipids.

[0135] In some specific embodiments, the phospholipids comprise 5%, 6%, 7%, 8%, 9%, or 10% by mass of phosphatidylinositol (PI) based on the total mass of the phospholipids.

[0136] In some exemplary embodiments, the phospholipids comprise, by weight, 30% phosphatidylethanolamine (PE), 27% phosphatidylcholine (PC), 25% sphingomyelin (SM), 10% serine phospholipid (PS), and 8% phosphatidylinositol (PI).

[0137] The phospholipid components of the present invention can exist in a liquid state, or in a semi-solid or solid state.

[0138] When the phospholipid component of the present invention is present in a liquid state, in some preferred embodiments, the concentration of the phospholipid component in the nutritional composition is between 10 nM and 500 nM.

[0139] In some preferred embodiments, the concentration of the phospholipid component in the nutritional composition is between 40 nM and 300 nM, for example: 43 nM, 44 nM, 45 nM, 87 nM, 88 nM, 89 nM, 115 nM, 116 nM, 117 nM, 187 nM, 188 nM, 189 nM, etc.

[0140] In some further preferred embodiments, the concentration of phosphatidylethanolamine (PE) in the nutritional composition is between 5 nM and 70 nM; the concentration of phosphatidylcholine (PC) is between 5 nM and 70 nM; the concentration of sphingomyelin (SM) is between 5 nM and 60 nM; the concentration of serine phospholipid (PS) is between 0.1 nM and 30 nM; and the concentration of phosphatidylinositol (PI) is between 0.1 nM and 30 nM.

[0141] In some more specific embodiments, the concentration of the phosphatidylethanolamine (PE) is between 10 nM and 60 nM, for example: 10 nM, 13 nM, 13.2 nM, 14 nM, 25 nM, 26 nM, 26.4 nM, 27 nM, 33 nM, 34 nM, 34.8 nM, 35 nM, 55 nM, 56 nM, 56.4 nM, 57 nM, etc.

[0142] In some more specific embodiments, the concentration of the phosphatidylcholine (PC) is between 5 nM and 70 nM, for example: 10 nM, 11 nM, 11.9 nM, 12 nM, 22 nM, 23 nM, 23.8 nM, 24 nM, 30 nM, 31 nM, 31.3 nM, 32 nM, 49 nM, 50 nM, 50.8 nM, 51 nM, etc.

[0143] In some more specific embodiments, the concentration of the sphingomyelin (SM) is between 5 nM and 55 nM, for example: 10 nM, 11 nM, 12 nM, 21 nM, 22 nM, 23 nM, 29 nM, 30 nM, 31 nM, 46 nM, 47 nM, 48 nM, etc.

[0144] In some more specific embodiments, the concentration of the serine phospholipid (PS) is between 0.1 nM and 25 nM, for example: 3 nM, 4 nM, 4.4 nM, 5 nM, 7 nM, 8 nM, 8.8 nM, 9 nM, 11 nM, 11.6 nM, 17 nM, 18 nM, 18.8 nM, 19 nM, etc.

[0145] In some more specific embodiments, the concentration of phosphatidylinositol (PI) is between 0.1 nM and 20 nM, for example: 3 nM, 3.5 nM, 4 nM, 5 nM, 7 nM, 8 nM, 9 nM, 9.3 nM, 10 nM, 14 nM, 15 nM, 16 nM, etc.

[0146] In some exemplary embodiments, in the nutritional composition, the concentrations of phosphatidylethanolamine (PE) are 13.2 nM, phosphatidylcholine (PC) are 11.9 nM, sphingomyelin (SM) are 11 nM, serine phospholipid (PS) are 4.4 nM, and phosphatidylinositol (PI) is 3.5 nM; in the nutritional composition, the concentrations of phosphatidylethanolamine (PE) are 26.4 nM, phosphatidylcholine (PC) are 23.8 nM, sphingomyelin (SM) are 22 nM, serine phospholipid (PS) are 8.8 nM, and phosphatidylinositol (PI) is 7 nM. nM; In the nutritional composition, the concentration of phosphatidylethanolamine (PE) is 34.8 nM, the concentration of phosphatidylcholine (PC) is 31.3 nM, the concentration of sphingomyelin (SM) is 29 nM, the concentration of serine phospholipid (PS) is 11.6 nM, and the concentration of phosphatidylinositol (PI) is 9.3 nM; In the nutritional composition, the concentration of phosphatidylethanolamine (PE) is 56.4 nM, the concentration of phosphatidylcholine (PC) is 50.8 nM, the concentration of sphingomyelin (SM) is 47 nM, the concentration of serine phospholipid (PS) is 18.8 nM, and the concentration of phosphatidylinositol (PI) is 15 nM.

[0147] (Sialotropic components)

[0148] N-Acetylneuraminic acid (NANA), also known as sialic acid, is an important component of gangliosides in the brain. N-Acetylneuraminic acid is widely distributed in various biological tissues and is an important component of glycoproteins, oligosaccharides, and glycolipids. It usually exists as a glycoside at the terminal ends of glycoproteins and glycolipids, and is particularly abundant in the brain, nerve tissue, blood, submandibular glands, mucins, and colostrum of mammals.

[0149] The main production methods for N-acetylneuraminic acid include natural raw material extraction, chemical synthesis, polymer decomposition, enzyme and immobilized enzyme methods, and microbial fermentation.

[0150] 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. The sialylated substances described in this invention mainly include sialic acid oligosaccharides, sialylated proteins, and sialylated lipids.

[0151] In some embodiments, the sialic acid oligosaccharides of the present invention include 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.

[0152] In some preferred embodiments, the sialic acid oligosaccharides of the present invention are 3'-sialylated lactose (3'-SL) and 6'-sialylated lactose (6'-SL).

[0153] This invention does not specifically limit the source of sialylated substances; typically, they can be introduced 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 fluid obtained from the mammary glands of mammals in lactation.

[0154] 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.

[0155] In some embodiments, the mass ratio of 3'-sialylated lactose (3'-SL) to 6'-sialylated lactose (6'-SL) in the sialylated substance component is (1~20): (15~1).

[0156] In some preferred embodiments, the mass ratio of 3'-sialylated lactose (3'-SL) to 6'-sialylated lactose (6'-SL) in the sialylated substance component is (1~15): (10~1).

[0157] In some more preferred embodiments, the mass ratio of 3'-sialylated lactose (3'-SL) to 6'-sialylated lactose (6'-SL) in the sialylated substance component is (8~13): (13~8).

[0158] In some further preferred embodiments, the mass ratio of 3'-sialylated lactose (3'-SL) to 6'-sialylated lactose (6'-SL) in the sialylated substance 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.

[0159] In some exemplary embodiments, the mass ratio of 3'-sialylated lactose (3'-SL) to 6'-sialylated lactose (6'-SL) can be 11:9.

[0160] The sialylated component of the present invention can exist in a liquid state, or in a semi-solid or solid state.

[0161] When the sialylated component of the present invention is present in a liquid state, in some preferred embodiments, the concentration of the sialylated component in the nutritional composition is 10-200 nM.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] When the 3'-sialylated lactose and 6'-sialylated lactose in the sialylated component are used in the above proportions and amounts, they can have a synergistic effect. When used in combination with other components of the nutritional composition, the nutritional composition can achieve a better synergistic effect in promoting nerve cell development.

[0168] When the sialic acid components, lipid components, and phospholipid components are used in combination, the nutritional composition achieves a better synergistic effect in promoting neurodevelopment.

[0169] (Synergistic effect)

[0170] To obtain a more synergistic composition for promoting neurodevelopment, in some embodiments, the molar ratio of the oil component, the phospholipid component, and the sialylated substance component in the nutritional composition is (1-5):(1-10):(0.01-5).

[0171] In some embodiments, the molar ratio of the oil component, the phospholipid component, and the sialic acidified substance component in the nutritional composition is (1-4): (1-8): (0.1-5).

[0172] In some preferred embodiments, the molar ratio of the oil component, the phospholipid component, and the sialic acidification component in the nutritional composition is (1-3): (1-6): (0.1-4).

[0173] In some more preferred embodiments, in the nutritional composition, the molar ratio of the oil component, the phospholipid component, and the sialic acidification component is (1-3): (1-5): (0.5-2).

[0174] In some exemplary embodiments, the molar ratio of the oil component, the phospholipid component, and the sialylated substance component in the nutritional composition may be 2.7:1.1:0.6; 2.7:2.2:0.6; 2.7:2.9:0.6; 2.7:4.7:0.6; 2.7:1.1:1.5; 2.7:2.2:1.5; 2.7:2.2:1.9; 2.7:2.9:0.6; 2.7:2.9:1.5; 2.7:2.9:1.9; 2.7:4.7:0.6; 2.7:4.7:1.5; 2.7:4.7:1.9.

[0175] To achieve a further and better synergistic effect in promoting neurodevelopment, in some more preferred embodiments, the molar ratio of the oil component, the phospholipid component, and the sialic acidified substance component in the nutritional composition is (2.5–3): (1–3): (0.5–2).

[0176] In some more preferred embodiments, the molar ratio of the oil component, the phospholipid component, and the sialylated substance component in the nutritional composition is 2.7:1.1:0.6; 2.7:2.2:0.6; 2.7:2.9:0.6; 2.7:4.7:0.6; 2.7:2.2:1.5; 2.7:2.9:1.5.

[0177] In some further preferred embodiments, the molar ratio of the oil component, the phospholipid component, and the sialic acidified substance component in the nutritional composition is (2.5–3): (2–3): (0.8–1.6).

[0178] In some further preferred embodiments, the molar ratio of the oil component, the phospholipid component, and the sialic acidification component in the nutritional composition is 2.7:2.2:1.5; 2.7:2.9:1.5.

[0179] In some more preferred embodiments, the molar ratio of the oil component, the phospholipid component, and the sialic acidified substance component in the nutritional composition is (2.5–3):(2.5–3):(0.8–1.6).

[0180] In some further preferred embodiments, the molar ratio of the oil component, the phospholipid component, and the sialic acidified substance component in the nutritional composition is 2.7:2.9:1.5.

[0181] In this invention, when the oil component, the phospholipid component, and the sialylated substance component are within the above-mentioned range, the composition further achieves a better synergistic effect in promoting neural development, such as promoting nerve cell proliferation and differentiation, promoting synapse construction, promoting myelin sheath development, and promoting neuronal maturation.

[0182] (Composition)

[0183] The nutritional composition of the present invention includes at least the lipid component, phospholipid component, and sialylated substance 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.

[0184] 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.

[0185] The nutritional composition of the present invention may exist in a liquid state, or in a semi-solid or solid state.

[0186] 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.

[0187] 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.

[0188] In some embodiments, the concentration of the sialylated substance component in the nutritional composition is below 200 nM.

[0189] In some preferred embodiments, the concentration of the sialylated substance component in the nutritional composition is below 100 nM. For example, the concentration of the sialylated substance 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.

[0190] In some exemplary embodiments, the concentration of the sialylated substance component in the nutritional composition is 24 nM, 60 nM, or 76 nM.

[0191] In the nutritional composition, the concentration of the phospholipid component is between 10 nM and 500 nM.

[0192] In some more preferred embodiments, the concentration of the phospholipid component in the nutritional composition is [value missing].

[0193] In some preferred embodiments, the concentration of the phospholipid component in the nutritional composition is between 40 nM and 300 nM. For example, the concentration of the phospholipid in the nutritional composition is between 40 and 200 nM, such as 43 nM, 44 nM, 45 nM, 87 nM, 88 nM, 89 nM, 115 nM, 116 nM, 117 nM, 187 nM, 188 nM, 189 nM, etc.

[0194] In some exemplary embodiments, the concentration of the phospholipid component in the nutritional composition is 44 nM, 88 nM, 116 nM, or 188 nM.

[0195] In the nutritional composition, the concentrations of the oil component, the phospholipid component, and the sialic acidified substance component within the above-mentioned ranges are within the above-mentioned ranges, and the three components can synergistically enhance each other and have the effect of promoting nerve development.

[0196] (use)

[0197] Furthermore, the above-mentioned composition of the present invention has been experimentally verified to have the effect of promoting nerve development, wherein the effect of promoting nerve development includes at least one of the following: promoting nerve cell proliferation and differentiation, promoting synapse construction, promoting myelin sheath development, and promoting neuronal maturation.

[0198] Specifically, the composition can increase the expression of brain-derived neurotrophic factor (BDNF), enhance the activity of neural stem cells, and promote their neuronalization; the composition can promote the expression of nerve growth factor (NGF), further promoting synapse formation; the composition can promote the expression of microtubule-associated protein MAP-2, further promoting neuronal maturation; the composition can promote the expression of myelin-associated glycoprotein MAG, further promoting myelination; the composition can promote the enhanced expression of neuron-specific β-III microtubule protein (labeled with Tuj-1 antibody) and synaptic vesicle marker synaptophysin, further increasing the integrity of nerve cells; the composition can promote the increase of the average length of neurons, further promoting axonal extension after neuronal differentiation.

[0199] Therefore, it can be used as a food, functional health product, or functional health additive.

[0200] <Second aspect>

[0201] 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.

[0202] The food products of this invention can be foods suitable for promoting brain nerve development in infants, adolescents, adults, and / or middle-aged and elderly people.

[0203] 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.

[0204] This invention does not impose any specific absolute limits on the content of oil components, phospholipid components, and sialic acidified substances (and their specific components) in food, as long as they meet the requirements of local food-related laws and regulations.

[0205] In some specific embodiments, the food can be obtained by processing the nutritional composition according to the first aspect described above.

[0206] 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.

[0207] 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.

[0208] For baked goods, this can be cakes or cookies that are mainly based on butter, eggs, and baking powder.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] Animal-derived ingredients, including meat products from cattle, sheep, fish, or poultry.

[0214] 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.

[0215] 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.).

[0216] 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.

[0217] 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.

[0218] 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.

[0219] 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.

[0220] This invention does not specifically limit the types of foods that can be prepared using the above-mentioned nutritional composition and have the effect of promoting brain nerve development, such as promoting nerve cell proliferation and differentiation, promoting synapse construction, promoting myelin sheath development, and promoting neuronal maturation.

[0221] In some implementations, the food is a solid dairy product or a liquid dairy product; for example, milk powder, cheese, yogurt, liquid milk, etc.

[0222] 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.).

[0223] Typically, the aforementioned foods include: infant formula, maternal formula, children's formula, or nutritional or dietary supplements.

[0224] <Third aspect>

[0225] The present invention also provides the use of a composition containing the three essential components—the oil component, the phospholipid component, and the sialylated substance component—in the preparation of a nutritional composition for promoting brain and nerve development for non-therapeutic purposes. The present invention further provides the use of a composition containing the three essential components—the oil component, the phospholipid component, and the sialylated substance component—in the preparation of a food product for promoting brain and nerve development.

[0226] In some specific embodiments, the nutritional composition or food can promote neural development.

[0227] In some specific implementations, the promotion of neural development includes at least one of the following: promoting nerve cell proliferation and differentiation, promoting synaptic formation, promoting myelin sheath development, and promoting neuronal maturation.

[0228] In some specific embodiments, the nutritional composition can enhance the activity of neural stem cells and promote their neuronalization; for example, the composition can increase the expression of brain-derived neurotrophic factor (BDNF).

[0229] In some specific embodiments, the nutritional composition can promote synapse formation; for example, the composition can promote the expression of nerve growth factor (NGF).

[0230] In some specific embodiments, the nutritional composition can promote myelination of nerve cells; for example, the composition can promote the expression of myelin-associated glycoprotein (MAG).

[0231] In some specific embodiments, the nutritional composition can promote neuronal maturation; for example, the composition can promote the expression of microtubule-associated protein 2 (MAP-2).

[0232] In some specific embodiments, the nutritional composition can increase the integrity of nerve cells; for example, the composition can promote enhanced expression of neuron-specific β-III tubulin (labeled with a Tuj-1 antibody) and the synaptic vesicle marker synaptophysin.

[0233] In some specific embodiments, the nutritional composition can promote axonal extension after neuronal differentiation; for example, the composition can promote an increase in the average length of neurons.

[0234] In some implementation schemes, the specific selection and proportioning of the oil component, the phospholipid component, and the sialic acidification substance component are as described in the preceding <First Aspect>.

[0235] Example

[0236] 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.

[0237] Test samples

[0238] In the nutritional combinations provided in the following examples, the concentration ratio (molar ratio) of DHA to ARA is fixed at 1:1.7, and the proportions of phospholipids (including PC, PE, PS, PI, and SM) and the internal proportions of each sialic acid oligosaccharide (SL) are set as shown in Tables 1-2 below:

[0239] Table 1. Phospholipid group ratio (mass ratio)

[0240]

[0241] Table 2. Ratio of sialic acid oligosaccharides (SL) (by mass)

[0242]

[0243] Various nutritional compositions were prepared by mixing DHA, ARA, phospholipids and sialic acid oligosaccharides (SL) in the proportions shown in the following examples, and the proportions of each group are shown in Table 3.

[0244] Table 3 Composition of the nutritional composition

[0245]

[0246] Note: "-" indicates that it has not been added.

[0247] The experimental materials and cell culture procedures used in the following implementation sections are as follows.

[0248] (1) Experimental materials

[0249] ① Experimental sample

[0250] Docosahexaenoic acid (DHA) lipids (CABIO, content (in C...) 22 H 32 O2 triglycerides), w / %≥35), arachidonic acid oil (ARA) (CABIO, content (as C 20 H32 O2 triglycerides (w / %≥38.0%)); soybean phosphatidylcholine (Shanghai Zhenzhun Biotechnology, purity 98.0%), L-α-phosphatidylethanolamine (Shanghai Zhenzhun Biotechnology, purity 99.0%), L-α-phosphatidylinositol (Shanghai Zhenzhun Biotechnology, purity 95.0%), sphingomyelin (Shanghai Zhenzhun Biotechnology, purity 98.0%), L-α-phosphatidylserine (Shanghai Zhenzhun Biotechnology, purity 96.0%); 3'-sialyl lactose (GlyCare™ 3SL 9001, DSM), 6'-sialyl lactose (GlyCare™ 6SL 9001, DSM).

[0251] ② Experimental cells and their sources

[0252] Neural stem cells were derived from mice within 24 hours of birth obtained from C57bL / 6 wild-type pregnant mice purchased from Shanghai Slack Animal Experiment Co., Ltd.

[0253] ③ Consumables and reagents

[0254] 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).

[0255] The ELISA kits for brain-derived neurotrophic factor (BDNF), myelin-associated glycoprotein (MAG), nerve growth factor (NGF), and microtubule-associated protein 2 (MAP-2) were all purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd.

[0256] (2) Cell culture

[0257] 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 culture 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 solution (PDL), with the cell density controlled at 1×10⁻⁶ cells / mL. 6 Approximately 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.

[0258] Test Example 1: Screening for the optimal concentration of DHA

[0259] 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.

[0260] 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.

[0261] Test Example 2: Expression of Brain-Derived Neuroinfluencing Factor BDNF

[0262] In the early stages of neural stem cell culture, the expression level of brain-derived neurotrophic factor (BDNF) was detected to assess the regulatory effects of different compositions on cell activity, proliferation, and initial differentiation trends. The results showed that specific composition ratios significantly increased BDNF expression, suggesting its positive role in enhancing neural stem cell activity and promoting the initial neuronalization stage.

[0263] (1) Experimental steps

[0264] 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:

[0265] Sample preparation: Neuronal cells were cultured according to the experimental design. After differentiation to the predetermined time point, the cells were washed three times with cold PBS buffer to remove residual culture medium. An appropriate amount of cell lysis buffer (containing protease inhibitors) was added, and the cells were lysed at 4°C for 30 minutes. Then, the cells were centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected as the protein sample and stored at -80°C for later use.

[0266] ELISA kit preparation: Using the BDNF-specific ELISA kit, bring all reagents to room temperature and prepare a gradient concentration of standards.

[0267] Testing steps:

[0268] 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.

[0269] 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.

[0270] 3) Add biotin-labeled MAP-2 detection antibody, incubate for 1 hour, and then repeat the washing step.

[0271] 4) Add streptavidin-horseradish peroxidase (HRP), incubate at room temperature for 30 minutes, and then wash the well plate.

[0272] 5) Add substrate solution (TMB), incubate in the dark for 15 minutes, then add stop solution to stop the reaction.

[0273] Data acquisition and analysis: The absorbance (OD) value of each well was read at a wavelength of 450 nm using an ELISA reader. According to the kit instructions, a standard curve was plotted using a series of standard concentrations, and the concentration of BDNF in the sample was calculated accordingly.

[0274] (2) Experimental results

[0275] ① Effects of the ratio of various nutritional compositions on the expression of brain-derived neurotrophic factor BDNF

[0276] 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 2 .

[0277] Depend on Figure 2 It was found that the more components in the combination, the higher the BDNF expression level, and the three-factor combination was superior to any single factor. The BDNF expression of the SL ratio groups (corresponding to Examples 5-8) was slightly better than that of the low SL ratio groups (corresponding to Examples 1-4) and the high SL ratio groups (corresponding to Examples 9-12). In addition, by fixing the phospholipid or SL ratio, it was found that BDNF expression showed a "first increase and then decrease" trend with the concentration of phospholipids or SL, reaching its maximum value when the phospholipid ratio was 2.9 and the SL ratio was 1.5, suggesting the existence of a dose-dependent optimal ratio. The results also suggest that DHA+ARA has a dominant role in neural stem cell differentiation (Comparative Example 1), while phospholipids (Comparative Examples 2-5) and SL (Comparative Examples 6-8) provide a synergistic enhancing effect.

[0278] Table 4 also shows the increase in BDNF expression levels in each group compared to the blank control. As can be seen from the table, taking Example 1 ((DHA, ARA) + low phospholipids + low SL) as an example, the increase in BDNF compared to the blank control was 79.2, which is 11.5 higher than the sum of the three single factors (79.2-47.2-6.3-14.2=11.5). Similarly, the BDNF expression levels of each nutritional combination in Examples 1-12 were all greater than the sum of the single factors, indicating a synergistic effect between DHA, ARA, phospholipids, and sialic acid oligosaccharides (SL), which can synergistically increase BDNF expression and better promote neural stem cell proliferation and differentiation. Among them, the BDNF combinations in Examples 6-8 showed the largest increase compared to the sum of the single factors, ranging from 19.2 to 20.2.

[0279] Table 4. Comparison of expression of brain-derived neurotrophic factor BDNF in different groups

[0280]

[0281] Note: "-" indicates that it has not been added.

[0282] Test Example 3: Expression of Nerve Growth Factor (NGF)

[0283] After neural stem cell differentiation enters the intermediate stage, the expression of nerve growth factor (NGF) was detected to assess the formation of synaptic connections between neurons. As a key factor in synapse formation, the expression level of NGF directly reflects the efficiency of intercellular connections and the state of functional establishment. Multiple formulations showed high NGF levels, validating the synergistic effect of the compositions in the synapse formation process.

[0284] (1) Experimental steps

[0285] The expression level of nerve growth factor (NGF) in neuronal cells was detected by enzyme-linked immunosorbent assay (ELISA). The specific steps were the same as those in test example 2 (1).

[0286] (2) Experimental results

[0287] ① Effects of the ratio of various nutrient compositions on the expression of nerve growth factor (NGF)

[0288] 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 NGF kit was used for quantitative analysis. Results are shown below. Figure 3 .

[0289] Depend on Figure 3It was found that the more components in the combination, the higher the NGF expression level, and the three-factor combination was superior to any single factor. The BDNF expression in the SL ratio groups (corresponding to Examples 5-8) was slightly better than that in the low SL ratio groups (corresponding to Examples 1-4) and the high SL ratio groups (corresponding to Examples 9-12). Furthermore, by fixing the phospholipid or SL ratio, NGF expression showed a trend of "first increasing and then decreasing" with the concentration of phospholipids or SL, reaching its maximum at a phospholipid ratio of 2.9 and an SL ratio of 1.5, suggesting a dose-dependent optimal ratio. The results also indicated that DHA+ARA plays a dominant role in neural stem cell differentiation (Comparative Example 1), while phospholipids (Comparative Examples 2-5) and SL (Comparative Examples 6-8) provide a synergistic enhancing effect.

[0290] Table 5 also shows the increase in NGF expression levels in each group compared to the blank control. As can be seen from the table, taking Example 1 ((DHA, ARA) + low phospholipids + low SL) as an example, the increase in NGF compared to the blank control was 358, which is 43 (358-253-18-44=43) compared to the sum of the three single factors. Similarly, the NGF expression levels in each nutritional composition of Examples 1-12 were all greater than the sum of the single factors, indicating a synergistic effect between DHA, ARA, phospholipids, and sialic acid oligosaccharides (SL), which can synergistically increase NGF expression, better promote synapse formation, enhance signal transmission between neurons, and thus promote the improvement of neural network function.

[0291] Table 5 Comparison of NGF expression in different groups

[0292]

[0293] Note: "-" indicates that it has not been added.

[0294] Test Example 4: Expression of microtubule-associated protein-2 (MAP-2)

[0295] In the later stages of differentiation, the expression of the microtubule-associated protein MAP-2 was further examined to determine whether the neuronal structure had stabilized and formed. MAP-2 is a key regulator of the neuronal cytoskeleton; increased expression of MAP-2 signifies enhanced neuronal structural integrity and functional maturation. Related experimental results indicate that a specific combination of ingredients can significantly increase MAP-2 expression and promote neuronal morphological development.

[0296] (1) Experimental steps

[0297] The expression level of microtubule-associated protein 2 (MAP-2) in neuronal cells was detected using enzyme-linked immunosorbent assay (ELISA). The specific steps were the same as those in test example 2 (1).

[0298] (2) Experimental results

[0299] ① Effect of the ratio of various nutrient compositions on the expression of microtubule-associated protein 2 (MAP-2)

[0300] 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, the MAP-2 assay kit was used for quantitative analysis. Results are shown below. Figure 4 .

[0301] Depend on Figure 4 It was found that the more components in the combination, the higher the MAP-2 expression level, and the three-factor combination was superior to any single factor. The BDNF expression in the SL ratio groups (corresponding to Examples 5-8) was slightly better than that in the low SL ratio groups (corresponding to Examples 1-4) and the high SL ratio groups (corresponding to Examples 9-12). Furthermore, by fixing the phospholipid or SL ratio, MAP-2 expression showed a trend of "first increasing and then decreasing" with the concentration of phospholipids or SL, reaching its maximum at a phospholipid ratio of 2.9 and an SL ratio of 1.5, suggesting a dose-dependent optimal ratio. The results also indicated that DHA+ARA plays a dominant role in neural stem cell differentiation (Comparative Example 1), while phospholipids (Comparative Examples 2-5) and SL (Comparative Examples 6-8) provide a synergistic enhancing effect.

[0302] Test Example 5: Expression of myelin-associated glycoprotein MAG

[0303] To further evaluate the signal transduction ability of neural networks, myelin-associated glycoprotein MAG was selected as an indicator to detect whether neuronal axons were effectively myelinated. MAG is a key marker of myelination, and its expression level can directly reflect the indirect regulatory ability of nutritional compositions on oligodendrocytes. Experiments showed that combinations containing DHA, ARA, phospholipids, and SL in a synergistic ratio significantly promoted MAG expression, suggesting accelerated myelination development.

[0304] (1) Experimental steps

[0305] 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 2 (1).

[0306] (2) Experimental results

[0307] ① Effects of the ratio of various nutrient compositions on the expression of myelin-associated glycoprotein MAG

[0308] 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 5 .

[0309] As shown in the figure, the higher the number of components in the combination, the higher the MAG expression level, and the three-factor combination is superior to any single factor. The MAG expression of the nutritional composition with a certain proportion of SL (corresponding to Examples 5-8) is slightly better than that of the low proportion of SL (corresponding to Examples 1-4) and the high proportion of SL (corresponding to Examples 9-12). In addition, by fixing the proportion of phospholipids or SL, it was found that MAG expression showed a trend of "first increasing and then decreasing" with the concentration of phospholipids or SL, reaching its maximum value when the proportion of phospholipids was 2.9 and the proportion of SL was 1.5, suggesting the existence of a dose-dependent optimal ratio. The results also suggest that DHA+ARA has a dominant role in neural stem cell differentiation (Comparative Example 1), while phospholipids (Comparative Examples 2-5) and SL (Comparative Examples 6-8) provide a synergistic enhancing effect.

[0310] Table 6 also shows the increase in MAG expression levels in each group compared to the blank control. As can be seen from the table, taking Example 1 ((DHA, ARA) + low phospholipids + low SL) as an example, the increase in MAG compared to the blank control was 42.8, which is 15.3 higher than the sum of the three single factors (42.8 - 21.9 - 3.1 - 2.5 = 15.3). Similarly, the MAG expression levels in each nutritional composition of Examples 1-12 were all greater than the sum of the single factors, indicating a synergistic effect between DHA, ARA, phospholipids, and sialic acid oligosaccharides (SL), which can synergistically increase MAG expression and better promote neuronal myelin development and maturation.

[0311] Table 6 Comparison of expression of myelin-associated glycoprotein MAG in different groups

[0312]

[0313] Note: "-" indicates that it has not been added.

[0314] Test Example 6: Fluorescence confocal imaging (Tuj-1 and Synaptophysin expression) to evaluate neuronal differentiation and maturation.

[0315] After completing the protein expression analysis, to verify the direct effect of the nutritional composition on neural stem cell differentiation, dual-labeled fluorescence confocal imaging using neuron-specific β-III tubulin (labeled with Tuj-1 antibody) and synaptophysin (a synaptic vesicle marker) was employed. The co-localization of these two markers comprehensively reflects cell differentiation and maturation status. The images showed that different combinations had varying effects on the number of neurons, synapse formation density, and network complexity, with specific ratios of DHA, ARA, phospholipids, and SL exhibiting significant advantages.

[0316] (1) Experimental steps

[0317] 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.

[0318] 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 with PBS aqueous solution again.

[0319] 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 against Tuj-1 and synaptophysin were added and incubated overnight at 4°C.

[0320] 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.

[0321] 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 Tuj-1 and synaptophysin, as well as the nuclear fluorescence signals of DAPI-stained cells.

[0322] ⑥ Data Acquisition and Analysis: High-resolution images were acquired using confocal fluorescence microscopy to record the expression distribution and cell morphology characteristics of Tuj-1 and synaptophysin (DAPI). Image processing software was used to quantitatively analyze fluorescence intensity, and the expression levels of Tuj-1 and synaptophysin were evaluated by comparing experimental groups.

[0323] ⑦ Data processing and graphing: The relevant graphs of the experimental results are arranged into a graph set using software.

[0324] (2) Experimental results

[0325] The images include nuclear staining (DAPI, blue), neuronal markers (Tuj-1, green), and synaptic markers (Synaptophysin, red), facilitating morphological comparison and analysis and enabling preliminary assessment of differentiation efficiency.

[0326] Figures 6A-6C Fluorescence confocal imaging revealed that different proportions of nutrient compositions significantly affected the differentiation efficiency and synapse formation of neural stem cells. Under untreated conditions (control group), neural stem cells exhibited a low natural differentiation rate and extremely weak expression of Tuj-1 and Synaptophysin. However, after intervention with the nutrient compositions, with an increase in the number of intervention types, cell morphology became more complete, cell density increased, overall fluorescence intensity was enhanced, and differentiation efficiency significantly improved (Examples 1-12). Further dose-dependent analysis showed that when the SL concentration was fixed, the expression of Tuj-1 and Synaptophysin exhibited a "first increase, then decrease" trend with increasing phospholipid concentration, reaching a peak at a phospholipid ratio of 2.9. Conversely, when the phospholipid concentration was fixed, the promoting effect was strongest at an SL concentration of 1.5, but decreased after further increasing to 1.9, although still higher than the control group, suggesting that both types of nutrients have optimal concentration ranges.

[0327] Test Example 7: Statistics on the Mean Length of Neurons Based on Fluorescence Microscopy

[0328] Based on the aforementioned confocal images, image analysis software was used to quantitatively measure the average length of neurons, objectively reflecting the morphological maturation of neurons.

[0329] (1) Experimental steps

[0330] 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.

[0331] (2) Experimental results

[0332] Based on previous experimental results, the sialic acid oligosaccharide (SL) ratio was 1.5, which showed the strongest promoting effect. Therefore, the average length of several groups of neurons was statistically analyzed, as shown in Table 7.

[0333] Table 7. Statistics on the average length of neurons based on fluorescence microscopy.

[0334]

[0335] Note: "-" indicates that it has not been added.

[0336] Statistical analysis showed that the neuronal lengths of Example 7 ((DHA, ARA) + higher phospholipids + medium SL) and Example 6 ((DHA, ARA) + medium phospholipids + medium SL) were significantly higher than those of other groups, indicating that the nutritional composition can more effectively promote the axonal extension of neural stem cells after differentiation into neurons, which is helpful for neuronal morphological development and the establishment of neural network structure, and has a significant supporting effect on neural development.

[0337] 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.

[0338] 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 including one or more of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids; a phospholipid component including one or more of phosphatidylcholine (PC) and / or its metabolic precursor, phosphatidylethanolamine (PE) and / or its metabolic precursor, phosphatidylserine (PS) and / or its metabolic precursor, phosphatidylinositol (PI) and / or its metabolic precursor, phosphatidylglycerol (PG) and / or its metabolic precursor, diphosphatidylglycerol (DPG) and / or its metabolic precursor, and sphingomyelin (SM) and / or its metabolic precursor; sialic acid material components, including N one or more of acetylneuraminic acid, sialylated oligosaccharides, sialylated proteins, and sialylated lipids; and, in the nutritional composition, the molar ratio of the lipid component, the phospholipid component, and the sialylated substance component is (1-5):(1-10):(0.01-5); Preferably, the molar ratio of the lipid component, the phospholipid component, and the sialylated substance component is (1-3):(1-5):(0.5-2); More preferably, the molar ratio of the lipid component, the phospholipid component, and the sialylated substance component is (1-3):(1-3):(0.5-2).

2. The nutritional composition of claim 1, wherein, The lipid component includes polyunsaturated fatty acids including 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), Preferably, the lipid component includes docosahexaenoic acid and arachidonic acid; More preferably, the molar ratio of docosahexaenoic acid and arachidonic acid in the lipid component is 1:(1-5).

3. The nutritional composition according to claim 1 or 2, wherein, The sialic acid material component includes sialylated oligosaccharides comprising 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. The sialic acid material component includes sialylated oligosaccharides comprising 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 sialylated substance component includes 3'-sialyllactose and 6'-sialyllactose; More preferably, the mass ratio of 3'-sialyllactose and 6'-sialyllactose in the sialylated substance component is (1-20):(15-1).

4. The nutritional composition according to any one of claims 1 to 3, wherein, The phospholipid component includes phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and sphingomyelin (SM). Preferably, in the phospholipid component, the phospholipids include 15-50% by mass of phosphatidylethanolamine (PE), 15-40% by mass of phosphatidylcholine (PC), 15-40% by mass of sphingomyelin (SM), 1-20% by mass of phosphatidylserine (PS), and 3-15% by mass of phosphatidylinositol (PI), based on the total mass of the phospholipids.

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

6. The food product of claim 5, wherein, The food includes any one or more of infant food, child food, adolescent food, pregnant and postpartum woman food, adult food, and elderly food.

7. The food product of claim 5 or 6, wherein, The food further includes 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 containing three essential components of an oil component, a phospholipid component, and a sialic acid substance component for the production of a nutritional composition for promoting neural development.

9. Use of a composition containing three essential components of an oil component, a phospholipid component, and a sialic acid substance component for the production of a food for promoting neural development.

10. Use according to claim 8 or 9, wherein, The promoting neural development includes at least one of promoting neural cell proliferation and differentiation, promoting synaptic connection, promoting myelination, and / or promoting maturation of neurons.

Images