Nutritional composition containing phospholipid and sialic acid oligosaccharide and capable of promoting construction of neural network

Abstract

The invention discloses a nutritional composition containing phospholipids and sialic acid oligosaccharides and capable of promoting construction of a neural network. The invention provides a nutritional composition, the nutritional composition comprises the following necessary components: a phospholipid component and a sialic acid oligosaccharide component, and in the nutritional composition, the molar ratio of the phospholipid component to the sialic acid oligosaccharide component is (1-10): (0.01-5). The composition provided by the invention has the advantages that the components are synergistic, the construction of a neural network 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] The present application relates to a nutritional composition, belonging to the field of food, more particularly, to a nutritional composition containing phospholipids and sialylated oligosaccharides for promoting neural network construction. BACKGROUND

[0002] During the early development of mammals, the establishment of the nervous system relies on the proliferation of neural stem cells, differentiation and the gradual formation of neuronal networks. Human milk, as the only or main source of nutrition for infants, contains a variety of active ingredients, among which phospholipids and human milk oligosaccharides (HMOs) play an irreplaceable role in brain development. Phospholipids are important components of neuronal membranes and myelin sheaths, while sialylated HMOs, especially 3'-sialylated oligosaccharides (3'-SL) and 6'-sialylated oligosaccharides (6'-SL), play an important role in synaptic plasticity and cognitive function establishment of neurons.

[0003] Previous studies have shown that the main components of phospholipids in breast milk include phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE) and sphingomyelin (SM), which play a key role in neuronal membrane stability, synaptic transmission efficiency and signal molecule function regulation. In addition, sphingomyelin, as an important component of myelin sheath, is believed to have unique value in the development of infant white matter and the formation of cognitive function (Guillermo et al., 2015).

[0004] Studies on sialylated oligosaccharides (SL) have shown that the concentration of 6'-SL in breast milk is positively correlated with the neural development indicators of infants, including language, memory, etc. (Liu et al. 2021). Animal experiments have further found that the supplementation of 3'-SL can significantly increase the sialic acid level in the hippocampus, enhance the expression of neural cell adhesion molecules (PSA-NCAM), fibroblast growth factor receptors (FGFR) and glutamate receptors, thereby promoting synaptic plasticity and learning and memory processes (Liu et al., 2021). These results suggest that sialylated oligosaccharides have potential positive effects on promoting neuronal function and synaptic development.

[0005] However, most of the above studies focus on the role of a single component. For example, some formula or patented solutions only focus on the efficacy of phospholipids in improving memory and learning ability, or only investigate the effect of HMOs, especially SL, on cognitive and behavioral indicators. Although these studies provide important clues, they all lack systematic verification of the effects of phospholipids and SL complex supplementation on cell level and synaptic network structure. Therefore, it is necessary to experimentally verify the synergistic effect of phospholipids and SL, and to study the mechanism of the combination of the two in neural stem cell differentiation, synapse generation and neural network structure integrity.

[0006] In this regard, the prior art has studied complex nutritional compositions, for example:

[0007] Reference 4 discloses a composition comprising 2'-FL and 3'-SL, which can synergistically promote brain development. Reference 5 discloses a synthetic nutritional composition tailored for Chinese infants, comprising specific 3'-SL and 6'-SL concentrations to provide optimal nutritional needs for infants in a specific region. Reference 6 provides a nutritional composition comprising sialic acid, sphingomyelin and phosphatidylcholine, which can affect the expression of hippocampal proteins. Reference 7 discloses a nutritional composition involving milk phospholipids and / or 2'-fucosyllactose for protecting and / or improving vision, such as improving visual fatigue, promoting visual development, and / or improving myopia.

[0008] However, it cannot be said that the above studies are perfect, and new compositions that have an effect on neural stem cell differentiation, synapse generation and neural network structure integrity still need to be developed.

[0009] References

[0010] Reference 1 Guillermo, Rosamond B et al. “Supplementation with complex milk lipids during brain development promotes neuroplasticity without altering myelination or vascular density.” Food & nutrition research vol. 5925765.

[0011] Reference 2 Liu, Shuang et al. “Six Oligosaccharides' Variation in Breast Milk: A Study in South China from 0 to 400 Days Postpartum.” Nutrients vol. 13,11 4017.

[0012] Reference 3 Zhu, Liuying et al. “Supplementation of 3'-Sialyllactose During the Growth Period Improves Learning and Memory Development in Mice.” Journal of agricultural and food chemistry vol. 72,44 (2024): 24518-24529.

[0013] Reference 4 CN120304556A

[0014] Reference 5 HK40099021A

[0015] Reference 6 CN120130546A

[0016] Reference 7 CN118765978A SUMMARY

[0017] Problem to be solved by the invention

[0018] As mentioned above, although there are literatures and patents that have disclosed the role of phospholipids or breast milk oligosaccharides in promoting cognitive function and neural system development of infants and young children, there are still the following shortcomings: first, existing researches are mostly single component supplementation, without considering the synergistic effect of phospholipids and SL. Phospholipids mainly improve cell membrane stability and myelin formation, while SL plays a role in synaptic plasticity and neurotransmitter signal regulation. However, it is unpredictable what effect the combination of the two will have, and there is a lack of research and verification of this in the existing technology; second, existing formula products and related patents lack research at the cellular and molecular level (such as neural stem cell proliferation, differentiation, neuron maturity, synaptic network structure, etc.), lack clear mechanistic basis, and lack optimal proportions.

[0019] Moreover, the prior art focuses on the fragmented description of a single indicator of neural development, lacks the construction of a synaptic network as the core, and lacks the scientific logic of forming a continuous chain of "synaptic connection and neural network construction" to establish the scientific logic of nutrients promoting synaptic and neural network construction. Therefore, there is a lack of application explainability and promotion value. Therefore, there is still a need to develop new compositions to make up for the above shortcomings.

[0020] Therefore, the technical problem to be solved by the present application is to verify the synergistic effect of phospholipids and SL through a scientific experimental system, and to determine the optimal application mode in promoting neural stem cell differentiation, neuron maturation and synaptic network establishment, thereby providing more accurate nutritional intervention programs for product ends, such as infant formula milk powder.

[0021] Solution for solving the problem

[0022] [1]. A nutritional composition, wherein the nutritional composition comprises the following essential components:

[0023] a phospholipid component and a sialylated oligosaccharide component,

[0024] and in the nutritional composition, the molar ratio of the phospholipid component and the sialylated oligosaccharide component is (1-10):(0.01-5);

[0025] Preferably, the molar ratio of the phospholipid component and the sialylated oligosaccharide component is (1-6):(0.5-3);

[0026] More preferably, the molar ratio of the phospholipid component and the sialylated oligosaccharide component is (1.5-3):(1-1.8).

[0027] [2]. The nutritional composition according to [1], wherein the sialylated oligosaccharide component comprises sialyllactose, and the sialyllactose comprises any one or more of 3'-sialyllactose, 6'-sialyllactose, sialyl-lactose-N-tetraose a, sialyl-lactose-N-tetraose b, sialyl-lactose-N-tetraose c and disialyl-lactose-N-tetraose,

[0028] Preferably, the sialylated oligosaccharide component comprises 3'-sialyllactose and 6'-sialyllactose;

[0029] More preferably, the mass ratio of 3'-sialyllactose and 6'-sialyllactose in the sialylated oligosaccharide component is (1-20):(15-1).

[0030] [3]. The nutritional composition according to [1] or [2], wherein the phospholipid component comprises any one or more of glycerophospholipids including 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, and diphosphatidylglycerol and / or its metabolic precursors (DPG), and / or sphingomyelin.

[0031] Preferably, the phospholipid component comprises phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and sphingomyelin (SM).

[0032] More preferably, the phospholipid component comprises 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 phospholipid component.

[0033] [4]. A food product, wherein the food product comprises or uses, as a raw material, the nutritional composition according to any one of [1] to [3].

[0034] [5]. The food product according to [4], wherein the food product comprises any one or more of infant food, child food, adolescent food, pregnant and lactating woman food, adult food, and elderly food.

[0035] [6]. The food product according to [4] or [5], wherein the food product can be in a solid form, a semi-solid form, or a liquid form.

[0036] [7]. The food product according to any one of [4] to [6], wherein the food product further comprises any one or more of a plant product component, an animal milk product component, an animal meat product component, a functional additive component, and any acceptable adjuvant.

[0037] [8]. Use of a composition containing two essential components of a phospholipid component and a sialyl-oligosaccharide component for the manufacture of a nutritional composition for promoting neural network formation.

[0038] [9]. Use of a composition containing two essential components of a phospholipid component and a sialyl-oligosaccharide component for the manufacture of a food product for promoting neural network formation.

[0039]

[10] . The use according to [8] or [9], wherein the promoting neural network construction comprises at least one of promoting synapse connection, promoting neural stem cell differentiation, improving cytoskeleton stability, and / or promoting neuron maturation.

[0040] Effects of the invention

[0041] The present application forms a complete chain in the perspective of synapse formation, neural network formation, and network structure stabilization and dimension expansion in the process of neural stem cell differentiation, and takes synapse network construction as the core target, specifically, forms a continuous chain of "synapse connection and neural network construction", establishes the scientific logic of nutrient substances promoting synapse and neural network construction, and enhances the explainability and popularization value of the application.

[0042] Compared with the prior art, the present application has the following outstanding differences and advantages: (1) the present application provides a composition for promoting neural network construction, which comprises a sialic acid oligosaccharide component and a phospholipid component, and can effectively promote neural network construction.

[0043] (2) The composition provided by the present application has broad prospects in promoting brain development, and plays an important role in various neural network construction cell mechanisms in the key stages of promoting synapse connection, promoting neural stem cell differentiation, improving cytoskeleton stability, promoting neuron maturation, etc. during the process of neural network construction.

[0044] (3) The composition provided by the present application can promote neural network construction, such as promoting synapse connection, promoting neural stem cell differentiation, improving cytoskeleton stability, and promoting neuron maturation, through in vitro model verification.

[0045] (4) Through a multi-dimensional evaluation system, the present application combines morphological indicators (fluorescence imaging and synapse number), molecular level indicators (Synaptophysin, MAP-2), and function-driven indicators (NGF, BDNF), forming a complete multi-level technical evaluation system.

[0046] (5) The present application uses a specific combination of phospholipids and sialic acid oligosaccharides, rather than a single component, emphasizes the synergistic effect, and verifies the promoting effect of the combination on synapse and neural network construction through in vitro experimental system.

[0047] (6) The composition of the present application is widely applicable to various populations such as infants, children, adolescents, pregnant women, adults, and the elderly, and is also conducive to the convenient addition or preparation of various foods or health products. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 Cell viability results after co-culturing neural stem cells with different concentrations of samples.

[0049] The image shows " " indicates P < 0.05, " " indicates P < 0.001.

[0050] Figure 2 The relative fluorescence intensity of Synaptophysin in cells co-cultured with neural stem cells in different proportions of nutrient compositions is statistically analyzed in fluorescence confocal imaging.

[0051] Figures 3A-3B Fluorescence confocal imaging of neuron-specific β-III tubulin (labeled with Tuj-1 antibody) and synaptophysin in cells co-cultured with neural stem cells in different proportions of nutrient compositions. Figure 3A 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 3B Fluorescence confocal imaging of neuron-specific β-III tubulin (labeled with Tuj-1 antibody) and synaptophysin in neural stem cells after co-culturing with comparative nutritional compositions.

[0052] Figure 4 The expression of nerve growth factor (NGF) in each group.

[0053] Figure 5 The expression of brain-derived neurotrophic factor BDNF in each group.

[0054] Figure 6 The expression of microtubule-associated protein 2 (MAP-2) in each group. Detailed Implementation

[0055] 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.

[0056] 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.

[0057] Unless otherwise indicated, the units used in the present specification are international standard units, and the numerical values, numerical ranges appearing in the present application should be understood to include inevitable systemic errors in industrial production.

[0058] In the present specification, the meaning indicated by "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0059] In the present specification, the "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", and the like refer to the specific elements (e.g., features, structures, properties, and / or characteristics) described in relation to the embodiments are included in at least one embodiment described herein, and can be present in other embodiments or can not be present in other embodiments. In addition, it should be understood that the elements can be combined in various embodiments in any suitable manner.

[0060] In the present specification, the numerical range indicated by "numerical value A ~ numerical value B" means a range including the end point values A, B.

[0061] In the present application, the term "about" is used to define the numerical ranges and parameters of the present application as approximate values, and the specific relevant values have been presented as accurately as possible. Unless otherwise explicitly stated, it should be understood that all ranges, numbers, values, and percentages used in the present application are modified by "about". Here, "about" generally means that the actual value is within ±3%, ±2%, ±1%, or ±0.5% of a certain value or range.

[0062] In the present application, "infant" is used to mean a human group of 0 to 6 months of age.

[0063] In the present application, "older infant" is used to mean a human group of 6 to 12 months of age.

[0064] In the present application, "toddler" is used to mean a human group of 12 to 36 months of age.

[0065] In the present application, "infant and toddler" is used to mean a human group of age less than 3 years.

[0066] In the present application, "child" is used to mean a human group of age greater than 3 years and less than 12 years, in the growth and development period.

[0067] In the present specification, the term "adult" means a person of age 18 years or older.

[0068] In the present specification, the term "adolescent" means a person of age 7 to 40 years.

[0069] In this specification, the terms "optional" or "optional" are used to indicate the use or omission of certain substances, components, procedures, application conditions, etc.

[0070] <First Aspect>

[0071] The first aspect of the present invention provides a nutritional composition comprising the following essential components: a phospholipid component and a sialic acid oligosaccharide component, wherein the molar ratio of the phospholipid component to the sialic acid oligosaccharide component in the nutritional composition is (1-10):(0.01-5).

[0072] In some preferred embodiments, the molar ratio of the phospholipid component to the sialic acid oligosaccharide component is (1-6):(0.5-3);

[0073] In some more preferred embodiments, the molar ratio of the phospholipid component to the sialic acid oligosaccharide component is (1.5-3):(0.5-2).

[0074] The nutritional composition of the present invention can promote the construction of neural networks. Specifically, the nutritional composition of the present invention can promote the construction of neural networks in many ways, such as synaptic connection, promoting neural stem cell differentiation, improving cytoskeleton stability and / or promoting neuronal maturation.

[0075] (Phospholipid components)

[0076] Phospholipids are a collective term for the various components of phospholipids.

[0077] Phospholipids, also known as phospholipids or phospholipid derivatives, 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, and are major components of biological membranes. A key characteristic is that hydrolysis produces a mixture containing fatty acids and phosphoric acid. In some embodiments, the phospholipids include glycerophospholipids and / or sphingomyelins.

[0078] In some preferred embodiments, the phospholipids include glycerophospholipids and sphingomyelins.

[0079] 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).

[0080] In some embodiments, the present invention does not particularly limit the metabolic precursors of the phosphate component. For example, the metabolic precursors may include at least one of glycerol-3-phosphate, phosphatidic acid, diglyceride, CDP-choline, CDP-ethanolamine, and CDP-diglyceride.

[0081] In some preferred embodiments, the glycerophospholipids include phosphatidylcholine (PC), phosphatidylethanolamine (also known as cephalin) (PE), serine phospholipids (PS), and phosphatidylinositol (PI).

[0082] In some preferred embodiments, the phospholipids include phosphatidylcholine (PC), phosphatidylethanolamine (PE), serine phospholipids (PS), phosphatidylinositol (PI), and sphingomyelin (SM).

[0083] 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.

[0084] In some embodiments, the phospholipid is a plant-derived phospholipid and / or an animal-derived phospholipid.

[0085] In some embodiments, the phospholipid may be a plant-derived phospholipid.

[0086] In some embodiments, the phospholipid may be an animal-derived phospholipid.

[0087] In some preferred embodiments, the phospholipids may be plant-derived phospholipids or animal-derived phospholipids.

[0088] 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.

[0089] 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.

[0090] This invention does not impose any particular limitation on the source of phospholipids.

[0091] In some embodiments, the phospholipid or plant-derived phospholipid may be soybean phospholipid.

[0092] In some preferred embodiments, the phospholipid or plant-derived phospholipid may be soybean-derived phosphatidylcholine (or soybean lecithin).

[0093] 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.

[0094] Animal-derived phospholipids and plant-derived phospholipids contain glycerophospholipids with similar or identical components and compositions.

[0095] 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.

[0096] In some embodiments, the phospholipid components 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).

[0097] In some preferred embodiments, the phospholipid components comprise, by weight, 20-45% phosphatidylethanolamine (PE); 20-35% phosphatidylcholine (PC); 20-30% sphingomyelin (SM); 5-15% serine phospholipid (PS); and 5-10% phosphatidylinositol (PI).

[0098] In some preferred embodiments, the phospholipid components comprise, by weight, 25-35% phosphatidylethanolamine (PE); 20-30% phosphatidylcholine (PC); 23-28% sphingomyelin (SM); 8-12% serine phospholipid (PS); and 5-10% phosphatidylinositol (PI).

[0099] In some specific embodiments, the phospholipid components comprise 25%, 27%, 30%, 33%, or 35% by mass of phosphatidylethanolamine (PE) based on the total mass of the phospholipid components.

[0100] In some specific embodiments, the phospholipid components comprise 20%, 23%, 25%, 27%, or 30% by mass of phosphatidylcholine (PC) based on the total mass of the phospholipid components.

[0101] In some specific embodiments, the phospholipid components comprise 23%, 24%, 25%, 26%, 27%, or 28% by mass of sphingomyelin (SM) based on the total mass of the phospholipid components.

[0102] 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 phospholipid components.

[0103] In some specific embodiments, the phospholipid components comprise 5%, 6%, 7%, 8%, 9%, or 10% by mass of phosphatidylinositol (PI), based on the total mass of the phospholipid components.

[0104] In some exemplary embodiments, the phospholipid components comprise, by weight, 30% phosphatidylethanolamine (PE), 27% phosphatidylcholine (PC), 25% sphingomyelin (SM), 10% serine phospholipid (PS), and 8% phosphatidylinositol (PI).

[0105] The phospholipid components of the present invention can exist in a liquid state, or in a semi-solid or solid state.

[0106] 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.

[0107] In some preferred embodiments, the concentration of the phospholipid component in the nutritional composition is between 40 nM and 300 nM, more preferably between 44 nM and 188 nM, for example: 44 nM, 45 nM, 87 nM, 88 nM, 89 nM, 115 nM, 116 nM, 117 nM, 187 nM, 188 nM, etc.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] When the various phospholipids in the phospholipid component are used in the above proportions and amounts, they can play a synergistic role. When used in combination with other components of the nutritional composition, the nutritional composition can achieve better synergistic effect in promoting neural network construction.

[0116] (Sialic acid oligosaccharide components)

[0117] Sialylation is a type of glycosylation involving the covalent addition of sialic acid to the terminal glycans of glycoproteins and glycolipids. This enzymatic process is tightly regulated by sialyltransferases (STs) and sialylases / neuraminidases (NEUs). Based on the type of glycosidic bond, sialylation can be classified into three types: α2-3-, α2-6-, and α2-8-sialylation. Sialylated oligosaccharides (SOS) are an important subclass of oligosaccharides, containing sialic acid residues in their molecular structure. Sialic acid is a nine-carbon monosaccharide, usually located at the end of the sugar chain, giving SOS a negative charge and important biological functions.

[0118] In some embodiments, the sialic acid oligosaccharides of the present invention include sialyl lactose.

[0119] Sialyllactose (SL) is the most basic and core member of sialic acid oligosaccharides, and one of the most important components of human milk oligosaccharides (HMOs). Its structure consists of sialic acid molecules and lactose molecules linked by glycosidic bonds.

[0120] In some embodiments, the sialylated lactose of the present invention includes any one or more of 3'-sialylated lactose, 6'-sialylated lactose, sialylated-lactose-N-tetrasaccharide a, sialylated-lactose-N-tetrasaccharide b, sialylated-lactose-N-tetrasaccharide c, and disialialylated-lactose-N-tetrasaccharide.

[0121] In some preferred embodiments, the sialic acid oligosaccharides of the present invention are 3'-sialylated lactose (3'-SL) and 6'-sialylated lactose (6'-SL).

[0122] This invention does not specifically limit the source of sialic acid oligosaccharides; typically, they can be derived from various milk-containing raw materials, such as animal milk raw materials (e.g., cow's milk, milk powder, etc.). In this invention, "animal milk" is used to refer to the liquid obtained from the mammary glands of mammals in lactation.

[0123] 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.

[0124] In some embodiments, the mass ratio of 3'-sialylated lactose (3'-SL) to 6'-sialylated lactose (6'-SL) in the sialic acid oligosaccharide component is (1~20): (15~1).

[0125] In some preferred embodiments, the mass ratio of 3'-sialylated lactose (3'-SL) to 6'-sialylated lactose (6'-SL) in the sialic acid oligosaccharide component is (1~15): (10~1).

[0126] In some more preferred embodiments, the mass ratio of 3'-sialylated lactose (3'-SL) to 6'-sialylated lactose (6'-SL) in the sialic acid oligosaccharide component is (8~13):(13~8).

[0127] In some further preferred embodiments, the mass ratio of 3'-sialylated lactose (3'-SL) to 6'-sialylated lactose (6'-SL) in the sialic acid oligosaccharide component is (8~12):(12~8). For example, the mass ratio of 3'-sialylated lactose (3'-SL) to 6'-sialylated lactose (6'-SL) can be 12:8, 11:9, 10:10, etc.

[0128] In some exemplary embodiments, the mass ratio of 3'-sialylated lactose (3'-SL) to 6'-sialylated lactose (6'-SL) can be 11:9.

[0129] The sialylated component of the present invention can exist in a liquid state, or in a semi-solid or solid state.

[0130] When the sialylated component of the present invention is present in a liquid state, in some preferred embodiments, the concentration of the sialylic oligosaccharide component in the nutritional composition is 10-200 nM.

[0131] In some preferred embodiments, the concentration of the sialylated component is 10-100 nM, and more specifically, the concentration of the sialylated component is 24-76 nM. Examples include: 23 nM, 24 nM, 25 nM, 59 nM, 60 nM, 61 nM, 75 nM, 76 nM, 77 nM, etc.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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. The 3'-sialylated lactose and 6'-sialylated lactose in the sialylated components, when used in the above proportions and amounts, can have a synergistic effect. Furthermore, when used in combination with other components of the nutritional composition, the nutritional composition achieves an even better synergistic effect in promoting neural network construction.

[0136] When sialic acid oligosaccharide components and phospholipid components are used in combination, the nutritional composition achieves a better synergistic effect in promoting neural network construction.

[0137] (Synergistic effect)

[0138] This invention promotes neuronal synergistic connections and network construction through the synergistic effect of phospholipid components (e.g., phospholipids) and sialic acid oligosaccharide components (e.g., sialyl lactose, especially 3′-SL and 6′-SL). The study was conducted in an in vitro neural stem cell differentiation model, and a hierarchical evidence system was established around synergistic formation efficiency and network complexity to verify the synergistic effect of phospholipids and SL and their concentration range.

[0139] To obtain a more synergistic composition for promoting neural network construction, in some embodiments, the molar ratio of the phospholipid component to the sialic acid oligosaccharide component in the nutritional composition is (1-10):(0.01-5).

[0140] In some embodiments, the molar ratio of the phospholipid component to the sialic acid oligosaccharide component in the nutritional composition is (1-8):(0.1-5).

[0141] In some preferred embodiments, the molar ratio of the phospholipid component to the sialic acid oligosaccharide component in the nutritional composition is (1-6):(0.1-4).

[0142] In some more preferred embodiments, in the nutritional composition, the molar ratio of the phospholipid component to the sialic acid oligosaccharide component is (1-5):(0.5-2) or (1.1-4.7):(0.6-1.9).

[0143] In some exemplary embodiments, the molar ratio of the phospholipid component and the sialic acid oligosaccharide component in the nutritional composition may be 1.1:0.6; 2.2:0.6; 2.9:0.6; 4.7:0.6; 1.1:1.5; 2.2:1.5; 2.9:1.5; 4.7:1.5; 1.1:1.9; 2.2:1.9; 2.9:1.9; 4.7:1.9.

[0144] To achieve a further enhanced synergistic effect in promoting neural network construction, in some more preferred embodiments, the molar ratio of the phospholipid component to the sialic acid oligosaccharide component in the nutritional composition is (1.8–3):(0.5–2).

[0145] In some more preferred embodiments, the molar ratio of the phospholipid component to the sialic acid oligosaccharide component in the nutritional composition is 2.2:0.6; 2.9:0.6; 2.2:1.5; 2.9:1.5; 2.2:1.9; 2.9:1.9.

[0146] In some specific embodiments, compared to other ratios or monomers, the above-mentioned preferred ratio composition, in a primary mouse neural stem cell culture system, showed increased Synaptophysin expression and enhanced Tuj-1 and Synaptophysin dual-labeling signals as demonstrated by fluorescence confocal imaging.

[0147] In some specific implementations, compared to the monomers, the above-mentioned preferred formulations increase the average length of neuronal synapses by approximately 15–20% in primary mouse neural stem cell culture systems.

[0148] In some specific implementations, compared to the monomers, the above-mentioned preferred composition significantly increases the expression levels of synapse-related proteins such as NGF and BDNF in primary mouse neural stem cells. Therefore, the composition shows a significant synergistic effect and ratio advantage in significantly improving synaptic density and network complexity.

[0149] In some preferred embodiments, the molar ratio of the phospholipid component to the sialic acid oligosaccharide component in the nutritional composition is (1.8~3):(1~1.8), (2~3):(1~1.8), (2.1~3):(1.3~1.8), (2.1~3):(1.4~1.7), (2.2~2.9):(1.4~1.6), or (2.5~3.0):(1.4~1.6). More preferably, the molar ratio of the sialic acid oligosaccharide component is (1.8~2.9):1.5. Further, the molar ratio of the sialic acid oligosaccharide component is (2~2.7):1.5 or (2.8~3.0):1.5.

[0150] In some further preferred embodiments, the molar ratio of the phospholipid component to the sialic acid oligosaccharide component in the nutritional composition is 2.2:1.5; 2.9:1.5.

[0151] In this invention, when the phospholipid component and the sialic acid oligosaccharide component are within the above-mentioned range, the composition further achieves a better synergistic effect in promoting neural network construction, such as promoting synapse construction, promoting myelin sheath development, and promoting neuronal maturation.

[0152] (Composition)

[0153] The nutritional composition of the present invention includes at least the phospholipid component and sialic acid oligosaccharide component described above. There are no particular limitations on the formation method of the composition; it can be formed by dissolving and mixing the substances containing these components as described above in a solvent, or by mixing various high-purity extracts.

[0154] 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.

[0155] The nutritional composition of the present invention may exist in a liquid state, or in a semi-solid or solid state.

[0156] In the nutritional composition, when the concentrations of the phospholipid component and the sialic acid oligosaccharide component are within the above-mentioned range, they can synergistically enhance each other and promote the construction of neural networks.

[0157] (use)

[0158] Furthermore, the above-mentioned composition of the present invention has been experimentally verified to promote neural network construction. The promotion of neural network construction includes at least one of promoting synaptic connections, promoting neural stem cell differentiation, enhancing cytoskeleton stability, and / or promoting neuronal maturation, thereby promoting neural development.

[0159] 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 enhanced expression of neuronal marker protein (Tuj-1) and synaptophysin, further increasing the integrity of nerve cells; the composition can promote the increase of average neuronal length, further promoting axonal extension after neuronal differentiation.

[0160] Therefore, it can be used as a food, functional health product, or functional health additive.

[0161] <Second aspect>

[0162] 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.

[0163] The food products of this invention can be foods suitable for promoting neural network construction in infants, adolescents, adults, and / or the elderly.

[0164] 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.

[0165] This invention does not impose any specific absolute limits on the phospholipid components, sialic acid oligosaccharide components (and their specific components) in food, as long as they meet the requirements of local food-related laws and regulations.

[0166] In some specific embodiments, the food can be obtained by processing the nutritional composition according to the first aspect described above.

[0167] 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.

[0168] 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.

[0169] For baked goods, this can be cakes or cookies that are mainly based on butter, eggs, and baking powder.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] Animal-derived ingredients, including meat products from cattle, sheep, fish, or poultry.

[0175] 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.

[0176] 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.).

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] The present invention does not specifically limit the types of foods that can be prepared using the above-mentioned nutritional compositions and have the effects of promoting neural network construction, such as promoting synaptic connections, promoting neural stem cell differentiation, improving cytoskeleton stability, and / or promoting neuronal maturation.

[0182] In some implementations, the food is a solid dairy product or a liquid dairy product; for example, milk powder, cheese, yogurt, liquid milk, etc.

[0183] 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.).

[0184] Typically, the aforementioned foods include: infant formula, maternal formula, children's formula, or nutritional or dietary supplements.

[0185] <Third aspect>

[0186] The present invention also provides the use of a composition containing the phospholipid component and the sialic acid oligosaccharide component, both essential components, in the preparation of a nutritional composition for promoting neural network construction for non-therapeutic purposes. The present invention further provides the use of a composition containing the phospholipid component and the sialic acid oligosaccharide component, both essential components, in the preparation of a food product for promoting neural network construction.

[0187] In some specific embodiments, the nutritional composition or food can promote neural network construction.

[0188] In some specific implementations, the promotion of neural network construction includes at least one of promoting synaptic connections, promoting neural stem cell differentiation, enhancing cytoskeleton stability, and / or promoting neuronal maturation.

[0189] In some specific embodiments, the nutritional composition can enhance the activity of neural stem cells and promote neuronal differentiation. For example, the composition can increase the expression of brain-derived neurotrophic factor (BDNF).

[0190] In some specific embodiments, the nutritional composition can promote synapse formation and synaptic connections; for example, the composition can promote the expression of nerve growth factor (NGF).

[0191] In some specific embodiments, the nutritional composition can promote neuronal maturation and enhance cytoskeleton stability. For example, the composition can promote the expression of microtubule-associated protein 2 (MAP-2), further aiding in the construction and expansion of neural networks.

[0192] In some specific embodiments, the nutritional composition can promote neuronal differentiation and synaptic connections. 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.

[0193] 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, further promoting neuronal maturation.

[0194] In some implementations, the phospholipid component and the sialic acid oligosaccharide component are specifically selected and proportioned as described in the preceding <First Aspect>.

[0195] Example

[0196] 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.

[0197] The nutritional composition provided by this invention, as tested in the sample, contains the following components:

[0198] (1) Phospholipid group, composed of phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and sphingomyelin (SM), mixed in a predetermined ratio, and the ratio of each phospholipid monomer is set as shown in Table 1 below;

[0199] (2) Sialic acid oligosaccharide (SL) is a mixture of 3'-sialic acid oligosaccharide (3'-SL) and 6'-sialic acid oligosaccharide (6'-SL) in a predetermined ratio. The ratio of the two oligosaccharides is set as shown in Table 2 below. The internal ratio of each substance is set as follows:

[0200] Table 1. Phospholipid group ratio (mass ratio):

[0201]

[0202] Table 2. Proportion of sialic acid oligosaccharides (SL) (mass ratio):

[0203]

[0204] Various nutritional compositions were prepared by mixing phospholipids and sialic acid oligosaccharides (SL) in the proportions (molar ratios) shown in the following examples, as shown in Table 3. One portion of the nutrient concentration was fixed at 40 nM; for example, in Example 1, the phospholipid concentration was 44 nM, and the 3'-SL+6'-SL concentration was 24 nM. The remaining concentrations were set proportionally, ultimately forming multiple nutritional compositions with different ratios.

[0205] Table 3 Composition of the nutritional composition

[0206]

[0207] Note: "-" indicates that it has not been added.

[0208] The experimental materials and cell culture procedures used in the following implementation sections are as follows.

[0209] (1) Experimental materials

[0210] ① Experimental sample

[0211] Soybean phosphatidylcholine (Shanghai Zhenzhun Biotechnology, 98.0% purity), L-α-phosphatidylethanolamine (Shanghai Zhenzhun Biotechnology, 99.0% purity), L-α-phosphatidylinositol (Shanghai Zhenzhun Biotechnology, 95.0% purity), sphingomyelin (Shanghai Zhenzhun Biotechnology, 98.0% purity), L-α-phosphatidylserine (Shanghai Zhenzhun Biotechnology, 96.0% purity)); 3'-sialic acid oligosaccharides (GlyCare™ 3SL 9001, DSM), 6'-sialic acid oligosaccharides (GlyCare™ 6SL 9001, DSM).

[0212] ② Experimental cells and their sources

[0213] 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.

[0214] ③ Consumables and reagents

[0215] 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).

[0216] The ELISA kits for nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd.

[0217] (2) Cell culture

[0218] 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.

[0219] Test Example 1: Screening of the optimal concentration of a nutrient composition

[0220] After neural stem cell culture, a pre-screening experiment was conducted to determine the effective concentration of the nutrient composition. The concentration range was set at 20 nM-400 nM per unit of nutrient. The results showed that cells survived at concentrations between 20 nM and 400 nM. Cell viability was assessed using the CCK-8 assay within this concentration range.

[0221] 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 1The 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, and the concentration of one nutrient was set at 40 nM. For example, in Example 1, the ratio of phospholipid concentration to 3'-SL+6'-SL concentration was 1.1:0.6, where the phospholipid concentration was 44 nM and the 3'-SL+6'-SL concentration was 24 nM.

[0222] Test Example 2: Synaptophysin Expression Levels and Tuj-1 & Synaptophysin Dual-Label Confocal Imaging to Evaluate Neuronal Differentiation and Maturation

[0223] Synaptophysin is a presynaptic vesicle membrane protein that reflects the number and distribution of synaptic vesicles. Higher expression levels indicate more active neuronal synapsis and a greater number of synapses. Further, dual-labeled fluorescence confocal imaging using β-III tubulin (labeled with Tuj-1 antibody, marking neuronal differentiation) and synaptophysin (labeling presynaptic vesicles) was used to observe neuronal differentiation and synaptic connections. The quantitative and imaging results corroborated each other, visually demonstrating the promoting effect of the composition on synaptic formation.

[0224] (1) Experimental steps

[0225] 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.

[0226] 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 with PBS aqueous solution three times for 5 minutes each time. After fixation, cells were permeabilized with 0.3% Triton X-100 aqueous solution and incubated at room temperature for 10 minutes, followed by washing with PBS aqueous solution three times again.

[0227] 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.

[0228] 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.

[0229] 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.

[0230] 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. 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.

[0231] ⑦ Data processing and graphing: The collected data is processed into corresponding charts using software, and the relevant graphs of the experimental results are arranged into a series of graphs using software.

[0232] (2) Experimental results

[0233] Synaptophysin expression levels were normalized using immunofluorescence staining combined with DAPI nuclear staining. Figure 2 It was found that the fluorescence intensity of Synaptophysin in the mixture of phospholipids and SL (Examples 1-12) was higher than that in the phospholipid single-factor group (Comparative Examples 1-4) and the SL single-factor group (Comparative Examples 5-7), indicating that the mixture significantly promoted neuronal differentiation and synaptic connections. Furthermore, when both nutrients were present, with the amount of SL fixed, Synaptophysin expression initially increased gradually with increasing phospholipid content, reaching a maximum at a phospholipid ratio of 2.9 (Examples 3, 7, 11), and then gradually decreased with further increases in the phospholipid ratio, exhibiting a curve of first increasing and then decreasing. Conversely, with the phospholipid concentration fixed, Synaptophysin expression initially increased and then decreased with increasing SL content, reaching its highest level at an SL ratio of 1.5 (Examples 5-8).

[0234] Figure 3A and Figure 3BFluorescence confocal images showed that the control group had fewer and fewer neuronal processes, while the Synaptophysin fluorescent dots were sparse and randomly distributed. Figure 3A In the mixed groups of phospholipids and SL (Examples 1-12), an increase in the number of processes and fluorescence intensity was observed, with a small number of synaptic points distributed along the processes. The fluorescence intensity in the single-factor groups was significantly lower than that in the mixed groups. The fluorescence intensity was most pronounced in Examples 6-7, specifically when the phospholipid group ratio was 2.2-2.9 and the SL ratio was 1.5, indicating a significant enhancement in synaptic connections and network construction. This composition effectively promotes the expression of presynaptic vesicle-related proteins, reflecting enhanced synapse formation activity and providing a structural basis for the stable establishment of neural networks. This highlights the unique advantage of phospholipids and SL synergistically promoting neuronal differentiation and network construction.

[0235] Test Example 3: Statistical Analysis of Mean Neuron Length Based on Fluorescence Microscopy

[0236] 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.

[0237] (1) Experimental steps

[0238] Representative fluorescence confocal images with intact stained neuronal morphology were selected, and 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 clearly visible and unobstructed axons were randomly selected from each field of view. Subsequently, the "line segment tool" was used to manually draw a measurement line segment from the center of the neuron cell body to the end of its corresponding axon, 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 average length was calculated, and one-way ANOVA was used for inter-group comparisons. All data are expressed as mean ± standard deviation.

[0239] (2) Experimental results

[0240] 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 4.

[0241] Table 4. Statistics on the average length of neurons based on fluorescence microscopy.

[0242]

[0243] Note: "-" indicates that it has not been added.

[0244] Statistical analysis showed that the neuron length in Example 6 (high phospholipid + medium SL) was significantly higher than that in Comparative Examples 3 and 6, and the length of the neurons in the composition was increased compared to the single neurons (16.7% and 12.6% higher than Comparative Examples 3 and 6, respectively). This indicates that the nutritional composition can more effectively promote the synaptic extension of neural stem cells after differentiation into neurons, which helps in the establishment of subsequent neural network structures and thus supports neural development.

[0245] Test Example 4: Expression of Nerve Growth Factor (NGF)

[0246] To further elucidate the molecular mechanism by which phospholipids and SL promote synaptic connections and neural network construction, this invention examined the expression level of nerve growth factor (NGF). Optimized formulations showed higher NGF expression levels, confirming the synergistic effect of the compositions under optimized formulations.

[0247] (1) Experimental steps

[0248] The expression level of nerve growth factor (NGF) in neuronal cells was detected using enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows:

[0249] ① 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 inhibitor) 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.

[0250] ② ELISA kit preparation: Using the NGF-specific ELISA kit, bring all reagents to room temperature and prepare a gradient concentration of standards.

[0251] ③ Testing steps:

[0252] 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.

[0253] 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.

[0254] 3) Add biotin-labeled NGF detection antibody, incubate for 1 hour, and then repeat the washing step.

[0255] 4) Add streptavidin-horseradish peroxidase (HRP), incubate at room temperature for 30 minutes, and then wash the well plate.

[0256] 5) Add substrate solution (TMB), incubate in the dark for 15 minutes, and then add stop solution to stop the reaction.

[0257] ④ Data acquisition and analysis: Use an ELISA reader to read the absorbance value (OD value) of each well at a wavelength of 450nm, plot a standard curve based on the concentration of the standard, and calculate the concentration of NGF in the sample.

[0258] (2) Experimental results

[0259] ① Effects of the ratio of various nutrient compositions on the expression of nerve growth factor (NGF)

[0260] 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 4 .

[0261] Depend on Figure 4 It was found that the expression level of NGF in the composition was higher than that of the single component. With a fixed phospholipid ratio, NGF expression showed a trend of "first increasing and then decreasing" with the SL ratio, and NGF expression in all examples was higher than that in the control group, reaching its maximum at an SL ratio of 1.5 (Examples 5-8). Furthermore, with a fixed SL ratio, NGF expression also showed a trend of "first increasing and then decreasing" with the phospholipid concentration, reaching its maximum at a phospholipid ratio of 2.9 (Examples 3, 7, 11), suggesting that there is a dose-dependent optimal ratio for each nutrient.

[0262] 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 (low phospholipids + low SL) as an example, the increase in NGF compared to the blank control was 95, and Example 1 showed an increase of 33 (95-18-44=33) compared to the sum of individual factors. Similarly, the NGF expression levels in each nutritional composition of Examples 1-12 were all greater than the sum of individual factors, indicating a synergistic effect between 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.

[0263] Table 5 Comparison of NGF expression in different groups

[0264]

[0265] Note: "-" indicates that no data was added or there is no data.

[0266] Test Example 5: Expression of Brain-Derived Neuroinfluencing Factor BDNF

[0267] The expression level of brain-derived neurotrophic factor (BDNF) can be used to assess the activity of neural stem cells and its effect on promoting neuronal differentiation from the perspective of cell regulation. A high expression level indicates a strong differentiation driving force, which is more conducive to synapse formation and connection.

[0268] (1) Experimental steps

[0269] The expression level of brain-derived neurotrophic factor (BDNF) in neurons was detected using enzyme-linked immunosorbent assay (ELISA). The specific steps were the same as those in Test Example 4.

[0270] (2) Experimental results

[0271] Effects of the ratio of various nutritional compositions on the expression of brain-derived neurotrophic factor BDNF

[0272] To assess the effects of multiple nutrient compositions relative to single nutrients, and the effects of different nutrient composition ratios on the proliferation and differentiation of neural stem cells, a BDNF kit was used for quantitative analysis. Results are shown below. Figure 5 .

[0273] Depend on Figure 6 It was found that the expression level of BDNF in the composition was higher than that of the single component. With a fixed phospholipid ratio, BDNF expression showed a trend of "first increasing and then decreasing" with the SL ratio, and BDNF expression was higher than the control group in all examples, reaching its maximum at an SL ratio of 1.5 (Examples 5-8). Furthermore, with a fixed SL ratio, BDNF expression also showed a trend of "first increasing and then decreasing" with the phospholipid concentration, and BDNF expression was higher than the control group in all examples, reaching its maximum at a phospholipid ratio of 2.9 (Examples 3, 7, 11), suggesting that there is a dose-dependent optimal ratio for each nutrient.

[0274] Table 6 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 (low phospholipids + low SL) as an example, the increase in BDNF compared to the blank control was 29.1, which is an increase of 8.6 compared to the sum of individual factors (29.1 - 6.3 - 14.2 = 8.6). Similarly, the BDNF expression levels in each nutritional composition of Examples 1-12 were all greater than the sum of individual factors, indicating a synergistic effect between phospholipids and sialic acid oligosaccharides (SL), which can synergistically increase BDNF expression and better promote neural stem cell differentiation and drive synaptic maturation.

[0275] Table 6. Comparison of expression of brain-derived neurotrophic factor BDNF in different groups

[0276]

[0277] Note: "-" indicates that no data was added or there is no data.

[0278] Test Example 6: Expression of microtubule-associated protein-2 (MAP-2)

[0279] MAP-2 is a key regulator of the neuronal cytoskeleton; its full expression enhances the integrity of the microtubule cytoskeleton, supports synaptic stability, and promotes the expansion of neural network dimensions. This study demonstrates that an optimized composition can significantly increase MAP-2 expression, improve cytoskeleton stability, and facilitate the construction and expansion of neural networks.

[0280] (1) Experimental steps

[0281] 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 4.

[0282] (2) Experimental results

[0283] Effects of the ratio of various nutrient compositions on the expression of microtubule-associated protein 2 (MAP-2)

[0284] 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 6 .

[0285] Depend on Figure 6 It was found that the composition exhibited a higher MAP-2 expression level than the single-factor formulation. With fixed phospholipid or SL ratios, MAP-2 expression showed a trend of "first increasing and then decreasing" with phospholipid or SL concentrations. Furthermore, BDNF expression was higher than the control group in all examples, reaching its maximum value at a phospholipid ratio of 2.9 (Examples 3, 7, 11) and an SL ratio of 1.5 (Examples 5-8), suggesting a dose-dependent optimal ratio.

[0286] Table 7 also shows the increase in MAP-2 expression levels in each group compared to the blank control. As can be seen from the table, taking Example 1 (low phospholipids + low SL) as an example, the increase in MAP-2 compared to the blank control was 3.3, and Example 1 showed an increase of 1.4 compared to the sum of single factors (3.3 - 0.5 - 1.4 = 1.4). Similarly, the MAP-2 expression levels in each nutritional composition of Examples 1-12 were all greater than the sum of single factors, indicating a synergistic effect between phospholipids and sialic acid oligosaccharides (SL), which can synergistically increase the expression level and effect of MAP-2, better promote the integrity of cellular microtubules and cytoskeleton stability, and drive neural network construction.

[0287] Table 7 Comparison of microtubule-associated protein 2 (MAP-2) expression in each group

[0288]

[0289] Note: "-" indicates that it has not been added.

[0290] The "method for promoting neuronal synaptic connections and network construction using phospholipids and sialic acid oligosaccharides" proposed in this invention has demonstrated the following technical effects in an in vitro neural stem cell experimental system:

[0291] Promotion of synapse generation and network construction: By detecting the expression of Synaptophysin and using Tuj-1 and Synaptophysin dual-labeled fluorescence confocal imaging, the data combined with imaging demonstrated that the composition can significantly increase the number of neuronal synapses and enhance the connection efficiency between synapses, ultimately forming a more complete and compact neural network structure.

[0292] Synergistic effect among various nutritional factors: The combination of phospholipids and sialic acid oligosaccharides (3′-SL and 6′-SL) showed a significantly higher improvement in the indicators of promoting synaptic connections (NGF), synaptic and neural network formation driving force (BDNF) and neural network dimension expansion (MAP-2) than the sum of the individual factors, demonstrating a synergistic effect.

[0293] 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.

[0294] 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, wherein, The nutritional composition contains the following essential components: Phospholipid components and sialic acid oligosaccharide components, Furthermore, in the nutritional composition, the molar ratio of the phospholipid component to the sialic acid oligosaccharide component is (1-10):(0.01-5); Preferably, the molar ratio of the phospholipid component to the sialic acid oligosaccharide component is (1-6):(0.5-3); More preferably, the molar ratio of the phospholipid component to the sialic acid oligosaccharide component is (1.5-3):(1-1.8).

2. The nutritional composition according to claim 1, wherein, The sialic acid oligosaccharide component includes sialylactose, which comprises any one or more of 3'-sialylated lactose, 6'-sialylated lactose, sialylated-lactose-N-tetrasaccharide a, sialylated-lactose-N-tetrasaccharide b, sialylated-lactose-N-tetrasaccharide c, and disialialylactose-N-tetrasaccharide. Preferably, the sialic acid oligosaccharide component includes 3'-sialylated lactose and 6'-sialylated lactose; More preferably, the mass ratio of 3'-sialylated lactose to 6'-sialylated lactose in the sialic acid oligosaccharide component is (1~20): (15~1).

3. The nutritional composition according to claim 1 or 2, wherein, The phospholipid components include glycerophospholipids and / or sphingomyelins, wherein the glycerophospholipids include any 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, and diphosphatidylglycerol (DPG) and / or its metabolic precursors. Preferably, the phospholipid components include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and sphingomyelin (SM). More preferably, the phospholipid component, based on the total mass of the phospholipid component, comprises 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).

4. A food product, wherein, The food product includes or uses the nutritional composition described in any one of claims 1-3 as a raw material.

5. The food product according to claim 4, wherein, The food products 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.

6. The food product according to claim 4 or 5, wherein, The food product may be in solid, semi-solid, or liquid form.

7. The food product according to any one of claims 4-6, wherein, The food also contains any one or more of the following ingredients: plant-based ingredients, animal dairy ingredients, animal meat ingredients, functional additives, and any acceptable excipients.

8. The use of a composition containing two essential components, a phospholipid component and a sialic acid oligosaccharide component, in the preparation of a nutritional composition that promotes neural network construction.

9. The application of a composition containing two essential components, a phospholipid component and a sialic acid oligosaccharide component, in the preparation of foods that promote neural network construction.

10. The application according to claim 8 or 9, wherein, The promotion of neural network construction includes at least one of promoting synaptic connections, promoting neural stem cell differentiation, enhancing cytoskeleton stability, and / or promoting neuronal maturation.

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