Nutritional composition for promoting brain development, food, and formula milk powder

Abstract

A nutritional composition for promoting brain development. The nutritional composition comprises the following essential components: docosahexaenoic acid, arachidonic acid, galactooligosaccharide, 1,3-dioleoyl-2-palmitoyl triglyceride, lutein, nucleotides, lactoferrin, and casein phosphopeptides. The nutritional composition and food containing same can effectively promote brain structure development and neural network formation. Specifically, on the brain structure, the brain weight can be particularly increased, and the maturation of the white matter region is promoted, and the beneficial development of lipid composition in the brain can also be promoted. Furthermore, in the neural network formation, related protein expression of neural network key components such as synapses and myelin sheaths can be promoted, thereby facilitating nerve cell activity and neural signal transmission, and further facilitating cognitive and neurobehavioral development.

Description

Nutritional composition, food and formula milk powder for promoting brain development Technical Field

[0001] The present invention belongs to the technical field of functional nutrient research, and specifically relates to a nutrient composition, food and formula milk powder for promoting brain development. Background Art

[0002] The first 1000 days of life, from conception to two years postnatally, represent a uniquely critical period in infant development. This period is a period of rapid growth and expansion of the infant's central nervous system (CNS), and neurodevelopmental processes are particularly sensitive to environmental influences. The anatomy of the brain is largely determined during this time, mediated by a complex interaction between the infant's genetic framework and their surroundings. Adequate nutrition during brain formation is crucial for maintaining the normal development of behavioral, cognitive, and socioemotional outcomes.

[0003] Breast milk is the ideal natural food for infants, promoting brain and nervous system development. For infants who are unable to breastfeed or whose mother's milk supply is insufficient, formula is the best alternative. Infant formula is based on cow's milk, goat's milk, or plant-based milks (such as soy milk), with additional protein, fat, and carbohydrates added to bring the nutritional profile of formula closer to that of breast milk. With the deepening of research on breast milk composition, advancements in deep milk processing, and breakthroughs in new raw material preparation technologies, the nutritional profile of infant formula is increasingly approaching that of breast milk. Optional ingredients for infant formula are becoming increasingly clear. For example, inositol, taurine, L-carnitine, docosahexaenoic acid (DHA), and eicosatetraenoic acid (AA, ARA) can be added based on the intended purpose of the formula. Furthermore, new functional active ingredients, such as the structural oil OPO, lactoferrin, oligosaccharides, and prebiotics, have been shown to benefit infant growth and development and can be added to formula. In addition to meeting the necessary nutritional composition, the infant formula milk powder currently available on the market also selectively adds optional ingredients and new functional ingredients of different types and dosages to meet the needs of different consumers. For example, Reference 1 discloses an infant formula goat milk powder, including whole goat milk powder, high-oil desalted goat whey powder, goat whey protein powder, oligofructose, oligogalactose, docosahexaenoic acid, arachidonic acid, animal Bifidobacterium Bb-12 and nutritional enhancers. Goat milk protein can effectively reduce the human body's allergy to milk protein; quantitatively added docosahexaenoic acid (DHA), arachidonic acid (ARA), and lutein have a promoting effect on the development of vision, can protect the retina, and prevent oxidative damage caused by light; DHA and ARA can also be beneficial to the brain development of infants and young children. While meeting the normal nutritional needs of infants and young children, attention is paid to the vision and brain development of infants and young children.

[0004] However, current functional claims for infant formulas primarily focus on the efficacy of specific nutrients. For example, choline can act as a methyl donor, regulating neuronal gene expression by modulating methylation, thereby modulating neural activity. Taurine is primarily involved in regulating the proliferation of neural precursor cells, the migration of newly generated neurons, and the formation of synapses. Numerous clinical studies have investigated DHA, and several observational studies have reported that high DHA levels in prenatal and postpartum breast milk can improve specific cognitive abilities in infants. However, breast milk, in reality, functions as an active nutrient system. Similarly, whether infant formulas formulated with multiple nutrients can effectively promote brain and neurobehavioral development in infants is a key focus of formula design and functional research. Furthermore, the impact of these multiple nutrient combinations on the sensory and solubility characteristics of infant formulas also needs to be considered.

[0005] Furthermore, the effectiveness of infant formula in promoting brain and neural development is currently primarily evaluated through clinical trials, using screening or diagnostic scales to assess infant behavioral development, such as fine motor skills, gross motor skills, language, and social development. Questionnaires, as a subjective evaluation method, are influenced by various factors, including the professional level of medical staff, the educational level of guardians, and the child's condition at the time of assessment. However, the overall role of formula in promoting brain development, and its effects on brain structure and physiological indicators, remains unclear.

[0006] References:

[0007] Reference 1: CN110063373A. Summary of the Invention

[0008] Problems to be solved by the invention

[0009] Although studies have shown that the various ingredients that can be added to infant formula have different effects on promoting brain development, such research cannot be said to be sufficient.

[0010] Currently, there are many studies on the functions of single nutritional components, but the functional investigation of infant formula as a combination of different nutrients, especially whether it can play a synergistic role in promoting brain structure development and the mechanism is still unclear. In addition, in terms of the overall formula promoting brain and neurobehavioral development, clinical practice often uses scales to judge the behavioral development of infants and young children and the development of the brain and nervous system. However, this method is affected by many factors such as the type of scale, the professionalism of medical staff, the educational level of guardians, and the condition of the child at the time of assessment, and cannot be objectively quantified. As a result, there is currently a lack of more accurate and objective functional research on overall formulas composed of different nutrients. At the same time, there is also concern about whether the combination of different nutrients will have adverse effects on the sensory aspects of infant formula.

[0011] To this end, the present invention aims to provide a nutritional composition that, through the combination of multiple ingredients, can more effectively promote the development of brain structure and the formation of neural networks, and to conduct more accurate research on this effect and its mechanism.

[0012] Solutions for solving problems

[0013] [1] A nutritional composition for promoting brain development, characterized in that the nutritional composition comprises the following essential components: docosahexaenoic acid, eicosatetraenoic acid, galacto-oligosaccharides, 1,3-dioleoyl-2-palmitoyl triglyceride, lutein, nucleotides, lactoferrin and casein phosphopeptide.

[0014] [2] The nutritional composition according to [1], characterized in that the nutritional composition further comprises choline, inositol, taurine and L-carnitine.

[0015] [3] The nutritional composition according to [1] or [2], characterized in that the nutritional composition further comprises probiotics, wherein the probiotics include Bifidobacterium animalis subsp. lactis.

[0016] [4] The nutritional combination according to any one of [1] to [3], characterized in that the nucleotides include disodium 5'-cytidylate, disodium 5'-uridylate, adenosine 5'-monophosphate, disodium 5'-guanylate and disodium 5'-inosinate.

[0017] [5] Use of the nutritional composition according to any one of [1] to [4] in preparing a food for promoting brain development.

[0018] [6] The use according to [5] is characterized in that the promotion of brain development includes at least one of increasing brain weight, promoting the maturation of white matter areas, promoting the development of lipid composition in the brain in a beneficial direction, and promoting the formation of neural networks.

[0019] [7] The use according to [6], characterized in that:

[0020] The said promoting the maturation of the white matter region of the brain includes promoting the myelination formation of the white matter region of the brain,

[0021] The promoting of the lipid composition in the brain to develop in a beneficial direction includes promoting the production of at least one of gangliosides, sphingomyelin, phosphatidylinositol and cardiolipin and / or reducing the production of lysophosphatidylethanolamine.

[0022] The promoting of neural network formation includes promoting the expression of at least one of postsynaptic density-95 and myelin proteolipid protein.

[0023] [8]. A food comprising the nutritional composition according to any one of [1] to [4].

[0024] [9] The food according to [8], characterized in that the food further comprises any one or more of the following ingredients: animal milk, protein components, fat components, carbohydrate components, vitamins and minerals.

[0025]

[0010] The food according to [8] or [9], characterized in that the food is infant formula.

[0026] Effects of the Invention

[0027] The present invention provides a nutritional composition capable of promoting brain development. By combining and strengthening multiple functional active components such as docosahexaenoic acid, eicosatetraenoic acid, galacto-oligosaccharides, 1,3-dioleoyl-2-palmitoyl triglyceride, lutein, nucleotides, lactoferrin and casein phosphopeptide, it can effectively promote brain structure development and neural network formation. In particular, the nutritional composition provided by the present invention and the food added thereto can increase brain weight and promote the maturation of white matter areas of the brain. At the same time, it can also promote the development of lipid composition in the brain in a beneficial direction, such as promoting the production of beneficial lipids and the reduction of harmful lipids in the brain. It can further promote the expression of proteins related to key points of neural networks such as synapses and myelin sheaths, so as to facilitate nerve cell activity, neural signal conduction and the formation of neural networks, and facilitate cognitive and neurobehavioral development.

[0028] The nutritional composition provided by the present invention can be used in a variety of food products, including infant formula, to meet a wide range of consumer needs. Furthermore, despite containing a combination of multiple functional active ingredients, the nutritional composition does not adversely affect the sensory properties or reconstitution properties of infant formula. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1: Comparison of body weight of rats after 28 days of intervention with different formulas and doses of formula powder.

[0030] Figure 2A: Comparison of FA values ​​in DTI structural analysis of rat brain after 28 days of intervention with different formulas and doses of formula powder.

[0031] Figure 2B: Comparison of MD values ​​of DTI structural analysis in rat brain after 28 days of intervention with different formulas and doses of formula powder.

[0032] Figure 3A: Comparison of relative concentrations of lipid GM1 in rat brain tissue after 28 days of intervention with different formulas and doses of formula powder.

[0033] Figure 3B: Comparison of relative concentrations of lipid SM in rat brain tissue after 28 days of intervention with different formulas and doses of formula powder.

[0034] Figure 4A: Comparison of relative concentrations of lipid PI in rat brain tissue after 28 days of intervention with different formulas and doses of formula powder.

[0035] Figure 4B: Comparison of relative concentrations of lipid CL in rat brain tissue after 28 days of intervention with different formulas and doses of formula powder.

[0036] Figure 4C: Comparison of relative concentrations of lipid LPE in rat brain tissue after 28 days of intervention with different formulas and doses of formula powder.

[0037] Figure 5: Differences in the expression of synapse-related proteins in rat brain tissue after 28 days of intervention with different formulas and doses of formula powder.

[0038] Figure 6: Differences in the expression of myelin-related proteins in rat brain tissue after 28 days of intervention with different formulas and doses of formula powder. DETAILED DESCRIPTION

[0039] The following describes the embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various structures described below, and various modifications can be made within the scope of the present invention. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention.

[0040] I. Definition of Terms

[0041] In the present invention, the term "a" or "an" or "the" may mean "one", and may also mean "one or more", "at least one" and "one or more than one".

[0042] In the present invention, the terms "comprising," "having," "including," or "containing" may be inclusive or open-ended, and do not exclude additional, unrecited components or method steps. At the same time, "comprising," "having," "including," or "containing" may also be closed-ended, excluding additional, unrecited components or method steps.

[0043] In the present invention, "infant" is used to refer to a human group aged 0 to 6 months.

[0044] In the present invention, "older infants" are used to refer to a human group of 6 to 12 months of age.

[0045] In the present invention, the term "infant" refers to a human group aged 12 to 36 months.

[0046] In the present invention, "infants" are used to refer to the human group under 3 years old.

[0047] In the present invention, the term "infant formula" encompasses infant formula, follow-on formula, and toddler formula. Generally, infant formula is used as a breast milk substitute from birth, follow-on formula is used as a breast milk substitute from 6 to 12 months after birth, and toddler formula is used as a breast milk substitute from 12 to 36 months after birth.

[0048] In the present invention, "animal milk" is used to refer to the liquid obtained from the mammary glands of mammals during lactation. The term "animal milk" should be interpreted broadly and covers both raw milk (i.e., liquid obtained directly from the mammary gland) and standardized milk products (such as, for example, skim milk or whole milk).

[0049] In the present invention, for the convenience of describing fatty acid glycerides, the following characters are used to refer to different types of fatty acids: P: palmitic acid (C16:0); O: oleic acid (C18:1).

[0050] In the present invention, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0051] Unless otherwise defined, other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0052] II. Research Basis of the Technical Solution of the Present Invention

[0053] Existing research on functional ingredients often focuses on the single efficacy of one or several additives in food, such as DHA and ARA, which can benefit infant brain development, and lutein, which can improve vision. However, in actual production, foods like infant formula often incorporate these nutrients into a milk base and combine them with other ingredients. This single-ingredient research ignores the effects of these ingredients when combined as a whole, or when combined and added to food to form a holistic whole with other nutrients.

[0054] In the process of studying the superposition of functional ingredients, the present invention unexpectedly discovered that by combining and strengthening multiple functional active components such as docosahexaenoic acid, eicosatetraenoic acid, galacto-oligosaccharides, 1,3-dioleoyl-2-palmitoyl triglyceride, lutein, nucleotides, lactoferrin and casein phosphopeptide, it is possible to more effectively promote the development of brain structure and the formation of neural networks, especially to promote the maturation of white matter areas of the brain, promote the production of beneficial lipids and the reduction of harmful lipids in the brain, and promote the expression of proteins related to key points of neural networks such as synapses and myelin sheaths.

[0055] III. Nutritional Composition

[0056] The present invention provides a nutritional composition, characterized in that the nutritional composition comprises the following essential components: docosahexaenoic acid, eicosatetraenoic acid, galacto-oligosaccharide, 1,3-dioleyl-2-palmityl triglyceride, lutein, nucleotides, lactoferrin and casein phosphopeptide.

[0057] The present invention unexpectedly discovered that by combining docosahexaenoic acid, eicosatetraenoic acid, galacto-oligosaccharide, 1,3-dioleoyl-2-palmitoyl triglyceride, lutein, nucleotides, lactoferrin and casein phosphopeptide, brain structure development and neural network formation can be effectively promoted.

[0058] In some embodiments, the nutritional composition further comprises choline, inositol, taurine, and L-carnitine.

[0059] The present invention has found that, on the basis of strengthening the combination of choline, inositol, taurine and L-carnitine, adding docosahexaenoic acid, eicosatetraenoic acid, galacto-oligosaccharides, 1,3-dioleyl-2-palmityl triglyceride, lutein, nucleotides, lactoferrin and casein phosphopeptide can more effectively promote brain development, especially promote the maturation of white matter areas, promote the development of lipid composition in the brain in a beneficial direction and promote the formation of neural networks.

[0060] In some specific embodiments, the nutritional composition comprises the following essential components: choline, inositol, taurine, L-carnitine, docosahexaenoic acid, eicosatetraenoic acid, galacto-oligosaccharides, 1,3-dioleyl-2-palmitoyl triglyceride, lutein, nucleotides, lactoferrin, and casein phosphopeptide.

[0061] In some specific embodiments, the nucleotides include disodium 5'-cytidylate, disodium 5'-uridine, adenosine 5'-monophosphate, disodium 5'-guanylate, and disodium 5'-inosinate.

[0062] In some embodiments, the nutritional composition further comprises probiotics, wherein the probiotics include Bifidobacterium animalis subsp. lactis. Exemplarily, the Bifidobacterium animalis subsp. lactis includes Bb-12 strain, HN109 strain, Bi-07 strain, and the like.

[0063] In some specific embodiments, the nutritional composition comprises the following essential components: choline, inositol, taurine, L-carnitine, docosahexaenoic acid, eicosatetraenoic acid, galacto-oligosaccharides, 1,3-dioleoyl-2-palmitoylglycerol, lutein, disodium 5'-cytidylate, disodium 5'-uridylphosphate, adenosine 5'-monophosphate, disodium 5'-guanylate, disodium 5'-inosinate, lactoferrin, casein phosphopeptide and Bifidobacterium animalis subsp. lactis Bb-12.

[0064] IV. Food

[0065] The present invention provides a food comprising the nutritional composition.

[0066] In some embodiments, the food comprises infant formula; illustratively, the infant formula comprises infant formula milk powder.

[0067] In order to meet the basic nutritional needs of consumers, in some embodiments, the food further comprises any one or more of the following ingredients: animal milk, protein components, fat components, carbohydrate components, vitamins and minerals.

[0068] The present invention has found that by adding the above-mentioned nutritional composition, or strengthening the multiple functional ingredients in the above-mentioned nutritional composition, and further combining it with other nutritional components in food such as vitamins and minerals, a more effective effect of promoting brain development can be achieved, especially promoting the maturation of white matter areas of the brain, promoting the development of lipid composition in the brain in a beneficial direction, and promoting the formation of neural networks.

[0069] In some embodiments, the source of the animal milk includes cows and / or sheep. In some specific embodiments, the animal milk is raw cow milk.

[0070] In some embodiments, the source of the protein component includes at least one of whole milk powder, skim milk powder, concentrated whey protein powder, whey protein powder, hydrolyzed whey protein powder, and demineralized whey powder.

[0071] In some embodiments, the source of the fat component includes at least one of structured mixed esters, sunflower oil, coconut oil, linseed oil, corn oil, rapeseed oil, and soybean oil.

[0072] In some embodiments, the source of the carbohydrate component comprises lactose.

[0073] In some embodiments, the vitamins include at least one of vitamin A, vitamin D, vitamin E, vitamin K, vitamin B1, vitamin B2, vitamin B6, vitamin B12, niacin, folic acid, pantothenic acid, calcium pantothenate, vitamin C, biotin, and niacinamide.

[0074] In some embodiments, the mineral comprises at least one of copper sulfate, magnesium sulfate, ferric pyrophosphate, zinc sulfate, calcium citrate, dibasic calcium phosphate, potassium iodate, sodium selenite, manganese sulfate, potassium chloride, calcium carbonate, tricalcium phosphate, sodium citrate, and ferrous sulfate.

[0075] In addition, in some embodiments, the food further comprises any acceptable excipients, including but not limited to solvents, antioxidants, antibacterial agents, thickeners, diluents, cosolvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, edible flavors, edible pigments, etc.

[0076] The present invention does not impose any particular limitation on the amount of the nutritional composition and its components used in food.

[0077] In some embodiments, based on dry weight, the content of docosahexaenoic acid in the food is greater than or equal to 40 mg / 100 g, preferably greater than or equal to 100 mg / 100 g, and preferably less than or equal to 190 mg / 100 g.

[0078] In some embodiments, based on dry weight, the content of eicosatetraenoic acid in the food is greater than or equal to 75 mg / 100 g, preferably greater than or equal to 170 mg / 100 g, and preferably less than or equal to 350 mg / 100 g.

[0079] In some embodiments, based on dry weight, the content of the galacto-oligosaccharide in the food is greater than or equal to 1 g / 100 g, preferably greater than or equal to 3 g / 100 g, and preferably less than or equal to 6.2 g / 100 g.

[0080] In some embodiments, based on dry weight, the content of 1,3-dioleoyl-2-palmitoyl triglyceride in the food is greater than or equal to 1 g / 100 g, preferably greater than or equal to 3 g / 100 g, and preferably less than or equal to 7 g / 100 g.

[0081] In some embodiments, based on dry weight, the lutein content in the food is greater than or equal to 200 μg / 100 g, preferably greater than or equal to 205 μg / 100 g, and preferably less than or equal to 220 μg / 100 g.

[0082] In some embodiments, based on dry weight, the content of the nucleotide in the food is greater than or equal to 20 mg / 100 g, preferably greater than or equal to 25 mg / 100 g, and preferably less than or equal to 50 mg / 100 g.

[0083] In some embodiments, based on dry weight, the content of lactoferrin in the food is greater than or equal to 35 mg / 100 g, preferably greater than or equal to 40 mg / 100 g, and preferably less than or equal to 500 mg / 100 g.

[0084] In some embodiments, based on dry weight, the content of the casein phosphopeptide in the food is greater than or equal to 30 mg / 100 g, preferably greater than or equal to 35 mg / 100 g, and preferably less than or equal to 50 mg / 100 g.

[0085] In some embodiments, based on dry weight, the choline content in the food is greater than or equal to 90 mg / 100 g, preferably greater than or equal to 200 mg / 100 g, and preferably less than or equal to 460 mg / 100 g.

[0086] In some embodiments, based on dry weight, the content of inositol in the food is greater than or equal to 20 mg / 100 g, preferably greater than or equal to 35 mg / 100 g, and preferably less than or equal to 200 mg / 100 g.

[0087] In some embodiments, based on dry weight, the taurine content in the food is greater than or equal to 15 mg / 100 g, preferably greater than or equal to 33 mg / 100 g, and preferably less than or equal to 80 mg / 100 g.

[0088] In some embodiments, based on dry weight, the content of L-carnitine in the food is greater than or equal to 5 mg / 100 g, preferably greater than or equal to 10 mg / 100 g, and preferably less than or equal to 50 mg / 100 g.

[0089] In some embodiments, based on dry weight, the food has a protein content of 8 to 20 g / 100 g, a fat content of 17 to 28 g / 100 g, and a carbohydrate content of 30 to 75 g / 100 g.

[0090] V. Uses to promote brain development

[0091] The nutritional composition provided by the present invention has the effect of promoting brain development. Therefore, the nutritional composition provided by the present invention can be used to prepare a food that promotes brain development. Furthermore, the food provided by the present invention can also promote brain development. In addition, in some embodiments, the purpose of promoting brain development in the present invention is not to prevent and / or treat a disease.

[0092] In some embodiments, the promoting brain development comprises at least one of increasing brain weight, promoting the maturation of white matter areas, promoting the lipid composition in the brain to develop in a beneficial direction, and promoting the formation of neural networks.

[0093] In some specific embodiments, said promoting the maturation of the white matter regions of the brain comprises promoting myelination of the white matter regions of the brain.

[0094] In some specific embodiments, promoting the lipid composition in the brain to develop in a beneficial direction includes promoting the production of at least one of gangliosides, sphingomyelin, phosphatidylinositol and cardiolipin and / or reducing the production of lysophosphatidylethanolamine.

[0095] In some specific embodiments, said promoting neural network formation comprises promoting the expression of at least one of postsynaptic density-95 and myelin proteolipid protein.

[0096] Example

[0097] The embodiments of the present invention will be described in detail below with reference to the examples. However, it will be understood by those skilled in the art that the following examples are only intended to illustrate the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the materials or instruments used were commercially available conventional products.

[0098] 1. Nutritional composition function evaluation method

[0099] 1.1 Experimental Animals

[0100] Fifty SPF male Sprague-Dawley rats, 3 weeks old (21-27 days old), were weaned and, after 7 days of adaptive feeding, randomly divided into six groups based on body weight: a group supplemented with 15% control formula, a group supplemented with 15% experimental formula, a group supplemented with 20% control formula, a group supplemented with 20% experimental formula, a group supplemented with 30% control formula, and a group supplemented with 30% experimental formula. The rats were fed for 4 weeks. Body weight and food intake were measured on days 0, 7, 14, 21, and 28 of group feeding. Experimental animals were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., with experimental unit use license number: SYXK (Beijing) 2022-0049 and experimental animal license number: SCXK (Beijing) 2021-0011. The animal experiments were approved by the Capital Medical University Ethics Committee, with animal ethics review number: AEEI-2023-035.

[0101] 1.2 Experimental animal feed

[0102] Basal feeds were supplemented with 15% control formula, 15% experimental formula, 20% control formula, 20% experimental formula, 30% control formula, and 30% experimental formula. The experimental formulas contained appropriate amounts of DHA, ARA, galacto-oligosaccharides, 1,3-dioleoyl-2-palmitoylglycerol, lutein, nucleotides, lactoferrin, and casein phosphopeptides. The control formulas in this experiment did not contain any of these ingredients. All feeds were provided by Keao Xieli (Tianjin) Feed Co., Ltd. The control and experimental formulas replaced corn starch in the feed at three different doses: 15%, 20%, and 30%.

[0103] 1.3 Biological sample collection and testing

[0104] On day 35 of group feeding, rats were anesthetized with intraperitoneal injection of tribromoethanol, and blood was collected from the heart. Rats were euthanized, and the brain, heart, liver, spleen, and kidneys were quickly collected. All organs were repeatedly rinsed with 0.9% saline, dried with filter paper, weighed, and stored at -80°C until further use.

[0105] 1) Brain MRI analysis

[0106] MRI technology was used to scan anesthetized SD rats for diffusion tensor imaging (DTI). DTI quantitatively analyzes the diffusion movement of water molecules in three-dimensional space by measuring the diffusion intensity of water molecules in all directions, thereby obtaining multiple parameter values. The most commonly used parameters are FA (fractional anisotropy), apparent diffusion coefficient (ADC) and mean diffusion rate (MD).

[0107] 2) HE staining of brain tissue

[0108] Paraffin-embedded tissue sections:

[0109] (1) Sampling: Fresh tissue was fixed in 4% paraformaldehyde for at least 24 hours. The tissue was removed from the fixative and trimmed flat with a scalpel in a fume hood. The trimmed tissue and the corresponding label were placed in a dehydration box.

[0110] (2) Dehydration: Place the dehydration box in the hanging basket and dehydrate in the dehydrator in a gradient of alcohol. 75% alcohol for 4 hours - 85% alcohol for 2 hours - 90% alcohol for 2 hours - 95% alcohol for 1 hour - anhydrous ethanol I for 30 minutes - anhydrous ethanol II for 30 minutes - benzene for 5-10 minutes - xylene I for 5-10 minutes - xylene II for 5-10 minutes - wax I for 1 hour - wax II for 1 hour - wax III for 1 hour.

[0111] (3) Embedding: Embed the wax-soaked tissue in an embedding machine. First, place the melted wax in the embedding frame. Before the wax solidifies, remove the tissue from the dehydration box and place it in the embedding frame according to the requirements of the embedding surface and affix the corresponding label. Cool in a -20℃ freezer. After the wax solidifies, remove the wax block from the embedding frame and trim the wax block.

[0112] (4) Sectioning: Place the trimmed wax block on a paraffin slicer and slice it to a thickness of 4 μm. Float the slices on a 40°C warm water slide to flatten the tissue. Pick up the tissue with a glass slide and bake it in a 60°C oven. Once the water is dried and the wax is melted, remove the slices and store them at room temperature for later use.

[0113] HE staining:

[0114] (1) Dewaxing of paraffin sections: sequentially place the sections in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 10 min, anhydrous ethanol II for 10 min, 95% alcohol for 5 min, 90% alcohol for 5 min, 80% alcohol for 5 min, 70% alcohol for 5 min, and wash with distilled water.

[0115] (2) Hematoxylin staining of cell nuclei: Stain sections with Harris hematoxylin for 3-8 minutes, wash with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, turn blue with 0.6% ammonia solution, and rinse with running water.

[0116] (3) Eosin staining of cytoplasm: Slice into eosin staining solution and stain for 1-3 minutes.

[0117] (4) Dehydration and sealing: Dehydrate the sections in 95% alcohol I for 5 min, 95% alcohol II for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, xylene I for 5 min, and xylene II for 5 min to make them transparent. Remove the sections from the xylene, air dry them slightly, and seal them with neutral gum.

[0118] (5) Microscopic examination, image acquisition and analysis.

[0119] 3) Lipidomics analysis of brain tissue

[0120] Brain tissue sample processing:

[0121] (1) Accurately weigh 50 mg of sample into a 2 mL centrifuge tube and add a 6 mm diameter grinding bead;

[0122] (2) Add 280 μL of extraction solution (methanol: water = 2:5) and then add 400 μL of MTBE;

[0123] (3) Grind in a frozen tissue grinder for 6 min (-10°C, 50 Hz);

[0124] (4) Low-temperature ultrasonic extraction for 30 min (5°C, 40 kHz), and the sample was placed at -20°C for 30 min;

[0125] (5) Centrifuge for 15 min (13000 g, 4°C), collect 350 μL of supernatant into an EP tube, and blow dry with nitrogen;

[0126] (6) Add 100 μL of extraction solution (isopropanol:acetonitrile = 1:1) to re-dissolve;

[0127] (7) vortex for 30 seconds and perform low-temperature ultrasonic extraction for 5 minutes (5°C, 40 kHz);

[0128] (8) Centrifuge for 10 min (13,000 g, 4°C), transfer the supernatant into a vial with an inner cannula, and analyze on an analyzer;

[0129] (9) In addition, 20 μL of supernatant was taken from each sample and mixed to serve as a quality control sample.

[0130] LC-MS detection:

[0131] The LC-MS analysis was performed using a Thermo Fisher Scientific UHPLC-QExactive HF-X system coupled with Fourier transform mass spectrometry. Chromatographic conditions included an Accucore C30 column (100 mm × 2.1 mm ID, 2.6 μm; Thermo); mobile phase A consisted of 50% acetonitrile in water (containing 0.1% formic acid and 10 mmol / L ammonium acetate); mobile phase B consisted of acetonitrile / isopropanol / water (10 / 88 / 2) (containing 0.02% formic acid and 2 mmol / L ammonium acetate); the injection volume was 5 μL, and the column temperature was 40°C.

[0132] Quality Control:

[0133] Quality control samples (QC) are prepared by mixing equal volumes of extracts from all samples. The volume of each QC is the same as that of the sample and is processed and tested using the same method as the analytical samples. During the instrument analysis process, a QC sample is inserted into every 5-15 analytical samples to examine the stability of the entire detection process.

[0134] 4) Immunoblotting to detect protein expression in rat tissues

[0135] Protein extraction:

[0136] Pre-cool RIPA protein extraction reagent, add protease inhibitor cocktail, centrifuge at 12,000 rpm (4°C) for 15 minutes, and collect the supernatant for protein quantification.

[0137] BCA protein quantification:

[0138] The protein concentration was determined according to the instructions of the BCA protein quantification kit.

[0139] (1) Prepare an appropriate amount of BCA working solution based on the number of samples, using reagents A and B in a ratio of 50:1. Mix thoroughly. BCA working solution is stable at room temperature for 24 hours.

[0140] (2) Completely dissolve the protein standard and dilute 10 μl to 100 μl to a final concentration of 0.5 mg / ml.

[0141] (3) Add 0, 1, 2, 4, 8, 12, 16, and 20 μl of the standard into the standard wells of a 96-well plate, and make up to 20 μl with standard diluent.

[0142] (4) Add the sample to be tested to the sample well and make up to 20 μl with standard diluent.

[0143] (5) Add 200 μl of BCA working solution to each well and incubate at 37°C for 30 min.

[0144] (6) Measure A570nm, draw a standard curve and calculate the protein concentration.

[0145] WB experiment:

[0146] (1) According to the molecular weight of the target protein, prepare 8% separation gel and 5% stacking gel.

[0147] (2) Sample amount of protein to be tested: 30 μg / well.

[0148] (3) Electrophoresis conditions: The stacking gel was subjected to a constant voltage of 90 V for approximately 20 min; the separation gel was subjected to a constant voltage of 120 V. The stopping time of electrophoresis was determined by pre-staining a protein marker.

[0149] (4) Wet transfer method, transfer conditions: 300 mA constant current; 0.45 μm pore size PVDF membrane, transfer time 60 min.

[0150] (5) Blocking: The membrane was completely immersed in 5% BSA-TBST and incubated on a horizontal shaker for 1 h (RT).

[0151] (6) Primary antibody incubation: dilute the primary antibody in 5% BSA-TBST and incubate overnight at 4°C on a horizontal shaker.

[0152] Table 1 Dilution ratio and molecular weight of each primary antibody

[0153] (7) The next day, wash the membrane: wash three times with TBST, each time for 10 minutes.

[0154] (8) Secondary antibody incubation: dilute the secondary antibody in 5% BSA-TBST, goat anti-rabbit IgG (H+L) HRP 1:10,000, and incubate at room temperature for 1 hour. Wash the membrane: wash the membrane three times with TBST, each time for 10 minutes.

[0155] (9) ECL is added dropwise to the protein side of the membrane and allowed to react for 3-5 minutes; film exposure: 10 seconds to 5 minutes (exposure time is adjusted according to different light intensities), development for 2 minutes, and fixation.

[0156] 5) Statistical analysis

[0157] SPSS21.0 software was used for statistical analysis of the data to obtain relevant information. In order to analyze the experimental data, descriptive statistical methods were used, and the quantitative indicators were expressed as mean ± standard deviation (mean ± SD). For the comparison of the six groups of measurement data, one-way analysis of variance was used for data that met the normality and had the same variance. If the comparison between the comparative proportion and the embodiment did not meet the normal distribution, the Mann-Whitney test was used. If it met the normal distribution, the t-test was used to compare the differences between the two groups, where p < 0.05 indicated that the significance level of the difference was statistically significant. GraphPad Prism 5.0 was used for mapping.

[0158] 2. Sensory testing and reconstitution testing methods of nutritional compositions

[0159] Sensory tests were performed on the comparative examples and example products in accordance with the "Infant Formula Sensory Evaluation Guidelines" (RHB 204-2004).

[0160] 3. Preparation of Experimental Formula Milk Powder

[0161] The raw milk and other proteins, fats, carbohydrates, vitamins, minerals and other functional ingredients produced by the applicant's own ranch are prepared into powder through processes such as sterilization, mixing, sterilization, homogenization, concentration and spray drying. Among them, proteins include skim milk powder, concentrated whey protein powder, whey protein powder, and hydrolyzed whey protein powder; lipids are mainly edible vegetable blended oils (1,3-dioleyl 2-palmityl triglyceride, sunflower oil, coconut oil, flaxseed oil); carbohydrates include lactose and oligosaccharides; vitamins include vitamin A, vitamin D, vitamin E, vitamin K, vitamin B1, vitamin B2, vitamin B6, vitamin B12, niacin, folic acid, pantothenic acid, vitamin C, biotin; minerals include Including copper sulfate, magnesium sulfate, ferric pyrophosphate, zinc sulfate, calcium citrate, calcium hydrogen phosphate, potassium iodate, sodium selenite, manganese sulfate; other functional active ingredients include choline, inositol, taurine, L-carnitine, docosahexaenoic acid (DHA), arachidonic acid (ARA), lutein, nucleotides (disodium 5'-cytidylate, disodium 5'-uridine, adenosine 5'-monophosphate, disodium 5'-guanylate, disodium 5'-inosinate), lactoferrin, casein phosphopeptide, Bifidobacterium animalis (Bb-12).

[0162] The composition of HMOs and lipid nutrients in the final experimental formula product is shown in the following table.

[0163] Table 2 Composition of HMOs and lipid nutrients in the experimental formula

[0164] 4. Preparation of Control Formula Milk Powder

[0165] Raw cow's milk and other proteins, fats, carbohydrates, vitamins, minerals, and other functional ingredients are prepared into powder through processes such as sterilization, mixing, sterilization, homogenization, concentration, and spray drying. Proteins include demineralized whey powder, whole milk powder, skim milk powder, and whey protein powder; lipids are mainly vegetable oils (corn oil, rapeseed oil, coconut oil, sunflower oil, and soybean oil); carbohydrates are mainly lactose; and other food additives include phospholipids, retinyl acetate, cholecalciferol, dl-α-tocopheryl acetate, phytonadione, thiamine hydrochloride, riboflavin, pyridoxine hydrochloride, cyanocobalamin, niacinamide, folic acid, D-calcium pantothenate, L-ascorbic acid, D-biotin, inositol, taurine, potassium chloride, calcium carbonate, tricalcium phosphate, sodium citrate, ferrous sulfate, zinc sulfate, magnesium sulfate, copper sulfate, manganese sulfate, potassium iodate, sodium selenite, choline chloride, and L-carnitine.

[0166] 5. Differences in nutritional indicators between experimental and control formulas

[0167] Table 3 Nutritional composition of experimental formula and comparative formula

[0168] 6. Example and Comparative Example Setup

[0169] Example 1: 15% of the experimental formula was mixed into the basic feed of rats.

[0170] Example 2: 20% of the experimental formula was mixed into the basic feed of rats.

[0171] Example 3: 30% of the experimental formula was mixed into the basic feed of rats.

[0172] Comparative Example 1: 15% of the control formula was mixed into the basic feed of rats.

[0173] Comparative Example 2: 20% of the control formula was mixed into the basic feed of rats.

[0174] Comparative Example 3: 30% of the control formula was mixed into the basic feed of rats.

[0175] Finally, the energy supply ratio and energy density of the three major macronutrients in the rat feed of each embodiment and comparative example are shown in the following table:

[0176] Table 4 Basic feed and experimental feed formula

[0177] 7. Body weight test of rats in each group

[0178] After 28 days of group feeding, no significant difference in body weight was found among the rats in each group, as shown in Figure 1. This indicates that the addition of different types of formula milk powder and different doses of formula milk powder had no significant effect on the increase in rat body weight.

[0179] 8. Organ development test of rats in each group

[0180] After 28 days of group feeding, the development of various organs of the rats was measured and characterized by organ index. Organ index = weight of each organ / body weight. The differences in brain index, heart index, liver index, spleen index and kidney index of rats in the experimental formula milk powder group and the control formula milk powder group were compared using t-test. The results are shown in the following table. It was found that except for the brain index of the experimental formula group (Example 1, Example 2 and Example 3), which was significantly higher than that of the control formula group (Comparative Example 1, Comparative Example 2 and Comparative Example 3) (p < 0.01, p < 0.05 and p < 0.001), there was no significant difference in liver index, spleen index and kidney index between the comparative example and the example.

[0181] Table 5 Organ indexes of rats after 28 days of intervention with different ratios and different milk powders

[0182] 8. Brain Structural Analysis of Rats in Each Group

[0183] The brain structure of rats was analyzed after intervention with different formulations and dosages. HE staining revealed mild overall brain tissue lesions in all six groups, primarily characterized by neuronal degeneration and apoptosis, with no significant differences between groups. This neuronal lesion was considered physiological. This study also used diffusion tensor imaging (DTI) to compare brain structural development in the rats. DTI measures the diffusion intensity of water molecules in all directions, quantitatively analyzing the diffusion movement of water molecules in three dimensions. This provides multiple parameters, including fractional anisotropy (FA), axial diffusion coefficient (AD), radial diffusion coefficient (RD), and mean diffusion coefficient (MD), reflecting the microstructure of the brain. Early in life, the efficiency of signal transmission within neural networks continuously improves, which is closely related to the myelination of white matter regions. The progression of myelination is paralleled by language learning. Abnormal brain development can lead to white matter damage and periventricular leukomalacia, which can lead to delayed white matter maturation and chronic myelination disorders, which in turn affect infant brain development. Increased FA and decreased MD are hallmarks of white matter maturation. This study compared the differences in FA and MD values ​​of rats after intervention with different formulas and different doses. It can be seen from Figures 2A and 2B that the higher the dose of added formula milk powder, the higher the FA value, which shows that the addition of formula milk powder can promote the myelination of brain white matter. After comparing different formulas with fixed formula doses, it was found that the FA value of the formula milk powder in the experimental group was higher than that in the control group, and Example 2 and Example 3 were significantly higher than Comparative Example 2 and Comparative Example 3, respectively (p < 0.01). The MD value showed that the comparative example was higher than the example, and Example 1, Example 2 and Example 3 were significantly lower than Comparative Example 1, Comparative Example 2 and Comparative Example 3, respectively (p < 0.01). This shows that the experimental formula milk powder can promote the maturation and myelination process of the white matter area of ​​the brain.

[0184] 9. Lipidome analysis of brain tissues of rats in each group

[0185] The normal development of brain structure is based on an adequate supply of nutrients. It is well established that the dry matter in the brain primarily consists of lipids, proteins, carbohydrates, and inorganic salts. Lipids are the most abundant, accounting for up to 50% of the brain's dry weight, and are crucial for the integrity of brain structure and functional development. These lipids are primarily phospholipids, which make up a quarter of the brain's dry matter and are a key component of the myelin sheath. Therefore, this study employed a non-targeted lipidomics analysis method to investigate the differences in lipid composition in the brain tissue of rats following 28 days of intervention with different formulas and dosages of milk powder. The results showed that the lipid metabolites in the brain tissues of the six groups of rats were highly similar, mainly including eight major categories and 96 subcategories: fatty acyls (FA), glycerolipids (GL), glycerophospholipids (GP), sphingolipids (SP), sterolipids (ST), prenol lipids (PR), glycolipids (SL), and polyketides (PK). Differences were found among the groups in these 96 subcategories.

[0186] Figures 3A and 3B show the differences in lipid subclasses within the sphingolipid class (SP) in rat brain tissue after 28 days of intervention with different formulations and doses. Ganglioside (GM1) is one of the major glycosphingolipids (GSLs) on the surface of central nervous system cells. GM1 protects neural tissue from various toxic substances or environmental insults, prevents neuronal apoptosis, neurodegeneration, and neuronal functional decline, thereby maintaining neuronal cell activity. It also induces neurotransmitter release in synaptosomes in the mouse cerebral cortex. GM1 has been shown to play a key role in memory and cognitive development. This study found that the GM1 content in Example 1 was significantly higher than that in Comparative Example 1 (p < 0.05), and the GM1 content in Example 3 was significantly higher than that in Comparative Example 3 (p < 0.01), indicating that the intervention of the experimental formula can promote the expression of GM1 in brain tissue; sphingomyelin (SM) is an important lipid in breast milk and has a promoting effect on the brain and nerve development of infants and young children. Studies have shown that it has the function of supporting the myelination of brain neurons on the one hand, and on the other hand, it plays an important role in cell-cell, cell-matrix interactions, cell adhesion, membrane receptor regulation and signal transduction. In this study, it was found that the experimental formula can significantly promote the expression of SM in rat brain tissue (p < 0.05), and is dose-dependent, but the dependent effect is not significant.

[0187] Figures 4A, 4B, and 4C show the differences in subclasses of lipids in the lipid glycerophospholipid GP in rat brain tissue after 28 days of intervention with different formulas and different doses. GP is an amphiphilic molecule that plays an active role in regulating ion channel function, transporters, receptors, and neuronal membrane function, transport, and proliferation. Among them, phosphatidylinositol (PI) accounts for about 5% of the total glycolipids in breast milk. It is the phospholipid that mainly contains inositol in cells. It mainly functions at the cell membrane level and is responsible for various physiological and biochemical processes, such as producing cellular responses to hormones and neurotransmitters to produce rapid physiological responses or stimulate cell proliferation. It may also play an important role in synaptic transmission. In this study, it was found that the experimental formula can promote the expression of PI in rat brain tissue, and the examples are significantly higher than the control examples; the other two GP lipids, cardiolipin (CL) and lysophosphatidylethanolamine (LPE), are related to the pathogenesis of the brain disease Alzheimer's disease (AD). Studies have shown that the reduction of total CL observed in AD models is associated with mitochondrial synaptic dysfunction and oxidative stress, and CL has a potential protective effect in the inflammatory response of AD. This study found that the experimental formula group can promote the expression of CL in brain tissue and reduce the expression of LPE. After t-test difference analysis, it was found that the CL in the brain tissue of rats in Example 3 was significantly higher than that in Comparative Example 3 (p < 0.001); and for LPE, after t-test analysis, it was found that Example 1 and Example 3 were significantly lower than Comparative Example 1 and Comparative Example 3, respectively (p < 0.001).

[0188] The above analysis results show that the experimental formula can promote the expression of beneficial lipids in brain tissue and reduce the expression of harmful lipids.

[0189] 10. Investigation of the formation of neural networks in the brains of rats in each group

[0190] During the formation of the brain's neural network, the development of synapses and myelin sheaths is directly related to the formation of the neural network and the conduction efficiency of neural signals, laying the foundation for the further development of the nervous system. In this study, the expression of synapse-related proteins and myelin-related proteins was specifically investigated to explore the effects of different formulations and different doses on the formation of the brain's neural network in rats after 28 days of intervention. The results showed that postsynaptic density protein (PSD)-95 showed significant differences between different formulations: the expression level of PSD-95 in the experimental formulation (Example) was higher than that in the control formulation (Comparative Example) (as shown in Figure 5). Although the difference was not significant, PSD-95 plays an important role in neurotransmitter signal conduction because it can regulate the maturation of synapses through interaction. Studies have also found that the destruction of PSD-95 is related to cognitive and learning defects observed in autism. Therefore, this result shows that the experimental formulation can promote the formation of neural synapses. We also found significant differences between the groups in myelin proteolipid protein (PLP), the main protein embedded in the phospholipid membrane that envelops the myelin sheath (as shown in Figure 6): the experimental formulas (Examples 1, 2, and 3) were significantly higher than the control formulas (Comparative Examples 1, 2, and 3) (p < 0.01). The increased expression of PLP indicates a high degree of myelination in the rat brain tissue, which directly affects the conduction of brain nerve signals and promotes neurobehavioral development.

[0191] 11. Sensory testing and dissolution testing of experimental formula, control formula and reference formula

[0192] The experimental formula and the control formula were as described above. The reference formula was adjusted based on the experimental formula by replacing the galacto-oligosaccharide with fructo-oligosaccharide, lactoferrin with osteopontin, and 1,3-dioleoyl-2-palmitoyl triglyceride with anhydrous butter. The rest of the composition of the reference formula was the same as that of the experimental formula.

[0193] The results are shown in Table 6. Although the experimental formula contained more breast milk-like functional ingredients to promote infant brain development, the overall sensory and dispensing properties of the product were not significantly different from those of the control group. The product even surpassed the control group in aspects such as color and dispensability. The reference formula, however, lagged behind the control group in dispensability and flavor.

[0194] Table 6 Sensory test results

[0195] Referring to the comparison between the reference formula and the control formula mentioned above, it shows a common phenomenon that a variety of functional ingredients can be added for nutritional or functional considerations, but this also leads to the deterioration of properties such as solubility, color or smell after the components with multiple performances are mixed.

[0196] Compared to the control group, the formula of the present invention not only further enhances functionality and nutritional value, but also suppresses the deterioration of solubility, color, and odor caused by the addition of multiple components. Therefore, the technical solution of the present invention achieves a better balance of the above properties.

[0197] Industrial applicability

[0198] The nutritional composition and food provided by the present invention can be utilized industrially.

Claims

1. A nutritional composition for promoting brain development, characterized in that: The nutritional composition comprises the following essential components: docosahexaenoic acid, eicosatetraenoic acid, galacto-oligosaccharide, 1,3-dioleyl-2-palmitoyl triglyceride, lutein, nucleotides, lactoferrin and casein phosphopeptide.

2. The nutritional composition according to claim 1, characterized in that The nutritional composition also includes choline, inositol, taurine, and L-carnitine.

3. The nutritional composition according to claim 1 or 2, characterized in that The nutritional composition further comprises a probiotic comprising Bifidobacterium animalis subsp. lactis.

4. The nutritional combination according to any one of claims 1 to 3, characterized in that The nucleotides include 5'-cytidylate disodium, 5'-uridine monophosphate disodium, adenosine 5'-monophosphate, 5'-guanylate disodium and 5'-inosinate disodium. 5 . Use of the nutritional composition according to claim 1 in preparing a food for promoting brain development.

6. The use according to claim 5, characterized in that The promoting of brain development includes at least one of increasing brain weight, promoting the maturation of white matter areas, promoting the lipid composition in the brain to develop in a beneficial direction, and promoting the formation of neural networks.

7. The use according to claim 6, characterized in that The said promoting the maturation of the white matter region of the brain includes promoting the myelination formation of the white matter region of the brain, The promoting of the lipid composition in the brain to develop in a beneficial direction includes promoting the production of at least one of gangliosides, sphingomyelin, phosphatidylinositol and cardiolipin and / or reducing the production of lysophosphatidylethanolamine. The promoting of neural network formation includes promoting the expression of at least one of postsynaptic density-95 and myelin proteolipid protein.

8. A food, characterized in that The food comprises the nutritional composition according to any one of claims 1 to 4.

9. The food according to claim 8, characterized in that The food further comprises any one or more of the following ingredients: animal milk, protein components, fat components, carbohydrate components, vitamins and minerals.

10. The food according to claim 8 or 9, characterized in that The food is infant formula.

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