A nutritional composition and its use for improving cognition

Abstract

The present application belongs to the field of functional substance research, and particularly relates to a nutritional composition and its use for improving cognition. The nutritional composition for improving cognition provided by the present application comprises essential active ingredients shown in (I) and (II) as follows: (I) branched-chain fatty acid C15:0; (II) neutral fucosylated human milk oligosaccharide; wherein the branched-chain fatty acid C15:0 comprises anteiso-C15:0, and the neutral fucosylated human milk oligosaccharide comprises 2'-FL. The branched-chain fatty acid C15:0 and the human milk oligosaccharide 2'-FL can exert a synergistic effect of improving cognition after being combined.

Description

Technical Field

[0001] This invention belongs to the field of functional substance research, specifically relating to a nutritional composition and its use in improving cognition. Background Technology

[0002] Cognitive function, as the core of higher neural activity in the brain, refers to a series of complex abilities an individual possesses to acquire, process, store, and utilize information, encompassing key psychological processes such as learning, memory, attention, decision-making, and problem-solving. For children and adolescents, healthy cognitive development directly impacts their learning efficiency (attention, memory), emotional and behavioral performance, social skills, and future ability to live independently. For middle-aged and elderly individuals, cognitive health is the baseline for maintaining independence and dignity. As the brain enters aging, cognitive decline, anxiety, depression, and behavioral disorders become increasingly prominent. Currently, methods to improve cognition mainly include medication and cognitive training. However, for children and adolescents, the therapeutic effects of medication are limited and may produce serious side effects, while behavioral training increases the financial burden on families and the psychological stress on parents. For middle-aged and elderly individuals, there are currently no specific drugs for cognitive impairment-related diseases such as Alzheimer's disease. Therefore, developing nutrients suitable for long-term use by people throughout their entire lifespan and possessing good cognitive-improving effects is of great significance for improving the health of people of all ages.

[0003] Branched-chain fatty acids (BCFAs) are fatty acids with one or more branched alkyl groups or other functional groups on their alkyl chains. When the branched alkyl group is located on the second carbon atom from the alkyl end, it is called an isomer or iso-fatty acid; when it is located on the third carbon atom from the alkyl end, it is called an anteiso-fatty acid. Due to their unique branched structure, BCFAs possess excellent physicochemical properties, including low freezing and pour points, good thermal and oxidative stability, good solubility, low foaming properties, and good air and water permeability in the membranes formed from them. BCFAs are widely distributed in nature, but in relatively low amounts. It has been reported that certain amounts of BCFAs with different structures are present in the milk and internal tissues of ruminants (such as cow and sheep milk and meat products) and human milk. Furthermore, BCFAs are abundant in sebum and the intestines of newborns. BCFA profiles of lanugo and meconium show that BCFAs are a major component of the gastrointestinal tract of normal, healthy, full-term newborns. Premature infants have significantly lower levels of branched-chain fatty acids (BCFAs) in their intestines compared to full-term healthy infants, resulting in a higher probability of developing necrotizing enterocolitis (NCD). Numerous studies have shown that BCFAs possess unique physiological regulatory functions, including inhibiting inflammation, anti-cancer effects, and improving gut health, which may help reduce the risk of NCD in premature infants. However, there are few reports on their role in improving cognition. Reference 1 discloses a nutritional composition comprising milk fat globule membranes and at least one nutrient selected from whey protein micelles, α-hydroxyisocaproic acid, citrulline, branched-chain fatty acids, and combinations thereof; this nutritional composition can be used to treat conditions such as low-grade inflammation and cognitive decline. While this reference mentions that the nutritional composition used to treat cognitive decline may contain branched-chain fatty acids, it does not disclose the specific role of branched-chain fatty acids, nor does it disclose the specific types of branched-chain fatty acids.

[0004] Human milk oligosaccharides (HMOs) are the third most abundant solid component in breast milk, after lactose and fat, and are even more abundant than proteins. HMOs are a class of complex sugars with various structures. Based on their molecular structure, HMOs can be classified into neutral fucosylated HMOs, neutral non-fucosylated HMOs, and acidic sialylated HMOs. Due to their unique structure, HMOs help infants establish a healthy gut microbiota, strengthen immune system development, and promote brain and cognitive development. Studies have shown that the total HMO content, 2'-fucosylated lactose (2'-FL), 6'-sialylated lactose (6'-SL), and 3'-sialylated lactose (3'-SL) content in early lactation (i.e., 1 month) are positively correlated with the cognitive, language, and motor development levels of infants in later stages (i.e., 6-24 months of age). Studies on exclusively breastfed infants have found that for every 1 μg / mL increase in 2'-FL content in breast milk during the first month postpartum, the infant's cognitive score at 24 months of age increased by 0.59. Furthermore, 6'-SL was positively correlated with the overall motor skills and developmental scores of 18-month-old infants, and 3'-SL was significantly correlated with the infant's language development level. In addition, animal experiments have demonstrated that the intake of HMOs combinations can significantly improve spatial memory in rodents and piglets and accelerate the learning speed of manipulative tasks. Moreover, some literature speculates that the mechanisms by which HMOs promote brain and neural development mainly include three aspects: 1) the gut-brain axis; 2) gut microbiome-independent effects; and 3) supporting brain development as a direct or indirect source of sialic acid.

[0005] References:

[0006] Reference 1: CN103596440A. Summary of the Invention

[0007] The problem the invention aims to solve

[0008] Currently, methods for improving cognition mainly include medication and cognitive training. However, for children and adolescents, the efficacy of medication is limited and may produce serious side effects, while behavioral training increases the economic burden on families and the psychological burden on parents. For middle-aged and elderly people, there are still no specific drugs for cognitive impairment-related diseases such as Alzheimer's disease. Therefore, developing nutrients suitable for long-term use by people throughout their entire lifespan and with good cognitive improvement effects is of great significance for improving the health level of people throughout their entire lifespan.

[0009] Branched-chain fatty acids and human milk oligosaccharides are both naturally sourced nutrients. Based on this, this invention takes prevention and improvement as its starting point. Through extensive research, it has been found that branched-chain fatty acids such as aC15:0 and human milk oligosaccharides such as 2'-FL have a synergistic effect on improving cognition. Furthermore, this invention screens for compositions in which the two synergistically promote cognitive improvement, with the aim of making them a product that can be consumed long-term by people throughout their entire life cycle to improve cognition.

[0010] Solution for solving the problem

[0011] [1]. A nutritional composition, wherein it is a nutritional composition having a cognitive-enhancing function, said nutritional composition comprising the essential active ingredients shown in (I) and (II) below:

[0012] (I) Branched-chain fatty acids C15:0;

[0013] (II) Neutral fucoidylated human milk oligosaccharides;

[0014] The branched-chain fatty acid C15:0 includes anteiso-C15:0, the neutral fucoidylated human milk oligosaccharide includes 2'-FL, and the anteiso-C15:0 and 2'-FL work synergistically to improve cognition.

[0015] The improvement in cognition includes aiding in memory improvement.

[0016] [2]. According to the nutritional composition of [1], wherein the mass ratio of the essential active ingredient shown in (I) to the essential active ingredient shown in (II) is 1:(1-150).

[0017] [3]. The nutritional composition according to [1] or [2], wherein the nutritional composition further comprises branched fatty acids having 10 or more carbon atoms and having methyl branches, excluding branched fatty acid C15:0.

[0018] [4]. The nutritional composition according to any one of [1]-[3], wherein the nutritional composition further comprises human milk oligosaccharides other than neutral fucoidylated human milk oligosaccharides.

[0019] [5]. Use of the nutritional composition according to any one of [1]-[4] in the preparation of a product that helps improve cognition; preferably, the help to improve cognition includes aiding in memory improvement.

[0020] [6]. According to the use described in [5], the improvement of cognition includes the improvement of cognitive impairment behavior.

[0021] [7]. According to the use described in [5] or [6], wherein the improvement of cognition includes improving the levels of neurotransmitters and / or neurotransmitter-degrading enzymes.

[0022] [8]. The use according to any one of [5]-[7], wherein the improvement of cognition includes improving the level of brain neurotrophic factors.

[0023] [9]. Use according to any one of [5]-[8], wherein the product contains any one or more of the following ingredients: plant-based ingredients, animal dairy ingredients, animal meat ingredients and functional additives.

[0024]

[11] . Use of the nutritional composition according to any one of [1]-[4] in the preparation of a product having any one or more of the following (a)-(c):

[0025] (a) Improve cognitive impairment behaviors;

[0026] (b) Improving the imbalance of neurotransmitter and / or neurotransmitter-degrading enzyme levels; preferably, the neurotransmitters include dopamine and / or serotonin, and the neurotransmitter-degrading enzymes include acetylcholinesterase;

[0027] (c) Improve abnormal expression levels of brain neurotrophic factors; preferably, the brain neurotrophic factors include brain-derived neurotrophic factors and / or glial cell-derived neurotrophic factors.

[0028] The effects of the invention

[0029] This invention proposes for the first time that the combination of branched-chain fatty acid C15:0 and human milk oligosaccharide 2'-FL has a synergistic effect on improving cognition. In particular, when the two are combined within a certain ratio range, they can synergistically improve cognitive behavior, improve the neurotransmitter system, and improve the expression level of neurotrophic factor genes in the brain. Both branched-chain fatty acids and human milk oligosaccharides are natural nutrients found in breast milk or cow's milk, which can be used long-term and have broad applicability to people throughout their entire life cycle, playing a good role in improvement and prevention. Attached Figure Description

[0030] Figure 1 The effects of different monomers and nutrient combinations on the survival rate of zebrafish.

[0031] Figure 2 Comparative analysis of 5-HT content in zebrafish brain tissue from 9 examples.

[0032] Figure 3 Comparative analysis of DA content in zebrafish brain tissue from 9 examples.

[0033] Figure 4 Comparative analysis of AChE content in zebrafish brain tissue from 9 examples. Detailed Implementation

[0034] The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various configurations described below, and various modifications can be made within the scope of the claims. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0035] <Terminology Definition>

[0036] In this invention, "comprising," "having," "including," or "containing" can mean included or open-ended, and does not exclude additional, uncited elements or method steps. At the same time, "comprising," "having," "including," or "containing" can also mean closed-ended, excluding additional, uncited elements or method steps.

[0037] In this invention, the meaning of "may" includes both performing a certain process and not performing a certain process.

[0038] In this invention, "optional" or "optionally" means that certain substances, components, execution steps, application conditions, etc., are used or not used.

[0039] In this invention, the numerical ranges represented by "value A ~ value B", "value A - value B", and "value A above / below" refer to the ranges including the endpoint values ​​A and B.

[0040] In this invention, the term "about" is used to define that the numerical ranges and parameters of this invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. Unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this invention are modified by the term "about". Here, "about" generally means that the actual value is within ±5%, ±3%, ±1%, or ±0.5% of a specific value or range. Furthermore, the values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0041] In this invention, terms such as "some specific / preferred embodiments," "other specific / preferred embodiments," and "implementation" refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to a particular embodiment that are included in at least one of the embodiments described herein, and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0042] In this invention, all unit names used are international standard unit names, and unless otherwise stated, the "%" used refers to weight or mass percentage content.

[0043] In this invention, "infants and toddlers" refers to the human group under the age of 3 years, including infants aged 0-6 months, older infants aged 6-12 months, and toddlers aged 12-36 months.

[0044] In this invention, "children" refers to the human group aged 3-6 years.

[0045] In this invention, "teenagers" refers to the human group aged 7-18.

[0046] In this invention, "pregnant women" includes women who are pregnant and women who are breastfeeding.

[0047] In this invention, "middle-aged and elderly" refers to the human group aged 41 and above, including middle-aged people aged 41-65 and elderly people aged 65 and above.

[0048] In this invention, "animal milk" refers to the fluid obtained from the mammary glands of a mammal in the process of lactation. The term "animal milk" should be interpreted broadly and encompasses both raw milk (i.e., the fluid obtained directly from the mammary glands) and standardized dairy products (such as skim milk or whole milk).

[0049] In this invention, "C15:0" represents a saturated fatty acid containing a total of 15 carbon atoms; where "C15" indicates that the fatty acid contains a total of 15 carbon atoms, and "0" indicates that the fatty acid contains 0 unsaturated double bonds; the same applies to other saturated fatty acids, such as C17:0, etc.

[0050] In this invention, "aC15:0" and "anteiso-C15:0" have the same meaning, both representing 12-methyltetradecanoic acid; wherein, "a" or "anteiso" indicates that the fatty acid contains a branched methyl group and the branched methyl group is located on the third carbon atom at the alkyl end of the fatty acid main chain; the same applies to other branched fatty acids, such as aC17:0, anteiso-C17:0, etc.

[0051] In this invention, "iC15:0" and "iso-C15:0" have the same meaning, both representing 13-methyltetradecanoic acid; wherein, "i" or "iso" indicates that the fatty acid contains a branched methyl group and that the branched methyl group is located on the second carbon atom at the alkyl end of the fatty acid main chain; the same applies to other branched fatty acids, such as iC17:0, iso-C17:0, etc.

[0052] In this invention, 2'-fucosyllactose (2'-FL) is a neutral trisaccharide composed of L-fucose, D-galactose, and D-glucose units, with the monosaccharide L-fucose linked to the disaccharide D-lactose via an α(1→2) bond. Its molecular formula is C2. 18 H 32 O 15 .

[0053] Unless otherwise defined, other technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0054] <Nutritional Combinations>

[0055] The nutritional composition provided by this invention comprises the essential active ingredients shown in (I) and (II) below:

[0056] (I) Branched-chain fatty acids C15:0;

[0057] (II) Neutral fucoidylated human milk oligosaccharides;

[0058] The branched-chain fatty acid C15:0 includes anteiso-C15:0, and the neutral fucoidylated human milk oligosaccharide includes 2'-FL.

[0059] The present invention does not impose any particular limitation on the source of branched-chain fatty acids C15:0. For example, it can be isolated and extracted from natural substances containing branched-chain fatty acids, such as bacterial biofilms, animal sebum, and mammary gland tissue, or it can be prepared by biological or chemical synthesis.

[0060] In some embodiments, the branched-chain fatty acid C15:0 in this invention is derived from lanolin. The lanolin undergoes a saponification reaction to prepare free lanolin alcohol and free lanolin acid soap. The branched-chain fatty acids in the lanolin are then obtained through multiple steps, such as alcohol-soap separation and fatty acid preparation and extraction.

[0061] In other embodiments, the branched-chain fatty acid C15:0 of ​​the present invention is provided in the form of edible ingredients, such as animal milk, animal dairy products, natto, etc. In some specific embodiments, the branched-chain fatty acid C15:0 of ​​the present invention is provided in the form of animal milk or animal dairy products, such as cow's milk, goat's milk, camel milk, cheese, yogurt, butter, etc.

[0062] This invention does not specifically limit the source of the neutral fucosylated human milk oligosaccharide. Typically, it can be synthesized by common chemical synthesis methods in the art, such as through a glycosylation reaction between lactose acceptor and fucosylation donor. It can also be obtained by means of microbial fermentation, for example, using exogenously added lactose as a substrate and 5'-guanine diphosphate nucleoside-fucosylate disodium salt formed through the microbial metabolic pathway as a precursor, and synthesizing 2'-fucosylated lactose under the action of fucosylation enzyme.

[0063] This invention has found that, compared to individual branched-chain fatty acid C15:0 such as anteiso-C15:0 and neutral fucosylated human milk oligosaccharides such as 2'-FL, the nutritional composition provided by this invention can more effectively improve cognition, that is, there is a synergistic effect between the two.

[0064] In some embodiments, the branched-chain fatty acid C15:0 also includes iso-C15:0.

[0065] In some embodiments, the branched-chain fatty acid C15:0 is anteiso-C15:0.

[0066] In some embodiments, the neutral fucosylated human milk oligosaccharide further includes any one or more of 3-fucosylated lactose, lactose-N-fucopentose I, and difucosylated lactose.

[0067] In some implementations, the neutral fucoidylated human milk oligosaccharide is 2'-FL.

[0068] In some embodiments, in the nutritional composition of the present invention, (I) branched-chain fatty acid C15:0 and (II) neutral fucosylated human milk oligosaccharides are the main active ingredients. That is, the nutritional composition of the present invention mainly relies on (I) branched-chain fatty acid C15:0 and (II) neutral fucosylated human milk oligosaccharides to exert specific physiological functions, such as helping to improve cognition. In other words, in some embodiments, the active ingredients of the nutritional composition (the ingredients that exert specific physiological functions, i.e., the ingredients that help improve cognition) are composed of the following components (I) and (II): (I) branched-chain fatty acid C15:0 and (II) neutral fucosylated human milk oligosaccharides.

[0069] In some embodiments, in the nutritional composition of the present invention, anteiso-C15:0 in (I) branched-chain fatty acid C15:0 and 2'-FL in (II) neutral fucosylated human milk oligosaccharide are the main active ingredients. That is, the nutritional composition of the present invention mainly relies on its contained anteiso-C15:0 and 2'-FL to exert specific physiological functions, such as helping to improve cognition. In other words, in some embodiments, the active ingredients of the nutritional composition (the components for exerting specific physiological functions, i.e., the components that help improve cognition) are composed of the following components (I) and (II): (I) branched-chain fatty acid C15:0 and (II) neutral fucosylated human milk oligosaccharide; wherein the branched-chain fatty acid C15:0 is anteiso-C15:0 and the neutral fucosylated human milk oligosaccharide is 2'-FL.

[0070] In some embodiments, the nutritional composition comprises an active ingredient (a component that performs a specific physiological function, i.e., a component that contributes to improving cognition) and an inactive ingredient (a substance that does not contribute to improving cognition, or at least does not have a synergistic effect with anteiso-C15:0 or 2'-FL). Exemplarily, the inactive ingredient may be other nutrients, any edible excipient, and / or substances that are generated during the production or acquisition of the active ingredient and cannot be effectively separated from the active ingredient or do not require separation. In some embodiments, the nutritional composition consists of the active ingredient and the inactive ingredient.

[0071] In some embodiments, the nutritional composition further comprises branched-chain fatty acids, other than branched-chain fatty acids C15:0, having 10 or more carbon atoms and being methyl-based, such as iso-C12:0, iso-C13:0, anteiso-C13:0, iso-C14:0, iso-C16:0, iso-C17:0, anteiso-C17:0, etc. In some embodiments, the nutritional composition further comprises human milk oligosaccharides other than neutral fucoidylated human milk oligosaccharides, such as at least one of lactose-N-neotetrasaccharide, lactose-N-tetrasaccharide, 3'-sialyllactose, and 6'-sialyllactose. All of the above substances can be present in the nutritional composition as inactive ingredients.

[0072] In some embodiments, the mass ratio of the essential active ingredient shown in (I) to the essential active ingredient shown in (II) in the nutritional composition is 1:(1-150); for example, it can be 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:1 00, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, or 1:150, etc.; preferably, the ratio of the two can be 1:(2-120); more preferably, the ratio of the two can be 1:(2-100); even more preferably, the ratio of the two can be 1:(2-50); preferably, the above ratio is the mass ratio of anteiso-C15:0 to 2'-FL. Within the above mass ratio range, the essential active ingredient shown in (I) and the essential active ingredient shown in (II) work synergistically to help improve cognition.

[0073] The present invention does not impose any particular limitation on the form of the nutritional composition; typically, it can be a liquid or a solid. From the perspective of ease of production, transportation, storage, and use, the nutritional composition of the present invention is preferably a powdered solid.

[0074] <Uses of Nutritional Combinations>

[0075] This invention provides the use of the above-described nutritional composition to help improve cognition. In some embodiments, the improvement of cognition is not intended to prevent or treat disease; that is, this invention provides the use of the above-described nutritional composition to help improve cognition for non-therapeutic purposes. Based on this, this invention also provides the use of the above-described nutritional composition in the preparation of products that help improve cognition.

[0076] In some implementations, the benefit of improved cognition includes aiding in improved memory.

[0077] In some implementations, the improvement in cognition includes improved spatial working memory.

[0078] In some implementations, the improvement in cognition includes the improvement of cognitive impairment behaviors; preferably, the cognitive impairment behaviors include spatial memory disorder.

[0079] In some specific implementations, the improvement in cognition includes the improvement of spatial memory disorder.

[0080] In some embodiments, the improvement in cognition includes improving the levels of neurotransmitters and / or neurotransmitter-degrading enzymes; preferably, the improvement in cognition includes improving the levels of neurotransmitters and neurotransmitter-degrading enzymes; more preferably, the neurotransmitters include dopamine and / or serotonin, and the neurotransmitter-degrading enzymes include acetylcholinesterase.

[0081] In some specific implementations, the improvement in cognition includes increasing the level of neurotransmitters; preferably, the improvement in cognition includes increasing the level of dopamine and / or serotonin.

[0082] In some specific implementations, the improvement in cognition includes reducing the level of neurotransmitter-degrading enzymes; preferably, the improvement in cognition includes reducing the level of acetylcholinesterase.

[0083] In some more specific implementations, the improvement in cognition includes increasing levels of dopamine and serotonin, and decreasing levels of acetylcholinesterase.

[0084] In some implementations, the improvement in cognition includes improving synaptic plasticity and / or neuronal plasticity.

[0085] In some embodiments, the improvement in cognition includes improving the levels of brain neurotrophic factors; preferably, the brain neurotrophic factors include brain-derived neurotrophic factor and / or glial cell-derived neurotrophic factor.

[0086] In some specific implementations, the improvement in cognition includes increasing the level of brain neurotrophic factors; preferably, the brain neurotrophic factors include brain-derived neurotrophic factor and / or glial cell-derived neurotrophic factor.

[0087] In some more specific embodiments, the improvement in cognition includes increasing the levels of brain-derived neurotrophic factor (BDNF) and / or glial cell-derived neurotrophic factor (GGF); preferably, the improvement in cognition includes increasing the mRNA levels of BDNF and / or GGF; and even more preferably, the improvement in cognition includes increasing the mRNA levels of both BDNF and GGF.

[0088] Based on this, the present invention also provides the use of the above-mentioned nutritional composition in the preparation of products having any one or more of the following (a)-(c):

[0089] (a) Improve cognitive impairment behaviors;

[0090] (b) Improving the imbalance of neurotransmitter and / or neurotransmitter-degrading enzyme levels; preferably, the neurotransmitters include dopamine and / or serotonin, and the neurotransmitter-degrading enzymes include acetylcholinesterase;

[0091] (c) Improve abnormal expression levels of brain neurotrophic factors; preferably, the brain neurotrophic factors include brain-derived neurotrophic factors and / or glial cell-derived neurotrophic factors.

[0092] The present invention does not specifically limit the products containing or prepared from the above-described nutritional composition. Each component of the nutritional composition is used in a source or form that meets local legal and regulatory requirements. For example, in some embodiments, anteiso-C15:0 is provided and added to the product in the form of an edible raw material (e.g., animal milk, animal dairy products, natto, etc.), and 2'-FL is provided and added to the product in the form of a natural source and / or a chemically synthesized source and / or a microbial fermentation source.

[0093] In some embodiments, the products of this invention are oral preparations, such as hard capsules, soft capsules, tablets, oral liquids, pills, granules, and powders.

[0094] In some embodiments, the product of the present invention is in the form of a liquid or a solid under normal temperature conditions.

[0095] In some embodiments, in the product of the present invention, the mass ratio of branched-chain fatty acid C15:0 to neutral fucoidylated human milk oligosaccharides can be 1:(1-150); preferably, the ratio can be 1:(2-120); more preferably, the ratio can be 1:(2-100); and even more preferably, the ratio can be 1:(2-50). In some embodiments, the above ratio is the content ratio of anteiso-C15:0 to 2'-FL in the product of the present invention.

[0096] This invention does not impose any specific absolute limits on the content of branched-chain fatty acid C15:0 and neutral fucoidylated human milk oligosaccharides in the product, as long as they meet the requirements of relevant local laws and regulations.

[0097] In some embodiments, the content of the branched-chain fatty acid C15:0 in the product may be 1 mg / 100g-250 mg / 100g, and the content of the neutral fucoidylated human milk oligosaccharide may be 20 mg / 100g-2000 mg / 100g.

[0098] In addition to the components described above in the nutritional composition, the product may also contain other ingredients, such as common nutrients like proteins / amino acids, carbohydrates, fats, vitamins, and minerals.

[0099] In addition, depending on the type of product and the end needs of the target audience, in some embodiments, the product contains any one or more of the following ingredients: plant-based ingredients, animal dairy ingredients, animal meat ingredients, functional additives, and any acceptable excipients.

[0100] Examples of plant-based ingredients include fruits such as figs, pomegranates, kiwis, oranges, pineapples, strawberries, apples, bananas, grapes, pears, cherries, blueberries, blackberries, blackcurrants, cranberries, raspberries, melons, amla, and bilberries, or their extracts; fruits and vegetables such as onions, cucumbers, tomatoes, cauliflower, carrots, 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, sorghum, yellow millet, buckwheat, soybeans, broad beans, peas, mung beans, red beans, and kidney beans, or their extracts; nuts such as walnuts, pistachios, cashews, hazelnuts, almonds, apricot kernels, pine nuts, peanuts, sunflower seeds, chestnuts, macadamia nuts, and ginkgo nuts, or their extracts; coffee or its extracts; and some medicinal and edible herbal medicines or their extracts.

[0101] Animal dairy product ingredients can include fresh milk from cows and sheep, as well as reprocessed dairy products such as whole milk powder, skim milk powder, whey protein concentrate, desalted whey powder, whey protein powder, hydrolyzed whey protein powder, and casein powder.

[0102] Examples of animal meat product ingredients include pork, beef, mutton, seafood, and poultry.

[0103] Examples of functional additives include vitamin supplements (such as 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, biotin, etc.), mineral supplements (such as iron, copper, manganese, zinc, cobalt, molybdenum, chromium, nickel, vanadium, fluorine, selenium, iodine, silicon, tin, etc.), nucleotide supplements (e.g.), dietary fiber (e.g., inulin, konjac flour, galactooligosaccharides, fructooligosaccharides, isomaltooligosaccharides, soybean polysaccharides, cyclodextrin, resistant dextrin, soybean fiber, etc.), and functional polyunsaturated fatty acid supplements (e.g., arachidonic acid oil powder, docosahexaenoic acid oil powder, etc.).

[0104] Any acceptable excipients may include solvents, antioxidants, antibacterial agents, thickeners, diluents, cosolvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, food flavorings, and food colorings.

[0105] Example

[0106] 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, specific conditions in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all materials and instruments used are commercially available conventional products.

[0107] Experimental Example 1: Toxicity Test Results of Modeling Drugs, Different Nutrients, and Combinations

[0108] 1. Experimental Methods

[0109] 1.1 Experimental Materials

[0110] aC15:0 (12-methyltetradecanoic acid) was purchased from Larodan, Malmö, Sweden; 2'-FL (2'-fucosylated lactose) was purchased from DSM; p-chlorophenylalanine was purchased from Shanghai Maclean Biotechnology Co., Ltd.; donepezil was purchased from Shanghai Maclean Biotechnology Co., Ltd.

[0111] E3 medium: 5 mM sodium chloride, 0.17 mM potassium chloride, 0.33 mM calcium chloride, 0.33 mM magnesium sulfate, purchased from Beijing Chemical Reagent Company. All reagents were of analytical grade.

[0112] 1.2 Laboratory Animals

[0113] Wild-type AB lineage zebrafish, bred and maintained by the Precision Nutrition and Active Health Center of Yantai University.

[0114] Zebrafish daily maintenance: Adult zebrafish are housed in an Aisheng zebrafish recirculating aquarium system, with a 14-hour / 10-hour light / dark cycle daily. The water temperature is maintained at 28±0.5℃, pH at 7.0-8.0, and conductivity at around 500 μs. They are fed fresh brine shrimp eggs twice daily.

[0115] Zebrafish spawning: Remove the mating box, insert a separator in the middle to separate the males and females, add circulating water to the system until it is 80% full, and finally place it in the isolation tank to prevent the adult fish from eating the embryos. After the adult zebrafish finish feeding at night, place them on both sides of the separator according to a male:female ratio of 1:2. The next morning, remove the separator. After the male and female fish chase each other, they will spawn and fertilize externally. After 0.5-1 hours, collect the adult fish into the circulating water system, mark the date, and use a filter to collect the embryos in a glass culture dish containing E3 water and culture them at 28±0.5℃.

[0116] Establishment of a cognitive impairment model in zebrafish juveniles: Wild-type AB strain zebrafish at 4 dpf were randomly selected and placed in 6-well plates, with 30 fish per well. A blank control group, a model group, a positive control group, and a sample treatment group were set up. The blank control group was cultured normally in E3 water. All other groups were treated with 4 mM p-chlorophenylalanine, and the positive control group was given 5 μg / mL donepezil. The sample treatment groups were given different concentrations of monomeric nutrients and combinations thereof.

[0117] 1.3 Experimental Apparatus

[0118] Zebrafish recirculating aquaculture system (ESEN-AW-S1, Beijing Aisheng Technology Development Co., Ltd.); Zebrafish behavioral analysis system (DanioVision, Noldus, Netherlands); Electronic balance (AR-2140, Shanghai Ohaus Instruments Co., Ltd.); Multifunctional microplate reader (SpectraMax, Molecular Devices); Ultrapure water system (TC-RO-100, Shanghai Likang Instruments Co., Ltd.); pH meter (pH9500, Shanghai Peirui Instruments Co., Ltd.); Conductivity meter (HM-100TDS, HM Digital, South Korea); Thermometer (high precision, Odasys Technology Co., Ltd.); Ultra-low temperature freezer (HYCD-205, Qingdao Haier Co., Ltd.); High-speed refrigerated centrifuge (Micro 21R, Thermo Fisher Scientific, USA); Mini centrifuge (LX-500, Anhui Zhongke Co., Ltd.); Real-time quantitative PCR system (7500 Fast, Thermo Fisher Scientific, USA); Ultra-micro spectrophotometer (ND5000, Wuxi Baitai Biotechnology Co., Ltd.).

[0119] 1.4 Experimental Intervention Design

[0120] The experimental design is shown in Table 1. The control group consisted of normal zebrafish without any treatment or intervention; the model group consisted of the p-chlorophenylalanine treatment group, specifically, 4 mM p-chlorophenylalanine was dissolved in E3 water, and then 3 ml of the solution was placed in the well of a 6-well plate containing zebrafish for feeding; the positive control group consisted of the p-chlorophenylalanine + donepezil treatment group, specifically, 4 mM p-chlorophenylalanine was dissolved in E3 water, and then 5 μg / mL donepezil positive control agent was dissolved in the same solution, and then 3 ml of the solution was placed in the well of a 6-well plate containing zebrafish for feeding. The nutrient intervention group was a p-chlorophenylalanine + nutrient treatment group. Specifically, 4 mM p-chlorophenylalanine was dissolved in E3 water, and then the test sample was dissolved in the solution. 3 ml of the solution was then placed in the well of a 6-well plate containing zebrafish and fed to the zebrafish. Among them, Comparative Examples 1-3 were 4 mM p-chlorophenylalanine + 0.001 mg / mL, 0.012 mg / mL and 0.02 mg / mL aC15:0 intervention groups; Comparative Examples 4-6 were 4 mM p-chlorophenylalanine + 0.04 mg / mL, 0.08 mg / mL and 0.12 mg / mL 2'-FL intervention groups; Examples 1-9 were intervention groups formed by combining 4 mM p-chlorophenylalanine + aC15:0 and 2'-FL in different concentration ratios (1:2-120).

[0121] Table 1. Experimental Intervention Design

[0122]

[0123] 2. Experimental Results

[0124] Before investigating cognitive function, the toxicity and lethality of zebrafish were examined at the concentrations of the monomers and combinations of the two nutrients. The study found that at three concentrations of aC15:0 (0.001 mg / mL, 0.012 mg / mL, 0.02 mg / mL) and three concentrations of 2'-FL (0.04 mg / mL, 0.08 mg / mL, 0.12 mg / mL), and at nine different combinations of aC15:0+2'-FL, no mortality or other toxicity was observed in zebrafish. For detailed results, see [link to results]. Figure 1 .

[0125] Experiment Example 2: Comparative Analysis of the Effects of Different Nutrients and Combinations on the Improvement of Cognitive Behavior in Zebrafish

[0126] 1. Experimental Methods

[0127] 1.1 The experimental materials are the same as those in Experiment Example 1.

[0128] 1.2 The experimental animals were the same as in Experiment 1.

[0129] 1.3 The experimental apparatus is the same as in Experiment Example 1.

[0130] 1.4 The experimental intervention design is the same as that in Experiment Example 1.

[0131] 1.5 Evaluation of Zebrafish Cognitive Structure

[0132] Wild-type AB strain zebrafish with a 4-day pf growth rate were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Different concentrations of nutrient monomers or combinations were administered in water. The positive control group was given donepezil 5 μg / mL. A normal control group and a model control group were also set up. Each group was treated at 28℃ for 24 h, and the volume of each well was 3 mL.

[0133] Cognitive structure tests were conducted on different groups of zebrafish juveniles. The activity area of ​​the zebrafish juveniles was a "cross" area. The cross area was divided into five parts: top, bottom, left, right, and center. Different colors were marked on the walls of the cross maze board for different areas: yellow (top), green (bottom), red (left), and blue (right). Repeatedly entering a single-colored area in the top, bottom, left, or right areas represented cognitive impairment; that is, the higher the percentage of time spent in the same analysis area in the top, bottom, left, or right areas, the higher the degree of cognitive impairment. First, the prepared juveniles were removed, and one juvenile was placed in each observation area of ​​the cross maze board. The "center" area was separated from other areas by a partition, and the juveniles were isolated in the "center" area. The bottom light of the monitoring area was turned on, and the cross maze board was placed in the behavioral monitoring area for 10 minutes to allow the juveniles to adapt to the environment. The camera's capture range was adjusted to coincide with the cross maze holes, and the zebrafish's movement trajectory was successfully captured. After that, the partition was removed, and the behavioral trajectory of the zebrafish juveniles was monitored. After 5 minutes of monitoring, the partition was removed, and the trajectory and the time spent in each analysis area were recorded.

[0134] Statistical results are expressed as mean ± SE. Statistical analysis was performed using GraphPad 9.0 software, and p < 0.05 was considered statistically significant.

[0135] 2. Experimental Results

[0136] Good cognitive function manifests as efficient information processing, accurate learning and memory, and flexible decision-making and execution. This invention first uses the cross maze method to investigate the effects of aC15:0 and 2'-FL monomers and their composition on improving cognitive impairment induced by p-chlorophenylalanine in zebrafish, particularly on improving spatial working memory. In the cross maze, zebrafish rely on their telencephalon (homogeneous with the mammalian hippocampus) and cholinergic neurotransmitter system to integrate visual-spatial cues (yellow, green, red, and blue regions) to form spatial memory. Juvenile zebrafish have an exploratory instinct towards novel environments. Under normal circumstances, they should enter the four regions (upper, lower, left, and right regions with different colors) with roughly the same frequency. When cognitive impairment occurs, the neural circuits are damaged, causing zebrafish to be unable to effectively distinguish spatial locations of different colors, resulting in repeated entry into the same region. This study used the percentage of zebrafish entering a single region at the highest frequency and the percentage of zebrafish entering a single region at the highest duration as indicators to evaluate the effects of aC15:0 and 2'-FL monomers and their combination on cognitive behavior improvement. The specific results are shown in Table 2.

[0137] Table 2 shows that the average percentage of zebrafish entering a single region in the control group was 37.9%, while the percentage in the model group (using p-chlorophenylalanine) reached 99%, indicating successful modeling and severe cognitive impairment in the model group zebrafish. The positive control group received donepezil intervention, and the results showed that the percentage of zebrafish entering a single region significantly decreased back to the control group level. Simultaneously, it can be seen that after intervention with branched-chain fatty acid aC15:0 at low, medium, and high doses (Comparative Examples 1-3), the proportion of zebrafish entering the single region at maximum frequency decreased, and after one-way ANOVA analysis, it was found to be significantly lower than that of the model group (p < 0.0001, as shown in Table 3). Furthermore, the degree of reduction increased with increasing aC15:0 dose, with the average value in the high-dose group decreasing to 68.69%. Similarly, after intervention with 2'-FL at low, medium, and high doses (Comparative Examples 4-6), the proportion of zebrafish entering the single region at maximum frequency also decreased to 68.83%-79.52% in all three groups, showing a trend of increasing reduction with increasing dose. One-way ANOVA analysis further confirmed this. ANOVA analysis revealed that the three comparative groups were significantly lower than the model group (p < 0.0001, as shown in Table 3). Examples 1-9 involved nine combinations of aC15:0 and 2'-FL at different dosages to intervene in zebrafish. The results showed that the percentage of zebrafish entering a single region in each group of examples was further reduced to the range of 34.57%-61.21%, and was significantly lower than the six comparative groups (significant differences are shown in Table 3). This indicates that the combined intervention of aC15:0 and 2'-FL is more effective than the intervention of the two nutrients alone. Further analysis revealed that the reduction in the percentage of zebrafish entering a single region after the combination of the two was greater than the sum of the reductions by the individual interventions of the two nutrients. This indicates that the two have a synergistic effect of 1+1>2, and that they have a synergistic effect in reducing the percentage of zebrafish entering a single region.

[0138] The percentage of the longest duration of entry into a single region is also an important indicator for evaluating cognitive impairment in zebrafish. As shown in Table 2, the average value of this value in normal zebrafish (blank control group) without any treatment is about 42.62%; while the percentage of the longest duration of entry into a single region in the model group after using p-chlorophenylalanine modeling is significantly increased to about 99.69%, which also indicates that the modeling was successful. After donepezil drug intervention, this indicator in the positive control group can return to the level of the blank control group. After intervention with branched-chain fatty acid aC15:0 at low, medium, and high doses (Comparative Examples 1-3), the proportion of zebrafish that remained in a single region for the longest time decreased to 82%-88%, and the difference was significantly lower than that of the model group (p < 0.0001, as shown in Table 4). After intervention with 2'-FL at low, medium, and high doses (Comparative Examples 4-6), the proportion of zebrafish that remained in a single region for the longest time also decreased in all three groups, ranging from approximately 74%-87%, which was significantly lower than that of the model group (p < 0.0001, as shown in Table 4). Examples 1-9 are the results of different combinations of aC15:0 and 2'-FL at different doses. Nine combinations were used to intervene in zebrafish. The results showed that the proportion of zebrafish entering a single area for the longest time in each group of the examples was further reduced to the range of 35%-68%, which was significantly lower than that of the six comparative examples (the difference is shown in Table 4). This indicates that the combined intervention of aC15:0 and 2'-FL is more effective than the intervention of the two nutrients alone. Further analysis showed that the reduction in the proportion of zebrafish entering a single area after the combination of the two was greater than the sum of the reductions of the two nutrients alone. This indicates that the two have a synergistic effect of 1+1>2, and that the two have a synergistic effect of reducing the proportion of zebrafish entering a single area for the longest time in the model.

[0139] The results above indicate that the combined intervention of branched-chain fatty acids aC15:0 and 2'-FL (with a ratio ranging from 1:2 to 120) has a significant effect on reducing cognitive impairment behaviors in zebrafish.

[0140] Table 2. Effects of different monomers and nutrient combinations on cognitive behavior in zebrafish.

[0141]

[0142] Table 3. Analysis of the differences in the percentage of the maximum frequency of zebrafish entering a single area among different groups.

[0143]

[0144]

[0145] Table 4. Analysis of the differences in the percentage of the maximum duration of zebrafish entering a single area among different groups.

[0146]

[0147]

[0148] Experiment Example 3: Effects of different nutrients and combinations on neurotransmitters in the zebrafish brain

[0149] 1. Experimental Methods

[0150] 1.1 The experimental materials are the same as those in Experiment Example 1.

[0151] 1.2 The experimental animals were the same as in Experiment 1.

[0152] 1.3 The experimental apparatus is the same as in Experiment Example 1.

[0153] 1.4 The experimental intervention design is the same as that in Experiment Example 1.

[0154] 1.5 Determination of Dopamine (DA) and Serotonin (5-HT) Content

[0155] Wild-type AB strain zebrafish with a dpf growth rate of 4 days were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Different concentrations of nutrient monomers or combinations were administered in water. The positive control group was given donepezil 5 μg / mL. A normal control group and a model control group were also set up. The volume of each well was 3 mL. After treatment at 28℃ for 24 h, 0.01 M 1×PBS was added to lyse the zebrafish, and the lysate was collected for subsequent detection.

[0156] Dopamine (DA) and serotonin (5-HT) were detected using enzyme-linked immunosorbent assay (ELISA). Purchase the kits and follow the instructions. The kits are: Fish Dopamine (DA) ELISA Kit (Shanghai Enzyme-Linked Biotechnology Co., Ltd., catalog number: ml103412) and Fish Serotonin (5-HT) ELISA Kit (Shanghai Enzyme-Linked Biotechnology Co., Ltd., catalog number: ml874512). The specific steps are as follows: Remove the required microplate strips from the aluminum foil bag after equilibration at room temperature for 20 min. Add 50 μL of the sample to be tested. Add 50 μL of biotin antigen working solution to each well (except the blank wells) and each standard and sample well. Seal the reaction wells with sealing film and incubate at 37°C for 30 min. Discard the liquid, pat dry on absorbent paper, fill each well with washing buffer, let stand for 30 s, discard the washing buffer, pat dry on absorbent paper, and repeat this washing process 5 times. Add 50 μL of avidin-HRP working solution to each well of the standard and sample solutions, seal the reaction wells with sealing film, and incubate at 37°C for 30 min. Repeat the washing process 5 times. Then add 50 μL each of colorimetric solutions A and B to each well and incubate at 37°C in the dark for 10 min. Add 50 μL of stop solution to each well. Within 10 min, measure the OD value of each well at a wavelength of 450 nm to determine the dopamine (DA) and serotonin (5-HT) content.

[0157] 1.6 Determination of acetylcholinesterase (AChE) content

[0158] Wild-type AB strain zebrafish with a dpf growth rate of 4 days were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Different concentrations of nutrient monomers or combinations were administered in water. The positive control group was given donepezil 5 μg / mL. A normal control group and a model control group were also set up. The volume of each well was 3 mL. After treatment at 28℃ for 24 h, 0.01 M 1×PBS was added to lyse the zebrafish, and the lysate was collected for subsequent detection.

[0159] The acetylcholinesterase (AChE) content was determined using an enzyme-linked immunosorbent assay (ELISA). The kit was purchased and the procedure followed according to the instructions. The zebrafish acetylcholinesterase (AChE) kit (Shanghai Enzyme-Linked Biotechnology Co., Ltd., catalog number: ml696511) was used. The specific steps were as follows: Remove the required microplate strips from the aluminum foil bag after equilibration at room temperature for 60 min. Add 50 μL of the sample to be tested. Add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well (except the blank wells). Seal the reaction wells with sealing film and incubate at 37°C for 60 min in a water bath or incubator. Discard the liquid, pat dry on absorbent paper, fill each well with washing buffer, let stand for 1 min, discard the washing buffer, and pat dry on absorbent paper. Repeat this washing process 5 times. Then add 50 μL each of substrate A and B to each well and incubate at 37°C in the dark for 15 min. Add 50 μL of stop solution to each well. Within 15 min, measure the OD value of each well at a wavelength of 450 nm to determine the acetylcholinesterase (AChE) content.

[0160] 2. Experimental Results

[0161] Neurotransmitter systems influence cognitive function, with dopamine (DA), serotonin (5-HT), and acetylcholine (ACh) being the three major neurotransmitter systems. In cognitive activity, the DA system regulates motivation and reward mechanisms through the mesolimbic pathway; the 5-HT system projects extensively to the prefrontal cortex and limbic system to maintain emotional homeostasis and inhibitory control; and the ACh system directly regulates attention, arousal, and memory encoding in the cortex and hippocampus. The activity of its hydrolase, acetylcholinesterase (AChE), dynamically balances the intensity and duration of cholinergic signals. This invention investigated the changes in the three major neurotransmitters in zebrafish brain tissue after cognitive impairment. The experimental results are shown in Table 5, and the significance analysis is shown in Tables 6-8.

[0162] As an important monoamine neurotransmitter in the central nervous system, serotonin (5-HT) participates in regulating higher brain functions such as emotion, cognition, and memory through its widely distributed receptor system (such as 5-HT1A and 5-HT4). Studies have shown that increasing central 5-HT levels can not only improve synaptic plasticity but also significantly alleviate cognitive impairment associated with cholinergic system dysfunction. In this invention, we found that the average 5-HT level in normal zebrafish brain tissue was approximately 102.41 ng / mL, while when zebrafish were given p-chlorophenylalanine to induce cognitive impairment, 5-HT decreased to 36.30 ng / mL, showing a highly significant difference (as shown in Table 6). This indicates successful modeling. Furthermore, we found that 5-HT levels could be restored to control levels after intervention with donepezil. Comparative Examples 1-3 showed that the 5-HT content in the brain tissue of zebrafish with cognitive impairment was increased to 50 ng / mL after low, medium, and high doses of branched-chain fatty acid aC15:0, and the level was significantly higher than that in the model group after significance analysis (p < 0.0001). Comparative Examples 4-6 showed that the 5-HT content in the brain tissue of zebrafish in the three groups was also increased to the range of 49.96 ng / mL-54.49 ng / mL after low, medium, and high doses of 2'-FL, and the level was significantly higher than that in the model group after significance analysis (p < 0.0001, as shown in Table 6). Examples 1-9 are nine compositions formed by combining aC15:0 and 2'-FL at different doses and their effects on zebrafish. The intervention results showed that the 5-HT content in zebrafish brain tissue in each group of the examples further increased to over 65 ng / mL, reaching a maximum of 120 ng / mL, and was significantly higher than that in the six comparative examples (Table 6). This indicates that the combined intervention of aC15:0 and 2'-FL was more effective than the intervention of either nutrient alone. Further analysis revealed that the increase in 5-HT content in zebrafish brain tissue after the combination of the two was greater than the sum of the increases from the individual interventions of the two nutrients. This indicates that the two had a synergistic effect of 1+1>2, meaning that they had a synergistic effect in increasing the 5-HT content in the brain tissue of the model zebrafish. Furthermore, Examples 8 and 9 reached or exceeded the effect of the positive control drug and were significantly higher than those of the other examples (such as...). Figure 2 (As shown).

[0163] Dopamine (DA), another neurotransmitter associated with reward and motivation, regulates signaling pathways such as cAMP / PKA and ERK by acting on D1-like and D2-like receptors. Increased DA levels in the hippocampus contribute to enhanced learning and memory functions. This invention found that the average DA level in normal zebrafish brain tissue was 175.26 ng / mL. After induction with p-chlorophenylalanine, the DA level in the brain tissue decreased to 111.26 ng / mL, showing a highly significant difference (as shown in Table 7). This indicates successful model establishment. Furthermore, intervention with donepezil restored DA levels to those in the control group. Comparative Examples 1-3 showed that intervention with branched-chain fatty acid aC15:0 at low, medium, and high doses increased the DA content in the brain tissue of zebrafish with cognitive impairment, reaching 130-140 ng / mL, and the level increased with increasing dose. Significant difference analysis showed that the DA content in the three groups was significantly higher than that in the model group (p < 0.01, p < 0.001, p < 0.0001). Comparative Examples 4-6 showed that intervention with 2'-FL at low, medium, and high doses increased the DA content in the brain tissue of zebrafish in the three groups to the range of 129 ng / mL-139 ng / mL. Significant difference analysis showed that the DA content in all three groups was significantly higher than that in the model group (p < 0.001, p < 0.0001). Examples 1-9 involved different doses of aC15:0 and 2'-FL. Nine combinations were developed and applied to zebrafish. Results showed that the DA content in the zebrafish brain tissue of each group was further increased to over 150 ng / mL, reaching a maximum of 249 ng / mL, and was significantly higher than that of the six comparative examples (as shown in Table 7). This indicates that the combined intervention of aC15:0 and 2'-FL was superior to the intervention of either nutrient alone. Further analysis revealed that the increase in DA content in zebrafish brain tissue after the combination was greater than the sum of the increases from the individual interventions of the two nutrients. This indicates a synergistic effect (1+1>2), demonstrating that the two nutrients synergistically increased the DA content in the model zebrafish brain tissue. Furthermore, Examples 8 and 9 surpassed the effect of the positive control drug and were significantly higher than the other examples (e.g., ...). Figure 3 (As shown).

[0164] Acetylcholinesterase (AChE), a key terminator of cholinergic synaptic signaling, is primarily responsible for hydrolyzing the neurotransmitter acetylcholine (ACh). Acetylcholine activates muscarinic (M-type) and nicotinic (N-type) receptors, thereby regulating signaling pathways such as cAMP / PKA-CREB and ultimately participating in the regulation of cognitive functions such as learning and memory. Inhibition of acetylcholinesterase activity can enhance cholinergic signal transduction via the ACh-M receptor-Gq / 11-PKC or Gi / o-AC-cAMP signaling axis, synergistically promoting CREB phosphorylation and the expression of synaptic plasticity-related genes, thus playing a crucial role in improving cognitive function and delaying the progression of neurodegenerative diseases. This invention found that the average AChE in the brain tissue of normal zebrafish was 11.63 ng / mL. When zebrafish were induced with p-chlorophenylalanine, the AChE in the brain tissue increased to 19.44 ng / mL, showing a highly significant difference (as shown in Table 8). This indicates that the model was successfully established. At the same time, it was found that the amount of AChE was further reduced to 13.08 ng / mL after intervention with donepezil. Comparative Examples 1-3 showed that the AChE content in the brain tissue of zebrafish with cognitive impairment decreased after low, medium, and high doses of branched-chain fatty acid aC15:0 intervention, and the degree of reduction increased with increasing dose. At the high dose, the AChE content was 15.48 ng / mL. After significance analysis, the three comparative examples were found to be significantly lower than the model group (p < 0.0001). Comparative Examples 4-6 showed that after low, medium, and high doses of 2'-FL intervention, the AChE content in the brain tissue of zebrafish in the three groups decreased to the range of 15.09 ng / mL-16.15 ng / mL. After significance analysis, all were found to be significantly lower than the model group (p < 0.0001). Examples 1-9 were nine combinations of aC15:0 and 2'-FL at different doses. The combination of these ingredients was used to intervene in zebrafish. Results showed that the AChE content in the zebrafish brain tissue of each group was further reduced to below 13.4 ng / mL, which was superior to the positive control. Examples 6-9 showed lower levels than the control group, and statistical analysis revealed that all nine examples were significantly lower than the six comparative examples (as shown in Table 8). This indicates that the combined intervention of aC15:0 and 2'-FL was more effective than the intervention of either nutrient alone. Further analysis showed that the reduction in AChE content in zebrafish brain tissue after the combination was greater than the sum of the reductions by the individual interventions of the two nutrients. This indicates that the combination produced a synergistic effect (1+1>2), demonstrating that the two nutrients synergistically reduced the AChE content in the model zebrafish brain tissue. Furthermore, Example 9 showed significantly lower levels than Examples 1-7 (as shown in Table 8). Figure 3 (As shown).

[0165] The above experimental results indicate that combined intervention with aC15:0 and 2'-FL can significantly promote the increase of neurotransmitter 5-HT and DA levels in brain tissue, significantly reduce the level of acetylcholinesterase, and the two have a synergistic effect.

[0166] Table 5. Effects of different monomers and nutrient combinations on neurotransmitters in zebrafish brains.

[0167]

[0168] Table 6. Significance analysis of the effects of different monomers and nutritional compositions on the neurotransmitter 5-HT in zebrafish brains.

[0169]

[0170]

[0171] Table 7. Significance analysis of the effects of different monomers and nutrient combinations on the neurotransmitter DA in zebrafish brains.

[0172]

[0173]

[0174] Table 8. Significance analysis of the effects of different monomers and nutrient combinations on AChE in zebrafish brains.

[0175]

[0176]

[0177] Experiment Example 4: Effects of different nutrients and their combinations on the expression levels of neurotrophic factor genes in the zebrafish brain

[0178] 1. Experimental Methods

[0179] 1.1 The experimental materials are the same as those in Experiment Example 1.

[0180] 1.2 The experimental animals were the same as in Experiment 1.

[0181] 1.3 The experimental apparatus is the same as in Experiment Example 1.

[0182] 1.4 The experimental intervention design is the same as that in Experiment Example 1.

[0183] 1.5 Determination of BDNF and GDNF gene expression levels

[0184] Wild-type AB strain zebrafish with a 4dpf cell count were randomly selected and randomly assigned to 6-well plates, with 30 zebrafish treated in each well (experimental group). After treatment at 28℃ for 24 h, all 30 zebrafish from each well were collected into 2 mL RNase-free centrifuge tubes, the water was aspirated, 350 μL of pre-chilled lysis buffer and 2 sterilized 3 mm steel beads were added, and the cells were lysed in a cell disruptor at 4℃.

[0185] Total RNA was extracted using the Cisco SPARKeasy RNA Rapid Extraction Kit. The simplified steps included: lysis followed by mixing with anhydrous ethanol, transferring to an adsorption column, centrifuging, and discarding the eluent; washing the adsorption column sequentially with washing buffer; and finally eluting RNA with RNase-free water. RNA concentration and purity were determined using a micro-spectrophotometer. An A260 / A280 ratio of 1.8–2.0 was considered acceptable. Acceptable samples were stored at -80°C for later use.

[0186] An equal volume of total RNA was used to synthesize cDNA using a Cisco reverse transcription kit. Genomic DNA was first removed by treatment with a gDNA Eraser at 42°C for 2 min. Then, SPARKscript II RT Plus Master Mix was added, and the reaction was terminated by heating at 85°C for 5 min. The cDNA product was stored at -20°C.

[0187] The expression levels of BDNF and GDNF genes were detected using a Cisco Real-Time Quantitative PCR kit. The reaction mixture (20 μL) contained: 10 μL of 2×SYBR qPCR Mix, 0.4 μL each of forward and reverse primers, 1 μL of cDNA template, and 8.2 μL of nuclease-free water. Each sample was tested in triplicate, normalized to an internal control gene, and analyzed using 2... -ΔΔCt The relative gene expression levels were calculated using a method. Primer sequences are shown in Table 9.

[0188] Experimental data are expressed as mean ± standard deviation. One-way ANOVA was performed using GraphPad Prism software, and Dunnett's test was used for comparisons between groups. P < 0.05 was considered statistically significant.

[0189] Table 9 Primer Sequences

[0190]

[0191] 2. Experimental Results

[0192] Cognitive load-induced changes in synaptic plasticity upregulate the expression of neurotrophic factor genes such as BDNF and GDNF. These factors not only promote neuronal survival and synaptic remodeling by activating MAPK and PI3K-Akt pathways through TrkB / RET receptors, but also positively regulate the functional integrity of the dopamine (DA), 5-HT, and aCh (ACh) systems, forming a positive feedback regulatory loop of "neurotransmitter-neurotroph." When cognitive load consistently exceeds the compensatory capacity of the nervous system, it may lead to DA depletion, abnormal 5-HT transport, AChE activity imbalance, and downregulation of BDNF / GDNF expression, resulting in accumulated oxidative stress, impaired mitochondrial function, and reduced synaptic plasticity. p-Chlorophenylalanine, as a selective tryptophan hydroxylase inhibitor, significantly inhibits the biosynthesis of serotonin (5-HT) in the central nervous system, blocking the key 5-HT-BDNF / GDNF signaling transduction axis. The disruption of this signaling axis theoretically leads to the suppression of downstream neurotrophic factor expression, thereby triggering a series of pathophysiological changes such as reduced neuronal plasticity, impaired synaptic function, and cognitive behavioral decline. In this invention, it was found that after modeling with p-chlorophenylalanine, the mRNA expression levels of both neurotrophic factors in the zebrafish brain tissue of the model group were significantly reduced (as shown in Table 10), and after significance analysis, a significant difference was found between the model group and the control group (as shown in Tables 11 and 12). However, after intervention with aC15:0 and 2'-FL, the mRNA expression levels of both neurotrophic factors were increased to varying degrees.

[0193] Table 10 shows that the mRNA expression level of brain-derived neurotrophic factor (BDNF) was significantly increased compared to the model group after intervention with different doses of aC15:0 and 2'-FL (comparative examples 1-6), reaching a level of 1.1-1.8, and the difference was significantly higher than that of the model group (as shown in Table 11). After intervention with combinations of aC15:0 and 2'-FL in different proportions, the mRNA expression level of BDNF was further increased to above 2.5, and even reached above 4. Significant difference analysis revealed that 9 examples were significantly higher than the model group (as shown in Table 11), and also significantly higher than 6 comparative examples (as shown in Table 11). Further analysis showed that the increase in BDNF mRNA expression level in zebrafish brain tissue after the combination of the two was significantly greater than the sum of the increases from intervention with either nutrient alone. This indicates that the two had a synergistic effect, with 1+1>2, and synergistically promoted the mRNA expression level of BDNF in the zebrafish brain tissue of the model group.

[0194] As shown in Table 10, the mRNA expression level of glial cell-derived neurotrophic factor (GDNF) was significantly increased compared to the model group after intervention with different doses of aC15:0 and 2'-FL (comparative examples 1-6), reaching a level of 1.4-2.0, and the difference was significantly higher than that of the model group (as shown in Table 12). After intervention with combinations of aC15:0 and 2'-FL in different proportions, the mRNA expression level of GDNF was further increased to above 2.8, even reaching 3.45. Significant difference analysis revealed that 9 examples were significantly higher than the model group (as shown in Table 12), and also significantly higher than 6 comparative examples (as shown in Table 12). Further analysis showed that the increase in GDNF gene expression in zebrafish brain tissue after the combination of the two was significantly greater than the sum of the increases from intervention by the two nutrients alone. This indicates that the two had a synergistic effect, with 1+1>2, and synergistically promoted the expression level of GDNF in the zebrafish brain tissue of the model group.

[0195] Table 10 Effects of different monomers and nutrient combinations on the gene expression levels of neurotrophic factors in the zebrafish brain

[0196]

[0197] Table 11. Significant differences in the expression levels of the neurotrophic factor BDNF gene in zebrafish brains due to interventions by different monomers and nutrient compositions.

[0198]

[0199]

[0200] Table 12. Significant differences in the expression levels of the neurotrophic factor GDNF gene in the zebrafish brain caused by interventions of different monomers and nutrient compositions.

[0201]

[0202]

[0203] The above results indicate that combined intervention with aC15:0 and 2'-FL can significantly promote the expression of neurotrophic factors BDNF and GDNF mRNA in brain tissue, and the two have a synergistic effect.

Claims

1. A nutritional composition, characterized in that, It is a nutritional composition with cognitive-enhancing functions, the nutritional composition comprising the essential active ingredients shown in (I) and (II) below: (I) Branched-chain fatty acids C15:0; (II) Neutral fucoidylated human milk oligosaccharides; The branched-chain fatty acid C15:0 includes anteiso-C15:0, the neutral fucoidylated human milk oligosaccharide includes 2'-FL, and the anteiso-C15:0 and 2'-FL work synergistically to improve cognition. The improvement in cognition includes aiding in memory improvement.

2. The nutritional composition according to claim 1, characterized in that, In the nutritional composition, the mass ratio of the essential active ingredient shown in (I) to the essential active ingredient shown in (II) is 1:(1-150).

3. The nutritional composition according to claim 1 or 2, characterized in that, The nutritional composition also contains branched-chain fatty acids, excluding branched-chain fatty acids C15:0, which have 10 or more carbon atoms and are branched by methyl groups.

4. The nutritional composition according to claim 1 or 2, characterized in that, The nutritional composition also contains human milk oligosaccharides other than neutral fucoidylated human milk oligosaccharides.

5. Use of the nutritional composition according to any one of claims 1-4 in the preparation of a product that helps improve cognition, wherein the improvement of cognition includes aiding in memory improvement.

6. The use according to claim 5, characterized in that, The benefits to improving cognition include improving cognitive impairment behaviors.

7. The use according to claim 5, characterized in that, The benefits to improved cognition include improving the levels of neurotransmitters and / or neurotransmitter-degrading enzymes.

8. The use according to claim 5, characterized in that, The benefits to improved cognition include improving the levels of neurotrophic factors in the brain.

9. The use according to any one of claims 5-8, characterized in that, The product contains any one or more of the following ingredients: plant-based ingredients, animal dairy ingredients, animal meat ingredients, and functional additives.

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