A nutritional composition and its use for assisting in improving neuro-oxidative damage

Abstract

This invention belongs to the field of nutrient research technology, specifically relating to a nutritional composition and its use in assisting the improvement of neuronal oxidative damage. The nutritional composition provided by this invention comprises the following two essential components: i) ergothioneine; ii) milk fat globule membrane; and, in the nutritional composition, the mass ratio of ergothioneine to milk fat globule membrane is 1:(1-150). The combination of ergothioneine and milk fat globule membrane can synergistically assist in improving neuronal oxidative damage, significantly and synergistically enhancing the survival rate of nerve cells under oxidative damage conditions. Furthermore, based on this, the combined use of the two also effectively reduces intracellular reactive oxygen species levels and inhibits neuronal apoptosis.

Description

Technical Field

[0001] This invention belongs to the field of nutrient research technology, and relates to a nutritional composition and its use in assisting in improving neuro-oxidative damage, and more specifically to a nutritional composition and its use in assisting in improving neuro-oxidative damage and / or neuroprotection. Background Technology

[0002] During oxidative stress-induced neuronal cell damage, excessive accumulation of intracellular reactive oxygen species (ROS) can lead to mitochondrial dysfunction, thereby activating the apoptosis pathway and ultimately resulting in progressive loss of neuronal function. Currently, commonly used neuroprotective ingredients (such as vitamin E and coenzyme Q10) have many limitations, including low bioavailability, limited efficacy when used alone, narrow applicable scenarios, and difficulty in achieving effective concentrations due to regulatory dosage restrictions. High doses may also pose potential side effects, making it difficult to meet overall clinical needs.

[0003] L-Ergothioneine (EGT) is a natural sulfur-containing amino acid derivative derived from histidine, first isolated from ergot fungi by French researchers in 1909. This substance is widely found in edible fungi, some legumes, and animal organs; the human body cannot synthesize it and must obtain it through diet. With further research, EGT has shown promising applications in cosmetics, food, and healthcare due to its unique antioxidant properties.

[0004] Studies have shown that although EGT has strong hydrophilicity, it can be efficiently absorbed by the gastrointestinal tract and distributed to various organs, including the brain. This process mainly relies on the active transport mechanism mediated by the EGT-specific transporter OCTN1 / SLC22A4. Animal experiments have shown that EGT can protect mice from stress-induced sleep disorders and β-amyloid-induced neuronal damage. In vitro observations have shown that EGT can have beneficial effects on brain function through its antioxidant activity and its role in promoting neurogenesis and neuronal maturation (cite reference 1). However, different in vitro cell studies have shown that EGT concentrations of 100 μM to 10 mM are required to effectively reduce cellular oxidative stress levels, resulting in relatively high overall dosages and limiting its application efficiency.

[0005] On the other hand, milk is a complex suspension system, with fat being one of its main components, accounting for 3%-5%. Milk fat exists in milk in the form of tiny droplets, spherical in shape, called milk fat globules, with a diameter of approximately 0.2-15 μm. Their surface is covered by a thin film 10-20 nm thick, known as milk fat globule membranes (MFGMs). This membrane structure plays a crucial role in maintaining the stability of milk fat globules and preventing their aggregation and decomposition. Reference 2 indicates that the MFGM content in cow's milk is approximately 3.6 g / L, with protein and lipids accounting for approximately 22.3% and 71.8%, respectively.

[0006] In recent years, MFGM has been widely listed in the ingredient lists of infant formula milk powder, and its unique nutritional value and functional properties have increasingly attracted the attention of the food industry, especially the infant formula milk powder industry. In breast milk, MFGM plays an important role in promoting infant brain development, improving cognitive ability, improving metabolic function, reducing gastrointestinal infections, and enhancing immunity (cite reference 3). Recent studies have shown that MFGM and its key components, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM), may exert neuroprotective effects through mechanisms such as regulating synaptic plasticity, reducing neuroinflammation, and maintaining cell membrane integrity; an animal study also evaluated the effects of infant formula rich in MFGM and milk fat on cognitive function under inflammatory neurotoxicity conditions (cite reference 4). However, to date, there is a lack of research reports in domestic and international literature on the direct effects of MFGM on oxidative stress damage in nerve cells.

[0007] While some studies have explored the combined use of EGT and milk-derived components, these studies primarily focus on antioxidant, anti-inflammatory, and cognitive function improvement effects (references 5-7). There are no reports of combining EGT and MFGM for the protection against oxidative damage in nerve cells, nor are there clear dose-response relationships or synergistic data between the two. Therefore, exploring the synergistic effects of MFGM and EGT in protecting against oxidative damage in nerve cells has significant research value and practical application potential.

[0008] References:

[0009] Cited literature 1: Ishimoto T, Kato Y. Ergothioneine in the brain [J]. FEBSLetters, 2022, 596: 1290-1298.

[0010] Cited literature 2: Fong BY, Norris CS, et al. Protein and lipid composition ofbovine milk-fat-globule membrane [J]. International Dairy Journal, 2007, 17(4): 275-288.

[0011] Reference 3: Liu Tingting, Zhang Guofang, Liu Libo. Research progress on milk fat globule membrane [J]. Dairy Science and Technology, 2019, 42(3): 45-50.

[0012] Cited literature 4: Basha S, KS P, et al. Emerging insights into dairy products and Alzheimer's disease: exploring the potential neuroprotective effects [J]. Critical Reviews in Food Science and Nutrition, 2026, 66(14): 2628-2655.

[0013] Reference 5: CN121890757A;

[0014] Reference 6: CN118634317B;

[0015] Reference 7: CN121714587A. Summary of the Invention

[0016] Problem to be Solved by the Invention

[0017] As mentioned earlier, although some studies have explored the physicochemical properties and biological functions of milk fat globule membranes, the focus has mainly been on promoting immune regulation, supporting brain development, and maintaining gut health. Therefore, there is still a need to expand its application scope.

[0018] Surprisingly, further research using cell models to evaluate the intervention effect of a nutritional composition consisting of ergothioneine and milk fat globule membrane on oxidative damage in nerve cells revealed that the combined application of these two ingredients synergistically improves oxidative damage in nerves and significantly enhances the survival rate of nerve cells under oxidative damage conditions. Furthermore, the composition also effectively reduces intracellular reactive oxygen species levels and inhibits nerve cell apoptosis. This discovery unexpectedly expands the potential applications of these two components, whether as a food for special medical purposes, or as a general food, nutritional supplement, or dietary supplement, making them suitable for improving oxidative damage in nerves and thus achieving neuroprotective functions.

[0019] Solution to Problem

[0020] To address the aforementioned technical problems, the present invention provides the following technical solution:

[0021] [1]. A nutritional composition comprising the following two essential components:

[0022] i) Ergothioneine;

[0023] ii) Milk fat globule membrane;

[0024] Furthermore, in the nutritional composition, the mass ratio of ergothioneine to the milk fat globule membrane is 1:(1-150).

[0025] [2]. The nutritional composition according to [1], wherein the mass ratio of the ergothioneine to the milk fat globule membrane is 1:(1-120).

[0026] [3]. The nutritional composition according to [1] or [2], wherein the ergothioneine is provided in the form of natural and / or synthetic and / or biofermentation sources.

[0027] [4]. The nutritional composition according to any one of [1]-[3], wherein the milk fat globule membrane is provided in the form of animal milk and / or dairy products containing it.

[0028] [5]. Use of the nutritional composition according to any one of [1]-[4] in the preparation of products that help improve neurooxidative damage.

[0029] [6]. According to the use described in [5], wherein the neurooxidative damage includes a decrease in the survival rate of nerve cells.

[0030] [7]. Use of the nutritional composition according to any one of [1]-[4] in the preparation of products with neuroprotective effects.

[0031] [8]. According to the use described in [7], wherein the neuroprotection includes increasing the survival rate of nerve cells.

[0032] [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, functional additives and any acceptable excipients.

[0033] Effect of the Invention

[0034] This invention is the first to propose that the combination of ergothioneine and milk fat globule membrane has a synergistic protective effect in improving neuronal oxidative damage. In particular, when the two are used in combination within a specific ratio range, they can synergistically enhance the survival rate of nerve cells under oxidative damage. Furthermore, based on this, the use of the composition of this invention also effectively reduces intracellular reactive oxygen species levels and inhibits nerve cell apoptosis.

[0035] Furthermore, the effective dose of ergothioneine combined with milk fat globule membrane is significantly lower than that of single-component formulations. This means that while maintaining or enhancing bioactivity, the amount of raw materials used is greatly reduced, effectively overcoming the technical drawbacks of existing single-component formulations, such as high dosage and high cost. This nutritional composition demonstrates good feasibility for industrial transformation and economic applicability, providing an important foundation for its widespread application in various product types, including general foods, sports nutrition foods, foods for special medical purposes, and health foods. Detailed Implementation

[0036] 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.

[0037] <Terminology Definition>

[0038] 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.

[0039] In this invention, the meaning of "may" includes both performing a certain process and not performing a certain process.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] In this invention, "children" refers to the human group aged 3-6 years.

[0046] In this invention, "teenagers" refers to the human group aged 7-18.

[0047] In this invention, "young adult" refers to the human group aged 19-40.

[0048] In this invention, "pregnant women" includes women who are pregnant and women who are breastfeeding.

[0049] 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.

[0050] In this invention, "animal milk" refers to the liquid obtained from the mammary glands of a mammal in the process of lactation.

[0051] 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.

[0052] (Nutritional composition)

[0053] The nutritional composition of the present invention comprises the following two essential components: i) ergothioneine and ii) milk fat globule membrane;

[0054] Furthermore, in the nutritional composition, the mass ratio of ergothioneine to the milk fat globule membrane is 1:(1-150).

[0055] Ergothioneine

[0056] L-Ergothioneine (EGT) is a class of sulfur-containing amino acid derivatives, first discovered in 1909 in the ergot fungus (Claviceps purpurea), with the molecular formula C9H. 15 N3O2S. EGT is naturally found in fungi (such as edible mushrooms) and some plants and animals. The human body cannot synthesize EGT and must obtain it through dietary intake. Many edible mushrooms, such as Boletusedulis, Pleurotus citrinopileatus, and Lentinula edodes, contain high levels of EGT and are the most important natural sources of it. EGT can scavenge free radicals in the body, helping to maintain health, and is considered a natural and safe antioxidant and dietary nutrient.

[0057] In some embodiments, the ergothioneine of this invention is provided in the form of natural sources and / or synthetic sources and / or bio-fermentation sources. For example, ergothioneine products isolated from fungi or certain plant and animal materials, chemically synthesized ergothioneine products, fermentation products of ergothioneine-producing microorganisms, etc. For example, the ergothioneine content in each source form can typically be from 0.002% to 99.9% by mass.

[0058] In some specific implementations, the ergothioneine is provided in the form of Pleurotus ostreatus concentrate powder.

[0059] In some implementations, the ergothioneine can be prepared in-house or obtained commercially.

[0060] Milk Fat Globule Membrane

[0061] Milk is a complex suspension, and fat is one of its main components. Milk fat exists in milk as small droplets, spherical in shape, hence the name milk fat globule (MFG). Milk fat globules are approximately 0.2-15 μm in diameter and are covered by a thin film of 10-20 nm, called the milk fat globule membrane (MFGM).

[0062] MFGM is generally considered to have a three-layer membrane structure and is a "natural" emulsifier that can prevent the coagulation and aggregation of fat globules in milk and protect fat from the action of enzymes.

[0063] Furthermore, in terms of composition, MFGM is a protein-lipid complex.

[0064] Existing research shows that milk fat globule membrane proteins are distributed asymmetrically within the milk fat globule membrane, accounting for approximately 25%-70% of the total MFGM. Based on their location, milk fat globule membrane proteins can be classified into: integrated proteins, peripheral proteins, and proteins loosely bound to the membrane.

[0065] There are many types of MFGM proteins, the most important of which include: lactolipoprotein (BTN, glycoprotein), mucin 1 (MUC1, glycoprotein), xanthine oxidoreductase / dehydrogenase (XO / XDH, glycoprotein), lactoglucosin (MFG-E8 or PAS6 / 7), mucin 15 (PAS III, glycoprotein), CD36 (or PAS IV, glycoprotein), adipocyte differentiation-associated protein (ADPH, non-glycoprotein), and fatty acid-binding protein (FABP, non-glycoprotein), etc.

[0066] For MFGM lipids, the lipid components of MFGM are mainly composed of polar lipids (such as phospholipids), and also contain some neutral lipids (such as triglycerides). Polar lipids are further divided into phospholipids and sphingolipids. Polar lipids in MFGM are often used as emulsifiers. Studies have found that the polar lipids in MFGM mainly include phosphatidylcholine (PC), phosphatidylethanolamines (PE), sphingomyelin (SM), phosphatidylinositols (PI), and phosphatidylserine (PS), with PC having the highest proportion, and PS and PI having relatively low proportions.

[0067] Furthermore, there are no particular limitations on the source of the milk fat globule membrane of the present invention. It can usually be obtained by extraction from animal milk or its products. In some preferred embodiments, such animal milk or its products can be cow milk, sheep milk, camel milk, horse milk or dairy products based on them (e.g., cheese, whey protein powder, milk protein powder), etc. More preferably, it can be extracted from cow milk (including bovine colostrum or bovine regular milk).

[0068] In some embodiments, the milk fat globule membrane is provided in the form of animal milk and / or dairy products containing it.

[0069] The present invention does not particularly limit the method for extracting MFGM from the above-mentioned animal milk or its products. For example, the method of acidification precipitation-centrifugation-isoelectric point enrichment-drying can be used, or the existing membrane filtration method can be used to separate MFGM.

[0070] Alternatively, MFGM can also be obtained from commercially available products, including Lacprodan, which is a commercial source of MFGM for this invention. ® MFGM-10, Lacprodan ® PL-20, Cor-Powder ® SM3, Cor-Powder ® SM2, lipid-rich MFGM fraction, or buttermilk powder concentrate BPC50, BPC60, G600, PC700, Hilmar ® WPC 7500MEGM ENRICHED WPC, etc.

[0071] This invention discovers that the combination of ergothioneine and milk fat globule membrane can exert a synergistic effect in helping to improve neurooxidative damage.

[0072] In some embodiments, ergothioneine and milk fat globule membrane are the main active ingredients of the nutritional composition. That is, the nutritional composition of the present invention mainly relies on ergothioneine and milk fat globule membrane to exert specific physiological functions, such as assisting in the improvement of neurooxidative damage. 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 assist in the improvement of neurooxidative damage) consist of ergothioneine and milk fat globule membrane.

[0073] In some embodiments, the nutritional composition comprises an active ingredient (a component that performs a specific physiological function, i.e., a component that helps improve neurooxidative damage) and an inactive ingredient (a substance that does not help improve neurooxidative damage, or at least does not have a synergistic effect with ergothioneine or milk fat globule membrane). Exemplarily, the inactive ingredient may be other nutrients, any food-acceptable 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.

[0074] In some embodiments, in the nutritional composition, the mass ratio of the essential component shown in i) to the essential component shown in ii) is 1:(1-150), for example, it can be 1:1, 1:2, 1:2.2, 1:3, 1:4, 1:4.4, 1:5, 1:8, 1:8.7, 1:10, 1:12, 1:15, 1:18, 1:20, 1:22, 1:25, 1:28, 1:30, 1:32, 1:32.6, 1:33, 1:35, 1:38, 1:40, 1:42, 1:43, 1:43.5, 1:44, 1:45, 1:48, 1:50, 1:55, 1:60, 1:65, 1:7 0, 1:75, 1:80, 1:85, 1:87, 1:90, 1:95, 1:100, 1:105, 1:108, 1:108.7, 1:109, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, etc.; preferably, the mass ratio of the two is 1:(1-120); more preferably, the mass ratio of the two is 1:(2-110); even more preferably, the mass ratio of the two is 1:(5-100); further preferably, the mass ratio of the two is 1:(5-20); preferably, the above ratio is the mass ratio of ergothioneine to milk fat globule membrane.

[0075] 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.

[0076] (Uses of the nutritional composition)

[0077] This invention provides the use of the above-mentioned nutritional composition to assist in improving neuro-oxidative damage. In some embodiments, the assistance in improving neuro-oxidative damage is not intended to prevent or treat disease, nor does the neuro-oxidative damage reach the level of disease; rather, the neuro-oxidative damage is closer to a decline or reduction in neuro-antioxidative function. That is, this invention provides the non-therapeutic use of the above-mentioned nutritional composition to assist in improving neuro-oxidative damage. Based on this, this invention also provides the use of the above-mentioned nutritional composition in the preparation of products that assist in improving neuro-oxidative damage.

[0078] In some implementations, the neurooxidative damage includes a decrease in the survival rate of nerve cells.

[0079] Based on this, the present invention also provides the use of the above-mentioned nutritional composition in the preparation of products with neuroprotective effects.

[0080] In some implementations, the neuroprotection includes increasing nerve cell survival.

[0081] The present invention does not specifically limit the products containing the above-mentioned nutritional composition or products that can be prepared using the above-mentioned nutritional composition; for example, they can be food products.

[0082] In some implementation schemes, the food is infant food, children's food, adolescent food, pregnant and postpartum food, young and middle-aged food, or middle-aged and elderly food.

[0083] In some embodiments, the food product of this invention is a confectionery, such as hard candy, gel candy, shortbread candy, compressed candy, and aerated candy. In some embodiments, the food product of this invention is a beverage, such as carbonated beverages, tea beverages, coffee beverages, fruit and vegetable juice beverages, and lactic acid bacteria beverages. In some embodiments, the food product of this invention is a dairy product, such as milk powder, cheese, fermented milk, and liquid milk. In some embodiments, the food product of this invention is a baked product, such as bread, cakes, and biscuits. In some embodiments, the food product of this invention is a dietary supplement, such as hard capsules, soft capsules, tablets, oral liquids, pills, granules, and powders.

[0084] In some embodiments, the food described in this invention is in the form of a liquid or a solid under normal temperature conditions.

[0085] In some embodiments, in the food described in this invention, the mass ratio of the necessary component shown in i) to the necessary component shown in ii) can be 1:(1-150); preferably, the mass ratio can be 1:(1-120); more preferably, the mass ratio can be 1:(2-110); even more preferably, the mass ratio can be 1:(5-100); further preferably, the mass ratio can be 1:(5-20); preferably, the above ratio is the mass ratio of ergothioneine to milk fat globule membrane.

[0086] This invention does not impose any specific absolute limits on the content of ergothioneine and milk fat globule membrane in food, as long as the requirements of local food-related laws and regulations are met.

[0087] In some embodiments, the ergothioneine content in the food, based on the total dry matter content of the food, may be 1 mg / 100g-1000 mg / 100g; and the milk fat globule membrane content may be 1 mg / 100g-15000 mg / 100g.

[0088] In addition to the components described above in the nutritional composition, the food may also contain other ingredients, such as common food ingredients like proteins / amino acids, carbohydrates, fats, vitamins, and minerals.

[0089] In addition, depending on the type of food and the end needs of the target audience, in some embodiments, the food may also contain one or more of the following ingredients: plant-based ingredients, animal dairy ingredients, animal meat ingredients, functional additives, and any acceptable excipients.

[0090] Examples of plant-based ingredients include fruits such as figs, pomegranates, kiwis, oranges, tangerines, pineapples, strawberries, apples, bananas, grapes, pears, cherries, blueberries, blackberries, blackcurrants, cranberries, raspberries, melons, amla, and bilberries, or their extracts; 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.

[0091] Animal dairy product ingredients can include fresh milk from mammals such as cows, sheep, and camels, 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.

[0092] Examples of animal meat product ingredients include pork, beef, mutton, seafood, and poultry.

[0093] 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, and vitamin B1). 12 Supplements include: Vitamin C, pantothenic acid, folic acid, niacin, biotin, etc.; mineral supplements (e.g., calcium, iron, copper, manganese, zinc, magnesium, fluorine, sodium, potassium, selenium, iodine, etc.); nucleotide supplements (e.g., disodium 5'-cytidine, disodium 5'-uridine, adenosine monophosphate, disodium 5'-guanylate, disodium 5'-inosine, etc.); dietary fiber (e.g., inulin, konjac flour, galactooligosaccharides, fructooligosaccharides, isomaltooligosaccharides, soybean polysaccharides, cyclodextrin, resistant dextrin, soybean fiber, etc.); and polyunsaturated fatty acid supplements (e.g., docosahexaenoic acid, eicosapentaenoic acid, eicosapentaenoic acid, etc.).

[0094] 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.

[0095] Example

[0096] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0097] The experimental instruments used in the experimental examples of this invention include: micropipette (Eppendorf, Germany); biosafety cabinet (HFsafe-1500LC, Likang Biomedical Technology Holding Co., Ltd.); RNase bag tips (EXTRAGENE, USA); microplate reader (Multiskan FC, Thermo Scientific, USA); flow cytometer (Novocyte 2000, Agilent Technologies (China) Co., Ltd.); low-temperature high-speed centrifuge (Micro17R, Thermo Scientific, USA); analytical balance (JA2003B, Shanghai Yueping Scientific Instruments Co., Ltd.); carbon dioxide incubator (bb150, Thermo Scientific, USA); culture plates (Corning, USA).

[0098] The experimental materials and reagents used in the experimental examples of this invention include: ergothioneine (EGT, Puyue Huahang (Shanghai) Biotechnology Co., Ltd.); milk fat globule membrane (MFGM, Heilongjiang Feihe Dairy Co., Ltd.); vitamin E (Shanghai Maclean Biochemical Technology Co., Ltd.); PBS buffer (AM9625, Gibco, USA); Cell Counting Kit-8 (CP736, DOJINDO Laborataries, Japan); Annexin V-FITC / PI apoptosis detection kit (KGA105, Jiangsu Kaiji Biotechnology Co., Ltd.); reactive oxygen species detection kit (KGT010-1, Jiangsu Kaiji Biotechnology Co., Ltd.); HT22 cell culture medium (CM-0697, Wuhan Pronosai Life Science Technology Co., Ltd.); 0.25% trypsin solution - containing EDTA (PB180226, Wuhan Pronosai Life Science Technology Co., Ltd.); 0.25% trypsin solution - without EDTA (PB180228, Wuhan Pronosai Life Science Technology Co., Ltd.); 30% hydrogen peroxide (10011218, Sinopharm Chemical Reagent Co., Ltd.).

[0099] The experimental cells used in the experimental examples of this invention are HT22 mouse hippocampal neurons (CL-0697, Wuhan Pronosai Life Science Technology Co., Ltd.), which were cultured and passaged by Huante Mammal Technology Center.

[0100] Experimental Example 1: Protective Effects of Different Nutrients and Combinations on Oxidative Damage in HT22 Cells

[0101] 1. Cell resuscitation and passage preparation

[0102] 1) After thawing the frozen HT22 cells, they were seeded into T25 culture flasks and cultured in LO2-specific medium.

[0103] 2) Pass the cells twice, and conduct subsequent detection experiments after the number of cells is sufficient.

[0104] 2. Protective effects of nutrient monomers and their combinations against oxidative damage in HT22 cells.

[0105] 1) Take HT22 cells that have grown to the logarithmic growth phase, digest and count them, and prepare a 1×10⁻⁶ concentration culture medium. 6 Cell suspensions of 10 cells / mL were seeded into 96-well cell culture plates. A total of 19 groups were set up: blank control group (single dose), model control group (single dose), positive control group (single dose), EGT single use group (4 doses), MFGM single use group (5 doses), and combination group (7 doses). The specific grouping is shown in Table 1.

[0106] 2) After the cells in each group have adhered to the culture vessel, remove the original culture medium and add 200 μL of complete culture medium containing the corresponding test sample.

[0107] EGT monotherapy group: culture media containing EGT concentrations of 5 μM (1.15 μg / mL), 10 μM (2.3 μg / mL), 25 μM (5.75 μg / mL) and 50 μM (11.5 μg / mL) were added respectively;

[0108] MFGM monotherapy group: culture medium containing MFGM concentrations of 25 μg / mL, 50 μg / mL, 75 μg / mL, 100 μg / mL and 125 μg / mL were added respectively;

[0109] Combined treatment groups: Seven combinations were prepared by adding culture media containing different ratios of EGT and MFGM: EGT 5μM + MFGM 125μg / mL; EGT 5μM + MFGM 100μg / mL; EGT 10μM + MFGM 100μg / mL; EGT 10μM + MFGM 75μg / mL; EGT 25μM + MFGM 50μg / mL; EGT 50μM + MFGM 50μg / mL; EGT 50μM + MFGM 25μg / mL.

[0110] Positive control group: culture medium containing 200 μM vitamin E was added;

[0111] Normal control group and model control group: culture medium containing no samples was added.

[0112] All cells were cultured at 37°C and 5% CO2.

[0113] 3) Set up four different batches of culture time. After each group of cells was cultured for 0h, 12h, 24h and 48h, hydrogen peroxide (final concentration 300μM) was added to each well except for the normal control group, and the cells were cultured for another 4h at 37℃ and 5%CO2.

[0114] 4) After completing the modeling process by culturing for another 4 hours, add 5 μL of CCK-8 dye to each well and continue culturing at 37℃ and 5% CO2 for another 2 hours. Remove the culture plate and gently shake it on a constant temperature shaker for 10 minutes. Measure the OD value of each well at 450 nm using a microplate reader. Calculate the survival rate of HT22 cells in each batch and group, using the normal control group as a reference.

[0115] Table 1. Experimental Design Scheme

[0116]

[0117] 3. The experimental results are shown in Tables 2-5.

[0118] Table 2. Effects of 0h intervention with nutrient monomers and combinations on HT22 cell survival (n=3)

[0119]

[0120] Table 3. Effects of nutrient monomers and combinations on HT22 cell survival rate after 12 h of intervention (n=3)

[0121]

[0122] Table 4. Effects of nutrient monomers and combinations on HT22 cell survival rate after 24 h of intervention (n=3)

[0123]

[0124] Table 5. Effects of nutrient monomers and combinations on HT22 cell survival rate after 48 h of intervention (n=3)

[0125]

[0126] The above results indicate that EGT monomer, MFGM monomer, and their combined concentrations at different levels all have significant protective effects against oxidative damage in HT22 cells, and this effect shows a clear dose- and time-dependent relationship. The protective effect reached its peak 24 hours after intervention in each group.

[0127] All combination groups (Examples 1-7) exhibited a good synergistic effect ("improvement compared to the model group" was stronger than "the sum of improvements compared to the two individual nutrient groups"), meaning that the improvement in HT22 cell survival rate after combination was better than the sum of the improvements from the individual effects of the two nutrients. Specifically, a synergistic effect was observed when the ratio of EGT to MFGM was between 1:2.2 and 1:108.7; the synergistic effect was most significant when the ratio was between 1:8.7 and 1:87 (the improvement rate of the combination in Examples 2-5 was significantly higher than that of other combination groups).

[0128] From a temporal perspective, the synergistic effect showed a rapid upward trend during incubation from 0 to 12 hours, reaching its maximum effect at 24 hours, and then plateauing after 48 hours. Specifically, significant synergistic protective effects were observed from 0 to 12 hours; while the protective effect reached its maximum at 24 hours, the synergistic effect weakened somewhat; and when nutrient administration was extended to 48 hours, both the single-use and combined-use groups showed good improvement in cell viability, with no significant difference between the two.

[0129] The above results provide important reference for the practical application of the two nutrients: it is recommended to introduce a sustained release or fractional supplementation mechanism in the formulation design to ensure that the effective concentration is maintained within the critical effect window of 12-24 hours, thereby achieving the best protective effect.

[0130] 4. Dose-response synergistic analysis of the effects of two nutrient combinations on oxidative damage in HT22 cells.

[0131] After selecting the optimal intervention time and group based on survival rate, the OD value of the normal control group in this batch was used as the survival benchmark, and the OD value of the model control group was used as the oxidative damage benchmark. The combination index (CI) and dose reduction index (DRI) were calculated according to the following formulas:

[0132] ① Protective effect value (fa)

[0133]

[0134] ② Fitting of dose-response relationship

[0135] For each nutrient, data points with fa ranging from 0.01 to 0.99 were selected, and the following relationship curve was plotted:

[0136]

[0137] Where fu = 1 - fa, m is the slope, and D m This is the intermediate dose (i.e., the dose required when fa=0.5).

[0138] ③ Calculation of equivalent dose

[0139] Based on the fitting results, calculate the dose D required to achieve the same protective effect when EGT and MFGM are intervened alone. x :

[0140]

[0141] ④ Calculation of Combination Index (CI) and Dose Reduction Index (DRI)

[0142] Let D1 and D2 be the actual doses of EGT and MFGM when used in combination, respectively. x1 and D x2 Let each be an equivalent dose required to achieve the same fa when used alone.

[0143] Mutually exclusive CI:

[0144] Non-mutually exclusive CI:

[0145] Dose reduction index:

[0146] Statistical analysis was performed on the dose-response synergy of each combination group, and the results are shown in Table 6.

[0147] Table 6. Evaluation results of the dose-response synergistic effect of each combination therapy on HT22 cell survival rate

[0148]

[0149] The results showed that the CI values ​​of each combination group were less than 1, indicating that all combination combinations exhibited strong synergistic effects within the tested dose range.

[0150] Among them, the synergistic effect of the combination in Example 5 was the most significant. Under this combination, the protective effect value fa = 1.0084, the mutual exclusion CI = 0.239, and the non-mutual exclusion CI = 0.25, all significantly lower than 0.9, indicating a strong synergistic effect. Furthermore, DRI... EGT =5.6, DRI MFGM =16.62, indicating that the combined use requires only 1 / 5.6 of the original EGT dose and 1 / 16.62 of the original MFGM dose to achieve a protective effect comparable to that of the single use. After 24 hours of intervention, the cell survival rate in the combined use group was close to 100%, with no significant difference compared to the normal control group. At the same time, its raw material cost was significantly lower than that of each single use group, making its overall benefits the most outstanding.

[0151] Experimental Example 2: Effects of different nutrients and their combinations on oxidative stress and apoptosis in HT22 cells

[0152] 1. The effect of individual and combined nutrients on the improvement of oxidative stress levels in HT22 cells

[0153] The cell culture, grouping, inoculation, nutrient intervention, and modeling process were the same as in Experiment 1.

[0154] After culturing for another 4 hours to complete the modeling process, cells were collected and digested with 0.25% trypsin, followed by centrifugation at 1000 rpm for 5 min, washing once with PBS, and adjusting the cell concentration to 1×10⁶ cells / year. 6 Cells / mL. The DCFH-DA fluorescent probe was diluted 1:1000 with serum-free culture medium to a final concentration of 10 μM. Cells were resuspended in the diluted DCFH-DA solution and incubated in a cell culture incubator at 37°C in the dark for 20 min, inverting and mixing every 3-5 min to ensure sufficient contact between the probe and cells. After incubation, cells were washed three times with serum-free cell culture medium to remove any uninfiltrated DCFH-DA. Intracellular reactive oxygen species (ROS) levels were detected by flow cytometry at an excitation wavelength (Ex) of 488 nm and an emission wavelength (Em) of 530 nm. The results are shown in Table 7.

[0155] 2. The effect of nutrients alone and in combination on apoptosis in HT22 cells

[0156] The cell culture, grouping, inoculation, nutrient intervention, and modeling process are the same as in Experiment 1.

[0157] After culturing for another 4 hours to complete the modeling process, the cells were digested with 0.25% trypsin (without EDTA) and collected. They were then centrifuged at 1000 rpm for 5 minutes, washed once with PBS, and the cell concentration was adjusted to 1×10⁶ cells / year. 6 Cells / mL. Add 5 μL Annexin V-FITC to the cell suspension, mix well, then add 5 μL Propidium Iodide (PI) and mix gently. Incubate at room temperature in the dark for 15 min, then use flow cytometry to detect cell apoptosis. The results are shown in Table 7.

[0158] Table 7. Results of intervention with nutrient monomers and combinations on ROS levels and apoptosis levels in HT22 cells (n=3)

[0159]

[0160] The results showed that EGT monomer, MFGM monomer and their combination could significantly improve the reactive oxygen species (ROS) level and apoptosis rate of HT22 cells under oxidative stress, and all showed obvious dose-dependent effects.

[0161] The combination of EGT and MFGM significantly reduced the apoptosis rate, suggesting that its protective effect is not limited to maintaining cell survival, but may also achieve high-quality cell protection by intervening in apoptosis signaling pathways (such as the mitochondrial apoptosis pathway). This composition has both antioxidant and anti-apoptotic functions, effectively rescuing cells from programmed cell death and providing a more thorough protective effect.

[0162] Meanwhile, the combined use of EGT and MFGM significantly reduced ROS levels, indicating that its protective mechanism focuses on intervening in oxidative stress at its source, rather than acting only on the terminal stage of cell death. In summary, the potential mechanism by which this composition mitigates oxidative damage may be that EGT exerts its antioxidant effect by directly scavenging free radicals, while MFGM stabilizes cell membrane structure and reduces ROS generation; the two work synergistically to achieve a protective effect on HT22 cells.

[0163] In summary, in this study's neural cell oxidative damage model, cell viability (OD value detected by CCK-8 assay), reactive oxygen species (ROS) level, and apoptosis rate (Annexin V-FITC / PI double staining method) together constitute a complete chain of evidence for "results-process-mechanism," and their logical relationship is as follows:

[0164] 1) Core indicator – Cell viability

[0165] The ultimate outcome of oxidative damage is cell death. Regardless of the oxidative stress events or activation of apoptotic signals that occur in between, the final standard for evaluating the effectiveness of a protective component is cell survival rate, which is the most direct and indisputable endpoint indicator.

[0166] 2) Auxiliary indicator ① - Reactive oxygen species (ROS) level

[0167] The direct effect of hydrogen peroxide (H2O2) modeling is the induction of massive intracellular ROS accumulation, which in turn triggers a series of damage cascades, including mitochondrial dysfunction, DNA damage, and lipid peroxidation. Therefore, ROS level is an indicator of the initiation stage of oxidative damage. If this composition can significantly reduce ROS levels, it suggests that its protective mechanism may intervene in oxidative stress at its source, rather than only performing salvage repair at the end of cell death.

[0168] 3) Auxiliary indicator ② – Apoptosis rate

[0169] A significant portion of cell death induced by oxidative damage occurs via apoptosis (programmed cell death) rather than simple necrosis. Apoptosis is a regulated process and a crucial target for nutrient intervention. If the composition significantly reduces the apoptosis rate, it indicates that its protective effect extends beyond simply maintaining cell survival; it may achieve higher-quality cell protection by blocking apoptosis signaling pathways (such as the mitochondrial apoptosis pathway).

[0170] Experimental conclusions

[0171] Under the experimental conditions of this study, both EGT monomers (5-50 μM) and MFGM monomers (25-125 μg / mL) showed significant protective effects against hydrogen peroxide-induced oxidative damage in HT22 cells, specifically by increasing cell viability, improving intracellular reactive oxygen species (ROS) levels, and effectively inhibiting apoptosis. Within each monomer dosage range, the combined use of EGT and MFGM showed a strong dose-response synergistic effect in improving the survival rate of nerve cells under oxidative damage. A synergistic effect was observed when the EGT to MFGM ratio was between 1:2.2 and 1:108.7 (1 μM: 0.5-25 μg / mL), with the most significant synergistic effect observed in the range of 1:8.7 to 1:87 (1 μM: 2-20 μg / mL) (the combined use in Examples 2-5 significantly improved the survival rate compared to other combined groups).

[0172] In terms of mechanism, the protective effect of EGT combined with MFGM may stem from a dual pathway: on the one hand, it removes free radicals and reduces oxidative stress levels from the source, and on the other hand, it blocks the cell apoptosis signaling pathway, thereby achieving higher quality cell protection.

Claims

1. A nutritional composition, characterized in that, The nutritional composition comprises the following two essential components: i) Ergothioneine; ii) Milk fat globule membrane; Furthermore, in the nutritional composition, the mass ratio of ergothioneine to the milk fat globule membrane is 1:(1-150).

2. The nutritional composition according to claim 1, characterized in that, The mass ratio of ergothioneine to milk fat globule membrane is 1:(1-120).

3. The nutritional composition according to claim 1 or 2, characterized in that, The ergothioneine is provided in the form of natural and / or synthetic and / or biofermentation sources.

4. The nutritional composition according to any one of claims 1-3, characterized in that, The milk fat globule membrane is provided in the form of animal milk and / or dairy products containing it.

5. Use of the nutritional composition according to any one of claims 1-4 in the preparation of products that help improve neuro-oxidative damage.

6. The use according to claim 5, characterized in that, The neurooxidative damage includes a decrease in the survival rate of nerve cells.

7. Use of the nutritional composition according to any one of claims 1-4 in the preparation of a product having neuroprotective effects.

8. The use according to claim 7, characterized in that, The neuroprotection includes increasing nerve cell survival.

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, functional additives, and any acceptable excipients.

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