Use of casein phosphopeptides to facilitate modulation of dopaminergic neurons

Abstract

The invention belongs to the technical field of food, and relates to novel application of casein phosphopeptides, in particular to application of the casein phosphopeptides in preparation of food beneficial to regulation of dopaminergic neurons of organisms, and regulation of the dopaminergic neurons of the organisms comprises enhancement of the strength of the dopaminergic neurons and / or enhancement of the strength of the dopaminergic neurons. Neurotransmitters and metabolites beneficial to operation of a dopamine energy system are regulated and controlled.

Description

Technical Field

[0001] This invention belongs to the food field and relates to the use of a casein phosphopeptide to help regulate dopaminergic neurons. Background Technology

[0002] Casein phosphopeptides (CPP) are bioactive polypeptides produced using biotechnology from cow's milk or casein. Since their discovery, they have received widespread attention in the food and health supplement industries. Numerous studies have confirmed that CPP can effectively promote the absorption and utilization of divalent mineral nutrients such as calcium, iron, and zinc in the human body. In addition, CPP also has various physiological functions such as anti-caries and blood pressure regulation.

[0003] Regarding the prevention of tooth decay, traditional views hold that chewing cheese after meals stimulates saliva production, and the alkaline buffering effect of saliva neutralizes the acidic substances in dental plaque, thereby reducing erosion of tooth enamel. Recent studies have found that CPP contained in cheese can bind calcium ions from food to the site of decay, inhibiting the enamel demineralization process and thus playing a role in preventing tooth decay.

[0004] Regarding blood pressure regulation, studies have shown that during fermentation in the milk matrix, *Lactobacillus helveticus* releases specific peptides from milk proteins through proteolytic hydrolysis. These peptides possess inhibitory activity against angiotensin-converting enzyme (ACE) or opioid peptide-like effects. CPP may play a synergistic role in this process, enhancing its antihypertensive effect in vivo.

[0005] Currently, CPP is widely used in a variety of products. In the field of nutrition and health foods, CPP is often used in fortified calcium, iron, and zinc supplements, dairy products (such as milk powder, school formula milk, high-calcium low-fat milk, etc.), children's foods (such as infant rice cereal, high-calcium biscuits, etc.), soy products (such as high-calcium soy milk powder, calcium-fortified tofu, etc.), as well as nutritional cereals, chewing gum, and other products. Its addition not only improves product formulations and enhances the bioavailability of minerals, but also promotes the upgrading and transformation of related products.

[0006] In the field of oral care, CPP has also been given new applications. For example, some studies have combined CPP with strontium ions to enhance the efficacy of oral care products. CPP can promote the adsorption and delivery of strontium ions on the tooth surface, synergistically enhancing their effects in resisting dentin hypersensitivity, promoting tooth mineralization, and preventing tooth erosion.

[0007] The application areas of casein phosphopeptides are constantly expanding in existing technologies. For example, reference 1, through a mouse model study, revealed that a combination of casein phosphopeptides and Bifidobacteria can work synergistically to significantly enhance immunity and improve the body's antioxidant capacity. This research data further confirms that even casein phosphopeptide monomers possess good immunomodulatory and antioxidant activities, opening up new avenues for their application in functional foods and health products.

[0008] References:

[0009] Reference 1: CN117441900B Summary of the Invention

[0010] The problem the invention aims to solve

[0011] Although existing technologies have been used to study the efficacy of casein phosphopeptides (CPP) and their compositions, current research on the efficacy of casein phosphopeptides mainly focuses on promoting mineral absorption and regulating immunity. Therefore, there is still room for further exploration of new efficacy studies for casein phosphopeptides.

[0012] Therefore, the primary objective of this invention is to provide a novel use of casein phosphopeptides, specifically, to provide a use of casein phosphopeptides in assisting the regulation of dopaminergic neurons.

[0013] Further research in this invention has demonstrated the verifiable effects of casein phosphopeptide (CPP) in regulating dopaminergic neurons. Using *Caenorhabditis elegans* as the research subject, this invention investigated CPP intervention and its effects on dopamine synthesis in the nematodes. Furthermore, the effects of CPP on neurotransmitters and metabolites associated with dopaminergic neurons in *Caenorhabditis elegans* were examined. It is concluded that different doses of CPP intervention enhance the strength of dopaminergic neurons, thereby regulating functions such as movement and foraging, controlling learning, memory, and behavioral plasticity, and modulating sensory acuity. Different doses of CPP intervention can significantly regulate dopaminergic-related neurotransmitters and metabolites such as L-glutamate, GABA, L-serine, and reduced glutathione, thereby maintaining the higher-level regulatory functions of the dopaminergic system and ensuring its long-term stable operation.

[0014] Solution for solving the problem

[0015] The present invention has found that the above-mentioned technical problems can be solved by the following technical solutions:

[0016] [1]. This invention provides the use of casein phosphopeptide in the preparation of food that helps regulate dopaminergic neurons in the body.

[0017] [2]. According to the use described in [1], wherein the casein phosphopeptide includes one or more of casein phosphopeptides derived from human milk, casein phosphopeptides derived from cow milk, casein phosphopeptides derived from sheep milk, and casein phosphopeptides derived from casein.

[0018] [3]. According to the use described in [1] or [2], wherein the regulation of dopaminergic neurons in the body includes: enhancing the strength of dopaminergic neurons, and / or regulating neurotransmitters and metabolites related to the operation of the dopaminergic system.

[0019] [4]. According to the use described in [3], wherein the enhancement of dopaminergic neuron strength includes increasing the number of neurons, the abundance of dopamine synthesis and transport-related proteins or one of them.

[0020] [5]. According to the use described in [3] or [4], wherein the neurotransmitter is selected from one or both of L-glutamate and GABA; and the metabolite is selected from one or both of L-serine and reduced glutathione.

[0021] [6]. The use according to any one of [1]-[5], wherein the food is a powdered reconstituteable food, a baked food, a beverage, milk and dairy products or pasta products.

[0022] [7]. According to any one of [1]-[6], wherein, at room temperature, the food is in liquid form, solid-liquid mixture form, solid block form or solid powder form.

[0023] [8]. The use according to any one of [1]-[7], wherein the food contains one or more of the following ingredients: plant product ingredients, animal dairy product ingredients, animal meat product ingredients, functional additives and any food-acceptable excipients.

[0024] [9]. According to any one of [1]-[8], wherein the content of casein phosphopeptide is 0.001% by mass or more based on the total mass of the food.

[0025] The effects of the invention

[0026] Based on the implementation of the above technical solution, the present invention can achieve the following technical effects:

[0027] 1) Experimental data from this invention show that different doses of CPP intervention enhance the strength of dopaminergic neurons, thereby having functions such as regulating movement and foraging, regulating learning, memory and behavioral plasticity, and regulating sensory acuity.

[0028] 2) Experimental data from this invention show that different doses of CPP intervention can significantly regulate dopaminergic neurotransmitters and related metabolites such as L-glutamate, GABA, L-serine and reduced glutathione, thereby maintaining the higher regulatory function of the dopaminergic system and ensuring its long-term stable operation.

[0029] 3) This invention provides a novel application of casein phosphopeptides, offering new scientific basis and research ideas for their further development in functional foods and health products. In particular, there is currently no relevant research or application in the field regarding the novel efficacy of the casein phosphopeptides involved in this invention. No description or utilization of this novel efficacy has been found in currently published national standards, textbooks, papers, patents, or specific products. This provides space and necessity for the innovation of this invention. Through in-depth research on the novel efficacy of casein phosphopeptides, it is hoped that new products with unique functions can be developed to meet the market demand for new applications of functional ingredients. Attached Figure Description

[0030] Figure 1 Effects of different concentrations of casein phosphopeptide on fluorescence intensity of dopaminergic neurons in nematodes

[0031] Figure 2 Effects of different concentrations of casein phosphopeptide on L-glutamate content in nematodes

[0032] Figure 3 Effects of different concentrations of casein phosphopeptide on the content of γ-aminobutyric acid in nematodes

[0033] Figure 4 Effects of different concentrations of casein phosphopeptide on the concentration of reduced glutathione in nematodes

[0034] Figure 5 Effects of different concentrations of casein phosphopeptide on L-serine concentration in nematodes Detailed Implementation

[0035] 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 and examples are also included in the technical scope of the present invention.

[0036] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0037] Unless otherwise stated, in this instruction manual, "more" in "multiple", "multi-variety", "multiple", etc., means a value of 2 or more.

[0038] In this specification, the terms "comprising," "having," "including," or "containing" can mean included or open-ended, and do 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 specification, the numerical range indicated by "above" or "below" refers to the numerical range that includes the stated number.

[0040] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0041] In this specification, the terms "optional" or "optional" are used to indicate the use or omission of certain substances, components, procedures, application conditions, etc.

[0042] Unless otherwise specified, “room temperature” in this instruction manual usually refers to a temperature of 23±2℃.

[0043] All unit names used in this manual are international standard unit names, and unless otherwise stated, the "%" indicates weight or mass percentage.

[0044] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0045] This invention primarily provides a novel use for casein phosphopeptides. Specifically, experimental research has revealed a beneficial effect of casein phosphopeptides in enhancing the strength and operational stability of dopaminergic neurons. Based on this research, existing applications of casein phosphopeptides are expanded. This invention is mainly based on the following insights:

[0046] Using *Caenorhabditis elegans* as a model, biological research has revealed that CPP intervention enhances the strength of dopaminergic neurons in the body, particularly increasing the abundance of proteins related to dopamine synthesis and transport. Furthermore, it was found that CPP intervention can regulate dopaminergic-related neurotransmitters and metabolites, thereby maintaining the higher-level regulatory functions of the dopaminergic system and ensuring its long-term stable operation.

[0047] Furthermore, based on the above new findings, this invention provides innovative applications of CPP in functional foods and health products.

[0048] Casein phosphopeptide

[0049] Casein phosphopeptides (CPP) are bioactive polypeptides produced from bovine milk casein using biotechnology. They can be used in various nutritional and health foods and can effectively promote the absorption and utilization of divalent mineral nutrients such as calcium, iron, and zinc by the human body.

[0050] Casein phosphopeptide (CPP) is produced by hydrolyzing casein with trypsin, followed by purification and refinement. Its molecular structure consists of twenty to thirty amino acid residues, including four to seven clustered phosphoseryl groups. Currently, commercially available CPP products have a purity of 12%-90%.

[0051] In some embodiments, the casein phosphopeptides of the present invention include one or more of the following: casein phosphopeptides derived from human milk, casein phosphopeptides derived from bovine milk, casein phosphopeptides derived from goat milk, and casein-derived casein phosphopeptides. Casein phosphopeptides derived from bovine milk are preferred. Accordingly, casein phosphopeptides can be isolated from bovine milk or commercially available finished products can be purchased directly.

[0052] In addition, it should be emphasized that the above-mentioned form of addition of casein phosphopeptide must comply with the permission of local laws and regulations. Within the permitted scope, it can be added as a single ingredient. When it is not allowed to be added as a single ingredient, other components containing the ingredient can be used to give the desired casein phosphopeptide in the form of use.

[0053] In this invention, there are no particular limitations on the final form of casein phosphopeptides. At room temperature, they can be liquid (with the help of the desired solvent), semi-solid (water-containing slurry), solid (such as powder, granules or blocks (after pressing)), etc.

[0054] Foods containing casein phosphopeptides (including health foods)

[0055] The food containing casein phosphopeptides described in this invention can be in any form. In some specific embodiments, at room temperature, the food described in this invention is in liquid form, solid-liquid mixture form, solid block form, or solid powder form.

[0056] In addition to casein phosphopeptides, the food products described in this invention may include other optional ingredients, depending on the needs of the final product. Examples of such ingredients include:

[0057] Plant-based ingredients include fruits such as fig, pomegranate, kiwi, orange, tangerine, pineapple, strawberry, apple, banana, grape, pear, cherry, blueberry, blackberry, raspberry, and bilberry, or their extracts; fruits and vegetables such as onion, cucumber, tomato, cauliflower, carrot, spinach, kale, Brussels sprouts, garlic, basil, and oregano, or their extracts; grains such as rice (indica rice, japonica rice, glutinous rice), cereals (wheat, barley, oats, rye), corn, sorghum, millet, sorghum, yellow millet, buckwheat, soybean, broad bean, pea, mung bean, red bean, and kidney bean, or their extracts; nuts such as walnut, cashew, hazelnut, almond, apricot kernel, pine nut, pistachio, peanut, sunflower seed, chestnut, macadamia nut, and ginkgo, or their extracts; coffee or its extracts; and some medicinal and edible herbal medicines or their extracts.

[0058] Animal dairy products include fresh milk from cows, sheep, etc., as well as processed dairy products such as milk powder, whey protein, or cheese.

[0059] Animal meat ingredients, including meat ingredients from beef, mutton, fish or poultry.

[0060] Functional additives include vitamins (vitamin A, beta-carotene, vitamin D3, vitamin E, vitamin K1, vitamin B1, vitamin B2, vitamin B6, vitamin B1). 12 One or more of the following: Vitamin C, pantothenic acid, folic acid, niacin, choline, inositol, and biotin; starch; modified starch; amino acids (L-lysine-L-glutamic acid, L-glutamic acid, L-arginine, L-tryptophan, L-glutamine, taurine, L-valine, L-isoleucine, or L-leucine, etc.); dietary fiber (inulin, konjac flour, galactooligosaccharides, fructooligosaccharides, isomaltooligosaccharides, soybean polysaccharides, cyclodextrin, resistant dextrin, or soybean fiber, etc.); micronutrient supplements (including metal ion salts of organic acids, e.g.) Examples of supplements include calcium citrate, L-calcium lactate, calcium hydrogen phosphate, potassium gluconate, sodium citrate, ferrous gluconate, zinc gluconate, sodium selenite, copper gluconate, manganese gluconate, and magnesium gluconate; fat supplements (e.g., saturated fatty acids, polyunsaturated fatty acids, monounsaturated fatty acids, OPO structured lipids, OPL structured lipids, LPL structured lipids, DHA, EPA, ARA, phospholipids, etc.); nucleotide supplements; and human milk oligosaccharides (e.g., 2'-FL, 3-FL, DFL, LNFPI, LNFP II, LNT, LNnT, 3'-SL, 6'-SL, DSLNT, etc.).

[0061] Any food additives that are acceptable, including but not limited to solvents, antioxidants, antibacterial agents, thickeners, diluents, solubilizers, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, food flavorings, and food colorings.

[0062] There is no particular limitation on the specific form of the food products of this invention. In some specific embodiments, the food products may be powdered instant food products (instant coffee, cereal powder, nut powder or lotus root powder, etc.), baked food products (bread, cake or biscuit baked food products, etc.), beverages (carbonated beverages, fruit and vegetable juice beverages, functional beverages, tea beverages or milk beverages, etc.), milk and dairy products (fresh milk, milk powder, whey powder, fermented milk, cheese or condensed milk, etc. derived from fresh cow (sheep) milk), pasta products (noodles, instant noodles, steamed buns, dumplings or wontons, etc.), etc.

[0063] In the food products described in this invention, the content of casein phosphopeptide complies with the requirements of relevant laws and regulations, and the content of casein phosphopeptide is sufficient to achieve the effective amount required to achieve the technical effects involved in this invention. In some specific embodiments of this invention, the content of casein phosphopeptide, based on the total mass of the food products, is 0.001% by mass or more, preferably 0.005% by mass or more, and more preferably 0.04% by mass or more. There is no particular limit to the upper limit of its content, and reference can be made to the provisions of laws and regulations in different regions or conventional usage.

[0064] Applications in regulating dopaminergic neurons in the body

[0065] This invention reveals for the first time a novel application of casein phosphopeptides in regulating dopaminergic neurons in the body. Based on this discovery, foods containing casein phosphopeptides are also suitable for this application, and their consumption can also contribute to the positive regulation of dopaminergic neurons in the body.

[0066] In some specific implementations, the regulation of dopaminergic neurons in the body includes: enhancing the strength of dopaminergic neurons, and / or regulating neurotransmitters and metabolites related to the operation of the dopaminergic system.

[0067] The enhancement of dopaminergic neuron strength includes increasing the number of neurons, the abundance of dopamine synthesis and transport-related proteins, or one or more of the following: L-glutamate, GABA, L-serine, and reduced glutathione.

[0068] In some specific implementation schemes, the active regulation of the body's dopaminergic neurons can thereby regulate movement and foraging, control learning, memory and behavioral plasticity, and regulate sensory acuity, and is conducive to maintaining the long-term stable operation of the dopaminergic system.

[0069] In addition, the food of the present invention is suitable for all people in principle, especially for children, young people and middle-aged people. The components of the food can also be adjusted accordingly for people with different characteristics.

[0070] Example

[0071] 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.

[0072] Example 1: Effects of CPP on dopaminergic neurons

[0073] 1. Method

[0074] (1) Solution preparation:

[0075] 1) Nematode lysis solution (Clorox solution): 5% NaClO: 1 M NaOH = 2:1, v / v, NaClO treated in the dark.

[0076] 2) 1M potassium phosphate buffer, pH 6.0: 108.3g KH2PO4, 35.6g K2HPO4 (46.63g K2HPO4·3H2O), 1L water, autoclaved for later use.

[0077] 3) S buffer: 129 mL 0.05 M K2HPO4 (1.12 g), 871 mL 0.05 M KH2PO4 (5.92 g), 5.85 g NaCl.

[0078] 4) Nematode cryopreservation solution: S buffer + 30% glycerol, prepared 1:1

[0079] 5) 1M MgSO4: 120.3676 g MgSO4 (246.47 g MgSO4·7H2O), 1L water, filtered through a 0.22μm bacterial filter and set aside.

[0080] 6) 1M CaCl2: 110.984g CaCl2, 1L water, filtered through a 0.22μm filter and set aside.

[0081] 7) 5 mg / ml cholesterol solution: Dissolve 0.5 g cholesterol in 100 mL ethanol, filter through a 0.22 μm filter, and store at 4 °C for later use.

[0082] 8) Solid NGM medium: Dissolve 3g NaCl, 2.5g peptone, and 17g agar in 975 mL of water, then sterilize at 121°C for 15 min. After sterilization, cool to 60°C, add 1 mL of 1 M CaCl2, 1 mL of 1 M MgSO4, 1 mL of 5 mg / mL cholesterol solution, and 25 mL of 1 M potassium phosphate buffer. Pour the solution into plates while still hot and store at 4°C for later use.

[0083] 9) M9 buffer: 6g Na2HPO4, 3g KH2PO4, 5g NaCl, 1L water. After sterilization, add 1mL 1M MgSO4.

[0084] 10) Levamisole hydrochloride: 0.0241 g levamisole hydrochloride, 10 ml water.

[0085] (2) Cryopreservation and resuscitation of Caenorhabditis elegans

[0086] Plates containing freshly depleted *E. coli* OP50 bacterial colonies and a large number of L1-L2 stage nematodes were selected. Nematodes were collected into 1.5 mL centrifuge tubes using M9 buffer, the supernatant was discarded, and the tubes were centrifuged at 3000 rpm for 3 min. The supernatant was discarded, 1 mL of cryopreservation buffer was added, and the mixture was vortexed and transferred to 1.8 mL cryovials. The bacterial strain and date were labeled, and the tubes were stored at -4℃ for 1 day, -20℃ for 1 day, and then at -80℃. After thawing the cryopreserved nematode samples at room temperature, the tubes were centrifuged at 2500 rpm for 2 min, the supernatant was discarded, and the precipitate at the bottom of the tubes was pipetted and spread onto NGM solid medium containing OP50.

[0087] (3) Synchronization of Caenorhabditis elegans

[0088] Inoculate OP50 from the slant agar into 100 mL of LB liquid medium, incubate overnight at 37°C and 220 rpm, then store at 4°C for later use. Take 100 μL (50 μL for a 35 mm plate) of OP50 bacterial culture and spread it evenly on a 60 mm NGM plate. Dry at 37°C for 8 hours. Store in a sealed container. Add 1.2 mL of M9 buffer to the NGM medium containing nematodes, gently agitate the plate to allow the nematodes to dissociate from the medium and enter the buffer. Use a pipette to transfer 1 mL of M9 buffer containing nematodes into a 1.5 mL centrifuge tube. Centrifuge (3000 rpm, 1 min), remove the supernatant, add 1 mL of lysis buffer and vortex to mix, process for 10 min, centrifuge (3000 rpm, 1 min), remove the supernatant. Then wash three times with M9 buffer to obtain nematode eggs.

[0089] (4) Sample preparation

[0090] CPP was dissolved in sterile water to prepare a 50 mg / mL stock solution, which was stored at 4°C protected from light. Before use, the stock solution was mixed with OP50 and diluted to working solutions with concentrations of 0.2, 1, and 2 mg / mL. The OP50 bacterial suspension containing CPP was then spread onto NGM plates and dried at 37°C for 8 hours. L1 stage larvae were transferred to NGM plates containing CPP and cultured for 48 hours. Nematodes were then collected for relevant indices testing.

[0091] (5) Measurement of fluorescence intensity of dopamine neurons

[0092] The development of dopaminergic neurons was assessed using the BZ555 nematode. After 48 h of intervention, the nematodes were fixed on slides with levamisole hydrochloride solution, and fluorescence images of 30 nematodes were randomly captured under a fluorescence microscope. Subsequently, the fluorescence intensity of neurons was calculated using ImageJ software.

[0093] 2. Results and Discussion

[0094] The fluorescence intensity of dopaminergic neurons in nematodes can reflect the number of neurons, the abundance of proteins related to dopamine synthesis and transport, etc.

[0095] The results of this embodiment show that ( Figure 1 Compared with the control group, CPP treatment significantly increased the fluorescence intensity of nematode neurons and showed a better dose-response effect.

[0096] The sensory endings (cilia) of dopaminergic neurons (especially CEP neurons) are embedded beneath the cuticle of nematodes, enabling them to directly "sense" the presence of bacteria (i.e., food) through mechanosensory perception. Within nematodes, the main functions of dopaminergic neurons are in regulating movement and foraging, modulating learning, memory, and behavioral plasticity, and regulating sensory acuity—all key aspects of behavior.

[0097] The dopaminergic neurons in nematodes act as a key “food presence sensor” and “context coordinator.” They broadcast a global modulatory signal to motor, sensory, and learning-related neural circuits (especially glutamatergic and GABAergic circuits) to match the animal’s behavior (such as slowed movement, heightened senses, and increased oviposition) with the current favorable environment (i.e., abundant food).

[0098] The results of this embodiment show that different casein phosphopeptide treatments can significantly enhance the fluorescence intensity of dopaminergic neurons in nematodes and promote the development of dopaminergic neurons, thereby having a certain regulatory effect on the movement and foraging, learning, memory and behavioral plasticity, and sensory acuity of nematodes.

[0099] Example 2: Effects of CPP on neurotransmitters and metabolites associated with dopaminergic neurons in nematodes

[0100] 1. Method

[0101] (1) Solution preparation:

[0102] 1) Nematode lysis solution (Clorox solution): 5% NaClO: 1 M NaOH = 2:1, v / v, NaClO treated in the dark.

[0103] 2) 1M potassium phosphate buffer, pH 6.0: 108.3g KH2PO4, 35.6g K2HPO4 (46.63g K2HPO4·3H2O), 1L water, autoclaved for later use.

[0104] 3) S buffer: 129 mL 0.05 M K2HPO4 (1.12 g), 871 mL 0.05 M KH2PO4 (5.92 g), 5.85 g NaCl.

[0105] 4) Nematode cryopreservation solution: S buffer + 30% glycerol, prepared 1:1

[0106] 5) 1M MgSO4: 120.3676 g MgSO4 (246.47 g MgSO4·7H2O), 1L water, filtered through a 0.22μm bacterial filter and set aside.

[0107] 6) 1M CaCl2: 110.984g CaCl2, 1L water, filtered through a 0.22μm filter and set aside.

[0108] 7) 5 mg / ml cholesterol solution: Dissolve 0.5 g cholesterol in 100 mL ethanol, filter through a 0.22 μm filter, and store at 4 °C for later use.

[0109] 8) Solid NGM medium: Dissolve 3g NaCl, 2.5g peptone, and 17g agar in 975 mL of water, then sterilize at 121°C for 15 min. After sterilization, cool to 60°C, add 1 mL of 1 M CaCl2, 1 mL of 1 M MgSO4, 1 mL of 5 mg / mL cholesterol solution, and 25 mL of 1 M potassium phosphate buffer. Pour the solution into plates while still hot and store at 4°C for later use.

[0110] 9) M9 buffer: 6g Na2HPO4, 3g KH2PO4, 5g NaCl, 1L water. After sterilization, add 1mL 1M MgSO4.

[0111] 10) Levamisole hydrochloride: 0.0241 g levamisole hydrochloride, 10 ml water.

[0112] (2) Cryopreservation of Caenorhabditis elegans

[0113] Select plates that have just depleted E. coli OP50 bacterial colonies and contain a large number of L1-L2 stage nematodes. Collect the nematodes into 1.5 mL centrifuge tubes using M9 buffer, centrifuge to remove the supernatant, centrifuge at 3000 rpm for 3 min, discard the supernatant, add 1 mL of cryopreservation solution, vortex to mix, transfer to 1.8 mL cryovials, label with bacterial strain and date, store at -4℃ for 1 day, at -20℃ for 1 day, and at -80℃.

[0114] (3) Resuscitation of Caenorhabditis elegans

[0115] After thawing the frozen nematode samples at room temperature, centrifuge at 2500 rpm for 2 min, discard the supernatant, and use a pipette to aspirate the precipitate at the bottom of the test tube and spread it onto NGM solid culture medium containing OP50.

[0116] (4) Preparation of OP50 seed solution

[0117] Inoculate OP50 from the slant into 100 mL of LB liquid medium, incubate overnight at 37°C and 220 rpm, and store at 4°C for later use.

[0118] (5) Production of NGM containing OP50.

[0119] Take 100 μL (50 μL for a 35 mm plate) of OP50 bacterial suspension and spread it evenly on a 60 mm NGM plate. Dry at 37°C for 8 hours. Store in a tightly closed container.

[0120] (6) Synchronization of Caenorhabditis elegans

[0121] Add 1.2 mL of M9 buffer to NGM medium containing nematodes. Gently agitate the culture dish to allow the nematodes to detach from the medium and enter the buffer. Use a pipette to transfer 1 mL of M9 buffer containing nematodes into a 1.5 mL centrifuge tube. Centrifuge (3000 rpm, 1 min), remove the supernatant, add 1 mL of lysis buffer, and vortex to mix. Process for 10 min, centrifuge (3000 rpm, 1 min), and remove the supernatant. Then wash three times with M9 buffer to obtain nematode eggs.

[0122] (7) Sample preparation

[0123] CPP was dissolved in sterile water to prepare a 50 mg / mL stock solution, which was stored at 4°C protected from light. Before use, the stock solution was mixed with OP50 and diluted to working solutions with concentrations of 0.2, 1, and 2 mg / mL. The OP50 bacterial suspension containing CPP was then spread onto NGM plates and dried at 37°C for 8 hours. L1 stage larvae were transferred to NGM plates containing CPP and cultured for 48 hours. Nematodes were then collected for relevant indices testing.

[0124] (8) Detection of neurotransmitters and metabolites

[0125] 50 μL of nematodes treated for 48 h were collected in a grinding tube, and the tissue was ground and broken up. Then, a 1:4 (v / v) volume of 50% acetonitrile solution with a concentration of 1% formic acid was added, and the mixture was sonicated for 10 min and vortexed for 2 min. The mixture was then sonicated on ice for 20 min, frozen at -20℃ for 60 min, and finally centrifuged at -4℃ (12000 rpm, 30 min). The supernatant was collected, freeze-dried, and reconstituted with 200 μL of 50% acetonitrile solution with a concentration of 1% formic acid. Finally, the mixture was filtered through a 0.22 μm organic filter membrane, and the neurotransmitter content was detected by liquid chromatography-mass spectrometry (LC-MS). LC-MS chromatographic analysis conditions: Discovery® HS F5-3 column (15 cm × 2.1 mm, 3 μm); combined mobile phases A (0.1% formic acid aqueous solution) and B (0.1% formic acid acetonitrile solution); gradient elution (0–2 min, 2% B; 2–10 min, 2%–28% B; 10–13 min, 28%–100% B; 13–14 min, 100% B; 14–16 min, 2% B); flow rate 0.40 mL / min, column temperature 35 °C, injection volume 2 μl.

[0126] 2. Results

[0127] (1) Effect of CPP on L-glutamate content in nematodes

[0128] See Figure 2 This study showed the effect of different concentrations of casein phosphopeptide on the L-glutamate content in nematodes.

[0129] Specifically, casein phosphopeptide content at different concentrations can significantly increase L-glutamate content in nematodes.

[0130] (2) Effect of CPP on the content of γ-aminobutyric acid (GABA) in nematodes

[0131] The regulatory effects of different casein phosphopeptide treatments on tyramine levels in nematodes, such as Figure 3 As shown, the casein phosphopeptide content significantly increased the GABA content in nematodes at different concentrations.

[0132] (3) Effect of CPP on the content of reduced glutathione (GSH) in nematodes

[0133] The results of this embodiment show that ( Figure 4 CPP intervention can significantly increase the concentration of GSH in nematodes. The concentration of GSH detected shows a dose-effect within the range of CPP intervention concentration, with the highest concentration at a CPP intervention concentration of 2 mg / mL.

[0134] (4) Effect of CPP on L-serine content in nematodes

[0135] The results of this embodiment show that ( Figure 5 CPP intervention can significantly increase the concentration of L-serine in nematodes. The concentrations detected showed a dose-effect within the CPP intervention range, with the highest concentration at a CPP intervention concentration of 2 mg / mL.

[0136] 3. Discussion and Conclusion

[0137] Dopamine, as a neuromodulator, primarily functions by regulating the output of other core neurotransmitter systems, particularly the excitatory glutamate system and the inhibitory GABA system.

[0138] L-glutamate is the primary excitatory neurotransmitter in the nematode nervous system. Its signaling is mediated by various ionotropic glutamate receptors (iGluRs), including AMPA / Kainate receptors (such as glr-1 and glr-2) and NMDA receptors (such as nmr-1 and nmr-2). Dopamine signaling does not operate independently in regulating behavioral plasticity and learning; rather, it functions by modulating the output of the glutamatergic circuit.

[0139] GABA (gamma-aminobutyric acid) is the main inhibitory neurotransmitter in nematodes. GABAergic neurons (such as VD / DD motor neurons) are essential for coordinating the contraction of the dorsal and ventral muscles of the body to produce sinusoidal motion.

[0140] In the decision-making behavior model of nematodes, after dopamine is released, it acts on cholinergic (excitatory) neurons via D1-like receptors (DOP-1) and simultaneously on GABAergic (inhibitory) neurons via D2-like receptors (DOP-3), with the two functions antagonistically interacting. The dopamine system achieves high-level control over the final behavioral output by simultaneously and precisely regulating the balance between excitatory "throttle" and inhibitory "brakes."

[0141] The results of this study indicate that casein phosphopeptide intervention can significantly increase the levels of L-glutamate and GABA in nematodes, suggesting that casein phosphopeptide can regulate the function of dopaminergic neurons in nematodes.

[0142] In addition, the advanced regulatory functions and long-term stable operation of the dopaminergic system require L-serine and reduced glutathione to provide auxiliary support and cell protection.

[0143] L-serine is a precursor to D-serine. D-serine is produced by an enzyme called serine racemase, encoded by the serr-1 gene in nematodes. The primary function of D-serine in the nervous system is as a compulsive co-agonist of NMDA-type glutamate receptors. Therefore, L-serine (through its derivative D-serine) acts as a regulator of dopaminergic activity, controlling the "gain" of the DA-glutamate interaction interface. The integrity of the L-serine pathway is a prerequisite for dopamine to perform its advanced learning and plasticity functions. The chemical nature of dopamine makes it extremely unstable and prone to auto-oxidation. More importantly, the routine enzymatic metabolism of dopamine (e.g., via monoamine oxidase MAO) produces toxic byproducts, including quinones, semiquinones, and large amounts of reactive oxygen species (ROS), such as superoxide and hydrogen peroxide. This high oxidative load places dopaminergic neurons in a state of continuous, high-level oxidative stress. Reduced glutathione (GSH) has a significant protective effect on the nerves of nematodes, mainly through its antioxidant properties, maintenance of intracellular redox balance, and regulation of cellular stress responses. As an important antioxidant in nematode cells, GSH can directly scavenge reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anions, preventing ROS from damaging neuronal cell membranes, proteins, and DNA.

[0144] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0145] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. Use of a casein phosphopeptide in the preparation of foods that help regulate dopaminergic neurons in the body.

2. The use according to claim 1, characterized in that, The regulation of dopaminergic neurons in the body includes: enhancing the strength of dopaminergic neurons, and / or neurotransmitters and metabolites that regulate the operation of the dopaminergic system.

3. The use according to claim 2, characterized in that, The enhancement of dopaminergic neuron strength includes increasing the number of neurons, the abundance of dopamine synthesis and transport-related proteins, or one or more of these factors.

4. The use according to any one of claims 1 to 3, characterized in that, The neurotransmitter is selected from one or both of L-glutamate and GABA; the metabolite is selected from one or both of L-serine and reduced glutathione.

5. The use according to any one of claims 1-4, characterized in that, The food products mentioned are powdered reconstituteable foods, baked goods, beverages, milk and dairy products, or pasta products.

6. The use according to any one of claims 1-5, characterized in that, The food contains one or more of the following ingredients: plant-based ingredients, animal dairy ingredients, animal meat ingredients, functional additives, and any food-acceptable excipients.

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