A kind of milk casein hydrolysate and its preparation method and application

Abstract

The present invention provides a milk casein hydrolyzate and its preparation method and application. The composition of the milk casein hydrolyzate of the present invention includes at least one of the peptide segment 1 with an amino acid sequence as shown in SEQ ID NO: 1, the peptide segment 2 with an amino acid sequence as shown in SEQ ID NO: 2, and the peptide segment 3 with an amino acid sequence as shown in SEQ ID NO: 3. The milk casein hydrolyzate has good neuronal protection and repair effects, can improve cholinergic neuron and dopaminergic neuron damage; can inhibit Aβ protein deposition, thereby alleviating neuronal synaptic disorders, neuronal apoptosis and brain damage caused by Aβ protein deposition; can improve cholinergic defects, thereby alleviating neurodevelopmental impairment, behavioral and motor disorders caused by cholinergic defects; can also repair damaged neurons in the hippocampus and hypothalamus, thereby maintaining a stable brain environment.

Description

Technical Field

[0001] The present invention relates to biotechnology, in particular to a milk casein hydrolyzate and a preparation method and application thereof. Background Art

[0002] Protein is one of the three major nutrients in the human body. It can be absorbed by the body in the form of amino acids, short peptides, or polypeptides, thereby exerting physiological functions. Active peptides exist in an inactive state within the amino acid sequence of proteins. They can be released through gastrointestinal digestion or proteolysis in vitro and exert physiological regulatory functions. Due to the many potential health benefits of food-borne bioactive peptides, they have recently been frequently used as functional food ingredients.

[0003] Casein is the primary protein found in the milk of mammals, including cows, sheep, and humans. It is highly safe and possesses excellent nutritional value and functional properties. The functional and nutritional health properties of casein can be attributed to its bioactive components, such as bioactive peptides. It has been reported that the hydrolysis of casein can produce casein hydrolysates with a variety of bioactive activities, including antihypertensive, antioxidant, anti-obesity, antibacterial, and immunomodulatory properties. Casein hydrolysates have very important application value. In order to fully utilize casein hydrolysates and maximize their biological functions, it is necessary to conduct in-depth research on the specific functions of casein hydrolysates. Summary of the Invention

[0004] The present invention provides a milk casein hydrolyzate, which can inhibit Aβ protein deposition, inhibit acetylcholinesterase activity, and repair damaged neurons in the hippocampus and hypothalamus, and has good neuron protection and repair effects.

[0005] The invention provides a method for preparing the milk casein hydrolysate, which has simple operation, mild conditions and is convenient for large-scale production.

[0006] The present invention also provides an application of the above-mentioned casein hydrolyzate or the casein hydrolyzate prepared by the above-mentioned preparation method in inhibiting Aβ protein deposition, improving cholinergic activity, repairing damaged neurons in the hippocampus and hypothalamus, and preparing related products with neuron protection and repair effects.

[0007] The present invention provides a milk casein hydrolyzate, wherein the milk casein hydrolyzate comprises at least one of peptide segment 1, peptide segment 2, and peptide segment 3;

[0008] The amino acid sequence of peptide segment 1 is shown in SEQ ID NO: 1;

[0009] The amino acid sequence of peptide segment 2 is shown in SEQ ID NO: 2;

[0010] The amino acid sequence of peptide segment 3 is shown in SEQ ID NO: 3.

[0011] The casein hydrolysate as described above, wherein, based on the mass of the casein hydrolysate, the mass content of peptide segment 1 is ≥1.1%, the mass content of peptide segment 2 is ≥0.7%, and the mass content of peptide segment 3 is ≥0.3%.

[0012] The milk casein hydrolysate as described above, wherein the degree of hydrolysis of the milk casein hydrolysate is 10%-15%;

[0013] and / or, the mass content of peptides with a molecular weight greater than 10,000 Da in the milk casein hydrolysate is ≤30%;

[0014] And / or, the mass content of peptides with a molecular weight of less than 1000 Da in the milk casein hydrolysate is ≥20%.

[0015] The milk casein hydrolyzate is obtained by enzymatically hydrolyzing the milk casein raw material with trypsin and neutral protease.

[0016] The present invention provides a method for preparing the above-mentioned casein hydrolysate, which comprises the following steps:

[0017] The milk casein raw material is enzymatically hydrolyzed by using trypsin and neutral protease to obtain milk casein hydrolyzate.

[0018] The preparation method as described above, wherein the trypsin has an enzyme activity of 150,000-250,000 U / g and is added in an amount of 0.15%-0.25% of the mass of the casein raw material;

[0019] and / or, the enzyme activity of the neutral protease is 150,000-250,000 U / g, and the addition amount is 0.10%-0.20% of the mass of the casein raw material;

[0020] and / or, during the enzymatic hydrolysis treatment, the enzymatic hydrolysis temperature is 50° C.-55° C., the enzymatic hydrolysis pH is 7.5-8.0, and the enzymatic hydrolysis time is 2.0-3.0 h;

[0021] And / or, the casein raw material is obtained by preparing casein powder and water in a mass ratio of 1:(8-12).

[0022] The present invention provides a use of the milk casein hydrolyzate or the milk casein hydrolyzate prepared by the above preparation method in inhibiting Aβ protein deposition.

[0023] The present invention provides a use of the milk casein hydrolyzate or the milk casein hydrolyzate prepared by the above preparation method in improving cholinergic activity.

[0024] The present invention provides a use of the milk casein hydrolyzate or the milk casein hydrolyzate prepared by the above preparation method in repairing damaged neurons in the hippocampus and hypothalamus.

[0025] The present invention also provides a use of the above-mentioned casein hydrolyzate or the casein hydrolyzate prepared by the above-mentioned preparation method in preparing related products with neuron protection and repair effects.

[0026] The present invention provides a milk casein hydrolyzate, which comprises at least one of a peptide segment 1 having an amino acid sequence as shown in SEQ ID NO: 1, a peptide segment 2 having an amino acid sequence as shown in SEQ ID NO: 2, and a peptide segment 3 having an amino acid sequence as shown in SEQ ID NO: 3. The present invention has the following beneficial effects: the milk casein hydrolyzate of the present invention has good neuronal protection and repair effects, can improve the degree of damage to cholinergic neurons and improve dopaminergic neuronal damage; can effectively inhibit Aβ protein deposition, thereby alleviating neuronal synaptic disorders, neuronal apoptosis and brain damage caused by Aβ protein deposition; can improve cholinergic deficiency by inhibiting acetylcholinesterase activity, increase cholinergic activity, thereby alleviating neurodevelopmental damage, behavioral and motor disorders caused by cholinergic deficiency; can also repair damaged neurons in the hippocampus and hypothalamus, thereby maintaining a stable brain environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a graph showing the paralysis lifespan of CL4176 nematodes treated with milk casein hydrolysate in Example 3;

[0028] Figure 2 This is a graph showing the cholinergic activity of CL4176 nematodes treated with milk casein hydrolysate in Example 4;

[0029] Figure 3 This is a graph showing the head swing frequency of CL4176 nematodes treated with casein hydrolyzate in Example 5;

[0030] Figure 4 This is a micrograph of Aβ deposition in CL4176 nematodes treated with milk casein hydrolysate in Example 6;

[0031] Figure 5 This is a comparison of Aβ deposition in CL4176 nematodes treated with milk casein hydrolysate in Example 6;

[0032] Figure 6 Schematic diagram of the chemotaxis experiment in Example 7;

[0033] Figure 7 This is a graph showing the chemotaxis index of CL2355 nematodes treated with milk casein hydrolyzate in Example 7;

[0034] Figure 8 These are micrographs of normal and swollen cholinergic neurons of LX929 nematodes in Example 8;

[0035] Figure 9 This is a graph showing the swelling of cholinergic neurons in LX929 nematodes treated with milk casein hydrolysate in Example 8;

[0036] Figure 10 These are micrographs of normal and damaged dopamine neurons of BZ555 nematodes in Example 9;

[0037] Figure 11 This is a graph showing the damage to dopamine neurons in BZ555 nematodes treated with casein hydrolysate in Example 9;

[0038] Figure 12 This is a staining image of rat hippocampus tissue sections treated with milk casein hydrolyzate in Example 10;

[0039] Figure 13 This is a staining image of rat hypothalamic tissue sections treated with milk casein hydrolyzate in Example 11. DETAILED DESCRIPTION

[0040] In order to enable those skilled in the art to better understand the scheme of the present invention, the present invention is further described in detail below. The specific embodiments listed below are only for describing the principles and features of the present invention. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts are within the scope of protection of the present invention.

[0041] The first aspect of the present invention provides a milk casein hydrolyzate, wherein the milk casein hydrolyzate comprises at least one of peptide segment 1, peptide segment 2, and peptide segment 3;

[0042] The amino acid sequence of peptide segment 1 is shown in SEQ ID NO: 1;

[0043] The amino acid sequence of peptide segment 2 is shown in SEQ ID NO: 2;

[0044] The amino acid sequence of peptide segment 3 is shown in SEQ ID NO: 3.

[0045] The casein hydrolysate provided by the present invention is a product prepared by hydrolyzing casein. Casein, a phosphorus-calcium-containing protein, is the primary protein in the milk of mammals, including cows, sheep, and humans. It is highly safe and has excellent nutritional value and functional properties. Through the hydrolysis process, casein is broken down into smaller peptide chains and amino acids, making it more easily digestible and absorbable.

[0046] In the present invention, the source of milk casein is not limited, and it can be derived from sheep or cattle, for example.

[0047] The composition of the milk casein hydrolyzate of the present invention includes at least one of peptide segment 1, peptide segment 2, and peptide segment 3. Specifically, the amino acid sequence of peptide segment 1 is HIRLSFNPTQLEEQ (SEQ ID NO: 1), which is a tetradecapeptide consisting of histidine (H), isoleucine (I), arginine (R), leucine (L), serine (S), phenylalanine (F), asparagine (N), proline (P), threonine (T), glutamine (Q), leucine (L), glutamic acid (E), glutamic acid (E), and glutamine (Q) from the N-terminus to the C-terminus; the amino acid sequence of peptide segment 2 is NLIRFF (SEQ ID NO: 2), which is a hexapeptide consisting of asparagine (N), leucine (L), isoleucine (I), arginine (R), phenylalanine (F), and phenylalanine (F) from the N-terminus to the C-terminus; the amino acid sequence of peptide segment 3 is GAWYYVPL (SEQ ID NO:3), which is an octapeptide composed of glycine (G), alanine (A), tryptophan (W), tyrosine (Y), tyrosine (Y), valine (V), proline (P), and leucine (L) from N-terminus to C-terminus.

[0048] Experimental verification has shown that when the composition of casein hydrolysate includes at least one of peptide segment 1, peptide segment 2, and peptide segment 3, the casein hydrolysate has good neuronal protection and repair effects, can improve the degree of damage to cholinergic neurons and improve dopaminergic neuronal damage; can effectively inhibit Aβ protein deposition, thereby alleviating neuronal synaptic disorders, neuronal apoptosis and brain damage caused by Aβ protein deposition; can improve cholinergic deficiency by inhibiting acetylcholinesterase activity, increase cholinergic activity, thereby alleviating neurodevelopmental damage, behavioral and movement disorders caused by cholinergic deficiency; can also repair damaged neurons in the hippocampus and hypothalamus, thereby maintaining a stable brain environment.

[0049] Furthermore, by regulating the content of functional peptides in casein hydrolysate, it is possible to achieve even better neuronal protection and repair. Specifically, based on the mass of casein hydrolysate, the mass content of peptide 1 is ≥1.1%, the mass content of peptide 2 is ≥0.7%, and the mass content of peptide 3 is ≥0.3%.

[0050] In the technical solution of the present invention, the degree of hydrolysis of the casein hydrolysate is 10%-15%. By regulating the degree of hydrolysis of the casein hydrolysate, the degree of hydrolysis of the casein can be ensured to be within an appropriate range, which is conducive to the enrichment of functional peptides.

[0051] In the technical solution of the present invention, the mass content of peptides with a molecular weight greater than 10,000 Da in the casein hydrolysate is ≤30%; the mass content of peptides with a molecular weight less than 1,000 Da in the casein hydrolysate is ≥20%. By regulating the molecular weight distribution of the casein hydrolysate to meet the above range, the casein hydrolysate is helped to have the advantages of small molecular weight and easy absorption. It can be understood that when the molecular weight distribution in the casein hydrolysate meets any one of the conditions of the mass content of peptides with a molecular weight greater than 10,000 Da being ≤30% or the mass content of peptides with a molecular weight less than 1,000 Da being ≥20%, the digestibility and absorptivity of the casein hydrolysate can be promoted; when the molecular weight distribution in the casein hydrolysate simultaneously meets the conditions of the mass content of peptides with a molecular weight greater than 10,000 Da being ≤30% and the mass content of peptides with a molecular weight less than 1,000 Da being ≥20%, the digestibility and absorptivity of the casein hydrolysate can be better promoted.

[0052] In a specific embodiment, the casein hydrolysate is obtained by enzymatically hydrolyzing the casein raw material using trypsin and neutral protease.

[0053] Trypsin, a serine protease extracted from the pancreas of cattle, sheep, and pigs, is also an endopeptidase that can cleave peptide bonds formed by the carboxyl groups of lysine or arginine. Its high specificity plays a key role in determining the amino acid arrangement of proteins. Neutral proteases are a class of proteolytic enzymes that have optimal activity under neutral pH conditions (usually between pH 6-8). They catalyze the cleavage of peptide bonds in protein molecules, breaking down large proteins into smaller peptide chains or amino acids.

[0054] In the present invention, by enzymatically hydrolyzing the casein raw material and limiting the type of enzyme preparation to meet the above range, functional peptides can be further enriched to improve the efficacy of casein hydrolysate in neuronal protection and repair.

[0055] In order to ensure that the composition of the casein hydrolysate includes at least one of peptide segment 1, peptide segment 2, and peptide segment 3, and to enrich the above functional peptide segments as much as possible, the second aspect of the present invention provides a method for preparing the above casein hydrolysate, comprising the following steps:

[0056] The milk casein raw material is enzymatically hydrolyzed by using trypsin and neutral protease to obtain milk casein hydrolyzate.

[0057] The preparation method provided by the present invention can quickly and simply prepare a large amount of milk casein hydrolyzate including at least one of peptide segment 1, peptide segment 2, and peptide segment 3. The preparation method of the present invention has mild conditions, is easy to implement, has low cost, and is convenient for expanding production.

[0058] In the technical solution of the present invention, to avoid excessive enzymatic hydrolysis that affects the components and functions of the casein hydrolysate, the enzymatic hydrolysate after enzymatic hydrolysis can be subjected to an enzyme inactivation treatment. The present invention does not limit the specific method of enzyme inactivation, and conventional enzyme inactivation methods in the art can be used for enzyme inactivation, for example, heating to 80°C for 30 minutes. After the enzyme inactivation treatment, the temperature can be lowered to 30°C-40°C to reduce the impact of high temperature on proteins or peptides.

[0059] In order to further enrich the functional peptides and obtain a solid form of casein hydrolysate for easy transportation and storage, the enzymatic hydrolysate after enzyme inactivation can be sequentially subjected to alcohol precipitation, filtration, concentration, and spray drying to obtain the target casein hydrolysate.

[0060] The present invention does not limit the operation mode of alcohol precipitation, filtration, concentration and spray drying, and can be carried out by conventional technical means in the field. For example, the alcohol precipitation operation can be achieved by adding an alcohol solvent to the enzymatic hydrolyzate after enzyme inactivation and standing at 60°C-70°C for 5-8 hours. After the addition of the alcohol solvent, the functional peptides are transferred to the alcohol solvent and the impurities are precipitated; after the alcohol precipitation treatment is completed, filtration can be performed to further remove impurities; after filtration, reduced pressure concentration can be performed at 65°C to further enrich the functional peptides; then, the spray drying can be set at an inlet temperature of 180°C and an outlet temperature of 90°C to finally obtain the target casein hydrolyzate.

[0061] Furthermore, to ensure a good match between the casein raw material and the enzyme preparation, promote the enzymatic hydrolysis reaction, and thus increase the mass content of functional peptides in the casein hydrolysate, the present invention regulates the enzyme activity and addition amount of the enzyme preparation. Specifically, the enzyme activity of trypsin is 150,000-250,000 U / g, and the addition amount is 0.15%-0.25% of the mass of the casein raw material; the enzyme activity of neutral protease is 150,000-250,000 U / g, and the addition amount is 0.10%-0.20% of the mass of the casein raw material.

[0062] In the present invention, the amount of trypsin or neutral protease added is calculated based on the dry weight of the casein raw material. For example, when the casein raw material is casein powder, the amount of trypsin added is 0.15%-0.25% of the mass of the casein powder; when the casein raw material is casein powder, the amount of neutral protease added is 0.10%-0.20% of the mass of the casein powder.

[0063] It is understandable that when the enzyme activity and addition amount of either trypsin or neutral protease meet the above ranges, the enzymatic hydrolysis reaction can be promoted; when the enzyme activity and addition amount of trypsin and neutral protease simultaneously meet the above ranges, the enzymatic hydrolysis reaction can be better promoted.

[0064] In the technical solution of the present invention, during the enzymatic hydrolysis treatment, the enzymatic hydrolysis temperature is 50° C.-55° C., the enzymatic hydrolysis pH is 7.5-8.0, and the enzymatic hydrolysis time is 2.0-3.0 h, which can better exert the enzymatic hydrolysis effects of trypsin and neutral protease.

[0065] Specifically, the casein raw material can be adjusted to the optimal enzymatic hydrolysis pH environment of the enzyme preparation, that is, a pH value of 7.5-8.0, to obtain an enzymatic hydrolysis stock solution that is conducive to enzymatic hydrolysis by the enzyme preparation. Subsequently, pre-mixed trypsin and neutral protease are added to the enzymatic hydrolysis stock solution at the appropriate enzymatic hydrolysis temperature of the enzyme preparation, that is, 50°C-55°C, and enzymatic hydrolysis is carried out for 2.0-3.0 hours to allow the enzyme preparation to fully act on the casein in the enzymatic hydrolysis stock solution.

[0066] In the technical solution of the present invention, the casein raw material is obtained by preparing casein powder and water in a mass ratio of 1: (8-12).

[0067] Mixing casein powder and water in the above mass ratio helps to evenly disperse the casein, facilitating the enzymatic hydrolysis reaction. If too much water is added, the reaction volume during the enzymatic hydrolysis process is likely to be too large, thereby increasing the load of the subsequent enrichment process; if too little water is added, the casein solution is likely to have poor fluidity, which is not conducive to the enzymatic hydrolysis reaction and thus reduces the enzymatic hydrolysis efficiency.

[0068] A third aspect of the present invention provides a use of the above-mentioned casein hydrolyzate or the casein hydrolyzate prepared by the above-mentioned preparation method in inhibiting Aβ protein deposition.

[0069] When the nervous system is underdeveloped or underdeveloped, excessive Aβ accumulation can occur, leading to synaptic dysfunction, neuronal apoptosis, and brain damage. Experimental studies have shown that the casein hydrolyzate of the present invention can effectively inhibit the abnormal aggregation of Aβ, thereby alleviating the paralysis, decreased motor function, and behavioral abnormalities caused by Aβ accumulation.

[0070] A fourth aspect of the present invention provides a use of the above-mentioned casein hydrolyzate or the casein hydrolyzate prepared by the above-mentioned preparation method in improving cholinergic activity.

[0071] Cholinergic deficiency alters brain neurons and signal transmission, including impaired acetylcholine (ACh) release, defective axonal transport, and elevated acetylcholinesterase (AChE) levels. Abnormal accumulation of AChE can accelerate the hydrolysis of the neurotransmitter ACh, ultimately affecting signal transmission between neurons and leading to learning and memory impairments. The casein hydrolysate of the present invention can significantly reduce AChE activity in the body, thereby enhancing cholinergic activity.

[0072] A fifth aspect of the present invention provides a use of the above-mentioned milk casein hydrolyzate or the milk casein hydrolyzate prepared by the above-mentioned preparation method in repairing damaged neurons in the hippocampus and hypothalamus.

[0073] The hippocampus is located between the hypothalamus and the falx cerebri of the brain. It is shaped like a seahorse and is an important component of the brain. Its main functions include controlling emotions, improving memory, regulating endocrine system and sleep, etc. The casein hydrolyzate of the present invention can improve the disordered arrangement of hippocampal pyramidal cells, promote more orderly arrangement of cells, and have a repair effect on hippocampal neuron damage.

[0074] The hypothalamus is a higher-order center of the autonomic nervous system beneath the cerebral cortex and a key part of the brain responsible for maintaining internal environmental balance and stability. The hypothalamus controls wakefulness and sleep through circadian rhythms and sleep homeostasis. The casein hydrolyzate of the present invention can improve the function of hypothalamic nerve cells, promoting their more orderly arrangement and repairing damaged hypothalamic neurons.

[0075] In a sixth aspect, the present invention provides a use of the above-mentioned casein hydrolyzate or the casein hydrolyzate prepared by the above-mentioned preparation method in the preparation of a medicine having neuron protection and repair effects.

[0076] The casein hydrolyzate of the present invention has good neuronal protection and repair effects, can improve the degree of damage to cholinergic neurons and improve dopaminergic neuronal damage; can effectively inhibit Aβ protein deposition, thereby alleviating neuronal synaptic disorders, neuronal apoptosis and brain damage caused by Aβ protein deposition; can improve cholinergic deficiency by inhibiting acetylcholinesterase activity, increase cholinergic activity, thereby alleviating neurodevelopmental damage, behavioral and movement disorders caused by cholinergic deficiency; and can also repair damaged neurons in the hippocampus and hypothalamus, thereby maintaining a stable brain environment.

[0077] A large amount of research data has proved that the casein hydrolyzate of the present invention has significant neuron protection and repair capabilities and can be used to prepare neuron protection or repair drugs, thereby broadening the application scope of casein hydrolyzate and providing new raw materials for neuron protection or repair drugs.

[0078] The technical solution of the present application will be further explained below with reference to specific embodiments.

[0079] In the following examples, animal experiments were conducted using Caenorhabditis elegans and laboratory rats. The animal experiments involved were supervised by the Laboratory Animal Ethics Committee of South China Agricultural University (Guangdong, China) and complied with animal protection, animal welfare, and ethical principles.

[0080] Among them, Caenorhabditis elegans is a classic and widely used model organism. Using C. elegans in scientific research offers numerous inherent advantages, including its small size (adult worms are approximately 1 mm long) and its transparency, allowing observation of its entire developmental cycle and multiple behavioral and developmental markers under a standard light microscope. Fluorescence imaging also eliminates the need for sample decolorization. It primarily feeds on bacteria, requiring only Escherichia coli OP50 to be inoculated in Nematode growth medium (NGM) to maintain C. elegans in the laboratory. It is also easy to maintain. The C. elegans nervous system is its most complex tissue, with the lineage and morphology of each neuron fully characterized. Furthermore, the function of its brain has been fully deciphered for the first time within a neural network circuit, with a complete map of all connections between neurons and from neurons to muscle and other tissues, such as the intestine and skin. Almost all neurons in the nematode Caenorhabditis elegans are labeled with fluorescent reporter genes. Using high-resolution imaging techniques, researchers can precisely track the effects of gene mutations on neuronal developmental phenotypes in C. elegans. This makes the C. elegans nervous system a leading model system for elucidating the nature of nervous system gene regulation, offering unique advantages for studying behavior and neurodevelopment. Adult hermaphroditic C. elegans possess 302 neurons, divided into two independent nervous systems: the large somatic nervous system (282 neurons) and the smaller pharyngeal nervous system (20 neurons). These two systems have distinct topologies and communicate through a pair of RIP rhythm-associated interneurons. In the somatic nervous system, neurons and their processes are typically located between the subcutaneous layer and the body wall muscles, sharing a basal lamina with the subcutaneous layer. Pharyngeal neurons, on the other hand, are located directly between the pharyngeal muscles. These neurons connect to the rest of the body through a nerve ring, controlling pharyngeal muscle movement and thus enabling food intake and transport.

[0081] The Caenorhabditis elegans used in this application specifically include: wild-type nematode N2, nematode CL4176, nematode CL2355, nematode LX929, nematode BZ555, and nematode CL2006.

[0082] Among them, wild-type nematodes N2 are usually cultured at 20°C. From eggs to final development into adults, it needs to go through four stages, L1-L4. This short process lasts about 3 days.

[0083] CL4176 nematodes are composed of the temperature-inducible system smg-1, human Aβ 1-42The CL4176 nematode is co-transfected with rol-6 mRNA. Normally, the CL4176 nematode moves in a circular motion. However, under certain conditions, it produces large amounts of the pathogenic Aβ protein. When Aβ accumulates to a certain level, the nematode loses its ability to move, becoming unable to swing. Unlike the wild-type N2 nematode, the CL4176 nematode is sensitive to ambient temperature and is typically cultured at 16°C to slow its growth and reduce Aβ accumulation. Therefore, by inducing a moderate temperature increase (25°C), the development and growth of the CL4176 nematode can be accelerated, leading to rapid Aβ accumulation and the appearance of a "paralyzed" phenotype, enabling the measurement of a paralyzed lifespan curve.

[0084] CL2355 nematode is a temperature-sensitive nematode. After high temperature induction (25°C), Aβ is expressed in large quantities in its neurons. 1-42 Pathogenic proteins. As Aβ accumulates in neurons, Aβ-induced neurotoxicity can lead to neuronal damage and even death. This loss of motor and chemical perception, controlled by these neurons, can lead to deficits in sensory chemotaxis and associative learning. Therefore, CL2355 nematodes can mimic the learning and memory impairments mediated by Aβ neurotoxicity in the brain of Alzheimer's disease (AD).

[0085] LX929 nematodes are transgenic nematodes. The cholinergic neurons in this model of nematodes are marked with green fluorescent protein and can be stably inherited during the passage process.

[0086] BZ555 nematodes are transgenic nematodes whose dopamine neurons specifically express green fluorescence (GFP).

[0087] CL2006 is a transgenic nematode purchased from the Caenorhabditis Genetics Center (CGC) in the United States. Its genotype is dvIs2 [pCL12 (unc-54 / human Abeta peptide 1-42 minigene) + pRF4]. Because it contains the muscle-specific promoter unc-54, it progressively expresses pathogenic Aβ in the body wall muscle cells of the nematode. 1-42 , leading to the accumulation of intramuscular Aβ deposits and the formation of fibrillar amyloid that can be detected by amyloid-specific dyes. 1-42 Expression and accumulation can lead to muscle fiber damage and abnormal motor function.

[0088] The rats used in the examples are rodents, similar to humans in terms of nervous system mechanisms. Therefore, rats are an excellent model organism for studying the neuroprotection and neurorepair effects of active ingredients. After modeling neuronal damage in rats, the effects of milk casein hydrolysate on neuronal repair in the hippocampus and hypothalamus can be studied. Specifically, specifically pathogen-free (SPF) male Sprague-Dawley (SD) rats (200-250 g) purchased from Beijing VitalRiver Laboratory Animal Technology Co., Ltd. were used for the experiments.

[0089] In addition, the preparation method of NGM plates is as follows: add 6.8 g of technical agar powder, 1.2 g of sodium chloride, 1.0 g of tryptone, and 0.08 g of streptomycin sulfate to every 390 mL of tertiary water, stir evenly, and sterilize at 121°C for 20 min. After sterilization, add 10 mL of 1 mol / L potassium phosphate buffer solution, 400 μL of 1 mol / L magnesium sulfate solution, 400 μL of 1 mol / L calcium chloride solution, and 400 μL of 5 mg / mL cholesterol and shake well. Pour into a culture dish to obtain an NGM plate.

[0090] The preparation method of OP50 bacterial liquid is as follows: take OP50 bacteria and streak them on a plate in LB medium, culture them at 37℃ for 48 hours, pick out a single OP50 colony and transfer it to sterilized LB liquid broth medium, place it in a shaker at 37℃ and 170 rpm / min and incubate it for 12 hours. When the OD600 is 0.4, the OP50 bacterial liquid is obtained.

[0091] For other experimental methods without specific conditions, conventional conditions or those recommended by the manufacturer were generally followed. All reagents used were commercially available or publicly available unless otherwise specified.

[0092] Example 1: Preparation of casein hydrolysate

[0093] (1) Pure water was added to the milk casein powder to obtain a milk casein solution, wherein the mass ratio of milk casein powder to pure water was 1:8. Trypsin with an enzyme activity of 200,000 U / g and an addition amount of 0.20 wt% (based on the mass of the milk casein powder) and neutral protease with an enzyme activity of 200,000 U / g and an addition amount of 0.20 wt% (based on the mass of the milk casein powder) were first mixed in 10 mL of pure water, and then added to the milk casein solution for enzymatic hydrolysis to obtain an enzymatic hydrolyzate. During the enzymatic hydrolysis, the enzymatic hydrolysis temperature was 50°C, the enzymatic hydrolysis pH was 7.5, and the enzymatic hydrolysis time was 2.0 h. After the enzymatic hydrolysis was completed, the enzymatic hydrolyzate was inactivated at 80°C for 30 min. After the inactivated enzymatic hydrolyzate was cooled to 30°C-40°C, it was successively subjected to alcohol precipitation, filtration, concentration, and spray drying to obtain milk casein hydrolyzate powder 1.

[0094] (2) Pure water was added to the casein powder to obtain a casein solution, wherein the mass ratio of the casein powder to the pure water was 1:10. Trypsin with an enzyme activity of 200,000 U / g and an addition amount of 0.15 wt% (based on the mass of the casein powder) and neutral protease with an enzyme activity of 200,000 U / g and an addition amount of 0.20 wt% (based on the mass of the casein powder) were first mixed in 10 mL of pure water and then added to the casein solution for enzymatic hydrolysis to obtain an enzymatic hydrolyzate. During the enzymatic hydrolysis, the enzymatic hydrolysis temperature was 55°C, the enzymatic hydrolysis pH was 7.5, and the enzymatic hydrolysis time was 2.5 h. After the enzymatic hydrolysis was completed, the enzymatic hydrolyzate was inactivated at 80°C for 30 min. After the inactivated enzymatic hydrolyzate was cooled to 30°C-40°C, it was sequentially subjected to alcohol precipitation, filtration, concentration, and spray drying to obtain a casein hydrolyzate powder 2.

[0095] (3) Pure water was added to the casein powder to obtain a casein solution, wherein the mass ratio of the casein powder to the pure water was 1:10. Trypsin with an enzyme activity of 200,000 U / g and an addition amount of 0.25 wt% (based on the mass of the casein powder) and neutral protease with an enzyme activity of 200,000 U / g and an addition amount of 0.10 wt% (based on the mass of the casein powder) were first mixed in 10 mL of pure water and then added to the casein solution for enzymatic hydrolysis to obtain an enzymatic hydrolyzate. During the enzymatic hydrolysis, the enzymatic hydrolysis temperature was 55°C, the enzymatic hydrolysis pH was 8.0, and the enzymatic hydrolysis time was 2.5 h. After the enzymatic hydrolysis was completed, the enzymatic hydrolyzate was inactivated at 80°C for 30 min. After the inactivated enzymatic hydrolyzate was cooled to 30°C-40°C, alcohol precipitation, filtration, concentration, and spray drying were performed in sequence to obtain a casein hydrolyzate powder 3.

[0096] (4) Pure water was added to the casein powder to obtain a casein solution, wherein the mass ratio of the casein powder to the pure water was 1:12. Trypsin with an enzyme activity of 200,000 U / g and an addition amount of 0.20 wt% (based on the mass of the casein powder) and neutral protease with an enzyme activity of 200,000 U / g and an addition amount of 0.16 wt% (based on the mass of the casein powder) were first mixed in 10 mL of pure water and then added to the casein solution for enzymatic hydrolysis to obtain an enzymatic hydrolyzate. During the enzymatic hydrolysis, the enzymatic hydrolysis temperature was 50°C, the enzymatic hydrolysis pH was 7.5, and the enzymatic hydrolysis time was 3.0 h. After the enzymatic hydrolysis was completed, the enzymatic hydrolyzate was inactivated at 80°C for 30 min. After the inactivated enzymatic hydrolyzate was cooled to 30°C-40°C, it was sequentially subjected to alcohol precipitation, filtration, concentration, and spray drying to obtain a casein hydrolyzate powder 4.

[0097] Example 2: Identification of casein hydrolysate

[0098] (1) Degree of hydrolysis of casein hydrolysate

[0099] The degree of hydrolysis of casein hydrolyzate powders 1-4 was determined using the OPA reaction. Since each peptide bond hydrolyzed releases a free amine group, and the free amine group reacts with OPA (o-phthalaldehyde) to form a yellow complex, the degree of hydrolysis can be characterized by measuring its absorbance at 340 nm using a spectrophotometer. The calculation formula for the degree of hydrolysis is as follows:

[0100]

[0101] Where h is the number of peptide bonds hydrolyzed, mmol / g; h tot is the total number of peptide bonds, mmol / g (the total number of peptide bonds in casein is 8.2 mmol / g); C serine is the millimolar number of serine amino groups in casein hydrolysate, mmol / L; V is the fixed volume of casein hydrolysate, L; N is the dilution multiple of casein hydrolysate; m is the mass of casein hydrolysate, g; w is the protein content in casein hydrolysate, %; β is a constant, which is 0.383 for casein; α is a constant, which is 1.039 for casein.

[0102] After three parallel experiments, the hydrolysis degrees of casein hydrolyzate powders 1-4 were measured to be 12.85%±0.80%, 11.03%±0.65%, 10.85%±0.51% and 13.17%±0.64%, respectively.

[0103] (2) Molecular weight distribution of casein hydrolysate

[0104] The molecular weight distribution of casein hydrolyzate powders 1-4 was determined by referring to the GPC / UV detection method in Appendix A of the national standard GB 31645-2018. The results are shown in Table 1.

[0105]

[0106] (3) LC-MS / MS mass spectrometry identification of casein hydrolysate

[0107] Casein hydrolyzate powders 1-4 were dissolved in ddH2O to obtain casein hydrolyzate solutions 1-4, which were then transferred to 10 kD ultrafiltration tubes and centrifuged at 4°C and 12,000 rcf for 10 min to collect polypeptide samples less than 10 kD. Dithiothreitol solution was added to the polypeptide samples to a final concentration of 10 mmol / L, and the samples were reduced in a 56°C water bath for 1 h. Iodoacetic acid solution was then added to a final concentration of 50 mmol / L. After the reaction was protected from light for 40 min, the samples were desalted using a self-filled desalting column, and the solvent was evaporated in a 45°C vacuum centrifugal concentrator to obtain samples 1-4 to be tested.

[0108] Samples 1-4 were analyzed by LC-MS / MS. Capillary liquid chromatography conditions were as follows: analytical column: 150 μm ID × 150 mm, packed with Acclaim PepMap RPLC C18, 1.9 μm, 100Å; mobile phase A: pure water (containing 0.1% (v / v) formic acid); mobile phase B: 80% acetonitrile (containing 0.1% (v / v) formic acid); flow rate: 600 nL / min; analysis time per component: 66 min. Mass spectrometry parameters: primary mass spectrometry parameters: resolution: 70,000, AGC target: 3e6, maximum IT: 100 ms, scan range: 300 to 1800 m / z; secondary mass spectrometry parameters: resolution: 75,000, AGC target: 1e5, maximum IT: 50 ms, top N: 20, NCE / stepped NCE: 28.

[0109] The mass spectrometry raw files obtained after LC-MS / MS analysis were searched against the Uniprot protein database using Maxquant (1.6.2.10). The peptide sequence information of the casein hydrolysates was detected by LC-MS / MS and analyzed comprehensively to obtain the characteristic peptides and their content ranges in casein hydrolysate powders 1-4, as follows:

[0110] In the casein hydrolyzate powders 1-4, the peptide HIRLSFNPTQLEEQ, the peptide NLIRFF, and the peptide GAWYYVPL are functional peptides, and the mass content of the peptide HIRLSFNPTQLEEQ is ≥1.1%, the mass content of the peptide NLIRFF is ≥0.7%, and the mass content of the peptide GAWYYVPL is ≥0.3%.

[0111] Example 3: Effect of milk casein hydrolyzate on the paralysis lifespan of CL4176 nematodes

[0112] The casein hydrolyzate powder 2 obtained in Example 1 was dissolved in sterile water to prepare a 20 mg / mL stock solution, which was sealed with parafilm and stored at 4°C in the dark. Before use, the stock solution was diluted with OP50 bacterial solution to concentrations of 0 mg / mL, 0.5 mg / mL, 1.5 mg / mL, and 2.5 mg / mL of casein hydrolyzate powder 2 to obtain working solutions. 200 μl of the working solution was evenly spread on an NGM plate, air-dried, and stored at 4°C until use. In subsequent experiments of this example, CL4176 nematodes were cultured on NGM plates coated with working solutions containing different concentrations of casein hydrolysate. The OP50 group was used for NGM plates coated with 0 mg / mL of casein hydrolysate; the 0.5 mg / mL group was used for NGM plates coated with 0 mg / mL of casein hydrolysate; the 1.5 mg / mL group was used for NGM plates coated with 1.5 mg / mL of casein hydrolysate; and the 2.5 mg / mL group was used for NGM plates coated with 2.5 mg / mL of casein hydrolysate. Each group contained 60 CL4176 nematodes, and three replicates were performed, for a total of 720 CL4176 nematodes.

[0113] The eggs of CL4176 nematodes were placed on the above-mentioned NGM plates coated with the working solution containing different concentrations of casein hydrolyzate and cultured in a 16°C incubator for 48 hours. That is, before the CL4176 nematodes entered the L4 stage (the lower abdomen had a crescent-shaped shape), they were transferred to 25°C for further culture. High temperature was used to induce the "paralysis" phenotype of CL4176 nematodes. At this time, the CL4176 nematodes only had a slight shake of the head, and the body no longer rolled and circled, and could not be induced to move when touched by the worm picker. When the first "paralyzed" nematode appeared, the time was started, and the number of paralyzed nematodes was recorded every 2 hours until all CL4176 nematodes were paralyzed. The time was stopped, and a lifespan curve was plotted based on the number of paralyzed nematodes every 2 hours. The horizontal axis is time and the vertical axis is the survival fraction (Fraction survival). Figure 1 , Figure 1 middle Indicates significant difference ( p<0.05, p<0.01, p<0.001 and p<0.001).

[0114] The average survival time of nematodes is calculated based on the lifespan curve. The specific calculation method is as follows:

[0115]

[0116] Where n is the total number of CL4176 nematodes, j is the age of CL4176 nematodes, hours; dj is the age interval (x j , x j+1 ). Furthermore, the median survival time refers to the time when the CL4176 nematode survival rate reaches 50%, and the maximum survival time refers to the time when the CL4176 nematode survival rate reaches 0%. Specific results can be found in Table 2, where different letters indicate significant differences.

[0117]

[0118] When the nervous system of Caenorhabditis elegans is underdeveloped or completely underdeveloped, excessive Aβ protein accumulation occurs, leading to synaptic dysfunction, neuronal apoptosis, and brain damage. When CL4176 worms are exposed to high temperatures (25°C), Aβ protein accumulation occurs. This massive accumulation ultimately leads to paralysis. Therefore, analyzing the lifespan of paralyzed CL4176 worms can be used to determine the effective concentration of casein hydrolysate.

[0119] Depend on Figure 1 As shown, compared with the OP50 group, the paralysis curve shifted significantly to the right at concentrations of 0.5 mg / mL to 2.5 mg / mL of casein hydrolysate, with the 0.5 mg / mL group showing the most significant rightward shift. Table 2 shows that compared with the OP50 group, casein hydrolysate increased the mean, median, and maximum survival times of CL4176 nematodes, with the 0.5 mg / mL concentration providing the greatest increase in mean, median, and maximum survival times. Therefore, casein hydrolysate concentrations of 0.5 mg / mL to 2.5 mg / mL can prolong the time it takes for CL4176 nematodes to enter the paralysis phenotype, effectively reducing the impact of paralysis caused by Aβ protein deposition, with the 0.5 mg / mL concentration providing the most effective effect.

[0120] Example 4: Effect of milk casein hydrolysate on cholinergic activity of CL4176 nematodes

[0121] Cholinergic deficiency alters brain neurons and signal transduction, including impaired acetylcholine (ACh) release, defective axonal transport, and elevated acetylcholinesterase (AChE) levels. Abnormal AChE accumulation can accelerate the hydrolysis of the neurotransmitter ACh, ultimately affecting signal transduction between neurons and leading to learning and memory impairments. Therefore, this example determined the cholinergic activity of CL4176 nematodes by measuring AChE activity, and further examined the effects of casein hydrolysate on the cholinergic activity of CL4176 nematodes. Since Example 3 demonstrated that casein hydrolysate concentrations of 0.5 mg / mL to 2.5 mg / mL can prolong the mean, median, and maximum survival times of CL4176 nematodes, the experimental concentration of casein hydrolysate in this example was also within the range of 0.5 mg / mL to 2.5 mg / mL.

[0122] NGM plates containing OP50, 0.5 mg / mL, 1.5 mg / mL, and 2.5 mg / mL were prepared using the same method as in Example 3. CL4176 nematode eggs were placed on NGM plates containing OP50, 0.5 mg / mL, 1.5 mg / mL, and 2.5 mg / mL, respectively. The plates were incubated in a 16°C incubator for 48 hours. Before the CL4176 nematodes entered the L4 stage (a crescent-shaped lower abdomen), the plates were transferred to 25°C and incubated for another 48 hours. At this point, the nematodes were rinsed from the culture plates into centrifuge tubes using M9 buffer (3 g potassium dihydrogen phosphate, 6 g sodium dihydrogen phosphate, 5 g sodium chloride, 1 mL 1 mol / L magnesium sulfate, and 1 L grade tertiary water). The nematodes were homogenized and ground using an automated tissue grinder. The nematodes were then centrifuged at 4°C (12,000 rpm for 10 minutes). The supernatant was collected and stored at 4°C. The protein content of nematode tissue protein in each supernatant was standardized using a protein quantification (TP) kit to determine the mass of nematode tissue protein in each supernatant. The supernatant was then assayed for AChE activity using the AChE assay kit (Cat. No. A024-1-1, manufactured by the Nanjing Jiancheng Bioengineering Research Institute). The supernatant was mixed with the substrate buffer and color development solution provided in the kit and incubated at 37°C for 6 minutes. The inhibitor and clearing agent provided in the kit were then added and mixed. The mixture was allowed to stand at room temperature for 15 minutes, and the absorbance (OD) value was measured to generate the experimental group data. To make the experimental results more accurate, it is also necessary to obtain the control group data, standard group data and blank group data according to the kit instructions. Among them, the control group mixed the substrate buffer and color development application solution provided by the kit, reacted at 37°C for 6 minutes, then added the supernatant and the inhibitor and clearing agent provided by the kit to mix, let it stand at room temperature for 15 minutes, and detected the absorbance OD value to obtain the control group data; the standard group replaced the supernatant with the 1 μmol / mL standard application solution provided by the kit, mixed with the substrate buffer and color development application solution, reacted at 37°C for 6 minutes, then added the inhibitor and clearing agent provided by the kit to mix, let it stand at room temperature for 15 minutes, and detected the absorbance OD value to obtain the standard group data; the blank group replaced the supernatant with double distilled water, mixed with the substrate buffer and color development application solution provided by the kit, reacted at 37°C for 6 minutes, then added the inhibitor and clearing agent provided by the kit to mix, let it stand at room temperature for 15 minutes, and detected the absorbance OD value to obtain the blank group data. This kit is designed based on the principle that acetylcholinesterase hydrolyzes acetylcholine to produce choline and acetic acid. Choline then reacts with a thiol-based colorimetric reagent to form a yellow compound called sym-trinitrobenzene (TNB). The absorbance of the yellow compound can be measured spectrophotometrically at a wavelength of 412 nm to reflect the amount of choline produced and, therefore, the activity of acetylcholinesterase. Each experiment was repeated three times, requiring at least 600 nematodes per assay.

[0123] Specifically, the activity of acetylcholinesterase can be determined based on activity units. For every mg of nematode tissue protein incubated at 37°C for 6 min, 1 μmol of substrate (acetylcholine) in the hydrolysis reaction system (the entire reaction system in which acetylcholinesterase hydrolyzes acetylcholine to produce choline and acetic acid) is 1 activity unit.

[0124] The calculation formula of AchE activity is as follows:

[0125]

[0126] Wherein, A1 is the absorbance OD value of the experimental group (OP50 group, 0.5 mg / mL group, 1.5 mg / mL group, or 2.5 mg / mL group); A2 is the absorbance OD value of the control group; A3 is the absorbance OD value of the standard group; A4 is the absorbance OD value of the blank group; B is the concentration of the AChE standard in the test kit, which is 1 μmol / mL; C is the concentration of nematode tissue protein, in mgprot / mL.

[0127] The effects of different concentrations of casein hydrolysate on the acetylcholinesterase activity of CL4176 nematodes can be seen in the following example: Figure 2 , Figure 2 Different letters represent significant differences, and the effects on the average AChE values can be seen in Table 3. Figure 2 As can be seen from Table 3, when the concentration of casein hydrolyzate is 0.5 mg / mL-2.5 mg / mL, the activity of AChE in CL4176 nematodes can be significantly reduced.

[0128]

[0129] Example 5: Effect of milk casein hydrolyzate on the motility of CL4176 nematodes

[0130] Extensive accumulation of Aβ protein can cause paralysis in CL4176 nematodes. During this process, the nematode's mobility gradually decreases as the toxicity of the pathogenic protein increases. This example determined the effect of 0.5 mg / mL of milk casein hydrolyzate on CL4176 nematode motility by measuring the frequency of head thrash in CL4176 nematodes.

[0131] NGM plates of the OP50 group and the 0.5 mg / mL group were obtained by referring to the method of Example 3. The eggs of CL4176 nematodes were placed in the NGM plates coated with the above-mentioned working solution of 0 mg / mL and 0.5 mg / mL casein hydrolysate, and cultured in a 16°C incubator for 48 h. That is, before the CL4176 nematodes entered the L4 period (there was a crescent-shaped lower abdomen), they were transferred to 25°C and continued to be cultured for 5 h, 10 h or 15 h. After the culture was completed, the CL4176 nematodes were picked up and placed in a new NGM plate that was not coated with E. coli OP50, and allowed to move freely for 1 min before observation. The criterion for judging head swing is: the nematode's head swings from the left to the right, which is recorded as one time, and then swings back to the left and recorded as one time. The number of times the CL4176 nematode's head swings within 30 s is recorded. Ten nematodes were randomly selected from each group for observation. This test was repeated three times. The specific results are shown in. Figure 3 ,in This indicates that there is a significant difference between the 0.5 mg / mL group and the OP50 group.

[0132] like Figure 3 As shown, compared with the OP50 group, the number of head swings in the 0.5 mg / mL group was significantly increased at the 10th and 15th hours after the start of observation (p<0.05), indicating that 0.5 mg / mL milk casein hydrolysate can improve the symptoms of acute paralysis in nematodes and enhance their locomotion ability.

[0133] Example 6: Effect of milk casein hydrolysate on Aβ protein deposition in CL4176 nematodes

[0134] The accumulation of Aβ protein can cause CL4176 nematodes to enter a paralysis process. In this example, the effect of 0.5 mg / mL milk casein hydrolyzate on the paralysis process was determined by detecting Aβ protein deposition in CL4176 nematodes.

[0135] NGM plates for the OP50 and 0.5 mg / mL groups were prepared using the same method as in Example 3. CL4176 nematode eggs were placed on NGM plates coated with the aforementioned 0 mg / mL and 0.5 mg / mL working solutions of casein hydrolysate and incubated in a 16°C incubator for 48 hours. Before the CL4176 nematodes entered the L4 stage (a crescent-shaped structure formed on the lower abdomen), the plates were transferred to 25°C and incubated for another 48 hours. At this point, the nematodes were rinsed from the culture dish with buffer into a centrifuge tube. The washed nematodes were centrifuged at 2500 rpm for 2 minutes in a microcentrifuge tube and fixed with 1 ml of fixative (4% (m / m) paraformaldehyde) for 15 minutes. The centrifuge tube containing the nematodes was then placed in a -80°C freezer for 30 minutes, removed and thawed in running tap water until the nematodes were almost thawed, i.e., just a little water appeared in the centrifuge tube. The tube was then placed in a -80°C freezer for another 48 hours. This cycle of freeze-thaw cycles was repeated three times. After the last time, the nematodes were taken out of the -80°C freezer and slowly thawed on ice. After thawing, the nematodes were centrifuged at 4000 rpm for 2 minutes to sediment the nematodes, the supernatant was discarded, and the nematodes were washed twice with M9 buffer. A 50% (m / v) ethanol solution containing 0.125% (m / m) thioflavin T was added to the washed nematodes and stained at room temperature for 10 minutes. After staining, the suspension was washed 2-3 times with M9 buffer until it became transparent and clear, and 0.5 mL of supernatant was retained for the last time. The nematodes stained with thioflavin T were observed under a fluorescence microscope using GFP fluorescence, and the Aβ deposition was quantified using ImageJ software. Figure 4 and Figure 5 .

[0136] Figure 4 This is a micrograph of Aβ protein deposition in CL4176 nematodes. Figure 5 This is a fluorescence intensity graph characterizing the deposition of Aβ protein in CL4176 nematodes, where CK represents the OP50 group and GMCH represents the 0.5 mg / mL group. This means there is a significant difference between the 0.5 mg / mL group and the OP50 group. Figure 4 and Figure 5 As shown in the results, compared with the OP50 group, the accumulation of Aβ was significantly decreased after treatment with 0.5 mg / mL milk casein hydrolysate (p<0.05), indicating that the effect of milk casein hydrolysate in alleviating the paralysis symptoms of CL4176 may be achieved by inhibiting the abnormal aggregation of Aβ.

[0137] Example 7: Effects of casein hydrolysate on learning and memory abilities of CL2355 nematodes

[0138] NGM plates for the OP50 and 0.5 mg / mL groups were prepared according to the method of Example 3. CL2355 nematode eggs were placed on NGM plates coated with the above-mentioned working solutions of 0 mg / mL and 0.5 mg / mL casein hydrolysate, and cultured in a 16°C incubator for 48 h. The plates were then transferred to 25°C and cultured for another 48 h. After the incubation period, the nematodes were washed with M9 buffer and collected. 80-100 nematodes were then placed in the center of a 120 mm diameter culture dish. The dish was divided into four quadrants, named Area A, Area B, Area C, and Area D. 10 μL of odor attractant (5 μL of 0.1% (m / v) benzaldehyde and 5 μL of 1% (m / m) sodium azide) was dripped onto the edges of Area A and Area D of the dish. 10 μL of solvent blank control (5 μL of 100% (m / v) sterile water + 5 μL of 1% (m / m) sodium azide) was added to the edges of Area B and Area C. The specific process can be found in [ 14 ]. Figure 6 After 30 minutes, the number of nematodes that moved from the center to each quadrant was recorded, and the chemotaxis index (CI) was calculated. The calculation formula of the chemotaxis index is as follows:

[0139]

[0140] Among them, D is the number of nematodes in the quadrant where the odor attractant is located, E is the number of nematodes in the quadrant where the solvent blank control is located, F is the total number of nematodes, The specific results can be seen in Figure 7 , Figure 7 CK represents the OP50 group, GMCH represents the 0.5 mg / mL group, There was a significant difference between the 0.5 mg / mL group and the blank control group.

[0141] Transgenic nematode CL2355 expresses Aβ protein in neurons. The aggregation toxicity of Aβ protein can lead to neuronal damage or even death and damage the corresponding cell functions. In particular, the motor and chemical perception abilities regulated by neurons will also be lost, leading to behavioral abnormalities. Figure 7 As shown in the results, the CI value of CL2355 nematodes treated with milk casein hydrolysate was significantly greater than that of the control group (p<0.05), indicating that milk casein hydrolysate can improve the chemotaxis dysfunction of CL2355 and alleviate the neurotoxicity caused by Aβ.

[0142] Example 8: Effects of milk casein hydrolysate on cholinergic neurons in LX929 nematodes

[0143] NGM plates of the OP50 group and the 0.5 mg / mL group were obtained by referring to the method of Example 3. The eggs of LX929 nematodes were placed in the NGM plates coated with the working solution of 0 mg / mL and 0.5 mg / mL milk casein hydrolysate, cultured in a 20°C incubator for 48 hours, and then continuously heat stressed at 35°C for 3 hours. The nematodes were picked onto a glass slide, anesthetized with 1% (m / m) sodium azide, and imaged with a fluorescence microscope (Axio Imager D2) to photograph the state of cholinergic neurons in the nematodes. The experiment was carried out three biological replicates, and at least 10 nematodes were anesthetized with 1% (m / m) sodium azide solution each time and fixed on a glass slide. They were photographed using an upright fluorescence microscope at 20× magnification. After the shooting was completed, the number of nematodes with normal and abnormal neuronal morphology was counted and plotted, as can be seen in detail. Figure 8 and Figure 9 .

[0144] Figure 8 These are micrographs of LX929 nematode cholinergic neurons when normal (Normal) and swollen (Blebbing) (indicated by white arrows). Figure 9 Figure 1 is a result graph characterizing the swelling of cholinergic neurons in LX929 nematodes, where CK represents the blank control group (OP50 group treated and not cultured in a 20°C incubator for 48 h, then continuously heat stressed at 35°C for 3 h), CK-Heat represents the heat stress group (OP50 group treated and cultured in a 20°C incubator for 48 h, then continuously heat stressed at 35°C for 3 h), and GMCH-Heat represents the 0.5 mg / mL group (0.5 mg / mL group treated and cultured in a 20°C incubator for 48 h, then continuously heat stressed at 35°C for 3 h). There was a significant difference between the heat stress group and the blank control group. This indicates that there was a significant difference between the 0.5 mg / mL group and the heat stress group.

[0145] Cholinergic neurons are neurons that can synthesize and release the neurotransmitter acetylcholine. Under normal circumstances, cholinergic neurons are relatively smooth and have no particularly obvious swelling protrusions; however, Figure 8 As shown in Figure 2, cholinergic neurons will swell significantly when they are exposed to external stress. Figure 9 As shown in the data, after heat stress treatment, the swollen neurons in the heat stress group increased compared with the blank control group; compared with the heat stress group, the swollen neurons in the 0.5 mg / mL group decreased, and there was a significant difference (p<0.05). Therefore, casein hydrolysate can improve the degree of damage to cholinergic neurons.

[0146] Example 9: Effects of milk casein hydrolysate on dopamine neurons in BZ555 nematodes

[0147] NGM plates of the OP50 group and the 0.5 mg / mL group were obtained by referring to the method of Example 3. The eggs of BZ555 nematodes were placed in the NGM plates coated with the working solution of 0 mg / mL and 0.5 mg / mL casein hydrolysate, cultured in a 20°C incubator for 48 hours, and then continuously heat stressed at 35°C for 3 hours. The nematodes were picked onto a glass slide, anesthetized with 1% (m / m) sodium azide, and imaged with a fluorescence microscope (Axio Imager D2) to photograph the state of dopaminergic neurons (CEP) in the nematodes. The experiment was carried out three biological replicates, and at least 10 nematodes were anesthetized with 1% (m / m) sodium azide solution each time and fixed on a glass slide. They were photographed using an upright fluorescence microscope at a magnification of 20×. After the photography was completed, the number of nematodes with normal and abnormal neuronal morphology was counted and plotted, as can be seen in detail. Figure 10 and Figure 11 .

[0148] Figure 10 This is a micrograph of the dopamine neurons of the nematode BZ555 when normal (Normal) and dendritic bubbling and abnormality (Dendritic bubbling and abnormality). Figure 11 This is a result diagram characterizing the degree of damage to dopamine neurons in BZ555 nematodes, where CK represents the blank control group (OP50 group treated and not cultured in a 20°C incubator for 48 h before continuous heat stress at 35°C for 3 h), CK-Heat represents the heat stress group (OP50 group treated and cultured in a 20°C incubator for 48 h before continuous heat stress at 35°C for 3 h), and GMCH-Heat represents the 0.5 mg / mL group (0.5 mg / mL group treated and cultured in a 20°C incubator for 48 h before continuous heat stress at 35°C for 3 h). There was a significant difference between the heat stress group and the blank control group. This indicates that there was a significant difference between the 0.5 mg / mL group and the heat stress group.

[0149] Dopamine is one of the most abundant monoamine neurotransmitters in the brain. The concentration of dopamine in the central nervous system is affected by many factors. Dopamine regulates a variety of behaviors related to wakefulness, such as movement, cognition, reward, and eating. Figure 10 and Figure 11 As shown in the data, the damage and repair of the dendrites of dopaminergic neurons in the head region can reflect the protective effect of casein hydrolysate on dopaminergic neurons; after heat stress treatment, the dendritic breakage or swelling of dopaminergic neurons in nematodes was significantly increased compared with the blank control group, while the dendritic breakage or swelling of nematodes fed with casein hydrolysate after heat stress was significantly reduced (p<0.05), indicating that casein hydrolysate can improve the damage of dopaminergic neurons.

[0150] Example 10: Effects of milk casein hydrolysate on rat hippocampal neurons

[0151] The para-chlorophenylalanine (PCPA) insomnia rat model is a widely recognized and classic experimental animal model for studying serotonin (5-HT) and its relationship with other neurotransmitters. PCPA, as a 5-HT synthesis inhibitor, selectively inhibits tryptophan hydroxylase activity. Sixty male rats were divided equally into six groups. The model group received an intraperitoneal injection of PCPA (300 mg / kg) at 9:00 AM on two consecutive days to establish an insomnia rat model. The control group received the same procedure, but received an intraperitoneal injection of the same volume of weakly alkaline 0.9% (m / m) sodium chloride solution as the PCPA injection. The insomnia rat model was successfully established when the rats' circadian rhythms were disrupted and they became irritable, active during the day, and exhibited increased aggressiveness, significantly different from the control group. After the model was successfully established, drug intervention was started. The experimental rats were gavaged with drugs at 8:00 am every day for 7 consecutive days. Among them, the insomnia rat model can be divided into the following groups according to the dosage and type of drug administration: the low-dose casein hydrolysate administration group (GMCH-L) has a concentration of 100 mg / kg, the medium-dose casein hydrolysate administration group (GMCH-M) has a concentration of 400 mg / kg, the high-dose casein hydrolysate administration group (GMCH-H) has a concentration of 700 mg / kg, and the diazepam administration group has a concentration of 3 mg / kg; among them, the casein hydrolysate is casein hydrolysate powder 2.

[0152] Hematoxylin and eosin (H&E) staining was used to assess hippocampal damage in insomnia rats: After drug administration, the rats were dissected, the hippocampus was removed and fixed in 4% (m / m) paraformaldehyde solution, dehydrated with a series of graded ethanol solutions, and embedded in paraffin. The dissected tissue was cut into 5 μm sections and stained with H&E. Finally, the histopathological changes were observed using an optical microscope (CX-41, Olympus). Figure 12 , Figure 12 There are 6 groups in total, each group is divided into left and right pictures, the magnification of the left picture is 100X, and the right picture is the image of the box in the left picture magnified to 400X; CK is the normal control group, Model is the insomnia rat model group, GMCH-L is the low-dose casein hydrolysate administration group, GMCH-M is the medium-dose casein hydrolysate administration group, GMCH-H is the high-dose casein hydrolysate administration group, and Diazepam is the diazepam administration group.

[0153] The hippocampus is located between the hypothalamus and the falx cerebri in the brain. It is shaped like a seahorse and is an important part of the brain. Its main functions include controlling emotions, improving memory, regulating endocrine and sleep, etc. Figure 12 As shown in the figure, the pyramidal cells in the CA region of the hippocampus of the normal control group were arranged neatly, tightly and evenly; the pyramidal cells in the CA region of the hippocampus of the insomnia rat model group were partially arranged slightly disordered, the structure of some neurons was unclear, and the normal morphology disappeared; after one week of treatment, the disordered arrangement of the hippocampal pyramidal cells in the low-dose, medium-dose and high-dose groups of casein hydrolysate was restored, and the cells were arranged more neatly, and the recovery effect of the high-dose group was more obvious; at the same time, the disordered arrangement of the hippocampal pyramidal cells in the positive control diazepam group was also restored, and the cells were arranged more neatly; this indicates that casein hydrolysate has a repair effect on hippocampal neuronal damage.

[0154] Example 11: Effects of milk casein hydrolysate on rat hypothalamic neurons

[0155] Referring to the method of Example 10, 60 male rats were divided into six groups: Model group, normal control group, low-dose casein hydrolysate administration group, medium-dose casein hydrolysate administration group, high-dose casein hydrolysate administration group and diazepam administration group.

[0156] Hematoxylin and eosin (H&E) staining was used to evaluate hypothalamic damage in insomnia rats: After the administration, the rats were dissected, the hypothalamus was removed and fixed in 4% (m / m) paraformaldehyde solution, dehydrated with a series of graded ethanol solutions, and embedded in paraffin. The dissected tissue was cut into 5 μm sections and stained with H&E. Finally, the histopathological changes were observed using an optical microscope (CX-41, Olympus). Figure 13 , Figure 13 There are 6 groups in total, with a magnification of 400X; CK represents the normal control group, Model represents the insomnia rat model group, GMCH-L represents the low-dose casein hydrolysate administration group, GMCH-M represents the medium-dose casein hydrolysate administration group, GMCH-H represents the high-dose casein hydrolysate administration group, and Diazepam represents the diazepam administration group.

[0157] The hypothalamus is a high-level center of the autonomic nervous system under the cerebral cortex. It is one of the important parts of the brain that maintains the balance and stability of the body's internal environment. The hypothalamus controls wakefulness and sleep through circadian rhythm and sleep homeostasis. Figure 13As shown, the hypothalamic nerve cells of rats in the normal control group had clear morphology, uniform distribution and intact structure; vacuoles were observed locally in the hypothalamic nerve cells of the insomnia rat model group (indicated by the arrows), the cells were loosely arranged and the number was reduced; after one week of treatment, most of the hypothalamic nerve cells of rats in the low-dose, medium-dose and high-dose groups were restored and arranged more neatly, and the recovery effects of the medium-dose and high-dose groups were more obvious; at the same time, most of the hypothalamic nerve cells in the positive control diazepam group were also restored and arranged more neatly; this indicates that casein hydrolysate has a repair effect on hypothalamic neuronal damage.

[0158] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A casein hydrolyzate, characterized in that The composition of the casein hydrolysate at least includes peptide segment 1, peptide segment 2, and peptide segment 3; The amino acid sequence of the peptide segment 1 is shown in SEQ ID NO: 1; The amino acid sequence of peptide segment 2 is shown in SEQ ID NO: 2; The amino acid sequence of peptide segment 3 is shown in SEQ ID NO: 3; The casein hydrolysate is obtained by enzymatically hydrolyzing the casein raw material with trypsin and neutral protease; The trypsin has an enzyme activity of 150,000-250,000 U / g, and the added amount is 0.15%-0.25% of the mass of the casein raw material; The enzyme activity of the neutral protease is 150,000-250,000 U / g, and the addition amount is 0.10%-0.20% of the mass of the casein raw material; In the enzymatic hydrolysis treatment, the enzymatic hydrolysis temperature is 50°C-55°C, the enzymatic hydrolysis pH is 7.5-8.0, and the enzymatic hydrolysis time is 2.0-3.0 h; The casein raw material is obtained by preparing casein powder and water in a mass ratio of 1:(8-12).

2. The casein hydrolyzate according to claim 1, wherein Based on the mass of the casein hydrolysate, the mass content of the peptide segment 1 is ≥1.1%, the mass content of the peptide segment 2 is ≥0.7%, and the mass content of the peptide segment 3 is ≥0.3%.

3. The casein hydrolyzate according to claim 1 or 2, characterized in that The degree of hydrolysis of the casein hydrolysate is 10%-15%; and / or, the mass content of peptides with a molecular weight greater than 10,000 Da in the milk casein hydrolysate is ≤30%; And / or, the mass content of peptides with a molecular weight of less than 1000 Da in the milk casein hydrolysate is ≥20%.

4. The method for preparing the casein hydrolyzate according to any one of claims 1 to 3, characterized in that: The steps include: Using trypsin and neutral protease to enzymatically hydrolyze the casein raw material to obtain the casein hydrolyzate; The trypsin has an enzyme activity of 150,000-250,000 U / g, and the added amount is 0.15%-0.25% of the mass of the casein raw material; The enzyme activity of the neutral protease is 150,000-250,000 U / g, and the addition amount is 0.10%-0.20% of the mass of the casein raw material; In the enzymatic hydrolysis treatment, the enzymatic hydrolysis temperature is 50°C-55°C, the enzymatic hydrolysis pH is 7.5-8.0, and the enzymatic hydrolysis time is 2.0-3.0 h; The casein raw material is obtained by preparing casein powder and water in a mass ratio of 1:(8-12).

5. Use of the casein hydrolyzate according to any one of claims 1 to 3 or the casein hydrolyzate prepared by the preparation method according to claim 4 in the preparation of a medicine having neuron protective and repairing effects.

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