Cream casein hydrolysate as well as preparation method and application thereof

Abstract

The invention provides a cheese protein hydrolysate as well as a preparation method and application thereof. The composition of the casein hydrolysate at least comprises at least one of a peptide fragment 1 with an amino acid sequence as shown in SEQ ID NO: 1, a peptide fragment 2 with an amino acid sequence as shown in SEQ ID NO: 2 and a peptide fragment 3 with an amino acid sequence as shown in SEQ ID NO: 3. The casein hydrolysate has good neuron protection and repair effects, and can improve cholinergic neuron and dopaminergic neuron damage; abeta protein deposition can be inhibited, so that neuronal synaptic disorder, neuronal apoptosis and brain injury caused by Abeta protein deposition are relieved; the cholinergic defect can be improved, so that the nerve development damage and the behavior and movement disorder caused by the cholinergic defect are relieved; and damaged neurons in hippocampus and hypothalamus can be repaired, so that the brain environment is kept stable.

Description

Technical Field

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

[0002] Protein is one of the three major nutrients for the human body. It can be absorbed by the body in the form of amino acids, or in the form of short peptides or polypeptides, and then play a physiological function. Active peptides exist in the amino acid sequence of proteins in an inactive state. They can be released through gastrointestinal digestion or in vitro protease hydrolysis and play a physiological regulatory function. Due to the many potential health benefits of food-borne bioactive peptides, they have been often used as functional food ingredients in recent years.

[0003] Casein is the main protein in the milk of mammals, including cows, sheep or humans. It is highly safe and has good nutritional value and functional properties. The functions and nutritional health properties of casein can be attributed to the bioactive ingredients it contains, such as bioactive peptides. It is reported that the hydrolysis process of casein can produce casein hydrolysates with multiple biological functional activities such as lowering blood pressure, anti-oxidation, anti-obesity, antibacterial and immunomodulatory. Casein hydrolysates have very important application value. In order to make full use of casein hydrolysates and exert their biological functions, it is necessary to conduct in-depth exploration and research on the specific functions of casein hydrolysates. Summary of the invention

[0004] The 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 casein hydrolysate, wherein the casein hydrolysate 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 casein hydrolysate as described above, wherein the degree of hydrolysis of the casein hydrolysate is 10%-15%;

[0013] and / or, the mass content of peptides with a molecular weight of >10000 Da in the casein hydrolysate is ≤30%;

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

[0015] The casein hydrolysate as described above, wherein the casein hydrolysate is obtained by enzymatically hydrolyzing the 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 casein raw material is enzymatically treated with trypsin and neutral protease to obtain casein hydrolysate.

[0018] The preparation method as described above, wherein the enzyme activity of trypsin is 150,000-250,000 U / g, and the added amount is 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 added amount is 0.10%-0.20% of the mass of the casein raw material;

[0020] and / or, 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;

[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 an application of the above-mentioned casein hydrolysate or the casein hydrolysate prepared by the above-mentioned preparation method in inhibiting Aβ protein deposition.

[0023] The present invention provides an application of the casein hydrolysate or the casein hydrolysate prepared by the preparation method in improving cholinergic activity.

[0024] The present invention provides an application of the above-mentioned casein hydrolysate or the casein hydrolysate prepared by the above-mentioned preparation method in repairing damaged neurons in the hippocampus and hypothalamus.

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

[0026] The present invention provides a milk casein hydrolysate, 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 hydrolysate of the present invention has good neuron protection and repair effects, can improve the degree of damage to cholinergic neurons and improve dopaminergic neuron damage; can effectively inhibit Aβ protein deposition, thereby alleviating neuronal synaptic disorders, neuronal apoptosis and brain damage caused by Aβ protein deposition; can improve the problem of cholinergic deficiency by inhibiting acetylcholinesterase activity, improve 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 paralysis lifespan curve of CL4176 nematodes treated with casein hydrolysate in Example 3;

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

[0029] Figure 3 This is a graph showing the head swing frequency of CL4176 nematodes treated with casein hydrolysate 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 casein hydrolysate in Example 6;

[0032] Figure 6 This is a schematic diagram of the chemotaxis experiment in Example 7;

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

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

[0035] Fig. 9 This is a diagram showing the swelling results of cholinergic neurons in LX929 nematodes treated with casein hydrolysate in Example 8;

[0036] Fig.10 The normal and damaged micrographs of dopamine neurons of BZ555 nematode in Example 9;

[0037] Fig.11 This is a diagram showing the damage results of dopamine neurons of BZ555 nematodes under the treatment of casein hydrolysate in Example 9;

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

[0039] Fig.13 This is a staining image of rat hypothalamic tissue sections treated with 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 implementation methods 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 implementation methods obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0041] The first aspect of the present invention provides a casein hydrolysate, wherein the casein hydrolysate 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 milk casein hydrolysate provided by the present invention is a product prepared by hydrolyzing milk casein. Milk casein is a phosphorus-calcium binding protein and is the main protein in mammals including cows, sheep or human milk. It has high safety and good nutritional value and functional properties. Through the hydrolysis process, milk casein is broken down into smaller peptide chains and amino acids, which are more conducive to digestion and absorption by the body.

[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 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 composed 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), 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 composed of asparagine (N), leucine (L), isoleucine (I), arginine (R), phenylalanine (F), phenylalanine (F) from the N-terminus to the C-terminus; the amino acid sequence of peptide segment 3 is GAWYYVPL (SEQ ID NO: 3). 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 neuron 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 can be promoted to show better efficacy in 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 scheme of the present invention, the mass content of peptides with a molecular weight of >10000 Da in the casein hydrolysate is ≤30%; the mass content of peptides with a molecular weight of <1000 Da in the casein hydrolysate is ≥20%. By regulating the molecular weight distribution of the casein hydrolysate to meet the above range, it helps the casein hydrolysate 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 satisfies any one of the conditions that the mass content of peptides with a molecular weight of >10000 Da is ≤30% or the mass content of peptides with a molecular weight of <1000 Da is ≥20%, the digestibility and absorption of the casein hydrolysate can be promoted; when the molecular weight distribution in the casein hydrolysate satisfies both the mass content of peptides with a molecular weight of >10000 Da and the mass content of peptides with a molecular weight of <1000 Da is ≥20%, the digestibility and absorption 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 with trypsin and neutral protease.

[0053] Among them, trypsin is a serine protease extracted from the pancreas of cattle, sheep and pigs. It is also an endopeptidase that can break the peptide bonds formed by the carboxyl groups of lysine or arginine. It has strong specificity and 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 can catalyze the breaking of peptide bonds in protein molecules and decompose large molecular 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, the functional peptide segments can be further enriched to improve the efficacy of casein hydrolysate in neuronal protection and repair.

[0055] In order to make the composition of the casein hydrolysate include 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 casein raw material is enzymatically treated with trypsin and neutral protease to obtain casein hydrolysate.

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

[0058] In the technical scheme of the present invention, in order to avoid excessive enzymatic reaction that affects the components and functions of the casein hydrolysate, the enzymatic solution after enzymatic hydrolysis can be subjected to enzyme inactivation treatment. The present invention does not limit the specific method of enzyme inactivation, and conventional enzyme inactivation means in the art can be used for enzyme inactivation, such as heating to 80°C for enzyme inactivation for 30 min. After 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 treatment 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 art. For example, the alcohol precipitation operation can be achieved by adding an alcohol solvent to the enzymatic hydrolyzate after the enzyme inactivation treatment and standing at 60°C-70°C for 5-8 hours. After the addition of the alcohol solvent, the functional peptide segment is 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 the filtration treatment, reduced pressure concentration can be performed at 65°C to further enrich the functional peptide segment; then, the spray drying can set the inlet temperature to 180°C and the outlet temperature to 90°C, and finally obtain the target casein hydrolyzate.

[0061] Furthermore, in order to promote a good matching effect between the casein raw material and the enzyme preparation, promote the enzymatic hydrolysis reaction to proceed better, and thus increase the mass content of the functional peptide segment in the casein hydrolyzate, 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 range, the enzymatic hydrolysis reaction can be promoted; when the enzyme activity and addition amount of trypsin and neutral protease meet the above range at the same time, the enzymatic hydrolysis reaction can be better promoted.

[0064] In the technical solution of the present invention, in the enzymatic hydrolysis treatment, the enzymatic hydrolysis temperature is 50° C.-55° C., the enzymatic hydrolysis pH value is 7.5-8.0, and the enzymatic hydrolysis time is 2.0-3.0 h, which can better exert the enzymatic hydrolysis effect 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, the pH value is 7.5-8.0, to obtain an enzymatic hydrolysis stock solution that is conducive to enzymatic hydrolysis by the enzyme preparation. Subsequently, at the appropriate enzymatic hydrolysis temperature of the enzyme preparation, that is, 50°C-55°C, pre-mixed trypsin and neutral protease are added to the enzymatic hydrolysis stock solution, and enzymatic hydrolysis is performed 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 the casein powder and water according to the above mass ratio helps to evenly disperse the casein and facilitate the enzymatic hydrolysis reaction. If too much water is added, it is easy to cause the reaction volume to be too large during the enzymatic hydrolysis treatment, thereby increasing the load of the subsequent enrichment process; if too little water is added, it is easy to cause the casein solution to have poor fluidity, which is not conducive to the occurrence of the enzymatic hydrolysis reaction, thereby reducing the enzymatic hydrolysis efficiency.

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

[0069] When the nervous system of the body is underdeveloped or not developed at all, excessive deposition of Aβ protein may occur, leading to synaptic disorders, neuronal apoptosis and brain damage. Experimental verification shows that the casein hydrolysate of the present invention can effectively inhibit the abnormal aggregation of Aβ, thereby alleviating paralysis, decreased motor ability and abnormal behavior caused by Aβ protein deposition.

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

[0071] The changes of cholinergic deficiency on brain neurons and signal transmission include impaired acetylcholine (ACh) release, axonal transport defects, increased acetylcholinesterase (AChE) levels, etc. Among them, the abnormal accumulation of AChE can accelerate the hydrolysis of neurotransmitter ACh, ultimately affecting the signal conduction between neurons, and further leading to learning and memory disorders. The casein hydrolyzate of the present invention can improve cholinergic activity by significantly reducing the activity of AChE in the body.

[0072] A fifth aspect of the present invention provides a use of the above-mentioned casein hydrolysate or the casein hydrolysate 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, is in the shape of a seahorse, and is an important component of the brain. Its main functions include controlling emotions, improving memory, regulating endocrine and sleep, etc. The casein hydrolyzate of the present invention can improve the disordered arrangement of hippocampal pyramidal cells, promote the cells to be arranged more neatly, and has a repairing effect on hippocampal neuron damage.

[0074] The hypothalamus is a high-level center of the autonomic nerves under the cerebral cortex and 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. The casein hydrolyzate of the present invention can improve hypothalamic nerve cells, promote the cells to be arranged more neatly, and has a repairing effect on hypothalamic neuron damage.

[0075] A sixth 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 the preparation of related products with neuron protection and repair effects.

[0076] The casein hydrolyzate of the present invention has good neuron protection and repair effects, can improve the degree of damage to cholinergic neurons and improve dopaminergic neuron damage; can effectively inhibit Aβ protein deposition, thereby alleviating neuronal synaptic disorders, neuronal apoptosis and brain damage caused by Aβ protein deposition; can improve the problem of cholinergic deficiency by inhibiting acetylcholinesterase activity, improve 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.

[0077] In the present invention, related products with neuron protection and repair effects include but are not limited to food, health products, and medicines. A large amount of research data proves that the casein hydrolyzate of the present invention has significant neuron protection and repair capabilities, and can be used to prepare conventional foods, such as milk powder; it can also be used to prepare conventional health products, such as neuron protection health products, neuron repair health products, neuron protection and repair health products; it can also be used to prepare neuron protection or repair medicines, etc., thereby broadening the application scope of casein hydrolyzate and providing new raw materials for related products of neuron protection or repair.

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

[0079] In the following examples, Caenorhabditis elegans and laboratory rats were used for animal experiments, and the animal experiments involved in the experiments 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, C. elegans is a classic and widely used model organism. The application of C. elegans in scientific research has many inherent advantages, including: small size, adult body length is about 1 mm, and the body is transparent, so its entire development cycle can be observed under an ordinary optical microscope or multiple behavioral and developmental indicators can be observed, and the sample does not need to be decolorized during fluorescence imaging; it mainly feeds on bacteria, and under laboratory conditions, only Escherichia coli OP50 (Escherichia coli OP50) needs to be inoculated on the nematode growth medium (NGM) culture medium to maintain C. elegans; it is easy to preserve. The nervous system of C. elegans is its most complex body tissue, and the lineage and morphology of each of its neurons have been described in detail. At the same time, its brain function has been completely cracked for the first time in the neural network circuit. All connections between neurons in nematodes and connections from neurons to nematode muscles and other tissues (such as intestines and skin) have complete connection maps. Almost all neurons of C. elegans have neuronal markers in the form of fluorescent reporter genes. Using high-resolution imaging technology, researchers can accurately track the effects of gene mutations on the developmental phenotypes of neuronal cells in C. elegans, which makes the nervous system of C. elegans the main model system for clarifying the nature of gene regulation in the nervous system. Therefore, C. elegans has unique advantages in studying behavior and neural development. Adult hermaphroditic C. elegans has 302 neurons, which belong to two independent nervous systems: the large somatic nervous system (282 neurons) and the small pharyngeal nervous system (20 neurons). The two systems have different topological structures and communicate information through a pair of RIP rhythm-related interneurons. In the somatic nervous system, neurons and their processes are usually located between the subcutaneous and body wall muscles and share the basal layer with the subcutaneous. Pharyngeal neurons are directly located between the pharyngeal muscles. These neurons are connected to other parts of the body through nerve rings to control the muscle movement of the pharynx, thereby achieving food intake and transportation.

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

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

[0083] CL4176 nematodes is a temperature-inducible system smg-1, human Aβ 1-42 and rol-6 mRNA co-transfection. Generally, CL4176 nematodes move in circles, but under certain conditions, CL4176 nematodes will produce a large amount of Aβ pathogenic protein. When Aβ protein accumulates to a certain stage, CL4176 nematodes will lose their original ability to move, that is, the body cannot swing. CL4176 nematodes are different from wild-type nematodes N2. They are sensitive to environmental temperature and are usually cultivated at 16°C to slow down the growth rate to reduce the accumulation of Aβ protein. Therefore, by appropriately inducing temperature rise (25°C), the development and growth of CL4176 nematodes can be accelerated, and Aβ protein can be rapidly accumulated to cause the appearance of a "paralysis" phenotype, so as to complete the determination of the paralysis lifespan curve.

[0084] CL2355 nematode is a temperature-sensitive nematode. After high temperature induction (25°C), Aβ is expressed in large quantities in neuronal cells. 1-42 Pathogenic protein. As Aβ gradually accumulates in neurons, Aβ-induced neurotoxicity can cause neuronal damage or even death, and the motor ability and chemical perception ability controlled by the relevant neurons are lost, leading to defects in sensory chemotaxis and associative learning abilities. Therefore, CL2355 nematodes can simulate the learning and memory disorders mediated by Aβ neuronal toxicity 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 generation process.

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

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

[0088] The rats used in the examples belong to rodent mammals and are similar to humans in terms of the mechanism of action of the nervous system. Therefore, rats are excellent model organisms for studying neuroprotection and neurorepair of active substances. After modeling neuronal damage in rats, the repair of neurons in the hippocampus and hypothalamus by casein hydrolysate can be studied. Specifically, specific pathogen-free (SPF) male Sprague-Dawley (SD) rats (200-250 g) purchased from Beijing VitalRiver Laboratory Animal Technology Co., Ltd. were used for the experiment.

[0089] In addition, the preparation method of NGM plates is as follows: add 6.8 g technical agar powder, 1.2 g sodium chloride, 1.0 g tryptone, and 0.08 g streptomycin sulfate to every 390 mL of grade 3 water, stir well and sterilize at 121°C for 20 min. After sterilization, add 10 mL 1 mol / L potassium phosphate buffer solution, 400 μL 1 mol / L magnesium sulfate solution, 400 μL 1 mol / L calcium chloride solution, and 400 μL 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 it on a plate in LB medium, culture it at 37℃ for 48 hours, then take it out, pick out a single OP50 colony and put it into sterilized LB liquid broth medium, place it in a shaker at 37℃ and 170 rpm / min and culture it for 12 hours, and obtain OP50 bacterial liquid when OD600 is 0.4.

[0091] For other experimental methods without specific conditions, conventional conditions or conditions recommended by the manufacturer are usually used. The reagents used are commercially available or publicly available unless otherwise specified.

[0092] Example 1: Preparation of casein hydrolysate

[0093] (1) Pure water is added to the milk casein powder to obtain a milk casein solution, wherein the mass ratio of the milk casein powder to the pure water is 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) are first mixed in 10 mL of pure water, and then added to the milk casein solution for enzymatic hydrolysis to obtain an enzymatic hydrolyzate. In the enzymatic hydrolysis treatment, the enzymatic hydrolysis temperature is 50°C, the enzymatic hydrolysis pH value is 7.5, and the enzymatic hydrolysis time is 2.0 h. After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is subjected to an enzyme inactivation treatment at 80°C for 30 min. After the enzyme inactivation treatment, the enzymatic hydrolyzate is cooled to 30°C-40°C, and then alcohol precipitation, filtration, concentration, and spray drying are performed in sequence to obtain a 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 subjected to an enzyme inactivation treatment at 80°C for 30 min. After the enzyme inactivation treatment, the enzymatic hydrolyzate was cooled to 30°C-40°C, and then alcohol precipitation, filtration, concentration, and spray drying were performed in sequence 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 subjected to an enzyme inactivation treatment at 80°C for 30 min. After the enzyme inactivation treatment, the enzymatic hydrolyzate was cooled to 30°C-40°C, and then 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 subjected to an enzyme inactivation treatment at 80°C for 30 min. After the enzyme inactivation treatment, the enzymatic hydrolyzate was cooled to 30°C-40°C, and then alcohol precipitation, filtration, concentration, and spray drying were performed in sequence to obtain a casein hydrolyzate powder 4.

[0097] Example 2: Identification of casein hydrolysate

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

[0099] The hydrolysis degree of casein hydrolyzate powder 1-4 was determined by OPA reaction. Since each hydrolysis of a peptide bond 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 with a spectrophotometer. The calculation formula of the hydrolysis degree 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, and the results can be seen in Table 1.

[0105]

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

[0107] The casein hydrolysate powders 1-4 were dissolved in ddH2O to obtain casein hydrolysate solutions 1-4, which were then transferred to a 10 kD ultrafiltration tube and centrifuged at 4°C and 12000 rcf for 10 min to collect polypeptide samples less than 10 kD. Dithiothreitol solution was added to the polypeptide samples to make a final concentration of 10 mmol / L, and the samples were reduced in a 56°C water bath for 1 h. Then, iodoacetic acid solution was added to make a final concentration of 50 mmol / L. After reacting in the dark 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 subjected to LC-MS / MS detection, where the 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 for each component: 66 min. Mass spectrometry conditions were as follows: primary mass spectrometry parameters: Resolution: 70000, AGCtarget: 3e6, MaximumIT: 100 ms, Scanrange: 300 to 1800 m / z; secondary mass spectrometry parameters: Resolution: 75000, AGCtarget: 1e5, MaximumIT: 50 ms, TopN: 20, NCE / steppedNCE: 28.

[0109] The mass spectrum raw files obtained after LC-MS / MS detection were used to search the Uniprot protein database using Maxquant (1.6.2.10). The peptide sequence information of casein hydrolysate was detected by LC-MS / MS and comprehensively analyzed 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 casein hydrolysate on paralysis lifespan of CL4176 nematodes

[0112] The casein hydrolysate powder 2 obtained in Example 1 was dissolved in sterile water to prepare a 20 mg / mL stock solution, which was sealed with a sealing film and stored at 4°C in the dark. Before use, the stock solution was diluted with OP50 bacterial solution to a concentration of 0 mg / mL, 0.5 mg / mL, 1.5 mg / mL and 2.5 mg / mL of the casein hydrolysate powder 2 to obtain a working solution, and 200 μl of the working solution was evenly spread on an NGM plate, dried and stored at 4°C for later use. In the subsequent experiments of this embodiment, CL4176 nematodes were cultured using NGM plates coated with working solutions containing different concentrations of casein hydrolysate, wherein NGM plates coated with working solutions containing 0 mg / mL casein hydrolysate were named OP50 group; NGM plates coated with working solutions containing 0.5 mg / mL casein hydrolysate were named 0.5 mg / mL group; NGM plates coated with working solutions containing 1.5 mg / mL casein hydrolysate were named 1.5 mg / mL group; NGM plates coated with working solutions containing 2.5 mg / mL casein hydrolysate were named 2.5 mg / mL group. Each group had 60 CL4176 nematodes, and 3 replicates were performed, for a total of 720 CL4176 nematodes.

[0113] The eggs of CL4176 nematodes were placed in 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 period (the lower abdomen had a crescent shape), they were transferred to 25°C for continued culture, and the high temperature was used to induce the "paralysis" phenotype of CL4176 nematodes. At this time, the CL4176 nematodes only had a slight swaying 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 life span curve was drawn based on the number of paralyzed nematodes every 2 hours. The horizontal axis was time, and the vertical axis was the survival fraction (Fraction survival). For details, see 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 according to 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 ) The number of paralyzed nematodes within 1 h was 1. In addition, the median survival time refers to the time when the survival rate of CL4176 nematodes is 50%, and the maximum survival time refers to the time when the survival rate of CL4176 nematodes is 0%. The specific results can be seen in Table 2, and different letters in the table represent significant differences.

[0117]

[0118] When the nervous system of Caenorhabditis elegans is underdeveloped or not developed at all, excessive deposition of Aβ protein will lead to synaptic disorders, neuronal apoptosis and brain damage. When CL4176 nematodes are induced by high temperature (25°C), Aβ protein deposition will occur. The accumulation of Aβ protein eventually leads to "paralysis". Therefore, the effective concentration of casein hydrolysate can be determined by analyzing the paralysis lifespan of CL4176 nematodes.

[0119] Depend on Figure 1 It can be seen that compared with the OP50 group, when the concentration of casein hydrolysate was 0.5 mg / mL-2.5 mg / mL, the paralysis curve shifted significantly to the right, and the 0.5 mg / mL group curve shifted to the right most significantly. As shown in Table 2, compared with the OP50 group, casein hydrolysate can increase the average survival time, median survival time and maximum survival time of CL4176 nematodes, and the concentration of 0.5 mg / mL casein hydrolysate can maximize the average survival time, median survival time and maximum survival time of nematodes. Therefore, when the concentration of casein hydrolysate is 0.5 mg / mL-2.5 mg / mL, it can prolong the time for CL4176 nematodes to enter the paralysis phenotype, effectively reduce the impact of paralysis caused by Aβ protein deposition, and 0.5 mg / mL casein hydrolysate has the best effect.

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

[0121] The changes of cholinergic deficiency on brain neurons and signal transmission include impaired acetylcholine (ACh) release, axonal transport defects, and increased acetylcholinesterase (AChE) levels. Among them, the abnormal accumulation of AChE can accelerate the hydrolysis of the neurotransmitter ACh, ultimately affecting the signal conduction between neurons, and further leading to learning and memory disorders. Therefore, this example determines the cholinergic activity of CL4176 nematodes by detecting the activity of AChE, and then determines the effect of casein hydrolyzate on the cholinergic activity of CL4176 nematodes. Since Example 3 has proved that the average survival time, median survival time and maximum survival time of CL4176 nematodes can be extended when the concentration of casein hydrolyzate is 0.5 mg / mL-2.5 mg / mL, the experimental concentration of casein hydrolyzate in this example is also selected in the range of 0.5 mg / mL-2.5 mg / mL.

[0122] The NGM plates of OP50 group, 0.5 mg / mL group, 1.5 mg / mL group and 2.5 mg / mL group were obtained by referring to the method of Example 3. The eggs of CL4176 nematodes were placed in the NGM plates of OP50 group, 0.5 mg / mL group, 1.5 mg / mL group and 2.5 mg / mL group, respectively, and cultured in a 16°C incubator for 48 h, that is, before the CL4176 nematodes entered the L4 period (crescent-shaped lower abdomen), they were transferred to 25°C and continued to be cultured for 48 h. At this time, the nematodes were rinsed from the culture dish into a centrifuge tube with M9 buffer prepared by 3 g potassium dihydrogen phosphate, 6 g disodium hydrogen phosphate, 5 g sodium chloride, 1 mL 1moL / L magnesium sulfate, and 1 L tertiary water, and the nematodes were homogenized by a fully automatic tissue grinder, the nematodes were ground and crushed, and then the supernatant was collected and stored at 4°C after low-temperature centrifugation (4°C, 12000 rpm / min, 10min). The protein content of nematode tissue protein in each group of supernatant was standardized using a protein quantification (TP) kit to obtain the mass of nematode tissue protein in each group of supernatant. The supernatant was then tested for AchE activity using the acetylcholinesterase test kit with the catalog number A024-1-1 produced by Nanjing Jiancheng Bioengineering Institute. The supernatant was mixed with the substrate buffer and color development application solution provided by the kit, reacted at 37°C for 6 minutes, and then the inhibitor and transparent agent provided by the kit were added and mixed, and the mixture was allowed to stand at room temperature for 15 minutes. The absorbance OD value was detected to obtain the experimental group data. In order 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 transparent 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 transparent 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 transparent 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. The above kit is designed based on the principle that acetylcholinesterase hydrolyzes acetylcholine to generate choline and acetic acid, and choline can react with a thiol colorimetric agent to generate a yellow compound of symmetric trinitrobenzene (TNB). The absorbance of the yellow compound can be measured at a wavelength of 412 nm using a spectrophotometer to reflect the amount of choline generated and thus the activity of acetylcholinesterase. Each group of experiments was repeated three times, and at least 600 nematodes were required each time.

[0123] Specifically, the activity of acetylcholinesterase can be determined based on the activity unit. 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] Among them, 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 AChE standard in the test box, which is 1 μmol / mL; C is the concentration of nematode tissue protein, the unit is mgprot / mL.

[0127] The effects of different concentrations of casein hydrolysate on the acetylcholinesterase activity of CL4176 nematodes can be seen in 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 hydrolysate 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 casein hydrolysate on the motility of CL4176 nematodes

[0130] The accumulation of Aβ protein will cause the CL4176 nematode to enter a paralysis process. During this process, the mobility of the CL4176 nematode will gradually slow down as the toxicity of the pathogenic protein increases. In this example, the head thrash frequency of the CL4176 nematode was detected to determine the effect of 0.5 mg / mL of casein hydrolyzate on the mobility of the CL4176 nematode.

[0131] The 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 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 head swinging 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 again. The number of head swings of the CL4176 nematode within 30 s was 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 at the 10th and 15th hours of the initial observation was significantly increased (p<0.05), indicating that 0.5 mg / mL milk casein hydrolysate can improve the symptoms of acute paralysis of nematodes and enhance movement ability.

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

[0134] The accumulation of Aβ protein in large amounts can cause the CL4176 nematode to enter a paralysis process. In this example, the deposition of Aβ protein in the CL4176 nematode was detected to determine the effect of 0.5 mg / mL of milk casein hydrolyzate on the paralysis process.

[0135] The 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 working solution of 0 mg / mL and 0.5 mg / mL casein hydrolyzate, and cultured in a 16°C incubator for 48 h, that is, before the CL4176 nematodes entered the L4 period (the lower abdomen had a crescent shape), they were transferred to 25°C and continued to be cultured for 48 h. At this time, the nematodes were rinsed from the culture dish into a centrifuge tube with a buffer solution, the washed nematodes were centrifuged at 2500 rpm for 2 min in a microcentrifuge tube, 1 ml of a fixative (4% (m / m) paraformaldehyde) was added and fixed for 15 min, and then the centrifuge tube containing the nematodes was placed in a -80°C refrigerator for quick freezing for 30 min, and then taken out and thawed in running tap water until the nematodes were about to thaw, that is, when a little water just appeared in the centrifuge tube, and then placed in a -80°C refrigerator for quick freezing, and repeated freezing and thawing three times in total. After the last time it was taken out of the -80℃ freezer, it was slowly thawed on ice, and after thawing, the nematodes were centrifuged at 4000 rpm for 2 min to settle, 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 for staining at room temperature for 10 min. After staining, it was washed 2-3 times with M9 buffer until the suspension was transparent and clear, and 0.5 mL of supernatant was retained for the last time. The nematodes stained with Thioflavin T were observed using GFP fluorescence under a fluorescence microscope, and the Aβ deposition was quantified using ImageJ software, which can be seen in detail. 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 diagram representing the deposition of Aβ protein in CL4176 nematodes, where CK represents the OP50 group, 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, compared with the OP50 group, the accumulation of Aβ was significantly decreased after treatment with 0.5 mg / mL casein hydrolysate (p<0.05), indicating that the effect of 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] The 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 CL2355 nematodes were placed on the NGM plates coated with the working solution of 0 mg / mL and 0.5 mg / mL milk casein hydrolysate, cultured in a 16°C incubator for 48 h, and then transferred to 25°C for further culture for 48 h. After the culture is completed, rinse and collect the nematodes with M9 buffer, then place 80-100 of the above nematodes in the center of a culture dish with a diameter of 120 mm, divide the culture dish into four quadrants, named as area A, area B, area C, and area D, drop 10 μL of odor attractant (5 μL 0.1% (m / v) benzaldehyde and 5 μL 1% (m / m) sodium azide) on the edge of area A and area D in the culture dish, and add 10 μL of solvent blank control (5 μL 100% (m / v) sterile water + 5 μL 1% (m / m) sodium azide) on the edge of area B and area C. The specific process can be seen in Figure 6 After 30 min, the number of nematodes moving 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, and F is the total number of nematodes. The specific results can be seen in Figure 7 , Figure 7 CK in the middle represents the OP50 group, GMCH represents the 0.5 mg / mL group, This indicates that there is a significant difference between the 0.5 mg / mL group and the blank control group.

[0141] The transgenic nematode CL2355 expresses Aβ protein in neurons. The aggregation toxicity of Aβ protein can cause neuronal damage or even death and impair the corresponding cell functions, especially the loss of movement and chemical perception ability regulated by neurons, leading to behavioral abnormalities. Figure 7 As shown, the CI value of CL2355 nematodes treated with casein hydrolysate was significantly greater than that of the control group (p<0.05), indicating that casein hydrolysate can improve the chemotaxis dysfunction of CL2355 and alleviate the neurotoxicity caused by Aβ.

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

[0143] The 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 casein hydrolysate, cultured in an incubator at 20°C 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, and photographed at 20× magnification using an upright fluorescence microscope. 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 Fig. 9 .

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

[0145] Cholinergic neurons are neurons that synthesize and release the neurotransmitter acetylcholine. Under normal circumstances, cholinergic neurons are relatively smooth and have no particularly obvious swelling or protrusions; however, Figure 8 As shown in Figure 2, cholinergic neurons will swell significantly when they are exposed to external stress. Fig. 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 casein hydrolysate on dopamine neurons in BZ555 nematodes

[0147] The 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 an incubator at 20°C 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, and photographed with 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. Fig.10 and Fig.11 .

[0148] Fig.10 This is a micrograph of the dopamine neurons of the nematode BZ555 when they are normal (Normal) and when the dendrites are swollen and abnormal (Dendritic bubbling and abnormality). Fig.11 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 and then continuously subjected to 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 and then continuously subjected to 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 and then continuously subjected to heat stress at 35°C for 3 h). There is a significant difference between the heat stress group and the blank control group. This indicates that there is a significant difference between the 0.5 mg / mL group and the heat stress group.

[0149] Dopamine is one of the most abundant monoamine transmitters 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. Fig.10 and Fig.11 As shown in the data, the damage and repair of the dendrites of dopaminergic neurons in the head area 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 casein hydrolysate on rat hippocampal neurons

[0151] The para-chlorophenylalanine (PCPA) insomnia rat model is currently recognized and widely used as a classic experimental animal model for the study of 5-hydroxytryptamine (5-HT) and its relationship with other neurotransmitters. As a 5-HT synthesis inhibitor, PCPA selectively acts on tryptophan hydroxylase and inhibits the activity of the enzyme. Sixty male rats were evenly divided into six groups. Among them, the rats were intraperitoneally injected with PCPA (300 mg / kg) at 9:00 am for two consecutive days to establish an insomnia rat model as the Model group, and the same operation was performed but the same volume of weak alkaline 0.9% (m / m) sodium chloride injection was intraperitoneally injected as PCPA as the normal control group. When the rats' circadian rhythm disappeared and began to become irritable, they were also active during the day, and their aggressiveness increased, which was significantly different from the normal control group, it indicated that the insomnia rat model had been successfully established. After the model was successfully established, drug intervention was started. The experimental rats were gavaged with drugs at 8:00 a.m. 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) had a concentration of 100 mg / kg, the medium-dose casein hydrolysate administration group (GMCH-M) had a concentration of 400 mg / kg, the high-dose casein hydrolysate administration group (GMCH-H) had a concentration of 700 mg / kg, and the diazepam administration group had a concentration of 3 mg / kg; among them, the casein hydrolysate was casein hydrolysate powder 2.

[0152] Hematoxylin and eosin (H&E) staining was used to evaluate hippocampal damage in insomnia rats: After drug administration, the experimental rats were dissected, and the hippocampus was removed and fixed in 4% (m / m) paraformaldehyde solution, dehydrated with a series of gradient ethanol solutions, and embedded in paraffin. The dissected tissue was cut into 5 μm sections, then stained with H&E, and finally an optical microscope (CX-41, Olympus) was used to observe the histopathological changes. Fig.12 , Fig.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. Fig.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 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 shows that casein hydrolysate has a repair effect on hippocampal neuron damage.

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

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

[0156] Hematoxylin and eosin (H&E) staining was used to evaluate hypothalamic damage in insomnia rats: After the administration, the experimental rats were dissected, and the hypothalamus was removed and fixed in 4% (m / m) paraformaldehyde solution, dehydrated with a series of gradient ethanol solutions, and embedded in paraffin. The dissected tissue was cut into 5 μm sections, then stained with H&E, and finally the histopathological changes were observed using an optical microscope (CX-41, Olympus). Fig.13 , Fig.13 There are 6 groups in total, and the magnification is 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. Fig.13As shown, the hypothalamic neurons of rats in the normal control group had clear morphology, uniform distribution and complete structure; vacuoles were observed locally in the hypothalamic neurons of the insomnia rat model group (indicated by arrows), and the cells were loosely arranged and reduced in number; after one week of treatment, most of the hypothalamic neurons 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 neurons in the positive control diazepam group were also restored and arranged more neatly; this indicates that casein hydrolysate has a repairing 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 the technical solutions described in the above embodiments can still be modified, or some or all of the technical features can be replaced by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A casein hydrolysate, characterized in that: The composition of the casein hydrolysate includes at least one of 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.

2. The casein hydrolyzate according to claim 1, characterized in that 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 of >10000 Da in the casein hydrolysate is ≤30%; And / or, the mass content of peptides with a molecular weight of less than 1000 Da in the casein hydrolysate is ≥20%.

4. The casein hydrolyzate according to claim 1 or 2, characterized in that The casein hydrolysate is obtained by enzymatically hydrolyzing the casein raw material with trypsin and neutral protease.

5. The method for preparing the casein hydrolyzate according to any one of claims 1 to 4, characterized in that: The steps include: The casein raw material is enzymatically treated with trypsin and neutral protease to obtain the casein hydrolyzate.

6. The preparation method according to claim 5, characterized in that: 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; And / or, the enzyme activity of the neutral protease is 150,000-250,000 U / g, and the added amount is 0.10%-0.20% of the mass of the casein raw material; And / or, 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; And / or, the casein raw material is obtained by preparing casein powder and water in a mass ratio of 1:(8-12).

7. Use of the casein hydrolysate according to any one of claims 1 to 4 or the casein hydrolysate prepared by the preparation method according to any one of claims 5 to 6 in inhibiting Aβ protein deposition.

8. Use of the casein hydrolysate according to any one of claims 1 to 4 or the casein hydrolysate prepared by the preparation method according to any one of claims 5 to 6 in improving cholinergic activity.

9. Use of the casein hydrolyzate according to any one of claims 1 to 4 or the casein hydrolyzate prepared by the preparation method according to any one of claims 5 to 6 in repairing damaged neurons in the hippocampus and hypothalamus.

10. Use of the casein hydrolyzate according to any one of claims 1 to 4 or the casein hydrolyzate prepared by the preparation method according to any one of claims 5 to 6 in preparing related products having neuron protection and repair effects.

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