A polypeptide having neuroprotective effects and hydrolyzed lacto-casein

Abstract

The present application provides a kind of polypeptide and hydrolyzed milk casein with neuroprotective effect.The polypeptide AWPQ of the present application can be absorbed by the intestinal tract after being eaten by the body, and penetrate the blood-brain barrier into the central nervous system, and then promote the growth and differentiation of nerve cells, and finally play its neuroprotective effect, and has good development prospects in the field of brain health, neuroprotection and nerve development related functional foods or bioactive peptides.

Description

Technical Field

[0001] This invention relates to biotechnology, and more particularly to a polypeptide and hydrolyzed casein with neuroprotective effects. Background Technology

[0002] Maintaining the health of the nervous system is a crucial component of daily healthcare, as its health directly impacts cognitive function, behavioral abilities, and lifelong health literacy, making it a core focus. In daily health maintenance, finding natural and safe nutritional supplements to provide precise nutritional support for the nervous system, reduce interference from adverse external factors, and help maintain its optimal state has become a core need for the public.

[0003] Currently, while there is a wide variety of nutritional supplements available, there is still a significant shortage of products specifically designed for the health and well-being of the nervous system. Existing products primarily focus on single nutrients (such as DHA and B vitamins), lacking highly targeted, bioactive, and easily absorbed natural active ingredients. Furthermore, some products suffer from complex compositions, low absorption efficiency, and insufficient adaptation to human physiological characteristics. These limitations make it difficult to fully meet the precise needs for specific active nutrients in the process of maintaining nervous system health, and thus fail to provide efficient nutritional support and scientific care for the nervous system. This also hinders the current trend of refined and natural health practices among the general public. Therefore, developing natural, safe, efficient products that are tailored to human physiological characteristics and can specifically provide nutritional support for nervous system health and development has become an urgent need and a hot research topic. Summary of the Invention

[0004] The purpose of this invention is to develop a specific polypeptide with clearly defined neuroprotective and neurodevelopment-supporting effects, optimize the casein enzymatic hydrolysis process to obtain hydrolyzed casein protein rich in this active polypeptide, with stable performance and adapted to the body's digestive and absorption characteristics, and apply it to the preparation of neuroprotective related products. This invention aims to solve the technical problems of insufficient targeting, unclear activity, and poor compatibility of existing neuroprotective nutrients, and has important theoretical significance and practical application value.

[0005] This invention provides a polypeptide with neuroprotective effects, wherein the polypeptide has the following amino acid sequence: Ala-Trp-Pro-Gln.

[0006] The present invention provides a hydrolyzed casein, wherein the above-mentioned polypeptide is included.

[0007] The hydrolyzed casein as described above, wherein the degree of hydrolysis of the hydrolyzed casein is 9.5%-12.5%; and / or,

[0008] In hydrolyzed casein, the mass content of peptides with a molecular weight ≤3000 Da is ≥90%, and the mass content of peptides with a molecular weight <1000 Da is ≥60%.

[0009] This invention provides a method for preparing the above-mentioned hydrolyzed casein, comprising the following steps:

[0010] The casein raw material was subjected to a first enzymatic hydrolysis using a first enzyme preparation to obtain a first enzymatic hydrolysis product;

[0011] The first enzymatic hydrolysis product is subjected to a second enzymatic hydrolysis using a second enzyme preparation to obtain hydrolyzed casein.

[0012] The first enzyme preparation includes at least one of serine protease, neutral protease, and glutaminase; the second enzyme preparation includes at least one of alkaline protease, flavor protease, and deaminase.

[0013] In the preparation method described above, the amount of the first enzyme preparation added, based on casein raw material, is 0.1-0.8 wt%; and / or,

[0014] Based on casein raw materials, the amount of the second enzyme preparation added is 0.1-0.8 wt%.

[0015] The preparation method described above, wherein the conditions for the first enzymatic hydrolysis treatment include: a temperature of 45-55°C, a time of 1.0-5.0 h, and a pH value of 7.0-7.5; and / or,

[0016] The conditions for the second enzymatic hydrolysis treatment include: a temperature of 45-55℃, a time of 1.5-5.0 h, and a pH of 8.0-8.5.

[0017] The preparation method described above, wherein the preparation method of casein raw material includes the following steps:

[0018] The raw milk was sequentially subjected to defatting, pasteurization and ultrafiltration concentration to obtain casein.

[0019] Casein and water are mixed to obtain casein raw material.

[0020] This invention provides the application of the above-mentioned polypeptide, the above-mentioned hydrolyzed casein, or the hydrolyzed casein prepared by the above-mentioned method in the preparation of neuroprotective products.

[0021] As described above, neuroprotection includes: improving neurological health and / or promoting neurodevelopment.

[0022] In the applications described above, neuroprotection manifests itself in any of the following ways:

[0023] (a) Improves the expression of synaptophysin in the hippocampus;

[0024] (b) Improve the expression of neuronal nucleoproteins in the hippocampus

[0025] (c) Promotes nerve cell growth and / or survival;

[0026] (d) Promotes nerve cell differentiation.

[0027] This invention provides a polypeptide with neuroprotective effects, having the following amino acid sequence: Ala-Trp-Pro-Gln. Experiments have shown that this polypeptide resists hydrolysis by peptidases in the intestinal microenvironment and is successfully transported across the membrane by intestinal epithelial cells into the body, thus enabling effective absorption after ingestion. Furthermore, this polypeptide can penetrate the blood-brain barrier, giving it the potential to enter the central nervous system and exert its functions. Finally, this polypeptide effectively enhances the survival and differentiation capabilities of nerve cells, promoting their growth, development, and differentiation, thus protecting nerve health and promoting nerve development after ingestion. Attached Figure Description

[0028] Figure 1 This is a graph showing the degree of hydrolysis of casein in one embodiment of the present invention;

[0029] Figure 2 This is a fluorescence micrograph of SYN protein expression in one embodiment of the present invention;

[0030] Figure 3 This is a statistical graph of the fluorescence results of SYN protein expression in one embodiment of the present invention;

[0031] Figure 4 This is a fluorescence micrograph of NeuN protein expression in one embodiment of the present invention;

[0032] Figure 5 This is a statistical graph of the fluorescence results of NeuN protein expression in one embodiment of the present invention;

[0033] Figure 6 This is a schematic diagram of a Caco-2 cell monolayer model in one embodiment of the present invention;

[0034] Figure 7 This is a graph showing the transmembrane resistance results of a Caco-2 cell monolayer model in one embodiment of the present invention;

[0035] Figure 8 This is a total ion chromatogram of the sample to be tested in one embodiment of the present invention;

[0036] Figure 9 This is a total ion chromatogram of the upper chamber component in one embodiment of the present invention;

[0037] Figure 10 This is a total ion chromatogram of the lower chamber component in one embodiment of the present invention;

[0038] Figure 11 This is a Venn diagram of the sample to be tested, the upper chamber components, and the lower chamber components in one embodiment of the present invention; wherein, Figure 11 (a) shows a statistical graph of common and unique peptides between the sample to be tested and the components in the upper chamber. Figure 11 (b) shows a statistical diagram of common and unique peptides between the upper and lower chamber components. Figure 11 (c) shows a statistical graph of common and unique peptides between the sample to be tested and the components in the lower chamber. Figure 11 (d) shows a statistical graph of common and unique peptides among the sample to be tested, the upper chamber component, and the lower chamber component;

[0039] Figure 12 This is a molecular docking simulation diagram of peptide AWPQ and BDNF receptor in one embodiment of the present invention; wherein, Figure 12 (a) shows a schematic diagram and a magnified view of the molecular docking simulation of the peptide AWPQ with the BDNF receptor. Figure 12 (b) A schematic diagram illustrating the interactions involved in the molecular docking simulation of the peptide AWPQ with the BDNF receptor;

[0040] Figure 13 This is a molecular docking simulation diagram of peptide AWPQ and NGF receptor in one embodiment of the present invention; wherein, Figure 13 (a) shows a schematic diagram and a magnified view of the molecular docking simulation between the peptide AWPQ and the NGF receptor. Figure 13 (b) A schematic diagram illustrating the interactions involved in the molecular docking simulation of the peptide AWPQ and the NGF receptor;

[0041] Figure 14 This is a mass spectrometry identification result of peptide AWPQ in one embodiment of the present invention;

[0042] Figure 15 This is a schematic diagram of an in vitro blood-brain barrier model in one embodiment of the present invention;

[0043] Figure 16 This is a diagram showing the transmembrane resistance results of an in vitro blood-brain barrier model in one embodiment of the present invention;

[0044] Figure 17 This is a diagram showing the leakage experiment results of an in vitro blood-brain barrier model in one embodiment of the present invention; wherein, Figure 17 (a) shows the leakage situation at the beginning of the experiment. Figure 17 (b) A diagram showing the leakage situation 4 hours after the experiment;

[0045] Figure 18This is a liquid chromatogram of peptide AWPQ in an upper or lower chamber solution according to an embodiment of the present invention;

[0046] Figure 19 This is a graph showing the survival rate of PC-12 cells in one embodiment of the present invention;

[0047] Figure 20 This is a graph showing the PC-12 cell differentiation rate in one embodiment of the present invention;

[0048] Figure 21 This is a micrograph of PC-12 cell differentiation in one embodiment of the present invention; wherein, Figure 21 (a) indicates the microscopic field of view of group CON. Figure 21 (b) indicates the microscopic field of view of the NGF group. Figure 21 (c) represents the microscopic field of view of the NGF+AWPQ group. Figure 21 (c) The red arrows indicate differentiated PC-12 cells;

[0049] Figure 22 This is a graph showing the PC-12 cell differentiation rate in one embodiment of the present invention;

[0050] Figure 3 , Figure 5 , Figure 19 , Figure 20 , Figure 22 In this context, letters such as a, b, c, d, e, f, and ab are significance markers; the same letter indicates no statistical difference between groups; different letters indicate significant differences between groups (p < 0.05); mixed letters indicate no significant difference between the groups containing single letters and significant differences between the groups with other letters (p < 0.05). Detailed Implementation

[0051] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below. The specific embodiments listed below are merely descriptions of the principles and features of the present invention, and the examples are only for explaining 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] The first aspect of the present invention provides a polypeptide with neuroprotective effect, which is obtained by linking four amino acids from the N-terminus to the C-terminus through peptide bonds, and its amino acid sequence is alanine-tryptophan-proline-glutamine (Ala-Trp-Pro-Gln, AWPQ).

[0053] Experiments have shown that the peptide AWPQ has the following advantages:

[0054] (1) The polypeptide AWPQ can resist the hydrolysis of peptidase in the intestinal microenvironment and is successfully transported across the membrane by intestinal epithelial cells into the body, indicating that it can be effectively absorbed by the body after being consumed.

[0055] (2) The polypeptide AWPQ can penetrate the blood-brain barrier of the body, indicating that it has the potential to enter the central nervous system and exert its function after being ingested by the body.

[0056] (3) The polypeptide AWPQ can effectively enhance the survival and differentiation of nerve cells, and promote the growth, development and differentiation of nerve cells, indicating that it has the effect of protecting nerve health and promoting nerve development after being consumed by the body.

[0057] In summary, the polypeptide AWPQ provided by this invention can be absorbed by the intestines after being ingested, and can penetrate the blood-brain barrier to enter the central nervous system, thereby promoting the growth, development, and differentiation of nerve cells and ultimately exerting its neuroprotective effect. Experiments have demonstrated that polypeptide AWPQ has promising development prospects in the fields of brain health, neuroprotection, and neurodevelopment or bioactive peptides.

[0058] Furthermore, based on the amino acid sequence of the polypeptide AWPQ provided by the present invention, those skilled in the art can obtain the polynucleotide sequence encoding the amino acid sequence using conventional techniques, and can also obtain an expression vector containing the polynucleotide sequence, thereby obtaining recombinant cells containing the polynucleotide sequence and / or the expression vector. It is understood that those skilled in the art, based on the known amino acid sequence of the functional peptide of the present invention, can obtain the polynucleotide encoding the amino acid sequence of the functional peptide, insert the polynucleotide into an expression vector to obtain a recombinant expression vector, and then transform the recombinant expression vector into a host cell to obtain a recombinant cell. Therefore, the present invention also provides a polynucleotide encoding the amino acid sequence of the above-mentioned functional peptide; the present invention provides a recombinant expression vector comprising the above-mentioned polynucleotide; and the present invention provides a recombinant cell comprising the above-mentioned recombinant expression vector.

[0059] Although the peptide AWPQ has good effects on the nervous system, the chemical synthesis or natural isolation of the peptide AWPQ still faces the challenges of high cost and long cycle. Therefore, obtaining natural substances containing peptide AWPQ, such as hydrolyzed casein, can effectively reduce costs and supplement the other nutrients needed for the body's growth.

[0060] Casein, as a rich and high-quality natural protein resource, boasts a balanced amino acid composition, is rich in various active precursors suitable for human health maintenance, and has a mild taste, low allergenicity, and is compatible with the human digestive and absorption system. Enzymatic hydrolysis can break down casein into small peptide fragments with different biological activities. This type of hydrolyzed casein not only retains the natural nutritional advantages of casein but also has the characteristics of being more easily absorbed by the human body, having biological activities more closely aligned with human physiological needs, and being widely available and relatively inexpensive to prepare, thus possessing broad application prospects in the field of nutrition and health care. However, current research on hydrolyzed casein mainly focuses on its antioxidant, immune-regulating, and basic nutritional supplementation effects in the general population, with relatively little research on its application in nervous system health. Furthermore, existing casein enzymatic hydrolysis processes often employ single-enzyme hydrolysis or conventional combined enzymatic hydrolysis methods, resulting in defects such as low hydrolysis efficiency, difficulty in precisely controlling the degree of hydrolysis, low content of active peptides in the hydrolysate, and unreasonable molecular weight distribution. This leads to unstable biological activity of the hydrolysate and difficulty in adapting it to the digestive and absorption characteristics of the human body, failing to fully realize its potential efficacy in supporting nervous system nutrition.

[0061] To address the aforementioned problems, a second aspect of the present invention provides a hydrolyzed casein, comprising the aforementioned polypeptide.

[0062] Experiments have shown that the hydrolyzed casein provided by this invention can increase the expression of synaptophysin (SYN) and neuronal nucleoprotein (NeuN) in the hippocampus, thereby promoting synapse formation and neural activity, and ultimately having a certain promoting effect on brain neural development.

[0063] In one embodiment of the present invention, the hydrolyzed casein may be amniotic hydrolyzed casein.

[0064] The degree of hydrolysis of hydrolyzed casein can range from 9.5% to 12.5%. Maintaining the degree of hydrolysis of hydrolyzed casein within a suitable range helps to promote the enrichment of neuroprotective peptides in hydrolyzed casein.

[0065] In hydrolyzed casein, the mass content of peptides with a molecular weight ≤3000 Da can be ≥90%, and the mass content of peptides with a molecular weight <1000 Da can be ≥60%. By regulating the molecular weight distribution of hydrolyzed casein to meet the above ranges, hydrolyzed casein can have the advantages of small molecular weight and easy absorption by the intestine.

[0066] Based on the above research, a third aspect of the present invention provides a method for preparing the above-mentioned hydrolyzed casein, comprising the following steps:

[0067] The casein raw material was subjected to a first enzymatic hydrolysis using a first enzyme preparation to obtain a first enzymatic hydrolysis product;

[0068] The first enzymatic hydrolysis product is subjected to a second enzymatic hydrolysis using a second enzyme preparation to obtain hydrolyzed casein.

[0069] The first enzyme preparation includes at least one of serine protease, neutral protease, and glutaminase; the second enzyme preparation includes at least one of alkaline protease, flavor protease, and deaminase.

[0070] This invention discovers that by using two enzymatic hydrolysis treatments and specifying the enzyme preparation to meet the aforementioned range, it is possible to enrich bioactive functional peptides that can be absorbed by the intestines and can tightly bind to brain-derived neurotrophic factor (BDNF) receptors (i.e., neurotrophic factor receptor tyrosine kinase B TrkB) and nerve growth factor (NGF) receptors (neurotrophic factor receptor tyrosine kinase A TrkA), thereby promoting the neuroprotective effect of hydrolyzed casein. Simultaneously, experiments have demonstrated that hydrolyzed casein obtained by using two enzymatic hydrolysis treatments and specifying the enzyme preparation to meet the aforementioned range can increase the expression of synaptophysin (SYN) and neuronal nucleoprotein (NeuN) in the hippocampus, thereby promoting synapse formation and neural activity, ultimately contributing to a certain degree of promotion of brain neural development.

[0071] Preferably, the first enzyme preparation can be a neutral protease, and the second enzyme preparation can be an alkaline protease and a flavor protease, which can yield hydrolyzed casein that more effectively promotes neural development.

[0072] In one specific embodiment, based on casein raw material, the amount of the first enzyme preparation added is 0.1-0.8 wt%; and based on casein raw material, the amount of the second enzyme preparation added is 0.1-0.8 wt%. A concentration below 0.1 wt% will lead to insufficient enzymatic hydrolysis, failing to effectively release functional peptides with neuroprotective activity; a concentration above 0.8 wt% is prone to over-hydrolysis, damaging the structure of functional peptides. The above ranges allow for mild, controllable stepwise enzymatic hydrolysis, maximizing the enrichment of hydrolyzed casein that enhances SYN and NeuN expression.

[0073] Furthermore, the conditions for the first enzymatic hydrolysis treatment include: a temperature of 45-55℃, a time of 1.0-5.0 h, and a pH of 7.0-7.5; the conditions for the second enzymatic hydrolysis treatment include: a temperature of 45-55℃, a time of 1.5-5.0 h, and a pH of 8.0-8.5.

[0074] The two enzymatic hydrolysis steps are both set at 45-55℃ to optimize the activity of the enzyme preparations used and protect the neuroprotective activity of the functional peptides. The first enzymatic hydrolysis (pH 7.0-7.5, 1.0-5.0 h) initially opens the protein structure, while the second enzymatic hydrolysis (pH 8.0-8.5, 1.5-5.0 h) selectively enriches neuroactive functional peptides. The two steps work synergistically to ensure neuroprotective and neurodevelopment-promoting effects.

[0075] Furthermore, the preparation method of casein raw material includes the following steps:

[0076] The raw milk was sequentially subjected to defatting, pasteurization and ultrafiltration concentration to obtain casein.

[0077] Casein and water are mixed to obtain casein raw material.

[0078] In one embodiment of the present invention, the hydrolyzed casein can be amniotic hydrolyzed casein, and the raw milk can be raw sheep milk.

[0079] Among these processes, defatting removes fats that interfere with enzymatic hydrolysis, pasteurization kills harmful microorganisms and protects protein activity, and ultrafiltration concentrates and enriches casein; then it is mixed with water to provide a suitable system for subsequent enzymatic hydrolysis.

[0080] Specifically, the defatting process is carried out at a temperature of 45-55℃, which efficiently removes fat and prevents it from interfering with subsequent enzymatic hydrolysis. Pasteurization is performed at 60-90℃ for 10-60 seconds, killing harmful microorganisms without damaging the activity of casein. Ultrafiltration concentration uses an ultrafiltration membrane with a molecular weight cutoff of 30-100 kDa, concentrating the feed solution at a concentration temperature of 10-20℃ and a transmembrane pressure of 0.1-1 MPa. The volume ratio of washing water to feed solution during the washing stage is controlled at 2-4:1 until the feed concentration reaches 5-10% (v / v). This process accurately enriches casein and removes small molecule impurities. A casein-to-water ratio of 1:6-12 forms a suitable concentration system, providing a favorable reaction environment for the subsequent two enzymatic hydrolysis steps.

[0081] The fourth aspect of the present invention provides the application of the above-described polypeptide, the above-described hydrolyzed casein, or the hydrolyzed casein prepared by the above-described preparation method in the preparation of neuroprotective related products.

[0082] It is understandable that the aforementioned neuroprotection may include at least one of the following: improving neurological health and promoting neurodevelopment.

[0083] The aforementioned neuroprotection can manifest itself in any of the following ways:

[0084] (a) Improves the expression of synaptophysin in the hippocampus;

[0085] (b) Improve the expression of neuronal nucleoproteins in the hippocampus

[0086] (c) Promotes nerve cell growth and / or survival;

[0087] (d) Promotes nerve cell differentiation.

[0088] For example, hydrolyzed casein, when administered to the body, can improve the expression of hippocampal synaptophysin (SYN) and neuronal nucleoprotein (NeuN). The peptide AWPQ, when administered to the body, can promote the growth and survival of nerve cells and facilitate the differentiation of nerve cells into neurites.

[0089] In the above technical solutions, the relevant products may include health foods.

[0090] Health foods can include those designed to help improve memory.

[0091] Furthermore, the aforementioned products may be in the form of at least one of the following: oral liquid, tablets, granules, capsules, pills, aqueous solutions, powders, soft capsules, and films.

[0092] The aforementioned products may also include carriers and / or physiologically acceptable excipients. Carriers include at least one of microcapsules, microspheres, nanoparticles, and liposomes. Excipients include at least one of fillers, flavoring agents, diluents, wetting agents, binders, disintegrants, lubricants, flavor and color modifiers, solvents, solubilizers, co-solvents, emulsifiers, antioxidants, metal complexing agents, inert gases, preservatives, local analgesics, pH adjusters, and isotonic or isotropic regulators.

[0093] The technical solution of this application will be further explained below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise specified, all reagents used are commercially available or obtained through public channels.

[0094] Example 1: Preparation of hydrolyzed casein

[0095] (1) Preparation of hydrolyzed casein No. 1:

[0096] Raw sheep milk was defatted, pasteurized, and concentrated via ultrafiltration to obtain casein. The defatting temperature was 50°C; the pasteurization conditions were 75°C for 20 seconds; and the ultrafiltration concentration was performed using a 50 kDa ultrafiltration membrane at a concentration temperature of 10°C and a transmembrane pressure of 0.15 MPa. The volume ratio of washing water to feed liquid was controlled at 4:1 during the washing stage until the concentration reached 5% (m / v). Water was then added to the casein to achieve a volume ratio of 1:10, yielding the casein raw material.

[0097] First, a first enzyme preparation was added to the casein raw material for a first enzymatic hydrolysis treatment to obtain a first enzymatic hydrolysate. The first enzyme preparation was a neutral protease, and the added mass of the neutral protease was 0.1% of the casein protein mass. The pH was adjusted to 7.5, and the reaction was carried out at 50°C for 1 h. Then, a second enzyme preparation was added to the first enzymatic hydrolysate for a second enzymatic hydrolysis treatment to obtain a second enzymatic hydrolysate. The second enzyme preparation was an alkaline protease, and the added mass of the alkaline protease was 0.12% of the casein protein mass. The pH was adjusted to 8.5, and the reaction was carried out at 50°C for 2.0 h.

[0098] The second enzymatic hydrolysis product was inactivated at 90℃ for 20 min, then placed at room temperature to cool to 50℃, filtered, concentrated at 65℃, and dried to obtain hydrolyzed casein No. 1.

[0099] (2) Preparation of hydrolyzed casein No. 2:

[0100] For the specific preparation method, please refer to the preparation of hydrolyzed casein No. 1, the only difference being: the first enzyme preparation is a neutral protease, the mass of which is added is 0.1% of the casein protein mass; the pH is adjusted to 7.5, and the reaction is carried out at 50℃ for 1.5 h; the second enzyme preparation is an alkaline protease, the mass of which is added is 0.15% of the casein protein mass; the pH is adjusted to 8.5, and the reaction is carried out at 50℃ for 1.5 h.

[0101] (3) Preparation of hydrolyzed casein No. 3:

[0102] For the specific preparation method, please refer to the preparation of hydrolyzed casein No. 1, the only difference being: the first enzyme preparation is a neutral protease, the added mass of which is 0.12% of the casein protein mass; adjust the pH to 7.5 and react at 50℃ for 1.5 h; the second enzyme preparation is an alkaline protease and a flavor protease, the added mass of which is 0.1% of the casein protein mass; adjust the pH to 8.5 and react at 50℃ for 2.0 h.

[0103] Example 2: Detection of hydrolyzed casein

[0104] 1. Determination of the degree of hydrolysis of hydrolyzed casein:

[0105] The degree of hydrolysis of hydrolyzed casein proteins 1-3 in Example 1 was determined using the OPA reaction. Since each hydrolysis of a peptide bond releases a free amino group, and this free amino 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 specific procedure is as follows:

[0106] Weigh 7.620 g sodium tetraborate and 0.201 g sodium dodecyl sulfonate (SDS) and dissolve them in 150 mL of distilled water. Weigh 0.160 g OPA (98%) and dissolve it in 4 mL of anhydrous ethanol. After both are completely dissolved, mix them together. Then add 0.176 g dithiothreitol (DTT, 99%) and dilute to 200 mL with distilled water to obtain the OPA reagent.

[0107] Take 0, 100, 200, 300, 400, and 500 μL of serine standard solution into small centrifuge tubes, add distilled water to make up to 500 μL, add 3 mL of OPA reagent, and after 2 min, use distilled water as a reference to measure A340 nm. Plot a curve with absorbance as the abscissa and serine concentration as the ordinate. Specific data can be found in Table 1.

[0108] Weigh 0.100 g each of hydrolyzed casein proteins 1-3 from Example 1, dissolve and bring the volume to 100 mL, filter, take 500 μL of the filtrate, add 3 mL of OPA reagent, react for 2 min, and measure A340 nm using distilled water as a reference. Repeat the measurement 5 times under the same conditions. The formula for calculating the degree of hydrolysis is as follows:

[0109]

[0110] Where h is the number of hydrolyzed peptide bonds, mmol / g; h tot Total peptide bond count, mmol / g (casein has 8.2 mmol / g); C serine 1. The number of serine amino groups in hydrolyzed casein millimoles, mmol / L; V is the volume of hydrolyzed casein dissolved to a fixed volume, L; N is the dilution factor of hydrolyzed casein; m is the protein content of hydrolyzed casein, g; w is the protein content in hydrolyzed casein, %; β is a constant, 0.383 for casein; α is a constant, 1.039 for casein.

[0111] Table 1: Hydrolysis Degree Detection Data

[0112]

[0113] The hydrolysis degree results can be found in detail. Figure 1 Among them, samples 1-3 correspond to hydrolyzed casein 1-3 respectively. The degree of hydrolysis of hydrolyzed casein 1 is 9.78%, that of hydrolyzed casein 2 is 11.21%, and that of hydrolyzed casein 3 is 12.49%.

[0114] 2. Detection of molecular weight distribution of hydrolyzed casein

[0115] The molecular weight distribution of hydrolyzed casein 1-3 in Example 1 was determined using the GPC / UV detection method in Appendix A of the national standard GB 31645-2018, and the results are shown in Table 2.

[0116] Table 2: Molecular weight distribution detection data

[0117]

[0118] As shown in Table 2, samples 1-3 correspond to hydrolyzed casein 1-3, respectively. The molecular weight distribution of hydrolyzed casein 1-3 is similar, and substances with a molecular weight ≤3000 Da account for more than 90% of the total in the three samples, while small molecule peptides and amino acids with a molecular weight <1000 Da account for more than 60% of the total in each sample.

[0119] Example 3: Functional Experiment of Hydrolyzed Casein

[0120] Synaptophysin (SYN) is a membrane-integrated glycoprotein found in presynaptic vesicles that regulates the interaction between synaptic vesicles and the cytoskeleton, promoting neurotransmitter release through phosphorylation. As a synapse-specific marker, SYN is widely distributed in the central nervous system. Neuronal nucleoprotein (NeuN) is an RNA-binding protein specifically expressed in the nucleus of neurons, belonging to the nucleoprotein family. NeuN plays a crucial role in neuronal development, differentiation, and functional maintenance. By binding to specific mRNAs, it participates in the translational regulation of these mRNAs, thereby influencing neuronal physiological activity and plasticity. To determine the effects of hydrolyzed casein on neuronal development, functional experiments were conducted using the expression levels of SYN and NeuN in animal brain tissue as biomarkers, as detailed below:

[0121] 1. Animal experimental grouping and dietary intervention

[0122] Three-week-old SPF-grade male C57BL / 6 mice (weight: 13.0±2.0 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Liaoning, China). Mice were acclimatized for one week with a standard diet, under an environment of 22±1℃ and 55±5% humidity, with 12-hour light and 12-hour dark cycles, during which free access to food and water was allowed. After the acclimatization period, 40 mice were randomly divided into four groups: a control group, a hydrolyzed casein group 1, a hydrolyzed casein group 2, and a hydrolyzed casein group 3, with 10 mice in each group. The feed formulations are shown in Table 1. Hydrolyzed casein groups 1, 2, and 3 were added to the basal diet at a ratio of 0.6%, ensuring that the total casein content of the feeds in the experimental groups was the same as that in the control group. Mice in each group were fed according to the corresponding feeds for a dietary intervention period of 5 weeks. Mice were sacrificed after the experiment.

[0123] Table 3: Animal Experiment Groups and Dietary Intervention Methods

[0124]

[0125] 2. Immunofluorescence staining

[0126] After harvesting the hippocampus from the mouse brain tissue, fixation with 4% paraformaldehyde was performed, followed by paraffin sectioning. The sections were then sequentially immersed in environmentally friendly dewaxing buffer I for 10 min, environmentally friendly dewaxing buffer II for 10 min, environmentally friendly dewaxing buffer III for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and anhydrous ethanol III for 5 min, followed by washing with distilled water. Antigen retrieval was then performed, taking care to prevent excessive evaporation of the buffer solution and avoiding drying the sections. After retrieval, the sections were allowed to cool naturally. The treated sections were then placed in phosphate-buffered saline (PBS) (pH 7.4) and washed three times on a decolorizing shaker for 5 min each time. After slightly drying the sections, a circular motion was drawn around the tissue using a histochemical pen, and bovine serum albumin (BSA) was added for blocking for 30 min. The prepared primary antibody was added to the sections, and the sections were incubated overnight at 4°C in a humidified chamber. The sections were then placed in PBS and washed three times on a decolorizing shaker for 5 min each time. The corresponding secondary antibody was then added, and the sections were incubated at room temperature in the dark for 50 min. The slides were placed in PBS and washed three times, 5 min each time, on a destaining shaker. 4',6-Dimamidin-2-phenylindole (DAPI) staining solution was added, and the slides were incubated at room temperature in the dark for 10 min. The slides were then placed in PBS and washed three times, 5 min each time, on a destaining shaker. Autofluorescence quencher solution B was added for 5 min, followed by rinsing with running water for 10 min. The slides were then mounted using an antifluorescence quenching mounting medium. Finally, the slides were observed and photographed using an upright fluorescence microscope.

[0127] Immunofluorescence staining results of SYN protein in the CA1 region of the mouse hippocampus are as follows: Figure 2 As shown; Figure 2 In the image, the horizontal columns are grouped as follows: column 1 is the blank group, columns 2-4 are hydrolyzed casein group 1, hydrolyzed casein group 2, and hydrolyzed casein group 3, respectively; the vertical rows are stained as follows: row 1 is the SYN-antifluorescent staining field, row 2 is the DAPI-stained field, and row 3 is a superimposed image of the SYN-antifluorescent staining field and the DAPI-stained field. Multiple non-overlapping fields of view in the mouse hippocampus were randomly selected, and the average fluorescence intensity of SYN protein in each field was statistically analyzed using ImageJ software. The results are shown below. Figure 3 As shown; Figure 3In the diagram, letters such as 'a' and 'b' are significance markers; the same letter indicates no statistically significant difference between groups; different letters indicate significant differences between groups (p < 0.05). Compared with the control group, the average fluorescence intensity of SYN protein in the hippocampus of mice in the hydrolyzed casein group 3 was the highest (P < 0.05), indicating that hydrolyzed casein has a certain promoting effect on the neural development of the mouse brain, suggesting that it has greater potential in promoting synapse formation or neural activity.

[0128] Immunofluorescence staining results of NeuN protein in the CA1 region of the mouse hippocampus are as follows: Figure 4 As shown; Figure 4 In the image, the horizontal columns are grouped as follows: column 1 is the blank group, columns 2-4 are hydrolyzed casein group 1, hydrolyzed casein group 2, and hydrolyzed casein group 3, respectively; the vertical rows are stained as follows: row 1 is the NeuN anti-fluorescent staining field, row 2 is the DAPI staining field, and row 3 is a superimposed image of the NeuN anti-fluorescent staining field and the DAPI staining field. The average fluorescence intensity of NeuN protein is shown in the figure. Figure 5 As shown; Figure 5 In the table, letters such as a, b, c, and d are significance markers; the same letter indicates no statistically significant difference between groups; different letters indicate significant differences between groups (p < 0.05). Compared with the control group, the mean fluorescence intensity of NeuN protein in the hippocampus of mice in hydrolyzed casein groups 1-3 was significantly increased (P < 0.05), indicating that dietary supplementation with hydrolyzed casein can significantly increase the expression level of NeuN in the mouse brain (P < 0.05) and promote neuronal survival.

[0129] Example 4: Screening of cognitive peptides in hydrolyzed casein

[0130] 1. Establishment and evaluation of the Caco-2 cell monolayer model

[0131] The cell density in the Caco-2 cell suspension was adjusted to 1×10⁻⁶. 5 pcs / cm 2 0.5 mL / well of Caco-2 cell suspension and 1.5 mL / well of complete culture medium were added to the upper chamber (aperture side, AP) and lower chamber (basal side, BL) of a Transwell 12-well plate, respectively. The plates were then incubated in a cell culture incubator. The culture medium in both chambers of the Transwell plate was changed every two days during the first week, and daily thereafter until day 21, resulting in a Caco-2 cell monolayer model. A schematic diagram can be seen below. Figure 6 .

[0132] To determine the integrity of the Caco-2 cell monolayer model, the transmembrane resistance (TEER) of Caco-2 cells was measured using a cell resistance meter on days 7, 14, and 21 of culture. A higher TEER indicates a more intact Caco-2 cell monolayer. A TEER value of 500 Ω•cm was considered optimal. 2 This indicates that a Caco-2 cell monolayer model has been established, and the TEER detection results are visible. Figure 7 .like Figure 7 As shown, during the first 7 days of culture, Caco-2 cells rapidly divided, causing the resistivity to increase rapidly to 342.20 ± 6.87 Ω•cm. 2 On day 14, Caco-2 cells gradually filled the entire Transwell chamber, and the resistivity reached 471.40 ± 6.80 Ω•cm. 2 As time progressed, the connections between cells became increasingly tight; on day 21, the resistivity of a Caco-2 cell monolayer reached 543.6 ± 15.32 Ω•cm. 2 The Caco-2 monolayer essentially filled the entire Transwell chamber, becoming very dense and meeting experimental requirements. Therefore, subsequent experiments selected a TEER value exceeding 500 Ω•cm. 2 The Caco-2 cell monolayer model was used.

[0133] To determine the functionality of the Caco-2 cell monolayer model, alkaline phosphatase (AKP) activities in Caco-2 cells in the upper and lower chambers of the Transwell plate were measured on days 7, 14, and 21 of culture. Since Caco-2 cells can form a dense monolayer model and secrete AKP, and this enzyme is a marker enzyme of the brush border of small intestinal epithelial cells, when the ratio of AKP activity in the upper and lower chambers of the Transwell plate is greater than 5 and remains stable above 5, it can be considered that the relevant marker proteins in Caco-2 cells have completed differentiation. At this point, the Caco-2 monolayer model is successfully established and can be used for subsequent experiments on the transport of casein hydrolysate. The results of AKP activity detection are shown in Table 4. As shown in Table 4, after 7 days of culture, the AKP activity ratio across the Caco-2 monolayer cells was low, at 1.69±0.01; after 14 days of culture, the AKP activity ratio rapidly increased to 4.20±0.05; and by day 21, the AKP activity ratio slowly increased to 5.06±0.15, reaching above 5, which basically conforms to the trend of alkaline phosphatase ratio changes reported in relevant literature in the Caco-2 cell monolayer model. Therefore, combined with the aforementioned TEER values ​​that meet the standards, this further demonstrates that the Caco-2 cell monolayer model was successfully constructed, and that the transmembrane transport experiment of casein hydrolysis can be started using the Caco-2 cell monolayer model as the experimental subject 22 days after its establishment.

[0134] Table 4: Detection data of alkaline phosphatase activity

[0135]

[0136] 2. Transmembrane transport and mass spectrometry identification of hydrolyzed casein

[0137] The hydrolyzed casein 3 from Example 1 was dissolved to a concentration of 1 mg / mL using Hank's Balanced Salt Solution (HBSS) to obtain the sample to be tested. 0.5 mL of the sample to be tested was added to the upper chamber of the Transwell in the successfully constructed Caco-2 cell monolayer model, followed by 1.5 mL of HBSS to the lower chamber. The mixture was incubated for 2 h, and the solutions from both chambers were collected. The upper chamber components, lower chamber components, and sample to be tested were identified using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS / MS), and the peptide composition of different components was analyzed to screen for common peptides in different components. Since the Caco-2 cell monolayer model can simulate the intestinal epithelial barrier and thus be used to study intestinal transmembrane transport processes, the screened common peptides appeared not only in the sample to be tested and the upper chamber components but also in the lower chamber components, indicating that these common peptides may remain stable during intestinal transmembrane transport, can be absorbed by the intestine in their intact form, and further exert physiological effects in vivo. The total ion chromatogram of the sample to be tested is visible. Figure 8 The total ion chromatogram of the upper chamber components is visible. Figure 9 The total ion chromatogram of the lower chamber components is visible. Figure 10 The peptide composition and quantity of the sample to be tested, the upper chamber component, and the lower chamber component are shown in Table 5.

[0138] Table 5: Peptide Composition and Quantity

[0139]

[0140] The experimental results showed that 755, 824, and 646 peptides were detected in the sample to be tested, the upper chamber component, and the lower chamber component, respectively, with peptide ranges of 3-25. In the sample to be tested, 10-15 peptides (362) were the most abundant, followed by <10 peptides (205) and >15 peptides (188). In the upper chamber component, the number of >15 peptides (190), 10-15 peptides (383), and <10 peptides (251) were all greater than in the sample to be tested, indicating that some larger peptides in the sample to be tested were further hydrolyzed into one or more smaller peptides by brush border membrane peptidases, leading to an increase in the number of peptides. Compared to the sample to be tested and the upper chamber component, the lower chamber component showed a decrease in the total number of peptides >15 (125), 10-15 (311), and <10 (210), indicating that the absorption of hydrolyzed casein in the intestine is affected by peptidase and transport systems, and only some peptides can cross the Caco-2 cell monolayer intact. The lower chamber component consists of two parts: one part of the hydrolyzed casein peptides that resist peptidase hydrolysis and cross the monolayer, and the other part of peptides derived from hydrolysis of hydrolyzed casein by brush border peptidase and intracellular peptidase. These results demonstrate that the intestine exhibits selectivity in peptide hydrolysis and absorption; only peptides that resist peptidase hydrolysis and are completely absorbed can exert their biological activity in vivo.

[0141] Furthermore, Venn diagram analysis was performed on the peptide composition of the sample to be tested, the upper chamber component, and the lower chamber component to compare the differences in peptide types among the three groups. (See details...) Figure 11 . Figure 11 (a) indicates that there are 677 common peptides between the sample to be tested and the upper chamber component, which means that 677 peptides in hydrolyzed casein can resist peptidase hydrolysis or be retained after slight degradation; the remaining 78 peptides are completely degraded by peptidase, producing new peptides or amino acids; correspondingly, 147 new peptides were detected in the upper chamber component, which are peptides derived from hydrolyzed casein by further hydrolysis by peptidase. Figure 11 (b) indicates that there are 579 common peptides between the upper and lower compartment components, meaning that 579 peptides in the upper compartment component can completely cross the Caco-2 cell monolayer membrane. These peptides are a mixture of peptides from hydrolyzed casein and peptides derived from peptidase hydrolysis. Of the remaining 245 peptides, some cannot cross the monolayer membrane and cannot be absorbed by the intestinal epithelium, while others are further hydrolyzed by intracellular peptidases during intracellular transport to produce new peptides and amino acids. The 67 new peptides detected in the lower compartment component are peptides produced by intracellular peptidase hydrolysis. Figure 11(c) indicates that there are 529 common peptides between the sample to be tested and the lower chamber component. Most of these 529 peptides are resistant to peptidase hydrolysis and can pass through the Caco-2 cell monolayer intact. The remaining 226 peptides are peptides that are degraded by peptidase or cannot be transported. 117 peptides in the lower chamber component were not detected in hydrolyzed casein. These are peptides derived from hydrolyzed casein by brush border peptidase and intracellular peptidase. Figure 11 (d) indicates that 505 peptides (absorbable peptides) were detected in all three components, suggesting that 505 peptides in hydrolyzed casein are resistant to peptidase hydrolysis and can be absorbed by the intestine in their intact form. These 505 common peptides range in size from 3 to 25, with 188 being 10 peptides or less. Peptide transport across the Caco-2 cell monolayer appears to be related to the number of amino acid residues; many bioactive peptides containing 3-10 amino acids have been reported to be absorbed intact by the intestine; peptides with more than 10 amino acid residues are more susceptible to peptidase activity and cannot be absorbed. In summary, peptide absorption involves both peptidase hydrolysis and transport, and the intact absorption of peptides is crucial for their biological activity.

[0142] 3. Molecular docking screening of hydrolyzed casein

[0143] PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ) is a bioactive peptide prediction server based on a novel N-to-1 neural network. This server analyzes the probability that a target peptide possesses biological activity. After analysis by PeptideRanker, peptides with a prediction value exceeding 0.5 are marked as potentially bioactive, and the higher the prediction value, the greater the likelihood of the peptide's bioactivity. Molecular docking technology involves attaching small molecules (ligands) to the active sites of the target molecule's three-dimensional structure (protein). By continuously optimizing the conformation of the receptor molecule, the optimal conformation for binding the small molecule ligand to the target macromolecule is found, thereby screening for active substances. Compared to traditional functional peptide screening methods, molecular docking is a rapid and efficient tool for predicting molecular activity and has been widely used in drug design and the screening of active substances. Neurotrophic factors are a class of growth factors essential for the development, survival, and maintenance of neurons. Brain-derived neurotrophic factor (BDNF) binds to and activates TrkB receptors, triggering downstream signaling pathways (such as PI3K / Akt, MAPK / Erk, and PLCγ), which are crucial for neuronal survival, axonal / dendritic growth, synaptic plasticity, and neural differentiation. Additionally, nerve growth factor (NGF) binds to and activates TrkA receptors, thereby promoting the survival, differentiation, and axonal growth of sensory and sympathetic neurons. PC-12 cells, a cell line derived from rat adrenal medullary pheochromocytoma, possess neuroendocrine characteristics and are a classic model widely used in neuroscience research. PC-12 cells have NGF receptors on their membranes; upon NGF induction, they cease division, grow axon-like neurites, and differentiate into cells with sympathetic neuronal characteristics.

[0144] To screen for cognitive peptides from 505 common peptides (absorbable peptides), the bioactivity prediction analysis of these 505 common peptides was first performed using the PeptideRanker server to screen for peptides with potential bioactivity. Then, from these absorbable and bioactive peptides, peptides that may promote neural differentiation or neurotrophic functions were screened. During screening, these peptides were molecularly docked with BDNF receptors (TrkB) and NGF receptors (TrkA), respectively. Peptides with the best docking affinity were selected, as they may mimic the effects of neurotrophic factors and activate downstream signaling pathways, thereby promoting neural differentiation and exerting neurotrophic functions. After screening, these potential cognitive peptides with the best docking affinity were synthesized in a solid-phase manner to further verify their effects on neural development.

[0145] Table 6 shows the bioactivity prediction results from the PeptideRanker server. The data indicates that 38 out of the 505 shared peptides have a prediction value greater than 0.50, meaning only 38 out of the 505 absorbable peptides possess bioactivity. Therefore, from these 38 absorbable and bioactive peptides, potential cognitive peptides that may promote neural differentiation or neurotrophic functions can be screened. The peptide-enzyme binding energy is a key indicator of complex stability. A higher energy release during binding (negative numerical value) means the system tends towards a lower energy state during complex formation, indicating a more stable complex structure. Table 6 shows the molecular docking binding energies of the 38 peptides with BDNF and NGF receptors (" / " in Table 6 indicates docking failure and binding energy could not be detected). Sorted by binding energy, AWPQ was selected from the 38 peptides. Its low binding energy with BDNF or NGF receptors suggests that AWPQ has the highest potential to mimic the physiological functions of neurotrophic factors. The toxicity of these 38 peptides was predicted using the online tool ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html), and the results showed that none of them were toxic. The molecular docking simulation results of peptide AWPQ with the BDNF receptor are shown below. Figure 12 ; Figure 12 (a) shows a schematic diagram and a magnified view of the molecular docking simulation of the peptide AWPQ with the BDNF receptor. Figure 12 (b) Schematic diagram illustrating the interactions involved in the molecular docking simulation of peptide AWPQ and the BDNF receptor. The simulation results of molecular docking between peptide AWPQ and the NGF receptor are shown below. Figure 13 ; Figure 13 (a) shows a schematic diagram and a magnified view of the molecular docking simulation between the peptide AWPQ and the NGF receptor. Figure 13 (b) shows a schematic diagram illustrating the interactions involved in the molecular docking simulation of peptide AWPQ and the NGF receptor. Therefore, peptide AWPQ, which may have neurodevelopment-promoting or neurotrophic activities, was chosen for subsequent functional studies.

[0146] Table 6: Molecular docking binding energy detection data

[0147]

[0148] 4. Solid-phase synthesis and identification analysis of cognitive peptides

[0149] The peptide AWPQ (Ala-Trp-Pro-Gln) was synthesized in vitro using a solid-phase synthesis method, and the synthesized peptide AWPQ was identified by mass spectrometry. The mass spectrometry identification results are shown below. Figure 14The analysis showed that the molecular weight of the solid-phase synthesized peptide AWPQ was the same as that of the same peptide AWPQ derived from hydrolyzed casein.

[0150] Example 5: Functional Verification of Cognitive Peptides

[0151] 1. Verification of the blood-brain barrier permeability of cognitive peptides:

[0152] (1) Construction of an in vitro blood-brain barrier model

[0153] The blood-brain barrier (BBB) ​​is a natural barrier between blood and brain tissue, restricting the free exchange of substances between them and maintaining the relative stability of the brain's internal environment and normal brain function. However, while the BBB prevents exogenous substances from entering the brain, it also restricts the entry of functional substances, thus affecting their efficacy. Currently, many researchers are studying the mechanisms of drug action by establishing in vitro BBB models. The Transwell cell model is the simplest, most feasible, and most common in vitro BBB cell model, which involves culturing one or more cell types on both sides of a Transwell microporous membrane to simulate the BBB structure. The semi-permeable microporous membrane of the Transwell structure allows for the exchange of small molecules across the membrane while preventing cell migration across the membrane, making it an ideal structure for constructing the BBB. Transwell cell models can be divided into monoculture Transwell models and coculture Transwell models. Monoculture Transwell models refer to seeding a single type of endothelial cell, such as rat brain microvascular endothelial cells or mouse brain hemangioendothelioma cells, onto a Transwell membrane.

[0154] To investigate the blood-brain barrier penetration properties of peptide AWPQ, a single-culture Transwell model was constructed using mouse cerebral angioendothelioma cells (bEnd.3) to simulate the in vitro blood-brain barrier. The specific procedures were as follows: Group A consisted of 1×10⁻⁶ cells... 5 Group B: 2 × 10⁻⁶ cells / mL 5 Group C: 3 × 10⁻⁶ cells / mL 5 Three groups of bEnd.3 cell suspensions with different cell densities were prepared. 0.5 mL / well of bEnd.3 cell suspension was added to the upper chamber of the Transwell, and 1.5 mL of complete culture medium was added to the lower chamber. The medium was changed every other day, and the cells were cultured for 7 days. A schematic diagram of the cell culture is shown below. Figure 15 During the culture period, the optimal culture time and cell density of the in vitro blood-brain barrier model were determined by measuring the TEER value. The results are shown in the figure. Figure 16 .like Figure 16As shown, on day 3, the TEER values ​​of all groups were significantly higher than those on day 2 (P<0.01); from day 4 to day 7, the daily TEER values ​​were significantly different from the previous day (P<0.01); after seeding cells at different densities, there was no significant difference in TEER values ​​among the three groups on the same day (P>0.05); finally, 3×10 5 An in vitro blood-brain barrier model was established at a cell density of cells / mL, and the TEER value reached its peak more quickly on day 3 of culture.

[0155] To confirm the integrity of the in vitro blood-brain barrier model, a 4-hour leakage experiment was conducted. The specific procedures were as follows: Blank wells were set up with complete culture medium containing no cells; experimental wells 1 or 2 contained a density of 3 × 10⁻⁶ cells / mL. 5 Cells were cultured at a density of 10 cells / mL for 3 days. Cell culture medium was added to the upper and lower sides of the Transwell, ensuring the liquid level in the upper chamber was at least 0.5 cm higher than that in the lower chamber, creating a significant liquid level difference. The transwell was then placed in a cell culture incubator and observed after 4 hours. If the liquid level difference between the upper and lower chambers remained greater than 0.5 cm, the in vitro blood-brain barrier model was successfully established with good barrier function, and leakage was observed. Figure 17 ; Figure 17 middle, Figure 17 (a) indicates the leakage situation at the beginning of the experiment. Figure 17 (b) shows the leakage situation 4 hours after the experiment. For example... Figure 17 As shown, after 4 hours, the upper chamber of the blank well showed obvious leakage, while experimental wells 1 and 2, which established the in vitro blood-brain barrier model, did not show obvious leakage, and the liquid level difference between the upper and lower chambers was still above 0.5 cm. This indicates that the blood-brain barrier model was successfully established and the barrier function was good, which is consistent with the TEER value experimental results in the previous part.

[0156] To further confirm the integrity of the in vitro blood-brain barrier model, a crystal violet staining experiment was performed. The specific procedure was as follows: Crystal violet staining was conducted using a density of 3 × 10⁻⁶... 5bEnd.3 cells per mL were seeded in the upper chamber of a Transwell and cultured for 7 days. Crystal violet staining was performed at the same time each day, and the staining was observed under an inverted microscope after staining. Five fields of view were randomly selected from each sample for observation and photography. It was observed that as time progressed, the number of bEnd.3 cells in the Transwell chamber gradually increased, and the intercellular spaces gradually decreased, with the most dense cell growth occurring on days 3 and 4. Therefore, the cell growth within the Transwell chamber followed a trend consistent with the TEER value; the TEER value reached its maximum when the cells filled the Transwell chamber membrane (day 3); subsequently, the TEER value gradually decreased as the viability of the bEnd.3 cells in the chamber decreased and they gradually died.

[0157] In summary, 3×10 5 After seeding bEnd.3 cells at a density of 10 cells / mL in Transwell plates and culturing for 3 days, the resistivity of a monolayer of bEnd.3 cells reached a stable high value of 51.968 ± 2.262 Ω•cm. 2 Furthermore, the 4-hour leakage test results were good, and the cell growth observed after crystal violet staining was consistent with the trend of resistance value changes. These results indicate that using 3×10... 5 A blood-brain barrier model was successfully established by seeding bEnd.3 cells at a density of 1 cell / mL in Transwell plates and culturing them for 3 days. The barrier function was good and can be applied to subsequent in vitro blood-brain barrier permeability experiments of cognitive peptides.

[0158] (2) Blood-brain barrier permeability test:

[0159] The successfully established in vitro blood-brain barrier model was equilibrated in HBSS for 30 min. Then, 0.5 mL of peptide AWPQ solution (obtained by dissolving solid-phase synthesized peptide AWPQ powder to 5 μg / mL using HBSS) was added to the upper chamber, and 1.5 mL of fresh HBSS buffer was added to the lower chamber. The mixture was incubated for 2 h. After incubation, the solutions from both chambers were collected and stored at -80℃ for subsequent reversed-phase high-performance liquid chromatography (RP-HPLC).

[0160] The peptide AWPQ solution, upper chamber solution, and lower chamber solution were analyzed by RP-HPLC using a Shimadzu high-performance liquid chromatography (HPLC) system. 20 μL of each solution was loaded into an RP-C18 column (250 × 4.6 mm, 5 μm, Shimadzu GL Sciences, Kyoto, Japan). Gradient elution was set with 0.1% trifluoroacetic acid aqueous solution (solution A) and 0.1% trifluoroacetic acid acetonitrile solution (solution B) as the mobile phase. The linear gradient program was: 0–20 min, solution B 0%–60%; 20–30 min, solution B maintained at 60%. The flow rate was 1 mL / min, the column temperature was 30℃, and the detection wavelength was 214 nm. The HPLC chromatograms of peptide AWPQ, peptide AWPQ in the upper chamber, and peptide AWPQ in the lower chamber are shown below. Figure 18 .

[0161] RP-HPLC analysis revealed that after treatment of the lower chamber of a Transwell plate with peptide AWPQ, AWPQ was detected in the corresponding lower chamber, indicating that peptide AWPQ can cross the blood-brain barrier in vitro. In summary, all the above experiments demonstrate that the cognitive peptide AWPQ can be absorbed intact by the intestine and can cross the blood-brain barrier in an in vitro model. Further in vitro and in vivo experiments will be conducted to further investigate its potential physiological functions in promoting neural development or neurotrophic effects.

[0162] 2. Research on the mechanism by which cognitive peptides promote PC-12 cell differentiation:

[0163] (1) Effects of cognitive peptides on PC-12 cell viability

[0164] PC-12 cells are a widely used cell model for studying nerve cell function, differentiation, and apoptosis. PC-12 cells can differentiate into neuron-like cells with neurites under the induction of nerve growth factor (NGF). This invention uses PC-12 as an in vitro cell model to study the neurotrophic effects of the peptide AWPQ in promoting cell viability, cell differentiation, and synaptic growth. The specific procedures are as follows:

[0165] Blank wells (complete culture medium without PC-12 cells) and control wells (containing 1×10⁶ cells each) were set up separately. 5 Complete culture medium containing 1 × 10⁶ PC-12 cells / mL and experimental wells (containing 1 × 10⁶ cells / mL). 5Complete medium containing PC-12 cells / mL was used for plating, with 6 replicates per group. After plating, the cells were incubated in a cell culture incubator for 24 hours. The original medium was then aspirated, and the wells were replaced with complete medium containing peptide AWPQ at final concentrations of 0 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, and 4 mM. After 24 hours of incubation, CCK-8 reagent was added to the wells, and the cells were incubated at 37°C in the dark for 2 hours. After incubation, the absorbance at 450 nm was measured using a microplate reader, and the survival rate of PC-12 cells after AWPQ intervention was calculated. The results are shown below. Figure 19 ; Figure 19 In the text, letters such as a, b, c, and ab are significance markers; the same letter indicates no statistically significant difference between groups; different letters indicate significant differences between groups (p < 0.05); mixed letters indicate no significant difference from the single-letter groups they contain, but significant differences from other letter groups. The cell viability rate is calculated using the following formula: Cell viability rate (%) = (OD value of experimental wells - OD value of blank wells) / (OD value of control wells - OD value of blank wells) × 100%.

[0166] like Figure 19 As shown, at concentrations of 0.5 mM, 1 mM, or 3 mM, peptide AWPQ had no significant effect on the viability of PC-12 cells (P>0.05). However, when the concentration was adjusted to 2 mM, peptide AWPQ significantly improved the viability of PC-12 cells (P<0.05). Therefore, 2 mM was subsequently used as the experimental concentration of AWPQ for subsequent cell differentiation experiments.

[0167] (2) Effects of cognitive peptides on PC-12 cell differentiation and neurite growth

[0168] Will contain 1×10 5 Complete culture medium containing PC-12 cells per mL was used to seed cells onto plates, with 6 replicates per group. After seeding, the plates were incubated in a cell culture incubator for 24 hours. The original culture medium was then aspirated, and the wells were replaced with complete culture medium containing NGF at final concentrations of 0 ng / mL, 0.3 ng / mL, 1 ng / mL, 3 ng / mL, 10 ng / mL, 30 ng / mL, and 100 ng / mL. Cells were co-cultured for 5 days, and photographs were taken using an inverted microscope. Changes in axon length and number were statistically analyzed. Cells containing one or more neural processes, with at least one neural process having a length greater than or equal to the cell body diameter, were considered positive cells. At least 5 random fields of view were selected for cell counting, with at least 100 cells in each field. Cell differentiation rate was calculated, and the results are shown below. Figure 20 ; Figure 20In the text, letters such as a, b, c, d, e, and f are significance markers; the same letter indicates no statistically significant difference between groups; different letters indicate significant differences between groups (p < 0.05). The cell differentiation rate is calculated using the following formula: Cell differentiation rate (%) = Number of positive cells / Total number of cells × 100%. Figure 20 As shown, compared with 0 ng / mL, 0.3 ng / mL NGF had no significant effect on the outward growth of PC-12 cells (P>0.05); at concentrations of 1 ng / mL, 3 ng / mL, 10 ng / mL, 30 ng / mL and 100 ng / mL, NGF significantly promoted PC-12 cell differentiation in a concentration-dependent manner (P<0.05).

[0169] Will contain 1×10 5 Complete medium containing 1000 cells / mL PC-12 was used to seed cells onto plates, with 6 replicates per group. After seeding, the plates were incubated in a cell culture incubator for 24 hours. The original medium was then aspirated, and the wells were replaced with complete medium containing 0.3 ng / mL NGF and 2 mM AWPQ peptide. Cells were co-cultured for 5 days. The number of differentiated cells was observed under a microscope, and the cell differentiation rate was calculated. This well was labeled NGF+AWPQ. A blank control group (CON) was prepared using the same procedure as the experimental group, but without the addition of NGF and AWPQ peptide. A negative control group (NGF) was prepared using the same procedure as the experimental group, but without the addition of AWPQ peptide. Microscopic images are shown. Figure 21 ,in, Figure 21 (a) indicates the microscopic field of view of group CON. Figure 21 (b) indicates the microscopic field of view of the NGF group. Figure 21 (c) represents the microscopic field of view of the NGF+AWPQ group. Figure 21 In (c), the red arrows represent differentiated PC-12 cells. The cell differentiation rate results are shown in the graph. Figure 22 ; Figure 22 In this context, letters such as 'a' and 'b' are significance markers; the same letter indicates no statistically significant difference between groups; different letters indicate a significant difference between groups (p < 0.05). Figure 21 and Figure 22 As shown, in the presence of 0.3 ng / mL NGF (which had no effect on cell differentiation), 2 mM peptide AWPQ significantly promoted the outward growth of PC-12 cell processes compared to the control group (P<0.05). These data indicate that peptide AWPQ has a significant effect on promoting the outward growth of PC-12 cell processes.

[0170] In summary, experiments conducted by this invention have shown that the hydrolyzed casein provided by this invention can increase the expression of synaptophysin (SYN) and neuronal nucleoprotein (NeuN) in the hippocampus, thereby promoting synapse formation and neural activity, ultimately contributing to brain neural development. Furthermore, the peptide AWPQ was obtained from the hydrolyzed casein provided by this invention. AWPQ can achieve transmembrane transport in small intestinal epithelial cells in vitro, indicating that it can be absorbed by the human body through diet; it can effectively penetrate the blood-brain barrier model in vitro, indicating its potential to enter the central nervous system and exert its function; and it also showed a significant ability to promote nerve cell growth, development, and differentiation in PC-12 in vitro cell viability and differentiation experiments, indicating its efficacy in protecting nerve health and promoting neural development.

[0171] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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. The use of a polypeptide or hydrolyzed casein comprising said polypeptide in the preparation of health foods for improving memory, characterized in that, The amino acid sequence of the polypeptide is Ala-Trp-Pro-Gln.

2. The application according to claim 1, characterized in that, The degree of hydrolysis of the hydrolyzed casein is 9.5%-12.5%; and / or, In the hydrolyzed casein, the mass content of peptides with a molecular weight ≤3000 Da is ≥90%, and the mass content of peptides with a molecular weight <1000 Da is ≥60%.

3. The application according to claim 1, characterized in that, The method for preparing the hydrolyzed casein includes the following steps: The sheep milk casein raw material was subjected to a first enzymatic hydrolysis using a first enzyme preparation to obtain a first enzymatic hydrolysis product; The first enzymatic hydrolysis product is subjected to a second enzymatic hydrolysis using a second enzyme preparation to obtain the hydrolyzed casein. The first enzyme preparation is a neutral protease; the second enzyme preparation is an alkaline protease and a flavor protease.

4. The application according to claim 3, characterized in that, Based on sheep milk casein raw material, the amount of the first enzyme preparation added is 0.1-0.8 wt%; and / or, Based on sheep milk casein raw material, the amount of the second enzyme preparation added is 0.1-0.8 wt%.

5. The application according to claim 3 or 4, characterized in that, The conditions for the first enzymatic hydrolysis treatment include: a temperature of 45-55℃, a time of 1.0-5.0 h, and a pH value of 7.0-7.5; and / or, The conditions for the second enzymatic hydrolysis treatment include: a temperature of 45-55℃, a time of 1.5-5.0 h, and a pH of 8.0-8.

5.

6. The application according to claim 3 or 4, characterized in that, The preparation method of the sheep milk casein raw material includes the following steps: The raw milk was sequentially subjected to defatting, pasteurization and ultrafiltration concentration to obtain casein. The casein and water are mixed to obtain the sheep milk casein raw material.

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