Brain polypeptide raw material with intelligence improving and brain strengthening functions, functional food and preparation method of brain polypeptide raw material

Abstract

The invention provides brain polypeptide raw materials capable of benefiting intelligence and strengthening brain, functional food and a preparation method of the brain polypeptide raw materials, and belongs to the technical field of functional food. The preparation method comprises the following steps: S1, pretreatment; s2, performing primary hydrolysis: adjusting the pH value of the pre-hydrolysate to 6.5-7.5, adding neutral protease with the addition amount being 3.5-4.5% of the mass of the bovine brain protein, performing enzymolysis at 50-60 DEG C for 1-3 hours, and performing enzyme deactivation to obtain first hydrolysate; s3, second hydrolysis: adjusting the pH value of the first hydrolysate to 6.0-7.0, adding papain with the addition amount being 1.0-3.0% of the mass of the bovine brain protein, carrying out enzymolysis at 45-65 DEG C for 1.5-2.5 h, and carrying out enzyme deactivation to obtain a second hydrolysate; and S4, performing post-treatment to obtain the brain polypeptide raw material powder. The brain polypeptide raw material can significantly remove DPPH free radicals and ABTS free radicals, inhibit the activity of acetylcholin esterase, repair PC12 nerve cells, inhibit the expression of a pro-apoptosis gene Bax and promote the expression of an anti-apoptosis gene Bcl-2, jointly exert the effects of benefiting intelligence, strengthening brain and protecting cranial nerves, and can be used as a functional factor to be applied to a functional product for benefiting intelligence and strengthening brain.

Description

TECHNICAL FIELD

[0001] The present application relates to a brain polypeptide raw material with intelligence and brain health, a functional food and a preparation method thereof, and belongs to the technical field of functional food. BACKGROUND

[0002] With the aggravation of population aging, diseases caused by neurotoxic oxidative damage are also increasing, and brain nerve oxidative damage and neurodegenerative diseases, such as brain polypeptide raw material, are small molecule active peptides extracted from animal brain tissue (such as cow brain or pig brain) by biological enzyme hydrolysis, which has various intelligence and brain health functions. ① Promote nerve cell survival and regeneration, brain polypeptide raw material can activate the secretion of neurotrophic factors (such as BDNF, NGF), promote the growth of neuron axon, and inhibit the apoptosis signal pathway; ② Improve brain metabolism and energy supply, increase ATP synthesis efficiency by enhancing mitochondrial function, and relieve brain nerve cell hypoxic oxidative damage.

[0003] Chinese patent CN118726526A discloses a preparation method of brain peptide for preventing dementia. The brain peptide for preventing dementia is prepared by using cold storage grinding to remove acid, molecular resonance assisted salting-out, molecular resonance assisted enzymolysis, secondary enzymolysis, and ultrafiltration drying. The extraction rate of brain peptide is improved by using trypsin and chymotrypsin in cooperation. Chinese patent CN120365355A discloses an active peptide for improving memory, a brain peptide enzymolysis extract, and a preparation method and application thereof. Trypsin and pepsin are used for microwave-assisted enzymolysis, carbon dioxide gas is introduced for aeration during microwave treatment, small molecule peptides with high activity are extracted, and AAVP, a peptide segment with the functions of improving sleep and improving memory, is screened out.

[0004] The core active peptide segment with clear intelligence and brain health functions in bovine brain peptide and its action mechanism still lack systematic research, so the development of bovine brain peptide products with high activity and high specificity is still an important research direction at present. At the same time, the existing preparation of intelligence and brain health peptides has problems such as complex equipment, low yield of active peptides, and single function. Therefore, it is of important practical significance and application value to develop an efficient, low-cost and comprehensive intelligence and brain health peptide preparation process. SUMMARY

[0005] The present application provides a brain polypeptide raw material with intelligence and brain health, a functional food and a preparation method thereof, which can effectively solve the above problems.

[0006] The present application provides a preparation method of a brain polypeptide raw material with intelligence and brain health protection of brain nerves, comprising the following steps: S1, pretreatment: after removing part of the white fat of the quarantine qualified bovine brain tissue, grinding, adding water in a mass ratio of 1:2~1:4, mixing, and heat treating at 54~56℃ for 0.5~1.5h, obtaining a pre-hydrolysis liquid; S2, first hydrolysis: adjusting pH of the pre-hydrolysis solution to 6.5-7.5, adding neutral protease in an amount of 3.5-4.5% of the mass of the bovine brain protein, and carrying out enzymatic hydrolysis at 50-60℃ for 1-3h, to obtain a first hydrolysis solution after enzyme inactivation; S3, second hydrolysis: adjusting pH of the first hydrolysis solution to 6.0-7.0, adding papain in an amount of 1.0-3.0% of the mass of the bovine brain protein, and carrying out enzymatic hydrolysis at 45-65℃ for 1.5-2.5h, to obtain a second hydrolysis solution after enzyme inactivation; S4, post-treatment: adjusting pH of the second hydrolysis solution to 4.0-5.0, and carrying out centrifugation, activated carbon decolorization and deodorization, diatomite filtration, ultrafiltration, nanofiltration desalination and concentration, and spray drying, to obtain the brain polypeptide raw material powder; wherein the ultrafiltration has a molecular weight cut-off of <5kDa, and the concentration has a mass content of the brain polypeptide raw material of 10-25%.

[0007] In some embodiments, in step S2, the neutral protease has an enzyme activity of 2800-3600 U / g; and in step S3, the papain has an enzyme activity of 4000-12000 U / g.

[0008] In some embodiments, in steps S2 and S3, the enzyme inactivation process is carried out at 84-86℃ for 15-20min.

[0009] In some embodiments, in step S4, the activated carbon-diatomite decolorization and deodorization step is carried out by cooling the second hydrolysis solution to 50-60℃, adding activated carbon for 0.5-1.5h, and then carrying out centrifugation and passing through a 0.45 μm water-based membrane and diatomite; wherein the activated carbon is added in an amount of 3.0-5.0% of the mass of the bovine brain protein.

[0010] In some embodiments, the centrifugation is carried out at 5000 rpm for 5-10min.

[0011] The present application provides a brain polypeptide raw material composition having the functions of benefiting intelligence, protecting the brain and protecting the brain nerves, which comprises an active peptide segment having a relative molecular weight of <5kDa, and includes at least one of the peptide sequences LDAF, DIAF, ALDF, IGEF, FGDI and LGFD.

[0012] In some embodiments, the brain polypeptide raw material composition having the functions of benefiting intelligence, protecting the brain and protecting the brain nerves is prepared by the method.

[0013] The present application provides a functional product comprising the brain polypeptide raw material composition having the functions of benefiting intelligence, protecting the brain and protecting the brain nerves as an active ingredient.

[0014] In some embodiments, the functional product has at least one of the following functions: (1) DPPH free radical scavenging rate is greater than or equal to 75%; (2) ABTS free radical scavenging rate is greater than or equal to 99%; (3) Acetylcholinesterase inhibition rate is greater than or equal to 85%; (4) Repair H2O2 induced PC12 nerve cell damage, cell survival rate is greater than or equal to 90%.

[0015] The application provides application of the brain polypeptide raw material composition in preparation of products for inhibiting acetylcholinesterase activity, scavenging free radicals or repairing oxidative damage of nerve cells.

[0016] The application has the following beneficial effects: The preparation method of the brain polypeptide raw material adopts step-by-step enzymolysis and conventional separation technology, has low equipment requirement, simple process and is suitable for large-scale production.

[0017] The brain polypeptide raw material prepared by the application has a DPPH free radical scavenging rate of more than 75%, an ABTS free radical scavenging rate of more than 99%, and can significantly inhibit acetylcholinesterase activity, and the inhibition rate is greater than or equal to 85%.

[0018] The brain polypeptide raw material prepared by the application has the advantages of intelligence and brain health, can repair H2O2 induced PC12 nerve cell damage, promote anti-apoptotic gene Bcl-2 expression, inhibit pro-apoptotic gene Bax expression, and play the role of intelligence and brain health through the PI3K / AKT pathway. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the application, and therefore should not be regarded as a limitation on the scope, and other related drawings can also be obtained by those skilled in the art without creative labor.

[0020] Figure 1 Results of PC12 nerve cell toxicity experiments of brain polypeptide raw materials with different processes and concentrations.

[0021] Figure 2 Results of construction of a hydrogen peroxide (H2O2) induced oxidative stress damage model of PC12 nerve cells.

[0022] Figure 3 Results of cell viability of H2O2 oxidative damage PC12 nerve cells after repair by brain polypeptide raw materials with different processes.

[0023] Figure 4 Effects of brain polypeptide raw materials with different processes on SOD enzyme activity of H2O2 oxidative damage PC12 nerve cells.

[0024] Figure 5 Effect of different process brain polypeptide raw materials on CAT enzyme activity of PC12 nerve cells damaged by H2O2 oxidation.

[0025] Figure 6 Effect of different process brain polypeptide raw materials on total antioxidant capacity of PC12 nerve cells damaged by H2O2 oxidation.

[0026] Figure 7 Effect of different process brain polypeptide raw materials on AChE activity of PC12 nerve cells damaged by H2O2 oxidation.

[0027] Figure 8 Effect of different process brain polypeptide raw materials on mRNA gene expression of P13K of PC12 nerve cells damaged by H2O2 oxidation.

[0028] Figure 9 Effect of different process brain polypeptide raw materials on mRNA gene expression of AKT of PC12 nerve cells damaged by H2O2 oxidation.

[0029] Figure 10 Effect of different process brain polypeptide raw materials on mRNA gene expression of Bax of PC12 nerve cells damaged by H2O2 oxidation.

[0030] Figure 11 Effect of different process brain polypeptide raw materials on mRNA gene expression of Bcl-2 of PC12 nerve cells damaged by H2O2 oxidation.

[0031] Figures 12 to 29 2D schematic diagram of interaction of different active peptide segments (LDAF, DIAF, ALDF, IGEF, FGDI, LGFD) simulated by molecular docking and TrkB, TREM2, AChE three receptor proteins.

[0032] There are overlapping parts of amino acid residues in the 2D schematic diagram of molecular docking interaction, which are specially described by numbers. Figure 15 Center 1: (APSA: 710) and 2: (LEUA: 608); Figure 21 Center 3: (LGYA: 710) and 4: (LEUA: 560); Figure 24 Center 5 (LEUA: 699) and 6 (ALAA: 586); Figure 27 Center 7 (LYSA: 643) and 8 (LEUA: 560); Figure 29The middle (PROA: 344) and 10 (GLYA: 345); the light blue circle is the solvent accessible surface, which refers to the area on the surface of the molecule that can be contacted by solvent molecules, and is helpful for analyzing the interaction between the molecule and the solvent, studying the solubility and stability of the molecule, and evaluating the binding site of the ligand and the receptor in the molecular docking. DETAILED DESCRIPTION

[0033] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application. Therefore, the following detailed description of the embodiments of the present application provided in the drawings is not intended to limit the scope of the claimed present application, but only represents selected embodiments of the present application.

[0034] Implementation column 1 1. Pretreatment: Take 500 g of quarantine qualified bovine brain tissue (protein content is about 12%, i.e. 60 g of protein), remove part of the visible white fat, and then shred. Add 1500 g of water (material to water ratio 1:3), mix uniformly, and then perform medium temperature heat treatment at 55℃ for 1.0 hour to obtain a pre-hydrolysis liquid.

[0035] 2. First hydrolysis: adjust the pH of the pre-hydrolysis liquid to 7.0, add 2.4 g of neutral protease (4.0% of the mass of bovine brain protein), and hydrolyze at 55℃ for 2 hours. Then raise the temperature to 85℃ for 15 min to inactivate the enzyme, and obtain a first hydrolysis liquid.

[0036] 3. Second hydrolysis: adjust the pH of the first hydrolysis liquid to 6.5, add 1.2 g of papain (2.0% of the mass of bovine brain protein), and hydrolyze at 55℃ for 2 hours. Then raise the temperature to 85℃ for 15 min to inactivate the enzyme, and obtain a second hydrolysis liquid.

[0037] 4. Post-treatment: adjust the pH of the second hydrolysis liquid to 4.5. First, centrifuge at 5000 rpm for 5 min to remove insoluble substances. Take the supernatant, add 2.4 g of activated carbon (4.0% of the mass of bovine brain protein), and decolorize and deodorize at 55℃ for 1 hour. Centrifuge again at 5000 rpm for 5 min to remove the residue, and then pass the supernatant through diatomite and a 0.45 μm water-based membrane in sequence for precision filtration. The obtained clear filtrate is subjected to ultrafiltration (molecular weight cut-off <5 kDa), and then desalting and concentration are performed by nanofiltration until the mass content of the brain polypeptide raw material reaches 10-25%. Finally, the concentrated liquid is subjected to spray drying to obtain a powdered brain polypeptide raw material product.

[0038] Example 2 1. Pretreatment: 500 g of bovine brain tissue (protein content 12%) was added with 1000 g of water (material to water ratio 1:2) after being shredded and degreased, and was heat treated at 54°C for 0.5 hour.

[0039] 2. First hydrolysis: the pH was adjusted to 6.5, 2.1 g of neutral protease (3.5% of the protein mass) was added, and hydrolysis was carried out at 50°C for 1 hour, and then enzyme inactivation was carried out at 84°C for 15 min.

[0040] 3. Second hydrolysis: the pH was adjusted to 6.0, 0.6 g of papain (1.0% of the protein mass) was added, and hydrolysis was carried out at 45°C for 1.5 hours, and then enzyme inactivation was carried out at 84°C for 15 min.

[0041] 4. Post-treatment: the subsequent steps were the same as in Example 1.

[0042] Example 3 Pretreatment: 500 g of bovine brain tissue (protein content 12%) was added with 2000 g of water (material to water ratio 1:4) after being shredded and degreased, and was heat treated at 56°C for 1.5 hour.

[0043] 2. First hydrolysis: the pH was adjusted to 7.5, 2.7 g of neutral protease (4.5% of the protein mass) was added, and hydrolysis was carried out at 60°C for 3 hours, and then enzyme inactivation was carried out at 86°C for 20 min.

[0044] 3. Second hydrolysis: the pH was adjusted to 7.0, 1.8 g of papain (3.0% of the protein mass) was added, and hydrolysis was carried out at 65°C for 2.5 hours, and then enzyme inactivation was carried out at 86°C for 20 min.

[0045] 4. Post-treatment: the subsequent steps were the same as in Example 1.

[0046] Comparative Examples In order to prove the key and superiority of the technical scheme of the present application, multiple groups of comparative examples were set up, the main difference being the raw material or enzyme hydrolysis combination, and the rest of the process was basically the same as in Example 1: Comparative Example A: the enzyme system was the same as in Example 1.

[0047] Comparative Examples 2 and B: the papain in the second step of hydrolysis was replaced by trypsin.

[0048] Comparative Examples 3 and C: the neutral protease in the first step was replaced by a complex lipase, and the papain in the second step was replaced by alkaline protease.

[0049] Other comparative examples (4-9, D-I): different enzyme combinations were used, such as alkaline protease + papain, neutral protease + alkaline protease, alkaline protease + acid protease, alkaline protease + flavor protease, alkaline protease + trypsin, pepsin + trypsin, etc.

[0050] Comparative examples A-I: the raw material was changed to pig brain tissue.

[0051] The enzymes used in the above examples and comparative examples are as follows: Neutral protease: model 0.8L, enzyme activity 80,000 U / g, Novozymes (China) Biotechnology Co., Ltd.; Papain: enzyme activity 400,000 U / g, Nanning Pangbo Biological Engineering Co., Ltd.; Trypsin: enzyme activity 200,000 U / g, Nanning Pangbo Biological Engineering Co., Ltd.; Compound lipase: enzyme activity 100,000 U / g, Dongheng Huadao Biological Technology Co., Ltd., alkaline protease: model 37071, enzyme activity 400,000 U / g, Novozymes (China) Biotechnology Co., Ltd.; Acid protease: enzyme activity 150,000 U / g, Shandong Longkete Enzyme Preparation Co., Ltd.; Flavor protease: model 1000L, enzyme activity 100,000 U / g, Novozymes (China) Biotechnology Co., Ltd.; Pepsin enzyme: enzyme activity 300,000 U / g, Shanghai Yugen Biological Technology Co., Ltd.

[0052] U / g refers to the enzyme activity per g of enzyme (used to characterize the ability of an enzyme to catalyze a certain chemical reaction). The meaning of the unit U / g of enzyme addition amount converted from the protein content (%) in each g of brain tissue is different.

[0053] As in Example 1: For neutral protease: the enzyme activity of neutral protease is 80,000 U / g; the neutral protease is added to the brain protein content of 2.4 g, according to the brain protein content of the cow (12%), the enzyme addition amount of neutral protease is 3200 U / g, the specific calculation process is: the enzyme activity of 2.4 g of neutral protease is: 80,000 U / g*2.4 g=192,000 U, converted to the enzyme addition amount according to the protein content in each g of brain tissue: 192,000 U / (500 g*12%) g=3200 U / g; For papain: the enzyme activity of papain is 400,000 U / g, the papain is added to the brain protein content of 1.2 g, according to the brain protein content of the cow (12%), the enzyme addition amount of papain is 8000 U / g; the enzyme activity of 1.2 g of enzyme is: 400,000 U / g*1.2=480,000 U, converted to the enzyme addition amount according to the protein content in each g of brain tissue: 480,000 U / (500 g*12%) g=8000 U / g.

[0054] In Example 1, the total enzyme addition amount of the two enzymes was 11,200 U / g. The enzyme addition amount of Examples 2, 3 and Comparative Examples was converted in the same way as the calculation method of Example 1 described above. The same name enzymes used in the above examples and comparative examples are the same enzymes of the same commercial company and brand, that is, the enzyme activities of the same name enzymes used in each example and comparative example are consistent.

[0055] Test Example The brain polypeptide raw material products prepared in the above examples and comparative examples were tested for performance: 1. Determination of DPPH and ABTS free radical scavenging rate of brain polypeptide raw material The brain polypeptide raw material contains functional groups that can specifically react with DPPH and ABTS free radicals on nerve cells, and can efficiently scavenge DPPH and ABTS free radicals on the surface and inside of nerve cells. The brain polypeptide raw material can rapidly bind to DPPH and ABTS free radicals on nerve cells, reduce them to stable products, reduce their concentration, reduce oxidative damage to nerve cells, maintain normal function of nerve cells, and resist oxidative stress and neurodegenerative diseases.

[0056] The determination method of DPPH and ABTS refers to "GB / T 39100-2020 Antioxidant Determination of Brain Polypeptide Raw Material".

[0057] The specific test results are shown in Table 1 below: Table 1. DPPH free radical scavenging rate of brain polypeptide raw material of different processes

[0058] Table 2. ABTS free radical scavenging rate of brain polypeptide raw material of different processes

[0059] As can be seen from Table 1, in different cases of the examples, the DPPH scavenging rate can reach more than 75%, which is a relatively high level, indicating that the examples are effective to a certain extent in DPPH scavenging, and the difference between the examples is small. The change range of DPPH scavenging rate of the comparative examples is large. The DPPH scavenging rate of Comparative Example 6 is 53.65%, which is significantly lower than that of the examples. The DPPH scavenging rate of Comparative Example C is 84.89%, which is significantly higher than that of the examples. The DPPH scavenging rate of the remaining comparative examples is lower than that of the examples, except that the DPPH scavenging rate of Comparative Example C is slightly higher than that of the examples.

[0060] As can be seen from Table 2, in different cases of the examples, the ABTS clearance rate can reach more than 99%, which has a relatively high level, indicating that the examples are effective for ABTS clearance, and the effect difference between the examples is small. The change range of the ABTS clearance rate of the comparative examples is small. Comparative Example 5 is 98.80%, which is slightly lower than the clearance rate of the examples; Comparative Example C is 99.88%, which is slightly higher than the clearance rate of the examples. The effect difference of the ABTS clearance rate between the examples and the comparative examples is not significant.

[0061] 2. Measurement of acetylcholinesterase of brain polypeptide raw material The brain polypeptide raw material contains aromatic and basic amino acid residues, which binds to the active center of acetylcholinesterase through π-π stacking and ionic bond, blocks the action of acetylcholine and enzyme, and inhibits its hydrolytic activity. This allows the concentration of acetylcholine in the synaptic cleft to be maintained, improving the efficiency of nerve signal transmission, and helping to improve cognitive function and delay the progression of neurodegenerative diseases. The inhibition rate of acetylcholinesterase is measured according to the method in Wang Wei's "Study on the Effect of Extracts from Fragrance Lotus on Improving Learning and Memory Ability in Alzheimer's Disease".

[0062] Table 3. Inhibition rate of acetylcholinesterase of brain polypeptide raw material of different processes

[0063] As can be seen from Table 3, in different cases of the examples, the acetylcholinesterase inhibition rate can reach more than 85%, which has a relatively high level, indicating that the examples are effective for acetylcholinesterase inhibition, and the effect difference between the examples is small. The change range of the acetylcholinesterase inhibition rate of the comparative examples is large. Comparative Example B is 47.88%, which is lower than the inhibition rate of the examples; Comparative Example 2 is 92.03%, which is slightly higher than the inhibition rate of the examples; However, the acetylcholinesterase inhibition rate of the examples is only slightly lower than that of Comparative Example 2, Comparative Example A and Comparative Example C, and is higher than that of other comparative examples. It is indicated that some conditions or methods in the comparative examples may produce stronger inhibition effect in some cases, but at the same time there are cases of low inhibition effect, and the inhibition rate is not as stable as Example 1.

[0064] In vitro antioxidant and acetylcholinesterase inhibition cannot be used as a single measure of the performance of brain polypeptide raw material. It is necessary to comprehensively screen and evaluate before entering the nerve cell experiment for comparison and overall evaluation. The DPPH free radical clearance rate of Table 1, the ABTS free radical clearance rate of Table 2 and the acetylcholinesterase inhibition rate of Table 3 are comprehensively evaluated, and Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example A, Comparative Example C, Comparative Example G and Comparative Example h are selected into the nerve cell experiment.

[0065] 3. PC12 nerve cell toxicity experiment of brain polypeptide raw material PC12 nerve cells are a nerve cell line derived from rat adrenal medulla chromaffin cell tumor, which has neuroendocrine nerve cell characteristics. Under the induction of nerve growth factor (NGF), it can differentiate into neuron-like nerve cells, and its morphology and function are similar to those of central nervous system neurons. Many nervous system diseases or cognitive disorders are related to oxidative damage and functional abnormalities of nerve cells. The repair and protection of brain polypeptide raw materials on oxidative damage of highly differentiated PC12 nerve cells indicate that they have potential effects on intelligence and brain health.

[0066] Neurocyte toxicity test is used to evaluate the harmful effects of exogenous substances on nerve cell growth, proliferation, metabolism, and nerve cell membrane integrity, thereby preliminarily judging the safety or biocompatibility of the substance. WST-8 in CCK-8 reagent is reduced to water-soluble orange yellow formazan product by dehydrogenase in nerve cells under the action of electron carrier, and its generation amount is proportional to the number of living nerve cells. The absorbance detected at 450 nm wavelength can reflect the activity of nerve cells and neurocyte toxicity.

[0067] The effect of brain polypeptide raw material on the proliferation activity of PC12 nerve cells was measured by CCK-8 method.

[0068] As shown in Figure 1 In the neurocyte toxicity test of different processes and concentrations of brain polypeptide raw materials, the nerve cell survival rates of Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example A, and Comparative Example C were all higher than 100% of the control group when the concentration of brain polypeptide raw material solution was 1.6 mg / mL, indicating that the brain polypeptide raw material had no toxicity to nerve cells.

[0069] The nerve cell survival rates of Comparative Example G and Comparative Example h were all higher than 100% of the control group when the concentration of brain polypeptide raw material solution was 1.2 mg / mL, and were all lower than 100% of the control group when the concentration of brain polypeptide raw material solution was 1.6 mg / mL, but the nerve cell survival rates were all greater than 80%, indicating that Comparative Example G and Comparative Example h had slight toxicity to nerve cells at 1.6 mg / mL. Comparative Example A, Comparative Example C and Comparative Example G had the highest nerve cell survival rate at 1.2 mg / mL, and the nerve cell survival rates of other examples and comparative examples were at a high level, so 1.2 mg / mL of brain polypeptide raw material will be used for subsequent experiments.

[0070] 4. Hydrogen peroxide (H2O2) oxidative stress damage model of PC12 nerve cells H2O2, as a reactive oxygen species (ROS), can induce the production of hydroxyl radicals in nerve cells, resulting in oxidative stress. This damages nerve cell membrane lipids, proteins, and DNA, leading to nerve cell apoptosis or necrosis. It is often used to construct models of oxidative damage in nerve cells to simulate the oxidative stress pathological process of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

[0071] This experiment used the CCK-8 method to determine the effect of H2O2 on the oxidative damage activity of PC12 nerve cells during modeling. Figure 2 As shown, our laboratory needs to subject nerve cells to severe oxidative damage to investigate the antioxidant, repair, and acetylcholinesterase inhibition effects of brain polypeptide raw materials on nerve cells. At a H2O2 solution concentration of 300 mM, the survival rate of PC12 nerve cells was less than 50%, indicating that significant oxidative damage to nerve cells was induced without causing a large number of nerve cell deaths. This demonstrates that PC12 nerve cells at 300 mM exhibit a good effect on oxidative stress and oxidative damage, indicating successful model establishment.

[0072] 4. The repair effect of brain polypeptide raw materials on PC12 nerve cells damaged by H2O2 oxidation. Brain peptides can inhibit neuronal apoptosis by regulating apoptosis proteins such as Bax and Bcl-2, thus maintaining neuronal survival; promote neuronal differentiation by activating signaling pathways such as PI3K / AKT, inducing neurite growth and synaptic protein synthesis; and enhance the activity of antioxidant enzymes, scavenging free radicals and protecting mitochondrial function. The repair effect of brain peptides on PC12 neurons is the result of a multi-mechanism synergy, achieving neuronal survival maintenance and functional reconstruction through a combined effect of "anti-apoptosis-promoting differentiation-antioxidation".

[0073] Experimental methods: The effect of brain polypeptide raw materials on the repair of PC12 nerve cells damaged by H2O2 oxidation was measured by the CCK-8 method.

[0074] Depend on Figure 3 As shown, in the repair of PC12 neurons damaged by H2O2 oxidation by different brain peptide raw materials, the survival rate of PC12 neurons in the model group was less than 80%, indicating that H2O2 solution can promote apoptosis of PC12 neurons and has weak resistance to H2O2. Among them, the neuronal survival rate of Example 1 reached 102%, and the survival rate of PC12 neurons in other comparative examples was also above 90%, indicating that brain peptide raw materials can inhibit neuronal apoptosis and protect PC12 neurons, with Example 1 showing the best protective effect.

[0075] 5. Effects of brain polypeptide raw materials on the antioxidant activity of PC12 nerve cells damaged by H2O2 oxidation. The effects of brain peptide raw materials on the catalase (CAT) activity of PC12 neurons damaged by H2O2 oxidation were determined using a SOD enzyme, CAT enzyme, and total antioxidant activity kit. The assays were performed according to the instructions for the SOD enzyme, CAT enzyme, and total antioxidant activity kits.

[0076] Depend on Figure 4 As shown: In the effect of different brain peptide raw materials on SOD enzyme activity of PC12 neurons damaged by H2O2 oxidation, the model group and the control group were compared. The SOD enzyme activity of PC12 neurons in the model group was significantly higher than that in the control group. This indicates that when PC12 neurons are damaged by H2O2 oxidation, the SOD enzyme activity will significantly increase, and it can specifically remove superoxide anions (O2). - It disproportionates into H2O2 and O2, laying the foundation for subsequent antioxidant reactions.

[0077] Comparison of Example 1 and Model Group: The SOD enzyme activity of PC12 nerve cells in Example 1 was significantly lower than that in the control group and the model group, indicating that its brain polypeptide raw material had the strongest antioxidant capacity.

[0078] Comparison with the model group: The SOD enzyme activity of comparative examples 2, 3, 4, A, C, G and h was significantly lower than that of the model group, and slightly lower or slightly higher than that of the control group. Among them, the SOD enzyme activity of comparative example A was close to that of Example 1.

[0079] Overall, this indicates that the brain peptide raw material has strong antioxidant capacity, can undergo redox reactions with H2O2, reduce the attack intensity of H2O2 on PC12 nerve cells, reduce SOD enzyme activity, and decrease the interaction between SOD enzyme and O2. - A redox reaction occurs, leading to excessive consumption of SOD enzymes. The control group showed slightly lower or slightly higher activity than the model group, indicating that the control group also possesses strong antioxidant capabilities, can protect nerve cells, and reduce SOD enzyme activity.

[0080] Depend on Figure 5 As shown: In the effect of different brain peptide raw materials on the CAT enzyme activity of PC12 neurons damaged by H2O2 oxidation, the model group and the control group were compared. The CAT enzyme activity of PC12 neurons in the model group was significantly higher than that in the control group. This indicates that when PC12 neurons are damaged by H2O2 oxidation, the CAT enzyme activity will increase significantly and can catalyze the decomposition of H2O2 into water and oxygen.

[0081] Comparison of Example 1 and Model Group: The CAT enzyme activity of PC12 nerve cells in Example 1 was significantly lower than that in the control group and the model group.

[0082] Comparison with the model group: The CAT enzyme activity of all comparative groups was significantly lower than that of the model group, and slightly lower or slightly higher than that of the control group. The CAT enzyme activity of all comparative groups was higher than that of Example 1. Lower CAT enzyme activity is better, indicating stronger antioxidant properties of the peptide. Lower CAT enzyme activity also reduces CAT enzyme consumption. The CAT enzyme activity of Example 1 was the lowest, exhibiting the strongest antioxidant properties.

[0083] Overall, this demonstrates that the brain peptide raw material possesses strong antioxidant capacity. By scavenging reactive oxygen species (ROS), it mitigates the attack of H2O2 on PC12 neurons, protects the structure of CAT enzymes within PC12 neurons, and maintains their catalytic activity. The CAT enzyme activity in Example 1 was significantly lower than that in the model group and the control group, indicating that the brain peptides of this example have strong antioxidant capacity, effectively scavenging ROS and protecting PC12 neurons. The CAT enzyme activities in the comparative examples were all significantly lower than those in the model group and close to those in the control group, indicating that the antioxidant capacity of the comparative brain peptides was slightly weaker than that of the examples, but they still possess the ability to protect neurons and reduce CAT enzyme activity.

[0084] Depend on Figure 6 As shown: In the effect of different brain peptide raw materials on the total antioxidant capacity of PC12 nerve cells damaged by H2O2 oxidation, the model group and the control group were compared. The antioxidant capacity of PC12 nerve cells in the model group was significantly lower than that in the control group, indicating that the antioxidant capacity of PC12 nerve cells will be significantly reduced when they are damaged by H2O2 oxidation.

[0085] Comparison of Example 1 and Model Group: The antioxidant capacity of PC12 nerve cells in Example 1 was significantly higher than that in the model group and slightly higher than that in the control group.

[0086] Comparison of comparative examples and model groups: The antioxidant activity of the comparative examples was significantly higher than that of the model group, and slightly higher or slightly lower than that of the control group. Among them, the antioxidant capacity of comparative example 3 was close to that of example 1, indicating that there are certain differences in the antioxidant capacity of brain peptide raw materials processed by different methods.

[0087] Overall, this indicates that the brain peptide raw material has a strong antioxidant capacity. When the ROS level in nerve cells increases, it can maintain the antioxidant capacity of nerve cells by timely neutralizing excess free radicals (ROS). The antioxidant activity of Comparative Example 4 was significantly higher than that of the model group and slightly lower than that of the control group, indicating that the antioxidant capacity of the comparative example brain peptide raw material was slightly weaker, but it could still neutralize excess free radicals (ROS) and maintain the antioxidant capacity of nerve cells.

[0088] 6. Effects of brain polypeptide raw materials on acetylcholinesterase (AChE) activity in PC12 neurons damaged by H2O2 oxidation. Acetylcholinesterase (AChE) is a key enzyme in the central nervous system that catalyzes the hydrolysis of acetylcholine. Abnormal AChE activity is closely associated with neurodegenerative diseases such as Alzheimer's disease (AD). H2O2 treatment can mimic β-amyloid (Aβ)-induced oxidative stress. Oxidative damage disrupts neuronal structure and accelerates the hydrolysis of neurotransmitters. Increased AChE activity is positively correlated with neuronal apoptosis. Oxidative stress disrupts the dynamic homeostasis of AChE, whose activity is regulated by post-translational modifications such as phosphorylation and ubiquitination. H2O2 may inhibit the degradative enzyme activity of AChE or promote its phosphorylation, leading to the accumulation and upregulation of AChE activity within neuronal cells.

[0089] The effect of brain peptide raw materials on the acetylcholinesterase (AChE) activity of PC12 neurons damaged by H2O2 oxidation was determined using an acetylcholinesterase (AChE) activity assay kit. Logarithmic-phase PC12 neurons were collected and cultured in DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) at a cell density of 1×10⁻⁶ cells / mL. 4 Neurons were seeded at a density of 1 / mL in 96-well plates and incubated at 37°C for 24 hours in a CO2 incubator to allow for cell adhesion. The culture medium was aspirated, and culture medium containing 1.2 mg / mL of the brain polypeptide raw material solution from the examples and comparative studies was added to create the experimental group; fresh culture medium was used as the control group; and culture medium without neurons was used as the blank group. All plates were incubated at 37°C for 24 hours. After incubation, 6-well plates were removed, and except for the control group, 1 mL of 300 mM H2O2 solution was added to each well. The plates were then incubated for 20 minutes in a constant temperature incubator, and the acetylcholinesterase (AChE) activity assay was performed according to the instructions of the kit.

[0090] Depend on Figure 7 As shown: In the effect of different brain peptide raw materials on the acetylcholinesterase (AChE) activity of PC12 neurons damaged by H2O2 oxidation, the model group and the control group were compared. The AChE activity of PC12 neurons in the model group was significantly higher than that in the control group. This indicates that H2O2 oxidation damage to PC12 neurons disrupts the dynamic balance of AChE activity, leading to disordered acetylcholine metabolism and abnormally increased AChE activity. Neurons may accelerate the hydrolysis of acetylcholine by upregulating AChE activity to avoid its excessive accumulation and excitotoxicity.

[0091] Comparison of Example 1 with the model group: The AChE activity of Example 1 was significantly lower than that of the model group and slightly lower than that of the control group, with the AChE activity of Example 1 being 90.28%.

[0092] The AChE activity of the comparative examples was significantly lower than that of the model group. The AChE activity of Comparative Example 4, Comparative Example A and Comparative Example G was slightly higher than that of the control group. The AChE activity of Comparative Example 2, Comparative Example 3, Comparative Example C and Comparative Example h was slightly lower than that of the control group. The AChE activity of Example 1, Comparative Example 2 and Comparative Example 3 was close. It was indicated that there was a certain difference in the antioxidant capacity of brain polypeptide raw materials prepared by different processes. The lower the AChE activity value, the better. Among them, Example 1 and Comparative Examples 2 and 3 were close, and all showed very good activity of reducing acetylcholinesterase.

[0093] In general, it was indicated that the brain polypeptide raw material had very strong antioxidant capacity, could undergo oxidation-reduction reaction with H2O2, reduce the attack intensity of H2O2 on PC12 nerve cells, inhibit the abnormal increase of AChE activity, avoid the excessive hydrolysis of acetylcholine, avoid the destruction of the dynamic balance of AChE activity, and cause abnormal movement regulation, synaptic dysfunction and central excitotoxicity risk.

[0094] The AChE activity of the PC12 nerve cells of the model group was significantly higher than that of the control group, indicating that H2O2 oxidative damage destroyed the structure of PC12 nerve cells, broke the dynamic balance of AChE activity, caused acetylcholine metabolism disorder, and abnormally increased AChE activity. The nerve cells may accelerate the hydrolysis of acetylcholine by up-regulating AChE activity to avoid excessive accumulation and cause excitotoxicity. This compensatory mechanism also has similar phenomena in the model of Alzheimer's disease (AD) and other neurodegenerative diseases. The increase of AChE activity under oxidative stress may exacerbate the decline of cholinergic system function.

[0095] The AChE activity of the examples and comparative examples was significantly lower than that of the model group. The AChE activity of Example 1, Comparative Example 2, Comparative Example 3, Comparative Example C and Comparative Example h was slightly lower than that of the control group. The AChE activity of Example 1 was 90.28%, indicating that the brain polypeptide raw material had very strong antioxidant capacity, could undergo oxidation-reduction reaction with H2O2, reduce the attack intensity of H2O2 on PC12 nerve cells, inhibit the abnormal increase of AChE activity, avoid the excessive hydrolysis of acetylcholine, avoid the destruction of the dynamic balance of AChE activity, and cause abnormal movement regulation, synaptic dysfunction and central excitotoxicity risk.

[0096] 7. Expression of related genes of PC12 nerve cells after oxidative damage by brain polypeptide raw material The phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) pathways are core pathways for nerve cell survival. PI3K catalyzes the conversion of phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-triphosphate (PIP3), which activates AKT phosphorylation. Phosphorylated AKT (p-AKT) can inhibit the activity of pro-apoptotic proteins (such as Bax) while activating the expression of anti-apoptotic proteins (such as Bcl-2), thereby inhibiting nerve cell apoptosis.

[0097] Bax is a pro-apoptotic gene that increases mitochondrial membrane permeability, releases neuronal cytochrome C, and activates the caspase cascade. Bcl-2 is an anti-apoptotic gene that maintains neuronal cell survival by inhibiting mitochondrial oxidative damage and caspase activation. Significance of expression balance: The Bax / Bcl-2 protein ratio is a key indicator determining the sensitivity of neuronal cells to apoptosis; an increased ratio promotes apoptosis, while a decreased ratio inhibits it.

[0098] The expression of genes related to H2O2 oxidative damage in PC12 neurons was measured using qPCR with brain peptide raw materials. Primer information for these neurons is shown in Table 4.

[0099] Table 4. Primers for PC12-related neural cell genes

[0100] Depend on Figure 8 As shown: In the effect of different brain polypeptide raw materials on the expression of PI3K mRNA in PC12 nerve cells damaged by H2O2 oxidation, the model group and the control group showed that the expression of PI3K gene in the model group (H2O2 oxidation damage) was significantly lower than that in the control group, indicating that H2O2 oxidation damage inhibited the expression of PI3K gene.

[0101] Comparison of Example 1 with the model group: The expression of PI3K gene after intervention with brain polypeptide raw materials in Example 1 was significantly higher than that in the model group, indicating that brain polypeptide raw materials can reverse H2O2 oxidative damage to a certain extent and promote the upregulation of PI3K gene expression.

[0102] Comparison between the comparative and model groups: The PI3K expression of the comparative group G was higher than that of Example 1, while some comparative groups were lower or close to it. The effects of different brain peptide raw materials on PI3K expression varied, but the brain peptide raw material of Example 1 showed a promoting effect on PI3K gene expression relative to the model group.

[0103] Overall, brain peptide raw materials can significantly upregulate the mRNA expression of the PI3K gene in PC12 neurons damaged by H2O2 oxidation. This may be achieved by activating PI3K and catalyzing the generation of PIP3, which then acts as a second messenger to activate AKT to the cell membrane, initiate anti-apoptotic signals, and exert the potential to improve intelligence and brain function.

[0104] As shown in FIG. 1, the mRNA gene expression of AKT in PC12 nerve cells damaged by H2O2oxidation was compared between the model group and the control group. The AKT gene expression in the model group (H2O2oxidation damage) was significantly higher than that in the control group, indicating that H2O2oxidation damage promoted the AKT gene expression and inhibited the apoptosis of nerve cells induced by oxidative stress. Figure 9 As shown in FIG. 1, the mRNA gene expression of AKT in PC12 nerve cells damaged by H2O2oxidation was compared between the model group and the control group. The AKT gene expression in the model group (H2O2oxidation damage) was significantly higher than that in the control group, indicating that H2O2oxidation damage promoted the AKT gene expression and inhibited the apoptosis of nerve cells induced by oxidative stress.

[0105] As shown in FIG. 1, the mRNA gene expression of AKT in PC12 nerve cells damaged by H2O2oxidation was compared between the model group and the control group. The AKT gene expression in the model group (H2O2oxidation damage) was significantly higher than that in the control group, indicating that H2O2oxidation damage promoted the AKT gene expression and inhibited the apoptosis of nerve cells induced by oxidative stress.

[0106] As shown in FIG. 1, the mRNA gene expression of AKT in PC12 nerve cells damaged by H2O2oxidation was compared between the model group and the control group. The AKT gene expression in the model group (H2O2oxidation damage) was significantly higher than that in the control group, indicating that H2O2oxidation damage promoted the AKT gene expression and inhibited the apoptosis of nerve cells induced by oxidative stress.

[0107] As shown in FIG. 1, the mRNA gene expression of AKT in PC12 nerve cells damaged by H2O2oxidation was compared between the model group and the control group. The AKT gene expression in the model group (H2O2oxidation damage) was significantly higher than that in the control group, indicating that H2O2oxidation damage promoted the AKT gene expression and inhibited the apoptosis of nerve cells induced by oxidative stress.

[0108] Figure 10 As shown in FIG. 1, the mRNA gene expression of AKT in PC12 nerve cells damaged by H2O2oxidation was compared between the model group and the control group. The AKT gene expression in the model group (H2O2oxidation damage) was significantly higher than that in the control group, indicating that H2O2oxidation damage promoted the AKT gene expression and inhibited the apoptosis of nerve cells induced by oxidative stress.

[0109] As shown in FIG. 1, the mRNA gene expression of AKT in PC12 nerve cells damaged by H2O2oxidation was compared between the model group and the control group. The AKT gene expression in the model group (H2O2oxidation damage) was significantly higher than that in the control group, indicating that H2O2oxidation damage promoted the AKT gene expression and inhibited the apoptosis of nerve cells induced by oxidative stress.

[0110] ​Comparison of comparative examples with the model group: The Bax gene expression of comparative examples C and G was higher than that of Example 1 and close to that of the model group; comparative examples 2, 3, 4, A and h were close to the control and significantly lower than that of the model group; indicating that the brain polypeptide raw materials of different processes have different effects on Bax expression, but the brain polypeptide raw material of Example 1 showed an inhibitory effect on Bax gene expression compared with the model group.

[0111] Overall, upon H2O2 stimulation, Bax translocates from the cytoplasm to the mitochondria, oligomerizes to form transmembrane channels, leading to mitochondrial membrane rupture and cytochrome C release. Overexpression exacerbates mitochondrial damage and promotes apoptosis in PC12 neurons under H2O2-induced damage. Brain peptides can downregulate Bax gene mRNA expression in H2O2-oxidatively damaged PC12 neurons, thereby exerting their potential for cognitive enhancement by inhibiting the Bax pathway.

[0112] Depend on Figure 11 As shown: In the effect of different brain polypeptide raw materials on the mRNA gene expression of Bcl-2 in PC12 nerve cells damaged by H2O2 oxidation, the model group and the control group showed that the Bcl-2 gene expression in the model group (H2O2 oxidation damage) was significantly lower than that in the control group, indicating that H2O2 oxidation damage inhibited the expression of Bcl-2 gene.

[0113] Comparison of Example 1 with the model group: In Example 1, the expression of Bcl-2 gene was significantly higher than that in the model group after intervention with brain polypeptide raw materials, indicating that brain polypeptide raw materials can reverse H2O2 oxidative damage to a certain extent and promote the upregulation of Bcl-2 gene expression.

[0114] Comparison of comparative examples with the model group: Comparative examples 4 and A were lower than or close to the control group, while the remaining comparative examples were higher than the control group and the model group. The effects of brain peptide raw materials from different processes on Bcl-2 expression were different, but the brain peptide raw material in Example 1 showed a promoting effect on the upregulation of Bcl-2 gene expression relative to the model group.

[0115] Overall, brain peptide raw materials can significantly upregulate the mRNA expression of the Bcl-2 gene in PC12 neurons damaged by H2O2 oxidation, and may exert the potential to improve intelligence and brain function by activating the Bcl-2 pathway.

[0116] In summary, the regulation of the PI3K / AKT and Bax / Bcl-2 pathways by brain peptide raw materials provides new targets for the prevention and treatment of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. For example, enhancing the activity of the PI3K / AKT pathway can protect dopaminergic neurons from oxidative stress damage, while reducing the Bax / Bcl-2 ratio can delay the process of neuronal apoptosis. Neuronal cell experimental data indicate that brain peptide raw materials have a clear potential for improving cognitive function and brain health.

[0117] 8. Molecular docking experiment Chromatographic conditions, mass spectrometry conditions and data analysis refer to "Tibetan Medicine Short-stalked Rabbit Ear Grass Regulates URAT1 Active Ingredient Screening Based on Target Cell Capture and Molecular Docking", mass spectrometry identification is entrusted to Beijing Baitai Piker Biological Technology Co., Ltd., and is given "Peptide Sequence Analysis Project Report Based on LC-MS / MS" for analysis.

[0118] The raw file collected by mass spectrometry contains more than 2-21 peptides 3000, and since 2 peptides are studied more, this screening does not contain 2 peptides. First, select the relative content of the peptide segment > 0.5% according to the peak area (abundance) of the peptide segment of 3 peptides and above, and 42 peptide segments of 3 peptides and above. In the data (http: / / distilldeep.ucd.ie / PeptideRanker / ) for activity scoring and screening, select the result > 0.60, and 6 peptide segments of 3 peptides and above, and further search in the brain polypeptide raw material database BIOPEP (http: / / bis.zju.edu.cn / biopepdbr / index.php), of which 6 peptide segments are new peptide segments that have not been reported. Further search for the toxicity of the 6 peptide segments, and the prediction website predicts that they are non-toxic (https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.php). After software Byonic database retrieval, the peptide segment list is shown below, and the following table is the 6 peptide sequences sorted by peak area (abundance) after screening by peptide sequence, number of amino acids, activity score, peak area (abundance) of peptide segment, relative molecular mass, toxicity experiment, etc. The specific information is shown in Table 5: Table 5. Brain polypeptide raw material sequence and information score

[0119] The molecular docking software is used to dock the above 4 peptide sequences with the three-dimensional structure of TrkB receptor (ID: 4AT3), TREM2 (ID: 5UD7) and AChE (ID: 4EY7) downloaded from PDB database (http: / / www.rcsb.org / ). The docking binding energy data of the 6 peptide sequences and different receptors are shown in Table 6: Table 6. moe (effectiveness measure) analysis of brain polypeptide raw material molecular docking

[0120] CDOCKER ENERGY is an energy value calculated in the CDOCKER docking process. It mainly reflects the overall binding energy of the ligand and the receptor in the docking process. This energy comprehensively considers various interactions between the ligand and the receptor, including van der Waals force, electrostatic interaction, etc. In a physical sense, a lower CDOCKER ENERGY value represents that the ligand binds to the receptor more tightly and more stably.

[0121] Figure 12 As shown, there are van der Waals force, π-cation, salt bridge, alkyl, conventional hydrogen bond, π-alkyl and carbon-hydrogen bond-based interactions between LDAF and TrkB receptor protein; Figure 13 As shown, there are van der Waals force, conventional hydrogen bond, salt bridge, carbon-hydrogen bond and attractive charge-based interactions between LDAF and TREM2 receptor protein; Figure 14 As shown, there are van der Waals force, carbon-hydrogen bond, salt bridge, conventional hydrogen bond and alkyl-based interactions between LDAF and AChE receptor protein; Figure 15 As shown, there are van der Waals force, carbon-hydrogen bond, salt bridge, alkyl, attractive charge, π-alkyl and conventional hydrogen bond-based interactions between DIAF and TrkB receptor protein; Figure 16 As shown, there are van der Waals force, carbon-hydrogen bond, salt bridge, attractive charge, alkyl and conventional hydrogen bond-based interactions between DIAF and TREM2 receptor protein; Figure 17 As shown, there are van der Waals force, carbon-hydrogen bond and conventional hydrogen bond-based interactions between DIAF and AChE receptor protein; Figure 18 As shown, there are van der Waals force, salt bridge, π-anion, attractive charge, alkyl, conventional hydrogen bond, π-alkyl and carbon-hydrogen bond-based interactions between LDAF and TrkB receptor protein; Figure 19 As shown, there are van der Waals force, conventional hydrogen bond, salt bridge, carbon-hydrogen bond and attractive charge-based interactions between LDAF and TREM2 receptor protein; Figure 20 As shown, there are van der Waals force, carbon-hydrogen bond, conventional hydrogen bond and alkyl-based interactions between LDAF and AChE receptor protein; Figure 21 As shown, there are van der Waals force, π-cation, attractive charge, alkyl, conventional hydrogen bond, π-alkyl and carbon-hydrogen bond-based interactions between IGEF and TrkB receptor protein; Figure 22 As shown, there are van der Waals force, salt bridge, conventional hydrogen bond, alkyl and carbon-hydrogen bond-based interactions between IGEF and TREM2 receptor protein; Figure 23 As shown, there are van der Waals force, carbon-hydrogen bond, attractive charge, conventional hydrogen bond and alkyl-based interactions between IGEF and AChE receptor protein; Figure 24As shown, there are van der Waals forces, carbon-hydrogen bonds, attractive charge, pi-pi T-shaped, conventional hydrogen bonds, and alkyl-based interactions between FGDI and TrkB receptor protein; Figure 25 As shown, there are van der Waals forces, conventional hydrogen bonds, salt bridges, carbon-hydrogen bonds, attractive charge, and alkyl-based interactions between FGDI and TREM2 receptor protein; Figure 26 As shown, there are van der Waals forces, carbon-hydrogen bonds, attractive charge, pi-anion, and conventional hydrogen bonds between FGDI and AChE receptor protein; Figure 27 As shown, there are van der Waals forces, salt bridges, pi-cation, conventional hydrogen bonds, alkyl, carbon-hydrogen bonds, and pi-alkyl-based interactions between LGFD and TrkB receptor protein; Figure 28 As shown, there are van der Waals forces, conventional hydrogen bonds, salt bridges, carbon-hydrogen bonds, attractive charge, and alkyl-based interactions between LGFD and TREM2 receptor protein; Figure 29 As shown, there are van der Waals forces, carbon-hydrogen bonds, conventional hydrogen bonds, and amide-pi stacking-based interactions between LGFD and AChE receptor protein.

[0122] TrkB receptor (PDBID: 4AT3) is an important subtype of tyrosine kinase receptor (RTK) as a member of neurotrophin receptor family, mainly expressed on the surface of central nervous system neurons and neural precursor cells, and can specifically bind to brain-derived neurotrophic factor (BDNF), which is a key molecule for regulating neural plasticity and synaptic regeneration.

[0123] TrkB activates downstream pathways by mimicking neurotrophic factors with brain polypeptide raw materials. When BDNF binds to the extracellular domain of TrkB, it induces receptor dimerization and triggers intracellular kinase domain autophosphorylation, thereby activating multiple signaling pathways such as PI3K / AKT. Activation of TrkB can significantly promote neuronal survival, the mechanisms of which include: 1) up-regulating anti-apoptotic protein Bcl-2 expression; 2) enhancing mitochondrial biosynthesis; 3) inhibiting caspase cascade. At the same time, TrkB signal can directly regulate synaptic plasticity, increase postsynaptic membrane AMPA receptor aggregation, and promote dendritic spine morphological remodeling to improve neural network connection efficiency.

[0124] TREM2 receptor (PDBID: 5UD7) belongs to the immunoglobulin superfamily, highly expressed in microglia cells, and can recognize pathological related molecules such as lipoprotein and β-amyloid protein (Aβ) as a nerve inflammation regulation hub. Its activation depends on the coupling with adapter protein DAP12: when the ligand binds to TREM2, DAP12 recruits Syk kinase to start downstream signals, and induces microglial cell function reprogramming. The wisdom and brain health of TREM2 are reflected in: 1) enhancing the phagocytosis of Aβ by microglia; 2) inhibiting excessive inflammatory factor release; 3) promoting myelin repair through the Wnt / β-catenin pathway. The loss of function of TREM2 in Alzheimer's disease will lead to metabolic disorders of microglia cells and accelerate neurodegeneration.

[0125] Acetylcholinesterase AChE (PDBID: 4EY7) as the core hydrolytic enzyme of the cholinergic system, can terminate nerve signal transmission by rapidly degrading synaptic acetylcholine (ACh). Its active center contains a substrate channel composed of a catalytic triad and aromatic amino acids. AChE inhibitors (such as donepezil) can prolong the action time of ACh by competitively binding to the active center, and improve cognitive function. In recent years, research has found that AChE also has non-catalytic function: its C-terminal peptide segment can promote β-amyloid protein aggregation, forming a positive feedback loop to exacerbate neurotoxicity.

[0126] Synergistic mechanism: TrkB and TREM2 cross-talk: BDNF-TrkB pathway can up-regulate TREM2 expression, and after TREM2 activation, the response of neurons to BDNF is enhanced through DAP12 signal, forming a positive cycle of wisdom and brain health.

[0127] AChE regulation network: AChE inhibitors can indirectly activate TrkB signal, while inhibiting AChE-mediated Aβ toxicity, and synergize with the clearance function of TREM2 to delay neurodegeneration.

[0128] Targeted therapy potential: The brain polypeptide raw materials designed in the application (containing sequences LDAF and DIAF, etc.) can bind to the TrkB ligand binding domain, the TREM2 immune recognition region and the AChE peripheral anion site at the same time, and realize the synergy through multi-target points: 1) enhance the BDNF / TrkB signal transduction efficiency; 2) improve the phagocytosis efficiency of microglia cells; 3) selectively inhibit the non-catalytic toxicity of AChE. The triple regulation network of "nerve repair-immune regulation-metabolic balance" constructed thereby provides a new intervention strategy for neurodegenerative diseases.

[0129] Because the brain tissue active brain polypeptide raw materials prepared in the embodiments of the application, the six peptide segments LDAF, DIAF, ALDF, IGEF, FGDI and LGFD are successfully docked with the three receptors TrkB receptor, TREM2 receptor and acetylcholinesterase AChE, and there is a certain binding energy.

[0130] Summary of experimental results: The brain polypeptide raw material with the optimal activity of benefiting intelligence and brain health is screened out through in vitro antioxidant and nerve cell experiments, so as to be used for the activity research of amino acid sequence and nerve protection: The activities of different enzyme compositions are evaluated by in vitro antioxidant, in vitro acetylcholinesterase, PC12 nerve cell proliferation activity, repair of hydrogen peroxide damaged PC12 nerve cells, CAT enzyme activity, SOD enzyme activity, total antioxidant capacity, acetylcholinesterase activity of hydrogen peroxide damaged PC12 nerve cells, P13K, AKT, Bax and Bcl-2 to evaluate the intelligence and brain health of different enzyme compositions. The brain polypeptide raw material prepared by the scheme of the embodiment of the application has the optimal comprehensive performance, and the brain polypeptide raw material has the optimal activity of benefiting intelligence and brain health.

[0131] The brain polypeptide raw material prepared by the embodiment of the application has the characteristics of high level of antioxidant capacity, significant in vitro acetylcholinesterase inhibition, promotion of PC12 nerve cell proliferation, significant repair of hydrogen peroxide damaged PC12 nerve cells, significant enhancement of total antioxidant capacity, significant acetylcholinesterase inhibition of hydrogen peroxide damaged PC12 nerve cells, promotion of P13K, AKT and Bcl-2 related survival gene expression, and inhibition of pro-apoptotic gene Bax expression. The brain polypeptide raw material has strong antioxidant activity, removes free radicals by combining with hydrogen peroxide, and reduces SOD enzyme and CAT enzyme oxidative stress.

[0132] In the embodiment of the application, six brain polypeptide raw materials with the activities of benefiting intelligence and brain health are screened out from all the identified peptide segments in the embodiment by mass spectrometry, virtual screening and molecular docking. They promote the repair of PC12 nerve cells, remove Aβ aggregates, inhibit acetylcholinesterase, promote the expression of survival genes, and inhibit the expression of pro-apoptotic genes to play the role of benefiting intelligence and brain health.

[0133] In summary, the preparation method of the brain polypeptide raw material provided in the application has at least the following action mechanisms and technical effects: (1) The application finds that the brain polypeptide raw material powder prepared by pretreating brain tissue by mincing, homogenizing and medium temperature heat treatment, and then subjecting to specific enzyme hydrolysis combination (first hydrolysis by neutral protease and second hydrolysis by papain) has a high level of effect of benefiting intelligence and brain health.

[0134] (2) The scheme of the application uses specific neutral protease and papain combination for enzyme hydrolysis to achieve the desired effect. It is not that any enzyme hydrolysis combination or other arbitrary protease hydrolysis combination can achieve the effect of the application.

[0135] For the combination of neutral protease and papain: Neutral protease exhibits stable activity in a mild range of pH 6-8, and can well adapt to the processing requirements in neutral environment. It has certain adaptability to temperature change, and can maintain good catalytic efficiency in the range of 30-50℃. Neutral protease is mostly metal protease, which plays a role by hydrolyzing the peptide bond inside the protein, and is suitable for conventional industrial fields such as food processing and detergent.

[0136] Papain has a wide pH adaptation range, and can maintain certain activity between pH 5-8, especially in acidic to neutral environment. It has certain resistance to high temperature, and can still play a catalytic role at a temperature of about 60℃. It also has certain resistance to organic solvents, and is suitable for special industrial scenes such as food, medicine and cosmetics.

[0137] (3) Based on the role and function of the brain polypeptide raw powder prepared above, it can be known that the brain polypeptide raw powder prepared by the application can be applied to products with intelligence and brain health, including but not limited to food and health care products.

[0138] (4) The method of the application uses complete bovine brain tissue as raw material, and uses enzyme hydrolysis-ultrafiltration method, first destroys the nerve cell structure by mincing and homogenizing, then uses enzyme hydrolysis method with specific enzyme hydrolysis combination to release brain polypeptide raw material, and finally separates brain polypeptide raw material with target molecular weight through ultrafiltration. The brain polypeptide raw material required can be prepared by simple operations such as mixing, enzyme hydrolysis and filtration, which combines the advantages of various technologies, has low requirements for equipment, simple process and easy operation, high extraction efficiency, and is convenient for industrial large-scale production.

[0139] In summary, the application scheme extracts and separates the active brain polypeptide raw material in the brain tissue, and obtains brain polypeptide raw powder with intelligence and brain health protection. The brain polypeptide raw material can be used as a raw material component of functional food, and can be applied to products with intelligence and brain health.

[0140] In addition, the application obtains brain polypeptide raw materials by enzymolysis of complete bovine brain tissue, and the brain polypeptide raw materials have the characteristics of high level of antioxidant capacity, significant in vitro acetylcholinesterase inhibition, promotion of PC12 nerve cell proliferation, significant repair of hydrogen peroxide damaged PC12 nerve cells, significant enhancement of total antioxidant capacity, significant acetylcholinesterase inhibition of hydrogen peroxide damaged PC12 nerve cells, promotion of P13K, AKT and Bcl-2 related survival gene expression, and inhibition of pro-apoptotic gene Bax expression. The brain polypeptide raw materials have strong antioxidant activity, and by combining with hydrogen peroxide, free radicals are removed, and SOD enzyme and CAT enzyme excessive oxidative stress is reduced. The amino acid sequence of the brain polypeptide raw materials is analyzed and identified by LC-MS / MS, and the brain polypeptide raw material amino acid sequence with intelligence and brain protection is screened by means of active prediction tool and molecular docking, and then artificially synthesized, and the intelligence and brain activity is verified and the mechanism is further clarified. Theoretical support is provided for the prediction, screening and structure-activity relationship research of the brain polypeptide raw materials. Among them, the mass spectrum analysis is performed on the prepared brain polypeptide raw materials to determine the amino acid linkage sequence, the molecular docking is used to determine the active site, and the synthesis verification is used to determine whether the corresponding activity is possessed, so as to provide a scientific theoretical basis for the production and application of the brain polypeptide raw materials, and has important theoretical value and practical significance for the research of the brain polypeptide raw materials.

[0141] The above only describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a brain polypeptide raw material with intelligence- promoting, brain-protecting and nerve-protecting functions, characterized in that, The method comprises the following steps: S1, pretreatment: after removing part of the white fat from the quarantine qualified bovine brain tissue, the bovine brain tissue is ground, mixed with water at a mass ratio of 1:2-1:4, and subjected to medium temperature heat treatment at 54-56°C for 0.5-1.5h to obtain a pre-hydrolysate; S2, first hydrolysis: the pH of the pre-hydrolysate is adjusted to 6.5-7.5, neutral protease is added in an amount of 3.5-4.5% of the mass of the bovine brain protein, and enzymolysis is performed at 50-60°C for 1-3h to obtain a first hydrolysate; the enzyme activity of the neutral protease is 2800-3600 U / g; S3, second hydrolysis: the pH of the first hydrolysate is adjusted to 6.0-7.0, papain is added in an amount of 1.0-3.0% of the mass of the bovine brain protein, and enzymolysis is performed at 45-65°C for 1.5-2.5h to obtain a second hydrolysate; the enzyme activity of the papain is 4000-12000 U / g; S4, post-treatment: the pH of the second hydrolysate is adjusted to 4.0-5.0, and the second hydrolysate is subjected to centrifugation, activated carbon decolorization and deodorization, diatomite filtration, ultrafiltration, nanofiltration desalting and concentration, and spray drying to obtain the brain polypeptide raw material powder; the molecular weight cut-off of the ultrafiltration is <5 kDa, and the mass content of the concentrated brain polypeptide raw material is 10-25%.

2. The method for preparing brain polypeptide raw materials with brain-boosting, intelligence-enhancing, and nerve-protecting properties according to claim 1, characterized in that, In steps S2 and S3, the enzyme inactivation process is inactivation at 84-86°C for 15-20 min.

3. The method for preparing brain polypeptide raw materials with brain-boosting, intelligence-enhancing, and nerve-protecting properties according to claim 1, characterized in that, In step S4, the activated carbon-diatomite decolorization and deodorization step is: the second hydrolysate is cooled to 50-60°C, activated carbon is added and treated for 0.5-1.5h, and then the activated carbon is removed by centrifugation and diatomite and a 0.45 μm water-based membrane.

4. The method for preparing brain polypeptide raw materials with brain-boosting, intelligence-enhancing, and nerve-protecting properties according to claim 3, characterized in that, The amount of activated carbon added is 3.0-5.0% of the mass of the bovine brain protein.

5. The method for preparing the brain polypeptide raw material with brain-boosting, intelligence-enhancing, and nerve-protecting properties according to claim 3, characterized in that, The centrifugation condition is centrifugation at 5000 rpm for 5-10 min.

6. A brain polypeptide raw material composition with brain-boosting, brain-protecting, and intelligence-enhancing properties, characterized in that, The brain polypeptide raw material composition comprises active peptide segments with a relative molecular weight of less than 5 kDa, including at least one of the peptide sequences LDAF, DIAF, ALDF, IGEF, FGDI and LGFD.

7. The brain polypeptide raw material composition for benefiting intelligence, protecting brain and nerve according to claim 6, characterized in that, Prepared by the method of any one of claims 1-5.

8. A functional product, characterized by The brain polypeptide raw material composition of any one of claims 6-7 as an active ingredient.

9. The functional product of claim 8, wherein, The functional product has at least one of the following functions: (1) DPPH free radical scavenging rate is greater than or equal to 75%; (2) ABTS free radical scavenging rate is greater than or equal to 99%; (3) Acetylcholinesterase inhibition rate is greater than or equal to 85%; (4) Repair of H2O2-induced PC12 nerve cell damage, cell survival rate is greater than or equal to 90%.

10. Use of the brain polypeptide raw material composition of any one of claims 6-7 in the preparation of a product for inhibiting acetylcholinesterase activity, scavenging free radicals or repairing oxidative damage of nerve cells.

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