Extraction method of yak brain myelopeptide

Abstract

The invention discloses an extraction method of yak brain myelopeptide, and belongs to the technical field of biology. Aiming at the technical bottlenecks of low peptide yield, large activity loss and the like in a traditional extraction process, brain tissue impurities are removed through ethanol degreasing pretreatment, and then a cell structure is destroyed through high-pressure treatment with specific pressure-temperature-time parameters, so that the release efficiency of active ingredients in brain tissues is remarkably improved; further carrying out directional enzymolysis by adopting trypsin to obtain a product of which the small molecule peptide accounts for more than or equal to 75 percent. Compared with a traditional process, the method has the advantages that the yield of active peptide and the proportion of small molecule peptide are remarkably increased, meanwhile, efficient production is achieved by shortening the reaction time and reducing the enzyme dosage, and the method is suitable for large-scale application in the fields of functional food, pharmaceutical preparations and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method for extracting yak brain myelin peptide, in particular to a method for preparing high-yield and high-activity yak brain myelin peptide by combining high-pressure pretreatment with enzymatic hydrolysis technology. Background Art

[0002] Peptides, due to their unique biological activities and functional properties, have broad application potential in medicine, food, cosmetics, and other fields. In recent years, with the increasing demand for health and functional foods, the research and development of peptides has become a hot topic. In particular, peptides derived from animal brain tissue have garnered widespread attention from academia and industry due to their neuroprotective, antioxidant, and anti-inflammatory properties.

[0003] Traditional methods for extracting yak brain myelin peptides mainly use enzymatic hydrolysis or hot water extraction, but these methods have problems such as low peptide yield, serious loss of active ingredients, and low proportion of small molecule peptides, which limit their application in food, medicine and functional products.

[0004] In recent years, high-pressure treatment (HPTP) has garnered widespread attention due to its ability to effectively disrupt cell structure and improve enzymatic hydrolysis efficiency. Combining HPTP with enzymatic hydrolysis can fully leverage the advantages of both technologies: HPTP can significantly increase the release efficiency of active substances in brain tissue, providing favorable substrate conditions for subsequent enzymatic hydrolysis reactions; and enzymatic hydrolysis can produce functionally active small-molecule peptides under mild conditions, significantly improving the yield and functional activity of the products.

[0005] Currently, there is still considerable room for optimization in the extraction of animal brain peptides, particularly in terms of increasing peptide yield, optimizing molecular weight distribution, and enhancing functional activity. Developing an efficient and stable extraction process is crucial for producing highly functional animal brain peptides. Furthermore, with the increasing demand for functional foods, developing an extraction process that can meet the demands of industrial production also has significant economic value. Summary of the Invention

[0006] The present invention provides a yak brain myelin peptide extraction process, which significantly improves the peptide extraction efficiency, optimizes the molecular weight distribution, and enhances its functional activity in neuroprotection, anti-oxidation, and anti-inflammatory aspects through high-pressure pretreatment combined with enzymatic hydrolysis technology.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A method for extracting yak brain myelin peptide, characterized by comprising the following steps:

[0009] S1. Pretreatment: Fresh yak brain was stripped of gray and white matter, and the medulla was collected. The brain was then soaked in a polar solvent and extracted with a non-polar solvent, centrifuged to remove lipids, and homogenized.

[0010] S2, high pressure treatment: the homogenized tissue is mixed with buffer and treated at a pressure of 150-400 MPa and 4-40°C for 8-15 minutes;

[0011] S3, enzymatic hydrolysis reaction: adding protease to the high pressure treatment solution, and performing enzymatic hydrolysis at pH 7.5-8.5 and 40-50°C for 4-8 hours; terminating the reaction by heat inactivation;

[0012] S4. Centrifugal separation: collect the supernatant by centrifugation to obtain yak brain myelin peptide extract.

[0013] In some embodiments of the present invention, in step S1: the polar solvent is a 60-90% ethanol solution, and the immersion time is 10-20 minutes; the non-polar solvent is a n-hexane-isopropanol mixture with a volume ratio of 2:8 to 5:5, and the liquid-to-solid ratio during extraction is 3:1 to 8:1 (mL / g); and the centrifugal separation conditions are 5000-10000g for 5-15 minutes.

[0014] In some embodiments of the present invention, in step S1: the polar solvent is 80% ethanol, and the immersion time is 15 minutes; the non-polar solvent is n-hexane-isopropanol (3:7, v / v), and the liquid-solid ratio is 5:1 (mL / g); and the centrifugal separation conditions are 8000g and 10 minutes.

[0015] In some embodiments of the present invention, the specific operations of step S1 include: a. immersing the isolated brain medulla in 80% ethanol (liquid-to-solid ratio 3:1) and ultrasonically treating it at 40 kHz for 15 minutes; b. transferring it to a mixture of n-hexane-isopropanol (3:7, v / v) (liquid-to-solid ratio 5:1) and shaking it at 200 rpm for 20 minutes; c. centrifuging at 8000g for 10 minutes, discarding the supernatant, and washing the precipitate three times with pre-chilled PBS (pH 7.4).

[0016] In some embodiments of the present invention, in step S2: the buffer is 20-100 mM phosphate buffer (pH 7.0-8.0); the high pressure treatment pressure is 200-300 MPa, the treatment time is 8-15 minutes, and the temperature is 30-35°C.

[0017] In some embodiments of the present invention, in step S2: the buffer is 50 mM phosphate buffer (Na2HPO4 / NaH2PO4, pH 7.8); the high pressure treatment pressure is 250 MPa, the treatment time is 10 minutes, and the temperature is 35°C.

[0018] In some embodiments of the present invention, the process parameters of step S2 are: mixing the tissue with 50 mM PBS buffer at a ratio of 1:3 (w / v); high pressure treatment at 250 MPa for 10 minutes, and maintaining 35° C. during the treatment process.

[0019] In some embodiments of the present invention, in step S3: the protease is trypsin, and the enzyme to substrate mass ratio is 1-5% (w / w); the enzymatic reaction is at pH 7.8-8.2, at a temperature of 45°C, and for 5-6 hours; the heat inactivation conditions are 90-100°C for 8-12 minutes; and the centrifugation conditions are 8000-12000g for 10-20 minutes.

[0020] In some embodiments of the present invention, in step S3: the mass ratio of trypsin to substrate is 3% (w / w), and 0.1 mM CaCl2 is added; the enzymatic reaction pH is dynamically controlled at 8.0±0.1, the temperature is 45±0.5°C, and the time is 6 hours; and heat inactivation is performed in a 95°C water bath for 10 minutes.

[0021] In some embodiments of the present invention, the enzymatic hydrolysis control of step S3 includes: adding trypsin (3% E / S) with a specific activity ≥2500 USP U / mg; the reaction system contains 0.1 mM CaCl2 and is shaken in a 45°C water bath (120 rpm) for 6 hours; monitoring and adjusting the pH to 8.0±0.1 every 30 minutes; and immediately cooling to 4°C in an ice bath after inactivation.

[0022] In some embodiments of the present invention, in step S4: the enzymatic hydrolysate is centrifuged at 8000-12000 g for 10-20 minutes, the precipitate and fat layer are removed, and the supernatant is collected as the crude peptide solution.

[0023] In some embodiments of the present invention, in step S4: the centrifugation conditions are 10000 g and 15 minutes.

[0024] In some embodiments of the present invention, the centrifugation operation in step S4 is: centrifugation at 10,000 g for 15 minutes to remove the precipitate, and the supernatant is sterilized through a 0.22 μm filter membrane.

[0025] Compared with the prior art, the beneficial effects of the present invention are mainly:

[0026] The present invention discloses a method for preparing highly active yak brain myelin peptides by high-pressure synergistic enzymatic hydrolysis. In view of the technical bottlenecks such as low peptide yield and large activity loss in traditional extraction processes (as shown in Comparative Examples 4 and 5), the release efficiency of active ingredients from brain tissue is significantly improved by combining ethanol degreasing pretreatment with high-pressure wall breaking (150-300MPa, 8-15 minutes, 25-40°C); further, trypsin-directed enzymatic hydrolysis (enzyme-substrate ratio 1-4%, pH 7.5-8.5, 40-50°C, 4-8 hours) is used to accurately control the molecular weight distribution and obtain a product in which small molecule peptides (<3kDa) account for ≥75%. This method achieves a total peptide yield of 50-59.24 mg / g wet weight tissue, which is more than 60% higher than the traditional process. The product has excellent antioxidant activity (DPPH clearance 61.57-70.11%) and neuroprotective function (restoring the survival rate of H2O2-damaged cells from 50.35% to 93.95%). The yak brain myelin peptide prepared by the present invention can be widely used in the fields of functional foods, neuroprotective drugs and anti-aging cosmetics. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be further described below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the following examples are only intended to illustrate the present invention and should not be construed as limiting the present invention. Specific conditions not specified in the examples are to be followed according to conventional conditions or the conditions recommended by the manufacturer. All reagents or instruments used, unless otherwise specified, are conventional products obtained from commercial channels.

[0028] Example 1

[0029] A method for extracting yak brain myelin peptide comprises the following steps:

[0030] S1. Pretreatment: Fresh yak brain was obtained and the gray and white matter were quickly dissected in a clean environment at 4°C. The medulla (including the corpus callosum, internal capsule, and other white matter regions) was collected. The medulla was initially delipidated by soaking in 80% ethanol for 15 minutes. The medulla was then extracted with a mixture of n-hexane and isopropanol (3:7, v / v) at 200 rpm for 20 minutes. The mixture was centrifuged at 8000 g for 10 minutes, the upper organic phase was discarded, and the precipitate was collected. The precipitate was washed three times with pre-chilled phosphate buffer (pH 7.4), centrifuged at 10,000 g for 5 minutes each time, and liquid nitrogen-chilled Tris-HCl buffer (20 mM, pH 7.4) was added at a ratio of 1:3 (w / v). The precipitate was homogenized in a high-speed homogenizer at 12,000 rpm for 2 minutes. S2. High-pressure treatment: Weigh 100 g of the yak brain homogenized in step S1, add PBS buffer at a liquid-to-solid ratio of 2:1 (mL / g wet weight tissue), and treat in a high-pressure homogenizer at 35°C and 200 MPa for 10 minutes. Then remove the sample and cool it to room temperature.

[0031] S3. Enzymatic Hydrolysis: Add pre-chilled 50 mM PBS buffer at a liquid-to-solid ratio of 5:1 (mL / g wet weight tissue) to the sample after high-pressure treatment in step S2. Add distilled water to a total volume of 200 mL and fine-tune the pH to 8.0 with 1 M NaOH / HCl. Add trypsin (specific activity 10,000 BAEE units / mg, Sigma Catalog No. T8003) at 3% (w / w) of the substrate mass. Simultaneously add 0.1 mM CaCl2 as an enzyme protectant. Incubate in a 45°C waterbath for 6 hours, monitoring the pH every 30 minutes and adjusting it to 8.0. Quickly transfer the reaction mixture to a 95°C waterbath for 10 minutes to completely inactivate the enzyme, then immediately cool to 4°C in an ice bath. S4. Centrifugation: Centrifuge at 10,000 g for 15 minutes. Discard the precipitate and fat layer, and collect the supernatant as the crude peptide solution.

[0032] Example 2

[0033] A method for extracting yak brain myelin peptide comprises the following steps: high pressure treatment for 15 minutes, and other parameters are the same as those in Example 1.

[0034] Example 3

[0035] A method for extracting yak brain myelin peptide comprises the following steps: the enzyme addition amount is 2%, and other parameters are the same as those in Example 1.

[0036] Example 4

[0037] A method for extracting yak brain myelin peptide comprises the following steps: high pressure treatment at a pressure of 200 MPa, a high pressure treatment time of 15 minutes, an enzyme addition amount of 1%, and other parameters being the same as those in Example 1.

[0038] Example 5

[0039] A method for extracting yak brain myelin peptide comprises the following steps: high pressure treatment at a pressure of 200 MPa, an enzyme addition amount of 2%, an enzyme treatment time of 4 hours, and other parameters are the same as those in Example 1.

[0040] Example 6

[0041] A method for extracting yak brain myelin peptide comprises the following steps: high pressure treatment at a pressure of 200 MPa, a high pressure treatment time of 8 minutes, an enzyme addition amount of 2%, an enzyme treatment time of 8 hours, and other parameters are the same as those in Example 1.

[0042] Example 7

[0043] A method for extracting yak brain myelin peptide comprises the following steps: high pressure treatment at a pressure of 150 MPa, a high pressure treatment time of 8 minutes, an enzyme treatment time of 4 hours, and other parameters are the same as those in Example 1.

[0044] Example 8

[0045] A method for extracting yak brain myelin peptide comprises the following steps: a high pressure treatment pressure of 150 MPa, and other parameters are the same as those in Example 1.

[0046] Example 9

[0047] A method for extracting yak brain myelin peptide comprises the following steps: high pressure treatment at a pressure of 150 MPa, a high pressure treatment time of 15 minutes, an enzyme addition amount of 2%, an enzyme treatment time of 8 hours, and other parameters are the same as those in Example 1.

[0048] Example 10

[0049] A method for extracting yak brain myelin peptide comprises the following steps: high pressure treatment at a pressure of 300 MPa, a high pressure treatment time of 8 minutes, an enzyme treatment time of 4 hours, and other parameters are the same as those in Example 1.

[0050] Example 11

[0051] A method for extracting yak brain myelin peptide comprises the following steps: high pressure treatment at a pressure of 300 MPa, an enzyme addition amount of 4%, and other parameters being the same as those in Example 1.

[0052] Example 12

[0053] A method for extracting yak brain myelin peptide comprises the following steps: high pressure treatment at a pressure of 300 MPa, a high pressure treatment time of 15 minutes, an enzyme addition amount of 4%, an enzyme treatment time of 8 hours, and other parameters are the same as those in Example 1.

[0054] Comparative Example 1

[0055] A method for extracting yak brain myelin peptide comprises the following steps: high pressure treatment for 20 minutes, and other parameters are the same as those in Example 1.

[0056] Comparative Example 2

[0057] A method for extracting yak brain myelin peptide comprises the following steps: the enzyme addition amount is 8%, and other parameters are the same as those in Example 1.

[0058] Comparative Example 3

[0059] A method for extracting yak brain myelin peptide comprises the following steps: enzyme treatment time is 12 hours, and other parameters are the same as those in Example 1.

[0060] Table 1 Extraction conditions for Examples 1-12 and Comparative Examples 1-3

[0061]

[0062] Comparative Example 4

[0063] The traditional single enzymatic hydrolysis method for extracting yak brain myelin peptides includes the following steps:

[0064] S1. Pretreatment: Gray and white matter were removed from fresh yak brains, and the medulla was collected and soaked in 80% ethanol for 15 minutes. The mixture was extracted with a mixture of n-hexane and isopropanol (3:7, v / v) and homogenized at 10,000-12,000 rpm for 2 minutes.

[0065] S2. Hot water extraction: Weigh 100 g of the homogenate and add phosphate buffer at a ratio of 1:2 between raw material and buffer, adjust the pH to 8.0, add trypsin (enzyme to substrate ratio: 3%), and incubate at 45°C for 6 hours, stirring every 30 minutes. Inactivate the enzyme in a boiling water bath for 10 minutes.

[0066] S3. Centrifugation: Centrifuge at 10,000 g for 15 minutes, discard the bottom precipitate and fat layer, and collect the supernatant as the crude peptide solution.

[0067] Comparative Example 5

[0068] The traditional hot water method for extracting yak brain myelin peptides includes the following steps:

[0069] S1. Pretreatment: Fresh yak brain was stripped of gray and white matter, and the medulla was collected and soaked in 80% ethanol for 15 minutes. The mixture was extracted with a mixture of n-hexane and isopropanol (3:7, v / v) and shaken for 20 minutes. The mixture was homogenized at 10,000-12,000 rpm for 2 minutes.

[0070] S2. Hot water extraction: Weigh 100 g of the homogenate, add phosphate buffer at a ratio of 1:2 between raw material and buffer, stir evenly, and extract at 95°C for 2 hours, stirring every 30 minutes.

[0071] S3. Centrifugation: After the extraction is completed, cool to room temperature, centrifuge at 10,000 g for 15 minutes, and collect the supernatant as the crude peptide solution.

[0072] Test Example 1 Physical and Chemical Analysis

[0073] (1) Determination of total peptide yield (BCA method)

[0074] Prepare standard solutions of bovine serum albumin (BSA) with concentrations of 0, 10, 20, 40, 60, 80, and 100 μg / mL, take 100 μL and add them to a 96-well plate, add 100 μL of BCA reagent, mix well, incubate at 37°C for 30 minutes, and measure the absorbance at 562 nm. Draw a standard curve with BSA concentration as the horizontal axis and absorbance as the vertical axis. Take 100 μL of the crude peptide solution prepared in each embodiment and comparative example and add it to a 96-well plate, add 100 μL of BCA reagent, mix well, and incubate at 37°C for 30 minutes. Measure the absorbance at 562 nm, calculate the peptide concentration according to the standard curve, and convert it into peptide yield:

[0075]

[0076] (2) Determination of small molecule peptide ratio (ultrafiltration + BCA method)

[0077] Take 1mL of crude peptide solution and centrifuge it at 12000g for 15 minutes using a 3kDa ultrafiltration centrifuge tube. Collect the flowthrough (<3kDa) and the retentate (≥3kDa) separately. Wash the retentate layer with 0.5mL of buffer, combine the flowthrough and record the total volume V1. Dissolve the retentate back to the original volume V2 (1mL) with the initial buffer. Take the flowthrough and retentate separately and determine the concentrations C1 and C2 (mg / mL) using the BCA method. Calculate the proportion of small molecule peptides:

[0078]

[0079] (3) Molecular weight distribution determination (SDS-PAGE)

[0080] The crude peptide solution prepared in each example and comparative example was diluted with 0.1% TFA to a concentration of 1 mg / mL, 10 μL was added to 5 μL of SDS-PAGE sample buffer containing β-mercaptoethanol, and mixed. 12% separation gel and 5% stacking gel were prepared, and the sample was added to the sample well. Marker (Precision Plus Protein TM Electrophoresis was performed at 100 V using a Coomassie Brilliant Blue marker. After electrophoresis, the gel was stained in Coomassie Brilliant Blue for 30 minutes at room temperature, rinsed with a destaining solution (methanol:acetic acid:water = 4:1:5), and photographed. Gel images were analyzed with ImageJ software, and the molecular weight distribution of the peptides was calculated based on the markers.

[0081] (4) Purity determination (gel chromatography + UV scanning)

[0082] A Sephadex G-75 gel chromatography column was used with 10 mM PBS + 0.15 M NaCl as the mobile phase at a flow rate of 0.5 mL / min. The crude peptide solution (concentration 1 mg / mL) prepared in each Example and Comparative Example was injected into the column, and the eluate was collected. The absorbance of the eluate at 260 nm and 280 nm was measured, and the 260 / 280 nm absorbance ratio was calculated to assess the purity of the peptide.

[0083] (5) DPPH clearance determination (colorimetric method)

[0084] DPPH was dissolved in anhydrous ethanol to prepare a 0.1 mM solution and stored in the dark. The crude peptide solution prepared in each example and comparative example was diluted with distilled water to a concentration of 2 mg / mL, and 100 μL was added to a 96-well plate. 100 μL of DPPH solution was added, mixed thoroughly, incubated in the dark for 30 minutes, and the absorbance at 517 nm (A 517 The control group was incubated with 100 μL of distilled water and 100 μL of DPPH solution for 30 minutes. DPPH clearance calculation:

[0085]

[0086] Table 2 Physicochemical properties of crude peptides extracted from bovine brain in Examples 1-12 and Comparative Examples 1-3

[0087]

[0088]

[0089] Experimental Example 2: Neuronal Cell Protective Effect

[0090] Set up the following groups:

[0091] Blank group: no H2O2 and no peptide added.

[0092] Model group: H2O2 (final concentration 200 μM) was added without peptide.

[0093] Example 1 group: H2O2 (200 μM) + peptide extracted from Example 1 of the present invention (100 μg / mL) were added.

[0094] Comparative Example 4: Add H2O2 (200 μM) + traditional enzymatic extraction of peptides (100 μg / mL) of Comparative Example 4.

[0095] Comparative Example 5: Add H2O2 (200 μM) + extract the peptide (100 μg / mL) using the hot water method of Comparative Example 5.

[0096] (1) Cell viability assay (CCK-8 method)

[0097] PC12 cells were seeded in 96-well plates, and 100 μL of cell suspension (1×10 4 cells / mL) and cultured for 24 hours until adhered. H2O2 and peptide samples were added according to the group and cultured for 24 hours. 10 μL CCK-8 reagent was added and incubated in the dark for 1-2 hours. The absorbance at 450 nm (A 450 Calculate cell viability:

[0098]

[0099] (2) ROS level detection (DCFH-DA probe fluorescence method)

[0100] Add the DCFH-DA probe (final concentration 10 μM) to the cell culture medium and incubate in the dark for 30 minutes. Wash the cells and resuspend them in PBS. Detect the fluorescence intensity of DCFH-DA using a fluorescence microscope or flow cytometer (excitation wavelength 488 nm, emission wavelength 525 nm). Calculate the ROS fluorescence intensity:

[0101]

[0102] (3) Cell apoptosis detection (Annexin V / PI staining flow cytometry analysis)

[0103] Digest the cells in the culture dish with trypsin, collect them into a centrifuge tube, centrifuge at 1000g for 5 minutes, and discard the supernatant. Resuspend the cells with Annexin V / PI staining buffer and adjust the concentration to 1×10 6 cells / mL. Add 5μL Annexin V-FITC and 5μL PI dye and stain for 15 minutes in the dark. Flow cytometry was used to analyze the cell apoptosis rate (early apoptotic cells: Annexin V + / PI - ; Late apoptotic cells: Annexin V + / PI + ). Calculate the apoptosis rate:

[0104]

[0105] (5) Inflammatory factor expression detection (ELISA test)

[0106] The cell culture supernatant was collected, and the concentration of TNF-α was detected according to the instructions of the ELISA kit, and the expression level of inflammatory factors was calculated (fold vs blank group).

[0107] Table 3 Protective effects of crude peptides from bovine brain extracted by different extraction methods on cells

[0108]

[0109] ROS (Reactive Oxygen Species) are markers of intracellular oxidative stress, including superoxide anions (O2 -), hydrogen peroxide (H2O2), etc. The fluorescence intensity is positively correlated with the intracellular ROS level. The ROS fluorescence intensity of the model group was set to 100%, representing a significant accumulation of ROS in the cells after H2O2 damage (oxidative stress state). In Example 1, the ROS was reduced to 55.6% (a decrease of 44.4%), indicating that the bovine brain myelin peptide extracted by the present invention can scavenge free radicals or activate antioxidant enzymes (such as SOD, CAT), effectively alleviating oxidative damage; the ROS of the comparative example 5 / 6 group was only reduced by 26.5% / 12.1%, indicating that the antioxidant activity of the traditional extraction process is significantly inferior to that of the present invention.

[0110] Cell survival and apoptosis rates reflect the protective effect of bovine brain myelin peptide against H2O2-induced cytotoxicity. Higher survival rates and lower apoptosis rates indicate better maintenance of cell membrane integrity and function. The survival rate of Example 1 was 93.95%, close to normal cell survival (blank group ≈ 100%), and the apoptosis rate was 6.23%, a 79.5% decrease compared to the model group, demonstrating its potent neuroprotective effect. In contrast, the protective effect of Comparative Example 5 / 6 groups was limited, suggesting that traditional processing leads to loss of active ingredients.

[0111] TNF-α is a core pro-inflammatory cytokine, and its expression level reflects the degree of neuroinflammation. The expression level of Example 1 (1.33) was close to that of the blank control (set as 1.0), indicating its ability to inhibit activation of the NF-κB signaling pathway. The anti-inflammatory effect of Comparative Example 5 / 6 (2.02 / 2.53) was limited, demonstrating the key role of high-pressure pretreatment in retaining anti-inflammatory peptides.

[0112] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the same. The basic principles and main features of the present invention have been described above using specific implementation schemes. Modifications or substitutions may be made based on the present invention, but such modifications or substitutions do not detract from the essence of the corresponding technical solutions from the scope of protection claimed by the present invention.

Claims

1. A method for extracting yak brain myelin peptide, characterized in that: The following steps are involved: S1. Pretreatment: Fresh yak brain was dissected of gray and white matter, and the medulla was collected, soaked in ethanol, extracted with a mixture of n-hexane and isopropanol, and defatted, and homogenized. S2, high pressure treatment: the homogenized brain tissue was mixed with buffer at a ratio of 1:1-1:3 (w / v) and treated at a pressure of 150-300 MPa and 25-40°C for 8-15 minutes; S3, enzymatic hydrolysis reaction: adding trypsin to the mixture after high pressure treatment at an enzyme to substrate ratio of 1-5% (w / w), and performing enzymatic hydrolysis at pH 7.5-8.5 and 40-50°C for 4-8 hours; heating to above 90°C to inactivate the enzyme; S4. Centrifugation: Centrifuge the enzymatic hydrolysate at 8000-12000 g for 10-20 minutes to remove the precipitate and fat layer, and collect the supernatant as the crude peptide solution.

2. The method for extracting yak brain myelin peptide according to claim 1, characterized in that: The pretreatment in step S1 includes: soaking in a 60-90% volume fraction ethanol solution for 10-20 minutes, shaking and extracting with a n-hexane-isopropanol mixture with a volume ratio of 2:8 to 5:5 for 20 minutes, with a liquid-to-solid ratio of (3:1) mL to (8:1) g during extraction, and homogenizing at 10000-15000 rpm for 1-10 minutes.

3. The method for extracting yak brain myelin peptide according to claim 1 or 2, characterized in that: The pretreatment in step S1 includes: soaking in 80% ethanol solution by volume for 15 minutes; extracting with a mixture of n-hexane and isopropanol with a volume ratio of 3:7 at a liquid-solid ratio of 5 mL:1 g for 20 minutes; centrifuging at 8000 g for 10 minutes, discarding the supernatant, and washing the precipitate three times with pre-cooled PBS at pH 7.4; and homogenizing in a high-speed homogenizer at 12000 rpm for 2 minutes.

4. The method for extracting yak brain myelin peptide according to claim 1, characterized in that: The high pressure treatment parameters in step S2 are: pressure 200-250 MPa, treatment time 10-12 minutes, and treatment temperature 30-35°C.

5. The method for extracting yak brain myelin peptide according to claim 1 or 4, characterized in that: The high pressure treatment parameters in step S2 are: pressure 250 MPa, treatment time 10 minutes, temperature 35°C.

6. The method according to claim 1, characterized in that The enzymatic reaction parameters in step S3 are: enzyme to substrate mass ratio 1-4%, pH 7.8-8.2, reaction temperature 43-47° C., and reaction time 6-8 hours.

7. The method according to claim 1 or 6, characterized in that The enzymatic reaction parameters in step S3 are: enzyme to substrate mass ratio 3%, pH 8.0, reaction temperature 45° C., and reaction time 6 hours.

8. The method for extracting yak brain myelin peptide according to claim 1, characterized in that: The inactivation treatment in step S3 is carried out in a 95° C. water bath for 8-12 minutes.

9. The method for extracting yak brain myelin peptide according to claim 1, characterized in that: The centrifugation condition in step S4 is: 8000-12000 g for 10-20 minutes.

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