Preparation method and application of yak blood peptide with anti-hypoxia and anti-fatigue functions

Abstract

The invention discloses a preparation method and application of yak blood peptide with anti-hypoxia and anti-fatigue functions. The method comprises the steps of raw material pretreatment, composite enzymolysis (adopting a mixture of enzyme digestion modified lumbrukinase and nattokinase), enzyme deactivation, separation and purification, ferrous chelation, Maillard reaction, concentration and drying and the like. The enzymolysis efficiency and the active peptide yield are remarkably improved through ultrasonic treatment and a composite enzymolysis technology, the stability and the anti-hypoxia capacity of peptide fragments are enhanced through ferrous chelation, and the antioxidant activity and the anti-fatigue activity are further improved through the Maillard reaction. Experiments show that the yak blood peptide prepared by the method can significantly improve the antioxidant enzyme activity, prolong the normal-pressure hypoxia survival time, prolong the swimming exhaustion time and significantly reduce the MDA content of brain tissue and myocardial tissue. The obtained product can be applied to anti-hypoxia and anti-fatigue medicines or health-care foods, and has the advantages of high process efficiency, remarkable activity, high safety and the like.

Description

Technical Field

[0001] This application relates to the field of bioactive peptides, specifically to a method for preparing and applying yak blood peptides with anti-hypoxia and anti-fatigue functions. Background Technology

[0002] With the accelerating pace of modern life, increasing work pressure, and rising demands for high-intensity exercise, hypoxia and fatigue are becoming increasingly common, leading to a sustained growth in market demand for products that combat hypoxia and fatigue. Furthermore, in special environments such as high-altitude operations, polar expeditions, and military training, the body's requirements for hypoxia tolerance and fatigue resistance are even more stringent. Therefore, developing safe and highly effective naturally sourced active substances for combating hypoxia and fatigue has become a research hotspot.

[0003] As a unique livestock species of the plateau, yak blood is rich in nutrients such as proteins, polypeptides, iron, amino acids, and bioactive factors, possessing potential physiological activity. Traditionally, the utilization of yak blood resources has mostly focused on simple processing or direct disposal, resulting in resource waste and insufficient development of its high-value bioactive components. Current technologies for extracting animal blood peptides often employ single enzymatic hydrolysis processes. Commonly used enzymes for preparing active peptides include papain, alkaline protease, pepsin, trypsin, acidic protease, neutral protease, and flavor protease. However, for the preparation of blood peptide products derived from livestock and poultry blood, the enzymatic hydrolysis effects of these commonly used commercial enzymes on livestock and poultry blood and its dry powder are not ideal, exhibiting problems such as low hydrolysis efficiency, low yield of active peptides, and limited functionality. Furthermore, the peptides suffer from poor stability and low bioavailability, making it difficult to meet the functional requirements for anti-hypoxia and anti-fatigue.

[0004] In the research of anti-hypoxia and anti-fatigue active substances, existing products mostly rely on chemical synthesis or single-component extraction, which has drawbacks such as significant side effects and limited efficacy. For example, while some chemically synthesized drugs can relieve fatigue in the short term, long-term use may burden liver and kidney function; natural extracts such as ginsenosides and rhodiolosides, although relatively safe, are limited by the source of raw materials and extraction processes, making it difficult to further enhance their activity. In addition, the preparation process of existing peptide products lacks precise modification and functional enhancement methods for peptides, such as the absence of techniques like metal ion chelation and Maillard reactions, resulting in the products' anti-hypoxia and anti-fatigue activities not being fully realized. Lumbrokinase and nattokinase have significant therapeutic effects on thrombosis, but their application in food and health products still lacks appropriateness.

[0005] The prior art CN112552394A discloses a yak antihypertensive peptide and its preparation method. The yak antihypertensive peptide is mainly prepared by a complex enzymatic hydrolysate of alkaline protease and serine protease, but does not mention lumbrokinase and nattokinase. Summary of the Invention

[0006] To address the shortcomings of existing animal blood peptide extraction processes, such as the prevalence of single-enzyme hydrolysis, low hydrolysis efficiency, low yield of active peptides, and limited functionality that fails to meet the functional requirements for anti-hypoxia and anti-fatigue, this application provides a method for preparing and applying yak blood peptides with anti-hypoxia and anti-fatigue functions. The method involves using yak blood as raw material, enzymatically modifying it with a combination of lumbrokinase and nattokinase, chelating it with FeCl2, and finally generating a Maillard reaction to extract yak blood peptides with anti-hypoxia and anti-fatigue properties.

[0007] This application provides a method for preparing yak blood peptides with anti-hypoxia and anti-fatigue functions, characterized in that the preparation method comprises: (1) Raw material pretreatment: Place yak blood or blood powder in a centrifuge tube and treat it in 20kHz ultrasound for 15 minutes to adjust the pH. (2) Enzymatic hydrolysis: Enzymatic hydrolysis was performed using a compound enzyme hydrolysate at a temperature of 40-50℃ for 4-6 hours to obtain hydrolysate products. (3) Enzyme inactivation: The hydrolysis product is heated at 90-95℃ for 15-20 min to inactivate the enzyme, and the inactivated product is obtained; (4) Separation and purification: The inactivated product is centrifuged at 4500-5000 rpm / min for 20-30 min at room temperature to obtain supernatant. The polysaccharide in the supernatant is precipitated and further separated by ultrafiltration tube to obtain purified yak blood peptide. (5) Ferrous chelation: The yak blood peptide latex FeCl2 was subjected to a chelation reaction, 4 times the volume of anhydrous ethanol was added for precipitation, centrifuged, and the precipitate was collected to obtain ferrous chelated yak blood peptide. (6) Maillard reaction: The ferrous chelated yak blood peptide and D-xylose were placed in a pressure-resistant bottle, dissolved in 20 times the volume of water, and the pH was adjusted to 7.5. The mixture was heated at a preset temperature for 60 min, cooled to room temperature for 10 min, cooled with ice water for 10 min, and centrifuged at 8000 rpm / min for 10 min to obtain the Maillard reaction product of ferrous chelated yak blood peptide.

[0008] (7) Concentration and drying: The ferrous chelated yak blood peptide Maillard reaction product is concentrated and spray-dried to obtain ferrous chelated yak blood peptide Maillard reaction product dry powder.

[0009] (8) Packaging and warehousing: The dry powder of the ferrous chelate yak blood peptide Maillard reaction product is aseptically packaged and stored.

[0010] Preferably, the compound enzymatic hydrolysate is a mixture of enzymatically modified lumbrokinase and nattokinase.

[0011] Preferably, the enzyme-modified lumbrokinase is an immobilized trypsin-modified lumbrokinase.

[0012] Preferably, the pH is adjusted to 6-8 in step (1).

[0013] Preferably, in step (4), the polysaccharides in the supernatant are precipitated using anhydrous ethanol.

[0014] Preferably, the preset temperature in step (6) is 110°C.

[0015] Preferably, the spray drying temperature in step (7) is 125-130℃ at the inlet and 90-110℃ at the outlet.

[0016] Preferably, the extraction method of the enzyme-modified lumbrokinase is as follows: (1) Weigh out Nongda No. 3 earthworms, clean them, soak them in water in a beaker for 2 hours, wash them, and homogenize them at low temperature to obtain Nongda No. 3 homogenate. (2) Add the Nongda No. 3 homogenate to PBS and benzoic acid solution at a ratio of 1:4 (w / v), allow it to autolyze for 8 hours, centrifuge and take the supernatant to obtain crude lumbrokinase extract; (3) Add ammonium sulfate to the crude extract of lumbrokinase for fractional precipitation, centrifuge at 25°C and 8000 rpm / min for 10 min to collect the precipitate, and dialyze to desalt and purify to obtain lumbrokinase dry powder; (4) Enzyme-modified lumbrokinase: Trypsin was immobilized with chitosan to obtain immobilized trypsin. The immobilized trypsin was added to lumbrokinase and incubated in a water bath at 45°C for 1 hour. After filtration, the enzyme-modified lumbrokinase was obtained.

[0017] Another aspect of this application provides the application of yak blood peptide with anti-hypoxia and anti-fatigue functions, wherein the yak blood peptide is used in anti-hypoxia and anti-fatigue drugs or health foods.

[0018] Preferably, the dosage forms of the anti-hypoxia and anti-fatigue drugs or health foods include tablets, capsules, granules, oral liquids, tinctures, powders, injections, and teas.

[0019] The beneficial effects of the embodiments in this application are as follows: (1) Highly efficient enzymatic hydrolysis process: The use of a composite enzymatic hydrolysate of lumbrokinase and nattokinase significantly improves the enzymatic hydrolysis efficiency and the yield of active peptides in yak blood protein. The lumbrokinase, after being modified with immobilized trypsin, has more precise cleavage sites, enabling the release of more active peptides with anti-hypoxia and anti-fatigue functions; nattokinase further assists in hydrolysis, enhancing peptide diversity and bioactivity. Compared with traditional single enzymatic hydrolysis processes, the composite enzymatic hydrolysis method of this application increases the yield of active peptides by more than 30%.

[0020] (2) Enhanced bioactivity: Through ferrous chelation reaction, yak blood peptides are combined with iron ions, which not only improves the stability of peptide segments, but also enhances their anti-hypoxia ability, making them particularly suitable for applications in low-oxygen environments such as plateaus.

[0021] (3) Maillard reaction optimization: Enhanced anti-hypoxia and anti-fatigue function. Through dual modification of ferrous chelation and Maillard reaction, the functionality of yak blood peptides was significantly improved. Ferrous chelation not only improved the stability of peptides, but also enhanced the body's oxygen-carrying capacity by supplementing bioavailable iron. The Maillard reaction product (melanoidin) has antioxidant and anti-fatigue activities, which further synergistically enhanced the anti-hypoxia and anti-fatigue effects of yak blood peptides.

[0022] (4) The raw material is natural yak blood. The enzymatic hydrolysis and modification process adopts physical and biological methods (ultrasonic treatment, enzymatic hydrolysis, ethanol precipitation, etc.) to avoid chemical pollution and ensure high safety. The product can be used in anti-hypoxia and anti-fatigue drugs or health foods, and supports multiple dosage forms such as tablets, capsules, and oral liquids to meet the needs of different scenarios (such as high-altitude operations, high-intensity exercise, and daily health care).

[0023] (5) Process Innovation and Technological Advantages: This method integrates multiple optimized processes, including ultrasonic pretreatment, combined enzymatic hydrolysis (using lumbrokinase and nattokinase), immobilized enzyme modification, metal chelation, and Maillard reaction, to form a highly efficient and controllable yak blood peptide preparation system. Compared with existing technologies (such as single enzymatic hydrolysis or unmodified peptide products), the products prepared by this method have more comprehensive functions and more significant activity, solving the problems of low enzymatic hydrolysis efficiency, single function, and poor stability of traditional products. Attached Figure Description

[0024] Figure 1 This is a schematic diagram showing the hydrolysis temperature of lumbrokinase in Example 1 of this application; Figure 2 This is a schematic diagram of the enzymatic hydrolysis time of lumbrokinase in Example 1 of this application; Figure 3 This is a schematic diagram of the pH experiment results for the preparation of lumbrokinase in Example 4 of this application. Detailed Implementation

[0025] The technical solutions of this application will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0026] Preparation Example 1: Lumbrokinase Extraction (1) Weigh 300g of Nongda No. 3 earthworms, wash them and soak them in water in a beaker for 2 hours to remove some mud and feces from the earthworms. After washing them, homogenize them at low temperature to obtain Nongda No. 3 homogenate. (2) Add 0.01 mol / L PBS (pH=7.8) solution to the homogenate at a ratio of 1:4 (w / v), add 1.2 g sodium benzoate, mix thoroughly, and autolyze in a water bath at 37°C for 8 h. Centrifuge the turbid liquid at 4000 rpm for 30 min using a refrigerated centrifuge, and take the supernatant to obtain crude lumbrokinase extract. (3) Ammonium sulfate fractionation: ammonium sulfate saturation was gradually precipitated in 10% increments from 10% to 90%. The supernatant was placed in a 25°C water bath, and solid ammonium sulfate was added to adjust the saturation. After standing for 6 hours, the mixture was centrifuged at 8000 rpm and 25°C for 10 minutes using a high-speed refrigerated centrifuge. The precipitate was collected, reconstituted with deionized water, and then desalted by dialysis using a dialysis bag with a molecular weight cutoff of 1000 Da for 48 hours to obtain crude lumbrokinase extract powder, which was then freeze-dried and stored. (4) Assay of lumbrokinase activity: Fibrinogen solution and 60℃ agarose solution were mixed at a ratio of 1:1 (vv). While stirring, an appropriate amount of thrombin solution was added. After stirring at low speed and mixing, the mixture was immediately poured into a plastic culture dish and placed horizontally at room temperature. After reacting for 1 hour, holes were punched. 10L of sample and 10L of standard were added, covered, and incubated at 37℃ for 16 hours. The two vertical diameters of the dissolution zone were measured with vernier calipers. A standard curve was plotted with the standard activity as the abscissa and the logarithm of the product of the vertical radii of the standard as the ordinate. The vertical radius of the sample was substituted to calculate the lumbrokinase activity. Three parallel tests were performed, and the average value was used. The enzyme activity of the product precipitated at a concentration of 40% to 50% ammonium sulfate saturation was 35.8% higher than that before autolysis, and the activities were 21825 U / mg and 29638 U / mg, respectively. (5) Determination of degree of hydrolysis: The Kjeldahl method in GB5009.5-2016 "National Food Safety Standard - Determination of Protein in Food" was used for determination. Protein concentration was monitored using the BCA method (Beyotime BCA Protein Concentration Assay Kit). Determination of protein hydrolysis degree: The amount of yak blood peptide prepared is expressed as DH, which refers to the percentage of peptide bonds cleaved during the hydrolysis of yak blood peptide protein out of the total peptide bonds in the protein. Refer to the formaldehyde titration method, see the formula: Where, M - concentration of NaOH standard solution, mol; V1 - volume of 0.05 mol / L NaOH standard solution consumed in the sample titration, mL; V2 - volume of 0.05 mol / L NaOH standard solution consumed in the blank experiment, mL; Cwo - protein concentration in yak blood peptide hydrolysate, g / L; V - volume of hydrolysate titrated with formaldehyde, mL; h tot - The number of millimoles of peptide bonds in the substrate protein; for blood proteins, this value is taken as 8.3 mmol / g. Figure 1As shown, the optimal hydrolysis temperature is controlled at 40-50℃, and the enzymatic hydrolysis time is as follows. Figure 2 As shown, the optimal time is 4-6 hours.

[0027] (6) DEAE Sepharose Fast Flow anion exchange column purification: 1) Sample preparation: DEAESepharose FastFlow anion exchange chromatography column was rinsed with deionized water until no ethanol residue or foam was present. The column was packed into a 16 mm diameter, 200 mm high chromatography column and allowed to settle naturally for 4 hours. The constant flow pump speed was adjusted to a flow rate of 1.0 mL / min, and the column was eluted for 1 hour until the column height stabilized. The height of the packing material was recorded. Several fractions with high activity were collected, dissolved in eluent, and prepared into a 5 mg / mL protein solution. The solution was then filtered through a 0.45 mm organic filter membrane, and the filtrate was collected for later use. 2) Sample loading and elution: The DEAE Sepharose FastFlow anion exchange packing material was rinsed with deionized water until no ethanol residue or foam was present, then packed into a 16 mm diameter, 200 mm high chromatography column. The packing material was allowed to settle naturally for 4 hours. The constant flow pump speed was adjusted to a flow rate of 1.0 mL / min, and the column was eluted for 1 hour until the column height stabilized. The packing height was recorded. Several fractions with high activity were collected, dissolved in eluent, and prepared into a 5 mg / mL protein solution. The solution was then filtered through a 0.45 mm organic filter membrane, and the filtrate was collected for later use.

[0028] (7) Isolation and purification of lumbrokinase: 1) Sample Preparation: Based on the molecular weight distribution of the active components in the ion exchange column, Sephadex G-75 was selected for further separation and purification of the active components. After Sephadex G-75 swelled overnight, it was degassed by a vacuum pump for 2 hours, and then repeatedly washed with deionized water until no foam was present before packing the column. The swollen Sephadex G-75 packing material was packed into a 1.6 x 100 cm glass chromatography column, and the packing material was allowed to settle naturally overnight. The gel column was equilibrated with PBS (pH=7.8) at a flow rate of 0.5 mm / min. The fraction with the best lumbrokinase activity among the separated components was prepared into a 2 mg / hm solution using the mobile phase, filtered through a 0.45 μm organic filter membrane, and then used for later use.

[0029] 2) Sample loading and elution: Open the peristaltic pump outlet and wait until the liquid level drops to tangent to the gel column surface. Accurately transfer 4 μL of filtrate into the chromatography column using a pipette. After the sample solution is completely adsorbed by the packing material, elute with PBS (pH=7.8) at a rate of 0.5 mL / min. Collect the eluent using an automatic collector, collecting one tube every 4 min, for a total of 80 tubes. After elution, measure the absorbance of the fraction at 280 nm using a UV spectrophotometer. Plot the elution curve, determine the kinase activity of the corresponding peak, and desalt using a 3 kDa ultrafiltration tube to obtain purified lumbrokinase, which is then lyophilized and stored.

[0030] (8) Preparation of liquid phase: 1) Weigh 20 mg of purified lumbrokinase lyophilized sample, dissolve it in 300 μL of 0.1 mol / L sodium sulfate + 0.1 mol / L disodium hydrogen phosphate + 0.1 mol / L sodium dihydrogen phosphate aqueous solution, centrifuge at 20000g for 15 min at 4℃, and take the supernatant into a 2 mL autosampler bottle for HPLC detection; 2) Chromatographic conditions: Instrument model: ThermoScitifc Ultimate 3000, Column: Tosoh TSKgelUP-SW3000 (4.6 mm ID. * 30°C, 2 pm), Mobile phase: A: 0.2 mol / L sodium sulfate aqueous solution, B: 0.2 mol / L disodium hydrogen phosphate + 0.2 mol / L sodium dihydrogen phosphate aqueous solution ratio: A:B = 1:1, elution isocratic for 45 min, Detector parameters: DAD detector, UV wavelength: 280 nm; 3) C18 column separation and purification of lumbrokinase: A gradient mobile phase A: ultrapure water, B: methanol was used. The eluent was collected and excess methanol was removed before freeze-drying for later use. SDS-PAGE electrophoresis was performed. Mass spectrometry parameters were acquired and peptides were matched according to the database.

[0031] Preparation Example 2 Preparation of lumbrokinase by enzyme digestion and modification (1) Immobilization of trypsin: Weigh 1.2g of chitosan, add 120mL of 1% acetic acid to prepare a transparent gel solution, then add 2mol / L NaOH to adjust the pH to about 5.5, add 0.1% glutaraldehyde to adjust the pH to 8.0, dry under vacuum, wash, add trypsin, stir magnetically for 30min, add glutaraldehyde, stir with an electric stirrer for 8h, filter and vacuum dry to obtain immobilized trypsin; (2) The immobilized trypsin was added to 10 mL of the purified lumbrokinase solution from Preparation Example 1, and the solution was kept in a water bath at 45°C for 1 h. The lumbrokinase was then filtered to obtain the enzyme-modified lumbrokinase.

[0032] Preparation Example 3 Assay of metal ion activity against lumbrokinase Metal ion solutions of 1, 5, 10, and 20 mmol / L were prepared by reacting CaCl2, FeCl3, FeCl2, MgSO4·H2O, and 20000 U / mL lumbrokinase, respectively. The reactions were carried out at room temperature for 24 h, and the lumbrokinase activity was measured as shown in Table 1. Table 1. Metal ion chelating lumbrokinase activity values

[0033] Among them, relative enzyme activity (%) = (enzyme activity under the given conditions / enzyme activity in the blank group) × 100%, yielding Fe 2+The enzyme activity was highest when prepared as a 5 mmol / L metal ion solution; therefore, a 5 mmol / L Fe solution was used in subsequent experiments. 2+ A chelation reaction is carried out.

[0034] Preparation Example 4 pH effect on lumbrokinase activity assay The activity of extracted lumbrokinase was tested by storing it at different pH values ​​for 24 hours. The results showed that the activity of lumbrokinase remained stable between pH 6 and 8. Figure 3 As shown.

[0035] Example 1 A method for preparing yak blood peptides with anti-hypoxia and anti-fatigue functions (1) Raw material pretreatment: Place 2 mL of purchased yak blood in a centrifuge tube, centrifuge at 3000 rpm for 10 min to remove blood cells, take the supernatant (plasma) and sonicate it at 20 kHz for 15 min, and adjust the pH to 7.0.

[0036] (2) Enzymatic hydrolysis: Add 2 mg of the enzyme-modified lumbrokinase (1000 FU / mg) prepared in Preparation Example 2 and 1 mg of nattokinase (2000 FU / mg, Sigma-Aldrich) in a compound enzyme hydrolysate and hydrolyze at 40℃ for 6 h to obtain the hydrolysate.

[0037] (3) Enzyme inactivation: The hydrolysis product is heated at 95℃ for 20 min to inactivate the enzyme and obtain the inactivated product.

[0038] (4) Separation and purification: The inactivated product was centrifuged at 5000 rpm / min for 30 min at room temperature to obtain the supernatant. The polysaccharides in the supernatant were precipitated and further separated by ultrafiltration tube to obtain purified yak blood peptides.

[0039] (5) Ferrous chelation: The FeCl2 in Preparation Example 3 was used for chelation reaction. Four times the volume of anhydrous ethanol was added for precipitation. The precipitate was collected by centrifugation to obtain ferrous chelated yak blood peptide.

[0040] (6) Maillard reaction: The ferrous chelated yak blood peptide and D-xylose were placed in a pressure-resistant bottle, dissolved in 20 times the volume of water, and the pH was adjusted to 7.5. The mixture was heated at 110°C for 60 min, cooled to room temperature for 10 min, cooled with ice water for 10 min, and centrifuged at 8000 rpm / min for 10 min. The supernatant was then collected to obtain the Maillard reaction product of the ferrous chelated yak blood peptide.

[0041] (7) Concentration and drying: The ferrous chelate yak blood peptide Maillard reaction product is concentrated and spray-dried. The spray drying temperature is set to 130℃ at the inlet and 110℃ at the outlet, and the time is 1h to obtain the dry powder of the ferrous chelate yak blood peptide Maillard reaction product.

[0042] (8) Packaging and warehousing: The dry powder of the ferrous chelate yak blood peptide Maillard reaction product is aseptically packaged and stored.

[0043] Example 2 A method for preparing yak blood peptides with anti-hypoxia and anti-fatigue functions (1) Raw material pretreatment: Place 2 mL of purchased yak blood in a centrifuge tube, centrifuge at 3000 rpm for 10 min to remove blood cells, take the supernatant (plasma) and sonicate it at 20 kHz for 15 min, and adjust the pH to 8.0.

[0044] (2) Enzymatic hydrolysis: Add 2 mg of the enzyme-modified lumbrokinase (1000 FU / mg) prepared in Preparation Example 1 and 1 mg of nattokinase (2000 FU / mg, Sigma-Aldrich) in a compound enzyme hydrolysate and hydrolyze at 50°C for 4 h to obtain the hydrolysate.

[0045] (3) Enzyme inactivation: The hydrolysis product is heated at 90℃ for 15 min to inactivate the enzyme, and the inactivated product is obtained.

[0046] (4) Separation and purification: The inactivated product was centrifuged at 5000 rpm / min for 30 min at room temperature to obtain the supernatant. The polysaccharides in the supernatant were precipitated and further separated by ultrafiltration tube to obtain purified yak blood peptides.

[0047] (5) Ferrous chelation: The yak blood peptide latex FeCl2 was chelated, and 4 times the volume of anhydrous ethanol was added for precipitation. After centrifugation, the precipitate was collected to obtain ferrous chelated yak blood peptide.

[0048] (6) Maillard reaction: The ferrous chelated yak blood peptide and D-xylose were placed in a pressure-resistant bottle, dissolved in 20 times the volume of water, and the pH was adjusted to 7.5. The mixture was heated at 110°C for 60 min, cooled to room temperature for 10 min, cooled with ice water for 10 min, and centrifuged at 8000 rpm / min for 10 min. The supernatant was then collected to obtain the Maillard reaction product of the ferrous chelated yak blood peptide.

[0049] (7) Concentration and drying: The ferrous chelate yak blood peptide Maillard reaction product is concentrated and spray-dried. The spray drying temperature is set to 125℃ at the inlet and 110℃ at the outlet, and the time is 1h to obtain the dry powder of the ferrous chelate yak blood peptide Maillard reaction product.

[0050] (8) Packaging and warehousing: The dry powder of the ferrous chelate yak blood peptide Maillard reaction product is aseptically packaged and stored.

[0051] Example 3 (without ultrasound) A method for preparing yak blood peptides with anti-hypoxia and anti-fatigue functions (1) Raw material pretreatment: Place 2 mL of purchased yak blood in a centrifuge tube, centrifuge at 3000 rpm for 10 min to remove blood cells, and take the supernatant (plasma).

[0052] (2) Enzymatic hydrolysis: Same as in Example 1.

[0053] (3) Enzyme inactivation: Same as in Example 1.

[0054] (4) Separation and purification: Same as in Example 1.

[0055] (5) Ferrous chelation: Same as in Example 1.

[0056] (6) Maillard reaction: Same as in Example 1.

[0057] (7) Concentration and drying: Same as in Example 1.

[0058] (8) Packaging and warehousing: Same as in Example 1.

[0059] Example 4 (without ferrous chelation) A method for preparing yak blood peptides with anti-hypoxia and anti-fatigue functions (1) Raw material pretreatment: Same as in Example 1.

[0060] (2) Enzymatic hydrolysis: Same as in Example 1.

[0061] (3) Enzyme inactivation: Same as in Example 1.

[0062] (4) Separation and purification: Same as in Example 1.

[0063] (5) Maillard reaction: Same as in Example 1.

[0064] (6) Concentration and drying: Same as in Example 1.

[0065] (7) Packaging and warehousing: The dry powder of the ferrous chelate yak blood peptide Maillard reaction product is aseptically packaged and stored.

[0066] Example 5 (No Maillard reaction) A method for preparing yak blood peptides with anti-hypoxia and anti-fatigue functions (1) Raw material pretreatment: Same as in Example 1.

[0067] (2) Enzymatic hydrolysis: Same as in Example 1.

[0068] (3) Enzyme inactivation: Same as in Example 1.

[0069] (4) Separation and purification: Same as in Example 1.

[0070] (9) Ferrous chelation: Same as in Example 1.

[0071] (5) Concentration and drying: Same as in Example 1.

[0072] (6) Packaging and warehousing: The dry powder of the ferrous chelate yak blood peptide Maillard reaction product is aseptically packaged and stored.

[0073] Example 6 (using nattokinase only) A method for preparing yak blood peptides with anti-hypoxia and anti-fatigue functions (1) Raw material pretreatment: Same as in Example 1.

[0074] (2) Enzymatic hydrolysis: Add 1 mg of nattokinase (2000 FU / mg, Sigma-Aldrich) compound enzymatic hydrolysate and hydrolyze at 40℃ for 6 h to obtain hydrolysate.

[0075] (3) Enzyme inactivation: Same as in Example 1.

[0076] (4) Separation and purification: Same as in Example 1.

[0077] (5) Ferrous chelation: Same as in Example 1.

[0078] (6) Maillard reaction: Same as in Example 1.

[0079] (7) Concentration and drying: Same as in Example 1.

[0080] (8) Packaging and warehousing: Same as in Example 1.

[0081] Comparative example (using papain) A method for preparing yak blood peptides with anti-hypoxia and anti-fatigue functions (1) Raw material pretreatment: Same as in Example 1.

[0082] (2) Enzymatic hydrolysis: Add 1 mg of papain (Sigma-Aldrich, purity ≥90%), hydrolyze at 40℃ for 6 h to obtain hydrolysate.

[0083] (3) Enzyme inactivation: Same as in Example 1.

[0084] (4) Separation and purification: Same as in Example 1.

[0085] (5) Ferrous chelation: Same as in Example 1.

[0086] (6) Maillard reaction: Same as in Example 1.

[0087] (7) Concentration and drying: Same as in Example 1.

[0088] (8) Packaging and warehousing: Same as in Example 1.

[0089] Experimental Example 1 Hypoxia resistance test (1) Experimental materials and animals: SPF-grade KM mice, male, weighing 18-22g, purchased from Chengdu Dashuo Experimental Animal Center; yak blood peptides prepared in each of Examples 1-4, Example 6 and the comparative example of this application.

[0090] (2) Experimental methods: 1) Mice were randomly divided into 8 groups of 10 mice each: a control group (equal volume of physiological saline), a Rhodiola rosea positive drug group, and a group receiving 700 mg / kg of yak blood peptide prepared in Examples 1-4, Example 6, and the comparative example. Mice were administered the solution by gavage once daily for 30 days, with free access to food and water. The dosage of Rhodiola rosea was calculated based on the human dosage (3 tablets twice daily), and the Rhodiola rosea granules were dissolved in distilled water to prepare a 36.4 mg / kg gavage solution, administered at a dose of 10 mL / kg. 2) Anti-hypoxia test: After 30 days of gavage, blood was collected from the inner canthal vein of the mouse orbit to measure MDA content (MDA test kit, Nanjing Jiancheng Biotechnology), SOD activity (SOD test kit, Nanjing Jiancheng Biotechnology), GSH-Px activity (GSH-Px test kit, Nanjing Jiancheng Biotechnology), and T-AOC (T-AOC test kit, Nanjing Jiancheng Biotechnology). According to the provisions of the "Technical Specifications for Inspection and Evaluation of Health Food (2023 Edition)", the mice were subjected to normobaric hypoxia treatment after 30 days of gavage to establish a normobaric hypoxia mouse model. When the experimental mice stopped breathing, they were sacrificed and the survival time was recorded. The brain and myocardial tissue of the mice were taken and stored at -80℃.

[0091] 3) Experimental data: The statistical method was one-way ANOVA + Tukey multiple comparisons. Three parallel experiments were performed and the average value was taken. The results are shown in Table 2. The survival time under normobaric hypoxia is shown in Table 3. The effect of yak blood peptide on the MDA content of brain and myocardial tissues of normobaric hypoxic mice is shown in Table 4. Table 2. Effects of yak blood peptides on MDA content, SOD, GSH-Px and T-AOC activity in mice.

[0092] Among them, #: significant difference compared with the control group (p<0.05), ##: extremely significant difference compared with the control group (p<0.01).

[0093] Table 3 Survival time under normal pressure hypoxia

[0094] Table 4. Effects of yak blood peptides on MDA content in brain and myocardial tissues of mice under normobaric hypoxia.

[0095] (3) Experimental results: 1) The MDA content obtained from Table 2: The MDA content of the positive control group, Example 1 group, and Example 2 group was lower than that of the control group, but the difference was not significant. The MDA content of Example 3 group, Example 4 group, Example 6 group, and comparative group was close to that of the control group, with no significant difference; the positive control group, Example 1 and Example 2 groups had a certain effect on reducing the oxidative damage index MDA, but the effect was not significant.

[0096] 2) Table 2 shows that the SOD activity of the positive control group, Example 1 group and Example 2 group was significantly higher than that of the control group. The SOD activity of other experimental groups was not significantly different from that of the control group. The positive control group and Example 1 and 2 groups can significantly improve SOD activity and enhance antioxidant capacity.

[0097] 3) As shown in Table 2, the GSH-Px activity in the positive control group, Example 1 group, and Example 2 group was significantly higher than that in the control group. The GSH-Px activity in other experimental groups was not significantly different from that in the control group. The positive control group and Example 1 and 2 groups could significantly enhance the GSH-Px activity, further supporting its antioxidant effect.

[0098] 4) T-AOC activity obtained from Table 2: The T-AOC activity of the positive control group was significantly higher than that of the control group. The T-AOC activity of the Example 1 group and Example 2 group was significantly higher than that of the control group. The T-AOC activity of other experimental groups was not significantly different from that of the control group. The positive control and Example 1 and 2 groups can significantly or extremely significantly improve the total antioxidant capacity (T-AOC).

[0099] 5) Table 2 shows that the positive control group, Example 1 group, and Example 2 group showed significantly or extremely significantly better performance than the control group in improving antioxidant indicators (SOD, GSH-Px, T-AOC). They reduced oxidative stress by enhancing antioxidant enzyme activity and total antioxidant capacity. The antioxidant effects of Example 3 group, 4 group, 6 group, and comparative group were not significantly different from those of the control group, indicating that their effects were limited. Although the trend of MDA content change was not significant, it was consistent with the increase in antioxidant enzyme activity, suggesting that oxidative damage may be alleviated.

[0100] 6) Table 3 shows that the hypoxic survival time of groups 1 and 2 of Examples was significantly prolonged (similar to the effect of positive control drugs), while there was no significant difference in other groups.

[0101] 7) As shown in Table 4, under normal pressure hypoxia, the results of groups 1-2 and the positive control group were consistent. The MDA content in brain tissue was significantly lower than that in the control group, and the MDA content in myocardial tissue was also significantly reduced. This can greatly enhance the tissue's antioxidant capacity (reduce lipid peroxidation product MDA) and improve its anti-hypoxia capacity.

[0102] Experimental Example 2 fatigue test (1) Experimental materials: 100 male Kunming mice, 7 weeks old, weighing 25g±2g, were selected and kept in a clean environment at 25℃ with natural light and good ventilation.

[0103] (2) Experimental grouping: The experimental animals were divided into 6 experimental groups: Example 1 group, Example 2 group, Example 3 group, Example 4 group, Example 6 group and comparative group, blank group. They were given the same amount of physiological saline. There were 10 mice in each group. Each group was fed separately. The mice in the experimental group were allowed to eat and drink freely for 2 days and then started to be administered by gavage. The administration was continued for 20 days.

[0104] (3) Experimental protocol: 30 minutes after the last administration of the drug, the mice in each group were placed in a constant-temperature water body at 25°C and allowed to swim freely. The time to exhaustion was recorded. The time to exhaustion was defined as the time from when the mouse started swimming until its head was completely submerged below the surface for 10 seconds. If the mouse remained motionless on the surface, it was stirred with a glass rod until it could no longer move. The swimming time extension rate was calculated using the formula: LR / % = (T1-T0) / T0 × 100%, where LR is the extension rate (%), T1 is the swimming time of the experimental group mice (min), and T0 is the swimming time of the control group mice (min).

[0105] (4) Experimental data: as shown in Table 5; Table 5. Data on the effect of yak blood peptide on swimming exhaustion time in mice.

[0106] (5) Test results: Anti-fatigue effect: The swimming exhaustion time of groups 1 and 2 in Examples 1 and 2 was significantly prolonged, with extension rates of 42.6% and 39.8% respectively, indicating that the compound enzymatic hydrolysis, ferrous chelation and Maillard reaction synergistically enhanced the anti-fatigue function.

[0107] Experimental Example 3 Maillard response sensory analysis A sensory evaluation team consisting of 10 people (aged 20-30) with rich sensory evaluation experience was formed. Before the experiment, relevant sensory training was required. Sensory comparison was conducted between Example 1 and Example 5. Example 1 mainly had caramel aroma, roast aroma and umami flavor; while Example 5 had metallic fishy smell, bitterness and blandness. Therefore, the Maillard reaction odor is more acceptable.

[0108] In summary, this application has successfully developed a method for preparing yak blood peptides with significant anti-hypoxia and anti-fatigue functions by innovatively integrating multiple optimized processes, including ultrasonic pretreatment, combined enzymatic hydrolysis (using a combination of enzymatically modified lumbrokinase and nattokinase), ferrous chelation, and Maillard reaction. This method not only significantly improves enzymatic hydrolysis efficiency and the yield of active peptides (by more than 30%), but also enhances the stability and oxygen-carrying capacity of peptides through ferrous chelation, and further improves antioxidant and anti-fatigue activities through the Maillard reaction. Animal experiments have confirmed that this product can significantly increase the activity of antioxidant enzymes such as SOD and GSH-Px, prolong survival time under normobaric hypoxia (up to 36.8 minutes) and swimming exhaustion time (prolonged by 42.6%), while significantly reducing MDA content in brain and myocardial tissues. This invention features a highly efficient and controllable process, and the product exhibits significant activity. It can be widely applied in the fields of anti-hypoxia and anti-fatigue drugs and health foods, providing a safe and effective solution for hypoxia and fatigue problems in scenarios such as high-altitude operations and high-intensity exercise.

[0109] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application. All equivalent changes and improvements made within the scope of this application shall still fall within the patent coverage of this application.

Claims

1. A method for preparing yak blood peptides with anti-hypoxia and anti-fatigue functions, characterized in that, The preparation method is as follows: (1) Raw material pretreatment: Place yak blood or blood powder in a centrifuge tube and treat it in 20kHz ultrasound for 15 minutes to adjust the pH. (2) Enzymatic hydrolysis: Enzymatic hydrolysis was performed using a compound enzyme hydrolysate at a temperature of 40-50℃ for 4-6 hours to obtain hydrolysate products. (3) Enzyme inactivation: The hydrolysis product is heated at 90-95℃ for 15-20 min to inactivate the enzyme, and the inactivated product is obtained; (4) Separation and purification: The inactivated product is centrifuged at 4500-5000 rpm / min for 20-30 min at room temperature to obtain supernatant. The polysaccharide in the supernatant is precipitated and further separated by ultrafiltration tube to obtain purified yak blood peptide. (5) Ferrous chelation: The yak blood peptide latex FeCl2 was subjected to a chelation reaction, 4 times the volume of anhydrous ethanol was added for precipitation, centrifuged, and the precipitate was collected to obtain ferrous chelated yak blood peptide. (6) Maillard reaction: The ferrous chelated yak blood peptide and D-xylose were placed in a pressure-resistant bottle, dissolved in 20 times the volume of water, and the pH was adjusted to 7.

5. The mixture was heated at a preset temperature for 60 min, cooled to room temperature for 10 min, cooled with ice water for 10 min, and centrifuged at 8000 rpm / min for 10 min to obtain the supernatant product of the ferrous chelated yak blood peptide Maillard reaction. (7) Concentration and drying: The ferrous chelate yak blood peptide Maillard reaction product is concentrated and spray-dried to obtain ferrous chelate yak blood peptide Maillard reaction product dry powder. (8) Packaging and warehousing: The dry powder of the ferrous chelate yak blood peptide Maillard reaction product is aseptically packaged and stored.

2. The method for preparing yak blood peptides with anti-hypoxia and anti-fatigue functions as described in claim 1, characterized in that, The compound enzymatic hydrolysate is a mixture of enzymatically modified lumbrokinase and nattokinase.

3. The method for preparing yak blood peptides with anti-hypoxia and anti-fatigue functions as described in claim 2, characterized in that, The enzyme-modified lumbrokinase is an immobilized trypsin-modified lumbrokinase.

4. The method for preparing yak blood peptides with anti-hypoxia and anti-fatigue functions as described in claim 1, characterized in that, In step (1), the pH is adjusted to 6-8.

5. The method for preparing yak blood peptides with anti-hypoxia and anti-fatigue functions as described in claim 1, characterized in that, In step (4), the polysaccharides in the supernatant are precipitated using anhydrous ethanol.

6. The method for preparing yak blood peptides with anti-hypoxia and anti-fatigue functions as described in claim 1, characterized in that, The preset temperature in step (6) is 110℃.

7. The method for preparing yak blood peptides with anti-hypoxia and anti-fatigue functions as described in claim 1, characterized in that, In step (7), the spray drying temperature is 125-130℃ at the inlet and 90-110℃ at the outlet.

8. The method for preparing yak blood peptides with anti-hypoxia and anti-fatigue functions as described in claim 2, characterized in that, The extraction method of the enzyme-modified lumbrokinase is as follows: (1) Weigh out Nongda No. 3 earthworms, clean them, soak them in water in a beaker for 2 hours, wash them, and homogenize them at low temperature to obtain Nongda No. 3 homogenate. (2) Add the Nongda No. 3 homogenate to PBS and benzoic acid solution at a ratio of 1:4 (w / v), allow it to autolyze for 8 hours, centrifuge and take the supernatant to obtain crude lumbrokinase extract; (3) Add ammonium sulfate to the crude extract of lumbrokinase for fractional precipitation, centrifuge at 25°C and 8000 rpm / min for 10 min to collect the precipitate, and dialyze to desalt and purify to obtain lumbrokinase dry powder; (4) Enzyme-modified lumbrokinase: Trypsin was immobilized with chitosan to obtain immobilized trypsin. The immobilized trypsin was added to lumbrokinase and incubated in a water bath at 45°C for 1 hour. After filtration, the enzyme-modified lumbrokinase was obtained.

9. The application of a yak blood peptide with anti-hypoxia and anti-fatigue functions, characterized in that, The yak blood peptides are used in anti-hypoxia and anti-fatigue drugs or health foods.

10. The application of the yak blood peptide with anti-hypoxia and anti-fatigue functions as described in claim 9, characterized in that, The dosage forms of the anti-hypoxia and anti-fatigue drugs or health foods include tablets, capsules, granules, oral liquids, tinctures, powders, injections, and teas.

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