Silka deer blood functional polypeptide extracted by ultrasonic wave and composite enzymatic hydrolysis and preparation process of sika deer blood functional polypeptide

Abstract

The invention relates to the technical field of ultrasonic wave synergistic composite enzymatic hydrolysis, in particular to a sika deer blood functional polypeptide extracted by ultrasonic wave synergistic composite enzymatic hydrolysis and a preparation process thereof, and a method for extracting the sika deer blood functional polypeptide by ultrasonic wave synergistic composite enzymatic hydrolysis. The method comprises the following steps: treating deer blood with an anticoagulant, separating blood cells, and freeze-thawing and splitting; crushing the cells through ultrasonic-assisted acid treatment; adding a compound enzyme composed of papain and the like according to a ratio of 3: 2: 1, carrying out enzymolysis by controlling the temperature and pH in stages, and carrying out nitrogen protection; performing enzyme deactivation and centrifugal ultrafiltration to obtain a crude product; purifying through an ion exchange column and a gel filtration column, grafting and modifying with gamma-polyglutamic acid, and freeze-drying to obtain the polypeptide. The method improves the yield and activity of the deer blood polypeptide and accurately controls the molecular weight through the cooperation of ultrasound and the compound enzyme; the modified polypeptide is enhanced in antioxidation and antihypertensive activity, and improved in stability and bioavailability; the process is green and environment-friendly, the cost is low, and the product can be used in multiple fields of health-care products, medicines and cosmetics.

Description

Technical Field

[0001] This invention relates to the field of ultrasonic synergistic enzymatic hydrolysis technology, specifically to an ultrasonic synergistic enzymatic hydrolysis method for extracting functional polypeptides from sika deer blood and its preparation process. Background Technology

[0002] Sika deer blood is rich in nutrients such as protein, amino acids, and trace elements, making it a biological resource with great development potential. In particular, its functional peptides possess various biological activities, including antioxidant, blood pressure-lowering, and immune-enhancing effects. However, traditional sika deer blood peptide extraction technologies have many limitations, hindering its industrial application and value enhancement.

[0003] Traditional processes such as direct hydrolysis and single enzymatic hydrolysis are inefficient. Direct hydrolysis often uses strong acids or alkalis, which not only destroys peptide activity but also introduces a large amount of harmful chemical residues, leading to a decrease in product safety. Single enzymatic hydrolysis, due to the limited enzyme action sites, is difficult to fully hydrolyze proteins, resulting in low peptide yields and uneven molecular weight distribution of the obtained peptides, with low content of active ingredients. Taking the traditional alkaline hydrolysis method as an example, hydrolysis for 6 hours at pH 12 and 100℃ results in extremely low yields of sika deer blood peptides and a significant decrease in antioxidant activity compared to before hydrolysis.

[0004] Current technologies lack synergistic optimization of the extraction process. While ultrasound-assisted extraction can accelerate cell disruption, when used alone, the thermal effect of ultrasound can denature proteins, leading to impaired peptide activity. During enzymatic hydrolysis, traditional methods do not consider the dynamic changes in factors such as dissolved oxygen, temperature, and pH, making it difficult to maintain optimal enzyme activity and affecting hydrolysis efficiency. For example, when the power of conventional ultrasonic extraction is too high (above 500W), the temperature of the extract can rise to above 40°C within 10 minutes, causing some heat-sensitive peptides to become inactive.

[0005] The extracted peptides suffer from poor stability and low bioavailability. Unmodified sika deer blood peptides are easily degraded by proteases in the gastrointestinal environment, and due to their structural characteristics, they tend to aggregate in aqueous solutions, resulting in poor solubility and absorption. Furthermore, current technologies lack precise methods for controlling peptide molecular weight, making it difficult to target and obtain highly active peptide fragments of specific molecular weights. Traditional extraction processes often use large amounts of chemical reagents, resulting in complex subsequent treatments, large wastewater discharges, and significant environmental pressure. Moreover, low extraction efficiency and yield lead to high production costs, limiting the market competitiveness of sika deer blood peptide products. Summary of the Invention

[0006] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides an ultrasonic-assisted enzymatic hydrolysis extraction process for functional polypeptides from sika deer blood and its preparation process.

[0007] (II) Technical Solution A method for extracting functional polypeptides from sika deer blood using ultrasound-assisted enzymatic hydrolysis includes the following steps: S1: Raw material pretreatment Fresh sika deer blood was mixed with an anticoagulant, centrifuged at 4°C to separate blood cells, washed three times with physiological saline, and then deionized water was added. The mixture was then subjected to a freeze-thaw cycle at -20°C three times to obtain a blood cell lysate. The anticoagulant consisted of sodium citrate, disodium EDTA-2Na, and sodium heparin. The structural formula of disodium EDTA-2Na is:

[0008] S2: Ultrasonic-assisted acid treatment Add glacial acetic acid to the lysis solution to adjust the pH to 3.5-4.0, place it in an ultrasonic generator for 15-20 minutes, and control the treatment temperature at 25-30℃ to form an acid treatment system. S3: Complex enzymatic hydrolysis reaction A compound enzyme preparation was added sequentially to the acid treatment system, and enzymatic hydrolysis was carried out at 50-55℃ and pH 6.5-7.0 for 2.5-3 hours. Then, the temperature was raised to 60-65℃ and pH 7.5-8.0 to continue enzymatic hydrolysis. Nitrogen gas was introduced for protection during the enzymatic hydrolysis process. The compound enzyme preparation was composed of papain, neutral protease and flavor protease in a mass ratio of 3:2:1. S4: Enzyme inactivation and separation The enzyme hydrolysate was heated to 95°C to inactivate the enzyme, cooled to room temperature, centrifuged at 4°C, the supernatant was collected, ultrafiltration was performed using an ultrafiltration membrane, the retentate was collected, and freeze-dried to obtain crude sika deer blood functional polypeptide. S5: Purification and Modification The crude product was dissolved in phosphate buffer at pH 7.4, loaded onto a DEAE-cellulose ion exchange column, and eluted with a gradient of 0-0.5 mol / L NaCl. The active peak was collected. The product was then purified by Sephadex G-50 gel filtration, and fractions with molecular weights of 1-3 kDa were collected. γ-polyglutamic acid was added to the purified peptide solution, and graft modification was performed using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as catalysts. The reaction was carried out at 40-45℃ and pH 8.0 for 3-4 hours. After the reaction, the product was dialyzed to remove salts and then lyophilized to obtain the modified sika deer blood functional peptide.

[0009] Preferably, the cavitation effect of the ultrasonic treatment in S2 results in a blood cell breakage rate of over 98%, and the concentration of generated hydroxyl radicals is 1.2 × 10⁻⁶. Up to 1.8× mol / L.

[0010] Preferably, the molecular weight distribution of the polypeptide fragments generated in the S3 enzymatic hydrolysis process is as follows: 1-3 kDa accounts for 65-70%, 3-5 kDa accounts for 20-25%, and >5 kDa accounts for 5-10%.

[0011] Preferably, the γ-polyglutamic acid grafting rate of the modified polypeptide in S5 is 30-40%.

[0012] Preferably, the modified polypeptide has an antioxidant activity ORAC value of 1200-1500 μmol TE / g and an inhibition rate IC50 of 0.08-0.12 mg / mL against angiotensin-converting enzyme ACE.

[0013] Preferably, the modified polypeptide has an isoelectric point of 4.8-5.2, a solubility of ≥95% in the pH range of 3-9, a zeta potential of -15 to -25 mV, and a stability of ≤5% / 24h in 0.9% (w / v) NaCl solution at 25°C.

[0014] Preferably, the total sugar content of the modified polypeptide is 8-12% (w / w), of which the sialic acid content is 1.5-2.5% (w / w).

[0015] Preferably, the application of a modified sika deer blood functional polypeptide prepared by a method of ultrasonic-assisted enzymatic hydrolysis extraction of sika deer blood functional polypeptide in the preparation of anti-aging health products is characterized in that the daily intake of the health product is 50-200mg, and continuous use for 3 months can increase the activity of superoxide dismutase (SOD) in serum by 25-35% and reduce the content of malondialdehyde (MDA) by 20-30%.

[0016] Preferably, the modified sika deer blood functional polypeptide prepared by a method of ultrasonic-assisted enzymatic hydrolysis extraction of sika deer blood functional polypeptide is used in the preparation of antihypertensive drugs. The polypeptide content in the drug is 20-50 mg / tablet. One tablet is taken three times a day for four consecutive weeks, which can reduce systolic blood pressure by 10-15 mmHg and diastolic blood pressure by 5-10 mmHg.

[0017] Preferably, a modified sika deer blood functional polypeptide prepared by a method of ultrasonic-assisted enzymatic hydrolysis extraction of sika deer blood functional polypeptide is used in the preparation of cosmetics. The amount of polypeptide added in the cosmetic is 0.1-0.5% (w / w). After application, the skin moisture content increases by 30-40% and elasticity increases by 25-35% within 24 hours, while the transepidermal water loss (TEWL) decreases by 15-20%.

[0018] (III) Beneficial Effects Compared with existing technologies, the beneficial effects of this invention are: 1. The synergistic effect of ultrasound and combined enzymatic hydrolysis results in an extremely high rate of blood cell disruption and a significantly improved peptide yield, nearly doubling that of traditional single enzymatic hydrolysis methods. The hydroxyl radicals generated by the cavitation effect of ultrasound promote protein hydrolysis, while precise control of power and frequency prevents peptide denaturation due to overheating, resulting in extremely high retention of antioxidant active ingredients.

[0019] 2. By stepwise enzymatic hydrolysis of a complex enzyme system and precise temperature and pH control, highly active polypeptide fragments of 1-3 kDa are obtained in a targeted manner; γ-polyglutamic acid grafting modification significantly improves the isoelectric point, solubility, and stability of the polypeptides, greatly enhances their antioxidant activity, significantly reduces the ACE inhibition rate IC50, and improves gastrointestinal digestive stability.

[0020] 3. The use of green anticoagulants and mild treatment conditions reduces harmful chemical residues; nitrogen protection and precise temperature control reduce oxidation risks; ultrafiltration purification simplifies the process, reduces production costs, and significantly reduces wastewater discharge.

[0021] 4. The prepared peptides exhibit outstanding effects in the fields of health products, pharmaceuticals, and cosmetics. When used in anti-aging health products, they can significantly enhance the body's antioxidant capacity; as an ingredient in antihypertensive drugs, they have a significant blood pressure-lowering effect; and when added to cosmetics, they can effectively improve skin hydration and elasticity. This process realizes the transformation of sika deer blood peptides from low-value raw materials to high-value-added products, providing an efficient, environmentally friendly, and highly active solution for industrial development, with both economic and social benefits. Attached Figure Description

[0022] Figure 1 This is a process flow diagram for preparing functional polypeptides from sika deer blood through ultrasonic-assisted compound enzymatic hydrolysis extraction. Figure 2 This is a bar chart showing the peptide yields of the examples and comparative examples; Figure 3 This is a bar and line graph comparing the percentage of 1-3 kDa and ORAC values ​​in the examples and comparative examples; Figure 4 This is a line graph showing the percentage yield of the examples and comparative examples. Detailed Implementation

[0023] according to Figures 1 to 4 The specific embodiments of the present invention are as follows: Example 1: Preparation of functional polypeptides from sika deer blood using standard processes 1. Raw material pretreatment Take 500 mL of fresh sika deer blood and add 50 mL of anticoagulant solution (containing 1.5% sodium citrate, 0.8% EDTA-2Na, and 0.2% heparin sodium), and gently invert to mix. Centrifuge at 3000 rpm for 15 minutes at 4°C, discard the supernatant plasma, and collect the lower layer of blood cells. Wash the blood cells three times with physiological saline (each time adding an equal volume of physiological saline and centrifuging at 2500 rpm for 10 minutes), and discard the supernatant. Add 2500 mL of deionized water to the blood cells, freeze at -20°C, thaw at room temperature, and repeat the freeze-thaw cycle three times to obtain the blood cell lysate.

[0024] 2. Ultrasonic-assisted acid treatment Slowly add glacial acetic acid to the lysis solution to adjust the pH to 3.8, then transfer it to an ultrasonic treatment container. Set the ultrasonic power to 350W, the frequency to 25kHz, and the treatment time to 18 minutes, maintaining the temperature at approximately 28°C during the process using circulating water cooling.

[0025] 3. Complex enzymatic hydrolysis reaction The dry weight of blood cells was measured to be 50g. 1.5g of a complex enzyme preparation (papain: neutral protease: flavor protease = 3:2:1) was added to the acid-treated system. The system temperature was first raised to 52℃, and the pH was adjusted to 6.8 with 0.1mol / L disodium hydrogen phosphate-citrate buffer. Enzymatic hydrolysis was continued for 2.5 hours. Then, the temperature was raised to 62℃, and the pH was adjusted to 7.8 with 0.1mol / L Tris-HCl buffer. Enzymatic hydrolysis continued for 1.5 hours. Nitrogen gas was continuously purged during the hydrolysis process to maintain dissolved oxygen levels <2mg / L.

[0026] 4. Enzyme inactivation and separation After enzymatic hydrolysis, the hydrolysate was rapidly heated to 95°C and held for 10 minutes to inactivate the enzyme. After cooling to room temperature, it was centrifuged at 8000 rpm for 20 minutes at 4°C, and the supernatant was collected. Ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and the retentate was collected and freeze-dried in a freeze dryer to obtain 17.5 g of crude sika deer blood functional polypeptide.

[0027] 5. Purification and Modification The crude product was dissolved in 50 mL of pH 7.4 phosphate buffer and loaded onto a DEAE-cellulose ion exchange column. Gradient elution with 0–0.5 mol / L NaCl solution was performed at a flow rate of 1 mL / min, and the active peak fraction was collected. The active peak solutions were combined and further purified using a Sephadex G-50 gel filtration column at a flow rate of 0.5 mL / min, collecting fractions with molecular weights between 1 and 3 kDa. 0.2% (w / v) γ-polyglutamic acid, 5% (w / w) EDC, and 3% (w / w) NHS were added to the purified peptide solution, and the reaction was carried out at 42 °C and pH 8.0 for 3.5 hours. After the reaction, the solution was placed in a dialysis bag and dialyzed with deionized water for 48 hours to remove small molecule impurities. The resulting product was lyophilized to obtain 12.3 g of modified sika deer blood functional peptides.

[0028] Example 2: Optimizing parameters to enhance antioxidant activity 1. Raw material pretreatment Take 500 mL of fresh sika deer blood and add 50 mL of anticoagulant solution (containing 1.5% sodium citrate, 0.8% EDTA-2Na, and 0.2% heparin sodium), and gently invert to mix. Centrifuge at 3000 rpm for 15 minutes at 4°C, discard the supernatant plasma, and collect the lower layer of blood cells. Wash the blood cells three times with physiological saline (each time adding an equal volume of physiological saline and centrifuging at 2500 rpm for 10 minutes), and discard the supernatant. Add 2500 mL of deionized water to the blood cells, freeze at -20°C, thaw at room temperature, and repeat the freeze-thaw cycle three times to obtain the blood cell lysate.

[0029] 2. Ultrasonic-assisted acid treatment Slowly add glacial acetic acid to the lysis solution to adjust the pH to 3.8, then transfer it to an ultrasonic treatment container. Set the ultrasonic power to 400W, the frequency to 25kHz, and the treatment time to 15 minutes, maintaining the temperature at approximately 28°C during the process using circulating water cooling.

[0030] 3. Complex enzymatic hydrolysis reaction The dry weight of blood cells was measured to be 50g. 1.6g of a complex enzyme preparation (papain: neutral protease: flavor protease = 3:2:1) was added to the acid-treated system. The system temperature was first raised to 52℃, and the pH was adjusted to 6.8 with 0.1mol / L disodium hydrogen phosphate-citrate buffer. Enzymatic hydrolysis was continued for 2.5 hours. Then, the temperature was raised to 65℃, and the pH was adjusted to 8 with 0.1mol / L Tris-HCl buffer. Enzymatic hydrolysis continued for 1.5 hours. Nitrogen gas was continuously purged during the hydrolysis process to maintain dissolved oxygen levels <2mg / L.

[0031] 4. Enzyme inactivation and separation After enzymatic hydrolysis, the hydrolysate was rapidly heated to 95°C and held for 10 minutes to inactivate the enzyme. After cooling to room temperature, it was centrifuged at 8000 rpm for 20 minutes at 4°C, and the supernatant was collected. Ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and the retentate was collected and freeze-dried in a freeze dryer to obtain 17.5 g of crude sika deer blood functional polypeptide.

[0032] 5. Purification and Modification The crude product was dissolved in 50 mL of phosphate buffer (pH 7.4) and loaded onto a DEAE-cellulose ion exchange column. Gradient elution with 0–0.5 mol / L NaCl solution was performed at a flow rate of 1 mL / min, and the active peak fraction was collected. The active peak solutions were combined and further purified using a Sephadex G-50 gel filtration column at a flow rate of 0.5 mL / min, collecting fractions with molecular weights between 1 and 3 kDa. 0.3% (w / v) γ-polyglutamic acid, 5% (w / w) EDC, and 3% (w / w) NHS were added to the purified peptide solution, and the reaction was carried out at 42 °C and pH 8.0 for 3.5 hours. After the reaction, the solution was placed in a dialysis bag and dialyzed with deionized water for 48 hours to remove small molecule impurities. The solution was then lyophilized to obtain 13.8 g of modified sika deer blood functional peptides.

[0033] Example 3: Scale-up verification of process stability 1. Raw material pretreatment Take 5000 mL of fresh sika deer blood and add 500 mL of anticoagulant solution (containing 1.5% sodium citrate, 0.8% EDTA-2Na, and 0.2% sodium heparin), and slowly invert to mix. Centrifuge at 3000 rpm for 15 minutes at 4°C, discard the supernatant plasma, and collect the lower layer of blood cells. Wash the blood cells three times with physiological saline (add 5 L of physiological saline each time, centrifuge at 2500 rpm for 10 minutes), and discard the supernatant. Add 25 L of deionized water to the blood cells, freeze at -20°C, thaw at room temperature, and repeat the freeze-thaw cycle three times to obtain the blood cell lysate.

[0034] 2. Ultrasonic-assisted acid treatment Slowly add glacial acetic acid to the lysis solution to adjust the pH to 3.8, then transfer it to an ultrasonic treatment container. Set the ultrasonic power to 350 W, the frequency to 25 kHz, and the treatment time to 18 minutes, maintaining the temperature at approximately 28°C during the process using circulating water cooling.

[0035] 3. Complex enzymatic hydrolysis reaction The dry weight of blood cells was determined to be 500 g. 15 g of a complex enzyme preparation (papain: neutral protease: flavor protease = 3:2:1) was added to the acid-treated system. The system temperature was first raised to 52℃, and the pH was adjusted to 6.8 with 0.1 mol / L disodium hydrogen phosphate-citrate buffer. Enzymatic hydrolysis was carried out for 2.5 hours. Then, the temperature was raised to 62℃, and the pH was adjusted to 7.8 with 0.1 mol / L Tris-HCl buffer. Enzymatic hydrolysis continued for 1.5 hours. Nitrogen gas was continuously purged during the hydrolysis process to maintain dissolved oxygen levels <2 mg / L.

[0036] 4. Enzyme inactivation and separation After enzymatic hydrolysis, the hydrolysate was rapidly heated to 95°C and held for 10 minutes to inactivate the enzyme. After cooling to room temperature, it was centrifuged at 8000 rpm for 20 minutes at 4°C, and the supernatant was collected. Ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and the retentate was collected and freeze-dried in a freeze dryer to obtain 175 g of crude sika deer blood functional polypeptide.

[0037] 5. Purification and Modification The crude product was dissolved in 500 mL of pH 7.4 phosphate buffer and loaded onto a DEAE-cellulose ion exchange column. Gradient elution with 0–0.5 mol / L NaCl solution was performed (flow rate 1 mL / min), and the active peak fraction was collected. The active peak solutions were combined and further purified using a Sephadex G-50 gel filtration column (flow rate 0.5 mL / min), collecting fractions with molecular weights of 1–3 kDa. 0.2% (w / v) γ-polyglutamic acid, 5% (w / w) EDC, and 3% (w / w) NHS were added to the purified peptide solution, and the reaction was carried out at 42 °C and pH 8.0 for 3.5 hours. After the reaction, the solution was placed in a dialysis bag and dialyzed with deionized water for 48 hours to remove small molecule impurities. Lyophilization yielded 125.6 g of modified sika deer blood functional peptides.

[0038] Comparative example: Traditional single enzymatic hydrolysis method step: Take 500 mL of sika deer blood, centrifuge to separate blood cells, and then directly add 0.8% (w / w) of single papain. Enzymatic hydrolysis was performed at 55°C and pH 7.0 for 4 hours. After enzyme inactivation, centrifugation, ultrafiltration, and freeze-drying, 10.2g of crude product was obtained.

[0039] Results: The peptide yield was only 20.4%, the proportion of 1-3kDa peptides was 42%, and the ORAC value was 820μmol TE / g, which was significantly lower than that of Example 1. Performance testing The modified sika deer blood functional peptides prepared in Example 1 achieved a yield of 34.6%, with 68.2% being 1-3 kDa active peptides. The ORAC value was 1238 μmol TE / g, and the IC50 for ACE inhibition activity was 0.10 mg / mL. In stability testing, after 7 days at 25°C and pH 7.0, the particle size change of the 5 mg / mL peptide solution was only 4.8%, and the activity retention rate reached 92.3%. Compared with the comparative example, this process increased the peptide yield by 69.6%, the ORAC value by 51.0%, and the ACE inhibition activity by 120%, fully verifying the superiority of the ultrasound-assisted enzymatic hydrolysis technology.

[0040] The peptide yields and raw material treatments for the examples and comparative examples are shown in the table below: Table 1

[0041] In summary, the yields of Examples 1-3 were all higher than those of the comparative examples (Example 1 was 1.72 times that of the comparative examples). Among them, the yield of Example 3 was stable (31.4%) after being scaled up 10 times, which verified the scalability of the process. The yield of Example 2 was slightly lower, but the activity was significantly improved, indicating that the parameter optimization focused on functionality rather than simply yield.

[0042] The results of the detection of docosahexaenoic acid and DHA-related indicators in the examples and comparative examples are compared in the table below: Table 2

[0043] In summary, the proportion of highly active peptides (1-3 kDa) in Examples 1-2 reached 67%-72%, and the ORAC value was significantly higher than that of the comparative example (Example 2 was 1.77 times that of the comparative example), indicating that compound enzymatic hydrolysis and ultrasound assistance can directionally generate highly active peptides; the comparative example had a high proportion of low-activity small molecule peptides (35%) due to single enzymatic hydrolysis, and the weakest antioxidant capacity.

[0044] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their likenesses.

Claims

1. A method for extracting functional polypeptides from sika deer blood using ultrasound-assisted enzymatic hydrolysis, characterized in that... Includes the following steps: S1: Raw material pretreatment Fresh sika deer blood was mixed with an anticoagulant, centrifuged at 4°C to separate blood cells, washed three times with physiological saline, and then deionized water was added. The mixture was then freeze-thawed three times at -20°C to obtain a blood cell lysate. The anticoagulant consisted of sodium citrate, disodium EDTA-2Na, and sodium heparin. S2: Ultrasonic-assisted acid treatment Add glacial acetic acid to the lysis solution to adjust the pH to 3.5-4.0, place it in an ultrasonic generator for 15-20 minutes, and control the treatment temperature at 25-30℃ to form an acid treatment system. S3: Complex enzymatic hydrolysis reaction A compound enzyme preparation was added sequentially to the acid treatment system, and enzymatic hydrolysis was carried out at 50-55℃ and pH 6.5-7.0 for 2.5-3 hours. Then, the temperature was raised to 60-65℃ and pH 7.5-8.0 to continue enzymatic hydrolysis. Nitrogen gas was introduced for protection during the enzymatic hydrolysis process. The compound enzyme preparation was composed of papain, neutral protease and flavor protease in a mass ratio of 3:2:

1. S4: Enzyme inactivation and separation The enzyme hydrolysate was heated to 95°C to inactivate the enzyme, cooled to room temperature, centrifuged at 4°C, the supernatant was collected, ultrafiltration was performed using an ultrafiltration membrane, the retentate was collected, and freeze-dried to obtain crude sika deer blood functional polypeptide. S5: Purification and Modification The crude product was dissolved in phosphate buffer at pH 7.4, loaded onto a DEAE-cellulose ion exchange column, and eluted with a 0-0.5 mol / L NaCl gradient. The active peak was collected. The product was then purified by Sephadex G-50 gel filtration, and fractions with molecular weights of 1-3 kDa were collected. γ-polyglutamic acid was added to the purified peptide solution, and graft modification was performed using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as catalysts. The reaction was carried out at 40-45℃ and pH 8.0 for 3-4 hours. After the reaction, the product was dialyzed to remove salts and then lyophilized to obtain the modified sika deer blood functional peptide.

2. The method for extracting functional polypeptides from sika deer blood using ultrasound-assisted enzymatic hydrolysis according to claim 1, characterized in that, The cavitation effect of the ultrasonic treatment in S2 resulted in a blood cell disruption rate of over 98%, and the concentration of generated hydroxyl radicals was 1.2 × 10⁻⁶. Up to 1.8× mol / L.

3. The method for extracting functional polypeptides from sika deer blood using ultrasound-assisted enzymatic hydrolysis according to claim 1, characterized in that, The molecular weight distribution of the polypeptide fragments generated in the S3 enzymatic hydrolysis process is as follows: 1-3 kDa accounts for 65-70%, 3-5 kDa accounts for 20-25%, and >5 kDa accounts for 5-10%.

4. The method for extracting functional polypeptides from sika deer blood using ultrasound-assisted enzymatic hydrolysis according to claim 1, characterized in that, The γ-polyglutamic acid grafting rate of the modified polypeptide in S5 is 30-40%.

5. The method for extracting functional polypeptides from sika deer blood using ultrasound-assisted enzymatic hydrolysis according to claim 1, characterized in that, The modified polypeptide has an antioxidant activity ORAC value of 1200-1500 μmol TE / g and an inhibition rate IC50 of 0.08-0.12 mg / mL against angiotensin-converting enzyme ACE.

6. The method for extracting functional polypeptides from sika deer blood using ultrasound-assisted enzymatic hydrolysis according to claim 1, characterized in that, The modified polypeptide has an isoelectric point of 4.8-5.2, a solubility of ≥95% in the pH range of 3-9, a zeta potential of -15 to -25 mV, and a stability of ≤5% / 24h in 0.9% (w / v) NaCl solution at 25°C.

7. The method for extracting functional polypeptides from sika deer blood using ultrasound-assisted enzymatic hydrolysis according to claim 1, characterized in that, The modified polypeptide has a total sugar content of 8-12% (w / w), of which the sialic acid content is 1.5-2.5% (w / w).

8. The application of the modified sika deer blood functional polypeptide prepared by the method according to any one of claims 1-7 in the preparation of anti-aging health products, characterized in that, The recommended daily intake of the health supplement is 50-200mg. Continuous use for 3 months can increase the activity of superoxide dismutase (SOD) in serum by 25-35% and reduce the content of malondialdehyde (MDA) by 20-30%.

9. The use of the modified sika deer blood functional polypeptide prepared by the method according to any one of claims 1-7 in the preparation of antihypertensive drugs, characterized in that, The drug contains 20-50 mg of polypeptide per tablet. It is taken 3 times a day, 1 tablet each time. Continuous use for 4 weeks can reduce systolic blood pressure by 10-15 mmHg and diastolic blood pressure by 5-10 mmHg.

10. The application of the modified sika deer blood functional polypeptide prepared by the method according to any one of claims 1-7 in the preparation of cosmetics, characterized in that, The cosmetic contains 0.1-0.5% (w / w) peptides, which increase skin moisture content by 30-40% and elasticity by 25-35% within 24 hours after application, and reduce transepidermal water loss (TEWL) by 15-20%.

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