Preparation method of yak hemoglobin antihypertensive peptide
Through the steps of collection, washing, crushing, dialysis and enzymatic decomposition, high-purity yak hemoglobin antihypertensive peptides are prepared, which solves the problems of resource waste and environmental pollution, and achieves efficient preparation of antihypertensive peptides and high ACE inhibitory activity.
Patent Information
- Application Number
- CN202510543337.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-28
- Publication Date
- 2025-08-15
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
In the prior art, the biologically active peptide of yak hemoglobin is not effectively utilized, resulting in waste of resources and environmental pollution, and a high-efficiency preparation method for antihypertensive peptides is lacking.
Through the steps of collection, washing, crushing, dialysis, enzymatic decomposition and ultrafiltration, high-purity yak hemoglobin antihypertensive peptides are prepared, including centrifugation, ultrasonic crushing, dialysis, enzymatic decomposition and decolorization treatment, and peptides with molecular weight in the range of 0.2-0.4 kDa have high ACE inhibitory activity.
High purity purification and enzymatic decomposition of yak hemoglobin were achieved, and the purity of the peptide and ACE inhibitory activity were significantly improved, with purity reaching 98.58% and ACE inhibitory activity reaching 75.89%.
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of small molecule peptide preparation, in particular to a method for preparing yak hemoglobin blood pressure-lowering peptide. Background Art
[0002] Antihypertensive peptides help improve cardiovascular health and have broad application prospects in antihypertensive treatment. Yak hemoglobin hydrolysate contains a variety of bioactive peptides, such as anticancer peptides, antioxidant peptides, antihypertensive peptides, and antidiabetic peptides, and has great development potential. During the yak slaughter process, protein hydrolysates derived from hemoglobin may be discarded, representing a potential source of bioactive peptides and causing environmental pollution. With the advancement of scientific and technological revolution, my country has begun to pay close attention to the development and protection of yak blood, listing it as a key research project in the Qinghai-Tibet Plateau. The market-oriented development of yak hemoglobin antihypertensive peptide products could significantly increase the economic benefits of the yak farming industry. To this end, it is necessary to enzymatically hydrolyze yak hemoglobin and utilize modern separation technologies to separate and purify the yak hemoglobin hydrolysate to develop new yak hemoglobin antihypertensive peptide products. Summary of the Invention
[0003] The object of the present invention is to provide a method for preparing yak hemoglobin antihypertensive peptide to solve the problems raised in the above background technology.
[0004] To achieve the above object, the present invention provides the following technical solutions:
[0005] Step 1: Yak blood collection and red blood cell washing: yak blood was collected from slaughtered yaks on site, and only the middle section of the blood was collected to avoid impurities and blood clots. The blood was filtered through 250-mesh gauze and then centrifuged at 4°C and 5000 rpm for 30 minutes. The blood was washed three times with physiological saline to remove residual impurities and obtain pure red blood cells.
[0006] Step 2: RBC disruption: Mix 30 mL of RBCs prepared in Step 1 with 30 mL of distilled water, let stand for 20 minutes, and ultrasonically disrupt on ice for 30 minutes. Filter to obtain a RBC suspension. Mix 30 mL of the RBC suspension with 30 mL of normal saline, gently stir, and then perform intermittent ultrasonic disruption in an ice-water bath for 30 minutes. After disruption, transfer the mixture to a sterile centrifuge tube and centrifuge at 3000 rpm for 10 minutes to separate cell debris. Filter the supernatant through a 0.45 μm filter membrane and collect the filtrate.
[0007] Step 3: Prepare hemoglobin by adding ammonium sulfate powder to the filtrate collected in step 2 to reach a saturation of 50%-80%, centrifuging at 4°C, 3800 rpm for 10 minutes, collecting the precipitated hemoglobin, and desalting the hemoglobin using a dialysis bag with a molecular weight cutoff of 3500 Da. The dialyzed hemoglobin solution is freeze-dried to complete dryness using a freeze dryer, and the purity of the yak hemoglobin is measured.
[0008] Step 4: enzymatically hydrolyze yak hemoglobin. The hemoglobin obtained in step 3 is prepared into 50 mL of hemoglobin solution (12.5 mg / mL) using purified water. 50-70 mg of alkaline protease with an activity of 200,000 U / g and 1-3 g of sorbitol are added. The pH value of the mixture is adjusted to 8.5-9.0 using 0.1 mol / L NaOH solution. The mixture is enzymatically hydrolyzed at 50° C. for 1-5 h to obtain an enzymatic solution, and the molecular weight distribution of small molecule peptides is measured.
[0009] Step 5: Decolorize the hemoglobin enzymatic hydrolysate obtained in step 4 by adding 0.2% sodium carboxymethyl cellulose to the solution, decolorize at 50° C. for 20 minutes, centrifuge at 5000 rpm and 4° C. for 10 minutes, and collect the supernatant.
[0010] Step 6, ultrafiltration, ultrafiltration is performed on the supernatant obtained in step 5, and the small molecule peptide solution that passes through the 3 kDa ultrafiltration membrane is collected. After freeze-drying, the yak hemoglobin antihypertensive peptide is obtained, and the ACE inhibitory activity of the antihypertensive peptide is detected.
[0011] Compared with the prior art, the present invention has the following beneficial effects:
[0012] 1. After purification by dialysis and freeze-drying, the purity of yak hemoglobin reaches 98.58%.
[0013] 2. After alkaline protease hydrolysis, the molecular weight of all peptides was less than 1 kDa, and the peptides with a molecular weight in the range of 0.2-0.4 kDa accounted for 44.4%.
[0014] 3. Alkaline protease hydrolysates with molecular weight <3kDa all have high ACE inhibitory activity, reaching 75.89%. DETAILED DESCRIPTION
[0015] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.
[0016] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work shall fall within the scope of protection of the present invention.
[0017] The following examples are implementation cases of the technical solutions of the present invention in actual applications, but are not limited thereto. The reagents and experimental equipment involved therein are all commercially available products.
[0018] Example 1: This example provides a method for preparing yak hemoglobin antihypertensive peptide, which specifically comprises the following steps:
[0019] S1. Yak blood collection and red blood cell washing. Yak blood was collected from slaughtered yaks on site. Only the middle section of the blood was collected to avoid impurities and blood clots. The blood was filtered through 250-mesh gauze and then centrifuged at 4°C and 5000 rpm for 30 min. The blood was washed three times with physiological saline to remove residual impurities and obtain pure red blood cells.
[0020] S2, red blood cell disruption: Mix 30 mL of red blood cells prepared in step S1 with 30 mL of distilled water, let stand for 20 minutes, and then ultrasonically disrupt on ice for 30 minutes. Filter to obtain a red blood cell suspension. Mix 30 mL of the red blood cell suspension with 30 mL of normal saline, gently stir, and then intermittently ultrasonicate in an ice-water bath for 30 minutes. After disruption, transfer the mixture to a sterile centrifuge tube and centrifuge at 3000 rpm for 10 minutes to separate cell debris. Filter the supernatant through a 0.45 μm filter membrane and collect the filtrate.
[0021] S3, preparing hemoglobin, adding ammonium sulfate powder to the filtrate collected in step S2 to reach 60% saturation, centrifuging at 3800 rpm at 4°C for 10 minutes, collecting the precipitated hemoglobin, desalting the hemoglobin using a dialysis bag with a molecular weight cutoff of 3500 Da, and lyophilizing the dialyzed hemoglobin solution to complete dryness using a freeze dryer. The purity of the yak hemoglobin is 95.73%.
[0022] S4, enzymatic hydrolysis of yak hemoglobin, the hemoglobin obtained in step S3 was prepared into 50 mL of hemoglobin solution (12.5 mg / mL) using purified water, 50 mg of 200,000 U / g alkaline protease and 3 g of sorbitol were added, the pH value of the mixture was adjusted to 8.5 using 0.1 mol / L NaOH solution, and the mixture was enzymatically hydrolyzed at 50°C for 3 h to obtain an enzymatic hydrolyzate with a molecular weight of less than 1 kDa, and the peptides with a molecular weight in the range of 0.2-0.4 kDa were the most numerous, accounting for 43.6%.
[0023] S5, decolorizing the hemoglobin enzymatic hydrolysate obtained in step 4, adding 0.2% sodium carboxymethyl cellulose to the solution, decolorizing at 50°C for 20 minutes, centrifuging at 5000 rpm and 4°C for 10 minutes, and collecting the supernatant.
[0024] S6, ultrafiltration, ultrafiltration of the supernatant obtained in step S5, collection of the small molecule peptide solution that passes through the 3 kDa ultrafiltration membrane, and freeze-drying to obtain yak hemoglobin antihypertensive peptide with an ACE inhibitory activity of 74.31%.
[0025] Example 2: This example provides a method for preparing yak hemoglobin antihypertensive peptide, which specifically includes the following steps:
[0026] S1. Yak blood collection and red blood cell washing. Yak blood was collected from slaughtered yaks on site. Only the middle section of the blood was collected to avoid impurities and blood clots. The blood was filtered through 250-mesh gauze and then centrifuged at 4°C and 5000 rpm for 30 min. The blood was washed three times with physiological saline to remove residual impurities and obtain pure red blood cells.
[0027] S2, red blood cell disruption: Mix 30 mL of red blood cells prepared in step S1 with 30 mL of distilled water, let stand for 20 minutes, and then ultrasonically disrupt on ice for 30 minutes. Filter to obtain a red blood cell suspension. Mix 30 mL of the red blood cell suspension with 30 mL of normal saline, gently stir, and then intermittently ultrasonicate in an ice-water bath for 30 minutes. After disruption, transfer the mixture to a sterile centrifuge tube and centrifuge at 3000 rpm for 10 minutes to separate cell debris. Filter the supernatant through a 0.45 μm filter membrane and collect the filtrate.
[0028] S3, preparing hemoglobin, adding ammonium sulfate powder to the filtrate collected in step S2 to reach 80% saturation, centrifuging at 3800 rpm at 4°C for 10 minutes, collecting the precipitated hemoglobin, desalting the hemoglobin using a dialysis bag with a molecular weight cutoff of 3500 Da, and lyophilizing the dialyzed hemoglobin solution to complete dryness using a freeze dryer. The purity of the yak hemoglobin is 98.58%.
[0029] 4. S4, enzymatic hydrolysis of yak hemoglobin, the hemoglobin obtained in step S3 was prepared into 50 mL of hemoglobin solution (12.5 mg / mL) using purified water, 60 mg of 200,000 U / g alkaline protease and 2 g of sorbitol were added, the pH value of the mixture was adjusted to 9.0 using 0.1 mol / L NaOH solution, and the mixture was enzymatically hydrolyzed at 50 ° C for 5 h to obtain an enzymatic solution with a molecular weight of less than 1 kDa, and the peptides with a molecular weight range of 0.2-0.4 kDa were the largest, accounting for 44.44%.
[0030] S5, decolorizing the hemoglobin enzymatic hydrolysate obtained in step 4, adding 0.2% sodium carboxymethyl cellulose to the solution, decolorizing at 50°C for 20 minutes, centrifuging at 5000 rpm and 4°C for 10 minutes, and collecting the supernatant.
[0031] S6, ultrafiltration, ultrafiltration of the supernatant obtained in step S5, collection of the small molecule peptide solution that passes through the 3 kDa ultrafiltration membrane, and freeze-drying to obtain yak hemoglobin antihypertensive peptide with an ACE inhibitory activity of 75.89%.
[0032] Example 3: This example provides a method for preparing yak hemoglobin antihypertensive peptide, which specifically includes the following steps:
[0033] S1. Yak blood collection and red blood cell washing. Yak blood was collected from slaughtered yaks on site. Only the middle section of the blood was collected to avoid impurities and blood clots. The blood was filtered through 250-mesh gauze and then centrifuged at 4°C and 5000 rpm for 30 min. The blood was washed three times with physiological saline to remove residual impurities and obtain pure red blood cells.
[0034] S2, red blood cell disruption: Mix 30 mL of red blood cells prepared in step S1 with 30 mL of distilled water, let stand for 20 minutes, and then ultrasonically disrupt on ice for 30 minutes. Filter to obtain a red blood cell suspension. Mix 30 mL of the red blood cell suspension with 30 mL of normal saline, gently stir, and then intermittently ultrasonicate in an ice-water bath for 30 minutes. After disruption, transfer the mixture to a sterile centrifuge tube and centrifuge at 3000 rpm for 10 minutes to separate cell debris. Filter the supernatant through a 0.45 μm filter membrane and collect the filtrate.
[0035] S3, preparing hemoglobin, adding ammonium sulfate powder to the filtrate collected in step S2 to reach 50% saturation, centrifuging at 3800 rpm at 4°C for 10 minutes, collecting the precipitated hemoglobin, desalting the hemoglobin using a dialysis bag with a molecular weight cutoff of 3500 Da, and freeze-drying the dialyzed hemoglobin solution to complete dryness using a freeze dryer. The purity of the yak hemoglobin is 90.33%.
[0036] 5. S4, enzymatic hydrolysis of yak hemoglobin, the hemoglobin obtained in step S3 was prepared into 50 mL of hemoglobin solution (12.5 mg / mL) using purified water, 70 mg of 200,000 U / g alkaline protease and 1 g of sorbitol were added, the pH value of the mixture was adjusted to 8.5 using 0.1 mol / L NaOH solution, and the mixture was enzymatically hydrolyzed at 50 ° C for 1 h to obtain an enzymatic solution with a molecular weight of less than 1 kDa. The peptides with a molecular weight in the range of 0.2-0.4 kDa were the most numerous, accounting for 41.83%.
[0037] S5, decolorizing the hemoglobin enzymatic hydrolysate obtained in step 4, adding 0.2% sodium carboxymethyl cellulose to the solution, decolorizing at 50°C for 20 minutes, centrifuging at 5000 rpm and 4°C for 10 minutes, and collecting the supernatant.
[0038] S6, ultrafiltration, ultrafiltration of the supernatant obtained in step S5, collection of the small molecule peptide solution that passes through the 3 kDa ultrafiltration membrane, and freeze-drying to obtain yak hemoglobin antihypertensive peptide with an ACE inhibitory activity of 71.3%.
[0039] Based on the above-mentioned ideal embodiment of the present invention, and through the above description, relevant personnel can make various changes and modifications without departing from the scope of the technical concept of the present invention. The technical scope of the present invention is not limited to the contents of the specification.
Claims
1. A method for preparing yak hemoglobin antihypertensive peptide, characterized in that: The following steps are involved: S1. Yak blood collection and red blood cell washing. Yak blood was collected from slaughtered yaks on site. Only the middle section of the blood was collected to avoid impurities and blood clots. The blood was filtered through 250-mesh gauze and then centrifuged at 4°C and 5000 rpm for 30 min. The blood was washed three times with physiological saline to remove residual impurities and obtain pure red blood cells. S2, red blood cell disruption: Mix 30 mL of red blood cells prepared in step S1 with 30 mL of distilled water, let stand for 20 minutes, and then ultrasonically disrupt on ice for 30 minutes. Filter to obtain a red blood cell suspension. Mix 30 mL of the red blood cell suspension with 30 mL of normal saline, gently stir, and then intermittently ultrasonicate in an ice-water bath for 30 minutes. After disruption, transfer the mixture to a sterile centrifuge tube and centrifuge at 3000 rpm for 10 minutes to separate cell debris. Filter the supernatant through a 0.45 μm filter membrane and collect the filtrate. S3, preparing hemoglobin, adding ammonium sulfate powder to the filtrate collected in step S2 to reach a saturation of 50%-80%, centrifuging at 3800 rpm at 4°C for 10 minutes, collecting the precipitated hemoglobin, desalting the hemoglobin using a dialysis bag, and freeze-drying the dialyzed hemoglobin solution using a freeze dryer until completely dry. S4, enzymatic hydrolysis of yak hemoglobin, the hemoglobin obtained in step S3 is prepared into 50 mL of hemoglobin solution (12.5 mg / mL) using purified water, 50-70 mg of alkaline protease and 1-3 g of sorbitol are added, the pH value of the mixture is adjusted to 8.5-9.0 using 0.1 mol / L NaOH solution, and enzymatic hydrolysis is performed at 50°C for 1-5 h to obtain an enzymatic hydrolyzate. S5, decolorizing the hemoglobin enzymatic hydrolysate obtained in step 4, adding 0.2% sodium carboxymethyl cellulose to the solution, decolorizing at 50°C for 20 minutes, centrifuging at 5000 rpm and 4°C for 10 minutes, and collecting the supernatant. S6, ultrafiltration, ultrafiltration of the supernatant obtained in step S5, collection of the small molecule peptide solution that passes through the 3 kDa ultrafiltration membrane, and freeze-drying to obtain the yak hemoglobin antihypertensive peptide.
2. The dialysis bag of step S3 of claim 1 has a molecular weight cutoff of 3500 Da.
3. The alkaline protease activity of step S4 of claim 1 is 200,000 U / g.