Yak plasma protein peptide powder with strong antioxidation and ACE (angiotensin converting enzyme) inhibition functions and preparation method of yak plasma protein peptide powder
Antioxidant and ACE-inhibiting peptide powders were prepared from yak plasma protein powder using multi-stage enzymatic hydrolysis and separation technology. This solved the problems of low utilization rate of yak blood and side effects of ACE inhibitors, and achieved efficient preparation of peptide powders with significant functions, suitable for food and health food.
Patent Information
- Application Number
- CN202511665534.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-13
- Publication Date
- 2026-01-23
AI Technical Summary
In existing technologies, the utilization rate of yak blood is low, leading to resource waste and environmental pollution. At the same time, commonly used ACE inhibitors have side effects, and the vicious cycle of oxidative stress and hypertension is difficult to control effectively.
A multi-stage enzymatic hydrolysis and separation technique was used to prepare peptide powder with strong antioxidant and ACE inhibitory functions from yak plasma protein powder. The process included water dissolution, high-temperature sterilization, enzymatic hydrolysis, multi-stage separation, concentration and drying, ensuring that the content of amino acid sequences EDGGGWWYNR and WYNR in the peptide powder was within a specific range.
The prepared yak plasma protein peptide powder has significant antioxidant and ACE inhibitory functions, is suitable for industrial production, has a clear composition, good water solubility, no unpleasant odor, and is suitable for special needs foods and health foods.
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Figure CN121380265A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of animal protein and processing, and particularly relates to a yak blood plasma protein peptide powder with strong antioxidant and ACE inhibiting functions and a preparation method. BACKGROUND
[0002] Yak is a rare livestock species that lives in hypoxic, cold and harsh high-altitude areas. In addition to yak meat being widely consumed, yak bones, yak milk and yak blood and other by-products also have very high utilization value. In particular, the protein content in yak whole blood is 15-18%, which is a high-quality animal protein resource. However, at present, yak blood is discarded or discharged in large quantities, not only the utilization rate is extremely low, but also it causes serious blood resource waste and environmental pollution. Yak blood is a high-quality raw material for preparing bioactive peptides. Through enzymatic technology, yak plasma proteins are converted into active peptides, which not only realizes the high-value utilization of yak by-products, but also is expected to discover peptides with special physiological regulation functions, thereby providing a new way for the resource utilization of yak blood.
[0003] Hypertension is a chronic metabolic disease that plagues hundreds of millions of people worldwide and is the main risk factor for inducing cardiovascular and cerebrovascular diseases. The occurrence of hypertension is closely related to the imbalance of the regulation of the renin-angiotensin-aldosterone system (RAAS) in the body. Angiotensin-converting enzyme (ACE) plays a key role in this system. It can convert inactive angiotensin I into angiotensin II with strong vasoconstriction, thereby causing blood pressure to rise. Therefore, blocking this adverse process through exogenous ACE inhibitors has become one of the effective strategies for the clinical prevention and treatment of hypertension. However, the currently used synthetic ACE inhibitors, although effective, still have side effects such as cough and angioedema. At the same time, the oxidative stress state in the body and the incidence and development of hypertension promote each other, forming a vicious cycle. Active oxygen free radicals (such as superoxide anion and hydroxyl radical) will attack cell membrane lipids, proteins and DNA, leading to endothelial dysfunction and vascular inflammation, and thus exacerbating the rise in blood pressure. In this case, supplementing exogenous antioxidants to maintain the redox balance of the body is of great significance for the synergistic control of blood pressure and the protection of cardiovascular health. Therefore, the above situation prompts people to seek safer and side-effect-free alternatives from natural food sources. SUMMARY
[0004] To solve the above technical problems, the application provides a yak blood plasma protein peptide powder with strong antioxidant and ACE inhibiting functions and a preparation method, which is prepared from quarantine-qualified yak blood plasma protein powder. The preparation method of the yak blood plasma protein peptide powder with strong antioxidant and ACE inhibiting functions comprises the following steps: raw material preparation, water dissolving, high-temperature sterilization, cooling and homogenization, enzymolysis, multi-stage separation, concentration and drying, and component determination. S1: obtaining yak plasma protein liquid by sequentially dissolving yak plasma protein powder in water, high-temperature sterilization and cooling homogenization treatment; S2: obtaining yak plasma protein enzymatic hydrolysate by enzymolysis of the yak plasma protein liquid; S3: obtaining yak plasma protein peptide powder with strong antioxidant and ACE inhibitory functions by sequentially performing multi-stage separation and concentration drying on the yak plasma protein enzymatic hydrolysate; S4: determining the components of the yak plasma protein peptide powder.
[0005] Further, the S1 specifically comprises the following steps: S11: dissolving quarantine qualified yak plasma protein powder in drinking water meeting health standards, and the concentration of protein is 7-10%; S12: cooking sterilization at 95-100°C for 10-20 min; S13: after cooling, treating with a high-speed homogenizer for 2-3 min to obtain yak plasma protein liquid.
[0006] Further, the enzyme hydrolysis in the S2 is carried out according to the weight percentage of 1.0-2.0% of proteinase added to the protein in the yak plasma protein liquid, and specifically comprises the following steps: S21: first-stage enzyme hydrolysis: adding 0.5-1.0% of alkaline proteinase based on the weight of raw material protein, and performing enzyme hydrolysis reaction at 42-50°C for 0.5-2 h; S22: second-stage enzyme hydrolysis: adding 0.5-1.0% of compound proteinase based on the weight of protein, and performing enzyme hydrolysis at 50-55°C for 0.5-2 h; S23: after the second-stage enzyme hydrolysis, inactivating the enzyme at 95-100°C for 10-20 min, and cooling to room temperature to obtain yak plasma protein enzymatic hydrolysate.
[0007] Further, the S3 comprises the following steps: S31: collecting the supernatant after first-stage centrifugal separation of the yak plasma protein enzymatic hydrolysate in a centrifuge at a rotation speed of 10,000 r / min to obtain first-stage separation yak plasma protein peptide supernatant; S32: performing second-stage membrane separation on the first-stage separation yak plasma protein peptide supernatant by using an ultrafiltration membrane with a pore size of 5000 daltons, and collecting second-stage separation yak plasma protein peptide solution; S33: performing third-stage membrane separation on the second-stage separation yak plasma protein peptide solution by using an ultrafiltration membrane with a pore size of 2000 daltons to separate yak plasma protein peptides with a molecular weight less than 2000 daltons, and collecting third-stage separation yak plasma protein peptide solution, and then freeze-drying to obtain third-stage separation yak plasma protein peptide freeze-dried powder; S34: The freeze-dried powder of the tertiary separation yak plasma protein peptide with a molecular weight less than 2000 daltons is dissolved into a 20 mg / mL solution with deionized water, and then subjected to a quaternary Sephadex G-15 gel separation, with deionized water as the eluent, and the elution peak is detected at 280 nm, and the second elution peak is collected to obtain a quaternary separation yak plasma protein peptide solution; S35: The quaternary separation yak plasma protein peptide solution is subjected to a quinary reverse phase chromatography separation by RP-HPLC reverse phase high performance liquid chromatography, and a quinary separation yak plasma protein peptide solution is obtained by collecting the 8-11 fraction; S36: The quinary separation yak plasma protein peptide solution is subjected to concentration, freezing and drying in sequence to obtain a yak plasma protein peptide powder with strong antioxidant and ACE inhibitory functions.
[0008] Further, the S4 is an ingredient determination of the yak plasma protein peptide powder by LC-MS / MS, and the ingredient determination result is that the amino acid sequence EDGGGWWYNR of the yak plasma protein peptide powder is 39.5-42.5%, and WYNR is 15.3-17.8%.
[0009] Further, the enzyme activity of the alkaline protease used in the enzyme hydrolysis process of the S2 is 100,000-150,000 U / g, the enzyme activity of the neutral protease is 250,000-400,000 U / g, the enzyme activity of the papain is 250,000-400,000 U / g, and the complex protease comprises the neutral protease and the papain and the ratio of the two is 1:1.
[0010] To solve the above-mentioned prior art problems, the application further provides a yak plasma protein peptide powder with strong antioxidant and ACE inhibitory functions, which is prepared by any one of the preferred yak plasma protein peptide powder preparation methods with strong antioxidant and ACE inhibitory functions, and the amino acid sequence EDGGGWWYNR of the yak plasma protein peptide powder is 39.5-42.5%, and WYNR is 15.3-17.8%.
[0011] Compared with the prior art, the application has the following beneficial effects: 1. In the research and development process, a series of complex enzyme hydrolysis product comparison experiments are repeatedly carried out by using various preparation methods, and it is found that most of the preparation methods can obtain peptides with smaller molecular weight during the enzyme hydrolysis of yak plasma protein, but most of the obtained peptides have weak antioxidant and ACE inhibitory functions, and only the above-mentioned preferred separation preparation scheme is suitable for yak plasma protein and can obtain a yak plasma protein peptide powder with strong antioxidant and ACE inhibitory functions; 2. The yak plasma protein peptide powder prepared by the method has clear components, the amino acid sequences EDGGGWWYNR and WYNR have significant strong antioxidant and ACE inhibiting effects, and the yak plasma protein peptide powder has good water solubility, is easy to be absorbed by the human body, has no bad smell, and has excellent characteristics, and can be widely applied to special food and health food. 3. The preparation method is simple in operation, suitable for industrial production, and does not add acid and alkali in the whole preparation process, so that green processing can be realized. BRIEF DESCRIPTION OF DRAWINGS
[0012] Figure 1 FIG. 1 is an ACE inhibitory rate curve schematic diagram of sample 1 in the present application; Figure 2 FIG. 2 is an ACE inhibitory rate curve schematic diagram of sample 2 in the present application; Figure 3 FIG. 3 is an ACE inhibitory rate curve schematic diagram of sample 3 in the present application; Figure 4 FIG. 4 is an ACE inhibitory rate curve schematic diagram of sample 4 in the present application; Figure 5 FIG. 5 is an ACE inhibitory rate curve schematic diagram of sample 5 in the present application; Figure 6 FIG. 6 is an ACE inhibitory rate curve schematic diagram of sample 6 in the present application; Figure 7 FIG. 7 is an ACE inhibitory rate curve schematic diagram of EDGGGWWYNR in the present application; Figure 8 FIG. 8 is an ACE inhibitory rate curve schematic diagram of WYNR in the present application. DETAILED DESCRIPTION
[0013] The preferred embodiments of the present application are described in detail below, and the preferred embodiments listed are not used to limit the scope of the present application. In order to solve the above-mentioned prior art problems, the present application provides a preparation method of yak plasma protein peptide powder with strong antioxidant and ACE inhibiting functions, which comprises the following steps: S1: yak plasma protein powder is sequentially subjected to water dissolution, high-temperature sterilization and cooling homogenization treatment to obtain yak plasma protein liquid; S2: the yak plasma protein liquid is subjected to enzymolysis to obtain yak plasma protein enzymolysis liquid; S3: the yak plasma protein enzymolysis liquid is sequentially subjected to multi-stage separation and concentration drying to obtain yak plasma protein peptide powder with strong antioxidant and ACE inhibiting functions; S4: the components of the yak plasma protein peptide powder are determined.
[0014] Optionally, the S1 specifically comprises the following steps: S11: The quarantine qualified yak blood plasma protein powder is dissolved in drinking water meeting health standards, and the concentration of protein is 7-10%; S12: Cook and sterilize at 95-100℃ for 10-20min; S13: After cooling, use a high-speed homogenizer for 2-3min to obtain yak blood plasma protein liquid.
[0015] Optionally, the enzyme hydrolysis in S2 is two-stage enzyme hydrolysis according to 1.0-2.0% of the weight percentage of proteinase added to the weight of protein in the yak blood plasma protein liquid, which specifically includes the following steps: S21: First-stage enzyme hydrolysis: add 0.5-1.0% of alkaline proteinase to the weight of raw material protein, and perform enzyme hydrolysis reaction at 42-50℃ for 0.5-2h; S22: Second-stage enzyme hydrolysis: add 0.5-1.0% of compound proteinase to the weight of protein, and perform enzyme hydrolysis at 50-55℃ for 0.5-2h; S23: After the second-stage enzyme hydrolysis, inactivate the enzyme at 95-100℃ for 10-20min, and cool to room temperature to obtain yak blood plasma protein hydrolysis liquid.
[0016] Optionally, S3 includes the following steps: S31: Perform first-stage centrifugal separation on the yak blood plasma protein hydrolysis liquid in a centrifuge with a rotation speed of 10,000r / min for 10min, and collect the supernatant to obtain first-stage separation yak blood plasma peptide supernatant; S32: Perform second-stage membrane separation on the first-stage separation yak blood plasma peptide supernatant by using an ultrafiltration membrane with a pore size of 5000 daltons, and collect second-stage separation yak blood plasma peptide solution; S33: Perform third-stage membrane separation on the second-stage separation yak blood plasma peptide solution by using an ultrafiltration membrane with a pore size of 2000 daltons to separate yak blood plasma peptides with a molecular weight less than 2000 daltons, and collect third-stage separation yak blood plasma peptide solution, and then freeze-dry to obtain third-stage separation yak blood plasma peptide freeze-dried powder; S34: Take the third-stage separation yak blood plasma peptide freeze-dried powder with a molecular weight less than 2000 daltons, dissolve it into a 20mg / mL solution with deionized water, and then perform fourth-stage Sephadex G-15 gel separation, with deionized water as eluent, and detect the elution peak at 280nm, collect the second elution peak, and obtain fourth-stage separation yak blood plasma peptide solution; S35: Perform fifth-stage reverse phase chromatography separation on the fourth-stage separation yak blood plasma peptide solution by using RP-HPLC reverse phase high performance liquid chromatography, and collect the 8-11 fraction to obtain fifth-stage separation yak blood plasma peptide solution; S36: the five-stage separation yak plasma protein peptide solution is sequentially subjected to concentration, freezing and drying to obtain yak plasma protein peptide powder having strong antioxidant and ACE inhibiting functions.
[0017] Optionally, the S4 is component determination of the yak plasma protein peptide powder by LC-MS / MS, and the component determination result is that the amino acid sequence EDGGGWWYNR of the yak plasma protein peptide powder is 39.5-42.5%, and the amino acid sequence WYNR is 15.3-17.8%.
[0018] The enzyme activity of the alkaline protease used in the enzymolysis process of the above preferred embodiment is 100,000-150,000 U / g, the enzyme activity of the neutral protease is 250,000-400,000 U / g, the enzyme activity of the papain is 250,000-400,000 U / g, and the protease complex comprises the neutral protease and the papain and the ratio of the two is 1:1.
[0019] To solve the above prior art problems, the application further provides yak plasma protein peptide powder having strong antioxidant and ACE inhibiting functions, which is prepared by any one of the above preferred yak plasma protein peptide powder preparation methods, and the amino acid sequence EDGGGWWYNR of the yak plasma protein peptide powder is 39.5-42.5%, and the amino acid sequence WYNR is 15.3-17.8%.
[0020] The experimental data and results of the preparation of the yak plasma protein peptide powder according to the above preferred embodiments are described in detail as follows: Preparation Experiment 1: S101: taking quarantine qualified yak plasma protein powder, the protein is dissolved in drinking water meeting the health standards, and the concentration of the protein is 7%; S102: sterilizing at 95℃ for 20 min; S103: after cooling, the yak plasma protein solution is obtained by treating with a high-speed homogenizer for 3 min; S104: first-stage enzymolysis: adding 0.5% alkaline protease based on the weight of the raw material protein, and performing enzymolysis reaction at 45℃ for 2 h; S105: second-stage enzymolysis: adding 1.0% protease complex based on the weight of the protein, and performing enzymolysis at 50℃ for 1 h; S106: after the second-stage enzymolysis, the enzyme is inactivated at 98℃ for 15 min, and the yak plasma protein enzymolysis solution is obtained after cooling to room temperature; S107: the yak plasma protein enzymolysis solution is subjected to first-stage centrifugal separation at 10,000 r / min for 10 min, and the supernatant is collected to obtain the first-stage separation yak plasma protein peptide supernatant; S108: The first separation yak plasma protein peptide supernatant is subjected to secondary membrane separation by using an ultrafiltration membrane with a pore size of 5000 daltons, and a secondary separation yak plasma protein peptide solution is collected; S109: The secondary separation yak plasma protein peptide solution is subjected to tertiary membrane separation by using an ultrafiltration membrane with a pore size of 2000 daltons to separate yak plasma protein peptides with a molecular weight less than 2000 daltons, and a tertiary separation yak plasma protein peptide solution is collected, followed by freeze-drying to obtain a tertiary separation yak plasma protein peptide freeze-dried powder; S110: The tertiary separation yak plasma protein peptide freeze-dried powder with a molecular weight less than 2000 daltons is dissolved in deionized water to form a 20 mg / mL solution, and then subjected to four-stage Sephadex G-15 gel separation, with deionized water as the eluent, and the elution peak is detected at 280 nm. The second elution peak is collected to obtain a four-stage separation yak plasma protein peptide solution; S111: The four-stage separation yak plasma protein peptide solution is subjected to five-stage reverse phase chromatography separation by RP-HPLC reverse phase high performance liquid chromatography, and a five-stage separation yak plasma protein peptide solution is collected by collecting fractions 8-11; S112: The five-stage separation yak plasma protein peptide solution is sequentially subjected to concentration, freezing and drying to obtain a yak plasma protein peptide powder with strong antioxidant and ACE inhibitory functions; S113 is the component determination of the yak plasma protein peptide powder by LC-MS / MS, and the component determination result is that the amino acid sequence EDGGGWWYNR of the yak plasma protein peptide powder is 40.2%, and WYNR is 17.3%.
[0021] Preparation Experiment 2: S201: Dissolve the quarantine qualified yak plasma protein powder in drinking water meeting the health standards, and the concentration of protein is 8%; S202: Cook and sterilize at 100°C for 10 min; S203: After cooling, treat with a high-speed homogenizer for 3 min to obtain a yak plasma protein solution; S204: First-stage enzymolysis: add 1.0% of alkaline protease based on the weight of the raw material protein, and perform enzymolysis reaction at 50°C for 1 h; S205: Second-stage enzymolysis: add 1.0% of complex protease based on the weight of the protein, and perform enzymolysis at 55°C for 1 h; S206: After the second-stage enzymolysis is completed, inactivate the enzyme at 98°C for 15 min, and cool to room temperature to obtain a yak plasma protein enzymolysis solution; S207: The yak plasma protein enzymolysis solution is subjected to first-stage centrifugal separation at a speed of 10,000 r / min for 10 min, and the supernatant is collected to obtain a first-stage separation yak plasma protein peptide supernatant; S208: The first separation yak plasma protein peptide supernatant is subjected to secondary membrane separation by using an ultrafiltration membrane with a pore size of 5000 daltons, and a secondary separation yak plasma protein peptide solution is collected; S209: The secondary separation yak plasma protein peptide solution is subjected to tertiary membrane separation by using an ultrafiltration membrane with a pore size of 2000 daltons to separate yak plasma protein peptides with a molecular weight less than 2000 daltons, and a tertiary separation yak plasma protein peptide solution is collected, followed by freeze-drying to obtain a tertiary separation yak plasma protein peptide freeze-dried powder; S210: The tertiary separation yak plasma protein peptide freeze-dried powder with a molecular weight less than 2000 daltons is dissolved in deionized water to form a 20 mg / mL solution, and then subjected to four-stage Sephadex G-15 gel separation, with deionized water as the eluent, and the elution peak being detected at 280 nm; the second elution peak is collected to obtain a four-stage separation yak plasma protein peptide solution; S211: The four-stage separation yak plasma protein peptide solution is subjected to five-stage reverse phase chromatography separation by using RP-HPLC reverse phase high performance liquid chromatography, and a five-stage separation yak plasma protein peptide solution is collected by taking 8-11 fractions; S212: The five-stage separation yak plasma protein peptide solution is sequentially subjected to concentration, freezing and drying to obtain a yak plasma protein peptide powder with strong antioxidant and ACE inhibitory functions; S213: The yak plasma protein peptide powder is subjected to component determination by LC-MS / MS, and the component determination result is that the amino acid sequence EDGGGWWYNR of the yak plasma protein peptide powder is 40.5%, and WYNR is 17.8%.
[0022] Preparation Experiment 3: S301: Quarantined yak plasma protein powder is dissolved in drinking water meeting health standards, and the concentration of the protein is 10%; S302: Cook and sterilize at 95°C for 20 min; S303: After cooling, a high-speed homogenizer is used for 3 min to obtain a yak plasma protein solution; S304: First-stage enzymolysis: 0.8% of alkaline protease by weight of the raw material protein is added, and the enzymolysis reaction is carried out at 48°C for 1.5 h; S305: Second-stage enzymolysis: 0.8% of complex protease by weight of the protein is added, and the enzymolysis is carried out at 52°C for 1.5 h; S306: After the second-stage enzymolysis is completed, the enzyme is inactivated at 98°C for 15 min, and then cooled to room temperature to obtain a yak plasma protein enzymolysis solution; S307: The yak plasma protein enzymatic hydrolysate was centrifuged at 10,000 r / min for 10 min to collect the supernatant, and a yak plasma protein peptide supernatant was obtained; S308: The yak plasma protein peptide supernatant was subjected to secondary membrane separation using an ultrafiltration membrane with a pore size of 5000 daltons, and a secondary separation yak plasma protein peptide solution was collected; S309: The secondary separation yak plasma protein peptide solution was subjected to tertiary membrane separation using an ultrafiltration membrane with a pore size of 2000 daltons to separate yak plasma protein peptides with a molecular weight less than 2000 daltons, and a tertiary separation yak plasma protein peptide solution was collected, followed by freeze-drying to obtain a tertiary separation yak plasma protein peptide freeze-dried powder; S310: The tertiary separation yak plasma protein peptide freeze-dried powder with a molecular weight less than 2000 daltons was dissolved in deionized water to obtain a 20 mg / mL solution, which was subjected to gel separation using a Sephadex G-15 gel, and the eluent was deionized water. The elution peak was detected at 280 nm, and the third elution peak was collected to obtain a quaternary separation yak plasma protein peptide solution; S311: The quaternary separation yak plasma protein peptide solution was subjected to quinary reverse phase chromatography separation using RP-HPLC, and the 8-11 fraction was collected to obtain a quinary separation yak plasma protein peptide solution; S312: The quinary separation yak plasma protein peptide solution was sequentially concentrated, frozen, and dried to obtain a yak plasma protein peptide powder with strong antioxidant and ACE inhibitory functions; S313: The yak plasma protein peptide powder was subjected to component determination by LC-MS / MS, and the component determination results were as follows: the amino acid sequence EDGGGWWYNR of the yak plasma protein peptide powder was 41.5%, and WYNR was 15.8%.
[0023] Preparation Experiment 4: S401: The quarantine qualified yak plasma protein powder was dissolved in drinking water meeting the health standards, and the protein concentration was 7%; S402: Cook and sterilize at 95°C for 20 min; S403: After cooling, the high-speed homogenizer was treated for 3 minutes to obtain a yak plasma protein solution; S404: First enzyme hydrolysis: 0.5% of alkaline protease was added based on the weight of the raw material protein, and the enzyme hydrolysis reaction was carried out at 45°C for 2h; S405: Secondary enzyme hydrolysis: 1.0% of compound protease was added based on the weight of the protein, and the enzyme hydrolysis was carried out at 50°C for 1h; S406: After the secondary enzyme hydrolysis, the enzyme was inactivated at 98°C for 15 min, and then cooled to room temperature to obtain a yak plasma protein enzymatic hydrolysate; S407: The yak plasma protein enzymatic hydrolysate is subjected to primary centrifugal separation at 10,000 r / min for 10 min, and the supernatant is collected to obtain a primary separation yak plasma protein peptide supernatant; S408: The primary separation yak plasma protein peptide supernatant is subjected to secondary membrane separation by using an ultrafiltration membrane with a pore size of 5000 daltons, and a secondary separation yak plasma protein peptide solution is collected; S409: The secondary separation yak plasma protein peptide solution is subjected to tertiary membrane separation by using an ultrafiltration membrane with a pore size of 2000 daltons to separate yak plasma protein peptides with a molecular weight less than 2000 daltons, and a tertiary separation yak plasma protein peptide solution is collected, followed by freeze-drying to obtain a tertiary separation yak plasma protein peptide freeze-dried powder.
[0024] Preparation Experiment 5: S501: Quarantined yak plasma protein powder is dissolved in drinking water meeting health standards, and the concentration of protein is 7%; S502: Cook and sterilize at 95°C for 20 min; S503: After cooling, a high-speed homogenizer is used for 3 minutes to obtain a yak plasma protein solution; S504: First enzyme hydrolysis: 0.5% of alkaline protease by weight of raw material protein is added, and the enzyme hydrolysis reaction is carried out at 45°C for 2h; S505: Second enzyme hydrolysis: 1.0% of compound protease by weight of protein is added, and the enzyme hydrolysis is carried out at 50°C for 1h; S506: After the second enzyme hydrolysis, the enzyme is inactivated at 98°C for 15 min, and then cooled to room temperature to obtain a yak plasma protein enzymatic hydrolysate; S507: The yak plasma protein enzymatic hydrolysate is subjected to primary centrifugal separation at 10,000 r / min for 10 min, and the supernatant is collected, and then freeze-dried to obtain a yak plasma protein enzymatic peptide powder.
[0025] The enzyme activity of alkaline protease used in the enzyme hydrolysis process in the above preparation experiments 1-5 is 100,000-150,000 U / g, the enzyme activity of neutral protease is 250,000-400,000 U / g, the enzyme activity of papain is 250,000-400,000 U / g, and the compound protease includes neutral protease and papain, and the ratio of the two is 1:1.
[0026] The antioxidant capacity determination experiment report and ACE inhibition capacity determination experiment report of the yak plasma protein peptide powder obtained according to the above preparation experiments 1-5 are described in detail as follows: Experiment Report 1: relates to the antioxidant capacity determination method and results of the yak plasma protein peptide powder, specifically including: Experimental samples: samples 1-3 are yak plasma protein peptide powders prepared by the steps S101-S112, S201-S212 and S301-S312 of the above preparation experiments 1-3, respectively, samples 4 and 5 are yak plasma protein peptide powders prepared by the preparation experiment 4 and the preparation experiment 5, sample 6 is a plasma protein powder without enzymolysis; sample EDGGGWWYNR is a polypeptide artificially synthesized according to the amino acid sequence EDGGGWWYNR, and sample WYNR is a polypeptide artificially synthesized according to the amino acid sequence WYNR.
[0027] The antioxidant activity of the yak plasma protein peptide powder was determined according to the following method: 1. DPPH free radical scavenging capacity: 100 μL of sample solution of different concentrations (crude peptide sample concentration is 5 mg / mL) was mixed with 100 μL of 0.1 mM DPPH ethanol solution, and the absorbance was measured at 517 nm after reaction at room temperature for 30 min in the dark; the same volume of deionized water was used instead of the sample solution as a blank group, and the absorbance was measured; the same volume of anhydrous ethanol was used instead of the DPPH ethanol solution as a control group, and the absorbance was measured; the clearance rate of a series of gradient concentration samples was determined, and the sample concentration was taken as the abscissa (X), and the corresponding DPPH free radical clearance rate was taken as the ordinate (Y), to draw the dose-effect curve; the data was subjected to non-linear regression fitting, and the value was calculated by fitting the curve; the DPPH free radical scavenging capacity was calculated according to the following formula: DPPH free radical scavenging capacity (%) = [1-(A measured-A control)] / A blank × 100; In the formula: A measured is the absorbance value of the sample group at a wavelength of 517 nm; A control is the absorbance value of the control group at a wavelength of 517 nm; A blank is the absorbance value of the blank group at a wavelength of 517 nm.
[0028] 2. ABTS free radical scavenging capacity: 17 mg of potassium persulfate was dissolved in 26 mL of double distilled water, 2.6 mL of the above potassium persulfate solution was taken, 10 mg of ABTS was added, and an ABTS stock solution was prepared, which was placed in the dark for 12-16 h; 0.8 mL of the ABTS stock solution was taken and diluted with 0.2 M phosphate buffer (pH 7.4) to obtain an ABTS stock solution with an absorbance of 0.70±0.02 at 734 nm; 0.04 mL of sample 1-8 solution of different concentrations was mixed with 4 mL of ABTS stock solution and shaken for 30 s, then placed in the dark for 6 min, and the absorbance was detected at 405 nm; the same volume of phosphate buffer was used instead of the sample solution as a blank group, and the absorbance was measured; the same volume of phosphate buffer was used instead of the ABTS stock solution as a control group, and the absorbance was measured; the clearance rate of a series of gradient concentration samples was determined, and the sample concentration was taken as the abscissa (X), and the corresponding ABTS free radical clearance rate was taken as the ordinate (Y), to draw the dose-effect curve; the data was subjected to non-linear regression fitting, and the The working solution was used as a control group to determine the absorbance; by determining the clearance rate of a series of gradient concentration samples, the sample concentration was used as the abscissa (X), and the corresponding ABTS free radical clearance rate was used as the ordinate (Y) to draw a dose-effect curve; the data was subjected to non-linear regression fitting, and the value was calculated by fitting the curve The ABTS free radical scavenging capacity was calculated according to the following formula: ABTS free radical scavenging capacity (%) = [1-(A measured-A control)] / A blank x 100 In the formula: A measured is the absorbance value of the sample group at a wavelength of 405 nm; A control is the absorbance value of the control group at a wavelength of 405 nm; and A blank is the absorbance value of the blank group at a wavelength of 405 nm.
[0029] The determination results are shown in Table 1, and the results prove that the yak plasma protein peptide powders prepared in Preparation Experiments 1, 2 and 3 and the synthesized pure peptides have good antioxidant capacity, the DPPH free radical scavenging capacity IC50 of EDGGGWWYNR and WYNR is only 49 μg / mL and 25 μg / mL, the IC50 of Sample 1 to Sample 3 is lower than 150 μg / mL; the ABTS free radical scavenging capacity IC50 of EDGGGWWYNR and WYNR is 43 μg / mL and 28 μg / mL, and the IC50 of Sample 1 to Sample 3 is lower than 130 μg / mL; the antioxidant capacity of the yak plasma protein peptide powders prepared in Preparation Experiments 1, 2 and 3 and the pure peptides of EDGGGWWYNR and WYNR is significantly higher than that of the yak plasma protein peptide powders prepared by other methods and yak plasma protein, which indicates that the yak plasma protein peptide powder developed in the present application has significant efficacy.
[0030] Table 1: Antioxidant activity test results of yak plasma protein peptide powder Sample DPPH radical scavenging capacity (IC 50 ) pg / mL ABTS radical scavenging capacity (IC50) 50 ) pg / mL Sample 1 146 129 Sample 2 128 116 Sample 3 139 109 Sample 4 986 949 Sample 5 1739 1507 Sample 6 5439 4309 EDGGGWWYNR 49 43 WYNR 25 28 ;
[0031] Experiment Report 2: relates to the ACE inhibition capacity determination method and results of the yak plasma protein peptide powder, specifically including: The determination method of ACE inhibition rate: the released Hip amount was quantitatively detected by high performance liquid chromatography at 228 nm, so as to calculate the ACE inhibition rate of the polypeptide.
[0032] 1. Preparation of reagents: Phosphate buffer solution at pH 8.3: prepared with ultrapure water, containing phosphate 50 mmol / L, NaCl 300 mmol / L, and adjusting pH to 8.3; ACE enzyme solution: 2 mL of phosphate buffer solution was added to 1 U of ACE, so that the concentration became 0.5 U / mL; HHL solution: Dissolve HHL in phosphate buffer to a final concentration of 5 mmol / L; Sample solution: Weigh an appropriate amount of sample and prepare a phosphate buffer solution of the required concentration; 2. Chromatographic conditions for ACE inhibition rate determination: Detection wavelength: 228 nm; Flow rate: 1 mL / min; Mobile phase A: ultrapure water (containing 0.1% trifluoroacetic acid); Mobile phase B: methanol (containing 0.1% trifluoroacetic acid); Injection volume: 10 μL, manual injection; 3. Methods for determining ACE inhibitory activity: Take 120 μL of HHL substrate solution, first add 20 μL of sample and mix well, then incubate at 37℃ for 10 min. Next, add 10 μL of ACE enzyme solution and react at 37℃ for 30 min. Finally, add 150 μL of 1mol / L HCl to terminate the reaction and obtain the reaction solution. The reaction solution is analyzed by HPLC, and a blank control group is set up. The formula for calculating the ACE inhibitory activity is as follows: ACE inhibitory activity % = (MN) / M × 100; In the formula: M is the peak area of hippuric acid in the control group; N is the peak area of hippuric acid in the added sample group; 4. Determination of half-inhibitory concentration: like Figures 1-8 As shown, the inhibitory activity of the yak plasma protein peptide powder was determined according to the in vitro detection method for ACE inhibitory peptides. A smooth curve was plotted with concentration as the abscissa and ACE inhibition rate as the ordinate. The IC50 value was calculated from the smooth curve. The experimental results are shown in Table 2.
[0033] Table 2: Experimental results of ACE inhibitory activity of yak plasma protein peptide powder Sample ACE Inhibitory Capacity (IC50) ug / mL Sample 1 198 Sample 2 167 Sample 3 158 Sample 4 2689 Sample 5 3588 Sample 6 4678 EDGGGWWYNR 71 WYNR 43 ;
[0034] The results showed that the yak plasma peptides and synthesized pure peptides prepared in Examples 1, 2 and 3 had good ACE inhibition ability. The IC50 values of DPPHACE inhibition ability of EDGGGWWYNR and WYNR were only 71 μg / mL and 43 μg / mL, respectively, and the IC50 values of samples 1 to 3 were less than 200 μg / mL. This indicates that the ACE inhibition ability of the yak plasma protein peptide powder developed in this invention is significantly higher than that of yak plasma protein peptide powder and yak plasma protein prepared by other methods.
[0035] The technical solutions described above are only preferred embodiments of the present application and are not used to limit the present application. Although the present application is described in detail with reference to the foregoing embodiments, those skilled in the art can modify the technical solutions recorded in the foregoing embodiments or equivalently replace some technical features thereof, and any modification, equivalent replacement and improvement, etc. solutions made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims
1. A method for preparing a yak blood plasma protein peptide powder with strong antioxidant and ACE inhibitory functions, characterized in that, It comprises the following steps: S1: the yak blood plasma protein powder is treated by dissolving in water, high-temperature sterilization and cooling homogenization in sequence to obtain yak blood plasma protein liquid; S2: the yak blood plasma protein liquid is subjected to enzymolysis to obtain yak blood plasma protein enzymolysis liquid; S3: the yak blood plasma protein enzymolysis liquid is subjected to multi-stage separation and concentration drying in sequence to obtain yak blood plasma peptide powder with strong antioxidant and ACE inhibitory functions; S4: the components of the yak blood plasma peptide powder are determined.
2. The method for preparing the yak blood plasma protein peptide powder with strong antioxidant and ACE inhibitory functions according to claim 1, characterized in that, The S1 specifically comprises the following steps: S11: the yak blood plasma protein powder qualified for quarantine is dissolved in drinking water meeting health standards, and the concentration of protein is 7-10%; S12: sterilization is performed by cooking at 95-100 DEG C for 10-20 min; S13: after cooling, the high-speed homogenizer is used for treatment for 2-3 min to obtain yak blood plasma protein liquid.
3. The method for preparing the yak blood plasma protein peptide powder with strong antioxidant and ACE inhibitory functions according to claim 1, characterized in that, The enzymolysis in the S2 is carried out according to the weight percentage of 1.0-2.0% of proteinase based on the weight of protein in the yak blood plasma protein liquid, and specifically comprises the following steps: S21: first-stage enzymolysis: 0.5-1.0% of alkaline protease based on the weight of raw material protein is added, and the enzymolysis reaction is carried out at 42-50 DEG C for 0.5-2 h; S22: second-stage enzymolysis: 0.5-1.0% of compound protease based on the weight of protein is added, and the enzymolysis is carried out at 50-55 DEG C for 0.5-2 h; S23: after the second-stage enzymolysis, the enzyme is inactivated at 95-100 DEG C for 10-20 min, and the temperature is cooled to room temperature to obtain yak blood plasma protein enzymolysis liquid.
4. The method for preparing the yak blood plasma protein peptide powder with strong antioxidant and ACE inhibitory functions according to claim 1, characterized in that, The S3 comprises the following steps: S31: the yak blood plasma protein enzymolysis liquid is subjected to first-stage centrifugal separation in a centrifuge with a rotation speed of 10,000 r / min for 10 min, and the supernatant is collected to obtain first-stage separation yak blood plasma peptide supernatant; S32: the first-stage separation yak blood plasma peptide supernatant is subjected to second-stage membrane separation by using an ultrafiltration membrane with a pore size of 5000 daltons, and second-stage separation yak blood plasma peptide solution is collected; S33: the second-stage separation yak blood plasma peptide solution is subjected to third-stage membrane separation by using an ultrafiltration membrane with a pore size of 2000 daltons to separate yak blood plasma peptide with a molecular weight less than 2000 daltons, and third-stage separation yak blood plasma peptide solution is collected, and then freeze-drying is carried out to obtain third-stage separation yak blood plasma peptide freeze-dried powder; S34: the third-stage separation yak blood plasma peptide freeze-dried powder with a molecular weight less than 2000 daltons is dissolved in deionized water to form a 20 mg / mL solution, and then subjected to fourth-stage Sephadex G-15 gel separation, the eluent is deionized water, and the elution peak is detected at 280 nm, and the second elution peak is collected to obtain fourth-stage separation yak blood plasma peptide solution; S35: the fourth-stage separation yak blood plasma peptide solution is subjected to fifth-stage reverse phase chromatography separation by using RP-HPLC reverse phase high performance liquid chromatography, and the fifth-stage separation yak blood plasma peptide solution is collected by taking 8-11 fractions; S36: the fifth-stage separation yak blood plasma peptide solution is subjected to concentration, freezing and drying in sequence to obtain yak blood plasma peptide powder with strong antioxidant and ACE inhibitory functions.
5. The method for preparing the yak blood plasma protein peptide powder with strong antioxidant and ACE inhibitory functions according to claim 1, characterized in that, The S4 is determined by LC-MS / MS, and the component determination result is that the amino acid sequence EDGGGWWYNR of the yak plasma protein peptide powder is 39.5-42.5%, and the amino acid sequence WYNR is 15.3-17.8%.
6. A kind of strong anti-oxidation and ACE inhibiting function's yak blood plasma protein peptide powder, it is characterized in that, The yak plasma protein peptide powder is prepared by the method in claims 1-5, and the amino acid sequence EDGGGWWYNR of the yak plasma protein peptide powder is 39.5-42.5%, and the amino acid sequence WYNR is 15.3-17.8%.