CN106755231B - Method for extracting angiotensin converting enzyme inhibitory peptide - Google Patents
Method for extracting angiotensin converting enzyme inhibitory peptide Download PDFInfo
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- CN106755231B CN106755231B CN201611072322.2A CN201611072322A CN106755231B CN 106755231 B CN106755231 B CN 106755231B CN 201611072322 A CN201611072322 A CN 201611072322A CN 106755231 B CN106755231 B CN 106755231B
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- 101710178392 Angiotensin-converting enzyme inhibitory peptide Proteins 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 20
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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Abstract
The invention provides an extraction method of angiotensin converting enzyme inhibitory peptide, which comprises the following steps: taking the enzymolysis liquid freeze-dried powder, and dissolving in borate buffer solution; mixing the immobilized angiotensin converting enzyme with the enzymolysis solution, oscillating in a constant-temperature water bath at 30 ℃, filtering, washing, and collecting a solid phase to obtain the immobilized angiotensin converting enzyme adsorbed with angiotensin converting enzyme inhibitory peptide; adding the immobilized angiotensin converting enzyme adsorbed with angiotensin converting enzyme inhibitory peptide into NaCl solution, washing the inhibitory peptide adsorbed by the angiotensin converting enzyme with water bath, collecting the eluent, and freeze-drying to obtain the angiotensin converting enzyme inhibitory peptide. The immobilized angiotensin converting enzyme has high activity and good stability, has excellent effect when being used for separating and purifying the corresponding angiotensin converting enzyme inhibitory peptide, and has simple immobilization process and better product stability.
Description
Technical Field
The invention belongs to the technical field of extraction of angiotensin converting enzyme inhibitory peptides, and particularly relates to separation and purification of angiotensin converting enzyme inhibitory peptides in raw materials with angiotensin converting enzyme inhibitory activity or enzymatic hydrolysate thereof by applying immobilized angiotensin converting enzyme.
Background
Hypertension changes and damages the structure and function of the brain, heart, blood vessels, kidneys and fundus oculi, causing concurrent diseases of these organs to result in functional failure. Hypertension results in several complications: coronary heart disease, cerebrovascular disease, hypertensive heart and brain diseases, chronic renal failure, atherosclerosis, etc. The current blood pressure lowering drugs mainly comprise diuretics, beta receptor blockers, calcium channel blockers, Angiotensin Converting Enzyme (ACE) inhibitors and angiotensin II receptor blockers.
Angiotensin Converting Enzyme (ACE) is a multifunctional dipeptide carboxypeptidase that is found in various tissues such as lung, kidney, brain, eyeball, small intestine, placenta, etc. In humans, blood pressure regulation is mainly controlled by the Renin-Angiotensin System (RAS) and the Kallikrein-Kinin System (KKS), which when unbalanced, cause abnormalities in human blood pressure, and ACE is a key enzyme of both systems.
At present, most of the drugs for treating hypertension are synthetic ACE inhibitory peptides, but because of obvious side effects, the development of natural healthy ACE inhibitory peptides without side effects is a focus of research in the future.
At present, Angiotensin Converting Enzyme Inhibitory Peptide (ACEIP) is usually isolated and purified by ultrafiltration, gel chromatography, ion exchange chromatography, reverse phase high performance liquid chromatography, affinity chromatography, and the like. The affinity separation method for separating and purifying protein peptide is a very efficient and rapid method, and the method for affinity separation and purification of protein by using immobilized enzyme technology is also developed rapidly. The carrier applying the immobilized enzyme affinity separation method can not only achieve the purpose of quickly separating and purifying the protein peptide, but also reduce the cost and is environment-friendly.
In the domestic and foreign reports on the immobilization research of angiotensin converting enzyme, chitosan, agarose microspheres and chitose are mostly adopted as immobilization carriers. The immobilization of ACE to SBA-15, the preparation of SBA-15, was not reported. The SBA-15 molecular sieve is a mesoporous material prepared in an acid synthesis system by adopting a triblock copolymer as a template agent, the pore size is 4.6-30 nm, and the pore volume can reach 0.85cm3And/g is the molecular sieve material with the largest pore diameter at present. The SBA-15 mesoporous molecular sieve has the characteristics of adjustable aperture, high pore wall thickness and high hydrothermal stability. The solvent-free method is adopted to react ZnO, (NH) with Sesamum tea and the like4)2SO4Is ground and mixed with a calcined main material SBA-15 molecular sieve manually and then is roasted to prepare Zn-SO4 2-SBA-15 modified mesoporous molecular sieve.
Disclosure of Invention
The invention aims to purify and separate Angiotensin Converting Enzyme (ACE) inhibitory peptides by using immobilized ACE to prepare natural and healthy angiotensin converting enzyme inhibitory peptides with high purity and no side effect.
In order to achieve the purpose, the invention provides an extraction method of angiotensin converting enzyme inhibitory peptide, which comprises the following steps:
s1, dissolving the enzymolysis liquid freeze-dried powder in 0.1mol/L borate buffer solution with the pH value of 8.3 to prepare enzymolysis liquid solution with the concentration of 20-100 g/L;
the enzymolysis liquid freeze-dried powder is prepared by hydrolyzing raw materials with angiotensin converting enzyme inhibitory activity by protease, and is prepared by ultrafiltration, classification and freeze-drying; preferably, selecting the soaked moth snail enzymolysis liquid freeze-dried powder;
the borate buffer solution contains NaCl with the concentration of 0.1-0.3 mol/L;
s2, mixing the immobilized angiotensin converting enzyme with the enzymolysis solution prepared in the step S1 according to the mass volume ratio of 1: 10-20 (g/mL), oscillating in a constant-temperature water bath at 30 ℃ for 50-120 min, performing suction filtration, washing with 0.1mol/L borate buffer solution with the pH of 8.3, and collecting a solid phase to obtain the immobilized angiotensin converting enzyme adsorbed with angiotensin converting enzyme inhibitory peptide;
s3, adding the immobilized angiotensin converting enzyme adsorbed with the angiotensin converting enzyme inhibitory peptide prepared in the step S3 into a NaCl solution with pH of 7 and concentration of 2mol/L, carrying out water bath at 30 ℃ for 30-120 min, eluting the inhibitory peptide adsorbed by the angiotensin converting enzyme, collecting the eluent, and freeze-drying to obtain the angiotensin converting enzyme inhibitory peptide.
Preferably, the specific preparation process of the enzymatic hydrolysate prepared by hydrolyzing the raw material having angiotensin-converting enzyme inhibitory activity with the protease in step S1 is as follows:
freeze-drying raw materials with angiotensin converting enzyme inhibitory activity to prepare freeze-dried powder, uniformly mixing the freeze-dried powder and water according to a mass-volume ratio of 1: 10-20 (g/mL), and preheating in a water bath at 45 ℃ for 5-15 min;
adjusting the pH value of the solution to 7.0, adding neutral protease with the enzyme addition amount of 2500U/g, starting reaction, dropwise adding a standard alkali solution or a standard acid solution in the reaction process, keeping the pH value of the reaction solution to be 7.0 unchanged, hydrolyzing for 2-3 h, inactivating the enzyme in a boiling water bath for 10-20 min, and cooling; centrifuging for 20-30 min at 4 ℃ and 10000rpm, and taking supernatant to obtain enzymatic hydrolysate.
The raw material with angiotensin converting enzyme inhibitory activity is preferably Bull-worm.
Further optimizing, the immobilized angiotensin converting enzyme in step S2 is SBA-15 immobilized angiotensin converting enzyme; the preparation method comprises the following steps:
mixing mesoporous molecular sieve SBA-15 and angiotensin converting enzyme solution according to the mass-volume ratio of 1: 5-20 (g/mL), oscillating in a water bath at 45-50 ℃ for 30-40 min in a water bath until the protein content in the reaction liquid is unchanged, and finishing the reaction; carrying out suction filtration, washing and removing the angiotensin converting enzyme which is not adsorbed and combined to obtain SBA-15 immobilized angiotensin converting enzyme;
the angiotensin converting enzyme solution is prepared by adopting 7.8-9.3 borate buffer solution, and the enzyme activity is 0.17U/mL.
Most preferably, the mesoporous molecular sieve SBA-15 is chelated with Zn prepared by a direct or indirect adsorption chelation mode2+SBA-15 molecular sieve of (a);
the specific operation of the direct chelation is as follows:
mixing mesoporous molecular sieve SBA-15 and ZnSO with the concentration of 0.05mol/L4Mixing the water solutions according to the mass-volume ratio of 1:10g/mL, and oscillating in water bath at 30-40 ℃ for 45-65 min to obtain the directly chelated Zn2+SBA-15 molecular sieve of (a);
the specific operation of the indirect chelation is as follows:
adding mesoporous molecular sieve SBA-15 into absolute ethyl alcohol, adding 3- (methacryloyloxy) propyl trimethoxy silane (GLYMO) into the absolute ethyl alcohol, oscillating in a water bath at 30 ℃ for 10-20 h, performing suction filtration after the reaction is finished, collecting solid substances, and cleaning;
the mass-volume ratio of the mesoporous molecular sieve SBA-15 to the absolute ethyl alcohol and the 3- (methacryloyloxy) propyl trimethoxy silane is 1: 350-400: 20-30 (g/mL/mL);
connecting iminodiacetic acid (IDA), mixing the obtained solid substance with iminodiacetic acid solution with the concentration of 0.6-0.8 mol/L according to the mass-volume ratio of 1:10(g/mL), carrying out water bath oscillation reaction at 30-40 ℃, carrying out suction filtration after the reaction is finished, and collecting the solid substance again;
mixing the solid matter collected again with ZnSO with a concentration of 0.05mol/L4The solution is mixed according to the mass-volume ratio of 1:10(g/mL), and is subjected to water bath oscillation at the temperature of 30-40 ℃ for 60-90 min to obtain the indirect chelated Zn2+The SBA-15 molecular sieve of (1).
In the process, the multi-purpose freeze-dried powder is used as an experimental material, firstly, the freeze-dried powder can be conveniently prepared into a solution with required concentration, and secondly, the freeze-dried powder is convenient to store and has more stable activity on a sample in a solid state than a sample in a liquid state.
After adopting the scheme, compared with the prior art, the invention has the following advantages:
1. based on the principle of affinity, the invention applies the immobilized angiotensin converting enzyme to the extraction, separation and purification of Angiotensin Converting Enzyme Inhibitory Peptide (ACEIP), and provides an excellent carrier and a method for the separation and purification of ACEIP.
2. The immobilized angiotensin converting enzyme has high activity and good stability, has excellent effect when used for separating and purifying corresponding angiotensin converting enzyme inhibitory peptides, preferentially adopts Zn-SBA-15 immobilized angiotensin converting enzyme, has simple immobilization process and better product stability; compared with the separation and purification of the ACEIP by the immobilized angiotensin converting enzyme, the separation and purification of the enzymolysis liquid can achieve better separation effect compared with the separation and purification of the single free enzyme.
3. The method is suitable for various raw materials with angiotensin-converting enzyme inhibitory activity, preferably the bulldog snails, and the raw materials are natural and healthy, and the prepared angiotensin-converting enzyme inhibitory peptide has high purity, is natural and healthy and has wide application range. The invention separates and purifies ACEIP from the bulldog snails for the first time, and provides a new way for the comprehensive utilization of the bulldog snails.
4. The free ACE is easy to denature and inactivate in the using process, great difficulty is brought to use and storage, and the immobilized enzyme adopted by the invention not only can realize recycling of the enzyme, but also can improve the stability of the immobilized enzyme.
Detailed Description
Example 1
1) The preparation method of the mesoporous molecular sieve SBA-15 carrier comprises the following specific steps:
preparing a mesoporous molecular sieve SBA-15 carrier: dissolving 1g of polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer (P123) in 50mL1.6mol/L hydrochloric acid solution, stirring in a water bath at 30 ℃ for 3h, then dropwise adding 5mL of Tetraethoxysilane (TEOS), and continuously stirring in a water bath at 40 ℃ for 20 h; then transferring the reactant into a reaction kettle, and putting the reaction kettle into an oven to react for 24 hours under the standing condition of 110 ℃; finally, collecting a solid product, washing with water, performing suction filtration, placing the solid product into an oven at 80 ℃ for 12h, taking out the solid product, placing the solid product into a muffle furnace at 550 ℃ for burning for 8h to obtain a mesoporous molecular sieve SBA-15; then 0.5g of SBA-15 is taken and the mass volume of the SBA-15: ZnSO is calculated4(0.05mol/L of ZnSO4Solution) 1:10 to ZnSO4Carrying out shaking reaction on the solution in a 35 ℃ constant-temperature water bath shaking pot for 50 min; taking out after 50min, and carrying out suction filtration on Zn on the surfaces of the microspheres by using a large amount of water2+And (5) cleaning.
2) Immobilization of angiotensin converting enzyme on Zn-SBA-15 vector prepared in the previous step
Weighing 0.5g of the Zn-SBA-15 vector prepared in the step 1), adding 10mL of angiotensin converting enzyme (the enzyme activity is 0.17U/mL, and the preparation is carried out by using 0.1mol/L borate buffer solution with the pH value of 8.3), and oscillating in a water bath in a constant-temperature water bath kettle at 50 ℃ for 30 min. After the reaction is finished, the enzyme which is not adsorbed and combined is filtered and cleaned, so that the immobilized angiotensin converting enzyme is obtained, and the enzyme activity is 0.2372U/g.
3) Weighing 100g of bulldog snail freeze-dried powder, mixing the powder with the following components in a ratio of 1: adding 10(g/mL) of the mixture into deionized water, preheating for 10min in a water bath at a certain temperature, adjusting the pH to be the optimum pH of neutral protease 7.0, adding 2500U/g of neutral protease, starting reaction, dropwise adding a standard alkali solution or an acid solution in the reaction process, maintaining the initial pH value to be 7.0, performing hydrolysis for 2h, inactivating the enzyme in the boiling water bath for 15min, performing freeze centrifugation for 20min at 4 ℃ and 10000rpm, taking supernatant, and performing freeze drying to obtain the protein hydrolysate of the oncomelania melanosporum enzymatic hydrolysate. Obtaining the lyophilized powder of the enzymolysis liquid of the vesicular moth snail enzymolysis liquid. Dissolving the prepared soaked moth snail enzymolysis solution freeze-dried powder in 0.1M borate buffer solution containing 0.2mol/L NaCl and having the pH value of 8.3 to prepare the soaked moth snail enzymolysis solution with the concentration of 50 g/L. Adding the prepared Zn-SBA-15 immobilized angiotensin converting enzyme into the enzymolysis liquid prepared in the step according to the mass-volume ratio of 1:20(g/mL), oscillating the solution in a water bath kettle at constant temperature of 30 ℃ for 60min, carrying out suction filtration and cleaning on the Zn-SBA-15 immobilized ACE adsorbing angiotensin converting enzyme inhibitory peptides from the solution by using 0.1mol/L borate buffer solution with pH of 8.3, adding the solution into 2mol/L NaCl solution with pH of 7, carrying out water bath at 30 ℃ for 60min, eluting the adsorbed angiotensin converting enzyme inhibitory peptides, collecting eluent and freeze-drying to obtain the angiotensin converting enzyme inhibitory peptides.
In the above lyophilized powder, 0.021g of angiotensin converting enzyme inhibitory peptide can be obtained from 1g of the enzymolysis solution, i.e. the yield is 2.1%.
Determination of ACE inhibitory Activity
The preparation method of the main reagent comprises the following steps:
ACE solution: 1U ACE was dissolved in 10mL cold 1M borate buffer (containing 0.3M NaCl) pH8.3 to make 0.1U/mLACE solution, dispensed, and stored at-20 deg.C for use.
HHL (equacy histidyl leucine) solution: an appropriate amount of HHL was taken and prepared into 5mmol/L HHL solution with 0.1M Borate buffer solution (containing 0.3M NaCl) with pH8.3 for use.
Hippuric acid standard solution: accurately weighing a hippuric acid standard sample, adding ultrapure water to dissolve the hippuric acid standard sample to prepare a hippuric acid standard solution with the concentration of 50 mug/mL for later use.
The reaction environment was pH8.3, 0.1M borate buffer (containing 0.3M NaCl). Taking 50 mu L of HHL with the concentration of 5mmol/L in a 1.5mL centrifuge tube, adding 20 mu L of sample solution, uniformly mixing, preheating in a thermostatic water bath with the temperature of 37 +/-0.5 ℃ for 5min, then adding 20 mu L of ACE with the concentration of 0.1U/mL, and fully and uniformly mixing. After incubation in a 37 ℃ thermostatic water bath for 60min, 10. mu.L of HCl 0.2M was added to stop the reaction. While 20 μ L of water was used as a blank in place of the sample. After the reaction solution was centrifuged, the amount of produced Hip was measured by HPLC (Waters).
Chromatographic conditions are as follows: ghall 12S05-2546C18 column (250 mm. times.4.6 mm); acetonitrile: water (0.05% TFA) 25:75, isocratic elution; the flow rate is 0.5 mL/min; the detection wavelength is 228 nm; the amount of the sample was 10. mu.L. ACE inhibition was calculated as follows.
In the formula: sample a-peak area of hippuric acid in the inhibitor added group;
control a-peak area of hippuric acid in blank control group.
The half inhibitory concentration (IC50) of the compound on ACE is 0.85g/L by adopting a high performance liquid phase method. The half inhibition concentration is 15.27g/L lower and 17 times lower than the half inhibition concentration (IC50) of ACE of the cystoselta snail enzymolysis liquid, which shows that the ACE inhibition rate of the component obtained after affinity separation is obviously improved compared with that of the original enzymolysis liquid. Meanwhile, the method of the invention achieves better separation effect on ACEIP in the enzymolysis liquid.
Example 2
1) Preparing a mesoporous molecular sieve SBA-15 carrier: dissolving 1g of polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer (P123) in 50mL of 1.6mol/L hydrochloric acid solution, stirring in a water bath at 30 ℃ for 3h, then dropwise adding 5mL of Tetraethoxysilane (TEOS), and continuously stirring in a water bath at 40 ℃ for 20 h; then transferring the reactant into a reaction kettle, and putting the reaction kettle into an oven to react for 24 hours under the standing condition of 110 ℃; finally, collecting a solid product, washing with water, performing suction filtration, placing the solid product into an oven at 80 ℃ for 12h, taking out the solid product, placing the solid product into a muffle furnace at 550 ℃ for burning for 8h to obtain a mesoporous molecular sieve SBA-15; adding 0.5g of SBA-15 into 175mL of absolute ethyl alcohol, adding 2mLGLYMO, oscillating in water bath at 30 ℃ for 15h, filtering after the reaction is finished, washing, connecting with IDA, adding 0.7mol/L IDA solution (the IDA solution is prepared by 1.5mol/L sodium carbonate solution) according to a ratio of 1:10(g/mL), oscillating in water bath at 35 ℃ for reaction for 70min, and after the reaction is finished and filtering, adding the components in a mass-volume ratio of 1:10(g/mL) of ZnSO was added to the solution at a rate of 0.05mol/L4The solution was shaken in a water bath at 30 ℃ for 75min to obtain the corresponding carrier.
2) Immobilization of angiotensin converting enzyme on Zn-SBA-15 vector prepared in the previous step
Weighing 0.5g of the Zn-SBA-15 vector prepared in the step 1), adding 10mL of angiotensin converting enzyme (the enzyme activity is 0.17U/mL, and the preparation is carried out by using 0.1mol/L borate buffer solution with the pH value of 8.3), and oscillating in a water bath in a constant-temperature water bath kettle at 50 ℃ for 30 min. After the reaction is finished, the enzyme which is not adsorbed and combined is filtered and cleaned, so that the immobilized angiotensin converting enzyme is obtained, and the enzyme activity is 0.3074U/g.
3)100g of blister moth snail freeze-dried powder is prepared by mixing 1: adding 10g/ml of the mixture into deionized water, preheating for 10min in a water bath at a certain temperature, adjusting the pH to be the optimum pH of neutral protease 7.0, adding 2500U/g of neutral protease, then starting the reaction, dropwise adding a standard alkali solution or an acid solution in the reaction process, maintaining the initial pH value to be 7.0, performing hydrolysis for 2h, performing enzyme deactivation in a boiling water bath for 15min, performing refrigerated centrifugation for 20min at 4 ℃ and 10000rpm, taking supernatant, and performing refrigerated drying to obtain the hydatid moth snail protein hydrolysate. The prepared bulldog snail enzymolysis solution freeze-dried powder is dissolved in 0.1M borate buffer solution with the pH value of 8.3 and containing 0.1mol/L NaCl to prepare the bulldog snail enzymolysis solution with the concentration of 50 g/L. Adding the prepared Zn-SBA-15 immobilized angiotensin converting enzyme into the enzymolysis liquid prepared in the step according to the mass-volume ratio of 1:15(g/ml), oscillating the solution in a water bath at constant temperature of 30 ℃ for 60min, then carrying out suction filtration and cleaning on the Zn-SBA-15 immobilized ACE adsorbing angiotensin converting enzyme inhibitory peptides from the solution by using 0.1mol/L borate buffer solution with pH of 8.3, then adding the solution into 2mol/L NaCl solution with pH of 7, carrying out water bath at 30 ℃ for 60min, eluting the adsorbed angiotensin converting enzyme inhibitory peptides, collecting eluent and freeze-drying to obtain the angiotensin converting enzyme inhibitory peptides.
In the above lyophilized powder, 0.0199g of angiotensin-converting enzyme inhibitory peptide can be obtained from 1g of the enzymolysis solution, i.e., the yield is 1.99%.
The half inhibitory concentration (IC50) of the compound on ACE is 1.0g/L by adopting a high performance liquid phase method. The half inhibition concentration is 15.27g/L lower and 15 times lower than the half inhibition concentration (IC50) of ACE of the cystoselta snail enzymolysis liquid, which shows that the ACE inhibition rate of the components obtained after affinity separation is obviously improved compared with that of the original enzymolysis liquid. Achieves better separation effect on ACEIP in the enzymolysis liquid. Meanwhile, the method of the invention achieves better separation effect on ACEIP in the enzymolysis liquid.
Experimental example 3
Steps 1) and 2) of this experiment were the same as 1) and 2) of example 1.
3)100g of blister moth snail freeze-dried powder is prepared by mixing 1: adding 10(g/mL) of the mixture into deionized water, preheating for 10min in a water bath at 45 ℃, adjusting the pH to be 7.0 which is the optimum pH value of neutral protease, adding 2500U/g of the neutral protease to start reaction, dropwise adding a standard alkali solution or an acid solution in the reaction process, maintaining the initial pH value to be 7.0, hydrolyzing for 2h, inactivating the enzyme in a boiling water bath for 15min, performing freeze centrifugation for 20min at 4 ℃, 10000rpm, taking supernatant, and performing freeze drying to obtain the protein hydrolysate of the oncomelania melanospongiensis enzymolysis liquid. Dissolving the soaked moth snail enzymolysis solution freeze-dried powder in 0.1M borate buffer solution containing 0.3mol/L NaCl and having pH of 8.3 to prepare the soaked moth snail enzymolysis solution with the concentration of 50 g/L. Adding the prepared Zn-SBA-15 immobilized angiotensin converting enzyme into the enzymolysis liquid prepared in the step according to the mass-volume ratio of 1:20(g/mL), oscillating the solution in a water bath kettle at constant temperature of 30 ℃ for 60min, carrying out suction filtration and cleaning on the Zn-SBA-15 immobilized ACE adsorbing angiotensin converting enzyme inhibitory peptide from the solution by using 0.1mol/L borate buffer solution with pH of 8.3, adding the solution into 2mol/L NaCl solution with pH of 7 for 60min at 30 ℃, eluting the adsorbed angiotensin converting enzyme inhibitory peptide, collecting eluent and freeze-drying to obtain the angiotensin converting enzyme inhibitory peptide.
In the above lyophilized powder, 0.02g of angiotensin converting enzyme inhibitory peptide can be obtained from 1g of the enzymolysis solution, i.e. the yield is 2%.
The half inhibitory concentration (IC50) of the compound on ACE is 1.2g/L by adopting a high performance liquid phase method. The half inhibition concentration is 15.27g/L lower and about 15 times lower than the half inhibition concentration (IC50) of ACE of the cystoselta snail enzymolysis liquid, and the lower the half inhibition concentration of the ACE indicates that the ACE inhibition activity of the peptide is better. The ACE inhibition rate of the component obtained after affinity separation is obviously improved compared with that of the original enzymolysis liquid. Meanwhile, the method of the invention achieves better separation effect on ACEIP in the enzymolysis liquid.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (3)
1. A method for extracting angiotensin converting enzyme inhibitory peptide is characterized by comprising the following specific steps:
s1, dissolving the enzymolysis liquid freeze-dried powder in a borate buffer solution with the pH of 8.3 and the concentration of 0.1mol/L to prepare an enzymolysis liquid solution with the concentration of 20-100 g/L;
the enzymolysis liquid freeze-dried powder is prepared by hydrolyzing raw materials with angiotensin converting enzyme inhibitory activity by protease, and is prepared by ultrafiltration, classification and freeze-drying;
the borate buffer solution contains NaCl with the concentration of 0.1-0.3 mol/L;
s2 preparation of Zn chelated food2+SBA-15 molecular sieve of (a): said chelated with Zn2+The SBA-15 is prepared by direct or indirect adsorption chelation;
the specific operation of the direct adsorption chelation is as follows:
mixing mesoporous molecular sieve SBA-15 and ZnSO with the concentration of 0.05mol/L4Mixing the water solutions according to the mass-volume ratio of 1:10g/ml, and oscillating in water bath at 30-40 ℃ for 45-65 min to obtain the direct chelated Zn2+SBA-15 molecular sieve of (a);
the specific operation of the indirect adsorption chelation is as follows:
adding mesoporous molecular sieve SBA-15 into absolute ethyl alcohol, adding 3- (methacryloyloxy) propyl trimethoxy silane into the absolute ethyl alcohol, oscillating in a water bath at 30 ℃ for 10-20 hours, performing suction filtration after the reaction is finished, collecting solid substances, and cleaning;
the mass-volume ratio of the mesoporous molecular sieve SBA-15 to the absolute ethyl alcohol and the 3- (methacryloyloxy) propyl trimethoxy silane is 1: 350-400: 20-30;
connecting iminodiacetic acid, mixing the obtained solid substance with iminodiacetic acid solution with the concentration of 0.6-0.8 mol/L according to the mass-volume ratio of 1:10, carrying out water bath oscillation reaction at 30-40 ℃, carrying out suction filtration after the reaction is finished, and collecting the solid substance again;
mixing the solid matter collected again with ZnSO with a concentration of 0.05mol/L4Mixing the solutions according to the mass-to-volume ratio of 1:10, and oscillating in a water bath at 30-40 ℃ for 60-90 min to obtain the indirect chelated Zn2+SBA-15 molecular sieve of (a);
s3, taking the Zn chelated compound prepared in the step S22+Mixing the SBA-15 molecular sieve and the angiotensin converting enzyme solution according to the mass-to-volume ratio of 1: 5-20, oscillating in a water bath at 45-50 ℃ for 30-40 min in a water bath until the protein content in the reaction liquid is unchanged, and finishing the reaction; filtering, washing, removing the non-adsorbed and non-bound angiotensin converting enzyme to obtain Zn chelated enzyme2+SBA-15 immobilized angiotensin converting enzyme (ace);
the angiotensin converting enzyme solution is prepared by adopting a borate buffer solution with the pH value of 7.8-9.3, and the enzyme activity is 0.17U/mL;
taking out said chelate with Zn2+Mixing the SBA-15 immobilized angiotensin converting enzyme with the enzymolysis solution prepared in the step S1 according to the mass-volume ratio of 1: 10-20, oscillating in a constant-temperature water bath at 30 ℃ for 50-120 min, performing suction filtration, washing with 0.1mol/L borate buffer solution with the pH of 8.3, and collecting a solid phase to obtain the immobilized angiotensin converting enzyme adsorbed with angiotensin converting enzyme inhibitory peptide;
s4, adding the immobilized angiotensin converting enzyme adsorbed with the angiotensin converting enzyme inhibitory peptide prepared in the step S3 into a NaCl solution with pH of 7 and concentration of 2mol/L, carrying out water bath at 30 ℃ for 30-120 min, eluting the adsorbed angiotensin converting enzyme inhibitory peptide, collecting eluent, and freeze-drying to obtain the angiotensin converting enzyme inhibitory peptide.
2. The method for extracting angiotensin-converting enzyme inhibitory peptide according to claim 1, wherein the enzymatic hydrolysate prepared by hydrolyzing the raw material having angiotensin-converting enzyme inhibitory activity with protease in step S1 is prepared by the following steps:
freeze-drying raw materials with angiotensin converting enzyme inhibitory activity to prepare freeze-dried powder, mixing the freeze-dried powder and water according to a mass-volume ratio of 1: 10-20, and preheating in a water bath at 45 ℃ for 5-15 min;
adjusting the pH value of the solution to 7.0, adding 2500U/g neutral protease, starting reaction, dropwise adding a standard alkali solution or a standard acid solution in the reaction process, keeping the pH value of the reaction solution unchanged, hydrolyzing for 2-3 h, inactivating the enzyme in a boiling water bath for 10-20 min, and cooling; centrifuging for 20-30 min at 4 ℃ and 10000rpm, and taking supernatant to obtain enzymatic hydrolysate.
3. The method for extracting angiotensin-converting enzyme inhibitory peptide according to claim 2, wherein the starting material having angiotensin-converting enzyme inhibitory activity is a hydatid moth snail.
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| In vitro controlled release of antihypertensive drugs intercalated into unmodified SBA-15 and MgO modified SBA-15 matrices;Iuliana Florentina Alexa 等;《International Journal of Pharmaceutics》;20120626;第436卷;第111-119页 * |
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