CN106755231A - A kind of extracting method of inhibiting peptide of tonin - Google Patents
A kind of extracting method of inhibiting peptide of tonin Download PDFInfo
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- CN106755231A CN106755231A CN201611072322.2A CN201611072322A CN106755231A CN 106755231 A CN106755231 A CN 106755231A CN 201611072322 A CN201611072322 A CN 201611072322A CN 106755231 A CN106755231 A CN 106755231A
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Abstract
The invention provides a kind of extracting method of inhibiting peptide of tonin, concretely comprise the following steps：Enzymolysis liquid freeze-dried powder is taken, is dissolved in borate buffer solution；Take immobilization ACE mix with enzymolysis liquor, 30 DEG C of water bath with thermostatic control concussions, suction filtrations, rinse well, collection solid phase obtains being adsorbed with the immobilization ACE of inhibiting peptide of tonin；The immobilization ACE that inhibiting peptide of tonin will be adsorbed with is added in NaCl solution, the peptide for inhibiting of water-bath eluting vascular angiotensin-converting enzyme absorption, collects eluent, is freezed, and obtains inhibiting peptide of tonin.Immobilization hypertensin conversion enzyme activity of the present invention is high, good stability, has excellent effect for isolating and purifying corresponding inhibiting peptide of tonin, and immobilization process is simple, and product stability is more preferable.
Description
Technical field
The extractive technique field of inhibiting peptide of tonin of the present invention, more particularly to a kind of application immobilization blood vessel
Angiotensin-converting enzyme is to the raw material of angiotensin converting enzyme inhibition activity or its enzymolysis liquid Angiotensin-Converting invertase
Peptide for inhibiting is isolated and purified.
Background technology
Hypertension can change and damage the 26S Proteasome Structure and Function on brain, heart, blood vessel, kidney and eyeground, cause these devices
The complication of official is so as to cause the exhaustion of function.Following several complication caused by hypertension：Coronary heart disease, cerebrovascular disease, height
Blood pressure cardio-cerebral diseases, chronic renal failure and atherosclerosis etc..Current blood-pressure drug mainly has diuretics, β to receive
Body retarding agent, calcium channel blocker, ACE (ACE) inhibitor and hypertensinⅡreceptorretarder.
Angiotensin-Converting (ACE) is a kind of to be present in the different tissues such as lung, kidney, brain, eyeball, small intestine, placenta
Multi-functional dipeptides carboxypeptidase.In human body, the regulation of blood pressure is mainly by RAS (Renin-
Angiotensin System, RAS) and kallikrein kinin system (Kallikrein-Kinin System, KKS)
Control, the exception of human blood-pressure will be caused when its is unbalance, and ACE is the key enzyme of the two systems.
At present, the medicine for the treatment of hypertension is mostly artificial synthesized ace inhibitory peptide, but because it has obvious secondary work
With, therefore the natural healthy ace inhibitory peptide that exploitation has no side effect turns into the focus studied at present.
At present, the side such as ultrafiltration, gel chromatography, ion-exchange chromatography, RPLC and affinity chromatography is commonly used
Method isolates and purifies inhibiting peptide of tonin (ACEIP).It is very high that wherein affine partition method isolates and purifies protein peptides
Efficiently method is imitated, and is also developed rapidly using the affine method for isolating and purifying albumen of enzyme immobilization technology.Using immobilised enzymes
The carrier of affine separation method can not only reach the purpose of fast separating and purifying protein peptides, while can be with reduces cost and right
It is environment-friendly.
The report of the domestic and international immobilization research to ACE, more using shitosan, agarose microbeads, first
Chitose is fixation support.ACE is fixed on SBA-15, SBA-15 is prepared and is not had been reported that.SBA-15 molecular sieves are to use three
Block copolymer is the mesoporous material that template is prepared in acid synthetic system, and aperture size is 4.6~30nm, hole body
Product is up to 0.85cm3/ g, is the maximum molecular screen material of current aperture.SBA-15 mesopore molecular sieves have that aperture is adjustable, hole wall
The characteristics of thick and hydrothermal stability is high.Tian Zhiming etc. uses solventless method by ZnO, (NH4)2SO4With the material of main part SBA- of calcining
15 molecular sieves are calcined after carrying out underhand polish mixing, are prepared for Zn-SO4 2-/ SBA-15 modified mesoporous molecular sieves.
The content of the invention
ACE is suppressed it is an object of the invention to application immobilization ACE (ACE)
Peptide carries out purifies and separates, prepare high-purity, have no side effect, the inhibiting peptide of tonin of natural health.
To reach above-mentioned purpose, the invention provides a kind of extracting method of inhibiting peptide of tonin, specifically
Step is：
S1, take enzymolysis liquid freeze-dried powder, it is 8.3,0.1mol/L borate buffer solutions to be dissolved in pH, be made concentration for 20~
The enzymolysis liquor of 100g/L；
The enzymolysis liquid freeze-dried powder is that the raw material with angiotensin converting enzyme inhibition activity is obtained through protease hydrolytic
Enzymolysis liquid, through ultrafiltration classification, lyophilized be obtained；It is preferred that bubble moth spiral shell enzymolysis liquid freeze-dried powder；
Contain the NaCl that concentration is 0.1~0.3mol/L in the borate buffer solution；
S2, the obtained enzymolysis liquor of immobilization ACE and step S1 is taken by mass volume ratio 1:10~
50~120min are shaken in the ratio mixing of 20 (g/mL), 30 DEG C of waters bath with thermostatic control, suction filtration, use pH for 8.3 0.1mol/L boric acid
Salt buffer solution is rinsed well, collects solid phase, obtains being adsorbed with the immobilization vasotonia of inhibiting peptide of tonin
Plain invertase；
S3, the immobilization ACE of inhibiting peptide of tonin will be adsorbed with obtained in step S3
Be added to pH be 7, concentration be 2mol/L NaCl solution in, 30 DEG C of 30~120min of water-bath, eluting vascular angiotensin-converting enzyme
The peptide for inhibiting of absorption, collects eluent, freezes, and obtains inhibiting peptide of tonin.
Under preferred embodiment, the raw material with angiotensin converting enzyme inhibition activity is through protease hydrolytic system described in step S1
The specific preparation process of enzymolysis liquid be：
The lyophilized prepared freeze-dried powder of the raw material with angiotensin converting enzyme inhibition activity is taken, the freeze-dried powder and water are pressed
1:The mass volume ratio of 10~20 (g/mL) is well mixed, and 5~15min is preheated in 45 DEG C of water-baths；
Regulation pH value of solution adds neutral proteinase to 7.0, and enzyme concentration is 2500U/g, starts reaction, leads in course of reaction
Cross dropwise addition standard alkali solution or standard acid, maintenance reaction liquid pH is 7.0 constant, after 2~3h of hydrolysis, boiling water bath go out enzyme 10~
20min, cooling；Under the conditions of 4 DEG C, 10000rpm, 20~30min is centrifuged, takes supernatant, obtain enzymolysis liquid.
The preferred bubble moth spiral shell of the raw material with angiotensin converting enzyme inhibition activity.
Further optimization, immobilization ACE described in step S2 turns for SBA-15 immobilizations angiotensins
Change enzyme；Specifically preparation method is：
Mesoporous molecular sieve SBA-15 is taken with ACE solution by mass volume ratio 1:The ratio of 5~20 (g/mL)
Example mixing, 45~50 DEG C, 30~40min is shaken in water-bath in water-bath, and the protein content into reaction solution is constant, reaction knot
Beam；Suction filtration, the ACE for cleaning, removing unadsorbed combination, obtain SBA-15 immobilization angiotensin-converters
Enzyme；
The ACE solution is configured using 7.8~9.3 borate buffer solutions, and enzyme activity is 0.17U/
mL。
Most have under mode, the mesoporous molecular sieve SBA-15 is chela obtained in by way of directly or indirectly absorption chelating
Conjunction has Zn2+SBA-15 molecular sieves；
The concrete operations of the direct chelating are：
It is the ZnSO of 0.05mol/L by mesoporous molecular sieve SBA-15 and concentration4The aqueous solution presses mass volume ratio 1:10g/mL
Ratio mixing, 30~40 DEG C of water-baths shake 45~65min, obtain directly chelating Zn2+SBA-15 molecular sieves；
The concrete operations of the indirect chelating are：
Mesoporous molecular sieve SBA-15 is added in absolute ethyl alcohol, to addition 3- (isobutene acyl-oxygen) in the absolute ethyl alcohol
10~20h is shaken in propyl trimethoxy silicane (GLYMO), 30 DEG C of water-baths, and reaction terminates rear suction filtration, collects solid matter, cleans；
The mesoporous molecular sieve SBA-15 and absolute ethyl alcohol, the mass body of 3- (isobutene acyl-oxygen) propyl trimethoxy silicane
Product is than being 1:350~400:20~30 (g/mL/mL)；
Connection iminodiacetic acid (IDA), by the iminodiacetic acid (salt) that gained solid matter and concentration are 0.6~0.8mol/L
Acid solution presses mass volume ratio 1:The ratio mixing of 10 (g/mL), 30~40 DEG C of water-bath concussion reactions, reaction terminates rear suction filtration, then
Secondary collection solid matter；
The solid matter that will be collected again is the ZnSO of 0.05mol/L with concentration4Solution presses mass volume ratio 1:10(g/
ML 60~90min is shaken in ratio mixing), 30~40 DEG C of water-baths, obtains chelating Zn indirectly2+SBA-15 molecular sieves.
Used as experiment material, one is that freeze-dried powder can conveniently be configured to needs to multiplex freeze-dried powder in said process of the invention
The solution of concentration, two be freeze-dried powder storage it is convenient and in the solid state compared with liquid sample it is active more stable.
After such scheme, compared with the prior art the present invention has advantages below：
1st, the present invention is based on affine principle, and immobilization ACE is applied into ACE suppression
The extraction separation and purification of peptide (ACEIP) processed, is that ACEIP is isolated and purified there is provided excellent carrier and method.
2nd, immobilization hypertensin conversion enzyme activity of the present invention is high, good stability, for isolating and purifying corresponding blood vessel
Angiotensin Converting enzyme inhibition peptide has excellent effect, it is preferred to use Zn-SBA-15 immobilization ACEs, it is fixed
Change process is simple, and product stability is more preferable；The separation of ACEIP is carried out to enzymolysis liquid by immobilization ACE
Purifying can reach more preferable separating effect compared to simple resolvase.
3rd, the present invention is applied to the various raw materials with angiotensin converting enzyme inhibition activity, preferably bubble moth spiral shell, former
Material natural health, the inhibiting peptide of tonin purity for preparing are high, natural health, applied widely.The present invention is first
ACEIP is isolated and purified in the secondary moth spiral shell from bubble, for the comprehensive utilization of bubble moth spiral shell provides new approach.
4th, dissociate ACE easy deactivations in use, brings very big difficulty, the present invention to use to use, preservation
Immobilised enzymes can not only realize the recycling of enzyme, can also improve the stability of immobilised enzymes.
Specific embodiment
Embodiment 1
1) a kind of preparation of mesoporous molecular sieve SBA-15 carrier, specific preparation method process is as follows：
The preparation of mesoporous molecular sieve SBA-15 carrier：Take 1g PEO-PPOX-PEO three blocks
Copolymer (P123) is dissolved in 30 DEG C of stirring in water bath 3h in 50mL1.6mol/L hydrochloric acid solutions, is then added dropwise over tetraethyl orthosilicate
(TEOS) 5mL, and persistently stir 20h in 40 DEG C of water-baths；Then reactant is transferred in reactor, 110 DEG C of baking oven is put into quiet
24h is reacted under the conditions of putting；Solid product is finally collected, 80 DEG C of placement 12h of baking oven are put into after washing suction filtration is clean, be put into after taking-up
550 DEG C of burning 8h of Muffle furnace, obtain mesoporous molecular sieve SBA-15；Then 0.5g SBA-15 are taken and with quality volume as SBA-15:
ZnSO4(the ZnSO of 0.05mol/L4Solution)=1:10 are added to ZnSO4Pot concussion reaction is shaken in solution, 35 DEG C of waters bath with thermostatic control
50min；After 50min take out with substantial amounts of water by way of suction filtration by the Zn of microsphere surface2+Clean.
2) ACE prepares the immobilization on Zn-SBA-15 carriers in upper step
Zn-SBA-15 carriers 0.5g prepared by 1) step is weighed to add the ACE of 10mL (enzyme activity is
0.17U/mL, is configured with the 0.1mol/L borate buffer solutions of pH 8.3 and formed), 50 DEG C, water-bath concussion in thermostat water bath
30min.Reaction terminates rear suction filtration and cleans the enzyme of unadsorbed combination, so as to obtain immobilization ACE, enzyme activity is
0.2372U/g。
3) 100g bubble moth spiral shell freeze-dried powders are weighed with 1：The ratio of 10 (g/mL) is added in deionized water, in uniform temperature water
10min is preheated in bath, regulation pH is the optimal pH 7.0 of neutral proteinase, add the enzyme concentration of neutral proteinase for after 2500U/g
Start reaction, by the way that standard alkali solution or acid solution is added dropwise in course of reaction, maintain initial pH value 7.0, after hydrolysis 2h, boiling water bath
Go out enzyme 15min, and 4 DEG C, 10000rpm, refrigerated centrifuge 20min take supernatant, and freeze-drying obtains bubble moth spiral shell enzymolysis liquid albumen
Hydrolysate.Obtain bubble moth spiral shell enzymolysis liquid enzymolysis liquid freeze-dried powder.Take obtained bubble moth spiral shell enzymolysis liquid freeze-dried powder and be dissolved in and contain
The bubble moth spiral shell enzymolysis liquid of 50g/L concentration is made into the borate buffer solution of the 0.1M of the pH8.3 of 0.2mol/L NaCl.Take
The Zn-SBA-15 immobilization ACEs of above-mentioned preparation, by mass volume ratio 1:20 (g/mL) add this step foregoing
In the enzymolysis liquid for being configured, water-bath concussion 60min, then will adsorb angiotensin-converter in 30 DEG C of isothermal vibration water-baths
The Zn-SBA-15 Immobilized ACEs of enzyme inhibition peptide are cleaned from solution with the 0.1mol/L borate buffer solutions suction filtration of pH8.3,
30 DEG C of water-bath 60min in the NaCl solution of the 2mol/L of pH 7 are then added into, the ACE that will be adsorbed
Peptide for inhibiting is eluted, and is collected eluent and is freezed, and obtains inhibiting peptide of tonin.
In above-mentioned bubble moth spiral shell freeze-dried powder, the angiotensin-converter of 0.021g can be obtained per enzymolysis liquid obtained in 1g raw materials
Enzyme inhibition peptide, i.e. its yield are 2.1%.
The measure of ACE inhibitory activity
Main agents compound method：
ACE solution：Borate buffer solution (NaCl containing 0.3M) 10mL that 1U ACE are dissolved in cold 1M pH 8.3 is prepared
Into 0.1U/mLACE solution, packing is stored in -20 DEG C, standby.
HHL (hippuroyl histidyl- leucine) solution：Take HHL appropriate, (contained with the borate buffer solution of 0.1M pH 8.3
0.3M NaCl) concentration is configured to for 5mmol/L HHL solution, it is standby.
Hippuric acid standard liquid：It is accurate to weigh hippuric acid standard sample, plus it is 50 μ g/mL that ultrapure water dissolves are configured to concentration
Hippuric acid standard liquid, it is standby.
Reaction system environment is pH 8.3,0.1M borate buffer solutions (containing 0.3M NaCl).In 1.5mL centrifuge tubes,
50 μ L, 5mmol/L HHL are taken, 20 μ L sample solution are added, is well mixed, preheat 5min in 37 DEG C of ± 0.5 DEG C of waters bath with thermostatic control, so
After add 20 μ L, 0.1U/mL ACE, fully mix.After 37 DEG C of water bath with thermostatic control insulation 60min, 10 μ L, 0.2M HCl are added to stop
Reaction.Replace sample as blank control group with 20 μ L water simultaneously.Detect that Hip is generated with HPLC (Waters) after reaction solution centrifugation
Amount.
Chromatographic condition：Ghall 12S05-2546C18 posts (250mm × 4.6mm)；Acetonitrile：Water (containing 0.05%TFA)=
25:75, Gradient elution；Flow velocity 0.5mL/min；Detection wavelength 228nm；The μ L of sample size 10.ACE inhibiting rates are counted as the following formula
Calculate.
In formula：A samples --- add the peak area of hippuric acid in inhibitor group；
A is compareed --- the peak area of hippuric acid in blank control group.
It is 0.85g/L to use high-efficient liquid phase technique to measure its 503nhibiting concentration (IC50) to ACE.This 503nhibiting concentration compared with
The 503nhibiting concentration (IC50) of the ACE of bubble moth spiral shell enzymolysis liquid is for 15.27g/L is low and up to 17 times, illustrates by after affine separation
The more former enzymolysis liquid of ACE inhibiting rates of the component for obtaining is significantly improved.Explanation simultaneously is by the method for the present invention in enzymolysis liquid
ACEIP has reached preferable separating effect.
Embodiment 2
1) preparation of mesoporous molecular sieve SBA-15 carrier：Take 1g PEOs-PPOX-PEO three embedding
Section copolymer (P123) is dissolved in 30 DEG C of stirring in water bath 3h in 50mL 1.6mol/L hydrochloric acid solutions, is then added dropwise over positive silicic acid
Ethyl ester (TEOS) 5mL, and persistently stir 20h in 40 DEG C of water-baths；Then reactant is transferred in reactor, baking oven 110 is put into
24h is reacted under DEG C static conditions；Solid product is finally collected, 80 DEG C of placement 12h of baking oven is put into after washing suction filtration is clean, after taking-up
550 DEG C of burning 8h of Muffle furnace are put into, mesoporous molecular sieve SBA-15 is obtained；0.5g SBA-15 add 175mL absolute ethyl alcohols to add
15h is shaken in 2mLGLYMO, 30 DEG C of water-baths, and reaction terminates rear suction filtration, connection ID A after cleaning, by 1:10 (g/mL) add 0.7mol/
L IDA solution (IDA solution is prepared with 1.5mol/L sodium carbonate liquors), 35 DEG C of water-bath concussion reaction 70min, reaction terminates suction filtration
Afterwards, with mass volume ratio be 1：10 (g/mL) add the ZnSO of 0.05mol/L475min is shaken in solution, 30 DEG C of water-baths, so that
Obtain corresponding carrier.
2) ACE prepares the immobilization on Zn-SBA-15 carriers in upper step
Zn-SBA-15 carriers 0.5g prepared by 1) step is weighed to add the ACE of 10mL (enzyme activity is
0.17U/mL, is configured with the 0.1mol/L borate buffer solutions of pH 8.3 and formed), 50 DEG C, water-bath concussion in thermostat water bath
30min.Reaction terminates rear suction filtration and cleans the enzyme of unadsorbed combination, so as to obtain immobilization ACE, enzyme activity is
0.3074U/g。
3) 100g bubbles moth spiral shell freeze-dried powder is with 1：The ratio of 10 (g/ml) is added in deionized water, in uniform temperature water-bath
Preheating 10min, regulation pH are the optimal pH 7.0 of neutral proteinase, add enzyme concentration to start after the neutral proteinase of 2500U/g
Reaction, by being added dropwise standard alkali solution or acid solution in course of reaction, maintains initial pH value 7.0, and after hydrolysis 2h, boiling water bath goes out enzyme
15min, 4 DEG C, 10000rpm, refrigerated centrifuge 20min take supernatant, and freeze-drying obtains bubble moth spiral shell protein hydrolysate.
Obtained bubble moth spiral shell enzymolysis liquid freeze-dried powder is taken to be dissolved in the borate buffer solution of the 0.1M of the pH8.3 containing 0.1mol/LNaCl
It is made into the bubble moth spiral shell enzymolysis liquid of 50g/L concentration.The Zn-SBA-15 immobilization ACEs of above-mentioned preparation are taken, is pressed
Mass volume ratio 1:15 (g/ml) are added in foregoing the configured enzymolysis liquid of this step, the water-bath shake in 30 DEG C of isothermal vibration water-baths
60min is swung, the Zn-SBA-15 Immobilized ACEs that then will adsorb inhibiting peptide of tonin use pH8.3's from solution
0.1mol/L borate buffer solutions suction filtration is cleaned, and is then added into 30 DEG C of water-baths in the NaCl solution of the 2mol/L of pH 7
60min, the inhibiting peptide of tonin of absorption is eluted, and is collected eluent and is freezed, and is obtained angiotensins and is turned
Change enzyme inhibition peptide.
In above-mentioned bubble moth spiral shell freeze-dried powder, the angiotensins that 0.0199g can be obtained per enzymolysis liquid obtained in 1g raw materials turns
It is 1.99% to change enzyme inhibition peptide, i.e. its yield.
It is 1.0g/L to use high-efficient liquid phase technique to measure its 503nhibiting concentration (IC50) to ACE.This 503nhibiting concentration compared with
The 503nhibiting concentration (IC50) of the ACE of bubble moth spiral shell enzymolysis liquid is for 15.27g/L is low and up to 15 times, illustrates by after affine separation
The more former enzymolysis liquid of ACE inhibiting rates of the component for obtaining is significantly improved.Preferable separation has been reached to the ACEIP in enzymolysis liquid to imitate
Really.Explanation simultaneously has reached preferable separating effect by the method for the present invention to the ACEIP in enzymolysis liquid.
Experimental example 3
The step of this experiment 1) and it is 2) 1) and 2) identical with embodiment 1.
3) 100g bubbles moth spiral shell freeze-dried powder is with 1：The ratio of 10 (g/mL) is added in deionized water, is preheated in 45 DEG C of water-baths
10min, regulation pH are the optimal pH 7.0 of neutral proteinase, add 2500U/g neutral proteinases to start reaction, in course of reaction
By being added dropwise standard alkali solution or acid solution, initial pH value 7.0 is maintained, after hydrolysis 2h, boiling water bath goes out enzyme 15min, 4 DEG C,
10000rpm, refrigerated centrifuge 20min, take supernatant, and freeze-drying obtains bubble moth spiral shell enzymolysis liquid protein hydrolysate.Water intaking
Bubble moth spiral shell enzymolysis liquid freeze-dried powder is made into 50g/ in being dissolved in the borate buffer solution of the 0.1M of the pH 8.3 containing 0.3mol/L NaCl
The bubble moth spiral shell solution liquid enzymolysis liquid of L concentration.The Zn-SBA-15 immobilization ACEs of above-mentioned preparation are taken, by quality
Volume ratio 1:20 (g/mL) are added in foregoing the configured enzymolysis liquid of this step, the water-bath concussion in 30 DEG C of isothermal vibration water-baths
60min, the Zn-SBA-15 Immobilized ACEs that then will adsorb inhibiting peptide of tonin use pH8.3's from solution
0.1mol/L borate buffer solutions suction filtration is cleaned, and is then added into 30 DEG C of water-baths in the NaCl solution of the 2mol/L of pH7
60min, the inhibiting peptide of tonin of absorption is eluted, and is collected eluent and is freezed, and is obtained angiotensins and is turned
Change enzyme inhibition peptide.
In above-mentioned bubble moth spiral shell freeze-dried powder, the angiotensin-converter of 0.02g can be obtained per enzymolysis liquid obtained in 1g raw materials
Enzyme inhibition peptide, i.e. its yield are 2%.
It is 1.2g/L to use high-efficient liquid phase technique to measure its 503nhibiting concentration (IC50) to ACE.This 503nhibiting concentration compared with
The 503nhibiting concentration (IC50) of the ACE of bubble moth spiral shell enzymolysis liquid is 15.27g/L low and up to 15 times or so, the 503nhibiting concentration of ACE
The ACE inhibitory activity of lower explanation peptide is better.Illustrate the more former enzymolysis liquid of ACE inhibiting rates by the component obtained after affine separation
Significantly improve.Explanation simultaneously has reached preferable separating effect by the method for the present invention to the ACEIP in enzymolysis liquid.
The above, the only present invention preferably specific embodiment, but protection scope of the present invention is not limited thereto,
Any one skilled in the art in the technical scope of present disclosure, technology according to the present invention scheme and its
Inventive concept is subject to equivalent or change, should all be included within the scope of the present invention.
Claims (5)
1. a kind of extracting method of inhibiting peptide of tonin, it is characterised in that concretely comprise the following steps：
S1, enzymolysis liquid freeze-dried powder is taken, be dissolved in during pH is the borate buffer solution that 8.3, concentration is 0.1mol/L, being made concentration is
The enzymolysis liquor of 20~100g/L；
The enzymolysis liquid freeze-dried powder is the raw material with angiotensin converting enzyme inhibition activity through enzyme obtained in protease hydrolytic
Solution liquid, through ultrafiltration classification, lyophilized is obtained；
Contain the NaCl that concentration is 0.1~0.3mol/L in the borate buffer solution；
S2, the obtained enzymolysis liquor of immobilization ACE and step S1 is taken by mass volume ratio 1:10~20
(g/mL) 50~120min are shaken in ratio mixing, 30 DEG C of waters bath with thermostatic control, suction filtration, use pH for 8.3 0.1mol/L borates
Cushioning liquid is rinsed well, collects solid phase, obtains being adsorbed with the immobilization angiotensins of inhibiting peptide of tonin
Invertase；
S3, the immobilization ACE addition that inhibiting peptide of tonin will be adsorbed with obtained in step S3
In being the NaCl solution that 7, concentration is 2mol/L to pH, 30 DEG C of 30~120min of water-bath, the absorption of eluting vascular angiotensin-converting enzyme
Peptide for inhibiting, collect eluent, freeze, obtain inhibiting peptide of tonin.
2. the extracting method of inhibiting peptide of tonin according to claim 1, it is characterised in that described in step S1
Raw material with angiotensin converting enzyme inhibition activity is through the specific preparation process of enzymolysis liquid obtained in protease hydrolytic：
The lyophilized prepared freeze-dried powder of the raw material with angiotensin converting enzyme inhibition activity is taken, the freeze-dried powder and water are pressed 1:10
The mass volume ratio mixing of~20 (g/mL), preheats 5~15min in 45 DEG C of water-baths；
Regulation pH value of solution adds the enzyme concentration of neutral proteinase to start reaction after 2500U/g to 7.0, passes through in course of reaction
Standard alkali solution or standard acid is added dropwise, maintenance reaction liquid pH is 7.0 constant, after 2~3h of hydrolysis, boiling water bath go out enzyme 10~
20min, cooling；Under the conditions of 4 DEG C, 10000rpm, 20~30min is centrifuged, takes supernatant, obtain enzymolysis liquid.
3. the extracting method of inhibiting peptide of tonin according to claim 2, it is characterised in that described with blood
The raw material of angiotensin conversion enzyme inhibition activity is bubble moth spiral shell.
4. the extracting method of inhibiting peptide of tonin according to claim 1, it is characterised in that described in step S2
Immobilization ACE is SBA-15 immobilization ACEs；Specifically preparation method is：
Mesoporous molecular sieve SBA-15 is taken with ACE solution by mass volume ratio 1:The ratio of 5~20 (g/mL) is mixed
Close, 45~50 DEG C, 30~40min is shaken in water-bath in water-bath, and the protein content into reaction solution is constant, and reaction terminates；Take out
The ACE of unadsorbed combination is filtered, cleaned, removing, SBA-15 immobilization ACEs are obtained；
The ACE solution is configured using 7.8~9.3 borate buffer solutions, and enzyme activity is 0.17U/mL.
5. the extracting method of inhibiting peptide of tonin according to claim 4, it is characterised in that described mesoporous point
Son sieve SBA-15 is to be chelated with Zn obtained in by way of directly or indirectly absorption chelating2+SBA-15 molecular sieves；
The concrete operations of the direct chelating are：
It is the ZnSO of 0.05mol/L by mesoporous molecular sieve SBA-15 and concentration4The aqueous solution presses mass volume ratio 1:The ratio of 10g/ml
45~65min is shaken in mixing, 30~40 DEG C of water-baths, obtains and directly chelates Zn2+SBA-15 molecular sieves；
The concrete operations of the indirect chelating are：
Mesoporous molecular sieve SBA-15 is added in absolute ethyl alcohol, to addition 3- (isobutene acyl-oxygen) propyl group in the absolute ethyl alcohol
10~20h is shaken in trimethoxy silane, 30 DEG C of water-baths, and reaction terminates rear suction filtration, collects solid matter, cleans；
The mesoporous molecular sieve SBA-15 and absolute ethyl alcohol, the mass volume ratio of 3- (isobutene acyl-oxygen) propyl trimethoxy silicane
It is 1:350~400:20~30 (g/mL/mL)；
Connection iminodiacetic acid, by iminodiacetic acid (salt) acid solution that gained solid matter and concentration are 0.6~0.8mol/L by
Mass volume ratio 1:The ratio mixing of 10 (g/ml), 30~40 DEG C of water-bath concussion reactions, reaction terminates rear suction filtration, collects solid again
Body material；
The solid matter that will be collected again is the ZnSO of 0.05mol/L with concentration4Solution presses mass volume ratio 1:The ratio of 10 (g/mL)
60~90min is shaken in example mixing, 30~40 DEG C of water-baths, obtains chelating Zn indirectly2+SBA-15 molecular sieves.
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| CN101294183A (en) * | 2007-04-24 | 2008-10-29 | 中国农业科学院饲料研究所 | Method for preparing protein peptide with immobilization proteolytic enzyme protolysate |
| WO2010050897A1 (en) * | 2008-10-28 | 2010-05-06 | Agency For Science, Technology And Research | Mesoporous material excipients for poorly aqueous soluble ingredients |
| CN105859868A (en) * | 2016-06-08 | 2016-08-17 | 广西科技大学 | Angiotensin converting enzyme inhibition peptide and preparing method thereof |
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| CN101294183A (en) * | 2007-04-24 | 2008-10-29 | 中国农业科学院饲料研究所 | Method for preparing protein peptide with immobilization proteolytic enzyme protolysate |
| WO2010050897A1 (en) * | 2008-10-28 | 2010-05-06 | Agency For Science, Technology And Research | Mesoporous material excipients for poorly aqueous soluble ingredients |
| CN105859868A (en) * | 2016-06-08 | 2016-08-17 | 广西科技大学 | Angiotensin converting enzyme inhibition peptide and preparing method thereof |
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