CN105803024A - Method of using coconut cake globulin as raw material to prepare ACE inhibiting peptide - Google Patents
Method of using coconut cake globulin as raw material to prepare ACE inhibiting peptide Download PDFInfo
- Publication number
- CN105803024A CN105803024A CN201610402642.3A CN201610402642A CN105803024A CN 105803024 A CN105803024 A CN 105803024A CN 201610402642 A CN201610402642 A CN 201610402642A CN 105803024 A CN105803024 A CN 105803024A
- Authority
- CN
- China
- Prior art keywords
- coconut
- globulin
- polypeptide
- ratio
- coconut cake
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
Abstract
The invention relates to a method of using coconut cake globulin as a raw material to prepare ACE inhibiting peptide. The method includes: selecting mature old coconut, and treating the same to obtain degreased coconut cake powder; extracting coconut cake globulin, and performing compound enzyme hydrolysis and centrifugal separation to prepare polypeptide original extract; separating out five polypeptide components from the polypeptide original extract, collecting the fifth polypeptide component, and further separating and purifying to obtain 12 polypeptide components; further subjecting the ninth component to carbon 18 analytical column-reversed phase high performance liquid chromatographic separation to obtain two pure peptides, wherein ACE inhibition rate of each pure peptide is higher than 70%. The coconut cake globulin is used as a substrate, the ACE inhibiting peptide higher than 95% in purity is prepared by adopting technologies of compound enzymolysis, gel chromatography separation and reversed phase high performance liquid chromatography purification, the ACE inhibiting peptide can be used as an ingredient of natural anti-hypertension drugs, can be added into food to prevent and treat food-borne hypertension, is conducive to realizing comprehensive utilization and deep processing of coconut and can powerfully promote development of coconut industry of China.
Description
Technical field
The invention belongs to agricultural product and adopt post-treatment technical field, a kind of method relating to ace inhibitory peptide, specifically relate to
And one prepares coconut cake globulin, and then the method preparing ace inhibitory peptide with coconut cake for raw material.
Background technology
Hypertension is one of principal disease threatening human health.According to statistics, countries in the world hypertension is averagely sent out
Sick rate is 10～20%, and the whole world reaches 12,000,000 because of hypertension death toll every year, and China dies from hypertension every year
And the number of relevant disease reaches 2,000,000, hypertension has become as one of worldwide health problem needing solution badly.
Angiotensin converting enzyme (Antiotensin I Converting Enzyme, ACE) is at regulation blood
Press key effect.Research shows, the renin-angiotensin system in human body
(rennin-angiotensin system, RAS) and kallikrein kinin system
(kallikrein-kinin system, KKS) is the balanced body maintaining normotensive two mutual antagonisms
System (Li et al., 2004).ACE can be catalyzed angiotensin I (angiotensin I) and be changed into tool
There is the Angiotensin II (angiotensin II) of potent raising blood pressure effect, will have blood pressure lowering simultaneously and make
Bradykinin degraded, be allowed to lose hypotensive effect, so that RAS and KKS equilibrium system is unbalance finally
Blood pressure is caused to raise.Therefore, the activity by suppressing internal ACE can play the effect of blood pressure lowering.At present
The activity of the decompression principle of the Altace Ramipril used clinically also mainly suppression ACE (Roye&Simpson,
2010；Shen Xiaowen, 2010).
Ace inhibitory peptide refers to suppress the polypeptide of ACE enzymatic activity.It is to pass through to the inhibiting mechanism of ACE
Be combined with the fixing site in ACE active center and make ACE lose enzyme activity, it is also possible to competitively with ACE
Substrate specificity combine, thus reduce the activity of ACE, and then play the effect of blood pressure lowering.Currently used
ACE inhibitor mostly is chemical synthetic drug, although evident in efficacy, but with high costs, and long-term taking can produce
Various side effect (such as injury of kidney, dry cough, diarrhoea, taste function disorder etc.).Food-borne ace inhibitory peptide because of
Blood pressure lowering effect is good, safety has no side effect becomes the focus of research at present.Along with people's self health care and safety
The raising of consciousness, the idea using non-drug therapy to reach preventing and treating diseases will be accepted by more people, from drink
Add food-borne ace inhibitory peptide on food prevent and treat having good prospects of hypertension.
Cortex cocois radicis (Cocos nucifera L.) is torrid areas most important woody oleiferous plants crop and food energy work
One of thing, problem has developed into the specialty industries that China hot-zone is well-known throughout the country.But, China coconut palm
The product of sub-processing industry is concentrated mainly on the common coconut food aspect such as coconut candy, coconut powder, nata de coco, merit
Energy property product proportion is the least；Enterprise lacks the capability of independent innovation, and product science and technology added value is few, it is difficult to carry
High economic benefit and resource utilization.In recent years, Southeast Asia Cortex cocois radicis main product state such as Vietnam, Indonesia etc.
Country, with cheap labour force and the advantage such as raw material, more advanced process technology, makes domestic Cortex cocois radicis processing enterprise
Face the increasing pressure and challenge.
Coconut flesh contains the protein of about 4～8%, and wherein albumin accounts for 21%, globulin accounts for 40.1%, alcohol
Molten albumen accounts for 3.3%, and acid-soluble glutelin accounts for 14.4%, and alkali solubility glutelin accounts for 4.8%.Nineteen thirty the U.S.
ScientistWith the globulin that Spychalski isolates a kind of molecular weight about 208kDa from coconut meat,
And named cocosin, open the research history of coconut protein.Follow-up research shows, ripe coconut palm
In sub-sarcocarp, globulin accounts for the 65% of total protein content, and in 7～8 months tender coconut fruits, globulin is always
Content in albumen may be up to 80%.Globulin according to sedimentation coefficient can be divided into 2S globulin, 7S,
9S globulin, 11S globulin and 15S globulin.Wherein 11S globulin accounts for the 86% of the total globulin of Cortex cocois radicis,
And 7S accounts for 14% (Garcia et al, 2005).Research worker also finds that cocosin is more than one
Aggressiveness globulin (hexamer), it is formed by connecting by a disulfide bond by acidic and alkaline subunit,
Herein in connection with glycosyl on alkaline subunit.In food industry, coconut powder, concentration coconut palm slurry, coconut juice protein beverage etc.
The main proteinaceous main component of coconut products is exactly coconut globulin.Thus, globulin is Cortex cocois radicis egg
Topmost protein in Bai, is also one of most important protein.
Coconut cake is the Main By product after coconut meat is squeezed the juice, containing 16～the protein of 24%, coconut cake yield simultaneously
Very big, Philippine's coconut cake yield of only 2014 just reaches 2,000,000 tons.Research shows, ball in coconut cake protein
The content of albumen is 54.35 ± 3.69g/100g.Coconut cake globulin is rich in aspartic acid and arginine, ammonia
It is 39.25% that base acid accounts for the ratio of total amino acids, and far above the recommendation of FAO/WHO, aminoacid forms more
Rationally；Its external digestion absorbance (in vitro digestibility index, IVPD) up to 88.26%,
Biological value (biological value, BV) is 58.41, far above soybean protein isolate, thus nutriture value
It is worth higher.Meanwhile, coconut cake globulin has extremely strong chelated iron ion ability, is to prepare food-borne iron supplementary
Or the good raw material of ferrous-fortifier, the interior free yls such as DPPH, OH, ATBS+ are had necessarily simultaneously
Scavenging activity, biological activity is higher.At present, coconut cake is only used as the food such as animal feed or cake
Adjuvant, protein isoreactivity composition is not exploited, and causes the significant wastage of resource.
Summary of the invention
It is an object of the invention to provide a kind of for the deficiencies in the prior art prepare with coconut cake globulin for raw material
The method of ace inhibitory peptide, prepares coconut globulin from coconut cake, with coconut cake globulin as substrate, uses compound
The technology such as enzymolysis, gel chromatography separation, reverse high-efficient liquid phase chromatogram purification are prepared purity and are reached more than 95%
Ace inhibitory peptide, not only improves and realizes the comprehensive utilization to Cortex cocois radicis and intensive processing, can turn waste into wealth again, have
Make every effort to promote the development into China's coconut industry.
The technical solution adopted in the present invention:
A kind of method preparing ace inhibitory peptide for raw material with coconut cake globulin, it specifically comprises the following steps that
1, raw material pre-treatment selects ripe old Cortex cocois radicis fruit, then shells, seed coat of pruning, clean, dig silk,
Squeeze the juice, obtain coconut cake.By coconut cake 40～50 DEG C of crushed after being dried, cross 40～60 mesh sieves, then by matter
Amount volume ratio (m/v) is ratio addition petroleum ether or the remaining Oleum Cocois of ether extraction of 1 10～1 20,
Obtain defat coconut cake powder.
2, extracting globulin is 1 10～1 30 by mass volume ratio (m/v) in defat coconut cake powder
Ratio adds the sodium chloride solution of 0.4～0.5mol/L, and under room temperature, concussion extracts 2～3h, filters, and collects
Filtrate；Repeatedly extract 3 times according to as above method, merge the filtrate collected.By filtrate 4～10 DEG C, 10000～
It is centrifuged 15～20min under conditions of 12000rpm, collects supernatant.Supernatant loads molecular cut off is
In 4000～8000 dialyzers, dialyse 12～24 hours in the distilled water of 4～6 DEG C, removal salt ion,
The impurity such as soluble sugar, small molecular protein；Simultaneously as can not dissolve in distilled water, coconut cake globulin
Meeting gradually Precipitation in dialysis procedure.Dialysis terminate after, by dialysis solution 4～10 DEG C, 80000～
It is centrifuged 15～20min under conditions of 12000rpm, collects precipitation, purity after vacuum lyophilization, can be obtained
Higher coconut cake globulin.
3, coconut cake globulin is the ratio of 1 15 in mass volume ratio (m/v) by combinative enzyme hydrolysis preparation
It is dissolved in 0.1～0.3M phosphate buffer, regulates pH to 8.0～pH9.0, the most by mass percentage
Being 0.5～the ratio of 1% (m/m) addition alkaline protease, at 45～55 DEG C, concussion hydrolysis 3～6h, regulates water
Solve pH value of solution to 7.0～pH 7.5, be the most by mass percentage 0.5～1% (m/m) ratio add in
Property protease, concussion hydrolysis 2～4h at 50～55 DEG C, regulate hydrating solution pH to 2.0, then by quality hundred
Proportion by subtraction is 0.3～the ratio of 0.6% (m/m) adds pepsin, and at 37 DEG C, concussion hydrolysis 2～4h, regulates water
Solution pH value of solution, to 7.0, is the ratio addition trypsin of 0.3～0.6% (m/m) the most by mass percentage,
Concussion hydrolysis 2～4h at 37 DEG C；Enzymolysis solution is heated at 95～100 DEG C 5～10min, enzyme denaturing.
4, high speed centrifugation separates enzymolysis solution after enzyme deactivation at the bar of 4～6 DEG C, 10000～12000rpm
It is centrifuged 25～30min under part, collects supernatant；Available polypeptide primary extract after lyophilization.
5, polypeptide primary extract is carried out Sephadex G-15 column chromatography for separation by gel chromatography separation.Column length
0.6～1.2 meter, a diameter of 16～20mm, fixing is Sephadex G-15 mutually, pure water eluting, flow velocity 0.6～
0.8mL/min, collects once for every 5 minutes, and spectrophotometric colo used at 220nm by eluent.Separable
Go out 5 polypeptide fractions.Detection indicate that, the 5th polypeptide fractions is the highest to the suppression ratio of ACE.Constantly weight
This step multiple, collects the 5th polypeptide fractions, can obtain the powder of this polypeptide fractions after lyophilization.Due to
Sephadex G-15 has the effect of desalination, thus this step also can remove the salinity in polypeptide extract.
6, the 5th polypeptide fractions of above-mentioned gel chromatography separation gained is existed by reverse high-efficient liquid phase chromatogram purification
Reverse high performance liquid chromatography (reversed-phase high performance liquid chromatography,
RP-HPLC) separate further and purification on: by the 5th polypeptide fractions in 0.6～1mg/mL ratio
It is dissolved in distilled water, crosses the film of 0.22 micron, collect filtrate；Filtrate is carried out reverse high performance liquid chromatography
Separate, detached dowel used be carbon 18 semi-preparative column (Zorbax semi-preparative C18column,
9.4mm × 250mm), binary gradient concentration eluting, mobile phase A is the acetonitrile containing 0.1% trifluoroacetic acid, stream
Dynamic phase B is ultra-pure water, flow velocity 2.0～2.5mL/min, and column temperature 30～40 DEG C detect wavelength 220nm,
May separate out 12 polypeptide fractions.Experiment shows, wherein the 9th polypeptide fractions is the highest to ACE suppression ratio.
9th component is separated (Zorbax analytical with carbon 18 analytical columns-reversed-phase high-performance liquid chromatography further
C18column, 4.6mm × 250mm), binary gradient concentration eluting, mobile phase A is containing 0.1% trifluoro second
The acetonitrile of acid, Mobile phase B are ultra-pure water, flow velocity 0.8～1.0mL/min, column temperature 30～40 DEG C, detection
Wavelength 220nm, is separated into 2 pure peptides.2 pure peptides are collected respectively, lyophilization, preserve.
Through LC-MC/MC Analysis and Identification, the aminoacid sequence of these 2 pure peptides and molecular weight be respectively as follows: 1,
Leu-Leu-Leu-Gly-Ala-Val-Asn-Tyr-Arg, molecular weight is 1017.6Da；2、
Arg-Pro-Phe-Asn-Leu-Phe-His-Lys, molecular weight is 1057.58Da.ACE is pressed down by the two
Rate processed is all more than 70%.
The present invention, with the coconut cake globulin of preparation from coconut cake as substrate, uses complex enzyme hydrolysis, gel chromatography to divide
Prepare purity from technology such as, reverse high-efficient liquid phase chromatogram purifications and reach the ace inhibitory peptide of more than 95%, due to system
Employ pepsin and trypsin during Bei, thus two polypeptide are in human gastrointestinal tract digestive system
The stability of digestive enzyme preferable, both may be used for the composition of natural antihypertensive drug, it is also possible to add to
In food, carry out food-borne prevention and treatment hypertension, be beneficial to realize to the comprehensive utilization of Cortex cocois radicis and profound add
Work, can turn waste into wealth again, the strong development promoting China's coconut industry.
Accompanying drawing explanation
Fig. 1 is Leu-Leu-Leu-Gly-Ala-Val-Asn-Tyr-Arg (molecular weight is 1017.6Da)
LC-MS/MS schemes.
Fig. 2 is Arg-Pro-Phe-Asn-Leu-Phe-His-Lys (molecular weight is 1057.58Da)
LC-MS/MS schemes.
Detailed description of the invention
Below in conjunction with embodiment, the detailed description of the invention of the present invention is described in further detail.Hereinafter implement
Example is used for illustrating the present invention, but is not intended to limit the scope of the present invention.
One, the preparation of ace inhibitory peptide
Embodiment one
1, raw material pre-treatment selects ripe old Cortex cocois radicis fruit, then shells, seed coat of pruning, clean, dig silk,
Squeeze the juice, obtain coconut cake.By coconut cake 45 DEG C of crushed after being dried, cross 60 mesh sieves, then by 1 10 (m/v)
Ratio add petroleum ether or the remaining Oleum Cocois of ether extraction, obtain defat coconut cake powder.
2, extract globulin and add 0.4mol/L in the ratio of 1 10 (m/v) in defat coconut cake powder
Sodium chloride solution, under room temperature concussion extract 2h, filter, collect filtrate.Filtering residue add again above-mentioned
Sodium chloride solution (1 10, m/v), extracts 3 times repeatedly according to as above method, merges the filtrate collected.Will
Filtrate, at 4 DEG C, centrifugal 15min under conditions of 12000rpm, collects supernatant.This supernatant is loaded and cuts
Staying molecular weight is in 8000 dialyzers, in the distilled water of 4 DEG C dialyse 12 hours, remove salt ion, can
The impurity such as dissolubility sugar, small molecular protein, and make globulin gradually Precipitation.After dialysis terminates, will dialysis
Liquid, at 4 DEG C, centrifugal 15min under conditions of 12000rpm, is collected precipitation, is obtained coconut palm after vacuum lyophilization
Bran globulin.
3, combinative enzyme hydrolysis preparation coconut cake globulin is dissolved in 0.1mol/L phosphate buffer (1 15,
M/v), in, regulate to pH9.0, add alkaline protease (0.1%, m/m), concussion hydrolysis 3h at 50 DEG C,
Then regulation hydrating solution is to pH 7.0, adds neutral protease (1%, m/m), concussion hydrolysis 2h at 55 DEG C.
Then regulation hydrating solution is to pH 2.0, adds pepsin (0.5%, m/m), concussion hydrolysis 2h at 37 DEG C；
Finally regulation hydrating solution is to pH 7.0, adds trypsin 0.5%, m/m), concussion hydrolysis 2h at 37 DEG C.
Hydrolyzed solution is heated at 100 DEG C 5min, enzyme denaturing.
4, high speed centrifugation separate by enzymolysis solution after enzyme deactivation 4 DEG C, under conditions of 12000rpm centrifugal
25min, collects supernatant.Available polypeptide primary extract after lyophilization.
5, polypeptide primary extract is carried out Sephadex G-15 column chromatography for separation by gel chromatography separation.Column length
1.0 meters, a diameter of 16mm, fixing is Sephadex G-15 mutually, pure water eluting, flow velocity 0.8mL/min, often
Within 5 minutes, collecting once, spectrophotometric colo used at 220nm by eluent.May separate out 5 polypeptide fractions.
Detection indicate that, the 5th polypeptide fractions is the highest to the suppression ratio of ACE.Constantly repeat this step, collect the 5th
Individual polypeptide fractions, can obtain the powder of this polypeptide fractions after lyophilization.
6, the 5th polypeptide fractions of above-mentioned gel chromatography separation gained is existed by reverse high-efficient liquid phase chromatogram purification
Reversely separate further and purification in high performance liquid chromatography: the 5th polypeptide fractions is dissolved in distilled water
In (1mg/mL), cross the film of 0.22 micron, collect filtrate.This filtrate is carried out reverse high performance liquid chromatography
Separate, detached dowel used be carbon 18 semi-preparative column (Zorbax semi-preparative C18column,
9.4mm × 250mm), binary gradient concentration eluting, mobile phase A is acetonitrile (containing 0.1% trifluoroacetic acid), stream
Dynamic phase B is ultra-pure water, flow velocity 2.5mL/min, and column temperature 40 DEG C detects wavelength 220nm, separable place
12 polypeptide fractions.9th component is separated with carbon 18 analytical columns-reversed-phase high-performance liquid chromatography further
(Zorbax analytical C18column, 4.6mm × 250mm), binary gradient concentration eluting, stream
Dynamic phase A be acetonitrile (containing 0.1% trifluoroacetic acid), Mobile phase B be ultra-pure water, flow velocity 1.0mL/min, column temperature
35 DEG C, detect wavelength 220nm, be separated into 2 pure peptides, collect respectively, lyophilization, preserve.
Embodiment two,
1, raw material pre-treatment selects ripe old Cortex cocois radicis fruit, then shells, seed coat of pruning, clean, dig silk,
Squeeze the juice, obtain coconut cake.By coconut cake 50 DEG C of crushed after being dried, cross 60 mesh sieves, then by 1 15 (m/v)
Ratio add petroleum ether or the remaining Oleum Cocois of ether extraction, obtain defat coconut cake powder.
2, extract globulin and add 0.4mol/L in the ratio of 1 10 (m/v) in defat coconut cake powder
Sodium chloride solution, under room temperature concussion extract 3h, filter, collect filtrate.Filtering residue add again above-mentioned
Sodium chloride solution (1 15, m/v), extracts 3 times repeatedly according to as above method, merges the filtrate collected.Will
Filtrate, at 4 DEG C, centrifugal 20min under conditions of 10000rpm, collects supernatant.This supernatant is loaded and cuts
Staying molecular weight is in 8000 dialyzers, in the distilled water of 4 DEG C dialyse 24 hours, remove salt ion, can
The impurity such as dissolubility sugar, small molecular protein, and make globulin gradually Precipitation.After dialysis terminates, will dialysis
Liquid, at 4 DEG C, centrifugal 20min under conditions of 10000rpm, is collected precipitation, is obtained coconut palm after vacuum lyophilization
Bran globulin.
3, combinative enzyme hydrolysis preparation coconut cake globulin is dissolved in 0.1mol/L phosphate buffer (1 15,
M/v), in, regulate to pH9.0, add alkaline protease (0.6%, m/m), concussion hydrolysis 6h at 50 DEG C,
Then regulation hydrating solution is to pH 7.0, adds neutral protease (0.6%, m/m), concussion hydrolysis at 55 DEG C
4h.Then regulation hydrating solution is to pH 2.0, adds pepsin (0.6%, m/m), concussion hydrolysis at 37 DEG C
2h；Finally regulation hydrating solution is to pH 7.0, adds trypsin 0.6%, m/m), concussion hydrolysis at 37 DEG C
2h.Hydrolyzed solution is heated at 95 DEG C 10min, enzyme denaturing.
4, high speed centrifugation separate by enzymolysis solution after enzyme deactivation 4 DEG C, under conditions of 10000rpm centrifugal
30min, collects supernatant.Available polypeptide primary extract after lyophilization.
5, polypeptide primary extract is carried out Sephadex G-15 column chromatography for separation by gel chromatography separation.Column length
0.8 meter, a diameter of 16mm, fixing is Sephadex G-15 mutually, pure water eluting, flow velocity 0.6mL/min, often
Within 5 minutes, collecting once, spectrophotometric colo used at 220nm by eluent.May separate out 5 polypeptide fractions.
Detection indicate that, the 5th polypeptide fractions is the highest to the suppression ratio of ACE.Constantly repeat this step, collect the 5th
Individual polypeptide fractions, can obtain the powder of this polypeptide fractions after lyophilization.
6, the 5th polypeptide fractions of above-mentioned gel chromatography separation gained is existed by reverse high-efficient liquid phase chromatogram purification
Reversely separate further and purification in high performance liquid chromatography.5th polypeptide fractions is dissolved in distilled water
In (1mg/mL), cross the film of 0.22 micron, collect filtrate.This filtrate is carried out reverse high performance liquid chromatography
Separate, detached dowel used be carbon 18 semi-preparative column (Zorbax semi-preparative C18column,
9.4mm × 250mm), binary gradient concentration eluting, mobile phase A is acetonitrile (containing 0.1% trifluoroacetic acid), stream
Dynamic phase B is ultra-pure water, flow velocity 2mL/min, and column temperature 40 DEG C detects wavelength 220nm, separable place 12
Individual polypeptide fractions.9th component is separated with carbon 18 analytical columns-reversed-phase high-performance liquid chromatography further
(Zorbax analytical C18column, 4.6mm × 250mm), binary gradient concentration eluting, stream
Dynamic phase A be acetonitrile (containing 0.1% trifluoroacetic acid), Mobile phase B be ultra-pure water, flow velocity 1.0mL/min, column temperature
35 DEG C, detect wavelength 220nm, be separated into 2 pure peptides, collect respectively, lyophilization, preserve.
Two, embodiment products therefrom is identified
Example one, embodiment two products therefrom are through LC-MC/MC Analysis and Identification, the amino of these 2 pure peptides
Acid sequence and molecular weight are respectively as follows:
I, Leu-Leu-Leu-Gly-Ala-Val-Asn-Tyr-Arg, molecular weight is 1017.6Da, its
LC-MS/MS schemes as shown in Figure 1.
II, Arg-Pro-Phe-Asn-Leu-Phe-His-Lys, molecular weight is 1057.58Da, its
LC-MS/MS schemes as shown in Figure 2.
Three, the inhibition of ACE is identified by pure peptide
The inhibition of ACE is used the mode of external test to carry out by pure peptide, and assay method is according to document
In (Jimsheena&Gowda, 2009), method is carried out:
The pure peptide of 50 microlitre (1mg/mL), the angiotensin converting enzyme (ACE, 25mill units/mL) of 50 microlitres
With 150 microlitres, hippuroyl-HIS-LEU (HHL) mix homogeneously of 8.3 mM/ls, 37 DEG C
After lower reaction 60 minutes, add 250 microlitres, the hydrochloric acid of 1 mol/L terminates reaction.It is subsequently adding 1.4 millis
After the ethyl acetate risen, fully mixing, it is centrifuged 5 minutes under 14100g.Draw the supernatant 1 milliliter to spacious
In mouth teat glass, it is dried 1 hour at 80 DEG C, is subsequently adding 2 milliliters of distilled water, survey under 228nm
Absorbance.Often group measures three repetitions, calculates average inhibition, the results are shown in Table 1.Suppression ratio is according to following public
Formula calculates:
Angiotensin converting enzyme suppression ratio (%)=(1-AS/AC)×100
In formula, As represents the absorbance adding pure peptide, and Ac represents the absorbance not adding pure peptide.
The table 1 pure peptide inhibition to ACE
The above results shows, the present invention, with coconut cake globulin as substrate, uses complex enzyme hydrolysis, gel chromatography to divide
The suppression ratio of I, the II couple of ACE of pure peptide prepared from technology such as, reverse high-efficient liquid phase chromatogram purifications is respectively
76.26% and 82.33%, it is above 70%, has the potential ability as treatment hypertension natural medicinal ingredients.
The above is only the preferred embodiment of the present invention, it is noted that common for the art
For technical staff, on the premise of without departing from the technology of the present invention principle, it is also possible to make some improvement and profit
Decorations, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (1)
1. the method preparing ace inhibitory peptide for raw material with coconut cake globulin, it is characterised in that it is concrete
Step is as follows:
1), raw material pre-treatment select ripe old Cortex cocois radicis fruit, then shell, seed coat of pruning, clean, dig
Silk, squeeze the juice, obtain coconut cake；By coconut cake 40～50 DEG C of crushed after being dried, cross 40～60 mesh sieves, then
Petroleum ether or the remaining Oleum Cocois of ether extraction is added in the ratio that mass volume ratio is 1 10～1 20,
To defat coconut cake powder；
2), extract globulin to add in the ratio that mass volume ratio is 1 10～1 30 in defat coconut cake powder
Entering the sodium chloride solution of 0.4～0.5mol/L, under room temperature, concussion extracts 2～3h, filters, and collects filtrate；
Repeatedly extract 3 times according to as above method, merge the filtrate collected；By filtrate 4～10 DEG C, 10000～
It is centrifuged 15～20min under conditions of 12000rpm, collects supernatant；Supernatant loads molecular cut off is
In 4000～8000 dialyzers, dialyse 12～24 hours in the distilled water of 4～6 DEG C；After dialysis terminates,
Dialysis solution is centrifuged under conditions of 4～10 DEG C, 80000～12000rpm 15～20min, collects precipitation,
The coconut cake globulin that purity is higher can be obtained after vacuum lyophilization；
3), coconut cake globulin is dissolved in by combinative enzyme hydrolysis preparation in the ratio that mass volume ratio is 1 15
In 0.1～0.3M phosphate buffer, regulate pH to 8.0～pH9.0, be the most by mass percentage 0.5～
The ratio of 1% adds alkaline protease, and concussion hydrolysis 3～6h at 45～55 DEG C, regulation hydrating solution pH is extremely
7.0～pH 7.5, it is the ratio addition neutral protease of 0.5～1% the most by mass percentage, 50～55 DEG C
Lower concussion hydrolysis 2～4h, regulates hydrating solution pH to 2.0, then is 0.3～0.6% by mass percentage
Ratio adds pepsin, and at 37 DEG C, concussion hydrolysis 2～4h, regulates hydrating solution pH to 7.0, finally press
Mass percent is the ratio addition trypsin of 0.3～0.6%, concussion hydrolysis 2～4h at 37 DEG C；By enzymolysis
Liquid heats 5～10min at 95～100 DEG C, enzyme denaturing；
4), high speed centrifugation separates enzymolysis solution after enzyme deactivation at the bar of 4～6 DEG C, 10000～12000rpm
It is centrifuged 25～30min under part, collects supernatant；Available polypeptide primary extract after lyophilization；
5), polypeptide primary extract is carried out Sephadex G-15 column chromatography for separation by gel chromatography separation: column length
0.6～1.2 meter, a diameter of 16～20mm, fixing is Sephadex G-15 mutually, pure water eluting, flow velocity 0.6～
0.8mL/min；Within every 5 minutes, collecting once, eluent with spectrophotometric colo, is isolated at 220nm
5 polypeptide fractions；Constantly repeat this step, collect the 5th polypeptide fractions, can be obtained this after lyophilization many
The powder of peptide composition；
6), reverse high-efficient liquid phase chromatogram purification is by the 5th polypeptide fractions of above-mentioned gel chromatography separation gained
Reverse high performance liquid chromatography separates and purification further: by the 5th polypeptide fractions by 0.6～1
The ratio of mg/mL is dissolved in distilled water, crosses the film of 0.22 micron, collects filtrate；Filtrate is carried out reversely
High performance liquid chromatography separates, and detached dowel used is carbon 18 semi-preparative column, binary gradient concentration eluting, and flow phase
A be the acetonitrile containing 0.1% trifluoroacetic acid, Mobile phase B be ultra-pure water, flow velocity 2.0～2.5mL/min, column temperature
30～40 DEG C, detect wavelength 220nm, isolate 12 polypeptide fractions；9th component is used carbon further
18 analytical columns-reversed-phase high-performance liquid chromatography separates, and binary gradient concentration eluting, mobile phase A is containing 0.1% 3
The acetonitrile of Fluoroethanoic acid, Mobile phase B are ultra-pure water, flow velocity 0.8～1.0mL/min, column temperature 30～40 DEG C,
Detection wavelength 220nm, is separated into 2 pure peptides, is collected respectively by 2 pure peptides, lyophilization, preserves.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610402642.3A CN105803024B (en) | 2016-06-08 | 2016-06-08 | A method of ace inhibitory peptide is prepared using coconut cake globulin as raw material |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610402642.3A CN105803024B (en) | 2016-06-08 | 2016-06-08 | A method of ace inhibitory peptide is prepared using coconut cake globulin as raw material |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105803024A true CN105803024A (en) | 2016-07-27 |
| CN105803024B CN105803024B (en) | 2019-05-10 |
Family
ID=56427793
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610402642.3A Expired - Fee Related CN105803024B (en) | 2016-06-08 | 2016-06-08 | A method of ace inhibitory peptide is prepared using coconut cake globulin as raw material |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105803024B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109680030A (en) * | 2019-01-28 | 2019-04-26 | 华南理工大学 | A kind of inoxidizability coconut polypeptide and the preparation method and application thereof |
| CN109810177A (en) * | 2019-03-25 | 2019-05-28 | 陕西师范大学 | A kind of walnut dregs polypeptide and its preparation method and application with ACE inhibitory activity |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1256088A (en) * | 1998-07-29 | 2000-06-14 | 不二制油株式会社 | Soybean protein hydrolysates, their production and use |
| CN104480177A (en) * | 2014-12-31 | 2015-04-01 | 广西壮族自治区农业科学院农产品加工研究所 | Preparation method of cannabis sativa protein ACE (Angiotensin Converting Enzyme) peptide inhibitor |
-
2016
- 2016-06-08 CN CN201610402642.3A patent/CN105803024B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1256088A (en) * | 1998-07-29 | 2000-06-14 | 不二制油株式会社 | Soybean protein hydrolysates, their production and use |
| CN104480177A (en) * | 2014-12-31 | 2015-04-01 | 广西壮族自治区农业科学院农产品加工研究所 | Preparation method of cannabis sativa protein ACE (Angiotensin Converting Enzyme) peptide inhibitor |
Non-Patent Citations (3)
| Title |
|---|
| 郑亚军: "椰肉乙醇提取物抗氧化性的研究", 《中国粮油学报》 * |
| 郑亚军: "脱脂椰肉中多酚化合物抗氧化性的研究", 《热 带 作 物 学 报》 * |
| 郑亚军: "超临界 CO 2 萃取原生态椰子油工艺及其抗氧化性的研究", 《中国粮油学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109680030A (en) * | 2019-01-28 | 2019-04-26 | 华南理工大学 | A kind of inoxidizability coconut polypeptide and the preparation method and application thereof |
| CN109810177A (en) * | 2019-03-25 | 2019-05-28 | 陕西师范大学 | A kind of walnut dregs polypeptide and its preparation method and application with ACE inhibitory activity |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105803024B (en) | 2019-05-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107141336B (en) | Yak bone protein peptide with DPP-IV inhibitory activity and preparation method thereof | |
| CN103333940B (en) | Method for preparing dipeptidyl peptidase IV (DPP-IV) inhibitory peptide through using hairtail | |
| CN103980347B (en) | Antihypertensive peptide of swim bladder of large yellow croaker and preparation method and use thereof | |
| CN103627760B (en) | Method for preparing biological active peptide with effect of reducing trioxypurine by using walnut protein | |
| JP2014500005A (en) | An industrial method for preparing corn antihypertensive peptides | |
| CN101240312A (en) | Method for preparing ACE inhibition peptide originate from fish skin | |
| CN107446978A (en) | A kind of stem of noble dendrobium small-molecular peptides and preparation method thereof | |
| CN105111282A (en) | Walnut peptide having ACE inhibitory activity and preparation method thereof | |
| CN104473171B (en) | A kind of fiery numb lozenge and preparation method thereof | |
| CN102808010A (en) | Method for preparing antihypertensive peptides through enzymolysis of ground meat proteins of tuna | |
| CN101015336A (en) | Prepn. of coconut extractive | |
| CN101037468A (en) | Preparation method of oyster active peptides | |
| CN101906135A (en) | Novel spirulina source antihypertensive peptide and preparation method thereof | |
| CN104610430B (en) | Antioxidation polypeptide prepared using ganoderma lucidum albumen and preparation method thereof | |
| CN103290086A (en) | A mung bean protein peptide having ACE inhibitory activity and a preparation method and applications thereof | |
| CN101012472B (en) | Method for preparation of egg white oligopeptide having high F value | |
| CN104774899A (en) | Method for preparing cervical cancer resistant polypeptide from tuna dark meat protein | |
| CN105803024A (en) | Method of using coconut cake globulin as raw material to prepare ACE inhibiting peptide | |
| CN104877007B (en) | Yak milk lactalbumin source ACE inhibitor peptides and preparation method thereof | |
| CN101906133A (en) | Preparation method and application of corn angiotensin converting enzyme inhibitory peptide | |
| CN109810177A (en) | A kind of walnut dregs polypeptide and its preparation method and application with ACE inhibitory activity | |
| CN101830967A (en) | Oligopeptide with angiotensin transferase inhibiting activity and preparation method thereof | |
| CN102517365A (en) | Preparation method for micro-molecular glycopeptide of jellyfish | |
| CN105646656B (en) | Griseus shark cartilage protein antioxidant peptide and application thereof | |
| CN104975059A (en) | Method for preparing ACE (angiotensin converting enzyme) inhibitory peptide by using cod meat |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190510 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |