CN105803024B - A method of ace inhibitory peptide is prepared using coconut cake globulin as raw material - Google Patents
A method of ace inhibitory peptide is prepared using coconut cake globulin as raw material Download PDFInfo
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- CN105803024B CN105803024B CN201610402642.3A CN201610402642A CN105803024B CN 105803024 B CN105803024 B CN 105803024B CN 201610402642 A CN201610402642 A CN 201610402642A CN 105803024 B CN105803024 B CN 105803024B
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- 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
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- 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 present invention relates to a kind of methods for preparing ace inhibitory peptide as raw material using coconut cake globulin, mature old coconut is chosen to be handled to obtain degreasing coconut cake powder, complex enzyme for hydrolyzing is carried out after extraction coconut cake globulin, centrifuge separation prepares polypeptide primary extract, 5 polypeptide fractions are isolated from polypeptide primary extract, it collects the 5th polypeptide fractions and further separates and be purified into 12 polypeptide fractions, 9th component is further used into the isolated 2 pure peptides of 18 analytical columns of carbon-reversed-phase high performance liquid chromatography, the two is to the inhibiting rate of ACE 70% or more.The present invention is using coconut cake globulin as substrate, purity is prepared up to 95% or more ace inhibitory peptide using technologies such as complex enzyme hydrolysis, gel chromatography separation, reversed high-efficient liquid phase chromatogram purifications, both the ingredient of natural drug for hypertension had been can be used for, it can also be added in food, carry out food-borne prevention and treatment hypertension, conducive to the comprehensive utilization and intensive processing realized to coconut, the strong development for promoting China's coconut industry.
Description
Technical field
The invention belongs to agricultural product to adopt post-processing technical field, be related to a kind of method of ace inhibitory peptide, and in particular to it is a kind of with
Coconut cake is the method that raw material prepares coconut cake globulin, and then prepares ace inhibitory peptide.
Background technique
Hypertension is to threaten one of the principal disease of human health.According to statistics, hypertension average attack rate in countries in the world is
10~20%, every year because hypertension death toll is up to 12,000,000, China dies of the number of hypertension and related disease every year in the whole world
Up to 2,000,000, hypertension has become one of worldwide health problem of urgent need to resolve.
Angiotensin converting enzyme (Antiotensin I Converting Enzyme, ACE) plays pass on adjusting blood pressure
Keyness effect.Studies have shown that the intracorporal renin-angiotensin system of people (rennin-angiotensin system, RAS) and
Kallikrein kinin system (kallikrein-kinin system, KKS) is to maintain normotensive two mutual antagonisms
Equilibrium system (Li et al., 2004).ACE can be catalyzed angiotensin I (angiotensin I) and be changed into potent
The Angiotensin II (angiotensin II) of blood pressure effect is increased, while the bradykinin with antihypertensive effect being degraded,
It is allowed to lose antihypertensive effect, to make RAS and KKS equilibrium system is unbalance to eventually lead to blood pressure raising.Therefore, by inhibiting internal
The activity of ACE can play the effect of blood pressure lowering.The decompression principle of the blood-pressure drug clinically used at present also mainly presses down
ACE processed activity (Roye&Simpson, 2010；Shen Xiaowen, 2010).
Ace inhibitory peptide refers to the polypeptide for being able to suppress ACE enzymatic activity.It is by active with ACE to the inhibiting mechanism of ACE
The fixation site at center in conjunction with and so that ACE is lost enzyme activity, can also be competitively in conjunction with the substrate specificity of ACE, to drop
The activity of low ACE, and then play the role of blood pressure lowering.Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe used at present is mostly chemical synthetic drug, although curative effect
Significantly, but it is with high costs, and long-term use can generate various side effects (such as injury of kidney, dry cough, diarrhea, taste function disorder
Deng).Food-borne ace inhibitory peptide is because blood pressure lowering effect is good, safety is without side-effects as the hot spot studied at present.With people self
The raising of health care and awareness of safety, the idea for using non-drug therapy to reach preventing and treating diseases will be received by more people, from drink
Food-borne ace inhibitory peptide is added on food to prevent and treat having good prospects for hypertension.
Coconut (Cocos nucifera L.) be the most important woody oleiferous plants crop in torrid areas and food energy crop it
One, problem has developed into China hot-zone specialty industries well-known throughout the country.However, the product master of China's problem
In terms of concentrating on the common coconut food such as coconut candy, coconut powder, nata de coco, functional product proportion very little；Enterprise lacks
The weary capability of independent innovation, product science and technology added value are few, it is difficult to increase economic efficiency and resource utilization.In recent years, Southeast Asia coconut palm
The country such as sub- main production country such as Vietnam, Indonesia is made with the advantages such as cheap labour and raw material, more advanced processing technology
Domestic coconut processing enterprise faces the increasing pressure and challenge.
Coconut flesh contains about 4~8% protein, and wherein albumin accounts for 21%, globulin and accounts for 40.1%, alcohol soluble protein
3.3% is accounted for, acid-soluble glutelin accounts for 14.4%, and alkali solubility glutelin accounts for 4.8%.Nineteen thirty American scientistWith
Spychalski isolates the globulin of molecular weight about 208kDa a kind of from coconut meat, and is named as cocosin, opens coconut
The research history of protein.It is subsequent studies have shown that globulin accounts for the 65% of total protein content in mature coconut flesh, and
In 7~8 months tender coconut fruits, content of the globulin in total protein may be up to 80%.Globulin can be divided into according to sedimentation coefficient
2S globulin, 7S globulin, 9S globulin, 11S globulin and 15S globulin.Wherein 11S globulin accounts for the total globulin of coconut
86%, and 7S globulin accounts for 14% (Garcia et al, 2005).Researcher also found that cocosin is a polymer ball
Albumen (hexamer), it is formed by connecting by acidic and alkaline subunit by a disulfide bond, on alkaline subunit herein in connection with
Glycosyl.In food industry, the main protein contained by coconut products of coconut powder, concentration coconut palm slurry, coconut juice protein beverage etc. it is main
Ingredient is exactly coconut globulin.Thus, globulin be in coconut protein most important protein and most important protein it
One.
Coconut cake is the Main By product after coconut meat juicing, and containing 16~24% protein, while coconut cake yield is very big, luxuriant and rich with fragrance
Lv Bin only coconut cake yield in 2014 just up to 2,000,000 tons.Studies have shown that in coconut cake protein globulin content be 54.35 ±
3.69g/100g.Coconut cake globulin is rich in aspartic acid and arginine, and the ratio that amino acid accounts for total amino acid is 39.25%, far
Recommendation higher than FAO/WHO, amino acid composition are relatively reasonable；Its external digestion absorptivity (in vitro
Digestibility index, IVPD) it is up to 88.26%, biological value (biological value, BV) is 58.41, far
Higher than soybean protein isolate, thus nutritive value is higher.Meanwhile coconut cake globulin has extremely strong chelated iron ion ability, is
The good raw material of food-borne iron supplementary or ferrous-fortifier is prepared, while being had to interior free yls such as DPPH, OH, ATBS+
Certain Scavenging activity, bioactivity are higher.Currently, coconut cake is only used as the auxiliary material of the food such as animal feed or cake, egg
White matter isoreactivity ingredient is not exploited, and causes the significant wastage of resource.
Summary of the invention
The purpose of the present invention is provide a kind of using coconut cake globulin as raw material preparation ACE suppression in view of the deficiencies of the prior art
The method of peptide processed, prepares coconut globulin from coconut cake, using coconut cake globulin as substrate, using complex enzyme hydrolysis, gel chromatography point
Purity is prepared up to 95% or more ace inhibitory peptide from technologies such as, reversed high-efficient liquid phase chromatogram purifications, not only improves realization to coconut palm
The comprehensive utilization and intensive processing of son, and can turn waste into wealth, the strong development for promoting China's coconut industry.
The technical solution adopted in the present invention:
A method of ace inhibitory peptide being prepared using coconut cake globulin as raw material, the specific steps of which are as follows:
1, the mature old coconut fruit of raw material pre-treatment selection, then decladding, prune kind of a skin, and cleaning, plane silk, juicing obtain
Coconut cake.By coconut cake in 40~50 DEG C of crushed after being dried, 40~60 meshes are crossed, are then 1 ﹕, 10~1 ﹕ by mass volume ratio (m/v)
Petroleum ether or the remaining coconut oil of extracted by ether is added in 20 ratio, obtains degreasing coconut cake powder.
2, it extracts globulin and is added 0.4 in the ratio that mass volume ratio (m/v) is 1 ﹕, 10~1 ﹕ 30 in degreasing coconut cake powder
The sodium chloride solution of~0.5mol/L, 2~3h is extracted in concussion at room temperature, and filtrate is collected in filtering；It is extracted repeatedly according to method as above
3 times, merge the filtrate of collection.Filtrate is centrifuged 15~20min under conditions of 4~10 DEG C, 10000~12000rpm, is collected
Supernatant.By supernatant be fitted into molecular cut off be 4000~8000 dialysis membranes in, in 4~6 DEG C of distilled water dialyse 12~
24 hours, the impurity such as removal salt ion, soluble sugar, small molecular protein；Simultaneously as cannot be dissolved in distilled water, coconut cake
Globulin can gradually Precipitation in dialysis procedure.After dialysis, by dialyzate in 4~10 DEG C, 80000~12000rpm
Under conditions of be centrifuged 15~20min, collect precipitating, can be obtained the higher coconut cake globulin of purity after vacuum freeze drying.
3, complex enzyme for hydrolyzing preparation coconut cake globulin is dissolved in 0.1 by mass volume ratio (m/v) for the ratio of 1 ﹕ 15~
In 0.3M phosphate buffer, pH to 8.0~pH9.0 is adjusted, is then added by mass percentage for the ratio of 0.5~1% (m/m)
Alkali protease, 3~6h of concussion hydrolysis, adjusts hydrating solution pH to 7.0~pH 7.5 at 45~55 DEG C, then presses quality percentage
Neutral proteinase is added than the ratio for 0.5~1% (m/m), 2~4h of concussion hydrolysis, adjusts hydrating solution pH at 50~55 DEG C
Pepsin is added for the ratio of 0.3~0.6% (m/m) to 2.0, then by mass percentage, 2~4h of concussion hydrolysis at 37 DEG C,
Hydrating solution pH to 7.0 is adjusted, is by mass percentage finally the ratio addition trypsase of 0.3~0.6% (m/m), 37 DEG C
Lower concussion hydrolyzes 2~4h；Enzymolysis liquid is heated into 5~10min, enzyme deactivation at 95~100 DEG C.
4, it is centrifuged at a high speed and enzymolysis solution after enzyme deactivation is centrifuged 25 under conditions of 4~6 DEG C, 10000~12000rpm
~30min collects supernatant；Polypeptide primary extract can be obtained after freeze-drying.
5, polypeptide primary extract is carried out Sephadex G-15 column chromatography for separation by gel chromatography separation.0.6~1.2 meter of column length,
Diameter is 16~20mm, and stationary phase is Sephadex G-15, pure water elution, 0.6~0.8mL/min of flow velocity, collection in every 5 minutes
Once, eluent uses spectrophotometric colo at 220nm.It may separate out 5 polypeptide fractions.Detection indicate that the 5th polypeptide
Inhibiting rate highest of the component to ACE.The step is constantly repeated, the 5th polypeptide fractions is collected, the polypeptide can be obtained after freeze-drying
The powder of component.Since Sephadex G-15 plays the role of desalination, thus this step can also remove the salt in polypeptide extract
Point.
6, reversed high-efficient liquid phase chromatogram purification is by resulting 5th polypeptide fractions of above-mentioned gel chromatography separation reversed efficient
It is carried out in liquid chromatogram (reversed-phase high performance liquid chromatography, RP-HPLC)
Further separation and purifying: the 5th polypeptide fractions are dissolved in distilled water in the ratio of 0.6~1mg/mL, cross 0.22 micron
Film, collect filtrate；Filtrate is subjected to reversed high performance liquid chromatography separation, splitter used is 18 semi-preparative column (Zorbax of carbon
Semi-preparative C18column, 9.4mm × 250mm), binary gradient concentration elution, mobile phase A is containing 0.1% 3
The acetonitrile of fluoroacetic acid, Mobile phase B are ultrapure water, and 2.0~2.5mL/min of flow velocity, 30~40 DEG C of column temperature, Detection wavelength 220nm can
Isolate 12 polypeptide fractions.Experiment shows that wherein the 9th polypeptide fractions are to ACE inhibiting rate highest.By the 9th component into one
Step 18 analytical columns of carbon-reversed-phase high performance liquid chromatography separation (Zorbax analytical C18column, 4.6mm ×
250mm), binary gradient concentration elutes, and mobile phase A is ultrapure water, flow velocity for the acetonitrile containing 0.1% trifluoroacetic acid, Mobile phase B
0.8~1.0mL/min, 30~40 DEG C of column temperature, Detection wavelength 220nm is separated into 2 pure peptides.2 pure peptides are collected respectively,
Freeze-drying saves.
It is analyzed and identified through LC-MC/MC, the amino acid sequence and molecular weight of this 2 pure peptides are respectively as follows: 1, Leu-Leu-Leu-
Gly-Ala-Val-Asn-Tyr-Arg, molecular weight 1017.6Da；2, Arg-Pro-Phe-Asn-Leu-Phe-His-Lys, point
Son amount is 1057.58Da.The two is to the inhibiting rate of ACE 70% or more.
The present invention is using the coconut cake globulin prepared from coconut cake as substrate, using complex enzyme hydrolysis, gel chromatography separation, reversed
The technologies such as high-efficient liquid phase chromatogram purification prepare purity up to 95% or more ace inhibitory peptide, due to having used stomach in preparation process
Protease and trypsase, thus two polypeptides are preferable to the stability of the digestive ferment in human gastrointestinal tract digestive system, both may be used
It with the ingredient for natural drug for hypertension, can also be added in food, carry out food-borne prevention and treatment hypertension, benefit
In comprehensive utilization and intensive processing of the realization to coconut, and can turn waste into wealth, the strong development for promoting China's coconut industry.
Detailed description of the invention
Fig. 1 is the LC-MS/MS of Leu-Leu-Leu-Gly-Ala-Val-Asn-Tyr-Arg (molecular weight 1017.6Da)
Figure.
Fig. 2 is the LC-MS/MS figure of Arg-Pro-Phe-Asn-Leu-Phe-His-Lys (molecular weight 1057.58Da).
Specific embodiment
With reference to embodiment, the embodiment of the present invention is furthur described in detail.Following embodiment is used for
Illustrate 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, the mature old coconut fruit of raw material pre-treatment selection, then decladding, prune kind of a skin, and cleaning, plane silk, juicing obtain
Coconut cake.By coconut cake in 45 DEG C of crushed after being dried, 60 meshes are crossed, petroleum ether then is added in the ratio of 1 ﹕ 10 (m/v) or ether mentions
Remaining coconut oil is taken, degreasing coconut cake powder is obtained.
2, the sodium chloride solution that 0.4mol/L is added in globulin in degreasing coconut cake powder in the ratio of 1 ﹕ 10 (m/v) is extracted,
2h is extracted in concussion at room temperature, and filtrate is collected in filtering.Above-mentioned sodium chloride solution (1 ﹕ 10, m/v) is added again in filter residue, according to
Method as above is extracted 3 times repeatedly, merges the filtrate of collection.Filtrate is centrifuged 15min under conditions of 4 DEG C, 12000rpm, is collected
Supernatant.It is to dialyse 12 hours in 4 DEG C of distilled water in 8000 dialysis membranes that this supernatant, which is fitted into molecular cut off, is removed
The impurity such as salt ion, soluble sugar, small molecular protein, and make globulin gradually Precipitation.After dialysis, dialyzate is existed
4 DEG C, 15min is centrifuged under conditions of 12000rpm, collect precipitating, obtain coconut cake globulin after vacuum freeze drying.
3, coconut cake globulin is dissolved in 0.1mol/L phosphate buffer (1 ﹕ 15, m/v) by complex enzyme for hydrolyzing preparation, is adjusted
To pH9.0, alkali protease (0.1%, m/m) is added, then concussion hydrolysis 3h at 50 DEG C adjusts hydrating solution to pH
7.0, it is added neutral proteinase (1%, m/m), concussion hydrolysis 2h at 55 DEG C.Then hydrating solution is adjusted to pH 2.0, and stomach is added
Protease (0.5%, m/m), concussion hydrolysis 2h at 37 DEG C；Hydrating solution is finally adjusted to pH 7.0, trypsase is added
(0.5%, m/m), concussion hydrolysis 2h at 37 DEG C.Hydrolyzate is heated into 5min, enzyme deactivation at 100 DEG C.
4, it is centrifuged at a high speed and enzymolysis solution after enzyme deactivation is centrifuged 25min under conditions of 4 DEG C, 12000rpm, in collection
Clear liquid.Polypeptide primary extract can be obtained after freeze-drying.
5, polypeptide primary extract is carried out Sephadex G-15 column chromatography for separation by gel chromatography separation.1.0 meters of column length, diameter
For 16mm, stationary phase is Sephadex G-15, and pure water elution, flow velocity 0.8mL/min, collection in every 5 minutes is primary, and eluent exists
Spectrophotometric colo is used at 220nm.It may separate out 5 polypeptide fractions.Detection indicate that the 5th suppression of the polypeptide fractions to ACE
Rate highest processed.The step is constantly repeated, the 5th polypeptide fractions is collected, the powder of the polypeptide fractions can be obtained after freeze-drying.
6, reversed high-efficient liquid phase chromatogram purification is by resulting 5th polypeptide fractions of above-mentioned gel chromatography separation reversed efficient
It is further separated and is purified in liquid chromatogram: the 5th polypeptide fractions are dissolved in distilled water (1mg/mL), it is micro- to cross 0.22
The film of rice collects filtrate.The filtrate is subjected to reversed high performance liquid chromatography separation, splitter used is 18 semi-preparative column of carbon
(Zorbax semi-preparative C18column, 9.4mm × 250mm), the elution of binary gradient concentration, mobile phase A is second
Nitrile (containing 0.1% trifluoroacetic acid), Mobile phase B are ultrapure water, and flow velocity 2.5mL/min, 40 DEG C of column temperature, Detection wavelength 220nm can divide
From 12 polypeptide fractions in place.9th component is further used into 18 analytical columns of carbon-reversed-phase high performance liquid chromatography separation (Zorbax
Analytical C18column, 4.6mm × 250mm), binary gradient concentration elution, mobile phase A is that acetonitrile (contains 0.1% trifluoro
Acetic acid), Mobile phase B be ultrapure water, flow velocity 1.0mL/min, 35 DEG C of column temperature, Detection wavelength 220nm is separated into 2 pure peptides, point
It does not collect, is freeze-dried, save.
Embodiment two,
1, the mature old coconut fruit of raw material pre-treatment selection, then decladding, prune kind of a skin, and cleaning, plane silk, juicing obtain
Coconut cake.By coconut cake in 50 DEG C of crushed after being dried, 60 meshes are crossed, petroleum ether then is added in the ratio of 1 ﹕ 15 (m/v) or ether mentions
Remaining coconut oil is taken, degreasing coconut cake powder is obtained.
2, the sodium chloride solution that 0.4mol/L is added in globulin in degreasing coconut cake powder in the ratio of 1 ﹕ 10 (m/v) is extracted,
3h is extracted in concussion at room temperature, and filtrate is collected in filtering.Above-mentioned sodium chloride solution (1 ﹕ 15, m/v) is added again in filter residue, according to
Method as above is extracted 3 times repeatedly, merges the filtrate of collection.Filtrate is centrifuged 20min under conditions of 4 DEG C, 10000rpm, is collected
Supernatant.It is to dialyse 24 hours in 4 DEG C of distilled water in 8000 dialysis membranes that this supernatant, which is fitted into molecular cut off, is removed
The impurity such as salt ion, soluble sugar, small molecular protein, and make globulin gradually Precipitation.After dialysis, dialyzate is existed
4 DEG C, 20min is centrifuged under conditions of 10000rpm, collect precipitating, obtain coconut cake globulin after vacuum freeze drying.
3, coconut cake globulin is dissolved in 0.1mol/L phosphate buffer (1 ﹕ 15, m/v) by complex enzyme for hydrolyzing preparation, is adjusted
To pH9.0, alkali protease (0.6%, m/m) is added, then concussion hydrolysis 6h at 50 DEG C adjusts hydrating solution to pH
7.0, it is added neutral proteinase (0.6%, m/m), concussion hydrolysis 4h at 55 DEG C.Then hydrating solution is adjusted to pH 2.0, is added
Pepsin (0.6%, m/m), concussion hydrolysis 2h at 37 DEG C；Hydrating solution is finally adjusted to pH 7.0, trypsase is added
(0.6%, m/m), concussion hydrolysis 2h at 37 DEG C.Hydrolyzate is heated into 10min, enzyme deactivation at 95 DEG C.
4, it is centrifuged at a high speed and enzymolysis solution after enzyme deactivation is centrifuged 30min under conditions of 4 DEG C, 10000rpm, in collection
Clear liquid.Polypeptide primary extract can be obtained after freeze-drying.
5, polypeptide primary extract is carried out Sephadex G-15 column chromatography for separation by gel chromatography separation.0.8 meter of column length, diameter
For 16mm, stationary phase is Sephadex G-15, and pure water elution, flow velocity 0.6mL/min, collection in every 5 minutes is primary, and eluent exists
Spectrophotometric colo is used at 220nm.It may separate out 5 polypeptide fractions.Detection indicate that the 5th suppression of the polypeptide fractions to ACE
Rate highest processed.The step is constantly repeated, the 5th polypeptide fractions is collected, the powder of the polypeptide fractions can be obtained after freeze-drying.
6, reversed high-efficient liquid phase chromatogram purification is by resulting 5th polypeptide fractions of above-mentioned gel chromatography separation reversed efficient
It is further separated and is purified in liquid chromatogram.5th polypeptide fractions are dissolved in distilled water (1mg/mL), it is micro- to cross 0.22
The film of rice collects filtrate.The filtrate is subjected to reversed high performance liquid chromatography separation, splitter used is 18 semi-preparative column of carbon
(Zorbax semi-preparative C18column, 9.4mm × 250mm), the elution of binary gradient concentration, mobile phase A is second
Nitrile (containing 0.1% trifluoroacetic acid), Mobile phase B are ultrapure water, and flow velocity 2mL/min, is separated by 40 DEG C of column temperature, Detection wavelength 220nm
Locate 12 polypeptide fractions.9th component is further used into 18 analytical columns of carbon-reversed-phase high performance liquid chromatography separation (Zorbax
Analytical C18column, 4.6mm × 250mm), binary gradient concentration elution, mobile phase A is that acetonitrile (contains 0.1% trifluoro
Acetic acid), Mobile phase B be ultrapure water, flow velocity 1.0mL/min, 35 DEG C of column temperature, Detection wavelength 220nm is separated into 2 pure peptides, point
It does not collect, is freeze-dried, save.
Two, embodiment products therefrom is identified
Example one, two products therefrom of embodiment are analyzed and identified through LC-MC/MC, the amino acid sequence of this 2 pure peptides and
Molecular weight is respectively as follows:
I, Leu-Leu-Leu-Gly-Ala-Val-Asn-Tyr-Arg, molecular weight 1017.6Da, LC-MS/MS figure are such as
Shown in Fig. 1.
II, Arg-Pro-Phe-Asn-Leu-Phe-His-Lys, molecular weight 1057.58Da, LC-MS/MS figure is as schemed
Shown in 2.
Three, pure peptide identifies the inhibitory effect of ACE
Pure peptide carries out the inhibitory effect of ACE by the way of external test, and measuring method is according to document (Jimsheena&
Gowda, 2009) method carries out in:
50 microlitres of pure peptides (1mg/mL), 50 microlitres of angiotensin converting enzyme (ACE, 25mill units/mL) and 150
Microlitre, 8.3 mM/ls of hippuroyl-histidine-Leucine (HHL) be uniformly mixed, react after sixty minutes, be added at 37 DEG C
250 microlitres, the hydrochloric acid of 1 mol/L terminate reaction.Then 1.4 milliliters of ethyl acetate is added, after mixing well, under 14100g
Centrifugation 5 minutes.1 milliliter of supernatant liquor is drawn into open glass tubes, it is 1 hour dry at 80 DEG C, 2 milliliters of steamings are then added
Distilled water surveys absorbance at 228nm.Three repetitions of every group of measurement calculate average inhibition, the results are shown in Table 1.Inhibiting rate is according to such as
Lower formula calculates:
Angiotensin converting enzyme inhibiting rate (%)=(1-AS/AC)×100
In formula, As indicates the absorbance value that pure peptide is added, and Ac indicates the absorbance value for not adding pure peptide.
Inhibitory effect of the pure peptide of table 1 to ACE
The above results show the present invention using coconut cake globulin as substrate, using complex enzyme hydrolysis, gel chromatography separation, reversed
The inhibiting rate of I, II couple of ACE of pure peptide of the technologies such as high-efficient liquid phase chromatogram purification preparation is respectively 76.26% and 82.33%, height
In 70%, there is the potential ability as treatment hypertension natural medicinal ingredients.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, several improvements and modifications can also be made, these improvements and modifications
Also it should be regarded as protection scope of the present invention.
Claims (1)
1. a kind of method for preparing ace inhibitory peptide as raw material using coconut cake globulin, which is characterized in that the specific steps of which are as follows:
1), raw material pre-treatment: kind of a skin is pruned in the mature old coconut fruit of selection, then decladding, and cleaning, plane silk, juicing obtain coconut palm
Bran；By coconut cake in 40~50 DEG C of crushed after being dried, 40~60 meshes are crossed, the ratio for being then 1 ﹕, 10~1 ﹕ 20 in mass volume ratio
Petroleum ether or the remaining coconut oil of extracted by ether is added, obtains degreasing coconut cake powder；
2), extract globulin: in degreasing coconut cake powder by mass volume ratio be 1 ﹕, 10~1 ﹕ 30 ratio addition 0.4~
The sodium chloride solution of 0.5mol/L, 2~3h is extracted in concussion at room temperature, and filtrate is collected in filtering；3 are extracted repeatedly according to method as above
It is secondary, merge the filtrate of collection；Filtrate is centrifuged 15~20min under conditions of 4~10 DEG C, 10000~12000rpm, in collection
Clear liquid；It is the dialysis 12~24 in 4~6 DEG C of distilled water in 4000~8000 dialysis membranes that supernatant, which is fitted into molecular cut off,
Hour；After dialysis, dialyzate is centrifuged 15~20min under conditions of 4~10 DEG C, 80000~12000rpm, it is heavy to collect
It forms sediment, the higher coconut cake globulin of purity is obtained after vacuum freeze drying；
3), prepared by complex enzyme for hydrolyzing: coconut cake globulin is dissolved in 0.1~0.3M phosphoric acid in the ratio that mass volume ratio is 1 ﹕ 15
In buffer, pH is adjusted to 8.0~9.0, then by mass percentage for 0.5~1% ratio be added alkali protease, 45
3~6h of concussion hydrolysis, is adjusted to 7.0~7.5 for hydrating solution pH at~55 DEG C, is by mass percentage then 0.5~1%
Neutral proteinase is added in ratio, and hydrating solution pH is adjusted to 2.0, then presses quality percentage by 2~4h of concussion hydrolysis at 50~55 DEG C
Pepsin is added than the ratio for 0.3~0.6%, 2~4h of concussion hydrolysis, is adjusted to 7.0 for hydrating solution pH, most at 37 DEG C
Trypsase is added for 0.3~0.6% ratio by mass percentage afterwards, 2~4h of concussion hydrolysis at 37 DEG C；By enzymolysis liquid 95
5~10min, enzyme deactivation are heated at~100 DEG C；
4) it, is centrifuged at a high speed: enzymolysis solution after enzyme deactivation is centrifuged to 25 under conditions of 4~6 DEG C, 10000~12000rpm~
30min collects supernatant；Polypeptide primary extract is obtained after freeze-drying；
5), gel chromatography separation: by polypeptide primary extract carry out Sephadex G-15 column chromatography for separation: 0.6~1.2 meter of column length, directly
Diameter is 16~20mm, and stationary phase is Sephadex G-15, pure water elution, 0.6~0.8mL/min of flow velocity；Collect one within every 5 minutes
Secondary, eluent, with spectrophotometric colo, isolates 5 polypeptide fractions at 220nm；The step is constantly repeated, collects the 5th
Polypeptide fractions obtain the powder of the polypeptide fractions after freeze-drying；
6), reversed high-efficient liquid phase chromatogram purification: by resulting 5th polypeptide fractions of above-mentioned gel chromatography separation in reversed efficient liquid
It is further separated and is purified in phase chromatography: the 5th polypeptide fractions are dissolved in distilled water in the ratio of 0.6~1mg/mL
In, 0.22 micron of film is crossed, filtrate is collected；Filtrate is subjected to reversed high performance liquid chromatography separation, splitter used is carbon 18 half
Column is prepared, the elution of binary gradient concentration, mobile phase A is ultrapure water, flow velocity for the acetonitrile containing 0.1% trifluoroacetic acid, Mobile phase B
2.0~2.5mL/min, 30~40 DEG C of column temperature, Detection wavelength 220nm isolates 12 polypeptide fractions；By the 9th component into one
Step 18 analytical columns of carbon-reversed-phase high performance liquid chromatography separation, the elution of binary gradient concentration, mobile phase A is containing 0.1% trifluoroacetic acid
Acetonitrile, Mobile phase B be ultrapure water, 0.8~1.0mL/min of flow velocity, 30~40 DEG C of column temperature, Detection wavelength 220nm is separated into 2
A pure peptide collects 2 pure peptides respectively, is freeze-dried, and saves.
Priority Applications (1)
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| 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 |
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| 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 |
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