CN106755243A - A kind of preparation method of konjaku albumen source ACE inhibitor peptides - Google Patents
A kind of preparation method of konjaku albumen source ACE inhibitor peptides Download PDFInfo
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- CN106755243A CN106755243A CN201611249647.3A CN201611249647A CN106755243A CN 106755243 A CN106755243 A CN 106755243A CN 201611249647 A CN201611249647 A CN 201611249647A CN 106755243 A CN106755243 A CN 106755243A
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
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Abstract
A kind of preparation method of konjaku albumen source ACE inhibitor peptides, konjaku protein freeze-dried powder is dissolved in pure water, is made into the protein solution of mass fraction 1 3%；Protein solution is heated to 35 55 DEG C, regulation pH value is 4.0 9.0, after adding compound protease well mixed, enzymolysis obtains enzymolysis liquid；Enzymolysis liquid is gone out after enzyme, regulation pH value to konjaku albumen isoelectric point；Centrifugation, collects supernatant, by supernatant by freeze-drying into freeze-dried powder, konjaku albumen source ACE inhibitor peptides crude product is obtained, by after separation, obtaining ace inhibitory peptide.The method that the present invention is provided is simple to operate, and efficiency of pcr product is high, and ACE inhibitions are good.
Description
Technical field
The invention belongs to ace inhibitory peptide preparation field, and in particular to a kind of preparation side of konjaku albumen source ACE inhibitor peptides
Method.
Background technology
Konjaku contains the nutrition of very abundant, rich in glucomannans, starch, protein, multivitamin and mineral matter
Element.Wherein, protein can obtain the biologically active polypeptide with the function such as anti-aging, anticancer, hypotensive, immune through enzymolysis,
It is the very good material for producing functional health-care food.Albumen in konjaku is mainly derived from fry starch of konjak, and testing result finds, evil spirit
Taro fly powder in protein content be up to 23.8%, can be produced after degraded branched-chain amino acid oligopeptides, ace inhibitory peptide, high F value oligopeptide,
The Several Active Peptides such as mannatide.
Natural ace inhibitory peptide is earliest that Ferriera has found in nineteen sixty-five from America thatch head cobra-venom.It is then many
Many researchs on ace inhibitory peptide are rapidly carried out, and are found that short chain from various different natural food albumen
Ace inhibitory peptide.Subsequent people are found that from the raw materials such as dregs of beans, corn, rapeseed dregs, prickly ash seed dregs, silver carp, PINPROL again
Ace inhibitory peptide.Ace inhibitory peptide is angiotensin converting enzyme inhibitor, can suppress the activity of Angiotensin-Converting.ACE
Peptide for inhibiting can effectively adjust blood pressure, while can also strengthen defence capability of the body to various diseases, maintain the physiology of itself to put down
Weighing apparatus.It has molecular weight small, exempt from digestion, directly absorb, bioavilability it is high, to human body without hypersensitive feature.So have must
A kind of method that ace inhibitory peptide is prepared from konjaku is provided.
The content of the invention
It is an object of the invention to provide a kind of preparation method of konjaku albumen source ACE inhibitor peptides, the method is simple to operate, produces
Thing yield is high, and ACE inhibitions are good.
For achieving the above object, the technical solution adopted by the present invention is as follows：
A kind of preparation method of konjaku albumen source ACE inhibitor peptides, konjaku protein freeze-dried powder is dissolved in pure water, is made into
The protein solution of mass fraction 1-3%；Protein solution is heated to 35-55 DEG C, regulation pH value is 4.0-9.0, adds compound egg
After white enzyme is well mixed, enzymolysis obtains enzymolysis liquid；Enzymolysis liquid is gone out after enzyme, regulation pH value to konjaku albumen isoelectric point；Centrifugation,
Supernatant is collected, by supernatant by freeze-drying into freeze-dried powder, konjaku albumen source ACE inhibitor peptides crude product is obtained, by separating
Afterwards, ace inhibitory peptide is obtained.
Further improvement of the invention is that the amount of compound protease is added in every gram of protein solution for 3000-6000U.
Of the invention further improvement is that the go out temperature of enzyme is 90-100 DEG C, and the time is 10-15min.
Further improvement of the invention is that centrifugation is that 10-30min is carried out under the rotating speed of 5000-10000r/min.
Further improvement of the invention is that separation is carried out using anion exchange resin 717.
Of the invention further improvement is that the temperature of freeze-drying is -50~-110 DEG C, and the time of freeze-drying is
10-24h。
Further improvement of the invention is that the temperature of enzymolysis is 35-55 DEG C, and the time is 3-6h.
Compared with prior art, the device have the advantages that：
The present invention is digested with konjaku protein freeze-dried powder as raw material with compound protease, and its enzymatic hydrolysis condition is：Substrate is dense
It is 2-10% to spend, enzyme concentration 1000-5000U/g, and 40-60 DEG C of hydrolysis temperature, pH value is 5-7, through the enzyme that goes out, centrifugation, isolated
Ace inhibitory peptide, detects that the inhibitory activity of konjaku ace inhibitory peptide is 15%-50% through ultraviolet spectrophotometry.The present invention is provided
Method it is simple to operate, efficiency of pcr product is high, and ACE inhibitions are good, be adequately and reasonably develop konjaku protein freeze-dried powder this
Plant high-quality resource and certain reference is provided.
Specific embodiment
The present invention is described in further details with reference to embodiment.
The present invention is comprised the following steps：
Enzymolysis process：
Konjaku protein freeze-dried powder is dissolved in pure water, protein solution is made into；Protein liquid is placed in constant temperature blender with magnetic force
Upper heating；Regulation pH, adds compound protease to be well mixed, and reaction conditions are：PH value is 4.0-9.0, substrate quality point
Number is 1-3%, and enzyme concentration is 3000-6000U/g, and enzymolysis time is 3-6h, and hydrolysis temperature is 35-55 DEG C；Enzymolysis liquid is placed in
Go out enzyme 10-15min in 90-100 DEG C of thermostat water bath, treats that enzymolysis liquid temperature is down to room temperature, adjusts its pH to the electricity such as konjaku albumen
Point；5000-10000r/min is centrifuged 10-30min, collects supernatant, takes a certain amount of supernatant and surveys its ACE inhibiting rate；
Determine ACE inhibiting rates：
Detected using Cushman detection methods:Take 75uL, HHL (hippuroyl-histidyl--leucine) and 25uL, ACE
Inhibitor (polypeptide liquid) is fully mixed.5min is incubated in 37 DEG C of water-baths, 25uL, 25mu/mL ACE borate solutions are added,
40min is reacted in 37 DEG C of water-baths.200uL, 1.0moL/L hydrochloric acid solution destruction reaction environment, terminating reaction are added after end
Process.Then ethyl acetate 1.7mL is added in each test tube, is well mixed；1.2mL ethyl acetate layers are drawn in another test tube
In, it is put into 100 DEG C of baking ovens, solvent flashing 40min.Deionized water 3mL is added after taking out cooling, in wavelength 228nm after mixing
Lower its absorbance of measure.
The ACE inhibiting rate detection methods of table 1
Computing formula is：
In formula：A1There is the absorbance of solution in-ace inhibitory peptide and ACE
A2- it is not added with the absorbance that ace inhibitory peptide has solution
A3There is the absorbance of blank solution in-ACE and HHL
Ultraviolet spectrophotometry detection activity：
Prepare reaction solution and blank solution, both other conditions all sames, to addition konjaku ace inhibitory peptide in reaction solution.With
Absorbance is ordinate, and hippuric acid standard items curvilinear equation is drawn by abscissa of concentration.Survey horse urine in blank solution and reaction solution
Acid content, calculates the inhibitory activity of ace inhibitory peptide.
Embodiment 1
Konjaku protein freeze-dried powder is dissolved in pure water, mass concentration is configured to for 1% protein solution；Protein liquid is put
In being heated to 35 DEG C in constant temperature blender with magnetic force, regulation pH value is 4.0, is subsequently adding compound protease and is well mixed, in constant temperature
Magnetic stirring apparatus carries out enzyme digestion reaction, and enzymolysis time is 3h, and hydrolysis temperature is 35 DEG C, obtains enzymolysis liquid；Wherein, every gram of albumen is molten
The amount that compound protease is added in liquid is 3000U.Enzymolysis liquid is placed in the enzyme 15min that gone out in 90 DEG C of thermostat water baths, enzymolysis liquid is treated
Temperature is down to room temperature, adjusts its pH value for konjaku albumen isoelectric point；8000r/min is centrifuged 20min, collects supernatant, takes certain
Amount supernatant surveys its ACE inhibiting rate>30%, by -50 DEG C of freeze-drying 24h of supernatant process into freeze-dried powder, obtain konjaku albumen
Source ACE inhibitor peptides crude product, by after the separation of anion exchange resin 717, obtaining ace inhibitory peptide, measures konjaku protein sources ACE suppressions
The inhibitory activity of peptide processed is 18.6%.
Embodiment 2
Konjaku protein freeze-dried powder is dissolved in pure water, mass concentration is configured to for 1.5% protein solution；By protein liquid
It is placed in constant temperature blender with magnetic force and is heated to 45 DEG C；Regulation pH value is 5.0, is subsequently adding compound protease and is well mixed, in perseverance
Warm magnetic stirring apparatus carries out enzyme digestion reaction, and enzymolysis time is 4h, and hydrolysis temperature is 40 DEG C, obtains enzymolysis liquid；Wherein, every gram of albumen
The amount that compound protease is added in solution is 4000U.Enzymolysis liquid is placed in the enzyme 15min that gone out in 95 DEG C of thermostat water baths, waits to digest
Liquid temperature degree is down to room temperature, adjusts its pH value for konjaku albumen isoelectric point；9500r/min is centrifuged 15min, collects supernatant, takes one
Quantitative supernatant surveys its ACE inhibiting rate>50%, by -110 DEG C of freeze-drying 10h of supernatant process into freeze-dried powder, obtain konjaku egg
White source ACE inhibitor peptides crude product, by after the separation of anion exchange resin 717, obtaining ace inhibitory peptide, measures konjaku protein sources ACE
The inhibitory activity of peptide for inhibiting is 31.2%.
Embodiment 3
Konjaku protein freeze-dried powder is dissolved in pure water, mass concentration is configured to for 2.0% protein solution；By protein liquid
It is placed in constant temperature blender with magnetic force and is heated to 55 DEG C, regulation pH value is 6.0, adds compound protease to be well mixed, in constant temperature magnetic
Power agitator carries out enzyme digestion reaction, and enzymolysis time is 3h, and hydrolysis temperature is 45 DEG C, obtains enzymolysis liquid；Wherein, every gram of protein solution
The amount of middle addition compound protease is 5000U.Enzymolysis liquid is placed in gone out in 97 DEG C of thermostat water baths enzyme 10min, liquid temperature to be digested
Degree is down to room temperature, adjusts its pH value for konjaku albumen isoelectric point；10000r/min is centrifuged 10min, collects supernatant, takes a certain amount of
Supernatant surveys its ACE inhibiting rate>60%, by -70 DEG C of freeze-drying 15h of supernatant process into freeze-dried powder, obtain konjaku protein sources
Ace inhibitory peptide crude product, by after the separation of anion exchange resin 717, obtaining ace inhibitory peptide, measures konjaku protein sources ACE suppression
The inhibitory activity of peptide is 37.0%.
Embodiment 4
Konjaku protein freeze-dried powder is dissolved in pure water, mass concentration is configured to for 2.5% protein solution；By protein liquid
It is placed in constant temperature blender with magnetic force and is heated to 40 DEG C, regulation pH value is 7.0, is subsequently adding compound protease and is well mixed, in perseverance
Warm magnetic stirring apparatus carries out enzyme digestion reaction, and enzymolysis time is 5h, and hydrolysis temperature is 50 DEG C, obtains enzymolysis liquid；Wherein, every gram of albumen
The amount that compound protease is added in solution is 6000U.Enzymolysis liquid is placed in the enzyme 15min that gone out in 90 DEG C of thermostat water baths, waits to digest
Liquid temperature degree is down to room temperature, adjusts its pH value for konjaku albumen isoelectric point；5000r/min is centrifuged 30min, collects supernatant, takes one
Quantitative supernatant surveys its ACE inhibiting rate>50%, by -80 DEG C of freeze-drying 20h of supernatant process into freeze-dried powder, obtain konjaku egg
White source ACE inhibitor peptides crude product, by after the separation of anion exchange resin 717, obtaining ace inhibitory peptide, measures konjaku protein sources ACE
The inhibitory activity of peptide for inhibiting is 33.0%.
Embodiment 5
Konjaku protein freeze-dried powder is dissolved in pure water, mass concentration is configured to for 3.0% protein solution；By protein liquid
It is placed in constant temperature blender with magnetic force and is heated to 50 DEG C, regulation pH value is 8.0, is subsequently adding the mixing of 6000U/g compound proteases equal
It is even, enzyme digestion reaction is carried out in constant temperature blender with magnetic force, enzymolysis time is 6h, and hydrolysis temperature is 55 DEG C, obtains enzymolysis liquid；Wherein,
The amount that compound protease is added in every gram of protein solution is 3000U.Enzymolysis liquid is placed in the enzyme that gone out in 95 DEG C of thermostat water baths
10min, treats that enzymolysis liquid temperature is down to room temperature, adjusts its pH value for konjaku albumen isoelectric point；7000r/min is centrifuged 20min, collects
Supernatant, takes a certain amount of supernatant and surveys its ACE inhibiting rate>40%, by -90 DEG C of freeze-drying 20h of supernatant process into freeze-dried powder,
Konjaku albumen source ACE inhibitor peptides crude product is obtained, by after the separation of anion exchange resin 717, obtaining ace inhibitory peptide, evil spirit is measured
The inhibitory activity of taro albumen source ACE inhibitor peptides is 23.3%.
Embodiment 6
Konjaku protein freeze-dried powder is dissolved in pure water, mass concentration is configured to for 3.0% protein solution；By protein liquid
It is placed in constant temperature blender with magnetic force and is heated to 50 DEG C, regulation pH value is 9.0, is subsequently adding the mixing of 6000U/g compound proteases equal
It is even, enzyme digestion reaction is carried out in constant temperature blender with magnetic force, enzymolysis time is 6h, and hydrolysis temperature is 55 DEG C, obtains enzymolysis liquid；Wherein,
The amount that compound protease is added in every gram of protein solution is 3000U.Enzymolysis liquid is placed in the enzyme that gone out in 100 DEG C of thermostat water baths
12min, treats that enzymolysis liquid temperature is down to room temperature, adjusts its pH value for konjaku albumen isoelectric point；10000r/min is centrifuged 10min, receives
Collection supernatant, takes a certain amount of supernatant and surveys its ACE inhibiting rate>40%, by -90 DEG C of freeze-drying 20h of supernatant process into lyophilized
Powder, obtains konjaku albumen source ACE inhibitor peptides crude product, by after the separation of anion exchange resin 717, obtaining ace inhibitory peptide.
Claims (7)
1. a kind of preparation method of konjaku albumen source ACE inhibitor peptides, it is characterised in that be dissolved in konjaku protein freeze-dried powder pure
In water, the protein solution of mass fraction 1-3% is made into；Protein solution is heated to 35-55 DEG C, regulation pH value is 4.0-9.0, plus
Enter compound protease it is well mixed after, enzymolysis obtains enzymolysis liquid；Enzymolysis liquid is gone out after enzyme, regulation pH value to the electricity such as konjaku albumen
Point；Centrifugation, collects supernatant, by supernatant by freeze-drying into freeze-dried powder, obtains konjaku albumen source ACE inhibitor peptides crude product,
By after separation, obtaining ace inhibitory peptide.
2. a kind of preparation method of konjaku albumen source ACE inhibitor peptides according to claim 1, it is characterised in that every gram of egg
The amount that compound protease is added in white solution is 3000-6000U.
3. the preparation method of a kind of konjaku albumen source ACE inhibitor peptides according to claim 1, it is characterised in that go out enzyme
Temperature is 90-100 DEG C, and the time is 10-15min.
4. the preparation method of a kind of konjaku albumen source ACE inhibitor peptides according to claim 1, it is characterised in that centrifugation is
10-30min is carried out under the rotating speed of 5000-10000r/min.
5. the preparation method of a kind of konjaku albumen source ACE inhibitor peptides according to claim 1, it is characterised in that separation is adopted
Carried out with anion exchange resin 717.
6. the preparation method of a kind of konjaku albumen source ACE inhibitor peptides according to claim 1, it is characterised in that freezing is dry
Dry temperature is -50~-110 DEG C, and the time of freeze-drying is 10-24h.
7. the preparation method of a kind of konjaku albumen source ACE inhibitor peptides according to claim 1, it is characterised in that enzymolysis
Temperature is 35-55 DEG C, and the time is 3-6h.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107997020A (en) * | 2018-01-30 | 2018-05-08 | 重庆凯年食品有限公司 | A kind of sausage seasoning formula |
| CN108949873A (en) * | 2018-04-19 | 2018-12-07 | 金华市景和科技有限公司 | The method of polypeptide active substance is extracted from konjaku |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105524966A (en) * | 2016-02-26 | 2016-04-27 | 天津现代职业技术学院 | Method for preparing ACE inhibitory peptides through bean pulp enzymolysis |
| CN105838765A (en) * | 2016-05-11 | 2016-08-10 | 陕西科技大学 | Method for preparing ACE inhibitory peptide through enzymolysis of bromelain on konjak protein |
-
2016
- 2016-12-29 CN CN201611249647.3A patent/CN106755243A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105524966A (en) * | 2016-02-26 | 2016-04-27 | 天津现代职业技术学院 | Method for preparing ACE inhibitory peptides through bean pulp enzymolysis |
| CN105838765A (en) * | 2016-05-11 | 2016-08-10 | 陕西科技大学 | Method for preparing ACE inhibitory peptide through enzymolysis of bromelain on konjak protein |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107997020A (en) * | 2018-01-30 | 2018-05-08 | 重庆凯年食品有限公司 | A kind of sausage seasoning formula |
| CN108949873A (en) * | 2018-04-19 | 2018-12-07 | 金华市景和科技有限公司 | The method of polypeptide active substance is extracted from konjaku |
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Application publication date: 20170531 |