CN110117633B - Preparation method of sunflower antioxidant active peptide - Google Patents
Preparation method of sunflower antioxidant active peptide Download PDFInfo
- Publication number
- CN110117633B CN110117633B CN201910323324.1A CN201910323324A CN110117633B CN 110117633 B CN110117633 B CN 110117633B CN 201910323324 A CN201910323324 A CN 201910323324A CN 110117633 B CN110117633 B CN 110117633B
- Authority
- CN
- China
- Prior art keywords
- sunflower
- protein
- enzymolysis
- volume fraction
- antioxidant
- 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.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
-
- 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
Abstract
The invention provides a preparation method of sunflower antioxidant active peptide, and relates to the technical field of preparation of sunflower peptide. The preparation method of the invention uses degreased sunflower meal as raw material to prepare sunflower protein isolate by a salt-dissolving acid-precipitation method. The sunflower protein isolate is hydrolyzed by protease to generate polypeptide mixture with antioxidant activity, and then is separated and purified by ultrafiltration, ion exchange chromatography, gel chromatography and reversed phase chromatography to obtain sunflower antioxidant active peptide product. The preparation method can be used for obtaining four new sunflower antioxidant oligopeptides with high purity and high activity.
Description
Technical Field
The invention belongs to the technical field of preparation of sunflower peptides, and particularly relates to a preparation method of sunflower antioxidant active peptide.
Background
Sunflower meal is a high-quality plant protein source, the essential amino acid content of the sunflower meal is rich, the amino acid composition is reasonable, the sunflower meal is protein with high nutritional value, and the sunflower protein has high digestibility and biological value. In addition, the sunflower protein has the functional characteristics of good oil absorption, foaming property, foaming stability, emulsifying activity, emulsifying stability and the like, and is an ideal additive for the food industry. However, sunflower meal is mainly used as feed so far, and is not widely applied like soybean protein, and few sunflower protein products are developed.
At present, the preparation method and application of sunflower peptide mainly comprise: chinese patent CN103250864A "a method for preparing sunflower seed polypeptide" uses low-temperature degreased sunflower seed meal as raw material, and is hydrolyzed by mixed enzyme solution of alkaline protease and papain, and the sunflower seed polypeptide powder is prepared by solid-liquid separation, concentration and spray drying; chinese patent CN103933007A entitled sunflower seed meal polypeptide effervescent tablet and its preparation method, mainly uses sunflower polypeptide with antioxidation and antiaging effects as functional raw material, and adds adjuvants such as citric acid, sodium bicarbonate, polyvinylpyrrolidone, microcrystalline cellulose, beta-cyclodextrin, etc. to make into polypeptide effervescent tablet; and Chinese patent CN106889303A "a production process of sunflower seed peptide powder" uses degreased sunflower seed dregs as raw material, and after protease hydrolysis, the sunflower seed peptide powder is obtained by concentration and drying. In various preparation methods of the sunflower peptide, sunflower meal is used as a raw material, and the prepared sunflower peptide has low purity and is difficult to purify.
Disclosure of Invention
In view of the above, the present invention aims to provide a preparation method of a sunflower antioxidant active peptide, wherein the prepared sunflower oligopeptide has characteristics of high purity and high antioxidant activity.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of sunflower antioxidant active peptide, which comprises the following steps: (1) washing the degreased sunflower meal twice with ethanol solution with volume concentration of 95%, and air-drying for 8-12 h to obtain sunflower protein concentrate;
(2) mixing and dissolving the sunflower protein concentrate and a salt solution for 2 hours, centrifuging, adjusting the pH value of a supernatant to be acidic, and centrifuging again to obtain a protein precipitate; the salt solution comprises 8-15% of NaCl and 0.2-0.35% of Na in volume fraction2SO3；
(3) Dissolving the protein precipitate in water, adjusting pH to 7, dialyzing with dialysis bag, and freeze drying to obtain sunflower protein isolate;
(4) carrying out enzymolysis on the sunflower protein isolate by using protease to obtain an enzymolysis product; the protease comprises one or two of Flavourzyme enzyme, Alcalase enzyme and Protamex enzyme;
(5) centrifuging the enzymolysis product, sequentially passing the supernatant through ultrafiltration membranes of 20-50 kDa, 6-10 kDa and less than 6kDa, and collecting ultrafiltration products of less than 6kDa to obtain an ultrafiltrate;
(6) and centrifuging the ultrafiltrate, and sequentially performing ion exchange chromatography, Sephadex G-25 gel filtration chromatography and ZORBAX SB-C18 reversed-phase chromatography to obtain the sunflower antioxidant active peptide.
Preferably, in the step (1), the mass-to-volume ratio of the degreased sunflower meal to the 95% ethanol solution is 1: (8-12).
Preferably, the temperature of the alcohol washing is 48-55 ℃, and the time of each alcohol washing is 1.5-3 h.
Preferably, the rotation speed of the centrifugation and the re-centrifugation in the step (2) is 3500-4500 rpm independently, and the time is 8-12 min.
Preferably, the sunflower protein isolate of step (3) has a crude protein content of greater than 85%.
Preferably, in the enzymolysis system during enzymolysis in the step (4), the mass concentration of the substrate is 5-7%, the mass concentration of the protease is 2-5%, the enzymolysis temperature is 50-60 ℃, the pH value is 7.0-8.5, and the enzymolysis time is 50-70 min.
Preferably, the ion exchange chromatography of step (6) is DEAE-Sepharose Fast Flow weak anion exchange chromatography, wherein φ is 1.6 × 20 cm.
Preferably, the ZORBAX SB-C18 reversed-phase chromatographic separation in step (6) is carried out twice, wherein the eluent A in the first time is a mixture of TFA and 2% acetonitrile with volume fractions of 0.065% and the eluent B is a mixture of TFA and 80% acetonitrile with volume fractions of 0.05% and the acetonitrile, respectively; in the second time, eluent A is a mixture of TFA and 10% acetonitrile with volume fractions of 0.065% and eluent B is a mixture of TFA and 60% acetonitrile with volume fractions of 0.05%.
The invention also provides the sunflower antioxidant active oligopeptide prepared by the preparation method.
Preferably, the antioxidant active oligopeptide comprises an amino acid sequence shown as SEQ ID NO. 1-4.
The invention provides a preparation method of sunflower antioxidant active peptide, which takes degreased sunflower meal as a raw material to prepare sunflower protein isolate by a salt-dissolving acid-precipitation method. The sunflower protein isolate is hydrolyzed by protease to generate polypeptide mixture with antioxidant activity, and then is separated and purified by ultrafiltration, ion exchange chromatography, gel chromatography and reversed phase chromatography to obtain sunflower antioxidant active peptide product. In the invention, the protein content of the sunflower protein isolate is more than 85%, and the sunflower protein isolate is used as a hydrolysis substrate, so that the difficulty of subsequent purification is reduced. After enzymolysis is finished, firstly, an ultrafiltration separation technology is adopted, an enzymolysis solution is filtered by ultrafiltration membranes with different pore diameters, an ultrafiltration fractionation product with the pore diameter smaller than 6,000Da is subjected to ion exchange chromatography, most protein in the solution has negative charges and can be well combined to an ion exchange medium, so that DEAE-Sepharose fast flow weak anion exchange chromatography is adopted, the binding force between the adsorbed protein and an ion exchanger group is weak, the protein is eluted under the condition of lower salt concentration, and a better desalting effect can be ensured; the gel chromatography desalination has the advantages of simple and rapid operation, difficult denaturation of macromolecular substances such as protein and the like in the desalination process, and the like, and the Sephadex G-25 carries out molecular weight classification on the protein with high antioxidant activity separated by DEAE-Sepharose Fast Flow weak anion exchange chromatography and completes desalination at the same time. Since the various amino acids constituting the peptide have different side chains, further separation and purification can be carried out by RP-HPLC using the difference in polarity, i.e., hydrophobicity, of the amino acids constituting the peptide component. Carrying out ZORBAX SB-C18(3mL) first reverse phase chromatographic separation on the collected active components after the Sephadex G-25 desalination, collecting the active components, carrying out ZORBAX SB-C18(3mL) second reverse phase chromatographic separation, and carrying out twice reverse phase chromatographic separation to obtain the sunflower antioxidant oligopeptide with high purity and high activity. Four new sunflower antioxidant oligopeptides can be obtained by the preparation method.
Drawings
FIG. 1 is a schematic diagram of the purification process of the sunflower peptide according to the invention;
FIG. 2 shows DEAE-Sepharose F F ion exchange chromatography elution profile of sunflower protein isolate;
FIG. 3 is a Sephadex G-25 gel chromatography elution profile;
FIG. 4 is a first ZORBAX SB-C18 elution profile;
FIG. 5 is a second ZORBAX SB-C18 elution profile;
FIG. 6 is a graph showing the results of mass spectrometry of the sequence of SEQ ID NO.1 of the present invention;
FIG. 7 is a graph showing the results of mass spectrometry of the sequence of SEQ ID NO.2 according to the present invention;
FIG. 8 is a graph showing the results of mass spectrometry of the sequence set forth in SEQ ID NO.3 of the present invention;
FIG. 9 is a diagram showing the results of mass spectrometry of the sequence of SEQ ID NO.4 of the present invention.
Detailed Description
The invention provides a preparation method of sunflower antioxidant active peptide, which comprises the following steps: (1) washing the degreased sunflower meal twice with ethanol solution with volume concentration of 95%, and air-drying for 8-12 h to obtain sunflower protein concentrate;
(2) mixing and dissolving the sunflower protein concentrate and a salt solution for 2 hours, centrifuging, adjusting the pH value of a supernatant to be acidic, and centrifuging again to obtain a protein precipitate; the salt solution comprises 8-15% of NaCl and 0.2-0.35% of Na in volume fraction2SO3；
(3) Dissolving the protein precipitate in water, adjusting pH to 7, dialyzing with dialysis bag, and freeze drying to obtain sunflower protein isolate;
(4) carrying out enzymolysis on the sunflower protein isolate by using protease to obtain an enzymolysis product; the protease comprises one or two of Flavourzyme enzyme, Alcalase enzyme and Protamex enzyme;
(5) centrifuging the enzymolysis product, sequentially passing the supernatant through ultrafiltration membranes of 20-50 kDa, 6-10 kDa and less than 6kDa, and collecting ultrafiltration products of less than 6kDa to obtain an ultrafiltrate;
(6) and centrifuging the ultrafiltrate, and sequentially performing ion exchange chromatography, Sephadex G-25 gel filtration chromatography and ZORBAX SB-C18 reversed-phase chromatography to obtain the sunflower antioxidant active peptide.
In the preparation method, alcohol washing is carried out on degreased sunflower meal twice by using ethanol solution with volume concentration of 95%, and air drying is carried out for 8-12 h, so as to obtain sunflower protein concentrate. In the invention, the mass volume ratio of the degreased sunflower meal to the 95% ethanol solution is preferably 1: (8-12), more preferably 1: (9-11), most preferably 1: 10. The alcohol washing temperature of the invention is preferably 48-55 ℃, more preferably 49-52 ℃, and most preferably 50 ℃. The time of each alcohol washing is preferably 1.5-3 h, more preferably 1.8-2.5 h, and most preferably 2 h. The degreased sunflower meal is subjected to alcohol washing and then is dried for 8-12 hours, and the preferable drying time is 10 hours. The air drying temperature is preferably 35-45 ℃, more preferably 38-42 ℃, and most preferably 40 ℃.
After sunflower protein concentrate is obtained, the sunflower protein concentrate and salt solution are mixed, dissolved for 2 hours, centrifuged, the pH value of supernatant is adjusted to be acidic, and centrifuged again to obtain protein precipitatePrecipitating; the salt solution comprises 8-15% of NaCl and 0.2-0.35% of Na in volume fraction2SO3. The mass volume ratio of the sunflower protein concentrate to the salt solution is preferably 1: (8-12), more preferably 1: (9-11), most preferably 1: 10. The salt solution is used for carrying out salt dissolution on the sunflower protein concentrate, and the temperature during salt dissolution is preferably 48-55 ℃, more preferably 49-52 ℃, and most preferably 50 ℃. The time for salt dissolution is preferably 1.5-3 h, more preferably 1.8-2.5 h, and most preferably 2 h. The pH value of the liquid in the salt dissolution process is preferably neutral, and more preferably 7. The salt solution is NaCl and Na2SO3The mass concentration of NaCl in the salt solution is preferably 9-12%, and more preferably 10%. Na in the salt solution of the invention2SO3The mass concentration of (b) is preferably 0.22 to 0.3%, more preferably 0.25%. The salt solution is centrifuged after the salt solution is finished, and the rotating speed of the centrifugation is preferably 3500-4500 rpm, more preferably 3800-4200 rpm, and most preferably 4000 rpm. The time for centrifugation is preferably 8-12 min, more preferably 9-11 min, and most preferably 10 min. After the centrifugation, the supernatant is collected, the pH value of the supernatant is adjusted to be acidic, and then the centrifugation is carried out again to obtain protein precipitate. The pH of the supernatant according to the invention is preferably adjusted to 4.5, and the protein is precipitated after it is acidic. The rotation speed of the re-centrifugation is preferably 3500-4500 rpm, more preferably 3800-4200 rpm, and most preferably 4000 rpm. The time for re-centrifugation is preferably 8-12 min, more preferably 9-11 min, and most preferably 10 min.
After obtaining the protein precipitate, the invention dissolves the protein precipitate in water, adjusts the pH value to 7, dialyzes by a dialysis bag, and obtains the sunflower protein isolate after freeze drying. The volume ratio of the protein precipitate to water is not particularly limited in the present invention. The method of dialysis and freeze-drying is not particularly limited in the present invention, and may be a method generally used in the art. The content of crude protein in the sunflower protein isolate is preferably more than 85 percent, which is more favorable for the later purification process.
After sunflower protein isolate is obtained, the sunflower protein isolate is subjected to enzymolysis by using protease to obtain an enzymolysis product; the protease comprises one or two of Flavourzyme enzyme, Alcalase enzyme and Protamex enzyme. Flavourzyme, Alcalase and Protamex enzymes of the present invention are all available from Novoverdin Biotech, Denmark. In the enzymolysis system during enzymolysis, the concentration of a substrate is preferably 5-7%, the addition amount of protease is preferably 2-5%, the enzymolysis temperature is preferably 50-60 ℃, the pH value is preferably 7.0-8.5, and the enzymolysis time is preferably 50-70 min. When the protease is Flavourzyme, the suitable conditions for enzymolysis are more preferably hydrolysis temperature of 53 ℃, pH of 7.0, substrate concentration of 5.5%, ratio of enzyme to substrate of 5%, and hydrolysis time of 60 min. At the moment, the antioxidant activity is 258.20U/mL, the content of soluble protein in hydrolysate is 14.56mg/mL, and the peptide segment of the hydrolysate with the relative molecular mass between 5117-370 accounts for 86.70%. When the protease is Alcalase, the suitable conditions for enzymolysis are as follows: the hydrolysis temperature is 60 ℃, the substrate concentration is 6%, the ratio of enzyme to substrate is 3%, the pH value of the enzymolysis liquid is 8.5, and the hydrolysis time is 60 min. Under the condition, the antioxidant activity of the hydrolysate is 126.80U/mL, the content of soluble protein in the hydrolysate is 43.78mg/mL, and the relative molecular mass of the polypeptide in the hydrolysate is mainly concentrated between 590-2,975 Da.
When the protease is Protamex enzyme, the suitable conditions for enzymolysis are that the concentration of a substrate is 6%, the ratio of the enzyme to the substrate is 2%, the pH value is 7.0, the temperature is 50 ℃, and the hydrolysis time is 60 min. At the moment, the antioxidant activity of the sunflower isolate protein hydrolysate is 174.36U/mL, the soluble protein content of the hydrolysate is 25.22mg/mL, and the relative molecular mass of the hydrolysate is mainly concentrated between 370 and 4,226 Da.
In the invention, the hydrolysate obtained by enzymolysis of the sunflower protein isolate by using the three proteases has different antioxidant activities, and the antioxidant activity of the Flavourzyme hydrolysate is the highest. The reason for this is probably that the peptide segment of the flavourzyme hydrolysate contains more hydrophobic amino acids, the hydrophobic amino acids can enhance the interaction of the antioxidant active peptide and fatty acid, improve the capture capability of lipid free radicals, and in addition, the peptide segment contains some special groups-hydrogen-supplying groups which can react with the free radicals, and can fully react with the free radicals to show stronger antioxidant activity. Thus, in the present examples, flavourzyme was used for enzymatic hydrolysis.
After obtaining an enzymolysis product, centrifuging the enzymolysis product, sequentially passing the supernatant through ultrafiltration membranes of 20-50 kDa, 6-10 kDa and less than 6kDa, and collecting the ultrafiltration product of less than 6kDa to obtain an ultrafiltrate. The centrifugal force of the centrifugal machine is preferably 8000-12000 g, more preferably 8500-10500 g, and most preferably 10000 g. The time for centrifugation is preferably 8-12 min, more preferably 9-11 min, and most preferably 10 min. In the invention, after the centrifugation, the supernatant is collected and subjected to ultrafiltration. The ultrafiltration of the supernatant of the invention is preferably carried out by ultrafiltration with an ultrafiltration membrane with a relative molecular mass cut off of 20,000-50,000Da, ultrafiltration with an ultrafiltration membrane with a relative molecular mass cut off of 6,000-10,000Da, and ultrafiltration with an ultrafiltration membrane with a relative molecular mass cut off of less than 6,000 Da. In the invention, after the enzymolysis liquid is separated by three ultrafiltration membranes, according to the size of molecules, four components with relative molecular mass sections can be obtained: more than 20,000Da, 20,000-10,000Da, 10,000-6,000Da and less than 6,000Da, the yield is 14.12%, 1.33%, 56.31% and 28.24%, respectively. The invention selects the short peptide with the relative molecular mass less than 6,000Da and the antioxidation activity for further separation, and the external liquid is stored at the temperature of minus 20 ℃ for standby after being frozen and pumped, and the short peptide is beneficial to the absorption and utilization of human bodies. The method adopts ultrafiltration separation technology, uses ultrafiltration membranes with different pore diameters to filter enzymolysis liquid, enables polypeptides with different relative molecular masses to be primarily separated, firstly uses an ultrafiltration membrane with 6,000-10,000Da cut-off relative molecular mass to carry out ultrafiltration, and finally uses an ultrafiltration membrane with less than 6,000Da cut-off relative molecular mass to carry out ultrafiltration on filtrate passing through the 6,000-10,000Da ultrafiltration membrane, and measures the protein content, the antioxidant activity and the like of ultrafiltration grading solutions at all levels. The ultrafiltration fractionation product of less than 6,000Da is subjected to ion exchange chromatography. As the optimum conditions for the hydrolysis of the flavourzyme are about pH7.0, namely the pH of the sunflower protein hydrolysate is about 7.0, the pH value of the optimum solution for the ion exchange chromatography is generally different from the pH value of the components by one to two units, so that the components to be separated can be combined on the ion exchange medium, the experiment selects 0.025mol/L of Tris-HCl with the pH value of 8.9 as a mobile phase for experiment, and most of the protein in the solution is negatively charged at the moment and can be better combined on the ion exchange medium. DEAE-Sepharose Fast Flow is a weak anion exchange filler with ethyl aminoethyl as an active group, and the filler has better mechanical strength and chemical stability and is suitable for high-Flow-rate operation. Therefore, the hydrolysis liquid is primarily separated by DEAE-Sepharose Fast Flow weak anion exchange chromatography, the binding force between the adsorbed protein and the ion exchanger group in the experiment is weak, and the protein is eluted under the condition of lower salt concentration, so that better desalting effect can be ensured in the next experiment.
And centrifuging the ultrafiltrate, and sequentially performing ion exchange chromatography, Sephadex G-25 gel filtration chromatography and ZORBAX SB-C18 reversed-phase chromatography to obtain the sunflower antioxidant active peptide. The rotating speed of the centrifugation is preferably 8000-12000 rpm, more preferably 9000-10500 rpm, and most preferably 10000 rpm. The time for centrifugation is preferably 8-12 min, more preferably 9-11 min, and most preferably 10 min. The invention centrifugates the ultrafiltrate, collects the supernatant fluid and passes through a 0.22 mu m microporous filter membrane for ion exchange chromatography. The ion exchange chromatography according to the invention is preferably DEAE-Sepharose fast Flow weak anion exchange chromatography, where φ is 1.6 x 20 cm. In the weak anion exchange chromatography, setting an initial buffer solution: 0.025mol/LpH 8.9.9 Tris-HCl, eluent: 0.025mol/L pH8.9Tris-HCl with 1mol NaCl, flow rate: 1.5mL/min, detection: UV214 nm. After the sample is loaded, the sample is washed by using an initial buffer solution until the baseline is stable, then gradient elution is carried out according to linear increasing of NaCl concentration, and an automatic part collector is used for collecting 4 min/tube at regular time.
The eluent after the ion exchange chromatography is preferably frozen, drained and concentrated, dissolved by 0.025mol/LpH8.9Tris-HCl, centrifuged at 10000r/min for 10min, and the supernatant is taken for gel filtration desalination chromatography (phi 1.6 x 100cm), wherein the height of the filled gel is about 90cm and is 200 mL. Eluent: 0.025mol/L pH8.9Tris-HCl, flow rate: 2mL/min, detection: UV214nm, 80mL (external water volume) was discharged each time, followed by 3 min/tube timed collection with an automatic partial collector.
The fractions collected after desalting by Sephadex G-25 were subjected to first ZORBAX SB-C18(3mL) reverse phase chromatography. Eluent A is 0.065% TFA and 2% acetonitrile; eluent B is 0.05 percent TFA and 80 percent acetonitrile; and (3) detection: UV214 nm. After loading, the unbound peptide was washed with eluent a until a214 was close to 0, and then eluted with a gradient from a to B in increasing acetonitrile concentrations for 45min at a flow rate: 1.0 mL/min; the automatic fraction collector collects 1 min/tube periodically.
In the invention, a sample subjected to first ZORBAX SB-C18(3mL) is freeze-dried and then subjected to second ZORBAXSB-C18(3mL) reverse phase chromatography. Eluent A: 0.065% TFA, 10% acetonitrile; eluent B: 0.05% TFA, 60% acetonitrile; and (3) detection: UV214 nm. After loading, the unbound peptide was washed with eluent a until a214 was close to 0, and then eluted with a gradient from a to B in increasing acetonitrile concentrations for 45min at a flow rate: 1.0 mL/min; the automatic fraction collector collects 1 min/tube periodically.
The invention also provides the antioxidant active oligopeptide prepared by the preparation method.
In the present invention, with respect to the above preparation method, each step comprises a test for antioxidant activity after obtaining the enzymatic hydrolysate. The detection method of the antioxidant activity is preferably an improved pyrogallol autoxidation method, and specifically comprises the following steps:
adding 4.5ml of Tris-HCl buffer solution with pH of 8.2 and 50mmol/L into a test tube according to the table 1, carrying out water bath heat preservation for 20min at the temperature of 25 ℃, adding 10 mu L of pyrogallol (a control tube is replaced by 10 mu L of 10mmol/L HCl solution), quickly shaking up, immediately pouring into a cuvette, carrying out time scanning at the wavelength of 325nm, measuring absorbance, repeating the measurement for three times, and controlling the average autoxidation rate to be 0.070 +/-0.002A/min (the absorbance value can be controlled by increasing or decreasing the addition of the pyrogallol).
The operation of measuring the antioxidant activity is the same as the operation of measuring the autoxidation rate of the pyrogallol. The addition amount of the sample can be properly increased or decreased according to the antioxidant activity of the sample to be detected, so that the rate of inhibiting the self-oxidation of the pyrogallol by the antioxidant active substance is controlled to be about 0.035A/min.
TABLE 1 sample anti-oxidation activity determination sample adding table
Definition of antioxidant activity units: the amount of substance inhibiting the pyrogallol autoxidation rate by 50% per minute in 1mL of the reaction solution was defined as one unit of activity, i.e., 0.035A/min as measured at a wavelength of 325 nm. If the rate of inhibiting the pyrogallol autoxidation is within the range of 35-65% per minute, the rate can be calculated according to the proportion generally, and if the value is not within the range, the adding amount of the sample is increased or decreased.
The amino acid sequence of the antioxidant active oligopeptide is shown in the table 2:
TABLE 2 sequence listing of antioxidative active oligopeptides
The following examples are provided to illustrate the preparation method and application of the sunflower antioxidant peptide of the present invention in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Preparation of sunflower protein isolate
Prepared by salt-dissolving and acid-precipitating sunflower degreased meal: washing with 95% ethanol twice (50 deg.C, 2 hr, pH 4.5), dissolving in ethanol at a ratio of 1:10(w/v), air drying at 40 deg.C overnight to obtain sunflower protein concentrate. With 10% NaCl, 0.25% Na2SO3Dissolving with salt (50 deg.C, 2h, pH 7.0), dissolving in water at a ratio of 1:10(w/v), centrifuging (4000r/min, 10min), adjusting the pH of the supernatant to 4.5, centrifuging (4000r/min, 10min) again to obtain precipitate, dissolving the precipitate in water, adjusting the pH to 7, dialyzing with dialysis bag, and freeze drying to obtain sunflower protein isolate. Defatted sunflower meal and sunflower separated eggThe white content is shown in table 3:
table 3 sunflower meal chemical composition (%)
2. Preparation of sunflower antioxidant active peptide
(1) Preparation of sunflower protein isolate enzymolysis liquid
Flavourzyme enzyme was purchased from Novoverdin Biotech, Denmark.
The Flavourzyme is used for enzymolysis of sunflower protein isolate under the appropriate conditions of hydrolysis temperature of 53 ℃, pH value of 7.0, substrate concentration of 5.5%, ratio of enzyme to substrate of 5% and hydrolysis time of 60 min. The enzymolysis liquid is centrifuged at 10,000 Xg, the antioxidant activity is 258.20U/mL, the content of soluble protein in the hydrolysate is 14.56mg/mL, and the peptide segment of the hydrolysate with the relative molecular mass of 5,117-370 accounts for 86.70%.
3 separation and purification of sunflower antioxidant active peptide
The sunflower peptide was purified according to the scheme shown in FIG. 1:
and (3) ultrafiltration: after the sunflower separation proteolytic liquid is centrifuged for 10min by 10,000 Xg, the supernatant is firstly ultrafiltered by an ultrafiltration membrane with the relative molecular mass cut off by 20,000-50,000Da, the filtrate is ultrafiltered by an ultrafiltration membrane with the relative molecular mass cut off by 6,000-10,000Da, the filtrate passing through the ultrafiltration membrane with the relative molecular mass cut off by 6,000-10,000Da is finally ultrafiltered by an ultrafiltration membrane with the relative molecular mass cut off by less than 6,000Da, the protein content, the antioxidant activity and the like of each stage of ultrafiltration fractionation solution are determined, and the ultrafiltration fractionation product with the relative molecular mass cut off by less than 6,000Da is freeze-dried for later use. The results of ultrafiltration are shown in Table 4:
TABLE 4 analysis of the results of ultrafiltration of the samples
After the enzymolysis liquid is separated by three ultrafiltration membranes, according to the size of molecules, four components with relative molecular mass sections can be obtained: more than 20,000Da, 20,000-10,000Da, 10,000-6,000Da and less than 6,000Da, the yield is 14.12%, 1.33%, 56.31% and 28.24%, respectively. The peptide with small relative molecular mass is beneficial to the absorption and utilization of human body, so that the short peptide with the relative molecular mass less than 6,000Da and having antioxidant activity is selected for further separation, and the external liquid is frozen, dried and stored at-20 ℃ for later use.
Ion exchange chromatography: the ultrafiltrate was centrifuged at 10000r/min for 10min, the supernatant was filtered through a 0.22 μm microfiltration membrane, and the filtrate was subjected to weak anion exchange chromatography (DEAE-Sepharose Fast Flow weak anion (. phi.1.6 x 20 cm)). Starting buffer solution: 0.025mol/LpH 8.9.9 Tris-HCl, eluent: 0.025mol/LpH 8.9.9 Tris-HCl with 1mol of NaCl, flow rate: 1.5mL/min, detection: UV214 nm. After the sample is loaded, the sample is firstly washed by using an initial buffer solution until the baseline is stable, then gradient elution is carried out according to linear increasing of NaCl concentration, 4 min/tube is periodically collected by using an automatic part collector, the antioxidant activity of each tube is measured, and the ion exchange result is shown in figure 2: the ion exchange condition under the condition is suitable, the repeatability is good, and a part of samples are eluted before gradient elution, which shows that a part of samples to be separated have no charge near pH8.9, cannot be subjected to ion exchange with ion exchanger groups, but are directly eluted by buffer solution, and the antioxidant activity of the part of proteins is not high. Most of the samples were eluted after gradient elution and there was a region of higher antioxidant activity. Most of the protein is eluted with the increasing of the salt concentration, the higher the salt concentration, that is to say, the higher the ionic strength, the more the ion exchange points are occupied, the components in the sample enter a limited adsorption equilibrium state one by one, and the more the protein is replaced. The binding force between the adsorbed protein and the ion exchanger group is weaker, and the protein is eluted under the condition of lower salt concentration, so that the better desalting effect can be ensured in the next step. Collecting 204-.
Sephadex G-25 gel filtration chromatography:
after the high-activity sample subjected to DEAE-Sepharose Fast Flow is freeze-dried, concentrated, dissolved in 0.025mol/LpH 8.9.9 Tris-HCl, centrifuged at 10000r/min for 10min, the supernatant is taken and subjected to gel filtration desalting chromatography (phi 1.6 x 100cm), and the height of the packed gel is about 90cm and 200 mL. Eluent: 0.025mol/L pH8.9Tris-HCl, flow rate: 2mL/min, detection: UV214nm, which was discharged 80mL (external water volume) each time, then collected periodically for 3 min/tube by an automatic partial collector, and the antioxidant activity of the collected liquid in each tube was measured, and the results are shown in FIG. 3: in general, the material to be separated is desalted using double distilled water. From the column type and bed height, the gel volume in the column was calculated to be 194.5mL, and the column had an external water volume of 85.5mL as determined by macromolecular blue dextran 2000, thus starting at 85mL after loading. Since the Sephadex G-25 separation range is 1,000-5,000Da, the protein polypeptide with larger relative molecular mass, namely the protein peptide with larger relative molecular mass than 5,000Da which is not entered into the gel and is excluded from the gel, is discharged from the chromatographic column immediately after the external water is discharged, and the 277-301mL is collected for the next experiment as required by the combination experiment.
First ZORBAX SB-C18(3mL) reverse phase chromatography: the active fraction collected after desalting by Sephadex G-25 was subjected to ZORBAX SB-C18(3mL) reverse phase chromatography. Eluent A is 0.065% TFA and 2% acetonitrile; eluent B is 0.05 percent TFA and 80 percent acetonitrile; and (3) detection: UV214 nm. After loading, the unbound peptide was washed with eluent a until a214 was close to 0, and then eluted with a gradient from a to B in increasing acetonitrile concentrations for 45min at a flow rate: 1.0 mL/min; collecting the liquid in the automatic part collector for 1 min/tube at regular time, measuring the antioxidant activity of the collected liquid in each tube, and the experimental result is shown in figure 4: the antioxidant activity of each tube is detected, a peak with the maximum antioxidant activity appears when the retention time is 33.320min, and the antioxidant activity is 73.04U/mL.
Second ZORBAX SB-C18(3mL) reverse phase chromatography: the ZORBAX SB-C18(3mL) sample was freeze dried and subjected to two ZORBAX SB-C18(3mL) reverse phase chromatographic separations. Eluent A: 0.065% TFA, 10% acetonitrile; eluent B: 0.05% TFA, 60% acetonitrile; and (3) detection: UV214 nm. After loading, the unbound peptide was washed with eluent a until a214 was close to 0, and then eluted with a gradient from a to B in increasing acetonitrile concentrations for 45min at a flow rate: 1.0 mL/min; the automatic partial collector collects the liquid for 1 min/tube at regular time, the antioxidant activity of the collected liquid of each tube is measured, and the experimental result is shown in figure 5: the peak with the maximum antioxidant activity appears at the retention time of 35.095min, the antioxidant activity is 79.42U/mL, and the subsequent 4 peaks are consistent with the previous reagent peak, so that mass spectrum sequencing is carried out after freeze-drying of the peak with the number of 35.095 min.
Example 2
Structure determination of sunflower antioxidant active peptide
(1) Sequencing of peptides
After spotting 0.5. mu.L of the sample on a MALDI target plate and drying it naturally, 0.5. mu.L of a 0.5g/L CHCA solution (solvent, 0.1% TFA + 50% ACN) was spotted and dried naturally at room temperature. Another spot of 0.5. mu.L of 0.5g/L CHCA solution (unpicked sample) was used as a blank, and the sample was subjected to mass spectrometry using 4700 tandem time-of-flight mass spectrometer (Applied Biosystems, USA), and the results are shown in FIGS. 6 to 9.
(2) Four new sunflower antioxidant active peptide structures
The sequences of four sunflower protein antioxidant active peptides obtained by separation are shown in the following table 5:
TABLE 5 sunflower protein antioxidant active peptide sequence table
(3) Sequence alignment of peptides
The sequences of the peptides obtained were aligned over the Internet (Web page: PIR. georgetown. edu. cn. andbodiopd. bjmu. edu. cn) using protein database (protein database) PIR-NREF and bioactive peptide database (bioactive peptide database). The four purified antioxidant active peptides are found to be novel active peptides by sequence alignment with known active peptides.
The invention provides a preparation method of sunflower antioxidant active peptide, and 4 new high antioxidant active oligopeptides can be prepared by the preparation method.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> university of ziqi hall
<120> preparation method of sunflower antioxidant active peptide
<160>4
<170>SIPOSequenceListing 1.0
<210>1
<211>7
<212>PRT
<213>Helianthus annuusL.
<400>1
Lys Gly Asp Trp His Ser Arg
1 5
<210>2
<211>8
<212>PRT
<213>Helianthus annuusL.
<400>2
His HisLys His Lys Lys Gly Arg
1 5
<210>3
<211>10
<212>PRT
<213>Helianthus annuusL.
<400>3
Pro Ala Ala Tyr Lys His Lys Lys Gly Arg
1 5 10
<210>4
<211>9
<212>PRT
<213>Helianthus annuusL.
<400>4
Lys His His Lys His Lys Lys Gly Arg
1 5
Claims (1)
1. The preparation method of the sunflower antioxidant active peptide is characterized by comprising the following steps: (1) washing the degreased sunflower meal twice with ethanol solution with volume concentration of 95%, and air-drying for 8-12 h to obtain sunflower protein concentrate; and during each alcohol washing, the mass-volume ratio of the degreased sunflower meal to the 95% ethanol solution is 1: (8-12); the temperature of the alcohol washing is 48-55 ℃, and the time of each alcohol washing is 1.5-3 h;
(2) mixing and dissolving the sunflower protein concentrate and a salt solution for 2 hours, centrifuging, adjusting the pH value of a supernatant to be acidic, and centrifuging again to obtain a protein precipitate; the salt solution comprises 8-15% of NaCl and 0.2-0.35% of Na in volume fraction2SO3(ii) a The rotation speed of the centrifugation and the re-centrifugation is 3500-4500 rpm independently, and the time is 8-12 min;
(3) dissolving the protein precipitate in water, adjusting pH to 7, dialyzing with dialysis bag, and freeze drying to obtain sunflower protein isolate; the content of crude protein in the sunflower protein isolate is more than 85 percent;
(4) carrying out enzymolysis on the sunflower protein isolate by using protease to obtain an enzymolysis product; the protease comprises one or two of Flavourzyme enzyme, Alcalase enzyme and Protamex enzyme; in the enzymolysis system during enzymolysis, the mass concentration of a substrate is 5-7%, the mass concentration of protease is 2-5%, the enzymolysis temperature is 50-60 ℃, the pH value is 7.0-8.5, and the enzymolysis time is 50-70 min;
(5) centrifuging the enzymolysis product, sequentially passing the supernatant through ultrafiltration membranes of 20-50 kDa, 6-10 kDa and less than 6kDa, and collecting ultrafiltration products of less than 6kDa to obtain an ultrafiltrate;
(6) centrifuging the ultrafiltrate, and sequentially performing ion exchange chromatography, Sephadex G-25 gel filtration chromatography and ZORBAX SB-C18 reversed phase chromatography to obtain sunflower antioxidant active peptide; the ion exchange chromatography is DEAE-Sepharose fast Flow weak anion exchange chromatography, wherein phi is 1.6 x 20 cm;
carrying out ZORBAX SB-C18 reversed-phase chromatographic separation twice, wherein the first eluent A is a mixed solution of TFA with the volume fraction of 0.065% and acetonitrile with the volume fraction of 2%, and the first eluent B is a mixed solution of TFA with the volume fraction of 0.05% and acetonitrile with the volume fraction of 80%; in the second time, eluent A is a mixed solution of TFA with the volume fraction of 0.065% and acetonitrile with the volume fraction of 10%, and eluent B is a mixed solution of TFA with the volume fraction of 0.05% and acetonitrile with the volume fraction of 60%;
the antioxidant active oligopeptide comprises an amino acid sequence shown as SEQ ID NO. 1-4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910323324.1A CN110117633B (en) | 2019-04-22 | 2019-04-22 | Preparation method of sunflower antioxidant active peptide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910323324.1A CN110117633B (en) | 2019-04-22 | 2019-04-22 | Preparation method of sunflower antioxidant active peptide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110117633A CN110117633A (en) | 2019-08-13 |
| CN110117633B true CN110117633B (en) | 2020-09-01 |
Family
ID=67521333
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910323324.1A Active CN110117633B (en) | 2019-04-22 | 2019-04-22 | Preparation method of sunflower antioxidant active peptide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110117633B (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105237622B (en) * | 2015-09-09 | 2018-11-30 | 齐齐哈尔大学 | Corn antioxidant active peptide and preparation method thereof |
-
2019
- 2019-04-22 CN CN201910323324.1A patent/CN110117633B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN110117633A (en) | 2019-08-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Shazly et al. | Fractionation and identification of novel antioxidant peptides from buffalo and bovine casein hydrolysates | |
| US9609883B2 (en) | Method for producing wheat glutamine peptide | |
| CN103992385B (en) | Pseudosciaena crocea swim bladder antioxidant collagen peptide and preparation method and application thereof | |
| CN110964087B (en) | Fugu active peptide and preparation method thereof | |
| CN111072756B (en) | Tetrodotoxin ACE inhibitory peptide and preparation method thereof | |
| Morato et al. | Optimization of casein hydrolysis for obtaining high contents of small peptides: use of subtilisin and trypsin | |
| CN1935016A (en) | Industrial production method for high-purity low molecular weight soybean obligopeptide powder | |
| CN113215212B (en) | Soybean protein peptide with antioxidant and ACE (angiotensin converting enzyme) inhibiting functions and preparation method thereof | |
| CN111100186B (en) | Puffer fish polypeptide with ACE inhibitory activity and preparation method thereof | |
| CN111004309B (en) | ACE inhibitory peptide prepared from Takifugu flavidus fish skin and preparation method thereof | |
| CN108610274A (en) | One group of ring gamma-polyglutamic acid Series Molecules and its preparation method and application | |
| Piot et al. | Application of ultrafiltration to the preparation of defined hydrolysates of bovine haemoglobin | |
| CN107119097B (en) | Mulberry leaf immune active peptide and preparation method thereof | |
| Bordenave et al. | Continuous hydrolysis of goat whey in an ultrafiltration reactor: generation of alpha-lactorphin | |
| CN110117633B (en) | Preparation method of sunflower antioxidant active peptide | |
| CN112111546B (en) | Industrial preparation method of phosvitin phosphopeptide and vitellin polypeptide | |
| CN114990181B (en) | Anti-aging soybean peptide and preparation method and application thereof | |
| WO2005078078A1 (en) | Process for producing lactoperoxidase | |
| Carreira et al. | Obtaining oligopeptides from whey: use of subtilisin and pancreatin | |
| CN115109817B (en) | Almond peptide with antioxidant and immune activities and preparation method and application thereof | |
| CN109021072B (en) | Rhodophyta antioxidant peptide and preparation method thereof | |
| CN115785201B (en) | Pea antioxidant peptide and preparation method and application thereof | |
| CN108504709B (en) | Corn oligopeptide and industrial production method thereof | |
| Mohammad et al. | Characteristics of enzymatic hydrolysis of protein from different food sources and potential separation techniques | |
| CN110951813A (en) | Extraction method of tuna protein peptide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |