CN110964087B - Fugu active peptide and preparation method thereof - Google Patents
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- CN110964087B CN110964087B CN201911415555.1A CN201911415555A CN110964087B CN 110964087 B CN110964087 B CN 110964087B CN 201911415555 A CN201911415555 A CN 201911415555A CN 110964087 B CN110964087 B CN 110964087B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6818—Sequencing of polypeptides
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
Abstract
The invention discloses a puffer fish active peptide and a preparation method thereof, belongs to the technical field of ACE inhibitory peptide, and the preparation method of the puffer fish active peptide comprises the following steps: s1: preparing the puffer fish enzymatic hydrolysate, S2: and (3) performing ultrafiltration separation on the puffer fish enzymatic hydrolysate, wherein the step S3: separating and purifying the polypeptide, S4, identifying the sequence of the polypeptide, S5, synthesizing the polypeptide and measuring the activity. A kind of active peptide of Fugu ocellatus is octapeptide, and its amino acid sequence is PPLLFAAL (Pro-Pro-Leu-Leu-Phe-Ala-Ala-Leu). The invention relates to a puffer fish active peptide and a preparation method thereof, which adopts puffer fish skin as a raw material to obtain an octapeptide amino acid sequence, and the prepared polypeptide has ACE inhibitory activity.
Description
Technical Field
The invention belongs to the technical field of ACE inhibitory peptides, and particularly relates to a puffer fish active peptide and a preparation method thereof.
Background
With the progress and development of society, the living standard of people is gradually improved, the proportion of fat and heat in diet is gradually increased, and the exercise amount is reduced, so that the incidence of cardiovascular diseases in China is rapidly increased, wherein hypertension is the main factor.
The puffer fish is a fish in the warm temperate zone and the tropical near-sea bottom layer, inhabits the middle and lower layers of the sea, has few species entering the fresh water river, and makes the whole body float on the water surface in a spherical shape when encountering external danger, and small thorns on the skin are erected to defend the puffer fish.
Angiotensin Converting Enzyme (ACE) inhibitory peptide is a small molecular polypeptide formed by proteolysis, has a remarkable blood pressure lowering effect, and more effectively is found to have no toxic or side effect and no influence on normal blood pressure compared with other common blood pressure lowering medicines. ACE plays an important role in blood pressure regulation, and through the removal of two amino acids (His-Leu) at the carbon end, originally inactive angiotensin I can be converted into active angiotensin II, so that vasoconstriction is caused, and the blood pressure is increased; the ACE also can lead the relaxing skin with the vasodilatation function to be inactivated and also can cause the situation of blood pressure rise, and the ACE inhibitory peptide can block two biochemical reaction processes caused by the ACE to play the role of reducing the blood pressure.
The prior art for preparing ACE inhibitory peptide from foods such as hazelnuts, yak milk, shellfish meat, medlar and the like does not refer to the technology for preparing ACE inhibitory peptide from puffer fish, particularly puffer fish skin.
Disclosure of Invention
The invention aims to provide a puffer fish active peptide and a preparation method thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a puffer fish active peptide, which is octapeptide with the amino acid sequence of PPLLFAAL (Pro-Pro-Leu-Leu-Phe-Ala-Ala-Leu).
Preferably, the octapeptide PPLLFAAL (Pro-Pro-Leu-Leu-Phe-Ala-Ala-Leu) has a molecular weight of 840.5 Da.
Preferably, the IC of the octapeptide PPLLFAAL (Pro-Pro-Leu-Leu-Phe-Ala-Ala-Leu)50The value was 0.1 to 0.3 umol/mL.
The invention also provides a preparation method of the puffer fish active peptide, which comprises the following steps: s1: preparing the puffer fish enzymatic hydrolysate, pulping puffer fish skins, adding water, stirring to form a protein solution, adjusting the pH value to be alkaline, adding alkaline protease for enzymolysis, keeping the pH value unchanged, performing enzyme deactivation treatment after the enzymolysis is finished, cooling to room temperature, adjusting the pH value to be neutral, centrifuging to obtain a supernatant, freeze-drying the supernatant to obtain crude puffer fish ACE inhibitory peptide, adding the freeze-dried crude puffer fish ACE inhibitory peptide into water to prepare the puffer fish enzymatic hydrolysate, S2: performing ultrafiltration separation on the puffer fish enzymatic hydrolysate, performing clarification separation on the puffer fish enzymatic hydrolysate, performing ultrafiltration treatment on the clarified puffer fish enzymatic hydrolysate, respectively collecting separation components with different sizes and molecular weights, freeze-drying, and determining the ACE inhibition rate of each separation component, S3: separating and purifying the polypeptide, primarily separating and purifying the separated components with the molecular weight less than 1kDa by using semi-preparative liquid chromatography, collecting 8 different absorption peak components, collecting eluent according to the time range of the absorption peaks, respectively naming the eluent as A1-A8 components, then carrying out ACE activity determination, selecting the A7 component and carrying out gel filtration separation, then carrying out high performance liquid chromatography on the A7 component after gel filtration separation for further separation and purification, separating to obtain high-purity polypeptide components, S4, carrying out sequence identification on the polypeptide, carrying out mass spectrum determination on the high-purity polypeptide components obtained by separation in the step S3, analyzing the result of the mass spectrum determination to obtain the octapeptide PPLLFAAL (Pro-Pro-Leu-Leu-Phe-Ala-Ala-Leu), S5, carrying out polypeptide synthesis and activity determination, and carrying out the octapeptide PPLLFAAL (Pro-Pro-Leu-Leu-Phe-Ala-Ala-Leu) according to the determined polypeptide sequence Solid phase synthesis is carried out to verify the ACE inhibitory activity of the synthesized polypeptide.
Preferably, the specific steps of step S1 are: pulping 80-120g of puffer fish skin, adding 250-350mL of distilled water, stirring to form a protein solution, adjusting the pH value to 7.5-9.0 by NaOH, adding alkaline protease with the enzyme amount of 450-350U/g, carrying out enzymolysis for 6-10h at 50-60 ℃, keeping the pH value unchanged, heating at 80-100 ℃ for 12-18min after the enzymolysis is finished, carrying out enzyme deactivation treatment, cooling to room temperature, adjusting the pH value to be neutral by HCl, centrifuging at the speed of 4000-6000r/min for 8-12min, taking supernatant, freeze-drying the supernatant to obtain crude puffer ACE inhibitory peptide, and adding the freeze-dried crude puffer ACE inhibitory peptide into distilled water to prepare 0.8-1.2mg/mL of puffer fish enzymatic hydrolysate.
Preferably, the specific steps of step S2 are: and (4) clarifying and separating the puffer fish enzymatic hydrolysate prepared in the step (S1) by adopting a ceramic membrane with the aperture of 50-100 mu m, then carrying out ultrafiltration treatment on the clarified puffer fish enzymatic hydrolysate by adopting polyether sulfone ultrafiltration membranes with the aperture of 50kDa, 30kDa, 10kDa, 5kDa and 1kDa, respectively collecting separation components with different molecular weights, freeze-drying and determining the ACE inhibition rate of each separation component.
Preferably, the semi-preparative liquid chromatography of step S3 is performed by using a 2.5cm × 20cm Sinochrom ODS-BP chromatographic column, the sample loading amount is 3-5mL, the buffer solution is subjected to isocratic elution by using a 10% methanol solution, the elution flow rate is 5mL/min, the detection is performed by using an ultraviolet detector, the ultraviolet detection wavelength is 220nm, 8 components are collected, and the freeze-drying is performed.
Preferably, the gel filtration separation of step S3 is performed by using a glass chromatography column with a diameter of 20X 1000mm, a SephadexG-15 packing, a height of 800-1000mm packing, a detection wavelength of 220nm, pure water as a mobile phase, a flow rate of 4.5-5.5mL/min as a mobile phase, and a temperature of room temperature.
Preferably, the high performance liquid chromatography of step S3 employs sunfire C of 4.6X 250mm18A chromatographic column, wherein the ultraviolet detection wavelength is 220nm, the mobile phase A is pure water containing TFA with the mass fraction of 0.4-0.6%, the mobile phase B is acetonitrile, the volume ratio of the mobile phase A to the mobile phase B is 75:25 in equilibrium, the flow rate of the mobile phase is 0.8-1.2mL/min, and the temperature is 25 ℃.
Preferably, step S4 is performed by LC-MS/MS mass spectrometry under the following conditions: the chromatographic column is PepMap RPLC C1875 μm i.d. × 150mm, 3 μm, 100) cation mode, scan range: m/z 300-1500 Da, emitter spray voltage 2-kV, and the result of mass spectrometry is analyzed by PEAKS STUDIO software to obtain the polypeptide amino acid sequence.
The invention has the beneficial effects that:
1. the octapeptide amino acid sequence is obtained by using the puffer fish skin as a raw material, and the prepared polypeptide has ACE inhibitory activity.
2. A hydrogen bond network is formed between the octapeptide PPLLFAAL (Pro-Pro-Leu-Leu-Phe-Ala-Ala-Leu) and the ACE, and the stability is high.
3. The octapeptide PPLLFAAL (Pro-Pro-Leu-Leu-Phe-Ala-Ala-Leu) Leu3 skeleton amide oxygen atom has electronegativity and can be combined with Zn of the binding site2+Forming stronger polar effect, thereby destroying the original function of Zn ions and improving the inhibition effect.
4. The octapeptide PPLLFAAL (Pro-Pro-Leu-Leu-Phe-Ala-Ala-Leu) has more hydrophobic residues, such as Leu3, Leu4, Phe5, etc., which can form stronger hydrophobic interactions and van der Waals potentials with the binding pocket residues.
Drawings
FIG. 1 is a graph showing the determination of ACE inhibitory ratio by three protease enzymatic hydrolysates.
FIG. 2 is the IC of ACE inhibition of fractions of different molecular weight fractions collected in step S2 of the present invention50Value measurement graph.
FIG. 3 is a graph showing the determination of ACE inhibitory activity of different molecular weight fractions collected in step S2 according to the present invention.
FIG. 4 is a semi-preparative liquid chromatography separation profile of the present invention.
FIG. 5 is a graph showing the ACE inhibitory activity of 8 absorption components collected by semi-preparative liquid chromatography in step S3 according to the present invention.
FIG. 6 is a diagram of the polypeptide fraction purified by SephadexG-15 in step 3 of the present invention.
FIG. 7 is a liquid chromatography chromatogram of a high purity polypeptide of the present invention.
FIG. 8 is a high purity polypeptide sequencing map of the present invention.
FIG. 9 is a graph of an assay for ACE inhibitory activity of the synthetic polypeptides of the present invention.
FIG. 10 is a pattern of binding of the synthetic polypeptides of the invention to ACE.
FIG. 11 shows the inhibition pattern and mechanism of action of the synthetic polypeptides of the invention with ACE.
Detailed Description
The invention will now be further described with reference to the accompanying drawings and detailed description.
The active peptide of the puffer fish provided by the embodiment is octapeptide, and the amino acid sequence is PPLLFAAL (Pro-Pro-Leu-Leu-Phe-Ala-Ala-Leu).
The octapeptide PPLLFAAL (Pro-Pro-Leu-Leu-Phe-Ala-Ala-Leu) has a molecular weight of 840.5 Da.
The embodiment also provides a preparation method of the pufferfish bioactive peptide, which comprises the following steps:
s1: the method comprises the steps of pulping 100g of puffer fish skin, adding 300mL of distilled water, stirring to form a protein solution, adjusting the pH value to 8.0 by NaOH, adding 500U/g of alkaline protease, performing enzymolysis for 10h at 55 ℃, keeping the pH value unchanged, heating for 15min at 90 ℃ after enzymolysis is finished, performing enzyme deactivation treatment, cooling to room temperature, adjusting the pH value to be neutral by HCl, centrifuging for 10min at 5000r/min, taking supernatant, freeze-drying the supernatant to obtain puffer fish ACE inhibitory peptide, storing at 4 ℃, adding the freeze-dried puffer fish ACE inhibitory peptide into distilled water, and preparing the puffer fish ACE inhibitory peptide into 1mg/mL of puffer fish enzymatic hydrolysate.
As shown in fig. 1, the protease was obtained as 3 commercial proteases: pepsin (the optimum pH and the temperature are pH5.7 and 60 ℃), neutral protease (the optimum pH and the temperature are pH7.0 and 50 ℃), alkaline protease (the optimum pH and the temperature are pH8.0 and 55 ℃) are used as enzyme preparation screening sources, the influence of the neutral protease on the ACE inhibition rate of the hydrolysis of the skin of the Takifugu flavidus under the optimum conditions is researched, and the protease suitable for producing the ACE inhibitory peptide by the proteolysis of the skin of the Takifugu flavidus is screened. The results show that the use of alkaline protease has the best enzymolysis effect. The enzymolysis conditions are as follows: alkaline protease enzyme concentration: 500U/g, the pH value is adjusted to 8.0 by sodium hydroxide (NaOH), the enzymolysis temperature is 55 ℃, and the enzymolysis time is 10 h.
S2: and (4) performing ultrafiltration separation on the puffer fish enzymatic hydrolysate prepared in the step (S1), namely performing clarification separation on the puffer fish enzymatic hydrolysate by adopting a ceramic membrane with the pore diameter of 50 mu m, performing ultrafiltration treatment on the clarified puffer fish enzymatic hydrolysate by adopting polyether sulfone ultrafiltration membranes with the pore diameters of 50kDa, 30kDa, 10kDa, 5kDa and 1kDa, respectively collecting separation components with different molecular weights, freeze-drying the separation components, and determining IC (integrated Circuit) of ACE (angiotensin converting enzyme) inhibition rate of each separation component50The value is obtained. The results showed that the component with a molecular weight of less than 1kDa showed the highest ACE inhibition as shown in FIG. 2, and the component with a molecular weight of less than 1kDa showed AC as shown in FIG. 3E inhibitory activity was highest and therefore, fractions with a molecular weight of less than 1kDa were selected for subsequent purification.
S3: separating and purifying polypeptide, performing primary separation and purification on separated components with molecular weight less than 1kDa by semi-preparative liquid chromatography, collecting 8 different absorption peak components, and collecting eluates according to the time range of the absorption peak, which are respectively named as A1-A8 components, as shown in figure 4. And then, performing ACE activity determination, as shown in figure 5, wherein A4, A5, A7 and A8 have high ACE inhibitory activity, selecting and performing gel filtration separation on the A7 component, performing high performance liquid chromatography on the A7 component after the gel filtration separation for further separation and purification, and separating to obtain the high-purity polypeptide component.
Specifically, the semi-preparative liquid chromatography of step S3 adopts a 2.5cm × 20cm Sinochrom ODS-BP chromatographic column, the sample loading amount is 5mL, the buffer solution adopts 10% methanol solution for isocratic elution, the elution flow rate is 5mL/min, and the detection is carried out in an ultraviolet detector, the ultraviolet detection wavelength is 220nm, 8 components are collected, and the freeze drying is carried out.
Specifically, the gel filtration and separation in step S3 is performed by using a glass chromatographic column with a diameter of 20 × 1000mm, the filler is sephadex g-15, the height of the filler is 900mm, the detection wavelength is 220nm, the mobile phase is pure water, the flow rate of the mobile phase is 5mL/min, the temperature is room temperature, and as shown in fig. 6, the gel filtration and separation is a graph of the purified polypeptide fraction passing through sephadex g-15.
Specifically, the high performance liquid chromatography of step S3 used SunfireC of 4.6X 250mm18A chromatographic column, the ultraviolet detection wavelength is 220nm, the mobile phase A is pure water containing TFA with the mass fraction of 0.5%, the mobile phase B is acetonitrile, the volume ratio of the mobile phase A to the mobile phase B is 75:25 in equilibrium, the flow rate of the mobile phase is 1mL/min, the temperature is 25 ℃, and as shown in FIG. 7, the chromatographic spectrum of the high-purity polypeptide liquid phase is shown.
S4, identifying the sequence of the polypeptide, carrying out mass spectrometry on the high-purity polypeptide component separated in the step S3, analyzing the mass spectrometry result to obtain the octapeptide PPLLFAAL (Pro-Pro-Leu-Leu-Phe-Ala-Ala-Leu), and specifically carrying out mass spectrometry by adopting LC-MS/MS, wherein the measurement conditions are as follows: the chromatographic column is PepMap RPLC C18, 75μm i.d.×150mm3 μm, 100 a) positive ion mode, scan range: m/z 300-1500 Da, emitter spray voltage 2-kV, and the result of mass spectrometry is analyzed by PEAKS STUDIO software to obtain the polypeptide amino acid sequence PPLLFAAL, and the sequence determination map is shown in FIG. 8.
S5 polypeptide synthesis and activity determination, the octapeptide PPLLFAAL (Pro-Pro-Leu-Leu-Phe-Ala-Ala-Leu) is synthesized according to the determined polypeptide sequence in solid phase, and the ACE inhibitory activity of the synthesized polypeptide is verified. The results show that the purified polypeptide has good ACE inhibitory activity, and the IC of the prepared octapeptide PPLLFAAL (Pro-Pro-Leu-Leu-Phe-Ala-Ala-Leu)50The value was 0.2 umol/mL. The ACE inhibitory activity of the synthetic polypeptides was determined as shown in figure 9.
The Discovery Studio software is used for carrying out simulation analysis on the docking mechanism of the polypeptide and the ACE, and the results are as follows:
as shown in fig. 10-11, a hydrogen bonding network is formed between the octapeptide PPLLFAAL and ACE. The two can form about 10 hydrogen bonds to interact (some sites in the figure have hydrogen bond competition relationship, such as Phe5 skeleton oxygen atom, can form 2 hydrogen bonds as hydrogen bond acceptor, and the competition relationship is shown as 3 in the figure). These hydrogen bonding effects are critical for stable binding of the polypeptide to ACE. Binding pocket residues that hydrogen bond with polypeptides include: glu384, Als356, Gln281, Tyr520, and the like. In addition, the skeleton amide oxygen atom of the polypeptide Leu3 has electronegativity and can be combined with Zn of a binding site2+Forming stronger polar action, thereby destroying the original functions of Zn ions. Therefore, it also plays a role in exerting the polypeptide inhibitory action. The polypeptide has more hydrophobic residues, such as Leu3, Leu4, Phe5, etc. These hydrophobic residues, in combination with pocket residues, form strong hydrophobic interactions and van der waals potentials. These more hydrophobic binding pocket residues include: phe457, Phe512, Val518, Val380, and the like.
Those not described in detail in this specification are within the skill of the art; the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art; the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (9)
1. A tetrodotoxin active peptide is characterized in that,
the active peptide is octapeptide, and the amino acid sequence is PPLLFAAL (Pro-Pro-Leu-Leu-Phe-Ala-Ala-Leu).
2. The tetrodotoxin active peptide of claim 1,
the octapeptide PPLLFAAL (Pro-Pro-Leu-Leu-Phe-Ala-Ala-Leu) has a molecular weight of 840.5 Da.
3. The tetrodotoxin active peptide of claim 1,
IC of the octapeptide PPLLFAAL (Pro-Pro-Leu-Leu-Phe-Ala-Ala-Leu)50The value was 0.1 to 0.3 umol/mL.
4. A method for preparing a puffer fish active peptide is characterized by comprising the following steps:
s1: preparation of fugu enzymatic hydrolysate
Pulping the skin of a puffer fish, adding water, stirring to form a protein solution, adjusting the pH value to be alkaline, adding alkaline protease for enzymolysis, adding the enzyme in an amount of 450-600U/g, performing enzymolysis for 6-10h at 50-60 ℃, keeping the pH value unchanged, performing enzyme deactivation treatment after the enzymolysis is finished, cooling to room temperature, adjusting the pH value to be neutral, centrifuging to obtain a supernatant, freeze-drying the supernatant to obtain crude puffer fish ACE inhibitory peptide, and adding the freeze-dried crude puffer fish ACE inhibitory peptide into water to prepare a puffer fish enzymatic hydrolysate;
s2: ultra-filtration separation of fugu enzymatic hydrolysate
Clarifying and separating the puffer fish enzymatic hydrolysate, then performing ultrafiltration treatment on the clarified puffer fish enzymatic hydrolysate, respectively collecting separation components with different molecular weights, freeze-drying, and determining the ACE inhibition rate of each separation component;
s3: separation and purification of polypeptide
Primarily separating and purifying the separated component with the molecular weight less than 1kDa by using a semi-preparative liquid chromatography, wherein the semi-preparative liquid chromatography adopts a 2.5cm × 20cm Sinochrom ODS-BP chromatographic column, the sample loading amount is 3-5mL, a buffer solution is subjected to isocratic elution by adopting a 10% methanol solution, the elution flow rate is 5mL/min, 8 different absorption peak components are obtained by collection, eluents are collected according to the time range of the absorption peaks and are respectively named as A1-A8 components, then ACE activity determination is carried out, the A7 component is selected and subjected to gel filtration separation, then the A7 component subjected to gel filtration separation is subjected to high performance liquid chromatography for further separation and purification, and the high performance liquid chromatography adopts 4.6 × 250mm sunfire C18A chromatographic column, wherein the ultraviolet detection wavelength is 220nm, the mobile phase A is pure water containing TFA with the mass fraction of 0.4-0.6%, the mobile phase B is acetonitrile, the volume ratio of the mobile phase A to the mobile phase B is 75:25 during balance, the flow rate of the mobile phase is 0.8-1.2mL/min, and the high-purity polypeptide component is obtained by separation at the temperature of 25 ℃;
s4 sequence identification of the polypeptide
Performing mass spectrometry on the high-purity polypeptide component separated in the step S3, and analyzing the mass spectrometry result to obtain octapeptide PPLLFAAL (Pro-Pro-Leu-Leu-Phe-Ala-Ala-Leu);
s5 polypeptide Synthesis and Activity determination
And carrying out solid phase synthesis on the octapeptide PPLLFAAL (Pro-Pro-Leu-Leu-Phe-Ala-Ala-Leu) according to the determined polypeptide sequence, and verifying the ACE inhibitory activity of the synthesized polypeptide.
5. The method for preparing tetrodotoxin active peptide according to claim 4,
the specific steps of step S1 are:
pulping 80-120g of puffer fish skin, adding 250-350mL of distilled water, stirring to form a protein solution, adjusting the pH value to 7.5-9.0 by NaOH, adding alkaline protease with the enzyme amount of 450-350U/g, carrying out enzymolysis for 6-10h at 50-60 ℃, keeping the pH value unchanged, heating at 80-100 ℃ for 12-18min after the enzymolysis is finished, carrying out enzyme deactivation treatment, cooling to room temperature, adjusting the pH value to be neutral by HCl, centrifuging at the speed of 4000-6000r/min for 8-12min, taking supernatant, freeze-drying the supernatant to obtain crude puffer ACE inhibitory peptide, and adding the freeze-dried crude puffer ACE inhibitory peptide into distilled water to prepare 0.8-1.2mg/mL of puffer fish enzymatic hydrolysate.
6. The method for preparing tetrodotoxin active peptide according to claim 4,
the specific steps of step S2 are:
and (4) clarifying and separating the puffer fish enzymatic hydrolysate prepared in the step (S1) by adopting a ceramic membrane with the aperture of 50-100 mu m, then carrying out ultrafiltration treatment on the clarified puffer fish enzymatic hydrolysate by adopting polyethersulfone ultrafiltration membranes with the apertures of 50kDa, 30kDa, 10kDa, 5kDa and 1kDa, respectively collecting separation components with different molecular weights, freeze-drying and determining the ACE inhibition rate of each separation component.
7. The method for preparing tetrodotoxin active peptide according to claim 4,
the semi-preparative liquid chromatography of the step S3 adopts a 2.5cm × 20cm Sinochrom ODS-BP chromatographic column, the sample loading amount is 3-5mL, the buffer solution adopts 10% methanol solution for isocratic elution, the elution flow rate is 5mL/min, the detection is carried out in an ultraviolet detector, the ultraviolet detection wavelength is 220nm, 8 components are collected and freeze-dried.
8. The method for preparing tetrodotoxin active peptide according to claim 4,
the gel filtration and separation in the step S3 adopts a glass chromatographic column with the diameter of 20 multiplied by 1000mm, the filler is SephadexG-15, the height of the filler is 800-1000mm, the detection wavelength is 220nm, the mobile phase is pure water, the flow rate of the mobile phase is 4.5-5.5mL/min, and the temperature is room temperature.
9. The method for preparing tetrodotoxin active peptide according to claim 4,
the step S4 adopts LC-MS/MS to carry out mass spectrometry, and the measurement conditions are as follows: the chromatographic column is PepMap RPLC C18,75μmi.d.×150mm,3μm，
Positive ion mode, scan range: m/z 300-1500 Da, emitter spray voltage 2-kV, and the result of mass spectrometry is analyzed by PEAKS STUDIO software to obtain the polypeptide amino acid sequence.
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| CN111499691B (en) * | 2020-04-27 | 2022-02-15 | 福建省水产研究所（福建水产病害防治中心） | ACE inhibitory peptide P1, application thereof and preparation method thereof |
| CN113603745B (en) * | 2021-08-09 | 2023-07-07 | 福建省水产研究所（福建水产病害防治中心） | Active fugu flavidus fish skin polypeptide and preparation method and application thereof |
| CN113617060B (en) * | 2021-08-09 | 2023-02-10 | 福建省水产研究所（福建水产病害防治中心） | Immobilized enzyme gel affinity adsorption column preparation method and method for extracting active peptide from puffer fish |
| CN113480607B (en) * | 2021-08-09 | 2022-12-27 | 福建省水产研究所（福建水产病害防治中心） | Active small molecule peptide and preparation method and application thereof |
| CN113480598B (en) * | 2021-08-09 | 2023-07-07 | 福建省水产研究所（福建水产病害防治中心） | Preparation method and application of bioactive tetrapeptides |
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| CN102190706B (en) * | 2011-04-07 | 2013-06-12 | 江苏省农业科学院 | Loach protein antihypertensive peptide and preparation method thereof |
| CN109320588B (en) * | 2018-10-18 | 2021-07-27 | 大连深蓝肽科技研发有限公司 | Apostichopus japonicus-derived ACE (angiotensin converting enzyme) inhibitory active peptide |
| CN110495579A (en) * | 2019-08-02 | 2019-11-26 | 青岛浩大生物科技工程有限责任公司 | A kind of technique improving globefish skin nutritive value |
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