CN114891065A - Blood sugar-reducing sea cucumber peptide with alpha-amylase inhibitory activity and preparation method and application thereof - Google Patents
Blood sugar-reducing sea cucumber peptide with alpha-amylase inhibitory activity and preparation method and application thereof Download PDFInfo
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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Abstract
The invention discloses a hypoglycemic sea cucumber peptide with alpha-amylase inhibitory activity, which consists of the following eight amino acids: phenylalanine-proline-serine-proline-leucineAmino acid-valine-alanine, abbreviated as FPSPPLVA, has a molecular weight of 826.45 Da. The hypoglycemic peptide consists of eight amino acids, including phenylalanine, proline, serine, proline, leucine, valine and alanine, and has molecular weight of 826.45Da, IC 50 The value was 414.23. mu.g/mL. Compared with common polypeptide, the hypoglycemic peptide belongs to micromolecule oligopeptide, has smaller molecular weight, is not easy to saturate a transport carrier, has low energy consumption and high transport speed, can be directly absorbed and utilized by a human body, does not increase the digestion burden of the gastrointestinal tract, and is particularly suitable for people with weakened gastrointestinal functions.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a blood sugar-reducing sea cucumber peptide with alpha-amylase inhibitory activity, and a preparation method and application thereof.
Background
With the continuous improvement of living standard, more and more people suffer from diabetes, seriously threaten the health of human beings, and are chronic non-infectious diseases which are ranked the third after cardiovascular and cerebrovascular diseases and malignant tumors. Postprandial hyperglycemia is a major feature of pre-diabetic patients, however, persistent hyperglycemia can lead to a variety of complications, and serious ones can be life threatening. Controlling and lowering blood glucose is the only way to treat diabetes. The conventional medicines for treating diabetes at present mainly comprise three types: synthetic insulin and insulin analogs, insulinotropic agents, and insulin sensitizers. The medicine can be used alone or in combination. Although effective in reducing blood sugar, it has great side effects and high price and affects people's normal life. The alpha-amylase inhibitor can inhibit the activity of saliva and pancreatic amylase in gastrointestinal tract, prevent or delay the absorption of main carbohydrate in food into reducing saccharide in small intestine, and avoid the fast increase of postprandial blood sugar concentration. Therefore, it is necessary to find a safe and effective alpha-amylase inhibitor without side effects, which is useful for the dietary treatment of diabetic patients.
The bioactive peptide is a general name of peptides which are composed of 2-20 amino acids and have special physiological activity, complex linear relation and ring structure, in recent years, food protein is widely researched as a functional peptide, and the marine bioactive peptide becomes a hot point of research in the medical field at present due to the advantages of multiple functions, wide sources, strong specificity, small toxic and side effects and the like. The sea cucumber peptide has rich protein content and complete amino acid types, the research on sea cucumber peptide is focused on the aspects of oxidation resistance, fatigue resistance and the like at present, the research on the blood sugar reducing effect of the sea cucumber peptide is less, the research on alpha-glucosidase and DPP-IV enzyme by a peptide mixture is focused, the inhibition effect on alpha-amylase is shallow, and the specific structure of the acting polypeptide is not clear.
The current research on active peptides has focused mainly on the selection of proteases and proteins, because of the random choice of proteins and proteases, which results in blindness. The computer technology is used for simulating the composite reaction between the small molecule ligand and the macromolecular receptor, and the computer-assisted small molecule virtual screening can be realized. The molecular docking technology is used for predicting the binding site of the active peptide and the target molecule, which is beneficial to the research of the interaction mode and the binding mechanism of the active peptide and the alpha-amylase, and provides a basis for clarifying the bioactive peptide of specific inhibitory site sequences on the peptide and the alpha-amylase, and can provide scientific basis for unknown bioactivity.
Through searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a hypoglycemic sea cucumber peptide with alpha-amylase inhibitory activity, a preparation method and application thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a hypoglycemic sea cucumber peptide with alpha-amylase inhibiting activity, which consists of the following eight amino acids: phenylalanine-proline-serine-proline-leucine-valine-alanine, abbreviated as FPSPPLVA, has a molecular weight of 826.45 Da.
Further, the alpha-amylase inhibition rate and IC of the blood sugar reducing sea cucumber peptide 50 It was 414.23. mu.g/mL.
Further, it is clear through molecular docking technology that the hypoglycemic sea cucumber peptide forms 5 hydrogen bonds with Glu63, Thr163, Lys200, His201 residues of alpha-amylase, and 6 hydrophobic interactions exist.
The preparation method of the hypoglycemic sea cucumber peptide with alpha-amylase inhibitory activity comprises the following steps:
A. enzymolysis: selecting papain, alkaline protease and bromelain, and mixing the selected materials in a ratio of 1.2: 1: 0.8, mixing the sea cucumbers according to the mass ratio of 1: 20, preparing a sea cucumber homogenate according to the feed-liquid ratio, and performing multi-enzyme synergistic hydrolysis on the sea cucumber homogenate according to the enzyme adding amount of 8000u/g protein;
B. and (3) ultrafiltration membrane separation: adding the enzymatic hydrolysate obtained in the step A into an ultrafiltration cup of an ultrafiltration system, installing an ultrafiltration membrane package with the molecular weight cutoff of 1kDa, carrying out ultrafiltration under the pressure of 20-30 psig, collecting filtrate, and freeze-drying;
C. preparing liquid phase chromatographic separation: dissolving the freeze-dried powder obtained in the step B in ultrapure water, and separating and purifying by using a preparative liquid chromatography;
D. high-speed countercurrent chromatographic separation: injecting the sample obtained in the step C into a sample loop for sample injection, finally starting an ultraviolet detector and an HSCCC workstation, detecting at 220nm, collecting fractions after secondary separation according to a spectrum, and obtaining the blood sugar-reducing sea cucumber peptide with alpha-amylase inhibitory activity.
Further, the enzymolysis parameters of the multi-enzyme synergistic hydrolysis in the step a are as follows: the pH value of enzymolysis is 7.8-8.2, the enzymolysis temperature is 50-55 ℃, and the enzymolysis time is 5-6 h.
Furthermore, the chromatographic column of the preparative liquid chromatogram in the step C is Agilent C-18, the detection wavelength of an ultraviolet detector of the preparative liquid chromatogram is 280nm, the mobile phase A is ultrapure water containing 0.1 percent of trifluoroacetic acid, and the mobile phase B is acetonitrile containing 0.1 percent of trifluoroacetic acid.
Further, the mass ratio of the freeze-dried powder to the ultrapure water in the step C is 1:10-1.2:10(mg: mL).
Further, the high-speed countercurrent chromatography separation in the step D is carried out twice as separation chromatography operations: the first separation chromatographic conditions are as follows: the volume ratio of water/n-butanol/ethyl acetate/acetic acid is 5: 3: 2: 0.5, the upper phase of the solvent system is a stationary phase, and the lower phase is a mobile phase; the rotation speed is 800-900rpm, the flow rate is 4mL/min, elution is carried out from head to tail, the detection wavelength is 220nm, the sample introduction volume is 10mL, and the sample concentration is 5 mg/mL; the chromatographic conditions for the second separation are as follows: the volume ratio of water/n-butanol/ethyl acetate/triethylamine is 4: 4: 1: 0.5, and other conditions are the same as the first separation.
The hypoglycemic sea cucumber peptide with alpha-amylase inhibitory activity is applied to foods, health products and medicines.
The invention has the advantages and positive effects that:
1. the hypoglycemic peptide consists of eight amino acids of phenylalanine-proline-serine-proline-leucine-valine-alanine, and has molecular weight of 826.45Da, IC 50 The value was 414.23. mu.g/mL. Compared with common polypeptide, the hypoglycemic peptide belongs to micromolecule oligopeptide, has smaller molecular weight, is not easy to saturate a transport carrier, has low energy consumption and high transport speed, can be directly absorbed and utilized by a human body, does not increase the digestion burden of the gastrointestinal tract, and is particularly suitable for people with weakened gastrointestinal functions. When the concentration of the hypoglycemic peptide is 3mg/mL, the inhibition rate of the hypoglycemic peptide on alpha-amylase reaches 84.57 percent.
2. The invention is different from the traditional single enzyme hydrolysis, and under the mild condition, papain, alkaline protease and bromelain are firstly adopted in the proportion of 1.2: 1: 0.8 mass ratio, strong specificity, easily controlled condition, no toxic and harmful substances, and no natural side effect.
3. After membrane ultrafiltration, the method is different from the traditional separation mode, the combination of the preparative liquid chromatography and the HSCCC is used for systematic separation of the sea cucumber peptide, the hypoglycemic sea cucumber peptide FPSPPLVA with effective alpha-amylase inhibition effect is obtained by screening, the action mechanism of the hypoglycemic sea cucumber peptide FPSPPLVA and the alpha-amylase is clarified by the molecular docking technology, the method has very important significance for the development of the sea cucumber peptide in the aspects of hypoglycemic foods, health products and medicines, and the method lays an experimental foundation for deep processing industrialization and high-value utilization of the sea cucumber.
4. The invention aims to provide the hypoglycemic sea cucumber peptide with alpha-amylase inhibitory activity and the preparation method thereof, and defines the action site of the hypoglycemic sea cucumber peptide and the alpha-amylase, has important significance for developing foods, health care products and medicines with hypoglycemic effect, lays an experimental foundation for deep processing industrialization and high-value utilization of sea cucumber, and fills the blank of the hypoglycemic sea cucumber peptide in the prior art.
Drawings
FIG. 1 is an HPLC chromatogram of FPSPPLVA of the hypoglycemic sea cucumber peptide of the present invention;
FIG. 2 is a mass spectrum of FPSPPLVA of the present invention;
FIG. 3 is a line graph of the inhibition of alpha-amylase by FPSPPLVA in accordance with the present invention;
FIG. 4 is a diagram showing the interaction of FPSPPLVA and alpha-amylase molecules in the present invention.
Detailed Description
The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.
The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.
A hypoglycemic sea cucumber peptide with alpha-amylase inhibiting activity, which consists of the following eight amino acids: phenylalanine-proline-serine-proline-leucine-valine-alanine, abbreviated as FPSPPLVA, has a molecular weight of 826.45 Da.
Preferably, the alpha-amylase inhibition rate and IC of the blood sugar reducing sea cucumber peptide 50 It was 414.23. mu.g/mL.
Preferably, the hypoglycemic sea cucumber peptide is clear to form 5 hydrogen bonds with Glu63, Thr163, Lys200 and His201 residues of the alpha-amylase by a molecular docking technology, and 6 hydrophobic effects exist.
The preparation method of the hypoglycemic sea cucumber peptide with alpha-amylase inhibitory activity comprises the following steps:
A. enzymolysis: selecting papain, alkaline protease and bromelain, and mixing the selected materials in a ratio of 1.2: 1: 0.8, mixing the sea cucumbers according to the mass ratio of 1: 20, preparing a sea cucumber homogenate according to the feed-liquid ratio, and performing multi-enzyme synergistic hydrolysis on the sea cucumber homogenate according to the enzyme adding amount of 8000u/g protein;
B. and (3) ultrafiltration membrane separation: adding the enzymatic hydrolysate obtained in the step A into an ultrafiltration cup of an ultrafiltration system, installing an ultrafiltration membrane package with the molecular weight cutoff of 1kDa, carrying out ultrafiltration under the pressure of 20-30 psig, collecting filtrate, and freeze-drying;
C. preparing liquid phase chromatographic separation: dissolving the freeze-dried powder obtained in the step B in ultrapure water, and separating and purifying by using a preparative liquid chromatography;
D. high-speed countercurrent chromatographic separation: injecting the sample obtained in the step C into a sample loop for sample injection, finally starting an ultraviolet detector and an HSCCC workstation, detecting at 220nm, collecting fractions after secondary separation according to a spectrum, and obtaining the blood sugar-reducing sea cucumber peptide with alpha-amylase inhibitory activity.
Preferably, the enzymolysis parameters of the multi-enzyme synergistic hydrolysis in the step A are as follows: the pH value of enzymolysis is 7.8-8.2, the enzymolysis temperature is 50-55 ℃, and the enzymolysis time is 5-6 h.
Preferably, the chromatographic column of the preparative liquid chromatogram in the step C is Agilent C-18, the detection wavelength of an ultraviolet detector of the preparative liquid chromatogram is 280nm, the mobile phase A is ultrapure water containing 0.1 percent of trifluoroacetic acid, and the mobile phase B is acetonitrile containing 0.1 percent of trifluoroacetic acid.
Preferably, the mass ratio of the freeze-dried powder to the ultrapure water in the step C is 1:10-1.2:10(mg: mL).
Preferably, the high-speed countercurrent chromatographic separation in the step D is carried out by two separation chromatographic operations: the first separation chromatographic conditions are as follows: the volume ratio of water/n-butanol/ethyl acetate/acetic acid is 5: 3: 2: 0.5, the upper phase of the solvent system is a stationary phase, and the lower phase is a mobile phase; the rotation speed is 800-900rpm, the flow rate is 4mL/min, elution is carried out from head to tail, the detection wavelength is 220nm, the sample introduction volume is 10mL, and the sample concentration is 5 mg/mL; the chromatographic conditions for the second separation are as follows: the volume ratio of water/n-butanol/ethyl acetate/triethylamine is 4: 4: 1: 0.5, and other conditions are the same as the first separation.
The hypoglycemic sea cucumber peptide with alpha-amylase inhibitory activity is applied to foods, health products and medicines.
Specifically, the preparation and detection examples are as follows:
the enzyme activity of papain used in the following examples was 800u/mg, the enzyme activity of alkaline protease was 120u/mg, and the enzyme activity of bromelain was 300 u/mg.
In the following examples, the separation conditions for HSCCC are specified as follows:
the first separation chromatographic conditions are as follows: water/n-butanol/ethyl acetate/acetic acid (volume ratio is 5: 3: 2: 0.5), the upper phase of the solvent system is a stationary phase, the lower phase is a mobile phase, the rotation speed of the instrument is 800-900rpm, the flow rate is 4mL/min, elution is carried out from head to tail, the detection wavelength is 220nm, the sample injection volume is 10mL, and the sample concentration is 5 mg/mL.
The chromatographic conditions of the second separation are as follows: water/n-butanol/ethyl acetate/triethylamine (volume ratio 4: 4: 1: 0.5), the upper phase of the solvent system is a stationary phase, the lower phase is a mobile phase, and the other conditions are the same as the first step.
In the following examples, the identification conditions for LC-MS/MS are as follows:
and (3) chromatographic separation: the liquid A used in the liquid phase is 0.1% formic acid aqueous solution, and the liquid B is 0.1% formic acid acetonitrile aqueous solution (acetonitrile is 84%). The Column was RP-C18(0.15 mm. times.150 mm, Column Technology Inc.), equilibrated with 95% solution A, and the sample was applied to Zorbax 300SB-C18 peptide tracks by an autosampler and separated by a liquid chromatography Column.
Mass spectrum identification: carrying out mass spectrometry on the enzymolysis product after capillary high performance liquid chromatography separation by using a mass spectrometer, wherein the analysis time is as follows: and (5) 60 min. The detection mode is as follows: a positive ion. The mass-to-charge ratio of the polypeptide and fragments of the polypeptide was collected as follows: 10 fragment patterns were acquired after each full scan.
And (3) data analysis: the corresponding database is searched by adopting software MaxQuant 1.5.5.1, and finally mass spectrometry is carried out by LC-MS/MS to obtain the hypoglycemic octapeptide FPSPPLVA, wherein the HPLC chromatogram and the mass spectrogram are shown in figures 1 and 2.
Example 1:
a preparation method of hypoglycemic sea cucumber peptide with alpha-amylase inhibition activity comprises the following steps:
(1) enzymolysis:
selecting papain, alkaline protease and bromelain, and mixing the selected materials in a ratio of 1.2: 1: 0.8, mixing the sea cucumbers according to the mass ratio of 1: 20, preparing the sea cucumber homogenate, and performing multi-enzyme synergistic hydrolysis on the sea cucumber homogenate according to the enzyme adding amount of 8000u/g protein. Wherein the enzymolysis parameters are as follows: the enzymolysis pH is 7.8, the enzymolysis temperature is 50 ℃, and the enzymolysis time is 5 h.
(2) And (3) ultrafiltration membrane separation:
adding the sea cucumber peptide solution obtained in the step (1) into an ultrafiltration cup of an ultrafiltration system, installing an ultrafiltration membrane package with the molecular weight cutoff of 1kDa, carrying out ultrafiltration under the controlled pressure of 20-30 psig, collecting filtrate, and freeze-drying.
(3) Preparing liquid phase chromatographic separation:
and (3) dissolving the freeze-dried powder obtained in the step (2) in ultrapure water, and separating and purifying the sea cucumber peptide subjected to ultrafiltration by using a preparative liquid chromatography. The chromatographic column is Agilent C-18, the detection wavelength of an ultraviolet detector is 280nm, the mobile phase A is ultrapure water containing 0.1 percent of trifluoroacetic acid, and the mobile phase B is acetonitrile containing 0.1 percent of trifluoroacetic acid. Samples were injected at a concentration of 10mg/mL for 20. mu.L.
(4) High-speed countercurrent chromatographic separation:
firstly, pumping the stationary phase into a separation column of a countercurrent chromatograph, starting a constant-temperature circulating water bath to adjust the temperature to 25 ℃, then starting a main machine of the countercurrent chromatograph, slowly adjusting the rotating speed of the main machine from low to high, and pumping the mobile phase from the head end to the tail end of a separation pipe. And (3) when the two-phase system achieves hydrodynamic balance and stability in the separation column, injecting the sample obtained in the step (3) prepared in advance into a sample loop for sample injection, finally starting an ultraviolet detector and a chromatographic workstation, detecting at 220nm, and collecting fractions after secondary separation according to a map to obtain the hypoglycemic sea cucumber peptide with alpha-amylase inhibitory activity.
Wherein, the high-speed counter-current chromatographic separation is carried out twice separation chromatographic operations: the first separation chromatographic conditions are as follows: the volume ratio of water/n-butanol/ethyl acetate/acetic acid is 5: 3: 2: 0.5, the upper phase of the solvent system is a stationary phase, and the lower phase is a mobile phase; the rotation speed is 800-900rpm, the flow rate is 4mL/min, elution is carried out from head to tail, the detection wavelength is 220nm, the sample introduction volume is 10mL, and the sample concentration is 5 mg/mL; the chromatographic conditions for the second separation are as follows: the volume ratio of water/n-butanol/ethyl acetate/triethylamine is 4: 4: 1: 0.5, and other conditions are the same as the first separation.
(5) And (3) drying:
and (4) concentrating the filtrate obtained in the step (4) to a proper amount, and freeze-drying to obtain the hypoglycemic sea cucumber peptide powder with good solubility.
The embodiment also provides the blood sugar reducing sea cucumber peptide prepared by the preparation method.
And (3) measuring the main components of the sea cucumber bioactive peptide powder with alpha-amylase inhibition activity obtained in the step (5) by LC-MS/MS and HPLC (figure 1 and figure 2), wherein the content of the peptide with the amino acid sequence of FPSPPLVA is 95.65%.
Example 2:
a preparation method of hypoglycemic sea cucumber peptide with alpha-amylase inhibition activity comprises the following steps:
(1) enzymolysis:
selecting papain, alkaline protease and bromelain, and mixing the selected materials in a ratio of 1.2: 1: 0.8, mixing the sea cucumbers according to the mass ratio of 1: 20, preparing the sea cucumber homogenate, and performing multi-enzyme synergistic hydrolysis on the sea cucumber homogenate according to the enzyme adding amount of 8000u/g protein. Wherein the enzymolysis parameters are as follows: the enzymolysis pH is 8.2, the enzymolysis temperature is 55 ℃, and the enzymolysis time is 6 h.
(2) And (3) ultrafiltration membrane separation:
adding the sea cucumber peptide solution obtained in the step (1) into an ultrafiltration cup of an ultrafiltration system, installing an ultrafiltration membrane package with the molecular weight cutoff of 1kDa, carrying out ultrafiltration under the controlled pressure of 20-30 psig, collecting filtrate, and freeze-drying.
(3) Preparing liquid phase chromatographic separation:
and (3) dissolving the freeze-dried powder obtained in the step (2) in ultrapure water, and separating and purifying the sea cucumber peptide subjected to ultrafiltration by using a preparative liquid chromatography. The chromatographic column is Agilent C-18, the detection wavelength of an ultraviolet detector is 280nm, the mobile phase A is ultrapure water containing 0.1 percent of trifluoroacetic acid, and the mobile phase B is acetonitrile containing 0.1 percent of trifluoroacetic acid. Samples were injected at a concentration of 10mg/mL for 20. mu.L.
(4) High-speed countercurrent chromatographic separation:
firstly, pumping the stationary phase into a separation column of a countercurrent chromatograph, starting a constant-temperature circulating water bath to adjust the temperature to 25 ℃, then starting a main machine of the countercurrent chromatograph, slowly adjusting the rotating speed of the main machine from low to high, and pumping the mobile phase from the head end to the tail end of a separation pipe. And (3) when the two-phase system achieves hydrodynamic balance and stability in the separation column, injecting the sample obtained in the step (3) prepared in advance into a sample loop for sample injection, finally starting an ultraviolet detector and a chromatographic workstation, detecting at 220nm, and collecting fractions after secondary separation according to a map to obtain the hypoglycemic sea cucumber peptide with alpha-amylase inhibitory activity.
Wherein, the high-speed counter-current chromatographic separation is carried out twice separation chromatographic operations: the first separation chromatographic conditions are as follows: the volume ratio of water/n-butanol/ethyl acetate/acetic acid is 5: 3: 2: 0.5, the upper phase of the solvent system is a stationary phase, and the lower phase is a mobile phase; the rotation speed is 800-900rpm, the flow rate is 4mL/min, elution is carried out from head to tail, the detection wavelength is 220nm, the sample introduction volume is 10mL, and the sample concentration is 5 mg/mL; the chromatographic conditions for the second separation are as follows: the volume ratio of water/n-butanol/ethyl acetate/triethylamine is 4: 4: 1: 0.5, and other conditions are the same as the first separation.
(5) And (3) drying:
and (4) concentrating the filtrate obtained in the step (4) to a proper amount, and freeze-drying to obtain the hypoglycemic sea cucumber peptide powder with good solubility.
The embodiment also provides the blood sugar reducing sea cucumber peptide prepared by the preparation method.
And (3) measuring the main components of the sea cucumber bioactive peptide powder with alpha-amylase inhibiting activity obtained in the step (5) by LC-MS/MS and HPLC (figure 1 and figure 2). The content of the peptide whose amino acid sequence was FPSPPLVA was 97.39%.
Example 3:
a preparation method of hypoglycemic sea cucumber peptide with alpha-amylase inhibition activity comprises the following steps:
(1) enzymolysis:
selecting papain, alkaline protease and bromelain, and mixing the selected materials in a ratio of 1.2: 1: 0.8, mixing the sea cucumbers according to the mass ratio of 1: 20, preparing the sea cucumber homogenate, and performing multi-enzyme synergistic hydrolysis on the sea cucumber homogenate according to the enzyme adding amount of 8000u/g protein. Wherein the enzymolysis parameters are as follows: the enzymolysis pH is 8.0, the enzymolysis temperature is 50-55 deg.C, and the enzymolysis time is 5-6 h.
(2) And (3) ultrafiltration membrane separation:
adding the sea cucumber peptide solution obtained in the step (1) into an ultrafiltration cup of an ultrafiltration system, installing an ultrafiltration membrane package with the molecular weight cutoff of 1kDa, carrying out ultrafiltration under the controlled pressure of 20-30 psig, collecting filtrate, and freeze-drying.
(3) Preparing liquid phase chromatographic separation:
and (3) dissolving the freeze-dried powder obtained in the step (2) in ultrapure water, and separating and purifying the sea cucumber peptide subjected to ultrafiltration by using a preparative liquid chromatography. The chromatographic column is Agilent C-18, the detection wavelength of an ultraviolet detector is 280nm, the mobile phase A is ultrapure water containing 0.1 percent of trifluoroacetic acid, and the mobile phase B is acetonitrile containing 0.1 percent of trifluoroacetic acid. Samples were injected at a concentration of 10mg/mL for 20. mu.L.
(4) High-speed countercurrent chromatographic separation:
firstly, pumping the stationary phase into a separation column of a countercurrent chromatograph, starting a constant-temperature circulating water bath to adjust the temperature to 25 ℃, then starting a main machine of the countercurrent chromatograph, slowly adjusting the rotating speed of the main machine from low to high, and pumping the mobile phase from the head end to the tail end of a separation pipe. And (3) when the two-phase system achieves hydrodynamic balance and stability in the separation column, injecting the sample obtained in the step (3) prepared in advance into a sample loop for sample injection, finally starting an ultraviolet detector and a chromatographic workstation, detecting at 220nm, and collecting fractions after secondary separation according to a map.
Wherein, the high-speed counter-current chromatographic separation is carried out twice separation chromatographic operations: the first separation chromatographic conditions are as follows: the volume ratio of water/n-butanol/ethyl acetate/acetic acid is 5: 3: 2: 0.5, the upper phase of the solvent system is a stationary phase, and the lower phase is a mobile phase; the rotation speed is 800-900rpm, the flow rate is 4mL/min, elution is carried out from head to tail, the detection wavelength is 220nm, the sample introduction volume is 10mL, and the sample concentration is 5 mg/mL; the chromatographic conditions for the second separation are as follows: the volume ratio of water/n-butanol/ethyl acetate/triethylamine is 4: 4: 1: 0.5, separating the mixture for the first time under other conditions to obtain the hypoglycemic sea cucumber peptide with alpha-amylase inhibitory activity.
(5) And (3) drying:
and (4) concentrating the filtrate obtained in the step (4) to a proper amount, and freeze-drying to obtain the hypoglycemic sea cucumber peptide powder with good solubility.
The embodiment also provides the sea cucumber peptide prepared by the preparation method.
And (3) measuring the main components of the sea cucumber bioactive peptide powder with alpha-amylase inhibition activity obtained in the step (5) by LC-MS/MS and HPLC (figure 1 and figure 2), wherein the content of the peptide with the amino acid sequence of FPSPPLVA is 98.15%.
In order to determine the effectiveness of the preparation method of the present invention, experiments were performed on the use and purification methods of protease, table 1 is a table showing the synergistic effect of different proteases, table 3 is a table showing the synergistic effect of separation and purification of sea cucumber peptides, and it can be seen from table 1 that when a sea cucumber homogenate is enzymatically hydrolyzed using a single enzyme, the α -amylase inhibition experiment shows that, under the same conditions of enzymatic hydrolysis parameters as in the examples, the α -amylase inhibition ratio of three hydrolysates, namely papain, alkaline protease and bromelain, does not exceed 20% at a concentration of 3mg/mL, which is consistent with the present invention when papain, alkaline protease and bromelain are simultaneously used according to a ratio of 1.2: 1: when the mass ratio of 0.8 is compounded, the inhibition rate of the obtained blood sugar-reducing sea cucumber peptide on alpha-amylase is up to more than 80% under the same concentration, and the inhibition rate of the obtained alpha-amylase is obviously higher than that of single treatment.
Since different enzymolysis conditions can affect the enzymolysis effect of the sea cucumber, in order to further determine the effectiveness of the invention, the comparative examples in table 1 are tested under respective optimum enzymolysis parameters, and the results are shown in table 2. As can be seen from the table, compared with Table 1, when three proteases are subjected to single enzymolysis under the optimal conditions, the inhibition rate of the alpha-amylase is slightly improved, but still far lower than the inhibition effect of the hypoglycemic sea cucumber peptide provided by the invention.
It can be seen that in the method of the present invention, the ratio of papain, alkaline protease and bromelain is 1.2: 1: the mass ratio of 0.8 is compounded, the synergistic effect is achieved when the composition is used, the effect of the three enzymes when the composition is used simultaneously is greatly higher than that of the three enzymes when the composition is used singly, and the three enzymes have the synergistic effect.
As can be seen from Table 3, when the preparative chromatography of the present invention is used in combination with HSCCC, the α -amylase inhibitory effect and the content of FPSPPLVA peptide chain are significantly improved, and the content of FPSPPLVA peptide chain is as high as 98.15%, compared to the case where no (comparative example 8) and a single purification method are used.
Therefore, the method has the advantages that the effect of synergy is achieved when the liquid chromatogram and the HSCCC are prepared and used simultaneously, the effect of the liquid chromatogram and the HSCCC is greatly higher when the liquid chromatogram and the HSCCC are prepared and used simultaneously than when the liquid chromatogram and the HSCCC are not used and used singly, and the synergistic effect is achieved when the liquid chromatogram and the HSCCC are prepared.
TABLE 1 synergistic interaction of proteases in the method of the invention
TABLE 2 Table of synergistic Effect under optimal conditions for different proteases
TABLE 3 Table of synergy of separation and purification in the method of the present invention
Experimental example 4: alpha-amylase inhibition activity determination of blood sugar reducing sea cucumber peptide
Mixing 125 mu L of 25mmol/L alpha-amylase solution and 150 mu L of hypoglycemic peptide in a test tube, uniformly mixing, reacting in a water bath at 37 ℃ for 10min, adding 625 mu L of 0.08% soluble starch water solution, continuing to react in the water bath at 37 ℃ for 15min, taking the test tube out of the water bath, adding 300 mu L of 1.0mol/L hydrochloric acid solution to terminate the reaction, adding 200 mu L of 0.01mol/L iodine solution to mix with the reaction solution for color development, diluting the final volume of the reaction solution to 5mL with water, adjusting the volume to zero by using a blank tube, and measuring the absorbance at the wavelength of 660 nm.
According to the alpha-amylase inhibitory activity experiment (figure 3), the blood sugar reducing sea cucumber peptide has good alpha-amylase inhibitory activity and is increased in a dose-dependent manner, when the concentration is 3mg/mL, the alpha-amylase inhibitory rate reaches 84.57%, the IC50 is calculated to be 414.23 mu g/mL, and then the blood sugar reducing sea cucumber peptide obtained by enzymolysis is further researched.
Experimental example 5: structure and site of action analysis
The receptor protein alpha-amylase and the polypeptide FPSPPLVA are subjected to semi-flexible docking by using the AutoDock, after 50 times of docking, the optimal docking conformation is selected, the docking binding energy is-9.61 KJ/mol, and the key amino acid and the interaction force of the receptor protein alpha-amylase and the polypeptide FPSPPLVA are determined.
FIG. 4 is the docking result of the interaction of the polypeptide FPSPPLVA (FA8) with the alpha-amylase (3BAJ) molecule.
As can be seen in fig. 4a and 4c, FA8 forms 5 hydrogen bonds with Glu63, Thr163, Lys200, His201, and there are 6 hydrophobic interactions. As can be seen from the number and length of hydrogen bonds and the result of hydrophobic interaction, the FA8 has obvious interaction with AAM and can effectively inhibit enzyme activity.
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, alterations and modifications are possible without departing from the spirit and scope of this disclosure and appended claims, and accordingly, the scope of this disclosure is not limited to the embodiments disclosed.
Claims (9)
1. A hypoglycemic sea cucumber peptide with alpha-amylase inhibition activity, which is characterized in that: the blood sugar reducing sea cucumber peptide consists of the following eight amino acids: phenylalanine-proline-serine-proline-leucine-valine-alanine, abbreviated as FPSPPLVA, has a molecular weight of 826.45 Da.
2. The hypoglycemic sea cucumber peptide with alpha-amylase inhibitory activity according to claim 1, wherein: alpha-amylase inhibition rate and IC of the hypoglycemic sea cucumber peptide 50 It was 414.23. mu.g/mL.
3. The hypoglycemic sea cucumber peptide having alpha-amylase inhibitory activity according to claim 1 or 2, characterized in that: it is clear through a molecular docking technology that the hypoglycemic sea cucumber peptide forms 5 hydrogen bonds with Glu63, Thr163, Lys200 and His201 residues of alpha-amylase, and 6 hydrophobic effects exist.
4. The method for producing a hypoglycemic sea cucumber peptide having α -amylase inhibitory activity according to any one of claims 1 to 3, wherein: the method comprises the following steps:
A. enzymolysis: selecting papain, alkaline protease and bromelain, and mixing the selected materials in a ratio of 1.2: 1: 0.8, mixing the sea cucumbers according to the mass ratio of 1: preparing a sea cucumber homogenate according to the material-liquid ratio of 20, and performing multi-enzyme synergistic hydrolysis on the sea cucumber homogenate according to the enzyme adding amount of 8000u/g protein;
B. and (3) ultrafiltration membrane separation: adding the enzymatic hydrolysate obtained in the step A into an ultrafiltration cup of an ultrafiltration system, installing an ultrafiltration membrane package with the molecular weight cutoff of 1kDa, carrying out ultrafiltration under the pressure of 20-30 psig, collecting filtrate, and freeze-drying;
C. preparing liquid phase chromatographic separation: dissolving the freeze-dried powder obtained in the step B in ultrapure water, and separating and purifying by using a preparative liquid chromatography;
D. high-speed countercurrent chromatographic separation: injecting the sample obtained in the step C into a sample loop for sample injection, finally starting an ultraviolet detector and an HSCCC workstation, detecting at 220nm, collecting fractions after secondary separation according to a spectrum, and obtaining the blood sugar-reducing sea cucumber peptide with alpha-amylase inhibitory activity.
5. The method for preparing a hypoglycemic sea cucumber peptide with alpha-amylase inhibitory activity according to claim 4, wherein the peptide comprises: the enzymolysis parameters of the multi-enzyme synergistic hydrolysis in the step A are as follows: the pH value of enzymolysis is 7.8-8.2, the enzymolysis temperature is 50-55 ℃, and the enzymolysis time is 5-6 h.
6. The method for preparing a hypoglycemic sea cucumber peptide with alpha-amylase inhibitory activity according to claim 4, wherein the peptide comprises: and C, preparing a chromatographic column of the liquid chromatogram by Agilent C-18, wherein the detection wavelength of an ultraviolet detector of the prepared liquid chromatogram is 280nm, a mobile phase A of the prepared liquid chromatogram is ultrapure water containing 0.1 percent of trifluoroacetic acid, and a mobile phase B of the prepared liquid chromatogram is acetonitrile containing 0.1 percent of trifluoroacetic acid.
7. The method for preparing a hypoglycemic sea cucumber peptide with alpha-amylase inhibitory activity according to claim 4, wherein the peptide comprises: and C, the mass ratio of the freeze-dried powder to the ultrapure water in the step C is 1:10-1.2: 10.
8. The method for producing a hypoglycemic sea cucumber peptide having α -amylase inhibitory activity according to any one of claims 4 to 7, wherein: and D, performing two times of separation chromatographic operations in the high-speed countercurrent chromatographic separation: the first separation chromatographic conditions are as follows: the volume ratio of water/n-butanol/ethyl acetate/acetic acid is 5: 3: 2: 0.5, the upper phase of the solvent system is a stationary phase, and the lower phase is a mobile phase; the rotation speed is 800-900rpm, the flow rate is 4mL/min, elution is carried out from head to tail, the detection wavelength is 220nm, the sample introduction volume is 10mL, and the sample concentration is 5 mg/mL; the chromatographic conditions for the second separation are as follows: the volume ratio of water/n-butanol/ethyl acetate/triethylamine is 4: 4: 1: 0.5, and other conditions are the same as the first separation.
9. The use of the hypoglycemic sea cucumber peptide with alpha-amylase inhibitory activity according to any one of claims 1 to 3 in food, health products, and pharmaceuticals.
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| CN117285596A (en) * | 2023-11-27 | 2023-12-26 | 中国科学院烟台海岸带研究所 | Hexapeptide RL6 with uric acid reducing function and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2006328017A (en) * | 2005-05-30 | 2006-12-07 | Nagata Sangyo Kk | PROTEIN HAVING alpha-AMYLASE INHIBITORY ACTIVITY |
| CN112795613A (en) * | 2021-04-06 | 2021-05-14 | 西北大学 | Peony seed meal-derived blood sugar-reducing polypeptide and application thereof |
| CN113025678A (en) * | 2021-04-22 | 2021-06-25 | 昆明理工大学 | Method for screening hypoglycemic peptide capable of inhibiting alpha-amylase activity |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006328017A (en) * | 2005-05-30 | 2006-12-07 | Nagata Sangyo Kk | PROTEIN HAVING alpha-AMYLASE INHIBITORY ACTIVITY |
| CN112795613A (en) * | 2021-04-06 | 2021-05-14 | 西北大学 | Peony seed meal-derived blood sugar-reducing polypeptide and application thereof |
| CN113025678A (en) * | 2021-04-22 | 2021-06-25 | 昆明理工大学 | Method for screening hypoglycemic peptide capable of inhibiting alpha-amylase activity |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117285596A (en) * | 2023-11-27 | 2023-12-26 | 中国科学院烟台海岸带研究所 | Hexapeptide RL6 with uric acid reducing function and application thereof |
| CN117285596B (en) * | 2023-11-27 | 2024-01-26 | 中国科学院烟台海岸带研究所 | Hexapeptide RL6 with uric acid reducing function and application thereof |
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