CN112553279A - Method for extracting hypoglycemic peptide from tea seed meal - Google Patents
Method for extracting hypoglycemic peptide from tea seed meal Download PDFInfo
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Images
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- 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
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
-
- 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
- C07K1/30—Extraction; Separation; Purification by precipitation
-
- 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
Abstract
A method for extracting hypoglycemic peptide from tea seed meal comprises the following steps: (1) pulverizing tea seed cake. (2) Tea seed meal is degreased. (3) And (3) extracting tea seed meal protein. (4) Preparing tea seed meal crude peptide. (5) Separating and screening the alpha-glucosidase inhibitory peptides. (6) And (3) structural identification of the alpha-glucosidase inhibitory peptide. Through the steps, the invention prepares three alpha-glucosidase inhibitory peptides, the amino acid sequences of which are a (GHSLESIK), b (GLTSLDRYK) and c (SPGYYDGR) in sequence, has very high inhibitory activity to alpha-glucosidase, and has very wide application prospect in the development of blood sugar reducing products. The raw materials related by the invention are cheap and have wide sources, and the prepared alpha-glucosidase inhibitory peptide has high activity and wide application prospect.
Description
Technical Field
The invention relates to a method for extracting hypoglycemic peptide from tea seed meal, belonging to the technical field of deep processing of agricultural products.
Background
In recent years, the number of people with diabetes in China is increasing. Medication is the primary means of controlling diabetes. For a long time, alpha-glucosidase inhibitors, insulin and some biguanide drugs are the most common hypoglycemic drugs in China. The drugs have large side effects or need to be injected in vitro, and the like, so the pain of patients is increased in the process of reducing blood sugar. Therefore, attempts to control and treat diabetes using dietary therapy are currently the focus of research. Among them, dietary polypeptides having a hypoglycemic effect are attracting much attention due to their wide sources and variety.
The tea seed meal is a main byproduct in tea oil processing, is low in price, is rich in components such as protein and the like, and is one of ideal sources of dietary polypeptide. Our studies have shown that mixtures of tea seed meal polypeptides have good α -glucosidase inhibitory activity, but the specific structure of the tea seed meal polypeptide that plays a major role is not clear. The action mechanism research of the alpha-glucosidase inhibitory peptide which restricts the tea seed meal source influences the application of the alpha-glucosidase inhibitory peptide in the development of hypoglycemic drugs.
Disclosure of Invention
The invention aims to provide a method for extracting hypoglycemic peptide from tea seed meal.
In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a method for extracting hypoglycemic peptide from tea seed meal is characterized in that: comprises the following steps:
step 1: crushing tea seed meal to obtain tea seed meal powder;
step 2: mixing tea seed meal powder with n-hexane according to the weight ratio of 1: 4-6, stirring the mixed solution, carrying out suction filtration to remove n-hexane, and drying the obtained powder at room temperature to obtain degreased tea seed meal powder;
and step 3: mixing the degreased tea seed meal powder with water according to the weight ratio of 1: 7-15, then adjusting the pH value to 9.5-10.5, heating the mixture to 45-60 ℃, then stirring for 1-2, then centrifuging for 10-20min at 9000r/min 7000-;
and 4, step 4: dispersing tea seed meal protein in water to prepare a tea seed meal protein solution with the mass fraction of 2-4%, then adding alkaline protease into the tea seed meal protein solution, carrying out enzymolysis under the conditions that the temperature is 50-60 ℃ and the pH value is 9.0-11.0, adjusting the pH value of a reaction system to 7.0 after the enzymolysis is finished, and heating in a boiling water bath for 10-15 min for enzyme deactivation; centrifuging the solution after enzyme deactivation for 10-20min at 7000-12000 r/min to obtain supernatant, namely the crude tea seed meal polypeptide sample;
and 5: sequentially passing the crude tea seed meal polypeptide sample through ultrafiltration membranes of 10KDa, 5KDa and 3KDa under the conditions that the working pressure is 0.1-0.2 Mpa and the working temperature is 0-40 ℃, collecting 4 samples, and freeze-drying to obtain a polypeptide component of more than 10KDa, a polypeptide component of 5-10KDa, a polypeptide component of 3-5KDa and a polypeptide component of less than 3 KDa;
step 6: respectively measuring the activity of the polypeptide component of >10KDa, the polypeptide component of 5-10KDa, the polypeptide component of 3-5KDa and the polypeptide component of <3KDa for inhibiting alpha-glucosidase;
and 7: performing fine grading separation on the polypeptide component with the highest inhibition rate; the fine-classification separation method comprises the following steps: separating the polypeptide fraction with the highest inhibitory rate obtained by screening in step 6 by preparative high performance liquid chromatography or semi-preparative high performance liquid chromatography, using acetonitrile containing 0.1% TFA as eluent at a flow rate of 1L/min, and detecting the peak at 220 nm; concentrating and volatilizing the organic solvent, freeze-drying, measuring the activity of each freeze-dried component for inhibiting alpha-glucosidase, and screening out the polypeptide component with the highest inhibition rate;
and 8: preparing the polypeptide component with the highest inhibition rate screened in the step 7 into a polypeptide solution with the concentration of more than or equal to 3mg/mL, centrifuging for 8-12min at 10000-; the peptide fragment sample is absorbed by an autosampler, then is combined with a C18 capture column, and then is eluted to an analysis column for separation; establishing an analytical gradient for 30 minutes using two mobile phases; the flow rate of the liquid phase was set at 300 nL/min; mass spectrometry IDA mode analysis, each scan cycle comprises one MS full scan followed by 40 MS/MS scans; the MS/MS collection conditions are set as that the parent ion signal is greater than 120cps, and the charge number is + 2- + 5; the exclusion time of repeated ion collection is set to be 18 s; two mobile phases A: H2O, 0.1% formic acid; and the mobile phase B comprises CAN and 0.1% formic acid.
The preferable technical scheme is as follows: in the step 1, the oil content of the tea seed meal is 3-4%, and the protein content is 29-32%;
the preferable technical scheme is as follows: the stirring speed in the step 2 and the step 3 is 200-400 r/min.
The preferable technical scheme is as follows: the conditions of freeze drying in step 3, step 5 and step 7 are as follows: the vacuum degree is 50Pa, the temperature of the cold trap is-50 ℃, and the temperature of the material is-20 ℃.
Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:
the raw materials related by the invention are cheap and have wide sources, and the prepared alpha-glucosidase inhibitory peptide has high activity and wide application prospect.
Drawings
FIG. 1 is a schematic diagram of the preparation process of the tea seed meal hypoglycemic peptide of the present invention.
FIG. 2 is a graph showing the inhibition of alpha-glucosidase by polypeptides of different molecular weights.
FIG. 3 shows the inhibitory activity of polypeptides on alpha-glucosidase at different stages of preparation.
FIG. 4 is a diagram of a model of molecular docking of three polypeptides with alpha-glucosidase.
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.
Please refer to fig. 1-4. It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are only used for matching with the disclosure of the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions under which the present invention can be implemented, so that the present invention has no technical significance, and any structural modification, ratio relationship change, or size adjustment should still fall within the scope of the present invention without affecting the efficacy and the achievable purpose of the present invention. In addition, the terms "upper", "lower", "left", "right", "middle" and "one" used in the present specification are for clarity of description, and are not intended to limit the scope of the present invention, and the relative relationship between the terms and the terms is not to be construed as a scope of the present invention.
Example 1: method for extracting hypoglycemic peptide from tea seed meal
A method for extracting hypoglycemic peptide from tea seed meal comprises the following steps:
(1) preparing tea seed meal powder. Crushing the tea seed meal by a crusher at 10000r/min, pausing for 1min every 2min, and after cumulative crushing for 4min, sieving the obtained powder by a 60-mesh sieve to obtain the tea seed meal powder at the undersize part.
(2) Preparing defatted tea seed powder. According to the following steps: 5, mixing the tea seed meal powder with n-hexane, stirring the mixed solution for 3 hours on a magnetic stirrer, and performing suction filtration to remove the n-hexane to obtain powder, and drying the powder at room temperature to obtain the degreased tea seed meal powder.
(3) Preparing the tea seed meal protein. The mass ratio of the degreased tea seed meal powder to water is 1: 10, adjusting the pH value to 10 after mixing; magnetically stirring the mixture at 50 ℃ for 1.5h, centrifuging at 8000r/min for 15min, adjusting pH of the obtained supernatant to 4.2 with 1mol/L HCl, centrifuging at 8000r/min for 15min after 1.5h, resuspending the obtained precipitate with deionized water, and freeze-drying to obtain the tea seed meal protein.
(4) Preparing a crude tea seed meal polypeptide sample. Preparing a tea seed meal protein solution with the mass fraction of 3%, carrying out enzymolysis by using alkaline protease, and adding the alkaline protease 9000U/g and the pH value of 11; the temperature is 55 ℃; and magnetically stirring for 3h for enzymolysis. After the reaction is finished, adjusting the pH value to 7.0 and heating in a boiling water bath for 12min for enzyme deactivation; centrifuging the obtained solution at 10000r/min for 15min, and obtaining the supernatant which is the crude tea seed meal polypeptide sample.
(5) And (3) grading the tea seed meal polypeptide. The obtained crude tea seed meal polypeptide sample sequentially passes through 10000Da, 5000Da and 3000Da ultrafiltration membranes under the conditions that the working pressure is 0.15Mpa and the working temperature is 20 ℃, then 4 samples are collected, and polypeptide components with the KD of more than 10KD, 5-10KD, 3-5KD and less than 3KD are obtained after freeze drying.
(6) The tea seed meal polypeptide has hypoglycemic activity. The hypoglycemic activity of the polypeptide is generally expressed using the inhibitory activity of alpha-glucosidase. Determining the inhibition rate of a polypeptide fraction of >10KDa, a polypeptide fraction of 5-10KDa, a polypeptide fraction of 3-5KDa and a polypeptide fraction of <3 KDa:
the assay was performed in a 96-well microplate, to which 112. mu.L of phosphate buffer (pH6.8) was first added. In addition, 20. mu.L of a 0.2U/mL alpha-glucosidase solution was added, followed by mixing 8. mu.L of the sample solution. After incubation at 37 ℃ for 30min, 20. mu.L of p-nitrophenyl-alpha-D-glucoside (pNPG) (5mmol/L) was added to initiate the reaction, followed by incubation at 37 ℃ for 30min, followed by addition of 80. mu.L of 0.02mol/L Na2CO3The reaction was stopped by dissolving. The amount of released p-nitrophenol (pNP) was measured at 405nm using an enzyme marker (Multiskan FC with Incubator, Japan). Inhibition assays were performed in triplicate for all tests.
Respectively measuring the inhibition activity of the classified tea seed meal polypeptide on alpha-glucosidase, and expressing the inhibition rate as follows:
the inhibition rate calculation method is as follows:
wherein A represents the absorbance of p-nitrophenol-alpha-D-glucopyranoside (pNPG), alpha-glucosidase and the polypeptide sample to be tested in a phosphate buffer; a. the0Is the absorbance without sample; a. the1Is the absorbance of the blank sample; a. the2The glucosidase and sample are included, but the pNPG is not included.
(7) The polypeptide fractions having the highest inhibition of α -glucosidase were fractionated, acetonitrile (0-40%, 40 min) containing 0.1% TFA was used as eluent, the flow rate was set to 1mL/min, and the peak of elution was detected at 220 nm.
After the determination by the method, the component with the highest inhibition rate to the alpha-glucosidase is used for further screening of the alpha-glucosidase inhibiting peptide.
(8) The fine separation of alpha-glucosidase inhibitory peptide in tea seed meal polypeptide. Separating the fractions screened in step (5) using preparative or semi-preparative High Performance Liquid Chromatography (HPLC). And optimizing HPLC separation according to the characteristics of the sample, separating main chromatographic peaks in the sample from each other, continuously collecting the chromatographic peaks of the HPLC respectively, and numbering the chromatographic peaks in sequence. The establishment method comprises the following steps: acetonitrile (0-40%, 40 min) containing 0.1% TFA was used as an eluent, the flow rate was set to 1mL/min, and the peak was detected at 220 nm. Concentrating volatile organic solvent, and freeze drying. And (4) measuring the activity of inhibiting the alpha-glucosidase of each freeze-dried component by using the method in the step (6), and screening the component with the highest inhibition rate for identifying the alpha-glucosidase inhibiting peptide with a specific structure.
(9) And (3) carrying out structural identification on the alpha-glucosidase inhibiting peptide. And (3) centrifuging the polypeptide solution prepared from the sample screened in the step (8) at 12000r/min for 10min, filtering the supernatant by using a 10kDa ultrafiltration tube, desalting the filtrate by using a C18 column, drying the desalted peptide fragment solution by using a centrifugal concentrator (working conditions: the rotating speed is 2000r/min, and the concentration time is 2min), and freezing and storing the dried peptide fragment solution at-20 ℃ for detection.
The mass spectrometry was performed using a TripleTOF 5600 mass spectrometry system from SCIEX corporation. The peptide fragment sample was absorbed by an autosampler, bound to a C18 capture column (5 μm,5 × 0.3mm), and then eluted to an analytical column (75 μm × 150mm,3 μm particulate size,
pore size, Eksigent). Using two mobile phases (mobile phase A: H)2O, 0.1% formic acid and mobile phase B ACN, 0.1% formic acid) an analytical gradient was established for 30 minutes (5% B at 0min, 5-35% B within 15min, 35-80% B within 1min, 80% B for 5min, 80-5% B within 0.1min, 5% B for 8.9 min). The flow rate of the liquid phase was set at 300 nL/min. Mass spectrometry IDA mode analysis comprised one MS full scan (m/z range of 350-. The MS/MS collection conditions are set to be that the parent ion signal is greater than 120cps and the charge number is + 2- + 5. The exclusion time for repeated ion acquisitions was set at 18 s.
Further, mass spectrum data generated by TripleTOF 5600 was retrieved by ProteinPilot (V4.5) using the database retrieval algorithm, paramon. The database used for the search was the proteome reference database of Camellia in UniProt. And obtaining a specific sequence of the alpha-glucosidase inhibitory peptide by database retrieval and comparison.
The first hypoglycemic peptide consists of the following amino acid residues: glycine-histidine-serine-leucine-glutamic acid-serine-isoleucine-lysine; the second hypoglycemic peptide consists of the following amino acid residues: glycine-leucine-threonine-serine-leucine-aspartic acid-arginine-tyrosine-lysine; the third hypoglycemic peptide consists of the following amino acid residues: threonine-proline-glycine-tyrosine-aspartic acid-glycine-arginine.
(8) The mechanism of action of the alpha-glucosidase inhibitory peptides. And searching the crystal structure of the alpha-glucosidase from the PDB database, and performing molecular docking simulation by using Auto-dock software.
The magnetic stirring speed in the steps (2), (3) and (4) is 200-;
the conditions for freeze-drying in steps (3), (5) and (6) are as follows: the vacuum degree is 50Pa, the temperature of the cold trap is-50 ℃, and the temperature of the material is-20 ℃.
Example 2: method for extracting hypoglycemic peptide from tea seed meal
(1) Preparing tea seed meal powder. Crushing the tea seed meal by a crusher at 10000r/min, pausing for 1min every 2min, and after cumulative crushing for 4min, sieving the obtained powder by a 60-mesh sieve to obtain the tea seed meal powder.
(2) Preparing defatted tea seed powder. According to the following steps: 5, mixing the tea seed meal powder with n-hexane, stirring the mixed solution on a magnetic stirrer for 3 hours (the rotating speed is 300r/min), carrying out suction filtration to remove the n-hexane, and drying at room temperature to obtain the degreased tea seed meal powder.
(3) Preparing the tea seed meal protein. Mixing the degreased tea seed meal powder with water according to the weight ratio of 1: 10, adjusting the pH value to 10.0 after mixing; magnetically stirring the mixture at 55 ℃ for 1.5h, centrifuging at 8000r/min for 15min, adjusting pH of the obtained supernatant to 4.5 with 1mol/L HCl, stirring the solution for 1.5h, centrifuging at 8000r/min for 15min, and freeze-drying the obtained precipitate to obtain tea seed meal protein.
(4) Preparing a crude tea seed meal polypeptide sample. Preparing 4% tea seed meal protein solution, performing enzymolysis by using alkaline protease, and adding the alkaline protease 6000U/g and the pH value of the solution is 10.0; and (3) carrying out enzymolysis by magnetic stirring at the temperature of 55 ℃ for 2-4 h. After the reaction is finished, adjusting the pH value to 7.0 and heating in a boiling water bath for 15min for enzyme deactivation; and centrifuging the obtained solution for 10-20min at 8000r/min to obtain supernatant, namely the hypoglycemic peptide crude sample.
(5) And (3) grading the tea seed meal polypeptide. The obtained crude tea seed meal blood sugar reducing polypeptide sample sequentially passes through 10000Da, 5000Da and 3000Da ultrafiltration membranes under the conditions that the working pressure is 0.12Mpa and the working temperature is 25 ℃, 4 samples are collected and are subjected to freeze drying, and polypeptide components with the KD of more than 10KD, 5-10KD, 3-5KD and less than 3KD are obtained. Steps 3-5 are shown in scheme 1.
(6) The tea seed meal polypeptide has hypoglycemic activity. The hypoglycemic activity of the polypeptide is generally expressed using the inhibitory activity of alpha-glucosidase. Thus, the activity of the fractionated tea seed meal polypeptides in inhibiting α -glucosidase was measured as follows:
the activity of the tea seed meal polypeptide for inhibiting alpha-glucosidase is determined by the following method: to a 96-well plate, 112. mu.L of phosphate buffer (pH6.8) was added. In addition, 20. mu.L of 0.2u/mL alpha-glucosidase solution was added, followed by mixing 8. mu.L of the sample solution. Culturing at 37 deg.C for 30min, adding 20 μ L p-nitrophenol-alpha-D-glucopyranoside (pNPG, 5mmol/L) for initiation reaction, culturing at 37 deg.C for 30min, and adding 80 μ L0.02 mol/LNa2CO3The solution stops the reaction.
The inhibition rate calculation method is as follows:
wherein, A represents the absorbance of p-nitrophenol-alpha-D-glucopyranoside (pNPG), alpha-glucosidase and the sample in phosphate buffer; a. the0Is the absorbance without sample; a. the1Is the absorbance of the blank sample; a. the2The glucosidase and sample are included, but the pNPG is not included.
According to the method, 4 tea seed meal polypeptides are obtained in the embodiment, namely polypeptide components with the KD of more than 10KD, 5-10KD, 3-5KD and <3KD are obtained, and the maximum inhibition rates of the polypeptide components on alpha-glucosidase are respectively 20.64% (6mg/mL), 33.55% (5mg/mL), 45.6% (5mg/mL) and 59.16% (6mg/mL), so that the polypeptide components have obvious hypoglycemic potential and can be used for developing different types of hypoglycemic products.
(1) The invention fully considers the relationship between the hydrolysis degree and the hydrolysis time, and gives consideration to the hydrolysis degree and the biological activity of the polypeptide to the maximum extent.
(2) The invention has simple operation and high polypeptide yield.
(3) The ultrafiltration technology is a membrane separation technology taking pressure as a driving force, separates macromolecules and micromolecules according to the molecular weight of the substances, has simple and convenient operation and low cost, does not need to add any chemical substances, and does not denature and inactivate the molecules. The ultrafiltration technology adopted by the invention ensures the biological activity of the polypeptide to the utmost extent.
(4) At present, China is stepping into an aging society, and meanwhile, the group of obese and sedentary office workers is getting bigger, and the high incidence of diabetes mellitus cannot be ignored. The biological industry develops vigorously, and the research on the tea seed meal hypoglycemic peptide has important significance and development prospect for subsequent processing into a series of functional foods, beverages or capsule type health-care foods or medicines.
Example 3: method for extracting hypoglycemic peptide from tea seed meal
A method for extracting hypoglycemic peptide from tea seed meal is characterized in that: comprises the following steps:
step 1: crushing tea seed meal to obtain tea seed meal powder;
step 2: mixing tea seed meal powder with n-hexane according to the weight ratio of 1: 4, stirring the mixed solution, carrying out suction filtration to remove n-hexane, and drying the obtained powder at room temperature to obtain degreased tea seed meal powder;
and step 3: mixing the degreased tea seed meal powder with water according to the weight ratio of 1: 7, then adjusting the pH value to 9.5, heating the mixture to 45 ℃, then stirring the mixture for 1, then centrifuging the mixture for 10min at 7000r/min to obtain a supernatant, adjusting the pH value of the obtained supernatant to 4.0, centrifuging the obtained supernatant for 14min at 7000r/min after 1h, resuspending the obtained precipitate with deionized water, and freeze-drying the resuspending the precipitate to obtain tea seed meal protein;
and 4, step 4: dispersing tea seed meal protein in water to prepare a tea seed meal protein solution with the mass fraction of 2%, then adding alkaline protease into the tea seed meal protein solution, carrying out enzymolysis under the conditions that the temperature is 50 ℃ and the pH value is 9.0, adjusting the pH value of a reaction system to 7.0 after the enzymolysis is finished, and heating in a boiling water bath for 10min for enzyme deactivation; centrifuging the solution after enzyme deactivation at 7000r/min for 10min to obtain supernatant, namely the crude tea seed meal polypeptide sample;
and 5: sequentially passing the crude tea seed meal polypeptide sample through ultrafiltration membranes of 10KDa, 5KDa and 3KDa under the conditions of working pressure of 0.1Mpa and working temperature of 0 ℃, collecting 4 samples, and freeze-drying to obtain a polypeptide component of more than 10KDa, a polypeptide component of 5-10KDa, a polypeptide component of 3-5KDa and a polypeptide component of less than 3 KDa;
step 6: respectively measuring the activity of the polypeptide component of >10KDa, the polypeptide component of 5-10KDa, the polypeptide component of 3-5KDa and the polypeptide component of <3KDa for inhibiting alpha-glucosidase;
and 7: performing fine grading separation on the polypeptide component with the highest inhibition rate; the fine-classification separation method comprises the following steps: separating the polypeptide fraction with the highest inhibitory rate obtained by screening in step 6 by preparative high performance liquid chromatography or semi-preparative high performance liquid chromatography, using acetonitrile containing 0.1% TFA as eluent at a flow rate of 1L/min, and detecting the peak at 220 nm; concentrating and volatilizing the organic solvent, freeze-drying, measuring the activity of each freeze-dried component for inhibiting alpha-glucosidase, and screening out the polypeptide component with the highest inhibition rate;
and 8: preparing the polypeptide component with the highest inhibition rate screened in the step 7 into a polypeptide solution with the concentration of more than or equal to 3mg/mL, then centrifuging for 8min at 10000r/min, taking the supernatant, filtering by using a 10kDa ultrafiltration tube, taking the filtrate, desalting by using a C18 column, and pumping the desalted peptide fragment solution through a centrifugal concentrator; the peptide fragment sample is absorbed by an autosampler, then is combined with a C18 capture column, and then is eluted to an analysis column for separation; establishing an analytical gradient for 30 minutes using two mobile phases; the flow rate of the liquid phase was set at 300 nL/min; mass spectrometry IDA mode analysis, each scan cycle comprises one MS full scan followed by 40 MS/MS scans; the MS/MS collection conditions are set as that the parent ion signal is greater than 120cps, and the charge number is + 2- + 5; the exclusion time of repeated ion collection is set to be 18 s; two mobile phases A: H2O, 0.1% formic acid; and the mobile phase B comprises CAN and 0.1% formic acid.
The preferred embodiment is: in the step 1, the oil content of the tea seed meal is 3 percent, and the protein content is 29 percent;
the preferred embodiment is: the stirring speed in the step 2 and the step 3 is 200 r/min.
The preferred embodiment is: the conditions of freeze drying in step 3, step 5 and step 7 are as follows: the vacuum degree is 50Pa, the temperature of the cold trap is-50 ℃, and the temperature of the material is-20 ℃.
The foregoing is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting thereof in any way, and any modifications or variations thereof that fall within the spirit of the invention are intended to be included within the scope thereof.
Claims (4)
1. A method for extracting hypoglycemic peptide from tea seed meal is characterized in that: comprises the following steps:
step 1: crushing tea seed meal to obtain tea seed meal powder;
step 2: mixing tea seed meal powder with n-hexane according to the weight ratio of 1: 4-6, stirring the mixed solution, carrying out suction filtration to remove n-hexane, and drying the obtained powder at room temperature to obtain degreased tea seed meal powder;
and step 3: mixing the degreased tea seed meal powder with water according to the weight ratio of 1: 7-15, then adjusting the pH value to 9.5-10.5, heating the mixture to 45-60 ℃, then stirring for 1-2, then centrifuging for 10-20min at 9000r/min 7000-;
and 4, step 4: dispersing tea seed meal protein in water to prepare a tea seed meal protein solution with the mass fraction of 2-4%, then adding alkaline protease into the tea seed meal protein solution, carrying out enzymolysis under the conditions that the temperature is 50-60 ℃ and the pH value is 9.0-11.0, adjusting the pH value of a reaction system to 7.0 after the enzymolysis is finished, and heating in a boiling water bath for 10-15 min for enzyme deactivation; centrifuging the solution after enzyme deactivation for 10-20min at 7000-12000 r/min to obtain supernatant, namely the crude tea seed meal polypeptide sample;
and 5: sequentially passing the crude tea seed meal polypeptide sample through ultrafiltration membranes of 10KDa, 5KDa and 3KDa under the conditions that the working pressure is 0.1-0.2 Mpa and the working temperature is 0-40 ℃, collecting 4 samples, and freeze-drying to obtain a polypeptide component of more than 10KDa, a polypeptide component of 5-10KDa, a polypeptide component of 3-5KDa and a polypeptide component of less than 3 KDa;
step 6: respectively measuring the activity of the polypeptide component of >10KDa, the polypeptide component of 5-10KDa, the polypeptide component of 3-5KDa and the polypeptide component of <3KDa for inhibiting alpha-glucosidase;
and 7: performing fine grading separation on the polypeptide component with the highest inhibition rate; the fine-classification separation method comprises the following steps: separating the polypeptide fraction with the highest inhibitory rate obtained by screening in step 6 by preparative high performance liquid chromatography or semi-preparative high performance liquid chromatography, using acetonitrile containing 0.1% TFA as eluent at a flow rate of 1L/min, and detecting the peak at 220 nm; concentrating and volatilizing the organic solvent, freeze-drying, measuring the activity of each freeze-dried component for inhibiting alpha-glucosidase, and screening out the polypeptide component with the highest inhibition rate;
and 8: preparing the polypeptide component with the highest inhibition rate screened in the step 7 into a polypeptide solution with the concentration of more than or equal to 3mg/mL, centrifuging for 8-12min at 10000-; the peptide fragment sample is absorbed by an autosampler, then is combined with a C18 capture column, and then is eluted to an analysis column for separation; establishing an analytical gradient for 30 minutes using two mobile phases; the flow rate of the liquid phase was set at 300 nL/min; mass spectrometry IDA mode analysis, each scan cycle comprises one MS full scan followed by 40 MS/MS scans; setting conditions of MS/MS collection to be that a parent ion signal is greater than 120cps and the charge number is +2 to + 5; the exclusion time of repeated ion collection is set to be 18 s; two mobile phases A: H2O, 0.1% formic acid; and the mobile phase B comprises CAN and 0.1% formic acid.
2. The method for extracting hypoglycemic peptide from tea seed meal according to claim 1, wherein the method comprises the steps of: in the step 1, the oil content of the tea seed meal is 3-4%, and the protein content is 29-32%.
3. The method for extracting hypoglycemic peptide from tea seed meal according to claim 1, wherein the method comprises the steps of: the stirring speed in the step 2 and the step 3 is 200-400 r/min.
4. The method for extracting hypoglycemic peptide from tea seed meal according to claim 1, wherein the method comprises the steps of: the conditions of freeze drying in step 3, step 5 and step 7 are as follows: the vacuum degree is 50Pa, the temperature of the cold trap is-50 ℃, and the temperature of the material is-20 ℃.
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| 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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