CN116479077A - Preparation method of high-activity tartary buckwheat albumin antihypertensive peptide - Google Patents
Preparation method of high-activity tartary buckwheat albumin antihypertensive peptide Download PDFInfo
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- CN116479077A CN116479077A CN202211364418.1A CN202211364418A CN116479077A CN 116479077 A CN116479077 A CN 116479077A CN 202211364418 A CN202211364418 A CN 202211364418A CN 116479077 A CN116479077 A CN 116479077A
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- Prior art keywords
- tartary buckwheat
- albumin
- peptide
- blood pressure
- ace
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Classifications
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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
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- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A61P9/00—Drugs for disorders of the cardiovascular system
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
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- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
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- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
- C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
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- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
- C07K5/1013—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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Abstract
The invention discloses a preparation method of high-activity tartary buckwheat albumin antihypertensive peptide, belonging to the field of foods and medicines. According to the invention, the tartary buckwheat albumin is extracted from tartary buckwheat, enzymolysis is carried out by adopting two proteases which are necessary for human digestion, and a pepsin-trypsin enzymolysis process is optimized, so that tartary buckwheat albumin peptide is obtained, and the ACE activity inhibition rate of the tartary buckwheat albumin peptide is detected by an in vitro detection method; separating the tartary buckwheat albumin peptide by gel filtration chromatography to obtain tartary buckwheat albumin peptides with different molecular weights, and respectively measuring the in vitro ACE activity inhibition rate of the tartary buckwheat albumin peptides; finally, nano-HPLC-MS/MS is adopted to identify the amino acid sequence of the peptide segment with the highest inhibition rate on ACE activity.
Description
Technical Field
The invention relates to a preparation method of high-activity tartary buckwheat albumin antihypertensive peptide, belonging to the field of foods and medicines.
Background
Hypertension is a common chronic cardiovascular disease, long-term hypertension is easy to cause cerebral apoplexy, coronary heart disease and other diseases, and most of hypertension patients need to take medicine for life. Angiotensin Converting Enzyme (ACE) has two main functions: 1. catalyzing the conversion of angiotensin I to angiotensin II; 2. inactivating the bradykinin. Angiotensin converting enzyme is an ideal target for treating diseases such as hypertension, heart failure, type 2 diabetes, diabetic nephropathy and the like due to the two functions. At present, the drug treatment of hypertension is relatively mature, such as perindopril, captopril and the like can inhibit ACE, but a series of side effects, such as drug resistance, cough, angioedema and the like, are easily caused by taking the drug. Therefore, the food-borne animal and plant proteins which are safe and have no toxic or side effect enter the field of vision of people, and as early as 1979 Oshima and the like hydrolyze gelatin by adopting bacterial collagenase to obtain antihypertensive peptides; more and more researches prove that the animal and plant proteins can prepare effective antihypertensive active peptide, provide a new thought for treating hypertension, and provide a theoretical basis for regulating and controlling blood pressure through methods such as diet therapy and the like.
Studies have shown that the inclusion of hydrophobic amino acids and prolines in biologically active peptide fragments has a critical effect on the ACE inhibitory activity of the peptide fragments. The koyama et al find that the buckwheat inhibition peptide Ser-Thr-Hyp has the effect of reducing blood pressure on spontaneous hypertension rats for the first time; li et al studied buckwheat protein hydrolysis and found that complete buckwheat not subjected to protein extraction was subjected to pepsin hydrolysis and then IC to ACE 50 IC after hydrolysis of whole buckwheat, which was not subjected to protein extraction, with pepsin, chymotrypsin and trypsin at 0.36mg protein/ml 50 The concentration of the protein is reduced to 0.14 mg/ml, which indicates that the ACE inhibition rate of the buckwheat protein can be greatly improved by adding chymotrypsin and trypsin after pepsin treatment. However, the enzymatic hydrolysate obtained by directly hydrolyzing whole buckwheat has an ACE inhibitory effect, but the influence of other components in the buckwheat on the ACE inhibitory ability cannot be completely eliminated.
Tartary buckwheat is one of the buckwheat, also called tartary buckwheat, and is a crop with higher edible value and medicinal value. Pharmacological studies show that the tartary buckwheat has the effects of reducing blood sugar, blood pressure and blood fat and resisting oxidization. Wherein, the regulation of lipid metabolism and improvement of sugar metabolism of the tartary buckwheat protein are all proved by researches. The tartary buckwheat protein contains 17 amino acids, including 8 essential amino acids for human body, and is a complete protein. The antihypertensive peptide prepared from tartary buckwheat at present has low ACE inhibition rate.
Disclosure of Invention
[ technical problem ]
The invention aims to solve the technical problem that the existing antihypertensive peptide from tartary buckwheat has low ACE inhibition rate.
Technical scheme
The invention provides a method for preparing a protein peptide derived from tartary buckwheat with a blood pressure reducing function, which comprises the following steps of:
(1) Extracting tartary buckwheat albumin from tartary buckwheat by an Osborne method;
(2) Taking 0.1-0.9g of the tartary buckwheat albumin obtained in the step (1), adding the tartary buckwheat albumin into a phosphate buffer solution, adjusting the pH of the system to 2-3, adding pepsin (2500U/mg) accounting for 3-6% of the mass of the tartary buckwheat albumin, performing enzymolysis for 2 hours at 37 ℃, adjusting the pH of the system to 7-8, adding trypsin (1500U/mg) accounting for 3-6% of the mass of the tartary buckwheat albumin, and performing enzymolysis for 1 hour at 37 ℃ to obtain an enzymolysis solution;
(3) Filtering the enzymolysis liquid through a microfiltration membrane to obtain filtrate, separating peptides with different molecular weights in the enzymolysis liquid filtrate through gel filtration chromatography, and screening out the tartary buckwheat albumin active peptide with the best ACE inhibition effect;
(4) The amino acid sequence of the tartary buckwheat albumin active peptide with the best inhibition effect is identified by using nano-HPLC-MS/MS.
In certain embodiments of the present invention, step (1) comprises the steps of:
(1) grinding: grinding radix Et rhizoma Fagopyri Tatarici in pulverizer, sieving with 60-80 mesh sieve;
(2) degreasing: adding petroleum ether into the tartary buckwheat powder, stirring for 6-10 hours, and replacing the petroleum ether for a plurality of times until the supernatant is clear, and air-drying for later use;
(3) extracting tartary buckwheat albumin: weighing 300g of defatted tartary buckwheat powder, adding 10 times of volume of water, stirring and extracting for 2 hours at 40 ℃, centrifuging for 15 minutes at 5000r/min, obtaining supernatant by centrifugation, namely albumin extract, regulating pH of the albumin extract to isoelectric point of albumin, precipitating for 1 hour, centrifuging, taking precipitate, and freeze-drying the precipitate to obtain the tartary buckwheat albumin.
In certain embodiments of the present invention, the pepsin used in step (2) may be selected from porcine pepsin.
In certain embodiments of the invention, the trypsin used in step (2) may be porcine trypsin.
In certain embodiments of the present invention, step (3): filtering the enzymolysis liquid by a microfiltration membrane with the thickness of 0.22 mu m, passing the filtrate through a Superdux peptide 10/300GL gel column, taking water as a mobile phase, controlling the flow rate to be 0.4mL/min, collecting the filtrate according to peaks under the condition of 280nm by using an AKTA avant system to obtain three peaks F1, F2 and F3, diluting the three peak components to the same concentration, and respectively measuring the inhibition rate of ACE, wherein the result shows that the F3 component has the highest inhibition rate of ACE.
In certain embodiments of the present invention, step (4) uses nano-HPLC-MS/MS to identify the amino acid sequence of the active peptide of tartary buckwheat albumin with the best inhibition effect, and ten active peptides of tartary buckwheat albumin for reducing blood pressure are obtained in total.
The invention provides an active peptide with blood pressure reducing function, wherein the amino acid sequences are FLR, LPRL, LFGK, TLFR, IPRL, VVLK and SFFK respectively.
The active peptide can be used for preparing antihypertensive products. In the product, auxiliary materials can be added, and the auxiliary materials are used for shaping, acting as carriers, improving stability, solubilization, assisting dissolution and/or sustained and controlled release.
[ advantageous effects ]
According to the invention, the tartary buckwheat albumin is extracted from tartary buckwheat, enzymolysis is carried out by adopting two proteases which are necessary for human digestion, and a pepsin-trypsin enzymolysis process is optimized, so that tartary buckwheat albumin peptide is obtained, and the ACE activity inhibition rate of the tartary buckwheat albumin peptide is detected by an in vitro detection method; separating the tartary buckwheat albumin peptide by gel filtration chromatography to obtain tartary buckwheat albumin peptides with different molecular weights, and respectively measuring the in vitro ACE activity inhibition rate of the tartary buckwheat albumin peptides; finally, nano-HPLC-MS/MS is adopted to identify the amino acid sequence of the peptide segment with the highest inhibition rate on ACE activity. The invention obtains 10 high-activity tartary buckwheat albumin peptides with blood pressure reducing function, and the amino acid sequences of the peptides are IFR, LRF, FLR, LPRL, FLK, LFGK, TLFR, IPRL, VVLK and SFFK respectively. Of these, FLR, LPRL, LFGK, TLFR, IPRL, VVLK and SFFK are peptides with ACE inhibitory activity which were first discovered in the present invention.
The high-activity tartary buckwheat albumin antihypertensive peptide prepared by the invention can be subjected to embedding treatment, so that the stability of the tartary buckwheat albumin antihypertensive peptide after entering a human body is enhanced; or making into oral liquid product to improve the added value of radix Et rhizoma Fagopyri Tatarici. The polypeptide sequence contained in the tartary buckwheat albumin antihypertensive peptide prepared by the process is clear and unique to the invention, and provides a theoretical basis for the high-activity tartary buckwheat albumin antihypertensive peptide as food or medicine.
The IC of the tartary buckwheat albumin peptide prepared by the method of the invention is not separated and purified 50 Has been as low as 0.255mg/mL; the inhibition rate of ACE is obviously improved after gel filtration chromatographic separation, and the inhibition rate reaches 71.83% at 0.1 mg/mL. The antihypertensive peptide separated from the tartary buckwheat albumin has better ACE activity inhibition effect.
Compared with the hydrolysis modes such as alkaline protease, the hydrolysis is mild, the damage degree to the tartary buckwheat albumin is low, and the process is simple; compared with other processes adopting pepsin and trypsin for hydrolysis, the hydrolysis time is short, the consumed protease amount is small, the molecular weight of the hydrolyzed tartary buckwheat protein peptide is small, and the tartary buckwheat protein peptide is easier to be absorbed by human bodies.
Drawings
Fig. 1 shows a preparation process route of the tartary buckwheat albumin antihypertensive peptide.
FIG. 2 shows the IC of the enzymatic hydrolysate to ACE under the optimal enzymatic hydrolysis conditions of Fagopyrum tataricum albumin 50 。
FIG. 3 is a graph showing the peak of the enzymatic hydrolysate of Tartary buckwheat albumin after pepsin-trypsin hydrolysis by gel filtration chromatography.
FIG. 4 shows the comparison of inhibition ratios of different components collected by gel filtration chromatography of an enzymatic hydrolysate of tartary buckwheat albumin after pepsin-trypsin hydrolysis.
Detailed Description
The water content and the protein content of the tartary buckwheat albumin are respectively measured by the methods specified in GB 5009.3-2016 and GB 5009.5-2016.
The measurement method of ACE inhibition rate is as follows: the experimental group was added with 100. Mu.L of a simulated substrate FAPGG (furylacrylic tripeptide), then with 50. Mu.L of a tartary buckwheat albumin hydrolysate for dilution 18 times, and finally with 50. Mu.L of ACE (60 mU/mL), the blank group was reacted at 37℃for 30min with 50. Mu.L of Tris-HCl buffer, and the inhibition effect was characterized by calculating the decrease in absorbance at 345nm after the simulated substrates were digested by Angiotensin Converting Enzyme (ACE) of the blank group and the experimental group, respectively, and the ACE inhibition rate was calculated as follows:
wherein A1 is absorbance reduction value of blank group; a2 is the absorbance decrease value of the experimental group.
Determination of peptide content: determining the peptide content in the tartary buckwheat albumin enzymolysis liquid before gel filtration chromatography by adopting a trichloroacetic acid method: weighing about 5g of hydrolysate, fixing the volume of trichloroacetic acid to 25mL, uniformly mixing, precipitating, centrifuging for 20min, reserving supernatant, and filtering with double-layer filter paper. 10mL of the filtrate was taken and added to a digestion vessel, and 3g of potassium sulfate, 0.2g of copper sulfate pentahydrate blue powder and 10mL of concentrated sulfuric acid were additionally added to digest in a graphite digestion instrument. After digestion, the peptide content of the hydrolysate was determined by the kjeldahl method.
Determination of the degree of hydrolysis: the hydrolysis degree of the tartary buckwheat albumin is measured by adopting a formaldehyde titration method: taking 5mL of hydrolysate, adding 60mL of distilled water with carbon dioxide removed, stirring uniformly, and adjusting pH=8.20. Neutral formaldehyde solution was prepared with 50mL of formaldehyde solution and 3mL of 0.5% (m/v) phenolphthalein solution, and ph=8.20 was adjusted. 20mL of neutral formaldehyde solution was added and titrated to pH=9.20 with 0.1mol/mL of standard NaOH solution. The volume of NaOH solution consumed is designated as V1. A blank experiment was performed by replacing the hydrolysate sample with an equal amount of distilled water and the volume V0 of NaOH consumed was recorded. The amino nitrogen content in the hydrolysate was calculated. The Degree of Hydrolysis (DH) of the protein was calculated.
Wherein V is 1 The volume of NaOH consumed in milliliters (mL) was titrated for the experimental group samples;
V 0 the volume of NaOH consumed in milliliters (mL) was titrated for the blank samples;
n is the concentration of standard NaOH solution used in the experiment, and the unit is mol per liter (mol/L);
v is the volume of the sample in milliliters (mL).
Gel filtration chromatography separation of the tartary buckwheat albumin enzymolysis product: filtering the Fagopyrum tataricum albumin enzymolysis liquid by a microfiltration membrane of 0.22 μm, passing the filtered enzymolysis liquid through a Superdux peptide 10/300GL gel column, taking water as a mobile phase, controlling the flow rate at 0.4mL/min, and respectively collecting the antihypertensive active peptide components by an AKTA avant system according to peaks under the condition of 280nm, wherein the three peaks F1, F2 and F3 are collected. The inhibition of ACE was measured by dilution of the three peak fractions to the same concentrations, respectively.
Determination of peptide sequence: the antihypertensive peptide with the best inhibition effect obtained after the gel filtration chromatography is subjected to nano-HPLC-MS/MS analysis, and the whole set of system is a Q-actual Plus mass spectrometer (Thermo Fisher Scientific, MA, USA) connected with EASY-nano LC 1200 in series. A total of 3. Mu.L of the sample (analytical column: acclaim PepMap C18, 75. Mu. m x 25 cm) was loaded, the sample was separated with a gradient of 60min, the column flow was controlled at 300nL/min, the column temperature was 40 ℃, the electrospray voltage was 2kV, the gradient was started from 2% of phase B, the gradient was increased to 35% with a nonlinear gradient in 47 min, the increase was 100% in 1 min, and the sample was maintained for 12 min. The mass spectrometer operates in a data dependent acquisition mode, automatically switching between MS and MS/MS acquisition. The mass spectral parameters were set as follows: (1) MS, scanning range (m/z): 200-1800; resolution is 70000; AGC target:3e6; the maximum injection time is 50ms; (2) HCD-MS/MS: resolution 17500; AGC target:1e5; the maximum injection time is 45ms; collision energy 28; dynamic exclusion time 30s.
The pepsin activity used in the examples below was 2500U/mg, from pigs.
The trypsin used in the examples described below had a viability of 1500U/mg and was derived from pigs.
The following are examples and comparative examples for preparing the antihypertensive active peptide of Fagopyrum tataricum, and the measurement results of the index are shown in Table 1 below.
EXAMPLE 1 preparation of enzymatic hydrolysate of Tartary buckwheat albumin
(1) Albumin extracted from Fagopyrum tataricum
Grinding: grinding radix Et rhizoma Fagopyri Tatarici in pulverizer, sieving with 60-80 mesh sieve;
degreasing: adding petroleum ether into the tartary buckwheat powder, stirring for 6-10 hours, and replacing the petroleum ether for a plurality of times until the supernatant is clear, and air-drying for later use;
extracting tartary buckwheat albumin: weighing 300g of defatted tartary buckwheat powder, adding 10 times of volume of water, stirring and extracting at 40 ℃ for 2 hours, centrifuging at 5000r/min for 15 minutes, obtaining supernatant which is albumin extract, regulating pH to isoelectric point of albumin, precipitating for 1 hour, centrifuging, taking precipitate, and freeze-drying to obtain tartary buckwheat albumin.
(2) Enzymolysis
Preparing a 1.6% concentration tartary buckwheat albumin suspension by using a phosphate buffer solution, regulating the pH of the suspension to 2 by using a 1M hydrochloric acid solution, adding pepsin accounting for 4% of the mass of tartary buckwheat albumin for enzymolysis for 2 hours, regulating the pH of the suspension to 7 by using a 1M sodium hydroxide solution, adding trypsin accounting for 5.5% of the mass of tartary buckwheat albumin for enzymolysis for 2 hours, and boiling to inactivate enzymes. Cooling and centrifuging to obtain a tartary buckwheat albumin enzymolysis liquid, and respectively measuring the inhibition rate, the hydrolysis degree and the peptide content of ACE. The results are shown in Table 1.
Example 2 preparation of Tartary buckwheat albumin enzymatic hydrolysate and separation of ACE inhibitory peptide
(1) Albumin is extracted from tartary buckwheat: the specific procedure is as in (1) above in example 1.
(2) Enzymolysis
Preparing a 2% concentration tartary buckwheat albumin suspension by using a phosphate buffer solution, regulating the pH of the suspension to 2 by using a 1M hydrochloric acid solution, adding pepsin accounting for 4% of the mass of tartary buckwheat albumin for enzymolysis for 2 hours, regulating the pH of the suspension to 7 by using a 1M sodium hydroxide solution, adding trypsin accounting for 5% of the mass of tartary buckwheat albumin for enzymolysis for 1 hour, and boiling for enzyme deactivation. Cooling and centrifuging to obtain a tartary buckwheat albumin enzymolysis liquid, and respectively measuring the inhibition rate, the hydrolysis degree and the peptide content of ACE. The results are shown in Table 1.
(3) Filtering the Fagopyrum tataricum albumin enzymolysis liquid by a microfiltration membrane of 0.22 μm, passing the filtered enzymolysis liquid through a Superdux peptide 10/300GL gel column, taking water as a mobile phase, controlling the flow rate at 0.4mL/min, and respectively collecting the antihypertensive active peptide components by an AKTA avant system according to peaks under the condition of 280nm, wherein the three peaks F1, F2 and F3 are collected. The inhibition of ACE was measured by dilution of the three peak fractions to the same concentrations, respectively. The results are shown in FIG. 4, where the F3 component has the highest ACE inhibition.
(4) Determination of peptide sequences
The antihypertensive peptide with the best inhibition effect obtained after the gel filtration chromatography is subjected to nano-HPLC-MS/MS analysis, and the whole set of system is a Q-actual Plus mass spectrometer (Thermo Fisher Scientific, MA, USA) connected with EASY-nano LC 1200 in series. A total of 3. Mu.L of the sample (analytical column: acclaim PepMap C18, 75. Mu. m x 25 cm) was loaded, the sample was separated with a gradient of 60min, the column flow was controlled at 300nL/min, the column temperature was 40 ℃, the electrospray voltage was 2kV, the gradient was started from 2% of phase B, the gradient was increased to 35% with a nonlinear gradient in 47 min, the increase was 100% in 1 min, and the sample was maintained for 12 min. The mass spectrometer operates in a data dependent acquisition mode, automatically switching between MS and MS/MS acquisition. The mass spectral parameters were set as follows: (1) MS, scanning range (m/z): 200-1800; resolution is 70000; AGC target:3e6; the maximum injection time is 50ms; (2) HCD-MS/MS: resolution 17500; AGC target:1e5; the maximum injection time is 45ms; collision energy 28; dynamic exclusion time 30s.
Comparative example 1
(1) Albumin is extracted from tartary buckwheat: the specific procedure is as in (1) above in example 1.
(2) Enzymolysis
Preparing a 2% concentration tartary buckwheat protein suspension by using a phosphate buffer solution, regulating the pH of the suspension to 2 by using a 1M hydrochloric acid solution, not adding pepsin, regulating the pH of the suspension to 7 by using a 1M sodium hydroxide solution after 2 hours of reaction, not adding trypsin, carrying out enzymolysis for 1 hour, boiling, centrifuging to obtain a supernatant, and respectively measuring the inhibition rate, the hydrolysis degree and the peptide content of ACE.
Comparative example 2
(1) Albumin is extracted from tartary buckwheat: the specific procedure is as in (1) above in example 1.
(2) Enzymolysis
Preparing a 2% concentration tartary buckwheat albumin suspension by using a phosphate buffer solution, regulating the pH of the suspension to 2 by using a 1M hydrochloric acid solution, adding 4% pepsin, reacting for 2 hours, boiling to inactivate enzyme, centrifuging to obtain a supernatant, and respectively measuring the inhibition rate, the hydrolysis degree and the peptide content of ACE.
Comparative example 3
(1) Albumin is extracted from tartary buckwheat: the specific procedure is as in (1) above in example 1.
(2) Enzymolysis
Preparing a 2% concentration tartary buckwheat albumin suspension by using a phosphate buffer solution, regulating the pH of the suspension to 7 by using a 1M sodium hydroxide solution, adding trypsin with the protein content of 5% for enzymolysis for 1h, and boiling to inactivate enzymes. Cooling and centrifuging to obtain a tartary buckwheat albumin enzymolysis liquid, and respectively measuring the inhibition rate, the hydrolysis degree and the peptide content of ACE.
Comparative example 4
(1) Albumin is extracted from tartary buckwheat: the specific procedure is as in (1) above in example 1.
(2) Enzymolysis
Preparing a 2% concentration tartary buckwheat albumin suspension by using a phosphate buffer solution, regulating the pH of the suspension to 2 by using a 1M hydrochloric acid solution, adding 4% pepsin, reacting for 2 hours, regulating the pH of the suspension to 7 by using a 1M sodium hydroxide solution, adding trypsin with the protein content of 6% for enzymolysis for 2 hours, and boiling to inactivate enzymes. Cooling and centrifuging to obtain a tartary buckwheat albumin enzymolysis liquid, and respectively measuring the inhibition rate, the hydrolysis degree and the peptide content of ACE.
Comparative example 5
(1) Albumin is extracted from tartary buckwheat: the specific procedure is as in (1) above in example 1.
(2) Enzymolysis
Preparing 2% concentration of tartary buckwheat albumin suspension by using phosphate buffer solution, wherein the enzymolysis temperature is 50 ℃, the pH value of the enzymolysis solution is 9.0, adding alkaline protease with the enzyme dosage of 20kU/g (calculated by substrate), and carrying out enzymolysis for 2 hours, and respectively measuring the inhibition rate, the hydrolysis degree and the peptide content of ACE.
TABLE 1 detection results of enzymatic liquids
As can be seen from comparative examples 1, 2, 3, no bioactive peptide could be produced without any enzyme, but pepsin or trypsin alone could produce some bioactive peptide, but less peptide with ACE inhibiting effect, lower inhibition rate, lower hydrolysis degree and peptide content, which may be related to less cleavage sites for tartary buckwheat protein when pepsin and trypsin are hydrolyzed alone.
As can be seen from examples 1, 2 and comparative example 4, in the preparation of antihypertensive active peptides, the enzymatic hydrolysate obtained under different enzymatic conditions also has a great difference in ACE inhibitory effect, peptide content and degree of hydrolysis under the same enzymatic action. In the enzyme hydrolysis reaction, the peptide content is gradually increased along with the increase of the trypsin dosage, the substrate concentration and the hydrolysis time, when the substrate concentration is 2%, and the trypsin dosage is 5% of the protein content, the obtained inhibition rate is maximum when the hydrolysis is carried out for 1h, which indicates that the peptide obtained under the hydrolysis condition has better inhibition effect on ACE, and the addition of more trypsin possibly damages the generated effective inhibition peptide, so that the viscosity of the reaction system is increased, and the reaction is unfavorable. The peptides effective for ACE are excessively hydrolyzed and the inhibition rate of ACE gradually decreases as the hydrolysis time increases and the hydrolysis degree increases. Therefore, the peptide with the best inhibition rate can be obtained only when the optimal amount of enzyme is added and the optimal time for hydrolysis is reached under the optimal substrate concentration condition. The bioactive peptide with the ratio of less than 3000Da in the enzymolysis liquid obtained under the optimal condition reaches 90%, wherein the peptide with the ratio of less than 1000Da reaches more than 66%, which indicates that the tartary buckwheat protein is thoroughly hydrolyzed under the condition, and researches show that the peptide with better ACE inhibition effect is mainly short peptide, which can explain the reason that the antihypertensive active peptide obtained by the invention has better inhibition effect.
As can be seen from comparative example 5, although bioactive peptide with ACE inhibition effect can be obtained by hydrolyzing the tartary buckwheat protein by alkaline protease, compared with the invention, the alkaline protease hydrolysis is strong, and the pepsin-trypsin continuous hydrolysis process adopted by the invention better evaluates the inhibition activity of the tartary buckwheat protein in vitro ACE. The way can better reflect the changes of the space structure, the amino acid sequence, the molecular mass and the like of the tartary buckwheat albumin peptide after entering the human body and being digested by gastrointestinal digestive enzymes.
As can be seen from fig. 4, the enzymolysis liquid prepared in the best enzymolysis condition in example 2 is separated into three components by gel filtration chromatography, wherein the inhibition rate of the F3 component to ACE is 71.83% when the peptide concentration is 0.1mg/mL, compared with the inhibition rate before separation, the inhibition rate is greatly improved, and the active ingredients are effectively enriched after gel column filtration, so that the tartary buckwheat protein peptide with high activity can be obtained, and can be identified.
10 bioactive peptides are identified from the F3 component in the step (4) of the example 2 by nano-HPLC-MS/MS analysis technology, the bioactive peptides are respectively predicted by adopting Peptide Ranker, the bioactive scores of the 10 tartary buckwheat protein peptides are all more than 0.5 according to the sequential arrangement of the scores, and the possibility of predicting the bioactive peptides is higher. According to research reports, most peptides with good ACE inhibition effect are peptides with stronger hydrophobicity, so that a polypeptide property calculator is used for calculating a hydrophobicity average value, the obtained polypeptide has higher hydrophobicity average value, and 10 tartary buckwheat protein polypeptides obtained by the invention can be presumed to have higher ACE inhibition activity. In addition, the research shows that the peptide C-terminal contains hydrophobic amino acid Tyr, phe, trp, lys, pro and Arg, which is helpful for greatly improving ACE inhibition rate, while the unreported 7 tartary buckwheat albumin peptides of the invention, the C-terminal of FLR, LFGK, TLFR, VVLK, SFFK, contains arginine or hydrophobic amino acid, so that higher ACE inhibition activity can be deduced, and in addition, the ACE inhibition activity of the peptide can be obviously enhanced due to the fact that LPRL and IPRL contain Leu at the C-terminal, and the detection of the ACE inhibition rate also proves that the estimation is performed.
The effect of the above predicted potential ACE inhibiting peptides was verified by solid phase synthesis from Sangon Biotech co., ltd. (Shanghai, china). The purity of the peptides was verified to be 99% by HPLC, and after confirming the molecular weight of the synthetic peptides using LC-MS/MS, the in vitro ACE inhibitory activity of the synthetic peptides was determined.
TABLE 2
The preparation method has the advantages of simple preparation process, less time consumption, small enzyme dosage and low cost; the in vitro ACE inhibition rate of the tartary buckwheat albumin peptide is higher, the obtained polypeptide is shorter, and a plurality of polypeptides are obtained by the first separation and identification of the tartary buckwheat albumin peptide. The invention has a great application prospect in terms of technology, action mechanism, acceptance, number of consumers and the like, and can be prepared into microcapsules by adopting a microcapsule embedding technology by taking the tartary buckwheat ACE inhibitory peptide obtained by the invention as a core material, so that the tartary buckwheat ACE inhibitory peptide can resist high-temperature high-acid and alkali environments, can be slowly released after entering a human body, and has strong stability; in addition, the tartary buckwheat polypeptide oral liquid product can be prepared, so that the commercial utilization value of the tartary buckwheat polypeptide oral liquid product is increased, and the added value of tartary buckwheat is improved.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. The method for preparing the protein peptide from the tartary buckwheat with the blood pressure reducing function is characterized by comprising the following steps of:
(1) Extracting tartary buckwheat albumin from tartary buckwheat by an Osborne method;
(2) Taking 0.1-0.9g of the tartary buckwheat albumin obtained in the step (1), adding the tartary buckwheat albumin into a phosphate buffer solution, adjusting the pH of the system to 2-3, adding pepsin accounting for 3-6% of the mass of the tartary buckwheat albumin, carrying out enzymolysis for 2 hours at 37 ℃, adjusting the pH of the system to 7-8, adding trypsin accounting for 3-6% of the mass of the tartary buckwheat albumin, and carrying out enzymolysis for 1 hour at 37 ℃ to obtain an enzymolysis solution;
(3) Filtering the enzymolysis liquid through a microfiltration membrane to obtain filtrate, separating peptides with different molecular weights in the enzymolysis liquid filtrate through gel filtration chromatography, and screening out the tartary buckwheat albumin active peptide with the best ACE inhibition effect;
(4) The amino acid sequence of the tartary buckwheat albumin active peptide with the best inhibition effect is identified by using nano-HPLC-MS/MS.
2. The method for preparing a protein peptide derived from tartary buckwheat having a blood pressure lowering function as claimed in claim 1, wherein the step (1) comprises the steps of:
(1) grinding: grinding radix Et rhizoma Fagopyri Tatarici in pulverizer, sieving with 60-80 mesh sieve;
(2) degreasing: adding petroleum ether into the tartary buckwheat powder, stirring for 6-10 hours, and replacing the petroleum ether for a plurality of times until the supernatant is clear, and air-drying for later use;
(3) extracting tartary buckwheat albumin: weighing 300g of defatted tartary buckwheat powder, adding 10 times of volume of water, stirring and extracting for 2 hours at 40 ℃, centrifuging for 15 minutes at 5000r/min, obtaining supernatant by centrifugation, namely albumin extract, regulating pH of the albumin extract to isoelectric point of albumin, precipitating for 1 hour, centrifuging, taking precipitate, and freeze-drying the precipitate to obtain the tartary buckwheat albumin.
3. The method for producing a protein peptide derived from Fagopyrum tataricum having a blood pressure lowering effect according to claim 1, wherein the pepsin used in the step (2) is porcine pepsin.
4. The method for producing a protein peptide derived from Fagopyrum tataricum having a blood pressure lowering effect as claimed in claim 1, wherein the trypsin used in the step (2) is porcine trypsin.
5. The method for producing a protein peptide derived from tartary buckwheat having a blood pressure lowering function as claimed in claim 1, wherein the step (3) is: filtering the enzymolysis liquid by a microfiltration membrane with the thickness of 0.22 mu m, passing the filtrate through a Superdux peptide 10/300GL gel column, taking water as a mobile phase, controlling the flow rate to be 0.4mL/min, collecting the filtrate according to peaks under the condition of 280nm by using an AKTA avant system, diluting each peak component to the same concentration, and respectively measuring the inhibition rate of ACE.
6. An active peptide with blood pressure lowering function, characterized in that the amino acid sequences are FLR, LPRL, LFGK, TLFR, IPRL, VVLK and SFFK, respectively.
7. The use of the active peptide with blood pressure lowering function as claimed in claim 6 for preparing a blood pressure lowering product.
8. A blood pressure lowering product comprising an active peptide, characterized by comprising at least one peptide from FLR, LPRL, LFGK, TLFR, IPRL, VVLK and SFFK.
9. The product of claim 8, further comprising an adjunct.
10. The product of claim 9, wherein the adjuvant is used to shape, act as a carrier, improve stability, solubilize, aid dissolution, and/or control release.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117247431A (en) * | 2023-11-17 | 2023-12-19 | 中国农业大学 | Tartary buckwheat peptide with DPP-IV inhibitory activity and application thereof |
| CN117264019A (en) * | 2023-11-22 | 2023-12-22 | 中国农业大学 | Tartary buckwheat protein source DPP-IV (dipeptidyl peptidase IV) inhibitory peptide and separation and purification method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117247431A (en) * | 2023-11-17 | 2023-12-19 | 中国农业大学 | Tartary buckwheat peptide with DPP-IV inhibitory activity and application thereof |
| CN117247431B (en) * | 2023-11-17 | 2024-01-30 | 中国农业大学 | Tartary buckwheat peptide with DPP-IV inhibitory activity and application thereof |
| CN117264019A (en) * | 2023-11-22 | 2023-12-22 | 中国农业大学 | Tartary buckwheat protein source DPP-IV (dipeptidyl peptidase IV) inhibitory peptide and separation and purification method thereof |
| CN117264019B (en) * | 2023-11-22 | 2024-02-09 | 中国农业大学 | Tartary buckwheat protein source DPP-IV (dipeptidyl peptidase IV) inhibitory peptide and separation and purification method thereof |
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