CN116082451B - Buckwheat-source cholecystokinin secretion peptide and preparation method and application thereof - Google Patents
Buckwheat-source cholecystokinin secretion peptide and preparation method and application thereof Download PDFInfo
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- CN116082451B CN116082451B CN202211645458.3A CN202211645458A CN116082451B CN 116082451 B CN116082451 B CN 116082451B CN 202211645458 A CN202211645458 A CN 202211645458A CN 116082451 B CN116082451 B CN 116082451B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- 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
- A61P3/04—Anorexiants; Antiobesity agents
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Diabetes (AREA)
- General Chemical & Material Sciences (AREA)
- Child & Adolescent Psychology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Genetics & Genomics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a buckwheat-source cholecystokinin secretion peptide, a preparation method and application thereof. The buckwheat-derived cholecystokinin (CCK) secretion peptide has any one or more of P1, P2, P3, P4 and P5, wherein P1 is RVTVQPDS, P2 is PAFKEEHL, P3 is QFDLDD, P4 is IPPLFP and P5 is SFHFPI. The invention also provides a preparation method of the peptide, which can be obtained by biological enzymolysis of buckwheat protein and then separation and purification of C18 chromatographic column, or can be synthesized artificially by adopting a chemical solid phase synthesis method. The active peptide can obviously promote the endocrine cells of the intestinal tract to secrete CCK, and has the advantages of safety, no toxic or side effect, resistance to enzymolysis of digestive enzymes of the gastrointestinal tract, easy absorption and the like. The peptide can be used as a functional component for developing medicines with the functions of promoting satiety, losing weight and delaying gastric emptying, and has important application value in the field of medicines.
Description
Technical Field
The invention belongs to the technical field of bioactive peptides, and particularly relates to a buckwheat-source cholecystokinin secretion peptide, and a preparation method and application thereof.
Background
Obesity has become a global public health problem with the development of economy and society and the improvement of the living standard of people. Various chronic diseases such as cardiovascular diseases, type II diabetes, atherosclerosis, fatty liver, gout, etc. are all associated with obesity. The imbalance in energy intake and energy expenditure is a direct cause of overweight obesity in an individual. Therefore, in order to prevent obesity, limiting excessive energy intake, i.e., limiting the intake of food, is an optimal means. The central hypothalamus and digestive tract of the body are key sites for controlling the regulation of ingestion, and a plurality of ghrelins related to the regulation of ingestion are distributed in the body. Cholecystokinin (cholecystokinin, CCK) is an important member of the classical brain-intestine peptides, and can regulate the organism to generate a satiety signal and reduce the ingestion of the organism, thereby achieving the effect of suppressing appetite. Increasing secretion of CCK in the intestinal tract is of great importance for the prevention and alleviation of obesity.
The regulation of CCK secretion by dietary factors has been widely demonstrated by scientists, which provides an important approach to improve or alleviate obesity and related chronic diseases by regulating intestinal CCK secretion through diet. The food-borne bioactive peptide is a common dietary factor and has the characteristics of easy digestion and absorption by human bodies, high edible safety and the like. Current sources of food-borne bioactive peptides mainly include animal proteins and vegetable proteins, and vegetable proteins are receiving increasing attention due to environmental, economic, sustainability and other factors. Buckwheat (Fagopyrum esculentum moench.) is one of the main crops, has important eating value, has very low gluten content, the main protein is globulin, the lysine content in the essential amino acid is high and the methionine content is low, and the amino acid pattern can be complemented with the main grains (such as wheat, corn and rice with lower lysine content). At present, the development and the utilization of buckwheat polysaccharide and polyphenol are more, but the development of buckwheat protein is lacking. The buckwheat-derived protein is used as a raw material, and the development of the bioactive peptide has important application value and development prospect.
Disclosure of Invention
Based on the current situation that the buckwheat-source bioactive peptide capable of promoting intestinal CCK secretion is lacking in the prior art, the invention provides the buckwheat-source cholecystokinin secretion peptide, and the preparation method and the application thereof.
The aim of the invention can be achieved by the following technical scheme:
In a first aspect of the present invention, there is provided a buckwheat-derived cholecystokinin-secreting peptide selected from one or more of the following active peptides:
The active peptide P1 is RVTVQPDS, and the amino acid sequence is Arg-Val-Thr-Val-Gln-Pro-Asp-Ser;
The active peptide P2 is PAFKEEHL, and the amino acid sequence is Pro-Ala-Phe-Lys-Glu-Glu-His-Leu;
The active peptide P3 is QFDLDD, and the amino acid sequence is Gln-Phe-Asp-Leu-Asp-Asp;
The active peptide P4 is IPPLFP, and the amino acid sequence is Ile-Pro-Pro-Leu-Phe-Pro;
the active peptide P5 is SFHFPI, and the amino acid sequence is Ser-Phe-His-Phe-Pro-Ile.
Buckwheat protein mainly contains globulin, wherein the amino acid sequence RVTVQPDS is present in buckwheat 13Sglobulin (Uniprot protein accession number: W6JQD 0); the amino acid sequences of the other active peptides PAFKEEHL, QFDLDD, IPPLFP and SFHFPI were derived from buckwheat UDP-glycose: glycosyltransferase (Uniprot protein accession number: A0A0A1HAP 0), aspartic proteinase (Uniprot protein accession number: Q5PT 35), ccmB (Uniprot protein accession number: A0A6M4SRV 2) and Protein Ycf2 (Uniprot protein accession number: A0A6N0GPR 0), respectively. Both of the active peptides RVTVQPDS, PAFKEEHL, QFDLDD, IPPLFP and SFHFPI can be prepared from buckwheat protein.
In a second aspect of the present invention, there is provided a polynucleotide encoding the buckwheat-derived cholecystokinin-secreting peptide.
In a third aspect of the invention, the preparation method of the buckwheat-derived cholecystokinin secretion peptide is provided, which is obtained by a separation and purification method from buckwheat protein, or is synthesized artificially by a genetic engineering method, or is prepared directly by chemical synthesis.
The method for directly obtaining the buckwheat protein through biological enzymolysis and then separation and purification comprises the following steps: based on the amino acid sequence of the given cholecystokinin secretion peptide, the cholecystokinin secretion peptide is obtained from buckwheat by adopting conventional enzymolysis, separation and purification methods in biological technology.
The artificial synthesis of the cholecystokinin secretion peptide by genetic engineering is a technical scheme which can be realized by a person skilled in the art, for example, the sequence synthesis of the polypeptide can be controlled by a proper DNA template based on a DNA recombination technology.
The active peptide is synthesized by a conventional solid phase synthesis method.
According to a fourth aspect of the present invention, there is provided a method for preparing an enzymatic hydrolysate containing the buckwheat-derived cholecystokinin-secreting peptide, wherein a two-step enzymatic hydrolysis method is employed to carry out enzymatic hydrolysis of buckwheat protein, comprising the steps of: sequentially hydrolyzing the buckwheat protein by pepsin and trypsin to obtain a buckwheat protein enzymolysis product, namely an enzymolysis product containing the buckwheat-derived cholecystokinin secretion peptide.
In one embodiment of the invention, the method for sequentially hydrolyzing the buckwheat protein by pepsin and trypsin comprises the following steps:
1) Extracting buckwheat protein from buckwheat;
2) Carrying out enzymolysis on the buckwheat protein by using the pepsin to obtain a first enzymolysis product;
3) And (3) carrying out enzymolysis on the first enzymolysis product by using the trypsin to obtain a buckwheat protein enzymolysis product.
In one embodiment of the present invention, a method for extracting buckwheat protein from buckwheat comprises the steps of:
Grinding semen Fagopyri Esculenti, degreasing, adding into distilled water, setting temperature to 50-60deg.C, adjusting pH to 4-6, and pre-treating with saccharifying enzyme for 0.5-2 hr; cooling to room temperature, adjusting pH to 10-12, extracting protein, centrifuging to obtain supernatant, adjusting pH to 4.4-4.6, standing, centrifuging, and washing with water; and finally drying to obtain the buckwheat protein.
In one embodiment of the invention, the mass ratio of pepsin or trypsin to the buckwheat protein is 1:10-100.
In one embodiment of the invention, the enzymolysis condition of pepsin or trypsin is 37 ℃ for 1-4h.
In one embodiment of the present invention, after the buckwheat protein enzymolysis product obtained in step 3), the method further comprises the following steps:
Separating the buckwheat protein enzymolysis product by using YMC ODS C18 chromatographic column, eluting with 5% methanol deionized water solution, 30% methanol deionized water solution, 80% methanol deionized water solution and 100% methanol solution as eluent at a certain flow rate, purifying by chromatography, and collecting different components; then detecting the influence of different components on CCK secretion of enteroendocrine cells, and selecting the component with the highest activity for stimulating CCK secretion as a target component, namely the product containing the buckwheat-source cholecystokinin secretion peptide.
In a fifth aspect, the present invention provides a product comprising an enzymatic hydrolysate of the cholecystokinin-secreting peptide of buckwheat origin prepared based on the above preparation method.
In a sixth aspect, the present invention provides an application of the buckwheat-derived cholecystokinin-secreting peptide and an enzymolysis product containing the buckwheat-derived cholecystokinin-secreting peptide in preparing a product with at least one of the following functions 1) -4):
1) Promoting CCK secretion;
2) Slowing gastric emptying;
3) Suppressing appetite and reducing food intake;
4) Preventing or assisting in treating obesity.
The buckwheat-source cholecystokinin secretion peptide and the enzymolysis product containing the buckwheat-source cholecystokinin secretion peptide have the function of promoting CCK secretion, and can slow down gastric emptying, inhibit appetite, reduce food intake and prevent or assist in treating obesity.
In a seventh aspect of the present invention, there is provided a product comprising said cholecystokinin-secreting peptide of buckwheat origin, or said enzymatic hydrolysate comprising said cholecystokinin-secreting peptide of buckwheat origin, said product having at least one of the following functions 1) -4):
1) Promoting CCK secretion;
2) Slowing gastric emptying;
3) Suppressing appetite and reducing food intake;
4) Preventing or assisting in treating obesity.
The product comprises a pharmaceutical product.
Compared with the prior art, the invention has the advantages and beneficial effects that:
The invention screens out the active peptide which has the function of promoting the secretion of intestinal CCK, and the active peptide has a novel peptide sequence structure, so far, other related reports are not yet seen; the active peptide can be prepared from food protein buckwheat protein, and has the advantages of safety and no toxic or side effect; the RVTVQPDS, PAFKEEHL, QFDLDD, IPPLFP and SFHFPI peptides can resist digestion of pepsin and pancreatin in the gastrointestinal tract, have good stability, and therefore play the activity of promoting CCK secretion to the maximum extent; the CCK secretion promoting peptide has the molecular weight smaller than 1000Da, has small molecular weight, can promote the secretion of intestinal CCK and is easy to be absorbed by organisms, so that the CCK secretion promoting peptide has a good nutrition function; the preparation method of RVTVQPDS, PAFKEEHL, QFDLDD, IPPLFP and SFHFPI active peptides or enzymolysis products containing RVTVQPDS, PAFKEEHL, QFDLDD, IPPLFP and SFHFPI sequence peptides provided by the invention is simple, easy to operate, convenient for industrial mass production and has wide application prospect.
Drawings
FIG. 1 is a graph showing the effect of RVTVQPDS, PAFKEEHL, QFDLDD, IPPLFP and SFHFPI sequence active peptides on STC-1 cell viability;
FIG. 2 is a graph showing the effect of RVTVQPDS, PAFKEEHL, QFDLDD, IPPLFP and SFHFPI sequence active peptides on CCK secretion by STC-1 cells, wherein C CCK represents the concentration of CCK secreted by the cells and C BCA represents the total protein secreted by the cells;
FIG. 3 shows the effect of buckwheat protein hydrolysate (B) on the secretion of CCK by STC-1 cells, C being a control group;
FIG. 4 is the effect of buckwheat protein hydrolysate on CCK secretion by mouse enteroendocrine cells;
FIG. 5 is a diagram showing the separation of buckwheat protein hydrolysate by YMC ODS C18 chromatographic column;
FIG. 6 is a graph showing the effect of isolated fractions on CCK secretion by STC-1 cells;
FIG. 7 is a MS/MS pattern and sequence analysis of RVTVQPDS peptides in isolated fraction F3/F4;
FIG. 8 is an MS/MS pattern and sequence analysis of QFDLDD peptides in isolated fraction F3;
FIG. 9 is an MS/MS pattern and sequence analysis of PAFKEEHL peptides in isolated fraction F3;
FIG. 10 is an MS/MS pattern and sequence analysis of SFHFPI peptides in isolated fraction F4;
FIG. 11 is an MS/MS pattern and sequence analysis of IPPLFP peptides in isolated fraction F4.
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples.
Examples 1RVTVQPDS, PAFKEEHL, QFDLDD, IPPLFP and SFHFPI Synthesis of active peptide and evaluation of CCK secretion-promoting Activity
1. Synthesis of RVTVQPDS, PAFKEEHL, QFDLDD, IPPLFP and SFHFPI active peptides
Active peptide Arg-Val-Thr-Val-Gln-Pro-Asp-Ser(RVTVQPDS);Pro-Ala-Phe-Lys-Glu-Glu-His-Leu(PAFKEEHL);Gln-Phe-Asp-Leu-Asp-Asp(QFDLDD);Ile-Pro-Pro-Leu-Phe-Pro(IPPLFP);Ser-Phe-His-Phe-Pro-Ile(SFHFPI) the active peptide is synthesized by "Zhejiang Hongtao technology Co., ltd.) using peptide solid phase synthesis method, and the purity of the synthesized peptide is verified to be more than 95% by high performance liquid phase method and mass spectrum technology.
2. RVTVQPDS, PAFKEEHL, QFDLDD, IPPLFP and SFHFPI effects on STC-1 cell Activity and CCK secretion
(1) Cultivation of STC-1 cells
STC-1 cells were cultured in DMEM medium containing 10% Fetal Bovine Serum (FBS), 1% non-essential amino acids (NEAA), 100U/mL penicillin and 0.1mg/mL streptomycin. Cells were incubated at 37℃in a cell incubator containing 5% CO 2 and subcultured by trypsin digestion when 80-90% density was reached.
(2) Cell Activity assay
The effect of RVTVQPDS, PAFKEEHL, QFDLDD, IPPLFP and SFHFPI active peptides on STC-1 cell viability was tested and evaluated by cell proliferation and cytotoxicity of WST-8 (2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfophenyl) -2H-tetrazolium monosodium salt). WST-8 was added to STC-1 cells treated in 96 well plates, and in the presence of an electron coupling reagent, WST-8 was reduced by some dehydrogenases in the mitochondria to yield Formazan in orange yellow, and absorbance was measured with a microplate reader at a detection wavelength of 450nm, as a percentage of the control group. The results are shown in FIG. 1, and the different peptides at the concentrations tested (5 mmol/L) showed no current change in cell viability compared to the control STC-1, indicating that RVTVQPDS, PAFKEEHL, QFDLDD, IPPLFP and SFHFPI active peptides were non-toxic to cells.
(3) Determination of the content of secreted hormone of STC-1 cells
RVTVQPDS, PAFKEEHL, QFDLDD, IPPLFP and SFHFPI active peptides were prepared in Hank's buffer to give 0.2,2,5mM peptide solutions, respectively. STC-1 cells were seeded at a density of 1.25X10 5 cells in 24 well plates. When the cells reached 80% -90% confluence, the cells were washed twice with Hank's buffer to remove the medium. The peptide solution was added to STC-1 cells and the cells were incubated in an incubator at 37℃for 2h. After the incubation, 1000g was centrifuged for 20min, and the supernatant was taken. CCK content was determined using a commercial CCK kit from Wohan cloud cloning technologies Co.
The total protein content of the supernatant measured using the Biyun biotechnology company BCA kit was used as a reference to reduce errors caused by handling between different batches. The calculation formula is as follows: c CCK/(CBCA×100)(×10-7), wherein C CCK represents CCK concentration (pg/mL) in the supernatant, and C BCA represents measured total protein concentration minus measured peptide concentration (mg/mL) in the supernatant.
The effect of RVTVQPDS, PAFKEEHL, QFDLDD, IPPLFP and SFHFPI active peptides on CCK secretion by STC-1 cells is shown in figure 2, where it can be seen that RVTVQPDS, PAFKEEHL, QFDLDD, IPPLFP and SFHFPI active peptides increase CCK secretion in a dose dependent manner. Numerous studies have demonstrated that CCK is an important member of the brain-intestinal peptide group, which slows gastric emptying, regulates the production of satiety signals in the body, and reduces the intake of food in the body, thereby achieving the effect of suppressing appetite. Increasing secretion of CCK in the intestinal tract is of great importance for the prevention and alleviation of obesity. The RVTVQPDS, PAFKEEHL, QFDLDD, IPPLFP and SFHFPI active peptides can obviously promote the secretion of CCK by the endocrine cell STC-1 of the intestinal tract, so that the peptides have important significance and application value for promoting satiety and losing weight.
EXAMPLE 2 preparation of buckwheat protein hydrolysate and Activity evaluation
(1) Preparation of buckwheat protein zymolyte
The buckwheat flour is milled and sieved by a sieve of 80, and then the buckwheat flour is defatted by hexane. Soaking defatted semen Fagopyri Esculenti powder in distilled water at a mass ratio of 12:1 in beaker, adjusting pH to 5.0 with 1mol/L HCl, and treating with saccharifying enzyme at 50deg.C for 1 hr. The pH was then adjusted to 11.0 with 1mol/L NaOH and the supernatant was centrifuged after 2h action with a magnetic stirrer. Regulating pH of supernatant to isoelectric point (pH 4.5) with 1mol/L HCl, standing for 1 hr, centrifuging, washing with water, precipitating to neutrality, redissolving with small amount of distilled water, lyophilizing to obtain buckwheat protein, and storing at 4deg.C for use.
1G of the lyophilized protein powder was dissolved in 20mL of a freshly prepared aqueous pepsin solution containing 25mg, the pH of the solution was adjusted to 2.0 with HCl (1 mol/L) and incubated at 37℃for 2h. After the incubation was completed, the pH of the solution was adjusted to 6.8 with NaOH (1 mol/L), and then 50mg of trypsin was added to continue the enzymatic hydrolysis for 2 hours. Inactivating enzyme in boiling water bath for 8min, centrifuging, collecting supernatant, and lyophilizing to obtain buckwheat protein hydrolysate.
(2) Activity evaluation
The effect of buckwheat protein zymolyte on the secretion of CCK by STC-1 cells was determined by the method described above using Hank's buffer to prepare a solution with a mass concentration of 5mg/mL, and the results are shown in FIG. 3. From the results, it can be seen that the buckwheat protein hydrolysate can significantly stimulate the secretion of CCK by STC-1 cells.
Further, the effect of buckwheat protein hydrolysate on hormone secretion by mouse enteroendocrine cells was evaluated at animal level. ICR mice were randomly divided into 2 groups (28 per group) after a1 week adaptation period. Control group: lavage physiological saline; buckwheat protein enzymolysis substance group: gastric lavage buckwheat protein hydrolysate (1.0 g/kg body weight). After the stomach is irrigated, eyeballs are respectively subjected to blood collection at 0,15 min,30min,60min,90min,120min and 150min, the eyeballs are placed into a centrifuge tube containing EDTA (final concentration is 1 mg/mL) and aprotinin (final concentration is 0.6 TIU/mL), supernatant is centrifugally collected, and the CCK hormone content in serum is obtained through an ELISA method. CCK in the serum of the gastric lavage saline group was maintained at a level of about 40pg/mL during this period. As shown in FIG. 4, the results of the gastric lavage of the buckwheat protein hydrolysate group show that the buckwheat protein hydrolysate greatly increases the CCK level in the mouse, and the concentration of CCK in the blood of the mouse reaches about 80pg/mL within 60-120 min.
EXAMPLE 3 preparation of buckwheat protein CCK secretion-promoting active peptide
Taking YMC ODS C18 filler, conventional swelling and column packing (methanol is used as a filling buffer solution), wherein the height of the column is 25cm, the inner diameter of the column is 1.6cm, a water layer is required to be reserved at the top of the column at any moment, the column can be used after balancing about 3-4 column volumes, and the buckwheat protein hydrolysate begins to be added when the liquid level is 2-3 mm. About 160mg of buckwheat protein zymolyte powder is weighed and dissolved in 2mL of distilled water, a 0.45 mu m microporous filter membrane is added into a chromatographic column after filtration, the eluent is methanol solutions (5%, 30%, 80% and 100%) with different volume concentrations, the elution speed is 2mL/min, and the elution peak is collected. The separation spectrum of the buckwheat protein hydrolysate is shown in fig. 5, and the separation spectrum of the buckwheat protein hydrolysate into 4 peptide components can be seen by a C18 chromatographic column.
The 4 peptide fractions were evaluated for activity by the method described above, and the results are shown in FIG. 6. It was found that of the 4 peptide components, the F3 and F4 components had the best ability to stimulate CCK secretion by STC-1 cells. Thus, the F3, F4 components are highly active CCK secretion-promoting peptides.
Example 4 identification of buckwheat proteins containing RVTVQPDS, PAFKEEHL, QFDLDD, IPPLFP and SFHFPI active peptides
Peptide sequences in the F3 and F4 components are identified by mass spectrometry. The sample was dissolved in distilled water to prepare a 1mg/mL sample. A reverse phase chromatography column (150 μm i.d. x 150mm,packed with AcclaimPepMap RPLC C18,1.9 μm,) Separating, wherein the mobile phase A is 0.1% formic acid aqueous solution, the mobile phase B is 0.1% formic acid/80% acetonitrile solution, and the gradient elution and separation gradient are carried out at the flow rate of 600 nL/min: 0-2min,4-8% B;2-45min,8-40% B;45-55min,40-60% B;55-56min,60-95% B;56-66min,95% B. The mass spectrum ion source type is electrospray ionization source (ESI), positive ion scan mode, spray voltage 2200V, capillary temperature 270 ℃. Primary mass spectrum parameter setting: scan range 100-2000m/z, maximum resolution 70000, auto gain parameter 3000000. Secondary mass spectrometry parameter setting: the scanning range is 50-2000m/z, the maximum resolution is 17500, and the automatic gain parameter is 100000.
The ion peaks mainly occurring in the F3 component were m/z=451.24, z=2, m/z=324.17, z=3, m/z=752.31, z=1, as detected by mass spectrometry; the ion peaks mainly occurring in the F4 component are m/z=451.24, z=2, m/z=374.19, z=2, m/z=683.41, z=1. The molecular ion peak is further subjected to secondary mass spectrometry, and the secondary mass spectrogram of the molecular ion peak is shown in figures 7-11. Through database matching, the peptides corresponding to the ion peaks are F3：Arg-Val-Thr-Val-Gln-Pro-Asp-Ser(RVTVQPDS);Pro-Ala-Phe-Lys-Glu-Glu-His-Leu(PAFKEEHL);Gln-Phe-Asp-Leu-Asp-Asp(QFDLDD);F4：Arg-Val-Thr-Val-Gln-Pro-Asp-Ser(RVTVQPDS);Ser-Phe-His-Phe-Pro-Ile(SFHFPI);Ile-Pro-Pro-Leu-Phe-Pro(IPPLFP).
Buckwheat protein mainly contains globulin, of which RVTVQPDS is present in buckwheat 13S globulin (Uniprot protein accession number: W6JQD 0); other active peptides PAFKEEHL, QFDLDD, IPPLFP and SFHFPI were derived from buckwheat UDP-glycose: glycosyltransferase (Uniprot protein accession number: A0A0 HAP 0), aspartic proteinase 9 (Uniprot protein accession number: Q5PT 35), ccmB (Uniprot protein accession number: A0A6M4SRV 2) and Protein Ycf2 (Uniprot protein accession number: A0A6N0GPR 0), respectively. Both of the active peptides RVTVQPDS, PAFKEEHL, QFDLDD, IPPLFP and SFHFPI can be prepared from buckwheat protein.
Wherein, the amino acid sequence of the buckwheat 13S globulin (Uniprot protein accession number: W6JQD 0) is shown as SEQ ID NO.1, and the specific steps are as follows:
MLLGVLLCIM VSLAASETRS RGSSTMRARQ CRLDQLTSSQ PNQKIQSEGGTIEVWDEEED 60
QFQCAGVAAM RVTVQPDSLS LPSYYSSPRL VYVEQGEGVF GLSLPGCPETYQSRGMEMRG 120
DEEEEEGFES GRRMTDAHQP TRRVRKGDVV ALPQGTVHWCFNDGQEDLVV VAVHNLNTDA 180
NQLDQSLKTF FLAGGVQGGS KEGKSQKLNF NNILSAFETKLLAEALGTEE ETVRKMQESD 240
ERGPIVKARK NMRQMVTPPR FGREQDEDET NGLEESFCNMRFRHNLGPRT EADIASRQAG 300
RIHSVDQNKL PILEFIDMSA EKGHLLPNAM LAPAWPLSGH RVFYVLRGEAQMQIVDDNGQ 360
TVLDDRVSEG SMVVIPQFYI STCRAGRDGL EYVSFETTAN PMSSPLNGHASVFKGMPIPV 420
LSNSYQISPR AAYELKQTRS HEHGLFSPFG GRS 453
The amino acid sequence of buckwheat UDP-glycose: glycosyltransferase (Uniprot protein accession number: A0A0A1HAP 0) is shown in SEQ ID NO.2, and is specifically as follows:
MSTATHILVF PHPAQGHMLP LLDLTNQLAI RGLRITILVT PKNLPILTPILTAHPSIQTL 60
VLSLPPHPKL PVGAENIRDI GNRGNFPLVS ALSKLAGPISDWFNSHPSPPHAIISDFFLG 120
WTHSLATQLG IPRIAFFSSG AFLCSIFSIL FSDIARFKAL HSVEFTDLPNSPAFKEEHLP 180
SVFRFYNESD PDSRSFKEGL LANMSSWGCV FNSFRALEGQYLDHFRKLTV HRRVHGVGPL 240
SLFRINSDQE SSCEPVLTWL DDCPDGSVLY VSFGSQKWLS EPQLQALGLALEKSQTRFIW 300
VVRGEPELDG LEERVAGRGL VVREWAPQVE ILGHRAVGGFLSHCGWNSAL ESVVAGVMIV 360
AWPMEADQYV NARILVEELG VGVHACEGPQ TVPDPDELGRVIKDAMGLNA AQKLRAKELK 420
EKAFEAVRDG GSSVTELDEF VNELVRVCPK 450
The amino acid sequence of buckwheat Aspartic proteinase (Uniprot protein accession number: Q5PT 35) is shown in SEQ ID NO.3, and is specifically as follows:
MTNISRISLL LLVLLVSPIS LSVANNDLVR VGLKKRKLDP TNRPASRFGCKKHLMQKYGL 60
GNGFGDDDTG IISLKNYMDA QYFGEIAIGT PSQTFTVIFD TGSSNLWVPSGKCYLSIACF 120
FHSKYKSSKS STYVKNGKSA EIHYGTGAIS GYFSQDNVKVGDLVVENQEF IEATREPSLT 180
FVAAKFDGIL GLGFQEISVG KAVPVWYNMV NQGLVNEPVFSFWLNRNADE EIGGEIVFGG 240
IDPAHHKGEH TYLPVTQKGY WQFDLDDVLV GGESTGFCSGGCSAIADSGT SLLAGPTPVV 300
AQINHAIGAS GVVSQECKTV VSQYGKQILD LLVSQTQPRK ICSQIGLCTFDGTRGVSMGI 360
ESVVDKNVDK SSGNLKDATC SACEMAVVWM QNQLKQNQTEDRILDYANQL CERLPSPMGE 420
SAVDCGSLST LPTVSFTLGG KTFALAPEQY ILQVGEGPAT QCISGFIALDVPPPRGPLWI 480
LGDIFMGQYH TVFDHGNMQV GFAEAA 506
The amino acid sequence of buckwheat CcmB (Uniprot protein accession number: A0A6M4SRV 2) is shown in SEQ ID NO.4, and is specifically as follows:
MRRLFFELYH KQIFFSTPIT SFSPFLSYIV VTPLMLGFEK DFSCHFHLGPIRIPPLFPFP 60
PAPFPRNEKE DGTLELYYLS AYCLPKILLL QLVGHRVIQI SRVFCSFPMLELLYQFGRSG 120
MDRLNILLGG PVLTLLCGIH SRSALGITSS SGWNSSQNLT TSPTLLPSTLSRTSIETEWF 180
HVLSSIGYSS PFVSLSPISV SISSQD 206
The amino acid sequence of buckwheat Protein Ycf (Uniprot protein accession number: A0A6N0GPR 0) is shown in SEQ ID NO.5, and is specifically as follows:
MTKKHSKNLD FWVFELKEIL REIKNSVGSL VKIFFHQERF IKLFDPRIWSILLSPNWRGS 60
IIKRYFTFTI TGVVFFVVAG LIYRINNRNM VEKKNIYLIG LLPIPMNSLG
GSSNINRLIV SLLYLPKGKK ISESCFLDPK ESTWVPPITK RFKSNVGSRWWRNWIVKKRD 180
SSQLKGSFDQ SRDRLNSIRN ADSEYHTSLN QREIQQRKER SIPWDPSFLQTEETEIGSGR 240
FPKCLSGYSR LFAEGEKQMN NQRLPEEIEE LLGNPTRSIR SFFSDRWSELHLGSNPTERS 300
TRDQKLLKKE QDFSFVPCRR SENQEMVNIF KIITYLQKTV SIHPISSDRGCDMVPKDEPN 360
MYSSNKISFL NKNPFFNLFR LFHDRNRGGY TLHHDFESEE RFQEMADLFTLSITEPDLVY 420
HKGFAFSIDS YGLDQKHFLN EVFNSRDESK KKSLLVLPPI FYDENESFYRRIRKKWLRIS 480
CGNYLKDTKE KIVVFASNNI MEAVNQYRLI RNLIQIQYST YGYIINVLSRFFFILINRSD 540
RNFEYGIQRD QIGNDTLNHR TIMKYTINQH LSNLKKNQKK RFDSLILISRSERSMNRDPD 600
AYRYKWFNGS KNFQEHFVSE QKSHFQVQVV FDRLRINQYWIDWSKVINKK NLSKSLSKLI 660
LFLSNSLPFF FVSLGNMPIH RSEIHIYELQ GPNDQLCNPL LKSLGLPIVHLKKWKADDHD 720
TSQKSKLLIN GGTISPFLFN KIPKWMVDSH TRKNRRKSFD NTDSYFSMISFDQDNWLNPV 780
KSFHRSSLIS SFSKANRLRF LNNPHHFFFY CNKRFPFYME KARIKNYDFTYRQFLNILFI 840
RNKQFSLCVG KKKHAFLERD TISPIESQVS NIFIPNYFPQ RGNERYNLYKSFHFPIRSDL 900
FVRRTIYSIA DISGTPLTEE EMVNLERTYC QPLSDMNLSD SERKNLHQYLNFNSNMGLIH 960
TPRSEKYLPS ETSQKLRLWR NWLKKGVEKG QMDRTFQRDSAFSTLSKWNL FQTYMPWFFT 1020
STGYKYLNFI FLDAFSDLLP MLSSSHKFVS ILNYIMHRSA CRILKLKWRALKRKLWGLWA 1080
PISEISSDIS WKCFRRLLRV EEMIHRNNES PLISTHLSSP NVREFLYSSLLLLLLAGCLV 1140
QVLLFSVSLD SSELQTEFER IKSLTIPSYT IEVYKLVDGY PKPEPNSFWLKNLFLVAREQ 1200
LEDFLTEILG FARFGGGPAY GVKLIQKIFF NLIDLISIIP NPINRITFSRNTRRLSHTSK 1260
EIYSWIRKGQ RFQTHDEIES WIETCDWFLD KERVYSFHFS TLRPEKGIDQILWSLTHIDH 1320
LVKSDYGYQM FEQAGAIYLR YLVDIHQKDL MNYEFNTSCLAERRIFLAHY QTITYSQTSC 1380
GANSFHFPSH GKPKPFSFRL ALYPSRGILV IGPIGTGRSY LVKSLATNSYLPFITVFLNK 1440
LDFKIVIDDL DPEDYMEALD DVDIGGIDDD SDPAKEYMYALKDVDDVDDR DSIYSNLDSD 1500
PELREEYTVD DEILRYIVEL EIDLAPINLQ FELARTMSPC IVWIPNIHDLYVDESESLGL 1560
LLNYLSGDSE RRSTRDILVI ASTHIPQKVD PALIAPNKLN TCIKIRRLLIPQQRKHVFTL 1620
SYTRGFHLEK KMFHTNGFGS IALGYNARDL VAITNEALSI SITQKKSIIDTNTIRFALHR 1680
QTWELRAHVR PVPDHGILFY QIGRAVAQNV LINNCCLIDP ISIYTKKQSCYEGDPYLYKW 1740
FFELGTSMKK LTILLYLLSC SAGSVAQDLW SLPGPDEKNW ITSYRLVETDSDLVDGLLEV 1800
VEGALVGSSL LRPEPRNPLE MMENGSRSIF DRRFLYESEF KEWAEGADPQQLAEDVVDHI 1860
VWAPRIWQPW GFLFDWIERP NELEFPYWAR SFRGKPIISD EVNDEYFDYAFYGEGDDGYD 1920
EEDELQENDW EFLQSETMEY PGRDRSSKEQ VFFRKGQFIW DPGDPLFYIFNDELSVFLFS1980
HREFFADEEM SKGLLLTSQR ETLYKRGFSK KAKEKYFEFF INRQRRRRLRTIRSLYNRSF 2040
RSNIQSASYQ YLSNLFLSNG RLLDQMTKTL FRKRWIFPDE LKIGFM 2086 the description of the embodiments above is provided to facilitate the understanding and use of the invention by those of ordinary skill in the art. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Claims (10)
1. A buckwheat-derived cholecystokinin secretion peptide, characterized by one or more of the following active peptides selected from the group consisting of:
The amino acid sequence of the active peptide P1 is Arg-Val-Thr-Val-Gln-Pro-Asp-Ser;
An active peptide P2, the amino acid sequence of which is Pro-Ala-Phe-Lys-Glu-Glu-His-Leu;
an active peptide P3, the amino acid sequence of which is Gln-Phe-Asp-Leu-Asp-Asp;
an active peptide P4, the amino acid sequence of which is Ile-Pro-Pro-Leu-Phe-Pro;
the amino acid sequence of the active peptide P5 is Ser-Phe-His-Phe-Pro-Ile.
2. A polynucleotide encoding the cholecystokinin-secreting peptide of buckwheat origin according to claim 1.
3. The method for preparing the cholecystokinin-promoting secretory peptide from buckwheat sources according to claim 1, which is characterized in that the cholecystokinin-promoting secretory peptide is obtained from enzymatic hydrolysis of buckwheat protein by a separation and purification method, or is synthesized artificially by a genetic engineering method, or is prepared directly by chemical synthesis.
4. A method for preparing an enzymatic hydrolysate containing the buckwheat-derived cholecystokinin-secreting peptide of claim 1, comprising the steps of: sequentially hydrolyzing the buckwheat protein with pepsin and trypsin to obtain a buckwheat protein enzymolysis product, namely an enzymolysis product containing the buckwheat-derived cholecystokinin secretion peptide according to claim 1.
5. The method of claim 4, wherein the method of extracting buckwheat protein from buckwheat comprises the steps of:
Grinding semen Fagopyri Esculenti, degreasing, adding into distilled water, setting temperature to 50-60deg.C, adjusting pH to 4-6, and pre-treating with saccharifying enzyme for 0.5-2 hr; cooling to room temperature, adjusting pH to 10-12, extracting protein, centrifuging to obtain supernatant, adjusting pH to 4.4-4.6, standing, centrifuging, and washing with water; and finally drying to obtain the buckwheat protein.
6. The method according to claim 4, wherein the mass ratio of pepsin or trypsin to the buckwheat protein is 1:10-100;
The enzymolysis condition of pepsin or trypsin is 37 ℃ for enzymolysis for 1-4 hours.
7. The method according to claim 4, further comprising the steps of, after obtaining the buckwheat protein hydrolysate:
Separating the buckwheat protein enzymolysis product by using YMC ODS C18 chromatographic column, eluting with 5% methanol deionized water solution, 30% methanol deionized water solution, 80% methanol deionized water solution and 100% methanol solution as eluent at a certain flow rate, purifying by chromatography, and collecting different components; then detecting the influence of different components on CCK secretion of enteroendocrine cells, and selecting the component with the highest activity for stimulating CCK secretion as a target component, namely the product containing the buckwheat-source cholecystokinin secretion peptide according to claim 1.
8. An enzymatic hydrolysate comprising the buckwheat-derived cholecystokinin-secreting peptide of claim 1 prepared based on the method of any of claims 4-7.
9. Use of a cholecystokinin-secreting peptide of buckwheat origin according to claim 1 or an enzymatic hydrolysate according to claim 8 for the preparation of a medicament for the prevention or adjuvant treatment of obesity.
10. A pharmaceutical product comprising the cholecystokinin-secreting peptide of buckwheat origin according to claim 1 or the enzymatic hydrolysate according to claim 8, said pharmaceutical product having a function of preventing or assisting in the treatment of obesity.
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| CN102361560A (en) * | 2008-12-31 | 2012-02-22 | 索莱有限责任公司 | Protein hydrolysate compositions having enhanced CCK releasing ability |
| CN115353549A (en) * | 2022-06-27 | 2022-11-18 | 上海理工大学 | Small peptide for promoting intestinal hormone secretion and preparation method and application thereof |
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| JP3997114B2 (en) * | 2002-06-10 | 2007-10-24 | 独立行政法人科学技術振興機構 | Arginine-containing peptide having cholecystokinin secretion promoting activity and food containing the same |
| MX2013009772A (en) * | 2011-02-23 | 2013-10-01 | Solae Llc | Protein hydrolysate compositions having enhanced cck and glp-1 releasing activity. |
| CN115353553B (en) * | 2022-06-27 | 2023-06-02 | 上海理工大学 | CCK secretion-promoting peptide targeting calcium sensitive receptor and preparation method and application thereof |
| CN115353551B (en) * | 2022-06-27 | 2024-01-26 | 上海理工大学 | Oat-derived GLP-1 secretion-promoting oligopeptide and preparation method and application thereof |
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| CN102361560A (en) * | 2008-12-31 | 2012-02-22 | 索莱有限责任公司 | Protein hydrolysate compositions having enhanced CCK releasing ability |
| CN115353549A (en) * | 2022-06-27 | 2022-11-18 | 上海理工大学 | Small peptide for promoting intestinal hormone secretion and preparation method and application thereof |
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