CN107141346B - Bioactive polypeptide ATLEDSPEVI, and preparation method and application thereof - Google Patents
Bioactive polypeptide ATLEDSPEVI, and preparation method and application thereof Download PDFInfo
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- CN107141346B CN107141346B CN201710480369.0A CN201710480369A CN107141346B CN 107141346 B CN107141346 B CN 107141346B CN 201710480369 A CN201710480369 A CN 201710480369A CN 107141346 B CN107141346 B CN 107141346B
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- atledspevi
- antioxidant
- polypeptide
- biologically active
- product
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4732—Casein
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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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
Abstract
The invention relates to the field of protein, and in particular relates to a bioactive polypeptide ATLEDSPEVI, a preparation method and application thereof, wherein the amino acid sequence of the bioactive polypeptide ATLEDSPEVI is Ala-Thr-Leu-Glu-Asp-Ser-Pro-Glu-Val-Ile. In-vitro antioxidant experiments and in-vitro immune function promotion experiments prove that the polypeptide ATLEDSPEVI has better antioxidant biological activity and immune regulation function, on one hand, the polypeptide has better antioxidant activity, can remove free radicals in a body and improve the quality of life; on the other hand, the bioactive polypeptide ATLEDSPEVI can enhance the in vitro proliferation capacity of lymphocytes and macrophages, promote the macrophages to secrete cytokines, improve the capability of an organism to resist infection of external pathogens, reduce the morbidity of the organism and has very important significance for developing foods, health-care products and medicines with immune regulation functions and anti-oxidation functions.
Description
Technical Field
The invention relates to the field of proteins, in particular to a bioactive polypeptide ATLEDSPEVI, and a preparation method and application thereof.
Background
In the process of fermenting the cow milk by the lactic acid bacteria, a part of protein in the cow milk is metabolized and utilized by the lactic acid bacteria, and a series of physiological and biochemical reactions occur, so that the protein is changed into polypeptide or free amino acid which is digested and absorbed by a human body or directly enters the blood circulation of the human body through the absorption and transportation of small intestinal epithelial cells. Among these polypeptides, some have a specific physiological function and are called "bioactive peptides".
Oxidation reactions and oxidative metabolism are vital to food and the human body, and free radicals and active oxygen cause a series of oxidation reactions. When excessive free radicals are formed, they exceed the protective effects of protective enzymes such as superoxide dismutase, catalase, resulting in a series of side effects such as lipid oxidation, apoptosis, etc. The oxidation reaction not only affects the shelf life of the fat-containing food, but also causes certain harm to the health of human bodies, such as rheumatoid arthritis, diabetes, arteriosclerosis and the like. In addition, Collins et al, 2005 discovered that cancer development was also associated with oxidative damage to DNA.
Early synthetic antioxidants such as Butylated Hydroxyanisole (BHA), 2, 6-di-tert-butyl-4-methylphenol (BHT) were used in food as lipid antioxidants, but these synthetic additives all have potential risks to humans. Therefore, it is particularly important to find safe antioxidants in natural food sources. In recent years, it has been found that some food-derived polypeptides, such as short peptides of corn, soybean peptides, milk polypeptides, etc., have a good antioxidant effect. The polypeptides can be obtained through various ways such as microbial fermentation, digestion and enzymolysis and the like, and most of the polypeptides with antioxidant activity consist of 2-20 amino acid residues, have the molecular weight of less than 6000Da and contain a certain amount of hydrophobic amino acids and aromatic amino acids.
Immunoactive peptides are a class of bioactive polypeptides that are first obtained from milk following opioid peptide discovery and demonstrate their physiological activity. Jolles et al found in 1981 for the first time that a hexapeptide with an amino acid sequence Val-Glu-Pro-Ile-Pro-Tyr can be obtained by hydrolyzing human milk protein with trypsin, and in vitro experiments prove that the hexapeptide can enhance the phagocytosis of mouse abdominal cavity macrophages to sheep erythrocytes. Migliore-Samour et al found that the casein-derived hexapeptide Thr-Thr-Met-Pro-Leu-Trp was able to stimulate phagocytosis of murine peritoneal macrophages by sheep red blood cells and to enhance resistance to Klebsiella pneumoniae. Lemna hexandra et al, fed rats with synthetic milk-derived immunoregulatory peptide (PGPIPN), found that phagocytosis of macrophages in the abdominal cavity of rats and immunoregulatory function related to erythrocytes were significantly enhanced.
Researches show that the immune active peptide can not only enhance the immunity of the organism, stimulate the proliferation of lymphocytes of the organism, enhance the phagocytic function of macrophages, promote the release of cell factors, improve the capability of the organism for resisting the infection of external pathogens, reduce the morbidity of the organism, but also can not cause the immune rejection reaction of the organism.
At present, there are many researches on bioactive polypeptides, for example, chinese patent CN105254738A discloses a milk-derived bioactive polypeptide DELQDKIH derived from β -casein, chinese patent CN105254739A discloses a milk-derived bioactive polypeptide GTQYTD derived from α s 1-casein, and chinese patent CN105254740A discloses a milk-derived bioactive polypeptide NQFYQKF derived from α s 2-casein.
Disclosure of Invention
The invention aims to provide a bioactive polypeptide ATLEDSPEVI, and a preparation method and application thereof.
The purpose of the invention can be realized by the following technical scheme:
in a first aspect of the invention, there is provided a biologically active polypeptide ATLEDSPEVI having the amino acid sequence Ala-Thr-Leu-Glu-Asp-Ser-Pro-Glu-Val-Ile as set forth in SEQ ID NO: 1 is shown.
Preferably, the biologically active polypeptide is milk-derived. In particular to kappa-casein, and is the amino acid residue from 165 th to 174 th positions of a kappa-casein variant A. The amino acid sequence of the kappa-casein variant A is shown as SEQ ID NO: 3, respectively.
The amino acid sequence and the corresponding nucleotide sequence of the kappa-casein are the existing technology, and the nucleotide fragment which codes the 165 th to 174 th amino acid residues of the kappa-casein variant A can code mature bioactive polypeptide ATLEDSPEVI.
Preferably, the bioactive polypeptide has antioxidant and immunoregulatory functions.
In a second aspect of the present invention, there is provided a nucleotide fragment encoding the biologically active polypeptide ATLEDSPEVI, the sequence of which is: 5'-gct act cta gaa gat tct cca gaa gtt att-3', as shown in SEQ ID NO: 2, respectively.
In the third aspect of the present invention, a preparation method of the bioactive polypeptide ATLEDSPEVI is provided, which can be artificially synthesized by a genetic engineering method, can be directly obtained from a dairy product by a separation and purification method, and can be directly prepared by chemical synthesis.
In the fourth aspect of the invention, the application of the bioactive polypeptide ATLEDSPEVI in preparing food, health products, medicines or cosmetics with antioxidant function is provided.
In the fifth aspect of the invention, the application of the bioactive polypeptide ATLEDSPEVI in preparing food, health-care products or medicines with immunoregulation function is provided.
In a sixth aspect, the invention provides an application of the bioactive polypeptide ATLEDSPEVI in preparing food, health care products or medicines with antioxidant function and immunoregulation function.
Specifically, the bioactive polypeptide ATLEDSPEVI can be used for preparing cosmetics for reducing free radical damage to skin, and medicines for resisting oxidation and/or regulating immunity of organism; and because the product of the bioactive polypeptide ATLEDSPEVI degraded by gastrointestinal tract still has bioactivity, the bioactive polypeptide ATLEDSPEVI can be used for preparing foods such as yoghourt and health-care products for regulating immunity, and can be orally taken for preparing medicines for resisting oxidation and/or regulating organism immunity.
In a seventh aspect of the invention, there is provided an antioxidant product comprising said biologically active polypeptide ATLEDSPEVI or a derivative of said biologically active polypeptide ATLEDSPEVI; the antioxidant product comprises antioxidant food, antioxidant health product, antioxidant medicine or antioxidant cosmetic; the derivative of the biologically active polypeptide ATLEDSPEVI refers to a polypeptide derivative obtained by performing modifications such as hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation on an amino acid side chain group, an amino terminal or a carboxyl terminal of the biologically active polypeptide ATLEDSPEVI.
In an eighth aspect of the invention, there is provided an immunomodulatory product comprising said biologically active polypeptide ATLEDSPEVI or a derivative of said biologically active polypeptide ATLEDSPEVI; the immunoregulation product comprises immunoregulation food, immunoregulation health-care products or immunoregulation medicines; the derivative of the biologically active polypeptide ATLEDSPEVI refers to a polypeptide derivative obtained by performing modifications such as hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation on an amino acid side chain group, an amino terminal or a carboxyl terminal of the biologically active polypeptide ATLEDSPEVI.
In the ninth aspect of the present invention, a product having both antioxidant function and immunoregulatory function is provided, which comprises the bioactive polypeptide ATLEDSPEVI or a derivative of the bioactive polypeptide ATLEDSPEVI; products with antioxidant and immunoregulatory effects include foods, health products or pharmaceuticals; the derivative of the biologically active polypeptide ATLEDSPEVI refers to a polypeptide derivative obtained by performing modifications such as hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation on an amino acid side chain group, an amino terminal or a carboxyl terminal of the biologically active polypeptide ATLEDSPEVI.
The bioactive polypeptide ATLEDSPEVI has the following beneficial effects: the milk-derived bioactive polypeptide ATLEDSPEVI has good antioxidant activity and activity for regulating the immunity of organisms; on one hand, the free radicals in the organism can be removed, and the harm of the free radicals to the human body is reduced; on the other hand, the bioactive polypeptide ATLEDSPEVI can also regulate the immunity of the organism, enhance the proliferation capacity of lymphocytes, improve the capability of the organism for resisting the infection of external pathogens, reduce the morbidity of the organism, and cannot cause the immune rejection reaction of the organism, thereby having very important significance for developing dairy products and health care products with the functions of resisting oxidation and regulating immunity.
Drawings
FIG. 1: mass chromatogram extraction (m/z 537.27);
FIG. 2: a primary mass spectrum of a fragment with a mass to charge ratio of 537.27;
FIG. 3: fragmentation of polypeptide az and by with mass-to-charge ratio of 537.27;
FIG. 4: [ DPPH. ] methanol Standard Curve;
FIG. 5: FeSO4A standard curve;
FIG. 6: an in vitro macrophage proliferation capacity test of the bioactive polypeptide ATLEDSPEVI;
FIG. 7: an IL-4 standard curve;
FIG. 8: the effect of the concentration of the cytokine IL-4 secreted by the biologically active polypeptide ATLEDSPEVI.
Detailed Description
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS Inmolecular BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATINSTRUCUTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) Methods Inenzymolygy, Vol.304, Chromatin (P.M. Wassarman and A.P.Wolffe, eds.), academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.
The invention is described in detail below with reference to the figures and specific embodiments.
Example 1 Artificial Synthesis of active peptide ATLEDSPEVI
Synthesis of bioactive peptide
1.3 g of RINK resin (degree of substitution 0.3mmol/g) was weighed into a 150ml reactor and soaked with 50ml of Dichloromethane (DCM).
After 2.2 hours, the resin was washed with 3 resin volumes of N-Dimethylformamide (DMF) and then drained, and this was repeated four times and the resin was drained until use.
3. The Fmoc protecting group on the resin was removed by adding a quantity of 20% piperidine (piperidine/DMF: 1:4, v: v) to the reactor and shaking on a decolourising shaker for 20 min. After deprotection, the resin was washed four times with 3 resin volumes of DMF and then drained.
4. And (3) detecting a small amount of resin by a ninhydrin (ninhydrin) method (detecting A and B, respectively, and reacting at 100 ℃ for 1min), wherein the resin is colored, which indicates that the deprotection is successful.
5. Weighing a proper amount of amino acid Ala and a proper amount of 1-hydroxy-benzotriazole (HOBT) into a 50ml centrifuge tube, adding 20ml of DMF to dissolve the amino acid Ala and the 1-hydroxy-benzotriazole (HOBT), then adding 3ml of N, N diisopropyl carbodiimide (DIC) to shake and shake for 1min, adding the solution into a reactor after the solution is clarified, and then placing the reactor into a30 ℃ shaking table to react.
After 6.2 hours, the column was capped with a suitable amount of acetic anhydride (acetic anhydride: DIEA: DCM ═ 1:1:2, v: v: v) for half an hour, then washed four times with 3 resin volumes of DMF and drained until needed.
7. The Fmoc protecting group on the resin was removed by adding a quantity of 20% piperidine (piperidine/DMF: 1:4, v: v) to the reactor and shaking on a decolourising shaker for 20 min. After deprotection was washed four times with DMF and then drained.
8. And (3) detecting a small amount of resin by a ninhydrin (ninhydrin) method (detecting A and B, respectively, and reacting at 100 ℃ for 1min), wherein the resin is colored, which indicates that the deprotection is successful.
9. Weighing a second proper amount of amino acid and a proper amount of HOBT in a 50ml centrifuge tube, adding 25ml of DMF to dissolve the amino acid and the HOBT, adding 2.5ml of DIC to shake and shake for 1min, adding the solution into a reactor after the solution is clarified, and then placing the reactor in a shaking table at 30 ℃ to react.
After 10.1 hours, a small amount of resin is taken for detection, and the detection is carried out by an indanthrone method (two drops are respectively detected A and B, and the reaction is carried out for 1min at 100 ℃), if the resin is colorless, the reaction is complete; if the resin is colored, the condensation is not complete and the reaction is continued.
11. After the reaction was completed, the resin was washed four times with DMF and then drained, and a certain amount of 20% piperidine (piperidine/DMF ═ 1:4, v: v) was added to the reactor, and the mixture was shaken on a decolorizing shaker for 20min to remove the Fmoc-protecting group from the resin. After the protection is removed, washing with DMF for four times, and then draining to detect whether the protection is removed.
12. And sequentially grafting amino acids Thr, Leu, Glu, Asp, Ser, Pro, Glu, Val and Ile according to the steps 9-11.
13. After the last amino acid had been grafted, the protection was removed, washed four times with DMF and the resin was drained with methanol. The polypeptide was then cleaved from the resin with 95 cleavage medium (trifluoroacetic acid: 1,2 ethanedithiol: 3, isopropylsilane: water: 95:2:2:1, v: v: v) (10 ml of cleavage medium per gram of resin) and centrifuged four times with glacial ethyl ether (cleavage medium: ethyl ether: 1:9, v: v).
To this end, bioactive peptide ATLEDSPEVI was synthesized.
Confirmation of biologically active peptides
1) UPLC analysis
UPLC conditions were as follows:
the instrument comprises the following steps: waters ACQUITY UPLC ultra-high performance liquid-electrospray-quadrupole-time-of-flight mass spectrometer
Specification of chromatographic column: BEH C18 chromatographic column
Flow rate: 0.4mL/min
Temperature: 50 deg.C
Ultraviolet detection wavelength: 210nm
Sample introduction amount: 2 μ L
Gradient conditions: solution A: water containing 0.1% formic acid (v/v), liquid B: acetonitrile containing 0.1% formic acid (v/v)
2) Mass spectrometric analysis
The mass spectrometry conditions were as follows:
ion mode: ES +
Mass range (m/z): 100-1000
Capillary voltage (Capillary) (kV): 3.0
Sampling cone (V): 35.0
Ion source temperature (. degree. C.): 115
Desolvation temperature (. degree. C.): 350
Desolventizing gas stream (L/hr): 700.0
Collision energy (eV): 4.0
Scan time (sec): 0.25
Inner scan time (sec): 0.02
According to the above analysis method, ultra high performance liquid chromatography-electrospray-quadrupole-time-of-flight mass spectrometry is used to perform chromatographic analysis and mass spectrometry on bioactive peptide ATLEDSPEVI, the mass chromatogram extraction diagram is shown in FIG. 1, the primary and secondary mass spectrograms of the peak are shown in FIGS. 2 and 3, the polypeptide mass-to-charge ratio of the peak is 537.27Da, and the retention time is 18.21 min.
3) Results
As can be seen from FIG. 3, the fragment sequence with the mass-to-charge ratio of 537.27Da, which is obtained by analysis and calculation of Progenetics QI software according to the cases of az and by fragmentation, is Ala-Thr-Leu-Glu-Asp-Ser-Pro-Glu-Val-Ile (ATLEDSPEVI) and is shown as SEQ ID NO: 1. the fragment corresponds to residue sequences of 165 th to 174 th sites of a kappa-casein variant A, the GenBank number of a kappa-casein amino acid sequence is AAA30433.1, and the sequence is shown in SEQ ID NO: 3.
example 2 antioxidant Activity assay of bioactive peptides
The bioactive polypeptide ATLEDSPEVI obtained in example 1 was tested for antioxidant activity by free radical scavenging (DPPH & method) and total antioxidant activity (Ferric Reducing AbilityPower FRAP method).
1. [ DPPH ] method for determining in vitro antioxidant activity of bioactive peptide ATLEDSPEVI
1) Experimental reagent and instrument
Reagent: 1, 1-Diphenyl-2-trinitrophenylhydrazine (1, 1-Diphenyl-2-piperidinylhydrazyl [ DPPH. ]), manufactured by Wako corporation of Japan; methanol, available from Shanghai national drug company; milk-derived bioactive polypeptide ATLEDSPEVI obtained in example 1.
The main apparatus is as follows: sunrise microplate reader, available from Tecan, austria; 96-well cell culture plates, manufactured by Millipore, usa; analytical balance, product of Meitelei-tolido.
2) Experimental methods
(1)1mmol/L of [ DPPH. ] methanol solution
0.349mg of [ DPPH ] is weighed by an analytical balance and dissolved in 1mL of methanol solution to prepare 1mmol/L of [ DPPH ] methanol solution, and the tinfoil is stored away from light and ready to use.
(2) Determination of [ DPPH. ] methanol Standard Curve
Add 100 μ L [ DPPH. cndot. ] methanol standard curve sample into 96-well plate according to table 1, let stand for 90min at room temperature, and detect the absorbance at 517nm with enzyme-linked immunosorbent assay.
TABLE 1 [ DPPH. methanol Standard Curve solution preparation
From the experimental results, a curve was fitted using Excel and a regression equation was calculated, and the results are shown in fig. 4 (regression equation: y ═ 0.192x +0.2271, R2＝0.9991)。[DPPH·]The linear relation of the methanol standard curve is good, the correlation coefficient is 0.999, and the result shows that [ DPPH ]]The precision and accuracy of the methanol standard curve meet the detection requirements. From the results, the absorbance value was compared with [ DPPH ]]The contents are in inverse proportion, [ DPPH ]]The lower the content, the higher the absorbance, i.e.the greater the ability of the sample to scavenge free radicals.
(3) Method for measuring antioxidant activity of bioactive peptide ATLEDSPEVIV by [ DPPH ]
1) Sample group: adding 80 μ L of 1mmol/L [ DPPH. cndot. ] methanol solution into a 96-well plate, and adding 20 μ L of samples to be tested (ATLEDSPEVI), positive control 1 (Trolox of 2.5 mg/mL), positive control 2 (Trolox of 0.025 mg/mL), and negative control (phytic acid) at different concentrations according to Table 2;
2) blank group: a blank was made on the same 96-well plate by adding 80. mu.L of a 1mmol/L [ DPPH. ] methanol solution and 20. mu.L of deionized water.
And (3) standing the sample to be detected for 90min at room temperature after the sample loading is finished, and detecting the light absorption value at 517nm by using an enzyme-labeling instrument. The radical scavenging rate was calculated according to the following formula and the experimental results are shown in table 2.
The formula:
TABLE 2 determination of antioxidant Activity of bioactive Polypeptides by the DPPH method
As can be seen from Table 2, 2.5mg/mL of Trolox as a positive control had the strongest ability to scavenge free radicals under the same conditions, almost all free radicals in solution were scavenged, followed by 0.025mg/mL of Trolox, phytic acid, active polypeptide. The rate of the polypeptide ATLEDSPEVI for eliminating [ DPPH ] free radicals is inverted bell-shaped along with the change of concentration, and reaches the highest value at the concentration of 2.5mg/mL, which is 23.60%.
2. Method for measuring in-vitro antioxidant capacity of polypeptide in fermented milk by FARP (false positive RP) method
1) Experimental reagents and instruments
A total antioxidant capacity detection kit (a pharmaceutical Reducing activity of Plasma FRAP method) purchased from Shanghai Biyuntian Biotech company; FeSO4Solution (10mmol/L), water-soluble vitamin E (Trolox solution) (10mmol/L), milk-derived bioactive polypeptide ATLEDSPEVI obtained in example 1.
The main apparatus is as follows: sunrise microplate reader, available from Tecan, austria; 96-well cell culture plates, manufactured by Millipore, usa; analytical balance, product of Meitelei-tolido; HWS26 model electric heating constant temperature water bath, manufactured by Shanghai-constant technology, Inc.
2) Experimental methods
(1) Preparation of FRAP working solution
According to the total antioxidant capacity detection kit, 7.5mL of TPTZ diluent, 750 mu L of TPTZ solution and 750 mu L of detection buffer solution are uniformly mixed, incubated in a water bath at 37 ℃ and used up within 2 hours.
(2)FeSO4Preparation and determination of Standard Curve
Adding 180 mu LFRAP working solution into a 96-well plate, adding 5 mu L FeSO according to the table 34And (3) lightly mixing the standard curve solution, incubating for 3-5min at 37 ℃, and measuring the light absorption value at 593nm by using an enzyme-labeling instrument.
TABLE 3 FeSO4Solution formulation for standard curve determination
FeSO4The concentration and the light absorption value are in a good proportional relation, FeSO4The higher the concentration, the higher the absorbance. FeSO of the invention4The results of the standard curve are shown in FIG. 5, the linear relation of the standard curve is good, the correlation coefficient is 0.998, and the FeSO4The precision and accuracy of the standard curve meet the detection requirements, and can be used for subsequent calculation。
(3) FRAP method for determining antioxidant capacity of bioactive polypeptide ATLEDSPEVI
Adding 180 mu L of FRAP working solution into a 96-well plate, and adding 5 mu L of ddH into a blank control well2And O, adding 5 mu L of a sample to be detected into the sample detection hole and 5 mu L of phytic acid into the positive control, slightly mixing uniformly, incubating at 37 ℃ for 3-5min, and measuring the light absorption value at 593nm by using an enzyme-linked immunosorbent assay. Total antioxidant capacity is expressed as FeSO4The concentration of the standard solution. The radical clearance was calculated according to the following formula and the results are shown in Table 4.
TABLE 4 FARP method for determining the total antioxidant capacity of bioactive polypeptides in Lactobacillus helveticus fermented milk
The in vitro total antioxidant activity of the polypeptide ATLEDSPEVI is measured by a total antioxidant activity method (a Ferric Reducing activity Power FRAP method), and the bioactive polypeptide ATLEDSPEVI is found to have better capability of Reducing an oxidation substance; under the condition of concentration of 4mg/mL, the total antioxidant level of the polypeptide ATLEDSPEVI reaches 0.0231 mmol/g; the total antioxidant capacity of the bioactive polypeptide ATLEDSPEVI is proved to be higher than that of phytic acid with weak antioxidant activity under the same concentration, and the difference is significant (p is more than 0.05). Thus, the biologically active polypeptide ATLEDSPEVI of the invention was identified as having significant antioxidant capacity.
Example 3 Activity experiment of bioactive peptide for improving immunity
First, MTT method for testing in vitro lymphocyte proliferation capacity experiment of bioactive polypeptide ATLEDSPEVI
1) Experimental materials and instruments:
reagents and materials: experimental animals balb/c mice (male 6-8 weeks old, animal experiment center of Shanghai university of transportation, college of agriculture and biology); milk-derived bioactive polypeptide ATLEDSPEVI obtained in example 1; mouse lymphocyte extract (ex solibao); RPMI1640 medium (purchased from GIBCO); 3- (4, 5-Dimethylthiazol-2) -2, 5-diphenyltetrazolium bromide salt (MTT, available from Amresco, Inc.); concanavalin (ConA, available from Sigma); bovine serum albumin (BSA, available from Genebase); pepsin (available from Sigma); pancreatin (Corolase PP, from AB).
The instrument comprises the following steps: LRH-250F Biochemical incubator, Shanghai Hengshi Co., Ltd; GL-22M high speed refrigerated centrifuge, shanghai luxiang instrument centrifuge instruments ltd; hera cell150CO 2 incubator, Heraeus; dragon wellscan mk3 microplate reader, Labsystems corporation; ALPHA 1-2-LD vacuum freeze drier, Christ company; ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometer, waters corporation.
2) The experimental method comprises the following steps:
taking mouse spleen under aseptic condition, extracting mouse lymphocyte with lymphocyte extract, and performing primary culture. The cell density was adjusted to 2.5X 10 with complete RPMI1640 medium6one/mL. To a 96-well cell culture plate were added in sequence: 100 μ L mouse lymphocyte suspension, 100 μ L RPMI1640 complete medium, 20 μ L concanavalin, 100 μ L sample. In addition, a blank control group (PBS with pH7.2-7.4 and 3 mol/L) and a negative control group (500 mu g/mL BSA) are arranged, and the research shows that the blank control group has no influence on the in vitro lymphocyte proliferation. Each set of 3 replicates. At 5% CO2Culturing at 37 deg.C for 68h, adding 20 μ L MTT into each well under aseptic condition, culturing for 4h, carefully removing supernatant, adding 100 μ L dimethyl sulfoxide into each well, incubating at 37 deg.C for 10min, shaking, and measuring absorbance at 570nm with microplate reader.
The in vitro lymphocyte proliferation capacity is expressed by a stimulation index and is calculated as follows:
in the formula: a. the1Absorbance at 570nm for the blank; a. the2Absorbance at 570nm for the negative control, A3The absorbance at 570nm for the experimental group.
3) Results and analysis of the experiments
The results are shown in Table 5. As can be seen from Table 5, under the condition that the mass concentration of the bioactive peptide ATLEDSPEVI is 100 μ g/mL, the stimulation index of the milk-derived bioactive peptide ATLEDSPEVI is greater than that of BSA, which indicates that ATLEDSPEVI can stimulate the proliferation of mouse lymphocytes in vitro to some extent. And ATLEDSPEVI reached a stimulation index of 1.166, which was significantly different from that of the negative control group (P < 0.05). Therefore, the active polypeptide ATLEDSPEVI can be considered to have the capacity of remarkably promoting the mouse lymphocyte proliferation, can be eaten as a health product or an additive, and can improve the immunity of animals and human bodies.
TABLE 5 Effect of biologically active polypeptide ATLEDSPEVI on in vitro lymphocyte proliferation
| Experiment grouping | Stimulation index SI |
| |
1 |
| ATLEDSPEVI | 1.166±0.059* |
Note: the number marked as significant difference (P <0.05) compared to the negative control.
Second, MTT method for measuring in vitro macrophage proliferation ability experiment of bioactive polypeptide ATLEDSPEVI
1) Experimental reagent and instrument
Reagent: experimental animals balb/c mice (male 6-8 weeks old) were collected at the animal Experimental center of the college of agriculture and biology of Shanghai university of transportation; milk-derived bioactive polypeptide ATLEDSPEVI obtained in example 1; 3- (4, 5-Dimethylthiazol-2) -2, 5-diphenyltetrazolium bromide (MTT) Amresco; LPS (lipopolysaccharide) Sigma company; bovine Serum Albumin (BSA) Genebase; triple solutions, aqueous solutions containing 10% SDS, 5% isobutanol and 0.012mol/L HCl.
The instrument equipment comprises: LRH-250F Biochemical incubator Shanghai Hengshi Co., Ltd; GL-22M high speed refrigerated centrifuge Shanghai Luxiang apparatus centrifuge Instrument Co., Ltd; hera cell150CO2Incubator Heraeus; dragon WellscanMK3 microplate reader Labsystems.
2) The test method comprises the following steps:
balb/c mice were injected intraperitoneally with 2ml of 2% (w/w) sterile starch solution for three consecutive days, and sacrificed by cervical dislocation 24 hours after the last injection. Peeling off the abdominal skin, sucking 4 ℃ Phosphate Buffer Solution (PBS) by using a syringe to repeatedly wash the abdominal cavity, centrifuging the washed solution by using a centrifuge tube for 10 minutes after collecting the washed solution, discarding the supernatant after centrifuging the washed solution (1000rpm and 4 ℃), washing the washed solution twice by using 4 ℃ RPMI1640 complete culture solution (containing 10% FBS), staining the washed solution by using 0.2% trypan blue solution to detect the vitality of the cells, and confirming that the collected viable macrophages account for more than 95%. After reading the cell counting plate, the cell concentration was adjusted to the appropriate concentration.
The cell suspension that had been blown to complete suspension was added to a 96-well cell culture plate at 37 ℃ with 5% CO in an appropriate volume2After culturing for 4 hours under the environment, removing liquid in the holes, carefully cleaning the bottom of the holes of the cell culture plate by using a complete culture solution RPMI1640 at 37 ℃, and washing the cells and cell fragments which are not attached to the walls to obtain the purified attached abdominal cavity macrophages. 0.2ml of RPMI1640 complete medium was added to each well, and the small peptide sample for experiment and LPS were dissolved in the medium in advance and then added to start cell culture.
The number of the added cells was 2X 105100 μ l/well of cell suspension/mL, 200 μ l/well of RPMI1640 complete medium (10% FBS) containing bioactive polypeptide (100, 500, 1000 μ g/mL) was added after adherent purification, and cultured continuously for 48 hours, and LPS was added to the inflammatory group at 24 hours to a final concentration of 100 ng/mL. 5% MTT 20. mu.l/well was added at 44 hours, and 100. mu.l/well triple lysis buffer was added to stop the incubation after 48 hoursAfter the overnight dissolution, the absorbance (OD570) of each well was measured at a wavelength of 570nm with a microplate reader, and the Growth index (Growth Indices) was calculated as follows:
among them, the blank group was a cell-treated group to which no small peptide and BSA were applied, and the BSA group was a negative control.
3) Results of the experiment
The results are shown in FIG. 6, in which the bioactive polypeptide (ATLEDSPEVI) was added at concentrations of 1000, 500, and 100. mu.g/mL in the experimental group, and the blank group was added with a corresponding amount of PBS as a blank control, indicating the proliferation of macrophages in the absence of LPS stimulation. Compared with a blank control group, the experimental group added with the polypeptide ATLEDSPEVI with different concentrations has the characteristic that the proliferation capacity of macrophages is gradually increased along with the increase of the experimental concentration, and has a significant difference (P <0.05) when the concentration is 1000 and 500 mu g/mL. Indicating that the biologically active polypeptide ATLEDSPEVI has the ability to promote macrophage proliferation.
Third, the experiment (ELISA method) of promoting macrophage to secrete cytokine of the biological activity polypeptide ATLEDSPEVI
1. Experimental reagent and equipment
1) Reagent: experimental animals balb/c mice (male 6-8 weeks old), Shanghai Slek Experimental animals, Inc.; mouse lymphocyte extract, shanghai solibao biotechnology limited; RPMI1640 medium, GIBCO; bovine Serum Albumin (BSA), Genebase; milk-derived bioactive polypeptide ATLEDSPEVI obtained in example 1; ELISA cytokine Rapid kit (IL-4), Wuhan doctor De bioengineering, Inc.
2) Instrumentation and equipment
Model CM-230 Mohr super Water, Shanghai Mole scientific instruments, Inc.; LRH-250F Biochemical incubator, Shanghai Hengshi Co., Ltd; GL-22M high speed refrigerated centrifuge, shanghai luxiang instrument centrifuge instruments ltd; hera cell150CO2Incubator, Heraeus corporation; dragon Wellscan MK3 microplate reader, Labsystems Inc.
2. Test method
1) Preparation of the Standard Curve
Making an IL-4 standard curve: IL-4 standard substances with the concentrations of 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.3pg/mL, 15.6pg/mL and 7.8pg/mL are respectively and sequentially added into the holes of the ELISA plate, then biotin-labeled anti-mouse IL-4 antibody (ELISA cytokine rapid kit) is added, the ELISA plate is covered, and the reaction is carried out for 90min at 37 ℃. And (3) throwing off liquid in the ELISA plate, and sequentially adding 0.1mL of avidin-peroxidase complex (ELISA cytokine rapid kit) into each hole. The reaction was carried out at 37 ℃ for 60 min. 0.01M PBS was washed 3 times, and 0.1mL of ABC working solution was added to each well and reacted at 37 ℃ for 30 min. Washing with 0.01M PBS for 5 times, adding 90ul of TMB color development solution into each well, and reacting at 37 deg.C in dark for 25 min. 0.1mL of TMB stop solution was added to each well, and the absorbance was measured at 450nm using a microplate reader. The standard curve for IL-4 detection was prepared as shown in FIG. 7. The IL-4 standard curve was fitted by first regression using the concentration as abscissa (unit pg/mL) and the absorbance at 450nm as ordinate to obtain a standard curve Y of 0.0038X +0.1224, R20.9979. Wherein X represents the IL-4 concentration in pg/mL and Y represents the absorbance at OD 450.
2) Macrophage secretion promoting cytokine detection of polypeptide ATLEDSPEVI
Taking mouse spleen lymphocytes under aseptic condition, adjusting cell concentration to 5 × 105The cells were inoculated in a 96-well plate, and the test group was cultured by adding ATLEDSPEVI, adjusting the final concentrations of ATLEDSPEVI to 100, 50 and 10. mu.g/mL, respectively, and then cultured with lymphocytes for 36 hours to measure the cytokine IL-4. The blank group was incubated for 36h without the addition of the biologically active polypeptide ATLEDSPEVI as a control.
3. Results of the experiment
The experimental results are shown in FIG. 8, and compared with the blank control group, the secretion amount of IL-4 is gradually increased along with the increase of the polypeptide concentration; when the polypeptide addition concentration reaches 50 and 100 mu g/mL, the IL-4 secretion amount is obviously greater than that of a blank group; therefore, the bioactive polypeptide ATLEDSPEVI has the function of promoting lymphocyte proliferation, and has the function of regulating humoral immunity of the organism through regulating the secretion of IL-4 cytokines.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, 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 embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
<110> Zhejiang ghui peptide Life health science and technology Limited
<120> a bioactive polypeptide ATLEDSPEVI, and its preparation method and application
<160>3
<170>PatentIn version 3.3
<210>1
<211>10
<212>PRT
<213> Artificial sequence
<220>
<223> biologically active polypeptide
<400>1
Ala Thr Leu Glu Asp Ser Pro Glu Val Ile
5 10
<210>2
<211>30
<212>DNA
<213> Artificial sequence
<220>
<223> bioactive polypeptide coding sequence
<400>2
gctactctag aagattctcc agaagttatt 30
<210>3
<211>190
<212>PRT
<213> Artificial sequence
<220>
<223> kappa-Casein variant A amino acid sequence
<400>3
Met Met Lys Ser Phe Phe Leu Val Val Thr Ile Leu Ala Leu Thr
5 10 15
Leu Pro Phe Leu Gly Ala Gln Glu Gln Asn Gln Glu Gln Pro Ile
20 25 30
Arg Cys Glu Lys Asp Glu Arg Phe Phe Ser Asp Lys Ile Ala Lys
35 40 45
Tyr Ile Pro Ile Gln Tyr Val Leu Ser Arg Tyr Pro Ser Tyr Gly
50 55 60
Leu Asn Tyr Tyr Gln Gln Lys Pro Val Ala Leu Ile Asn Asn Gln
65 70 75
Phe Leu Pro Tyr Pro Tyr Tyr Ala Lys Pro Ala Ala Val Arg Ser
80 85 90
Pro Ala Gln Ile Leu Gln Trp Gln Val Leu Ser Asn Thr Val Pro
95 100 105
Ala Lys Ser Cys Gln Ala Gln Pro Thr Thr Met Ala Arg His Pro
110 115 120
His Pro His Leu Ser Phe Met Ala Ile Pro Pro Lys Lys Asn Gln
125 130 135
Asp Lys Thr Glu Ile Pro Thr Ile Asn Thr Ile Ala Ser Gly Glu
140 145 150
Pro Thr Ser Thr Pro Thr Thr Glu Ala Val Glu Ser Thr Val Ala
155 160 165
Thr Leu Glu Asp Ser Pro Glu Val Ile Glu Ser Pro Pro Glu Ile
170 175 180
Asn Thr Val Gln Val Thr Ser Thr Ala Val
185 190
Claims (9)
1. A bioactive polypeptide ATLEDSPEVI, characterized in that its amino acid sequence is Ala-Thr-Leu-Glu-Asp-Ser-Pro-Glu-Val-Ile.
2. A nucleotide fragment encoding the biologically active polypeptide ATLEDSPEVI of claim 1, wherein the nucleotide fragment has the sequence set forth in SEQ ID NO: 2, respectively.
3. The method of claim 1, wherein the biologically active polypeptide ATLEDSPEVI is synthesized by genetic engineering methods or is prepared directly by chemical synthesis.
4. The use of the biologically active polypeptide ATLEDSPEVI of claim 1, wherein the biologically active polypeptide ATLEDSPEVI is used in the preparation of a food, a health product, a pharmaceutical or a cosmetic product with antioxidant activity.
5. The use of the biologically active polypeptide ATLEDSPEVI of claim 1, wherein the biologically active polypeptide ATLEDSPEVI is used in the preparation of a food, a health product or a pharmaceutical product with immunomodulatory activity.
6. The use of the biologically active polypeptide ATLEDSPEVI of claim 1, wherein the biologically active polypeptide ATLEDSPEVI is used in the preparation of a food, a health product or a pharmaceutical product with antioxidant and immunomodulatory effects.
7. An antioxidant product comprising the biologically active polypeptide ATLEDSPEVI of claim 1; the antioxidant product comprises antioxidant food, antioxidant health product, antioxidant medicine or antioxidant cosmetic.
8. An immunomodulatory product comprising the biologically active polypeptide ATLEDSPEVI of claim 1; the immunoregulation product comprises immunoregulation food, immunoregulation health-care products or immunoregulation medicines.
9. A product having antioxidant and immunomodulating properties, comprising the biologically active polypeptide ATLEDSPEVI of claim 1; the product with antioxidant and immunoregulatory effects comprises food, health product or medicine.
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| CN107840878A (en) * | 2017-11-14 | 2018-03-27 | 上海交通大学 | A kind of biologically active polypeptide NNQFLPYPYYAKPA and its preparation method and application |
| CN107840882A (en) * | 2017-11-14 | 2018-03-27 | 上海交通大学 | A kind of biologically active polypeptide DIPNPIGSE and its preparation method and application |
| CN107903314A (en) * | 2017-11-14 | 2018-04-13 | 上海交通大学 | A kind of biologically active polypeptide EVIESPPEINTV and its preparation method and application |
| CN107827972A (en) * | 2017-12-11 | 2018-03-23 | 浙江辉肽生命健康科技有限公司 | A kind of biologically active polypeptide SPEVIESPPEIN and its preparation method and application |
| CN109234343B (en) * | 2018-09-30 | 2020-12-01 | 中国海洋大学 | Methionine oligopeptide and preparation method and application thereof |
| CN112625113B (en) * | 2020-12-16 | 2022-03-29 | 熊猫乳品集团股份有限公司 | Bioactive peptide AGYDVEKNNSRIKLGLK, and preparation method and application thereof |
| CN112778410B (en) * | 2021-01-19 | 2022-05-31 | 浙江辉肽生命健康科技有限公司 | Bioactive peptide SAPRHGSLGFLPRK, and preparation method and application thereof |
| CN112724237B (en) * | 2021-01-19 | 2022-05-31 | 浙江辉肽生命健康科技有限公司 | Bioactive peptide GGSDGYGSGRGF, and preparation method and application thereof |
| CN116444612A (en) * | 2022-11-30 | 2023-07-18 | 内蒙古伊利实业集团股份有限公司 | Milk active peptide APADPGRPTGY and preparation method and application thereof |
| CN116444609A (en) * | 2022-11-30 | 2023-07-18 | 内蒙古伊利实业集团股份有限公司 | Milk active peptide LPPPLPSRWPL and preparation method and application thereof |
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| DE10050091A1 (en) * | 2000-10-09 | 2002-04-25 | Biotechnolog Forschung Gmbh | New casein-derived peptide with growth promoting activity, useful as additive for eukaryotic cell cultures and for recombinant protein production |
| EP2253324A1 (en) * | 2009-04-30 | 2010-11-24 | Consejo Superior De Investigaciones Cientificas | Use of a casein-derived peptide and compositions thereof as antihypertensive |
| CN107163129B (en) * | 2014-12-19 | 2019-12-24 | 浙江辉肽生命健康科技有限公司 | Preparation and application of kappa-casein-derived bioactive peptide |
| CN105254714B (en) * | 2015-10-16 | 2019-09-10 | 中国农业大学 | A kind of anti-oxidation peptide and application from casein |
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