CN107880105B - Bioactive polypeptide VPITPTLNR, and preparation method and application thereof - Google Patents
Bioactive polypeptide VPITPTLNR, and preparation method and application thereof Download PDFInfo
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- CN107880105B CN107880105B CN201711310260.9A CN201711310260A CN107880105B CN 107880105 B CN107880105 B CN 107880105B CN 201711310260 A CN201711310260 A CN 201711310260A CN 107880105 B CN107880105 B CN 107880105B
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- vpitptlnr
- antioxidant
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- biologically active
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- C—CHEMISTRY; METALLURGY
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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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- 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
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- 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
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- A—HUMAN NECESSITIES
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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Abstract
The invention relates to the field of protein, in particular to a bioactive polypeptide VPITPTLNR, a preparation method and application thereof, wherein the amino acid sequence of the bioactive polypeptide VPITPTLNR is Val-Pro-Ile-Thr-Pro-Thr-Leu-Asn-Arg. In vitro antioxidant experiments and in vivo anti-aging experiments prove that the polypeptide VPITPTLNR has good antioxidant biological activity and anti-aging activity, and on one hand, the bioactive polypeptide VPITPTLNR has good antioxidant activity, can remove free radicals in organisms and improve the quality of life; on the other hand, the activity of an anti-peroxidase system in vivo can be improved, and the function of resisting exogenous stimulation of the organism is enhanced, so that the probability of aging, aging and illness of the organism is reduced, and the method has very important significance for developing foods, health-care products and medicines with antioxidant and anti-aging functions.
Description
Technical Field
The invention relates to the field of proteins, in particular to a bioactive polypeptide VPITPTLNR, 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".
It is particularly important to find safe bioactive peptides in natural food sources. In recent years, some food-derived polypeptides, such as short peptides of corn, soybean peptides, milk polypeptides, etc., have been found to have good biological activity. The polypeptides can be obtained through various ways such as microbial fermentation, digestion and enzymolysis and the like, and most of the polypeptides with biological 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.
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. In the course of research on natural antioxidants, antioxidant peptides derived from food proteins have become one of the most popular studies. The antioxidant is high in safety, is easier to absorb and utilize than macromolecular nutrient substances such as protein and the like, can promote the absorption of micronutrients such as calcium, iron and the like, has better antioxidant activity and has wide application prospect.
Aging is a natural phenomenon, and the process is often accompanied by the changes of antioxidant level, organ tissues and immune factors, wherein the cytokines are changed in a complex way, such as proinflammatory cytokines IL-6, IL-4, TNF- α and the like show a growing trend, and IL-6 and TNF-a are considered to play important roles in the process of the senile diseases.
The anti-aging peptide has the advantages that the anti-aging peptide is a novel anti-aging agent, has incomparable advantages with amino acid in the aspect of physiological function, can promote or inhibit enzymes in organisms, improve the absorption and utilization of minerals and other nutrient elements, clear away free radicals in the bodies, enhance the self anti-oxidation capability of the organisms and delay aging. Therefore, the nutrition and health care effects of bioactive peptides have become the focus of research on the subjects of scholars at home and abroad. Experiments and researches by meaningful people find that the milk-derived bioactive small peptide can effectively prolong the life of the drosophila and delay the aging of the drosophila, and has better antioxidation effect, and presumably is rich in thiopeptides. The results of Zhou Zhi Hui et al show that the bovine colostrum extract can obviously improve the SOD activity in serum of the elderly, reduce lipid peroxides of the SOD, enhance the oxidation resistance of organisms and have certain anti-aging function.
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 VPITPTLNR, 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 VPITPTLNR having an amino acid sequence Val-Pro-Ile-Thr-Pro-Thr-Leu-Asn-Arg as shown in SEQ ID NO: 1 is shown.
Preferably, the bioactive polypeptide is milk-derived, is specifically derived from α s 2-casein, and is the amino acid residue at the 132 th to 140 th positions of α s 2-casein variant A, and the amino acid sequence of α s 2-casein variant A is shown as SEQ ID NO. 3.
α s 2-casein and the corresponding nucleotide sequence are the existing technology, the nucleotide fragment coding the 132 th to 140 th amino acid residues of the α s 2-casein variant A can code the mature biological active polypeptide VPITPTLNR.
Preferably, the bioactive polypeptide has an antioxidant function and an anti-aging function.
In a second aspect of the present invention, there is provided a nucleotide fragment encoding the biologically active polypeptide VPITPTLNR, the sequence of which is: 5'-gtt ccc att act ccc act ctg aac aga-3', as shown in SEQ ID NO: 2, respectively.
In the third aspect of the present invention, a preparation method of the bioactive polypeptide VPITPTLNR 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 VPITPTLNR 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 VPITPTLNR in preparing food, health-care products or medicines with the anti-aging function is provided.
In a sixth aspect, the invention provides an application of the bioactive polypeptide VPITPTLNR in preparing food, health care products or medicines with antioxidant function and anti-aging function.
Specifically, the bioactive polypeptide VPITPTLNR can be used for preparing cosmetics for reducing free radical damage to skin, and medicines for resisting oxidation and/or aging; and because the product of the bioactive polypeptide VPITPTLNR degraded by gastrointestinal tract still has bioactivity, the bioactive polypeptide VPITPTLNR can also be used for preparing foods such as yoghourt and the like, antioxidant health care products, and oral medicines with antioxidant and/or anti-aging effects.
In a seventh aspect of the invention, there is provided an antioxidant product comprising said biologically active polypeptide VPITPTLNR or a derivative of said biologically active polypeptide VPITPTLNR; the antioxidant product comprises antioxidant food, antioxidant health product, antioxidant medicine or antioxidant cosmetic; the derivative of the biologically active polypeptide VPITPTLNR 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 VPITPTLNR.
In an eighth aspect of the invention, there is provided an anti-aging product comprising the biologically active polypeptide VPITPTLNR or a derivative of the biologically active polypeptide VPITPTLNR; the anti-aging product comprises anti-aging food, anti-aging health care product or anti-aging drug; the derivative of the biologically active polypeptide VPITPTLNR 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 VPITPTLNR.
In the ninth aspect of the present invention, a product having both antioxidant function and anti-aging function is provided, which comprises the bioactive polypeptide VPITPTLNR or the derivative of the bioactive polypeptide VPITPTLNR; products with antioxidant and antiaging effects include food, health product or medicine; the derivative of the biologically active polypeptide VPITPTLNR 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 VPITPTLNR.
The bioactive polypeptide VPITPTLNR has the following beneficial effects: the milk-derived bioactive polypeptide VPITPTLNR has good antioxidant activity and anti-aging activity; on one hand, the bioactive polypeptide VPITPTLNR has good antioxidant activity, can remove free radicals in organisms and improve the quality of life; on the other hand, the activity of an anti-peroxidase system in vivo can be improved, and the function of resisting exogenous stimulation of the organism is enhanced, so that the probability of aging, aging and illness of the organism is reduced, and the method has very important significance for developing foods, health-care products and medicines with antioxidant and anti-aging functions.
Drawings
FIG. 1: mass chromatogram extraction (m/z 505.8033);
FIG. 2: a secondary mass spectrum of a fragment with a mass to charge ratio of 505.8033;
FIG. 3: fragmentation of polypeptide az and by with mass-to-charge ratio of 505.8033;
FIG. 4: [ DPPH. ] methanol Standard Curve;
FIG. 5: tocopherol Trolox standard curve;
FIG. 6: hydrogen peroxide (H)2O2) And (5) acute experiments.
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, 1989 and 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 VPITPTLNR
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 Val and a proper amount of 1-hydroxy-benzotriazole (HOBT) into a 50ml centrifuge tube, adding 20ml of DMF to dissolve the amino acid Val 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. Amino acids Pro, Ile, Thr, Pro, Thr, Leu, Asn and Arg are grafted in sequence 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 VPITPTLNR 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 analysis method, the ultra-high performance liquid chromatography-electrospray-quadrupole-time-of-flight mass spectrometry is used for carrying out chromatographic analysis and mass spectrometric analysis on the bioactive peptide VPITPTLNR, the mass chromatogram extraction diagram is shown in figure 1, the secondary mass spectrogram of the peak and the az and by fracture conditions are shown in figures 2 and 3, the polypeptide mass-to-charge ratio of the peak is 505.8033Da, and the retention time is 25.7 min.
3) Results
As can be seen from FIG. 3, according to the cases of az and by fragmentation, the sequence of the fragment with the mass-to-charge ratio of 505.8033Da is Val-Pro-Ile-Thr-Pro-Thr-Leu-Asn-Arg (VPITPTLNR) obtained by analysis and calculation of Mascot software, and is marked as SEQ ID NO: 1, the fragment corresponds to the residue sequence at the 132 th to 140 th positions of α s 2-casein variant A, the GenBank number of the α s 2-casein amino acid sequence is AAA30479.1, and the sequence is shown as SEQ ID NO: 3.
Example 2 antioxidant Activity assay of bioactive peptides
Method for measuring in-vitro antioxidant activity of bioactive peptide VPITPTLNR by adopting [ DPPH ] method
1. Experimental reagents and instruments:
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 VPITPTLNR 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. The experimental method comprises the following steps:
(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 VPITPTLNR 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 (VPITPTLNR), 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.
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 VPITPTLNR 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, namely 25.93%.
Second, ABTS method for measuring in vitro antioxidant ability of biological active peptide VPITPTLNR
1. Experimental reagents and instrumentation:
total Antioxidant Capacity Assay Kit (Total Antioxidant Capacity Assay Kit with ABTS method) purchased from Shanghai Bintian bioscience, Inc.; ABTS solution, oxidant solution, water-soluble vitamin E (Trolox solution) (10mmol/L), milk-derived bioactive polypeptide VPITPTLNR 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. The experimental method comprises the following steps:
(1) preparation of ABTS working solution
According to the instruction of the total antioxidant capacity detection kit, mixing the ABTS solution and the ABTS oxidant solution in a ratio of 1:1, and storing for 12-16h in a dark place for use. The prepared ABTS mother liquor is stored at room temperature in a dark place and is stable within 2-3 days. Before use, diluting the ABTS working mother liquor by 38-42 times with PBS, so that after the absorbance of the ABTS working liquor is subtracted from the corresponding PBS blank control, the A734 is 0.7 +/-0.05, and the ABTS working liquor is stored in dark place and is ready for use.
(2) Making determination of standard curve of tocopherol (Trolox)
200 mu L of ABTS working solution is added into each detection hole of a 96-well plate, 10 mu L of tocopherol (Trolox) solution diluted by PBS is added into the detection hole of the standard curve according to the requirements of the table 3, 10 mu L of PBS is added into the blank control hole, and the mixture is gently mixed. After incubation at room temperature for 4min, the absorbance was measured at 734 nm.
TABLE 3 solution formulation for tocopherol (Trolox) standard curve determination
According to the experimental results, Excel is used for fitting a regression curve and obtaining a regression equation, and the results are shown in figure 5. The Trolox standard curve has good linear relation, and the correlation coefficient reaches 0.998, which shows that the accuracy and precision of the standard curve meet the detection requirements and can be used for subsequent result calculation. As can be seen from the figure, the Trolox standard curve has a good inverse relationship with the absorbance, and the higher the concentration of the Trolox solution is, the lower the absorbance at 734nm is, i.e. the stronger the free radical scavenging capability of the tested sample is.
(3) Determination of antioxidant capacity of bioactive polypeptide VPITPTLNR by ABTS method
And adding 200 mu L of ABTS working solution into each detection hole of a 96-well plate, adding 10 mu L of a sample to be detected into the sample detection hole, adding 10 mu L of PBS into the blank control hole, and gently mixing. After incubation at room temperature for 4min, the absorbance was measured at 734nm using a microplate reader. And calculating the total antioxidant capacity of the sample according to the standard curve. The total antioxidant capacity is expressed in terms of the concentration of Trolox standard solution, the radical scavenging rate is calculated according to the following formula, and the experimental results are shown in table 4.
Total antioxidant capacity (mmol/g) ═ CTrolox/CS
In the formula: cTroloxTrolox Standard solution concentration (mmol/L) identical to the absorbance of the sample
CSConcentration of synthetic polypeptide samples (mg/mL)
TABLE 4 ABTS assay Total antioxidant Capacity results for bioactive polypeptide VPITPTLNR
The Total Antioxidant activity of the polypeptide VPITPTLNR in vitro is measured by a Total Antioxidant activity method (Total Antioxidant Capacity Assay Kit with ABTS method), and the result shows that the light absorption value of the bioactive polypeptide VPITPTLNR is reduced to a certain extent compared with that of a blank group, and the bioactive polypeptide has better Capacity of reducing oxidized substances. As can be seen from Table 4, the total antioxidant capacity of the polypeptide VPITPTLNR is increased with the increase of the concentration of the polypeptide, and the total antioxidant level of the polypeptide VPITPTLNR reaches 0.1996mmol/g at the concentration of 5mg/mL, namely, the total antioxidant capacity of the polypeptide is equal to the total antioxidant capacity of 1mmol/L Trolox at the concentration of 5mg/m L. Thus, the biologically active polypeptide VPITPTLNR of the invention was identified as having significant antioxidant capacity.
Example 3 anti-aging Activity assay of bioactive peptides
Experiment for improving survival ability of drosophila by bioactive polypeptide VPITPTLNR
1. Experimental reagents and instruments:
reagent: oregon K wild type drosophila melanogaster, university of shanghai transport college genetics laboratory; agar powder, national drug group chemical reagents limited; milk-derived bioactive polypeptide VPITPTLNR obtained in example 1.
The instrument equipment comprises: model CM-230 Mohr super Water, Shanghai Mole scientific instruments, Inc.; model G136T Zealway intelligent high temperature sterilization pot, xiamen micro instrument science and technology ltd; BJ-CD SERIES Bioincubators, Shanghai Bingbo industries, Inc.; GRX-9073 hot air sterilization cabinet, Shanghai-constant technology, Inc.
2. The experimental method comprises the following steps:
taking fruit flies as an experimental model: collecting newly emerged fruit fly imagoes within 8 hours, randomly transferring the imagoes into each experimental group after anesthesia, wherein each sex of each group is 100, each group is provided with 3 parallels, a control group is given with a common corn flour culture medium, and the experimental groups are VPITPTLNR bioactive peptide-corn culture media containing 0.05mg/ml, 0.5mg/ml and 1mg/ml respectively. Fresh medium was changed every 2 days, and the number of deaths of flies of different genders was observed and recorded every day until all flies died. And (4) drawing a survival curve of the fruit flies, and calculating the average life and the maximum life of the fruit flies of different sexes (taking 5 fruit flies dead finally for statistics).
3. Experimental results and analysis:
TABLE 5-1 VPITPTLNR Effect on the longevity of male Drosophila
From table 5-1, it can be seen that the average life span of the male drosophila in the low dose group is not significantly changed relative to the blank control group, but the average life span of the male drosophila in the medium dose group and the high dose group is improved, namely 16.01% and 9.73% respectively, but only the medium dose group generates a significant difference (p <0.05), which indicates that the average life span of the male drosophila in the medium dose group is significantly improved. Meanwhile, the half death time of the drosophila flies in the medium-dose group and the high-dose group is improved, but no obvious difference exists in the aspect of the longest life. As can be seen from table 5-2, the low-, medium-and high-dose groups of female drosophila all improved in mean life, but did not produce significant differences. However, the longest life of the medium-dose group and the high-dose group is improved, is respectively prolonged by 7 days and 6 days compared with the blank control group, and generates a significant difference (P < 0.05).
The results of this experiment demonstrate that bioactive polypeptide VPITPTLNR can increase the average and maximum life span of Drosophila at certain concentrations, but is related to concentration and sex. The phenomenon related to the concentration and strain of the test substance is probably because VPITPTLNR is involved in part of the biological metabolism of the fruit flies, or the effect of prolonging the life of the fruit flies is achieved by improving the antioxidant system of the fruit fly tissues. The metabolism of fruit flies in different strains can be differentiated, so that the results are different. The sex difference is probably because the female fruit flies have certain conservation and resistance to the external environment, so VPITPTLNR is not obvious in prolonging the life of the female fruit flies.
II, acute oxidation experiment of bioactive polypeptide VPITPTLNR hydrogen peroxide
1. Experimental reagents and instruments:
reagent: oregon K wild type drosophila melanogaster, university of shanghai transport college genetics laboratory; agar powder, national drug group chemical reagents limited; hydrogen peroxide, shanghai Lingfeng Chemicals, Inc.; milk-derived bioactive polypeptide VPITPTLNR obtained in example 1.
The instrument equipment comprises: model CM-230 Mohr super Water, Shanghai Mole scientific instruments, Inc.; milliporem Milllex GP0.22 μm filter membrane, Millipore, USA; GL-22M high-speed refrigerated centrifuge, Shanghai Luxiang apparatus centrifuge instruments Inc.
2. The experimental method comprises the following steps:
collecting newly emerged fruit fly imagoes within 8 hours, randomly transferring the imagoes into each experimental group after anesthesia, taking a peptide concentration culture medium with a better result in a life test, setting a blank control group and the experimental group, and giving a common corn flour culture medium to the control group. Each group of male and female sex fruit flies was 50, and the fruit flies were cultured for three weeks. Then, 5 male and 5 female fruit flies each time were transferred to a new container containing a paper disc containing 300. mu.L of a 5% sucrose solution and 1ml of 30% hydrogen peroxide, and the blank and experimental groups were exposed to the toxic peroxide environment generated by this hydrogen peroxide, and 10 replicates of each group were set up and observed for their antioxidant capacity. The number of fruit fly deaths and sex were recorded every 4 hours until all of the flies had died.
3. Experimental results and analysis:
as can be seen from fig. 6(a), for male fruit flies fed at VPITPTLNR, the survival rate of the male fruit flies was higher than that of the fruit flies not fed at VPITPTLNR at each time point, and the survival time was improved compared with that of the blank control group, indicating that the antioxidant capacity of the male fruit flies was improved after VPITPTLNR was fed. In fig. 6(B), the survival rate of the female fruit flies fed with VPITPTLNR in the high-concentration hydrogen peroxide environment for 15h is obviously higher than that of the control group, which shows that the antioxidant capacity of the female fruit flies is improved in this period. However, the survival curves of the later experimental group and the control group are basically coincident, which shows that the antioxidant capacity of the female drosophila fed with VPITPTLNR is gradually weakened, and the female drosophila fed with VPITPTLNR has no difference with the control group after a certain time. The experimental result shows that VPITPTLNR can improve the antioxidant capacity of fruit flies. According to H2O2As a result of acute toxicity test, it is presumed that VPITPTLNR might increase the H pair of fruit fly by regulating catalase CAT activity2O2Resistance to injury.
Experiment for influence of bioactive polypeptide VPITPTLNR on content of SOD and MAD in fruit fly
1. Experimental reagents and instruments:
reagent: oregon K wild type drosophila melanogaster, university of shanghai transport college genetics laboratory; agar powder, national drug group chemical reagents limited; MDA lipid peroxide kit, south kyo kaiky biotechnology limited; SOD superoxide dismutase kit, Nanjing, biological technology Limited; milk-derived bioactive polypeptide VPITPTLNR obtained in example 1.
The instrument equipment comprises: model CM-230 Mohr super Water, Shanghai Mole scientific instruments, Inc.; tissue homogenizers, Shanghai Yuanxiang Biotech, Inc.; model G136T Zealway intelligent high temperature sterilization pot, xiamen micro instrument science and technology ltd; BJ-CD SERIES Bioincubators, Shanghai Bingbo industries, Inc.; GRX-9073 hot air sterilization cabinet, Shanghai-constant technology, Inc.; an Infine type microplate reader, Ati Diken Co., Ltd.
2. The experimental method comprises the following steps:
collecting newly emerged fruit fly imagoes within 8 hours, randomly transferring the imagoes into each experimental group after anesthesia, wherein each sex of each group is 100, each group is provided with 3 parallels, a control group is given with a common corn flour culture medium, and the experimental groups are VPITPTLNR bioactive peptide-corn culture media containing 0.05mg/ml, 0.5mg/ml and 1mg/ml respectively. Changing fresh culture medium once every 2 days, feeding for 30 days, weighing 40mg of drosophila melanogaster per group, adding 0.5ml of normal saline, grinding homogenate in ice bath, intermittently maintaining for 10s, repeating for 3 times, making homogenate, and determining the SOD activity and MDA concentration content of drosophila melanogaster per group according to the kit instructions. The MDA detection kit is used for detecting the concentration content of the lipid peroxidation product MDA in the drosophila melanogaster, and the wavelength of a spectrophotometer is 532 nm.
3. Experimental results and analysis:
TABLE 6 VPITPTLNR Effect on Drosophila SOD, MDA
As can be seen from table 6, the SOD content in the drosophila hermaphrodite of the polypeptide-treated group was increased compared to the blank control group, and for the male drosophila group, the SOD content in the drosophila hermaphrodite was significantly different when the peptide concentration reached 1mg/ml, while for the female drosophila group, the SOD content was significantly different when the peptide concentration was 0.5mg/ml and 1 mg/ml. It is demonstrated that the SOD content in vivo can be increased by taking certain polypeptide, and the organism can be protected from oxidation injury. As can be seen from the MDA contents in Table 6, the MDA contents in both male and female drosophila of the experimental group were reduced. The MDA content of the drosophila groups at concentrations of 0.5mg/ml and 1mg/ml showed a significant reduction with respect to the MDA content of 1.37. + -. 0.21. mu. mol/L in the male placebo group, whereas in the female drosophila group, the MDA content in the drosophila bodies showed a significant reduction with 1mg/ml peptide treatment. Because MDA is generated by lipid peroxidation in vivo, the reduction of the content thereof indirectly indicates that the activity of the antioxidant enzyme system of the drosophila is improved, thereby protecting tissues and organs of organisms without generating a large amount of lipid peroxides.
The experimental results show that the experimental results of SOD and MDA prove that the bioactive polypeptide VPITPTLNR is helpful for improving the activity of an antioxidant enzyme system in a body, thereby effectively improving the antioxidant capacity of the body, reducing the stimulation of harmful factors on the body, and reducing the probability of aging, aging and illness of the body.
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.
Sequence listing
<110> Zhejiang ghui peptide Life health science and technology Limited; shanghai platinum Biotech Ltd
<120> a bioactive polypeptide VPITPTLNR, and its preparation method and application
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>9
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>1
Val Pro Ile Thr Pro Thr Leu Asn Arg
1 5
<210>2
<211>27
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
gttcccatta ctcccactct gaacaga 27
<210>3
<211>222
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>3
Met Lys Phe Phe Ile Phe Thr Cys Leu Leu Ala Val Ala Leu Ala Lys
1 5 10 15
Asn Thr Met Glu His Val Ser Ser Ser Glu Glu Ser Ile Ile Ser Gln
20 25 30
Glu Thr Tyr Lys Gln Glu Lys Asn Met Ala Ile Asn Pro Ser Lys Glu
35 40 45
Asn Leu Cys Ser Thr Phe Cys Lys Glu Val Val Arg Asn Ala Asn Glu
50 55 60
Glu Glu Tyr Ser Ile Gly Ser Ser Ser Glu Glu Ser Ala Glu Val Ala
65 70 75 80
Thr Glu Glu Val Lys Ile Thr Val Asp Asp Lys His Tyr Gln Lys Ala
85 90 95
Leu Asn Glu Ile Asn Gln Phe Tyr Gln Lys Phe Pro Gln Tyr Leu Gln
100 105 110
Tyr Leu Tyr Gln Gly Pro Ile Val Leu Asn Pro Trp Asp Gln Val Lys
115 120 125
Arg Asn Ala Val Pro Ile Thr Pro Thr Leu Asn Arg Glu Gln Leu Ser
130 135 140
Thr Ser Glu Glu Asn Ser Lys Lys Thr Val Asp Met Glu Ser Thr Glu
145150 155 160
Val Phe Thr Lys Lys Thr Lys Leu Thr Glu Glu Glu Lys Asn Arg Leu
165 170 175
Asn Phe Leu Lys Lys Ile Ser Gln Arg Tyr Gln Lys Phe Ala Leu Pro
180 185 190
Gln Tyr Leu Lys Thr Val Tyr Gln His Gln Lys Ala Met Lys Pro Trp
195 200 205
Ile Gln Pro Lys Thr Lys Val Ile Pro Tyr Val Arg Tyr Leu
210 215 220
Claims (9)
1. A bioactive polypeptide VPITPTLNR, characterized in that its amino acid sequence is Val-Pro-Ile-Thr-Pro-Thr-Leu-Asn-Arg.
2. A nucleotide fragment encoding the biologically active polypeptide VPITPTLNR 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 VPITPTLNR is synthesized by genetic engineering methods or is prepared directly by chemical synthesis.
4. The use of the biologically active polypeptide VPITPTLNR of claim 1, wherein the biologically active polypeptide VPITPTLNR 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 VPITPTLNR of claim 1, wherein the biologically active polypeptide VPITPTLNR is used in the preparation of a food, a health product or a pharmaceutical product with anti-aging properties.
6. The use of the biologically active polypeptide VPITPTLNR of claim 1, wherein the biologically active polypeptide VPITPTLNR is used in the preparation of a food, a health product or a pharmaceutical product with antioxidant and anti-aging properties.
7. An antioxidant product comprising the biologically active polypeptide VPITPTLNR of claim 1; the antioxidant product comprises antioxidant food, antioxidant health product, antioxidant medicine or antioxidant cosmetic.
8. An anti-aging product comprising the biologically active polypeptide VPITPTLNR of claim 1; the anti-aging product comprises anti-aging food, anti-aging health care products or anti-aging drugs.
9. A product having antioxidant and anti-aging properties comprising the biologically active polypeptide VPITPTLNR of claim 1; the product with antioxidant and antiaging effects comprises food, health product or medicine.
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1374885A1 (en) * | 2002-06-27 | 2004-01-02 | Ingredia | Use of at least one peptide of alpha-s2 casein with inhibiting activity of ACE for the preparation of medicaments and foodstuffs |
WO2005081628A2 (en) * | 2004-03-01 | 2005-09-09 | Peptera Pharmaceutical Ltd. | Casein derived peptides and therapeutic uses thereof |
CN106146644A (en) * | 2016-09-07 | 2016-11-23 | 哈尔滨工业大学 | Antithrombotic peptide and directional enzymatic preparation method thereof |
-
2017
- 2017-12-11 CN CN201711310260.9A patent/CN107880105B/en active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1374885A1 (en) * | 2002-06-27 | 2004-01-02 | Ingredia | Use of at least one peptide of alpha-s2 casein with inhibiting activity of ACE for the preparation of medicaments and foodstuffs |
WO2005081628A2 (en) * | 2004-03-01 | 2005-09-09 | Peptera Pharmaceutical Ltd. | Casein derived peptides and therapeutic uses thereof |
CN106146644A (en) * | 2016-09-07 | 2016-11-23 | 哈尔滨工业大学 | Antithrombotic peptide and directional enzymatic preparation method thereof |
Non-Patent Citations (4)
Title |
---|
Hiroshi Uenishi等.Isolation and identification of casein-derived dipeptidyl-peptidase 4 (DPP-4)-inhibitory peptide LPQNIPPL from gouda-type cheese and its effect on plasma glucose in rats.《International Dairy Journal》.2012,第22卷(第1期),摘要,表2,第27页3.3. * |
Isolation and identification of casein-derived dipeptidyl-peptidase 4 (DPP-4)-inhibitory peptide LPQNIPPL from gouda-type cheese and its effect on plasma glucose in rats;Hiroshi Uenishi等;《International Dairy Journal》;20120131;第22卷(第1期);摘要,表2,第27页3.3 * |
Novel angiotensin I-converting enzyme inhibitory peptides produced in fermented milk by specific wild Lactococcus lactis strains;J. C. Rodríguez-Figueroa等;《J. Dairy Sci.》;20120815;第95卷(第10期);第5536-5543页 * |
The dipeptidyl peptidase-4 (DPP-4) inhibitor teneligliptin functions as antioxidant on human endothelial cells exposed to chronic hyperglycemia and metabolic high-glucose memory;Gemma Pujadas等;《Endocrine》;20160816;第56卷(第3期);摘要 * |
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