CN107759666B - Bioactive polypeptide AQTQSLVYPFPGPIHN, and preparation method and application thereof - Google Patents
Bioactive polypeptide AQTQSLVYPFPGPIHN, and preparation method and application thereof Download PDFInfo
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- CN107759666B CN107759666B CN201711249188.3A CN201711249188A CN107759666B CN 107759666 B CN107759666 B CN 107759666B CN 201711249188 A CN201711249188 A CN 201711249188A CN 107759666 B CN107759666 B CN 107759666B
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- Prior art keywords
- aqtqslvypfpgpihn
- antioxidant
- aging
- polypeptide
- biologically active
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Abstract
The invention relates to the field of protein, and in particular relates to a bioactive polypeptide AQTQSLVYPFPGPIHN, a preparation method and application thereof, wherein the amino acid sequence of the bioactive polypeptide AQTQSLVYPFPGPIHN is Ala-Gln-Thr-Gln-Ser-Leu-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn. In vitro antioxidant experiments and in vivo anti-aging experiments prove that the polypeptide AQTQSLVYPFPGPIHN has good antioxidant biological activity and anti-aging activity, and on one hand, the bioactive polypeptide AQTQSLVYPFPGPIHN 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 AQTQSLVYPFPGPIHN, 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 AQTQSLVYPFPGPIHN, 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 AQTQSLVYPFPGPIHN having an amino acid sequence of Ala-Gln-Thr-Gln-Ser-Leu-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn, as shown in SEQ ID NO: 1 is shown.
Preferably, the bioactive polypeptide is milk-derived, is specifically derived from β -casein, and is β -amino acid residues from 53 th to 68 th positions of a casein variant A1, and an amino acid sequence of the β -casein variant A1 is shown as SEQ ID NO. 3.
β -amino acid sequence of casein and corresponding nucleotide sequence are the existing technology, and the nucleotide fragment encoding amino acid residues 53-68 of β -casein variant A1 can encode mature biological active polypeptide AQTQSLVYPFPGPIHN.
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 AQTQSLVYPFPGPIHN, the sequence of which is: 5'-gcc cag aca cag tct cta gtc tat ccc ttc cct ggg ccc atc cataac-3', as shown in SEQ ID NO: 2, respectively.
In the third aspect of the present invention, a preparation method of the bioactive polypeptide AQTQSLVYPFPGPIHN 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 AQTQSLVYPFPGPIHN 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 AQTQSLVYPFPGPIHN 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 AQTQSLVYPFPGPIHN in preparing food, health care products or medicines with antioxidant function and anti-aging function.
Specifically, the bioactive polypeptide AQTQSLVYPFPGPIHN can be used for preparing cosmetics for reducing free radical damage to skin and medicines with antioxidant and/or anti-aging functions; and because the product of the bioactive polypeptide AQTQSLVYPFPGPIHN degraded by gastrointestinal tract still has bioactivity, the bioactive polypeptide AQTQSLVYPFPGPIHN 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 AQTQSLVYPFPGPIHN or a derivative of said biologically active polypeptide AQTQSLVYPFPGPIHN; the antioxidant product comprises antioxidant food, antioxidant health product, antioxidant medicine or antioxidant cosmetic; the derivative of the biologically active polypeptide AQTQSLVYPFPGPIHN 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 AQTQSLVYPFPGPIHN.
In an eighth aspect of the invention, there is provided an anti-aging product comprising the biologically active polypeptide AQTQSLVYPFPGPIHN or a derivative of the biologically active polypeptide AQTQSLVYPFPGPIHN; the anti-aging product comprises anti-aging food, anti-aging health care product or anti-aging drug; the derivative of the biologically active polypeptide AQTQSLVYPFPGPIHN 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 AQTQSLVYPFPGPIHN.
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 AQTQSLVYPFPGPIHN or the derivative of the bioactive polypeptide AQTQSLVYPFPGPIHN; products with antioxidant and antiaging effects include food, health product or medicine; the derivative of the biologically active polypeptide AQTQSLVYPFPGPIHN 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 AQTQSLVYPFPGPIHN.
The bioactive polypeptide AQTQSLVYPFPGPIHN has the following beneficial effects: the milk-derived bioactive polypeptide AQTQSLVYPFPGPIHN has good antioxidant activity and anti-aging activity; on one hand, the bioactive polypeptide AQTQSLVYPFPGPIHN 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 884.959);
FIG. 2: a secondary mass spectrum of a fragment with a mass to charge ratio of 884.959;
FIG. 3: fragmentation of polypeptide az and by with mass-to-charge ratio of 884.959;
FIG. 4: [ DPPH. ] methanol Standard Curve;
FIG. 5: tocopherol Trolox standard curve;
FIG. 6: the effect of biologically active polypeptide AQTQSLVYPFPGPIHN on caenorhabditis elegans under heat stress;
FIG. 7: the effect of biologically active polypeptide AQTQSLVYPFPGPIHN on caenorhabditis elegans under oxidative stress.
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, 1987 and 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 AQTQSLVYPFPGPIHN
Synthesis of bioactive peptide
1. 3g 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. The amino acids Gln, Thr, Gln, Ser, Leu, Val, Tyr, Pro, Phe, Pro, Gly, Pro, Ile, His and Asn 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 AQTQSLVYPFPGPIHN 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 AQTQSLVYPFPGPIHN, 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 884.959Da, and the retention time is 75.6 min.
3) Results
As can be seen from FIG. 3, according to the cases of az and by fragmentation, the fragment sequence with the mass-to-charge ratio of 884.959Da is obtained by analysis and calculation of Mascot software, and is Ala-Gln-Thr-Gln-Ser-Leu-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn (AQTQSLVYPFPGPIHN) and is marked as SEQ ID NO: 1, the fragment corresponds to the residue sequence of 53-68 th positions of β -casein variant A1, the GenBank number of the β -casein amino acid sequence is AAA30431.1, 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 AQTQSLVYPFPGPIHN 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 AQTQSLVYPFPGPIHN 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 AQTQSLVYPFPGPIHN 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 (AQTQSLVYPFPGPIHN), 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 scavenging [ DPPH. ] free radicals by the polypeptide AQTQSLVYPFPGPIHN is inverted bell-shaped with concentration change, and reaches the highest value at the concentration of 2.5mg/mL, which is 25.80%.
Second, ABTS method for measuring in vitro antioxidant ability of biological active peptide AQTQSLVYPFPGPIHN
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 AQTQSLVYPFPGPIHN 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 AQTQSLVYPFPGPIHN 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 4ABTS assay Total antioxidant Capacity results for bioactive polypeptide AQTQSLVYPFPGPIHN
The Total Antioxidant activity of the polypeptide AQTQSLVYPFPGPIHN 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 AQTQSLVYPFPGPIHN 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 AQTQSLVYPFPGPIHN is increased with the increase of the concentration of the polypeptide, and the total antioxidant level of the polypeptide AQTQSLVYPFPGPIHN reaches 0.1803mmol/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 AQTQSLVYPFPGPIHN of the invention was identified as having significant antioxidant capacity.
Example 3 anti-aging Activity assay of bioactive peptides
Acute heat stress survival rate experiment of bioactive polypeptide AQTQSLVYPFPGPIHN
1. Experimental reagents and instruments:
reagent: caenorhabditis elegans, subsidiary of the institute for combined Chinese and Western medicine, university of Compound Dane; coli OP50, subsidiary of the university of fudan; agar powder, national drug group chemical reagents limited; yeast powder, national drug group chemical reagents limited; milk-derived bioactive polypeptide AQTQSLVYPFPGPIHN obtained in example 1.
The instrument equipment comprises: likang RO15 pure water system, Likang biomedical science and technology, Inc.; model G136T Zealway intelligent high temperature sterilization pot, xiamen micro instrument science and technology ltd; THZ-32 type desk type constant temperature oscillator, shanghai smart dense testing equipment ltd; TDL-40B centrifuge, Shanghai' an pavilion scientific instrument factory; luxiang apparatus GL-22M high speed refrigerated centrifuge, Shanghai Luxiang apparatus instruments Ltd; boxun BJ-CD SERIES biosafety cabinet, Shanghai Boxun industries, Inc.; nikko inverted Electron microscope, Nikon corporation.
2. The experimental method comprises the following steps:
(1) preparation of NGM plate
Taking colibacillus strains to streak on an LB plate, picking single colonies in 10ml of LB liquid culture medium, culturing for 24h at 37 ℃ and 200rpm under shaking until OD600 is 0.4 for inoculating NGM plates to feed nematodes. 100 mu L of bacterial liquid is applied to a 60mm NGM plate, and the distance between the edge of the bacterial liquid and the edge of the plate is about 0.5 cm. The coated NGM plates were ready for use overnight at room temperature (21-25 ℃).
(2) Nematode culture
The nematodes used in the experiment are hermaphrodite and grow under standard culture conditions (temperature 20 ℃, humidity 40-60%).
(3) Synchronization treatment of nematodes
1) Bleaching with sodium perchlorate
Preparing a pregnant insect growth plate (more than 80% of insects in the plate are in a reproductive period) 2-3 plates, washing 5ml of M9 buffer solution for 2 times, sucking the buffer solution into a 15ml centrifuge tube, centrifuging at 1000r/min for 3min, and discarding the supernatant. 5ml of fresh contemporaneous bleaching solution was added and shaken vigorously at room temperature for 2.5min to erode the adult worms. Centrifuged and the supernatant discarded. Ensuring that the total treatment time cannot exceed 5min and preventing insect eggs from being damaged. And adding M9 buffer solution to resuspend the precipitate, mixing uniformly, centrifuging, discarding supernatant, and repeating the process for 3 times.
2) Time-limited spawning method
Selecting a plurality of nematodes in the egg laying period in the same plate, wherein the specific quantity is based on the number of the nematodes needing to be synchronized. Under general conditions, one nematode can lay eggs for about 6 within 1 h. After 0.5h incubation in the plates, the nematodes were picked out of the plates and the eggs in the plates were in the same growth phase.
(4) Index measurement
The experiments are divided into a blank group and a polypeptide group, the L4 stage nematodes after the synchronization treatment are placed in corresponding NGM plates, each plate is not less than 40, the experiments are carried out at 35 ℃, the number of the killed and survived nematodes is counted every 1h, the nematode death judgment standard is that no movement and swallowing action exist, no reaction still exists after light touch, the removal standard is that ① escapes to the flat plate wall or the cover to be died, ② eggs hatch in vivo to form bag-shaped worms, and ③ eggs are drilled into agar.
3. Experimental results and analysis:
TABLE 5 Effect of biologically active polypeptide AQTQSLVYPFPGPIHN on nematodes under Heat stress
As can be seen from Table 5, under the condition of heat stress, half of the death time and the average life span of the experimental group and the blank group have no significant difference, and the longest life span of the experimental group is prolonged by 1 h. Meanwhile, as can be seen from fig. 6, the nematodes are within 0h to 10h, and the blank group is not obviously different from the experimental group; after 10h, the survival rate of the experimental group was slightly higher than that of the blank group. This indicates that feeding polypeptide AQTQSLVYPFPGPIHN has an insignificant effect on extending nematode longevity under heat stress conditions.
Second, acute oxidative stress survival rate experiment of bioactive polypeptide AQTQSLVYPFPGPIHN
1. Experimental reagents and instruments:
reagent: caenorhabditis elegans, subsidiary of the institute for combined Chinese and Western medicine, university of Compound Dane; coli OP50, subsidiary of the university of fudan; agar powder, national drug group chemical reagents limited; yeast powder, national drug group chemical reagents limited; 30% hydrogen peroxide solution, national pharmaceutical group chemical reagents ltd; milk-derived bioactive polypeptide AQTQSLVYPFPGPIHN obtained in example 1.
The instrument equipment comprises: likang RO15 pure water system, Likang biomedical science and technology, Inc.; model G136T Zealway intelligent high temperature sterilization pot, xiamen micro instrument science and technology ltd; THZ-32 type desk type constant temperature oscillator, shanghai smart dense testing equipment ltd; TDL-40B centrifuge, Shanghai' an pavilion scientific instrument factory; luxiang apparatus GL-22M high speed refrigerated centrifuge, Shanghai Luxiang apparatus instruments Ltd; boxun BJ-CD SERIES biosafety cabinet, Shanghai Boxun industries, Inc.; nikko inverted Electron microscope, Nikon corporation.
2. The experimental method comprises the following steps:
(1) preparation of NGM plate
Taking colibacillus strains to streak on an LB plate, picking single colonies in 10ml of LB liquid culture medium, culturing for 24h at 37 ℃ and 200rpm under shaking until OD600 is 0.4 for inoculating NGM plates to feed nematodes. 100 mu L of bacterial liquid is applied to a 60mm NGM plate, and the distance between the edge of the bacterial liquid and the edge of the plate is about 0.5 cm. The coated NGM plates were ready for use overnight at room temperature (21-25 ℃).
(2) Nematode culture
The nematodes used in the experiment are hermaphrodite and grow under standard culture conditions (temperature 20 ℃, humidity 40-60%).
(3) Synchronization treatment of nematodes
1) Bleaching with sodium perchlorate
Preparing a pregnant insect growth plate (more than 80% of insects in the plate are in a reproductive period) 2-3 plates, washing 5ml of M9 buffer solution for 2 times, sucking the buffer solution into a 15ml centrifuge tube, centrifuging at 1000r/min for 3min, and discarding the supernatant. 5ml of fresh contemporaneous bleaching solution was added and shaken vigorously at room temperature for 2.5min to erode the adult worms. Centrifuged and the supernatant discarded. Ensuring that the total treatment time cannot exceed 5min and preventing insect eggs from being damaged. And adding M9 buffer solution to resuspend the precipitate, mixing uniformly, centrifuging, discarding supernatant, and repeating the process for 3 times.
2) Time-limited spawning method
Selecting a plurality of nematodes in the egg laying period in the same plate, wherein the specific quantity is based on the number of the nematodes needing to be synchronized. Under general conditions, one nematode can lay eggs for about 6 within 1 h. After 0.5h incubation in the plates, the nematodes were picked out of the plates and the eggs in the plates were in the same growth phase.
(4) Index measurement
Grouping experiments: blank and polypeptide groups. The L4 stage nematodes after the synchronization treatment were placed in corresponding NGM plates and tested in the presence of 20mM H2O2The number of the nematode is not less than 10, the number of the killed and survived nematodes is counted every half hour, the judgment standard of nematode death is that no movement and swallowing action exist, no reaction still exists after the nematode is touched, the elimination standard is that ① escapes to the flat wall or the cover to be died, ② eggs hatch in vivo, and sacked insects are drilled into agar, ③.
3. Experimental results and analysis:
TABLE 6 Effect of biologically active polypeptide AQTQSLVYPFPGPIHN on nematodes under oxidative stress
As can be seen from Table 6, the mean life span of the nematodes under oxidative stress in the experimental group is significantly improved (P <0.05), and the polypeptide AQTQSLVYPFPGPIHN group shows an extremely significant difference (P < 0.05). Half of the death time of each group is correspondingly prolonged to a certain extent, and the mixed peptide group shows a remarkable improvement compared with other experimental groups (P < 0.05). As shown in fig. 7, the survival rate of the experimental group was significantly higher than that of the blank group under oxidative stress condition. This indicates that the survival rate of nematodes is significantly improved under oxidative stress conditions, probably because polypeptide AQTQSLVYPFPGPIHN is effective in helping nematodes resist oxidative damage, scavenge free radicals produced in the body and reduce the accumulation of peroxides, rather than by enhancing their heat tolerance. The prolongation of the life of the organism is due to the improvement of the resistance of cells to stress conditions to a certain extent, so that the delay of aging is greatly related to the survival rate under the stress conditions. The experimental result proves that the polypeptide AQTQSLVYPFPGPIHN can obviously increase the pressure stress and oxidative stress capability of the nematode, improve the survival rate of the nematode and show that the polypeptide AQTQSLVYPFPGPIHN with a certain concentration has the anti-aging effect on the nematode.
Third, the pressure acute stress in vivo experiment of the bioactive polypeptide AQTQSLVYPFPGPIHN
1. Experimental reagents and instruments:
reagent: caenorhabditis elegans, subsidiary of the institute for combined Chinese and Western medicine, university of Compound Dane; coli OP50, subsidiary of the university of fudan; agar powder, national drug group chemical reagents limited; yeast powder, national drug group chemical reagents limited; 30% hydrogen peroxide solution, national pharmaceutical group chemical reagents ltd; malondialdehyde (MDA) assay kit, Nanjing was built into Biotechnology Ltd; a Reactive Oxygen Species (ROS) determination kit, Nanjing, established to Biotechnology Limited; superoxide dismutase (SOD) kit, Nanjing, established Biotech limited; milk-derived bioactive polypeptide AQTQSLVYPFPGPIHN obtained in example 1.
The instrument equipment comprises: likang RO15 pure water system, Likang biomedical science and technology, Inc.; model G136T Zealway intelligent high temperature sterilization pot, xiamen micro instrument science and technology ltd; THZ-32 type desk type constant temperature oscillator, shanghai smart dense testing equipment ltd; TDL-40B centrifuge, Shanghai' an pavilion scientific instrument factory; luxiang apparatus GL-22M high speed refrigerated centrifuge, Shanghai Luxiang apparatus instruments Ltd; boxun BJ-CD SERIES biosafety cabinet, Shanghai Boxun industries, Inc.; nikko inverted electron microscope, Nikon corporation; JY 92-IID ultrasonic cell disruptor, Shanghai Bilang instruments, Inc.
2. The experimental method comprises the following steps:
(1) preparation of NGM plate
Taking colibacillus strains to streak on an LB plate, picking single colonies in 10ml of LB liquid culture medium, culturing for 24h at 37 ℃ and 200rpm under shaking until OD600 is 0.4 for inoculating NGM plates to feed nematodes. 100 mu L of bacterial liquid is applied to a 60mm NGM plate, and the distance between the edge of the bacterial liquid and the edge of the plate is about 0.5 cm. The coated NGM plates were ready for use overnight at room temperature (21-25 ℃).
(2) Nematode culture
The nematodes used in the experiment are hermaphrodite and grow under standard culture conditions (temperature 20 ℃, humidity 40-60%).
(3) Synchronization treatment of nematodes
1) Bleaching with sodium perchlorate
Preparing a pregnant insect growth plate (more than 80% of insects in the plate are in a reproductive period) 2-3 plates, washing 5ml of M9 buffer solution for 2 times, sucking the buffer solution into a 15ml centrifuge tube, centrifuging at 1000r/min for 3min, and discarding the supernatant. 5ml of fresh contemporaneous bleaching solution was added and shaken vigorously at room temperature for 2.5min to erode the adult worms. Centrifuged and the supernatant discarded. Ensuring that the total treatment time cannot exceed 5min and preventing insect eggs from being damaged. And adding M9 buffer solution to resuspend the precipitate, mixing uniformly, centrifuging, discarding supernatant, and repeating the process for 3 times.
2) Time-limited spawning method
Selecting a plurality of nematodes in the egg laying period in the same plate, wherein the specific quantity is based on the number of the nematodes needing to be synchronized. Under general conditions, one nematode can lay eggs for about 6 within 1 h. After 0.5h incubation in the plates, the nematodes were picked out of the plates and the eggs in the plates were in the same growth phase.
(4) Index measurement
The experiments are divided into a non-treatment experimental group, a heating treatment experimental group and an oxidation treatment experimental group.
Wherein, the non-treatment experimental groups are divided into a blank non-treatment group and a polypeptide non-treatment group. Selecting a plurality of L4 stage nematodes, carrying out synchronization treatment, washing and centrifuging the nematodes by using an M9 buffer solution, removing supernatant, adding a PBS buffer solution, carrying out ultrasonic disruption for 2min, centrifuging to obtain tissue homogenate supernatant, and immediately carrying out determination on indexes of SOD, MDA and ROS according to the kit instructions.
The heat treatment experimental groups are divided into a blank heat treatment group and a polypeptide heat treatment group. The L4 stage nematodes after the synchronization treatment are placed in corresponding NGM plates, each plate is not less than 40, and the experiment is carried out at 35 ℃. After 2h of culture, the indexes are measured, and the detection method is the same as that of the untreated experiment group.
The oxidation treatment experimental groups are divided into a blank oxidation treatment group and a polypeptide oxidation treatment group. The L4 stage nematodes after the synchronization treatment were placed in corresponding NGM plates and tested in the presence of 20mM H2O2Is carried out in NGM plates, the number of each plate being not less than 10. After 1h of culture, various indexes of the strain are measured, and the measuring method is the same as that of the untreated experiment group.
3. Experimental results and analysis:
TABLE 7 Effect of bioactive polypeptide AQTQSLVYPFPGPIHN on nematode SOD, MDA and ROS
As can be seen from table 7, compared to the blank non-treatment test group, MDA and ROS values in the blank heat treatment test group and the blank oxidation treatment test group were significantly increased, and SOD was significantly decreased (P <0.05), which indicates that neither heat stress nor oxidation stress can cause damage to nematode organisms. It can also be found from the table that both MDA and SOD in the polypeptide non-treated group showed significant difference compared to the blank group.
Under 35 ℃ treatment conditions, only MDA was reduced in the polypeptide group (P <0.05), whereas ROS and SOD were unchanged. This further confirms that milk-derived small peptides do not improve their heat resistance under stress conditions. Under the condition of 20mM H2O2 treatment, the values of SOD and ROS in the polypeptide group both show significant changes (P <0.05), wherein the MDA value of the polypeptide group shows very significant reduction (P < 0.01). The analysis shows that the bioactive peptide AQTQSLVYPFPGPIHN can effectively improve the SOD content in the body of the nematode, reduce the generation of lipid peroxide and active oxygen, and show good functions of resisting oxidation and scavenging free radicals.
Induced enzymes such as SOD can remove excessive free radicals in time, maintain the metabolic balance of the free radicals in vivo, and is an index for measuring the health condition of organisms; MDA can reflect the reaction degree of lipid peroxide in the organism and indirectly reflect the damage degree of the organism; the reactive oxygen species, ROS, are produced by aerobic cells during metabolism and can cause apoptosis through oxidative stress. Therefore, the combined use of the three indexes can well reflect the oxidation resistance and the damage degree of the organism. According to the results of the experiment, the bioactive peptide AQTQSLVYPFPGPIHN can improve the activity of antioxidant enzymes in the nematode body to a certain extent, effectively protect the organism against oxidative damage and remove free radicals under the oxidative stress condition, but can not obviously improve the heat resistance.
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 panda dairy group, Inc.; zhejiang ghui peptide Life health science and technology Limited
<120> a bioactive polypeptide AQTQSLVYPFPGPIHN, and its preparation method and application
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>16
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>1
Ala Gln Thr Gln Ser Leu Val Tyr Pro Phe Pro Gly Pro Ile His Asn
1 5 10 15
<210>2
<211>48
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
gcccagacac agtctctagt ctatcccttc cctgggccca tccataac 48
<210>3
<211>209
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>3
Arg Glu Leu Glu Glu Leu Asn Val Pro Gly Glu Ile Val Glu Ser Leu
1 5 10 15
Ser Ser Ser Glu Glu Ser Ile Thr Arg Ile Asn Lys Lys Ile Glu Lys
2025 30
Phe Gln Ser Glu Glu Gln Gln Gln Thr Glu Asp Glu Leu Gln Asp Lys
35 40 45
Ile His Pro Phe Ala Gln Thr Gln Ser Leu Val Tyr Pro Phe Pro Gly
50 55 60
Pro Ile His Asn Ser Leu Pro Gln Asn Ile Pro Pro Leu Thr Gln Thr
65 70 75 80
Pro Val Val Val Pro Pro Phe Leu Gln Pro Glu Val Met Gly Val Ser
85 90 95
Lys Val Lys Glu Ala Met Ala Pro Lys His Lys Glu Met Pro Phe Pro
100 105 110
Lys Tyr Pro Val Gln Pro Phe Thr Glu Ser Gln Ser Leu Thr Leu Thr
115 120 125
Asp Val Glu Asn Leu His Leu Pro Pro Leu Leu Leu Gln Ser Trp Met
130 135 140
His Gln Pro His Gln Pro Leu Pro Pro Thr Val Met Phe Pro Pro Gln
145 150 155 160
Ser Val Leu Ser Leu Ser Gln Ser Lys Val Leu Pro Val Pro Glu Lys
165 170 175
Ala Val Pro Tyr Pro Gln Arg Asp Met Pro Ile Gln Ala Phe Leu Leu
180 185190
Tyr Gln Gln Pro Val Leu Gly Pro Val Arg Gly Pro Phe Pro Ile Ile
195 200 205
Val
Claims (9)
1. A bioactive polypeptide AQTQSLVYPFPGPIHN, characterized in that its amino acid sequence is Ala-Gln-Thr-Gln-Ser-Leu-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn.
2. A nucleotide fragment encoding the biologically active polypeptide AQTQSLVYPFPGPIHN 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 AQTQSLVYPFPGPIHN is synthesized by genetic engineering methods or is prepared directly by chemical synthesis.
4. The use of the biologically active polypeptide AQTQSLVYPFPGPIHN of claim 1, wherein the biologically active polypeptide AQTQSLVYPFPGPIHN 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 AQTQSLVYPFPGPIHN of claim 1, wherein the biologically active polypeptide AQTQSLVYPFPGPIHN 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 AQTQSLVYPFPGPIHN of claim 1, wherein the biologically active polypeptide AQTQSLVYPFPGPIHN 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 AQTQSLVYPFPGPIHN 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 AQTQSLVYPFPGPIHN 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 AQTQSLVYPFPGPIHN of claim 1; the product with antioxidant and antiaging effects comprises food, health product or medicine.
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Address after: 325800 LITE-ON East Road, Ling Xi Town, Cangnan County, Zhejiang 650-668 Applicant after: Panda Dairy Group Limited by Share Ltd Applicant after: Zhejiang peptide life health science and Technology Co Ltd Address before: 325800 LITE-ON East Road, Ling Xi Town, Cangnan County, Wenzhou, Zhejiang 650-668 Applicant before: ZHEJIANG PANDA DAIRY CORPORATION Applicant before: Zhejiang peptide life health science and Technology Co Ltd |
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