CN107880102A - A kind of biologically active polypeptide PEVIESPPEIN and its preparation method and application - Google Patents
A kind of biologically active polypeptide PEVIESPPEIN and its preparation method and application Download PDFInfo
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- CN107880102A CN107880102A CN201711288373.3A CN201711288373A CN107880102A CN 107880102 A CN107880102 A CN 107880102A CN 201711288373 A CN201711288373 A CN 201711288373A CN 107880102 A CN107880102 A CN 107880102A
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- China
- Prior art keywords
- peviesppein
- biologically active
- active polypeptide
- polypeptide
- derivative
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-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
The present invention relates to albumen field, and in particular to its amino acid sequence of a kind of biologically active polypeptide PEVIESPPEIN and its preparation method and application, biologically active polypeptide PEVIESPPEIN is Pro Glu Val Ile Glu Ser Pro Pro Glu Ile Asn.Tested by antioxidation in vitro, internal Antisenility Experiment, demonstrating polypeptide PEVIESPPEIN has preferable antioxidation biology activity and activity of fighting against senium, on the one hand, the biologically active polypeptide PEVIESPPEIN of the present invention has preferable antioxidation activity, free radical that can be in removing machine body, improve the quality of life；On the other hand, the vigor of internal anti-peroxidation enzyme system can be improved, strengthen the function of body resistance external source sexual stimulus, so as to reduce organism aging process, aging and sick probability, exploitation is of great significance with anti-oxidation function, the food of anti-senescence function, health products and medicine tool.
Description
Technical field
The present invention relates to albumen field, more particularly, to a kind of biologically active polypeptide PEVIESPPEIN and preparation method thereof
And application.
Background technology
During cow's milk is through lactobacillus-fermented, a part of protein in cow's milk is metabolized by lactic acid bacteria to be utilized, concurrently
A series of biochemical reactions have been given birth to, protein is changed into polypeptide or free amino acid, is digested or passes through
The absorption and transport of intestinal epithelial cell is directly entered the blood circulation of human body.In these polypeptides, some has special
Physiological function, it is referred to as " biologically active peptide ".
It is particularly important that safe biologically active peptide is found in natural food source.In the last few years, it has been found that some foods
The polypeptides matter in thing source has good bioactivity, such as corn small peptide, Soybean Peptide, cow's milk polypeptide.These polypeptides can
To be obtained by number of ways such as microbial fermentation, digestion enzymolysis, and the polypeptide with bioactivity is by 2~20 mostly
Amino acid residue forms, and molecular weight is less than 6000Da, contains a certain amount of hydrophobic amino acid, aromatic amino acid.
Oxidation reaction and oxidative metabolism are all vital for food and human body, and free radical and active oxygen cause
A series of oxidation reaction.When the free radical of excess is formed, they can exceed protective enzyme such as superoxide dismutase, peroxide
Change the protective effect of hydrogen enzyme, so as to cause a series of side effect such as lipid oxidation, Apoptosis to produce.This kind of oxidation is anti-
Should, the shelf-life of the food containing fat is not only influenceed, certain harm also is caused to the health of human body, such as rheumatic arthritis, sugar
Urinate disease, artery sclerosis etc..In addition, Collins et al. researchs in 2005 find that oxidative damage of the generation of cancer also with DNA has
Close.
Some artificial synthesized antioxidant such as butylated hydroxy anisoles (BHA), 2,6- di-t-butyl -4- methylbenzenes in early days
Phenol (BHT) is applied in food, as the antioxidant of lipid, but these artificial synthesized additives have for human body it is latent
Risk.In the research process of natural, from the anti-oxidation peptide of food proteins become popular research it
One.It is not only safe, is easier to be absorbed and used than macro-nutrients such as protein, and such as calcium, iron can be promoted micro-
The absorption of nutrient is measured, also with preferable antioxidation activity, is had a extensive future.
Aging is a natural phenomena, and process is often accompanied by the change of antioxidant levels, organ-tissue, immune factor, its
The change of complexity, the trend that such as proinflammatory cytokine IL-6, IL-4, TNF-α presentation increase, IL-6 occur for middle cell factor
It is all considered to play an important role in the generating process of geriatric disease with TNF-a.With science of heredity and molecular biology
Development, the research of biological decay mechanism achieve gratifying progress.Researcher by using some model organisms, as mouse,
The term single gene mutating experiment of drosophila and C. Elegans Automatic Screening etc., it is found that some genes can dramatically increase life-spans of these organisms and reach
As many as 6 times.
Anti-aging peptide in terms of physiological function there is amino acid can not compare excellent as a kind of emerging antidotal agent
Gesture, it can produce promotion or inhibitory action to the enzyme in organism, improve absorption and the profit to mineral matter and other nutrients
With, removing interior free yl, the resistance to oxidation of enhancing body itself, with anti-aging.Therefore, the nutrition and health care of biologically active peptide
Effect has turned into the emphasis of domestic and foreign scholars subject study.Qiu Juan et al. pass through experimental studies have found that, milk-derived bioactive micro peptide
Life span of drosophila melanogaster can effectively be extended, delay its aging, and also there is preferable antioxidation, thus it is speculated that be probably wherein to be rich in coloured glaze
Base peptides.SOD vigor in serum, reduces its lipid in discovery bovine colostrum extract energy conspicuousness raising the elderly's body such as the brightness in week
Peroxide and enhancing body resistance to oxidation, have certain anti-senescence function.
The research on biologically active polypeptide has much at present, for example Chinese patent CN105254738A discloses one kind and come
The milk-derived biologically active polypeptide DELQDKIH of beta-casein is come from, Chinese patent CN105254739A discloses one kind and derived from
The milk-derived biologically active polypeptide GTQYTD of α s1- caseins, Chinese patent CN105254740A, which are disclosed, a kind of derives from α s2-
The milk-derived biologically active polypeptide NQFYQKF of casein.
United States Patent (USP) US8106152B2 discloses a kind of antimicrobial compositions, and it includes a kind of peptide, the polypeptide amino acid
Sequence is Met Ala Ile Pro Pro Lys Lys Asn Gln Asp Lys Thr Glu Ile Pro Thr Ile Asn
Thr Ile Ala Ser Gly Glu Pro Thr Ser Thr Pro Thr Ile Glu Ala Val Glu Ser Thr
Val Ala Thr Leu Glu Ala Ser(P)Pro Glu Val Ile Glu Ser Pro Pro Glu Ile Asn Thr
Val Gln Val Thr Ser Thr Ala Val；Polypeptide is a part for bovine casein in the patent.Disclosed in the patent
It is a macromolecular substances, its property digested and assimilated is poor, while macromolecular bioactivity has anti-inflammatory work(disclosed in the patent
Can, do not disclose whether it there are other performances.
The content of the invention
It is an object of the invention to provide a kind of biologically active polypeptide PEVIESPPEIN and its preparation method and application.
The purpose of the present invention can be achieved through the following technical solutions：
First aspect present invention, there is provided a kind of biologically active polypeptide PEVIESPPEIN, its amino acid sequence are Pro-Glu-
Val-Ile-Glu-Ser-Pro-Pro-Glu-Ile-Asn, such as SEQ ID NO：Shown in 1.
Preferably, the biologically active polypeptide is milk-derived.κ-casein is derive specifically from, and is κ-ss-casein variants
The amino acid residue that A is the 171st~181.κ-ss-casein variants A amino acid sequence such as SEQ ID NO：Shown in 3.
The amino acid sequence and nucleotides sequence of κ-casein are classified as existing technology, and coding κ-ss-casein variants A the 171st~
The biologically active polypeptide PEVIESPPEIN of the nucleotide fragments energy encoding mature of 181 amino acids residues.
Preferably, the biologically active polypeptide has anti-oxidation function and anti-senescence function.
Second aspect of the present invention, there is provided encode the nucleotide fragments of the biologically active polypeptide PEVIESPPEIN, its sequence
It is classified as：5 '-cca gaa gtt att gag agc cca cct gag atc aac-3 ', such as SEQ ID NO：Shown in 2.
Third aspect present invention, there is provided the preparation method of the biologically active polypeptide PEVIESPPEIN, base can be passed through
Because the method for engineering is artificial synthesized, can be directly obtained from dairy products by the method isolated and purified, can direct passing through
Learn synthetically prepared.
Fourth aspect present invention, there is provided the biologically active polypeptide PEVIESPPEIN has anti-oxidation function in preparation
Food, health products, the application in medicine or cosmetics.
Fifth aspect present invention, there is provided the biologically active polypeptide PEVIESPPEIN has anti-senescence function in preparation
Food, the application in health products or medicine.
Sixth aspect present invention, there is provided the biologically active polypeptide PEVIESPPEIN is being prepared while had anti-oxidant
Application in the food of function and anti-senescence function, health products or medicine.
Specifically, biologically active polypeptide PEVIESPPEIN of the invention, which can be used for preparing, reduces free radical to skin
The cosmetics of injury, preparation have anti-oxidant and/or anti-aging medicine；And due to the biologically active polypeptide of the present invention
Product after PEVIESPPEIN is degraded by intestines and stomach still has bioactivity, therefore can be also used for preparing the food such as Yoghourt
Product, oxidation-resisting health-care product, and the oral preparation that is used for have anti-oxidant and/or anti-aging medicine.
Seventh aspect present invention, there is provided a kind of oxidation resistant product, including the biologically active polypeptide PEVIESPPEIN or
The derivative of the biologically active polypeptide PEVIESPPEIN；Described oxidation resistant product includes antioxidant food, anti-oxidation health
Product, anti-oxidation medicine or antioxidation cosmetic product；The derivative of the biologically active polypeptide PEVIESPPEIN, refer to living in biology
Property polypeptide PEVIESPPEIN amino acid side groups on, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonylation, first
Base, acetylation, phosphorylation, esterification or glycosylation etc. are modified, obtained polypeptide derivative.
Eighth aspect present invention, there is provided a kind of anti-aging product, including the biologically active polypeptide PEVIESPPEIN or
The derivative of the biologically active polypeptide PEVIESPPEIN；Described anti-aging product includes antisenility cistanche food, anti-ageing healthcare
Product or antiaging agent；The derivative of the biologically active polypeptide PEVIESPPEIN, refers in biologically active polypeptide
On PEVIESPPEIN amino acid side groups, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate,
Acetylation, phosphorylation, esterification or glycosylation etc. are modified, obtained polypeptide derivative.
Ninth aspect present invention, there is provided product a kind of while that there is anti-oxidation function and anti-senescence function, including institute
State biologically active polypeptide PEVIESPPEIN or described biologically active polypeptides PEVIESPPEIN derivative；With anti-oxidation function
Include food, health products or medicine with the product of anti-senescence function；The derivative of the biologically active polypeptide PEVIESPPEIN,
Refer on biologically active polypeptide PEVIESPPEIN amino acid side groups, aminoterminal or c-terminus carry out hydroxylating, carboxyl
Change, be carbonylated, methylating, acetylation, phosphorylation, the modification such as esterification or glycosylation, obtained polypeptide derivative.
Biologically active polypeptide PEVIESPPEIN's of the present invention has the beneficial effect that：The milk-derived biologically active polypeptide of the present invention
PEVIESPPEIN has preferable antioxidation activity and activity of fighting against senium；On the one hand, biologically active polypeptide of the invention
PEVIESPPEIN has preferable antioxidation activity, free radical that can be in removing machine body, improves the quality of life；The opposing party
Face, it is possible to increase the vigor of internal anti-peroxidation enzyme system, the function of enhancing body resistance external source sexual stimulus are old so as to reduce body
Change, aging and sick probability, to developing with anti-oxidation function, the food of anti-senescence function, health products and medicine with ten
Divide important meaning.
The application polypeptide is structurally and functionally (amino acid sequence is Met Ala in such as background technology with prior art
Ile Pro Pro Lys Lys Asn Gln Asp Lys Thr Glu Ile Pro Thr Ile Asn Thr Ile Ala
Ser Gly Glu Pro Thr Ser Thr Pro Thr Ile Glu Ala Val Glu Ser Thr Val Ala Thr
Leu Glu Ala Ser(P)Pro Glu Val Ile Glu Ser Pro Pro Glu Ile Asn Thr Val Gln Val
Thr Ser Thr Ala Val polypeptide) it is essentially different：Biologically active polypeptide PEVIESPPEIN of the present invention is a kind of
Small molecule bioactive fragment, belongs to core fragment；Biologically active polypeptide PEVIESPPEIN of the present invention, which has more, to be digested and assimilated
Property.Biologically active polypeptide PEVIESPPEIN of the present invention has anti-oxidant and aging activity simultaneously, on functional activity with existing skill
Polypeptide disclosed in art has significant difference.
Brief description of the drawings
Fig. 1：Mass chromatography extraction figure (m/z=612.3138)；
Fig. 2：Mass-to-charge ratio is the second order mses figure of 612.3138 fragment；
Fig. 3：Mass-to-charge ratio is 612.3138 polypeptide az, by crack conditions；
Fig. 4：[DPPH] methanol standard curve；
Fig. 5：Tocopherol Trolox standard curves；
Fig. 6：Influences of the biologically active polypeptide PEVIESPPEIN to C. Elegans Automatic Screening under heat stress；
Fig. 7：Influences of the biologically active polypeptide PEVIESPPEIN to C. Elegans Automatic Screening under oxidative stress.
Embodiment
Before specific embodiments of the present invention are further described, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, the protection domain being not intended to be limiting of the invention.
When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, two ends of each number range
Any one numerical value can be selected between point and two end points.Unless otherwise defined, in the present invention all technologies for using and
Scientific terminology is identical with the meaning that those skilled in the art of the present technique are generally understood that.Except used in embodiment specific method, equipment,
Outside material, according to grasp of the those skilled in the art to prior art and the record of the present invention, it can also use and this
Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real
The existing present invention.
Unless otherwise indicated, disclosed in this invention experimental method, detection method, preparation method using this technology lead
Domain conventional molecular biology, biochemistry, chromatin Structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of association area.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc.
MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
The active peptide PEVIESPPEIN's of embodiment 1 is artificial synthesized
First, the synthesis of biologically active peptide
1. RINK resin 3g (substitution value 0.3mmol/g) are weighed in 150ml reactor, with 50ml dichloromethane
(DCM) soak.
2. after 2 hours, resin is washed with nitrogen-dimethylformamide (DMF) of 3 times of resin volumes, is then drained, so
It is repeated four times, resin is drained rear stand-by.
3. a certain amount of 20% piperidines (piperidines/DMF=1 is added into reactor:4,v:V), it is placed on decolorization swinging table and shakes
20min is shaken, the Fmoc blocking groups on resin are sloughed with this.Washed four times with the DMF of 3 times of resin volumes after having taken off protection,
Then drain.
4. take the detection of a small amount of resin ninhydrin (nine well ninhydrins) method (each two drop of inspection A, inspection B, 100 DEG C of reactions
1min), resin has color, illustrates to be deprotected successfully.
5. weigh amino acid Pro in right amount and 1- hydroxyls-benzene a pair of horses going side by side triazole (HOBT) is in right amount in 50ml centrifuge tube, addition
20ml DMF is dissolved, and then adds 3ml N, and N DICs (DIC) vibration shakes up 1min, treats that solution is clear
It is added to after clear in reactor, then reactor is placed in 30 DEG C of shaking table and reacted.
6. after 2 hours, with a certain amount of acetic anhydride end socket (acetic anhydride:DIEA:DCM=1:1:2,v:v:V) half an hour,
Then washed four times, drained stand-by with the DMF of 3 times of resin volumes.
7. a certain amount of 20% piperidines (piperidines/DMF=1 is added into reactor:4, v:V), it is placed on decolorization swinging table and shakes
20min is shaken, the Fmoc blocking groups on resin are sloughed with this.Washed four times with DMF after having taken off protection, then drained.
8. take the detection of a small amount of resin ninhydrin (nine well ninhydrins) method (each two drop of inspection A, inspection B, 100 DEG C of reactions
1min), resin has color, illustrates to be deprotected successfully.
9. weighing second amino acid next in right amount and HOBT being in right amount in 50ml centrifuge tube, 25ml DMF generals are added
It dissolves, and the DIC vibrations for then adding 2.5ml shake up 1min, are added to after solution clarification in reactor, then by reactor
It is placed in 30 DEG C of shaking table and reacts.
10. after 1 hour, taking a small amount of resin to detect, (each two drop of inspection A, inspection B, 100 DEG C of reactions are detected with ninhydrin method
1min), if resin is colourless, illustrate that reaction is complete；If resin has color, illustrate that condensation is incomplete, continue to react.
After 11. question response is complete, washs resin four times with DMF, then drain, a certain amount of 20% is added into reactor
Piperidines (piperidines/DMF=1:4,v:V), it is placed on decolorization swinging table and rocks 20min, the Fmoc protection groups on resin is sloughed with this
Group.Washed four times with DMF after having taken off protection, then drain whether detection protection sloughs.
12. according to step 9-11 connect successively amino acid Glu, Val, Ile, Glu, Ser, Pro, Pro, Glu, Ile and
Asn。
13. after last amino acid is connected, protection is sloughed, is washed four times with DMF, is then taken out resin with methanol
It is dry.Then with 95 cutting liquid (trifluoroacetic acids:1,2 dithioglycols:3, isopropyl base silane:Water=95:2:2:1, v:v:V) by polypeptide
Cut down from resin (every gram of resin adds 10ml cutting liquids), and with ice ether (cutting liquid：Ether=1:9,v:V) centrifugation is heavy
Drop four times.
So far, artificial synthesized biologically active peptide PEVIESPPEIN.
2nd, the confirmation of biologically active peptide
1) UPLC is analyzed
UPLC conditions are as follows：
Instrument：Waters ACQUITY UPLC ultra high efficiency liquid phase-electron spray-level Four bar-time of-flight mass spectrometer
Chromatographic column specification：BEH C18 chromatographic columns
Flow velocity：0.4mL/min
Temperature：50℃
Ultraviolet detection wavelength：210nm
Sample size：2μL
Gradient condition：A liquid：Water containing 0.1% formic acid (v/v), B liquid：Acetonitrile containing 0.1% formic acid (v/v)
2) mass spectral analysis
Mass Spectrometry Conditions are as follows：
Ionic means：ES+
Mass range (m/z)：100-1000
Capillary voltage (Capillary) (kV)：3.0
Sampling spiroid (V)：35.0
Ion source temperature (DEG C)：115
Remove solvent temperature (DEG C)：350
Go solvent stream (L/hr)：700.0
Collision energy (eV)：4.0
Sweep time (sec)：0.25
Interior sweep time (sec)：0.02
According to above analysis method, using ultra high efficiency liquid phase-electron spray-level Four bar-flight time mass spectrum, to bioactivity
Peptide PEVIESPPEIN carries out chromatography and mass spectral analysis, and its mass chromatography extraction figure is as shown in figure 1, extract the two level at this peak
As shown in Figures 2 and 3, the polypeptide mass-to-charge ratio that can obtain this peak is 612.3138Da, and retention time is for mass spectrogram and az, by crack conditions
54.1min。
3) result
From the figure 3, it may be seen that situation about being broken according to az, by, calculates by Mascot software analysis, obtains mass-to-charge ratio
612.3138Da fragment sequence be Pro-Glu-Val-Ile-Glu-Ser-Pro-Pro-Glu-Ile-Asn
(PEVIESPPEIN) SEQ ID NO, are designated as：1.The fragment is relative with κ-ss-casein variants A the 53rd~68 residue sequence
Should, the GenBank numberings of κ-casamino acid sequence are AAA30433.1, and sequence is shown in SEQ ID NO：3.
The antioxidation activity experiment of the biologically active peptide of embodiment 2
First, [DPPH] method measure biologically active peptide PEVIESPPEIN antioxidation activity in vitro
1. experiment reagent and instrument：
Reagent：1,1- diphenyl -2- trinitrophenyl-hydrazines (1,1-Diphenyl-2-picrylhydrazyl [DPPH]),
Japanese Wako companies production；Methanol, Shanghai traditional Chinese medicines company provide；The milk-derived biologically active polypeptide that embodiment 1 obtains
PEVIESPPEIN。
Key instrument：Sunrise ELIASAs, Austrian Tecan Products；96 porocyte culture plates, the U.S.
Millipore companies manufacture；Assay balance, Meitelei-tolido Products.
2. experimental method：
(1) 1mmol/L [DPPH] methanol solution
0.349mg [DPPH] is weighed with assay balance to be dissolved in 1mL methanol solutions, prepares obtained 1mmol/L
[DPPH] methanol solution, tinfoil are kept in dark place, i.e., with i.e. use.
(2) measure of [DPPH] methanol standard curve
100 μ L [DPPH] methanol standard curve samples are separately added into by table 1 in 96 orifice plates, are stored at room temperature 90min, are used
ELIASA detects light absorption value at 517nm.
[DPPH] methanol of table 1 calibration curve solution is prepared
According to experimental result, using Excel matched curves and regression equation is calculated, as a result sees Fig. 4 (regression equations：Y=-
0.192x+0.2271, R2=0.9991).The linear relationship of [DPPH] methanol standard curve is good, coefficient correlation 0.999,
Show that [DPPH] methanol standard curve preci-sion and accuracy meets testing requirements.In terms of result, absorbance with
[DPPH] content is in inverse relation, and [DPPH] content is fewer, and light absorption value is higher, i.e. the ability of sample removing free radical is got over
By force.
(3) [DPPH] method measure biologically active peptide PEVIESPPEIN antioxidation activity
1) sample sets：80 μ L concentration are added in 96 orifice plates for 1mmol/L [DPPH] methanol solution, by table 2 respectively to add
Enter the testing sample (PEVIESPPEIN), positive control 1 (2.5mg/mL Trolox), positive control 2 of 20 μ L various concentrations
(0.025mg/mL Trolox), and negative control (phytic acid)；
2) blank group：On same 96 orifice plate, to add 80 μ L concentration as 1mmol/L [DPPH] methanol solutions and 20 μ L
The sample of deionized water does blank control.
After detected sample is loaded, 90min is stored at room temperature, light absorption value is detected at 517nm with ELIASA.Under
Formula calculates free radical scavenging activity, and experimental result is shown in Table 2.
Table 2 [DPPH] method determines the antioxidation activity result of biologically active polypeptide
From table 2 it can be seen that the Trolox as the 2.5mg/mL of positive control have under the same conditions it is most strong clear
Except the ability of free radical, free radical all in solution can be almost removed, is secondly 0.025mg/mL Trolox, phytic acid, work
Property polypeptide.Polypeptide PEVIESPPEIN removes [DPPH] free radical rate and is presented bell with change in concentration, is 2.5mg/ in concentration
Reach peak at mL, be 23.17%.
2nd, ABTS methods measure biologically active peptide PEVIESPPEIN antioxidant activity in vitro
1. experiment reagent and instrument：
TAC detection kit (Total Antioxidant Capacity Assay Kit with ABTS
Method), purchased from the green skies biotechnology company in Shanghai；ABTS solution, oxidizing agent solution, watermiscible vitamin E (Trolox solution)
(10mmol/L), the milk-derived biologically active polypeptide PEVIESPPEIN that embodiment 1 obtains.
Key instrument：Sunrise ELIASAs, Austrian Tecan Products；96 porocyte culture plates, the U.S.
Millipore companies manufacture；Assay balance, Meitelei-tolido Products.
2. experimental method：
(1) preparation of ABTS working solutions
According to TAC detection kit specification, by ABTS solution and ABTS oxidizing agent solutions 1:1 mixing, keeps away
Used after light storage 12-16h.The ABTS mother liquors prepared at room temperature deposit by lucifuge, stable in 2-3 days.It is before use, dilute with PBS
Release 38-42 times of ABTS working stocks so that after the absorbance of ABTS working solutions subtracts corresponding PBS blank controls, A734 be 0.7 ±
0.05, ABTS working solution tinfoil is kept in dark place, now with the current.
(2) the making measure of tocopherol (Trolox) standard curve curve
200 μ L ABTS working solutions are added in each detection hole of 96 orifice plates, by the requirement of table 3 in standard curve detection hole
Tocopherol (Trolox) solution that 10 μ L are diluted with PBS is added, 10 μ L PBS is added in blank control wells, gently mixes.Room temperature
After being incubated 4min, light absorption value is detected at 734nm.
The solution of the tocopherol of table 3 (Trolox) standard curve determination is prepared
Experimental result, with Excel fit regression curves and regression equation is drawn, as a result as shown in Figure 5.Trolox
Standard curve linear relationship is good, and its coefficient correlation reaches 0.998, and the degree of accuracy and accuracy for showing the standard curve all meet
Testing requirements, calculated available for subsequent result.It can be seen that well anti-is presented with light absorption value in Trolox standard curves
Than relation, the concentration of Trolox solution is higher, and its light absorption value under 734nm is lower, i.e. the Scavenging ability of institute's test sample product
It is stronger.
(3) ABTS methods measure biologically active polypeptide PEVIESPPEIN oxidation resistance
200 μ L ABTS working solutions are added in each detection hole of 96 orifice plates, adding 10 μ L in sample detection hole treats test sample
Product, 10 μ L PBS are added in blank control wells, are gently mixed.After being incubated at room temperature 4min, extinction is detected at 734nm with ELIASA
Value.The TAC of sample is calculated according to standard curve.TAC representation is with Trolox standard liquids
Concentration represent that calculate free radical scavenging activity according to the following formula, experimental result is shown in Table 4.
TAC (mmol/g)=CTrolox/CS
In formula：CTrolox--- with sample light absorption value identical Trolox concentration of standard solution (mmol/L)
CS--- the concentration (mg/mL) of synthesis polypeptide sample
Table 4ABTS methods measure biologically active polypeptide PEVIESPPEIN TAC result
Pass through TAC method (Total Antioxidant Capacity Assay Kit with ABTS methods)
Polypeptide PEVIESPPEIN external total antioxidant activity is determined, it is found that biologically active polypeptide PEVIESPPEIN is compared
Its light absorption value decrease to some degree of blank group, there is the ability of preferable reduction-oxidation material.As shown in Table 4, find more
Peptide PEVIESPPEIN TAC raises with the rise of peptide concentration, in the case of concentration is 5mg/mL, polypeptide
PEVIESPPEIN total antioxidation level reaches 0.1798mmol/g, i.e., under 5mg/mL concentration, its TAC with
1mmol/L Trolox TAC mutually maintains an equal level.Therefore, the biologically active polypeptide PEVIESPPEIN tools of invention can be assert
There is significant oxidation resistance.
The activity of fighting against senium experiment of the biologically active peptide of embodiment 3
First, biologically active polypeptide PEVIESPPEIN acute heat stress survival experiment
1. experiment reagent and instrument：
Reagent：C. Elegans Automatic Screening (Caenorhabditis elegans), attached Chinese and Western binding institute of Fudan University；Large intestine
Bacterial strain E.coli OP50, attached Chinese and Western binding institute of Fudan University；Agar powder, Chemical Reagent Co., Ltd., Sinopharm Group；Ferment
Female powder, Chemical Reagent Co., Ltd., Sinopharm Group；The milk-derived biologically active polypeptide PEVIESPPEIN that embodiment 1 obtains.
Instrument and equipment：Power health RO15 pure water systems, Li Kang biologic medicals Science and Technology Ltd.；G136T types Zealway intelligence
High-temperature sterilization pot, Xiamen Zhi Wei instruments Science and Technology Ltd.；THZ-32 type Desk type constant-temperatureoscillator oscillators, Shanghai precision experimental facilities have
Limit company；TDL-40B centrifuges, Anting Scientific Instrument Factory, Shanghai；Lu Xiang instrument GL-22M high speed freezing centrifuges, Shanghai Lu Xiang instrument
Instrument Ltd.；Win fast BJ-CD SERIES Biohazard Safety Equipments, Shanghai Bo Xun Industrial Co., Ltd.s；Nikko is inverted electronic display
Micro mirror, Nikon Corp..
2. experimental method：
(1) prepared by NGM flat boards
Taking strain Escherichia coli, picking single bacterium is fallen within 10ml LB fluid nutrient mediums, 37 DEG C in LB plate streakings,
200rpm, shaken cultivation 24h, it is used to be inoculated with NGM flat boards nursing nematode to OD600=0.4.100 μ L bacterium solutions are taken to be applied to 60mm
NGM flat boards, notice that bacterium solution edge should be apart from plate edge 0.5cm or so.The NGM flat boards having been coated with are in room temperature (21-25 DEG C) mistake
It can be used after night.
(2) culture of nematodes
Nematode used is hermaphroditic in this experiment, Standard culture conditions (20 DEG C of temperature, humidity 40%~
60%) growth is cultivated under.
(3) the synchronization processing of nematode
1) sodium perchlorate bleaching
Prepare pregnant worm growth plate (more than 80% insect is in reproduction period i.e. in plate) 2-3 plates, take 5ml M9 wash buffers 2
It is secondary, buffer solution is sucked in 15ml centrifuge tubes, 1000r/min centrifugation 3min, supernatant discarding.5ml is added newly to bleach with synchronization
Liquid, 2.5min is acutely vibrated at room temperature, adult polypide is corroded.Centrifugation, supernatant discarding.Ensure total processing time no more than
5min, prevent from damaging worm's ovum.Resuspension will be precipitated by adding M9 buffer solutions, be centrifuged after mixing, supernatant discarding, be repeated this process 3 times.
2) prescribe a time limit spawning method
Some nematodes in the egg-laying season of picking are in same flat board, and the particular number of picking is with required synchronization nematode
Number is foundation.Under general condition, an egg-laying season nematode can lay eggs 6 or so within 1h.After 0.5h being cultivated in flat board,
Choose nematode in flat board, then the ovum in flat board is in same growth period.
(4) index determining
Experiment packet：Blank group and polypeptide group.L4 phase nematodes after synchronization is handled are placed in corresponding NGM plates, per plate
No less than 40, experiment is placed in 35 DEG C of progress, and nematode death, viable count, nematode death criterion are counted every 1h：Without shifting
Dynamic and swallowing act, still without any reaction after touching.Rejecting standard：1. flee to flat board wall or cover and thirst；2. worm's ovum exists
Hatching forms bag sample worm in vivo：3. pierce in agar.
3. experimental result and analysis：
Influences of the biologically active polypeptide PEVIESPPEIN of table 5 to nematode under heat stress
As can be seen from Table 5, half death time and average life span be simultaneously under the conditions of heat stress, in experimental group and blank group
There was no significant difference, and MaLS also only extends 1h in experimental group.Meanwhile from Fig. 6 it can also be seen that, nematode in 0h extremely
Within 10h, blank group has no significant difference with experimental group；After 10h, experimental group survival rate is slightly above blank group.This explanation
Under the conditions of heat stress, feeding polypeptide PEVIESPPEIN has inapparent effect for extending the nematode life-span.
2nd, biologically active polypeptide PEVIESPPEIN Acute oxidative stress survival experiment
1. experiment reagent and instrument：
Reagent：C. Elegans Automatic Screening (Caenorhabditis elegans), attached Chinese and Western binding institute of Fudan University；Large intestine
Bacterial strain E.coli OP50, attached Chinese and Western binding institute of Fudan University；Agar powder, Chemical Reagent Co., Ltd., Sinopharm Group；Ferment
Female powder, Chemical Reagent Co., Ltd., Sinopharm Group；30% hydrogenperoxide steam generator, Chemical Reagent Co., Ltd., Sinopharm Group；Implement
The milk-derived biologically active polypeptide PEVIESPPEIN that example 1 obtains.
Instrument and equipment：Power health RO15 pure water systems, Li Kang biologic medicals Science and Technology Ltd.；G136T types Zealway intelligence
High-temperature sterilization pot, Xiamen Zhi Wei instruments Science and Technology Ltd.；THZ-32 type Desk type constant-temperatureoscillator oscillators, Shanghai precision experimental facilities have
Limit company；TDL-40B centrifuges, Anting Scientific Instrument Factory, Shanghai；Lu Xiang instrument GL-22M high speed freezing centrifuges, Shanghai Lu Xiang instrument
Instrument Ltd.；Win fast BJ-CD SERIES Biohazard Safety Equipments, Shanghai Bo Xun Industrial Co., Ltd.s；Nikko is inverted electronic display
Micro mirror, Nikon Corp..
2. experimental method：
(1) prepared by NGM flat boards
Taking strain Escherichia coli, picking single bacterium is fallen within 10ml LB fluid nutrient mediums, 37 DEG C in LB plate streakings,
200rpm, shaken cultivation 24h, it is used to be inoculated with NGM flat boards nursing nematode to OD600=0.4.100 μ L bacterium solutions are taken to be applied to 60mm
NGM flat boards, notice that bacterium solution edge should be apart from plate edge 0.5cm or so.The NGM flat boards having been coated with are in room temperature (21-25 DEG C) mistake
It can be used after night.
(2) culture of nematodes
Nematode used is hermaphroditic in this experiment, Standard culture conditions (20 DEG C of temperature, humidity 40%~
60%) growth is cultivated under.
(3) the synchronization processing of nematode
1) sodium perchlorate bleaching
Prepare pregnant worm growth plate (more than 80% insect is in reproduction period i.e. in plate) 2-3 plates, take 5ml M9 wash buffers 2
It is secondary, buffer solution is sucked in 15ml centrifuge tubes, 1000r/min centrifugation 3min, supernatant discarding.5ml is added newly to bleach with synchronization
Liquid, 2.5min is acutely vibrated at room temperature, adult polypide is corroded.Centrifugation, supernatant discarding.Ensure total processing time no more than
5min, prevent from damaging worm's ovum.Resuspension will be precipitated by adding M9 buffer solutions, be centrifuged after mixing, supernatant discarding, be repeated this process 3 times.
2) prescribe a time limit spawning method
Some nematodes in the egg-laying season of picking are in same flat board, and the particular number of picking is with required synchronization nematode
Number is foundation.Under general condition, an egg-laying season nematode can lay eggs 6 or so within 1h.After 0.5h being cultivated in flat board,
Choose nematode in flat board, then the ovum in flat board is in same growth period.
(4) index determining
Experiment packet：Blank group and polypeptide group.L4 phase nematodes after synchronization is handled are placed in corresponding NGM plates, experiment
In H containing 20mM2O2NGM flat boards in carry out, 10 are no less than per plate quantity, counts that nematode is dead, viable count per half an hour,
Nematode death criterion：Without mobile and swallowing act, still without any reaction after touching.Rejecting standard：1. flee to flat board wall
Or cover and thirst；2. worm's ovum is hatched into bag sample worm in vivo：3. pierce in agar.
3. experimental result and analysis：
Influences of the biologically active polypeptide PEVIESPPEIN of table 6 to nematode under oxidative stress
As can be seen from Table 6, there is the average life span under oxidative stress of experimental group nematode conspicuousness to improve (P ＜ 0.05),
Extremely significant difference (P is presented in polypeptide PEVIESPPEIN groups<0.05).The each group half death time is also accordingly to a certain degree
On extended, compared with other experimental groups hybrid peptide group present conspicuousness improve (P<0.05).As shown in fig. 7, should in oxidation
Under the conditions of swashing, experimental group survival rate is obviously higher than blank group survival rate.This explanation is under the conditions of oxidative stress, the survival of nematode
Rate is significantly improved, it may be possible to because polypeptide PEVIESPPEIN can effectively help nematode to resist oxidative damage, removes internal
Caused free radical and the accumulation for reducing peroxide, rather than realized by strengthening its heat hardiness.The extension of organism life-span
It is due to improve resistance of the cell to stress conditions to a certain extent, thus under anti-aging and pressure stressed condition
Much relations be present in survival rate.The pressure that this results show polypeptide PEVIESPPEIN can significantly increase nematode stress be with
Oxidative stress ability, the survival rate of nematode is improved, illustrate that certain density polypeptide PEVIESPPEIN has anti-aging for nematode
The effect of.
3rd, biologically active polypeptide PEVIESPPEIN pressure acute stress experiment in vivo
1. experiment reagent and instrument：
Reagent：C. Elegans Automatic Screening (Caenorhabditis elegans), attached Chinese and Western binding institute of Fudan University；Large intestine
Bacterial strain E.coli OP50, attached Chinese and Western binding institute of Fudan University；Agar powder, Chemical Reagent Co., Ltd., Sinopharm Group；Ferment
Female powder, Chemical Reagent Co., Ltd., Sinopharm Group；30% hydrogenperoxide steam generator, Chemical Reagent Co., Ltd., Sinopharm Group；The third two
Aldehyde (MDA) determines kit, and bio tech ltd is built up in Nanjing；Active oxygen (ROS) determines kit, and biology is built up in Nanjing
Science and Technology Ltd.；Superoxide dismutase (SOD) kit, bio tech ltd is built up in Nanjing；What embodiment 1 obtained
Milk-derived biologically active polypeptide PEVIESPPEIN.
Instrument and equipment：Power health RO15 pure water systems, Li Kang biologic medicals Science and Technology Ltd.；G136T types Zealway intelligence
High-temperature sterilization pot, Xiamen Zhi Wei instruments Science and Technology Ltd.；THZ-32 type Desk type constant-temperatureoscillator oscillators, Shanghai precision experimental facilities have
Limit company；TDL-40B centrifuges, Anting Scientific Instrument Factory, Shanghai；Lu Xiang instrument GL-22M high speed freezing centrifuges, Shanghai Lu Xiang instrument
Instrument Ltd.；Win fast BJ-CD SERIES Biohazard Safety Equipments, Shanghai Bo Xun Industrial Co., Ltd.s；Nikko is inverted electronic display
Micro mirror, Nikon Corp.；The D ultrasonic cell disruptors of JY92- II, Shanghai is than bright Instrument Ltd..
2. experimental method：
(1) prepared by NGM flat boards
Taking strain Escherichia coli, picking single bacterium is fallen within 10ml LB fluid nutrient mediums, 37 DEG C in LB plate streakings,
200rpm, shaken cultivation 24h, it is used to be inoculated with NGM flat boards nursing nematode to OD600=0.4.100 μ L bacterium solutions are taken to be applied to 60mm
NGM flat boards, notice that bacterium solution edge should be apart from plate edge 0.5cm or so.The NGM flat boards having been coated with are in room temperature (21-25 DEG C) mistake
It can be used after night.
(2) culture of nematodes
Nematode used is hermaphroditic in this experiment, Standard culture conditions (20 DEG C of temperature, humidity 40%~
60%) growth is cultivated under.
(3) the synchronization processing of nematode
1) sodium perchlorate bleaching
Prepare pregnant worm growth plate (more than 80% insect is in reproduction period i.e. in plate) 2-3 plates, take 5ml M9 wash buffers 2
It is secondary, buffer solution is sucked in 15ml centrifuge tubes, 1000r/min centrifugation 3min, supernatant discarding.5ml is added newly to bleach with synchronization
Liquid, 2.5min is acutely vibrated at room temperature, adult polypide is corroded.Centrifugation, supernatant discarding.Ensure total processing time no more than
5min, prevent from damaging worm's ovum.Resuspension will be precipitated by adding M9 buffer solutions, be centrifuged after mixing, supernatant discarding, be repeated this process 3 times.
2) prescribe a time limit spawning method
Some nematodes in the egg-laying season of picking are in same flat board, and the particular number of picking is with required synchronization nematode
Number is foundation.Under general condition, an egg-laying season nematode can lay eggs 6 or so within 1h.After 0.5h being cultivated in flat board,
Choose nematode in flat board, then the ovum in flat board is in same growth period.
(4) index determining
Experiment is divided into without processing experimental group, heats experimental group and oxidation processes experimental group.
Wherein, no processing experimental group is divided into blank without treatment group and polypeptide without treatment group again.Some of L4 phase nematodes are chosen,
After synchronization processing, rinsed and centrifuged with M9 buffer solutions, removal supernatant, addition PBS, ultrasonication 2min,
Centrifugation obtains its tissue homogenate supernatant, and illustrates the measure of progress SOD, MDA and ROS index according to kit immediately.
Heat experimental group and be divided into blank heat treatment group and polypeptide heat treatment group again.L4 phase lines after synchronization is handled
Worm is placed in corresponding NGM plates, 40 is no less than per plate, experiment is placed in 35 DEG C of progress.After cultivating 2h, its indices is determined, is examined
Survey method is identical with without processing experimental group.
Oxidation processes experimental group is divided into blank oxidation processes group and polypeptide oxidation processes group again.L4 after synchronization is handled
Phase nematode is placed in corresponding NGM plates, is tested in H containing 20mM2O2NGM flat boards in carry out, 10 are no less than per plate quantity.Culture
After 1h, its indices is determined, assay method is identical with without processing experimental group.
3. experimental result and analysis：
Influences of the biologically active polypeptide PEVIESPPEIN of table 7 to nematode SOD, MDA and ROS
As can be seen from Table 7, it is real without processing experimental group ratio, blank heat treatment experiment group and blank oxidation processes with blank
The equal conspicuousness increase of MDA and ROS values in group is tested, SOD is substantially reduced (P ＜ 0.05), this explanation either heat stress, or oxidation
Nematode body stress can be caused to damage.From table it is also found that polypeptide without MDA in treatment group and SOD and blank group
Compared to presentation significant difference.
Under 35 DEG C for the treatment of conditions, polypeptide group only MDA reduces (P<0.05), and ROS is unchanged with SOD.This is further
Its temperature capacity under stressed condition can not be improved by having proved milk-derived small peptide.Under 20mM H2O2 treatment conditions, polypeptide
There is conspicuousness change (P ＜ 0.05) in SOD and ROS values in group, and the wherein MDA values of polypeptide group are even more and pole is presented to significantly reduce (P
<0.01).Think that the online polypide interior energies of biologically active peptide PEVIESPPEIN effectively improve SOD contents, reduce lipid peroxy
The generation of compound and active oxygen, show good anti-oxidant, removing free radical function.
The induced enzymes such as SOD can remove itself excessive free radical in time, keep the metabolic balance of interior free yl, be to weigh
The index of body health situation；MDA can reflect body inner lipid peroxide reactions degree, reflect that body is damaged journey indirectly
Degree；Reactive oxygen species ROS is that aerobic cell is caused in metabolic process, can cause Apoptosis by response to oxidative stress.Cause
This, three indexs combined uses can reflect the oxidation resistance of body well and be damaged degree.According to this experiment
As a result, biologically active peptide PEVIESPPEIN can improve the Antioxidant Enzymes activity in nematode body to a certain extent, and aoxidize
Under stressed condition, body resistance oxidative damage is effectively protected, removes free radical, but its heat hardiness can not be significantly improved.
The above-mentioned description to embodiment is understood that for ease of those skilled in the art and using invention.
Person skilled in the art obviously can easily make various modifications to these embodiments, and described herein general
Principle is applied in other embodiment without by performing creative labour.Therefore, the invention is not restricted to above-described embodiment, ability
Field technique personnel do not depart from improvement that scope made and modification all should be the present invention's according to the announcement of the present invention
Within protection domain.
Sequence table
<110>Zhejiang Hui Tai life and healths Science and Technology Ltd.；Shanghai Bo Hui bio tech ltd
<120>A kind of biologically active polypeptide PEVIESPPEIN and its preparation method and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 11
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Pro Glu Val Ile Glu Ser Pro Pro Glu Ile Asn
1 5 10
<210> 2
<211> 33
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
ccagaagtta ttgagagccc acctgagatc aac 33
<210> 3
<211> 190
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 3
Met Met Lys Ser Phe Phe Leu Val Val Thr Ile Leu Ala Leu Thr Leu
1 5 10 15
Pro Phe Leu Gly Ala Gln Glu Gln Asn Gln Glu Gln Pro Ile Arg Cys
20 25 30
Glu Lys Asp Glu Arg Phe Phe Ser Asp Lys Ile Ala Lys Tyr Ile Pro
35 40 45
Ile Gln Tyr Val Leu Ser Arg Tyr Pro Ser Tyr Gly Leu Asn Tyr Tyr
50 55 60
Gln Gln Lys Pro Val Ala Leu Ile Asn Asn Gln Phe Leu Pro Tyr Pro
65 70 75 80
Tyr Tyr Ala Lys Pro Ala Ala Val Arg Ser Pro Ala Gln Ile Leu Gln
85 90 95
Trp Gln Val Leu Ser Asn Thr Val Pro Ala Lys Ser Cys Gln Ala Gln
100 105 110
Pro Thr Thr Met Ala Arg His Pro His Pro His Leu Ser Phe Met Ala
115 120 125
Ile Pro Pro Lys Lys Asn Gln Asp Lys Thr Glu Ile Pro Thr Ile Asn
130 135 140
Thr Ile Ala Ser Gly Glu Pro Thr Ser Thr Pro Thr Thr Glu Ala Val
145 150 155 160
Glu Ser Thr Val Ala Thr Leu Glu Asp Ser Pro Glu Val Ile Glu Ser
165 170 175
Pro Pro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val
180 185 190
Claims (10)
1. a kind of biologically active polypeptide PEVIESPPEIN, it is characterised in that its amino acid sequence is Pro-Glu-Val-Ile-
Glu-Ser-Pro-Pro-Glu-Ile-Asn。
A kind of 2. biologically active polypeptide PEVIESPPEIN according to claim 1, it is characterised in that the bioactivity
Polypeptide is milk-derived.
3. encode the nucleotide fragments of biologically active polypeptide PEVIESPPEIN described in claim 1, it is characterised in that the core
The sequence of acid fragments such as SEQ ID NO：Shown in 2.
4. biologically active polypeptide PEVIESPPEIN as claimed in claim 1 preparation method, it is characterised in that pass through gene work
The method of journey is artificial synthesized, or is directly obtained from dairy products by the method isolated and purified, or directly by chemical synthesis system
It is standby.
5. biologically active polypeptide PEVIESPPEIN as claimed in claim 1 application, it is characterised in that the bioactivity is more
Applications of the peptide PEVIESPPEIN in the food with anti-oxidation function, health products, medicine or cosmetics are prepared.
6. biologically active polypeptide PEVIESPPEIN as claimed in claim 1 application, it is characterised in that the bioactivity is more
Applications of the peptide PEVIESPPEIN in the food with anti-senescence function, health products or medicine is prepared.
7. biologically active polypeptide PEVIESPPEIN as claimed in claim 1 application, it is characterised in that the bioactivity is more
Applications of the peptide PEVIESPPEIN in the food with anti-oxidation function and anti-senescence function, health products or medicine is prepared.
A kind of 8. oxidation resistant product, it is characterised in that including biologically active polypeptide PEVIESPPEIN as claimed in claim 1 or
The derivative of the biologically active polypeptide PEVIESPPEIN；Described oxidation resistant product includes antioxidant food, anti-oxidation health
Product, anti-oxidation medicine or antioxidation cosmetic product；The derivative of the biologically active polypeptide PEVIESPPEIN, refer to living in biology
Property polypeptide PEVIESPPEIN amino acid side groups on, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonylation, first
Base, acetylation, phosphorylation, esterification or glycosylation modified, obtained polypeptide derivative.
A kind of 9. anti-aging product, it is characterised in that including biologically active polypeptide PEVIESPPEIN as claimed in claim 1 or
The derivative of the biologically active polypeptide PEVIESPPEIN；Described anti-aging product includes antisenility cistanche food, anti-ageing healthcare
Product or antiaging agent；The derivative of the biologically active polypeptide PEVIESPPEIN, refers in biologically active polypeptide
On PEVIESPPEIN amino acid side groups, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate,
Acetylation, phosphorylation, esterification or glycosylation modified, obtained polypeptide derivative.
10. a kind of product with anti-oxidation function and anti-senescence function, it is characterised in that including raw as claimed in claim 1
Thing active peptides PEVIESPPEIN or described biologically active polypeptides PEVIESPPEIN derivative；With anti-oxidation function and resist
The product of aging function includes food, health products or medicine；The derivative of the biologically active polypeptide PEVIESPPEIN, refers to
On biologically active polypeptide PEVIESPPEIN amino acid side groups, aminoterminal or c-terminus carry out hydroxylating, carboxylated,
Be carbonylated, methylate, acetylation, phosphorylation, esterification or glycosylation modified, obtained polypeptide derivative.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002030958A2 (en) * | 2000-10-09 | 2002-04-18 | GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF) | Casein peptide fragments with growth-influencing activity on cell cultures |
| WO2005081628A2 (en) * | 2004-03-01 | 2005-09-09 | Peptera Pharmaceutical Ltd. | Casein derived peptides and therapeutic uses thereof |
| CN105254714A (en) * | 2015-10-16 | 2016-01-20 | 中国农业大学 | Casein-derived antioxidant peptide and preparation method thereof |
-
2017
- 2017-12-07 CN CN201711288373.3A patent/CN107880102A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002030958A2 (en) * | 2000-10-09 | 2002-04-18 | GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF) | Casein peptide fragments with growth-influencing activity on cell cultures |
| WO2005081628A2 (en) * | 2004-03-01 | 2005-09-09 | Peptera Pharmaceutical Ltd. | Casein derived peptides and therapeutic uses thereof |
| CN105254714A (en) * | 2015-10-16 | 2016-01-20 | 中国农业大学 | Casein-derived antioxidant peptide and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| Y. JIN ET AL.: "Peptide profiling and the bioactivity character of yogurt in the simulated gastrointestinal digestion", 《JOURNAL OF PROTEOMICS》 * |
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