CN107880106A - A kind of biologically active polypeptide VPITPTLNRE and its preparation method and application - Google Patents
A kind of biologically active polypeptide VPITPTLNRE and its preparation method and application Download PDFInfo
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- CN107880106A CN107880106A CN201711311343.XA CN201711311343A CN107880106A CN 107880106 A CN107880106 A CN 107880106A CN 201711311343 A CN201711311343 A CN 201711311343A CN 107880106 A CN107880106 A CN 107880106A
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- Prior art keywords
- vpitptlnre
- biologically active
- active polypeptide
- polypeptide
- derivative
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4732—Casein
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
-
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Birds (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Toxicology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Zoology (AREA)
- Epidemiology (AREA)
- Food Science & Technology (AREA)
- Nutrition Science (AREA)
- Mycology (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Dermatology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to albumen field, and in particular to its amino acid sequence of a kind of biologically active polypeptide VPITPTLNRE and its preparation method and application, biologically active polypeptide VPITPTLNRE is Val Pro Ile Thr Pro Thr Leu Asn Arg Glu.Tested by antioxidation in vitro, internal Antisenility Experiment, demonstrating polypeptide VPITPTLNRE has preferable antioxidation biology activity and activity of fighting against senium, on the one hand, the biologically active polypeptide VPITPTLNRE 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 VPITPTLNRE and preparation method thereof and
Using.
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.
The content of the invention
It is an object of the invention to provide a kind of biologically active polypeptide VPITPTLNRE 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 VPITPTLNRE, its amino acid sequence are Val-Pro-
Ile-Thr-Pro-Thr-Leu-Asn-Arg-Glu, such as SEQ ID NO：Shown in 1.
Preferably, the biologically active polypeptide is milk-derived.Specific source α s2- caseins, and become for α s2- caseins
The amino acid residue that body A is the 132nd~141.α s2- ss-casein variants A amino acid sequences such as SEQ ID NO：Shown in 3.
The amino acid sequence and corresponding nucleotides sequence of α s2- caseins are classified as existing technology, and coding for alpha s2- caseins become
The biologically active polypeptide VPITPTLNRE of the nucleotide fragments energy encoding mature of the amino acids residues of body A the 132nd~141.
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 VPITPTLNRE, its sequence
It is classified as：5 '-gtt ccc att act ccc act ctg aac aga gag-3 ', such as SEQ ID NO：Shown in 2.
Third aspect present invention, there is provided the preparation method of the biologically active polypeptide VPITPTLNRE, 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 VPITPTLNRE is being prepared with anti-oxidation function
Application in food, health products, medicine or cosmetics.
Fifth aspect present invention, there is provided the biologically active polypeptide VPITPTLNRE is being prepared with anti-senescence function
Application in food, health products or medicine.
Sixth aspect present invention, there is provided the biologically active polypeptide VPITPTLNRE is being prepared while had anti-oxidant work(
Can be with the application in the food, health products or medicine of anti-senescence function.
Specifically, biologically active polypeptide VPITPTLNRE of the invention, which can be used for preparing, reduces free radical to skin wound
Harmful cosmetics, prepare there is anti-oxidant and/or anti-aging medicine；And due to the biologically active polypeptide of the present invention
Product after VPITPTLNRE 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 VPITPTLNRE or
The derivative of the biologically active polypeptide VPITPTLNRE；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 VPITPTLNRE, refers in bioactivity
On polypeptide VPITPTLNRE amino acid side groups, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonylation, methyl
Change, 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 VPITPTLNRE or
The derivative of the biologically active polypeptide VPITPTLNRE；Described anti-aging product includes antisenility cistanche food, anti-ageing healthcare
Product or antiaging agent；The derivative of the biologically active polypeptide VPITPTLNRE, refers in biologically active polypeptide
On VPITPTLNRE amino acid side groups, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate, second
Acylation, 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 VPITPTLNRE or described biologically active polypeptides VPITPTLNRE derivative；With anti-oxidation function and
The product of anti-senescence function includes food, health products or medicine；The derivative of the biologically active polypeptide VPITPTLNRE, refers to
On biologically active polypeptide VPITPTLNRE amino acid side groups, aminoterminal or c-terminus progress hydroxylating, carboxylated, carbonyl
Base, methylate, acetylation, phosphorylation, the modification such as esterification or glycosylation, obtained polypeptide derivative.
Biologically active polypeptide VPITPTLNRE's of the present invention has the beneficial effect that：The milk-derived biologically active polypeptide of the present invention
VPITPTLNRE has preferable antioxidation activity and activity of fighting against senium；On the one hand, biologically active polypeptide of the invention
VPITPTLNRE 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.
Brief description of the drawings
Fig. 1：Mass chromatography extraction figure (m/z=570.3278)；
Fig. 2：Mass-to-charge ratio is the second order mses figure of 570.3278 fragment；
Fig. 3：Mass-to-charge ratio is 570.3278 polypeptide az, by crack conditions；
Fig. 4：[DPPH] methanol standard curve；
Fig. 5：FeSO4Standard curve；
Fig. 6：Influences of the biologically active polypeptide VPITPTLNRE to the C. Elegans Automatic Screening life-span；
Fig. 7：Influences of the biologically active polypeptide VPITPTLNRE 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, 1987 and 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 VPITPTLNRE'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.
After 2.2 hours, resin is washed with nitrogen-dimethylformamide (DMF) of 3 times of resin volumes, is then drained, so weight
It is multiple 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 Val 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.
After 6.2 hours, with a certain amount of acetic anhydride end socket (acetic anhydride:DIEA:DCM=1:1:2,v:v:V) half an hour, so
Washed four times, drained stand-by with the DMF of 3 times of resin volumes afterwards.
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.
After 10.1 hours, take 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. amino acid Pro, Ile, Thr, Pro, Thr, Leu, Asn, Arg and Glu are connected successively according to step 9-11.
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 VPITPTLNRE.
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 VPITPTLNRE carries out chromatography and mass spectral analysis, and its mass chromatography extraction figure is as shown in figure 1, extract the two level matter at this peak
As shown in Figures 2 and 3, the polypeptide mass-to-charge ratio that can obtain this peak is 570.3278Da, and retention time is for spectrogram and az, by crack conditions
24.3min。
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
570.3278Da fragment sequence is Val-Pro-Ile-Thr-Pro-Thr-Leu-Asn-Arg-Glu (VPITPTLNRE), note
For SEQ ID NO：1.The fragment and α s2- ss-casein variants A the 132nd~141 residue sequence is corresponding, α s2- caseins
The GenBank numberings of amino acid sequence are AAA30479.1, 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 VPITPTLNRE 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
VPITPTLNRE。
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 VPITPTLNRE 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 (VPITPTLNRE), 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.
Formula：
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.It is 29.53% that polypeptide VPITPTLNRE, which removes [DPPH] free radical rate, and with the drop of VPITPTLNRE concentration
Low, Scavenging ability weakens.
2nd, FARP methods measure biologically active peptide VPITPTLNRE antioxidant activity in vitro
1) experiment reagent and instrument
TAC detection kit (Ferric Reducing Ability of Plasma FRAP methods), is purchased from
The green skies biotechnology company in Shanghai；FeSO4Solution (10mmol/L), watermiscible vitamin E (Trolox solution) (10mmol/
L), the milk-derived biologically active polypeptide VPITPTLNRE 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；HWS26 type electric-heated thermostatic water baths, Shanghai
One permanent Science and Technology Ltd.'s manufacture.
2) experimental method
(1) preparation of FRAP working solutions
According to TAC detection kit, TPTZ 7.5mL dilutions, the μ L solution of TPTZ 750, detection are buffered
The μ L of liquid 750 are well mixed, and are incubated in 37 DEG C of water-baths, are finished in 2 hours h.
(2)FeSO4The making measure of standard curve curve
180 μ LFRAP working solutions are first added in 96 orifice plates, 5 μ L FeSO are added by table 34Calibration curve solution, gently mix
It is even, after 37 DEG C are incubated 3-5min, light absorption value is determined at 593nm with ELIASA.
The FeSO of table 34The solution of standard curve determination is prepared
FeSO4Concentration and light absorption value are in good proportional relation, FeSO4Concentration is higher, and light absorption value is higher.FeSO of the present invention4
Standard curve result is shown in Fig. 5, and the linear relationship of standard curve is good, coefficient correlation 0.998, FeSO4The precision of standard curve
Degree and the degree of accuracy meet testing requirements, are calculated available for follow-up.
(3) FRAP methods measure biologically active polypeptide VPITPTLNRE oxidation resistance
180 μ L FRAP working solutions are first added in 96 orifice plates, 5 μ L ddH are added in blank control wells2O, sample detection hole
5 μ L phytic acid are added in 5 μ L testing samples of interior addition, positive control, are gently mixed, after 37 DEG C are incubated 3-5min, are existed with ELIASA
Light absorption value is determined at 593nm.TAC representation is with FeSO4The concentration of standard liquid represents.Count according to the following formula
Free radical scavenging activity is calculated, experimental result is shown in Table 4.
The FARP methods of table 4 measure biologically active polypeptide VPITPTLNRE TAC result
By TAC method (Ferric Reducing Ability Power FRAP methods) to polypeptide
VPITPTLNRE external total antioxidant activity is determined, it is found that biologically active polypeptide VPITPTLNRE has preferably also
The ability of primary oxide matter；In the case of concentration is 4mg/mL, polypeptide VPITPTLNRE total antioxidation level reaches
0.0231mmol/g；Illustrate biologically active polypeptide VPITPTLNRE TAC, higher than under comparable sodium have it is weak
The phytic acid of antioxidation activity, there is conspicuousness (p>0.05) difference.Therefore, the biologically active polypeptide of invention can be assert
VPITPTLNRE has significant oxidation resistance.
The activity of fighting against senium experiment of the biologically active peptide of embodiment 3
First, experiments of the biologically active polypeptide VPITPTLNRE to C. Elegans Automatic Screening aging effects
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；5-fluor-uracil, Sigma Co., USA；The milk-derived life that embodiment 1 obtains
Thing active peptides VPITPTLNRE.
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.Some of L4 phase nematodes are chosen, after synchronization processing, are respectively placed in corresponding
In NGM plates；Every group of nematode population is no less than 60, is now designated as 0 day, transfers them to daily in new plate, to the reproduction later stage
Do not retransfer.Record nematode is dead daily and eliminates the bar number of experiment.Wherein contain in each NGM plates of life experiment
12.5mg/L 5-fluor-uracils are to suppress nematode reproduction.Nematode death criterion：Without mobile and swallowing act, after touching still
Without any reaction.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 VPITPTLNRE of table 5 to the nematode life-span
It can be seen from table 5 and Fig. 6 when feeding polypeptide VPITPTLNRE mass concentration is 300mg/L, in experimental group
The average life span of nematode extends about 10.01% respectively, meanwhile, its half death time has even more obtained conspicuousness raising (P
＜ 0.05), the MaLS time also extends 4 days respectively compared to blank group.It is even more to be intuitive to see in Fig. 6, when identical
Between point, nematode survival rate was extended apparently higher than blank group, nematode life-span in experimental group.It is demonstrated experimentally that experiment use is more
Peptide mass concentration is suitable.It can effectively delay nematode aging, improve survival rate, and also further demonstrate that polypeptide simultaneously
The effect that VPITPTLNRE extends the life-span is realized not by nematode reproductive capacity is suppressed, because it has good anti-oxidation function
Stronger Scavenging ability.
2nd, biologically active polypeptide VPITPTLNRE 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 VPITPTLNRE 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 VPITPTLNRE 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 VPITPTLNRE groups<0.05).The each group half death time also it is corresponding to a certain extent
It has been extended that, hybrid peptide group is presented conspicuousness and improves (P compared with other experimental groups<0.05).As shown in fig. 7, in oxidative stress
Under the conditions of, experimental group survival rate is obviously higher than blank group survival rate.This explanation is under the conditions of oxidative stress, the survival rate of nematode
It is significantly improved, it may be possible to because polypeptide VPITPTLNRE can effectively help nematode to resist oxidative damage, remove production in vivo
The accumulation of raw free radical and reduction peroxide, rather than realized by strengthening its heat hardiness.The extension of organism life-span exists
It is due to improve resistance of the cell to stress conditions to a certain extent, thus anti-aging and depositing under pressure stressed condition
Much relations be present in motility rate.This results show polypeptide VPITPTLNRE can significantly increase the oxidative stress ability of nematode,
The survival rate of nematode is improved, illustrates that certain density polypeptide VPITPTLNRE has the effect of anti-aging for nematode.
3rd, biologically active polypeptide VPITPTLNRE 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 VPITPTLNRE.
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 VPITPTLNRE 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 VPITPTLNRE effectively improve SOD contents, reduce lipid peroxidation
The generation of thing 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 VPITPTLNRE 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 VPITPTLNRE and its preparation method and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Val Pro Ile Thr Pro Thr Leu Asn Arg Glu
1 5 10
<210> 2
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
gttcccatta ctcccactct gaacagagag 30
<210> 3
<211> 222
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 3
Met Lys Phe Phe Ile Phe Thr Cys Leu Leu Ala Val Ala Leu Ala Lys
1 5 10 15
Asn Thr Met Glu His Val Ser Ser Ser Glu Glu Ser Ile Ile Ser Gln
20 25 30
Glu Thr Tyr Lys Gln Glu Lys Asn Met Ala Ile Asn Pro Ser Lys Glu
35 40 45
Asn Leu Cys Ser Thr Phe Cys Lys Glu Val Val Arg Asn Ala Asn Glu
50 55 60
Glu Glu Tyr Ser Ile Gly Ser Ser Ser Glu Glu Ser Ala Glu Val Ala
65 70 75 80
Thr Glu Glu Val Lys Ile Thr Val Asp Asp Lys His Tyr Gln Lys Ala
85 90 95
Leu Asn Glu Ile Asn Gln Phe Tyr Gln Lys Phe Pro Gln Tyr Leu Gln
100 105 110
Tyr Leu Tyr Gln Gly Pro Ile Val Leu Asn Pro Trp Asp Gln Val Lys
115 120 125
Arg Asn Ala Val Pro Ile Thr Pro Thr Leu Asn Arg Glu Gln Leu Ser
130 135 140
Thr Ser Glu Glu Asn Ser Lys Lys Thr Val Asp Met Glu Ser Thr Glu
145 150 155 160
Val Phe Thr Lys Lys Thr Lys Leu Thr Glu Glu Glu Lys Asn Arg Leu
165 170 175
Asn Phe Leu Lys Lys Ile Ser Gln Arg Tyr Gln Lys Phe Ala Leu Pro
180 185 190
Gln Tyr Leu Lys Thr Val Tyr Gln His Gln Lys Ala Met Lys Pro Trp
195 200 205
Ile Gln Pro Lys Thr Lys Val Ile Pro Tyr Val Arg Tyr Leu
210 215 220
Claims (10)
1. a kind of biologically active polypeptide VPITPTLNRE, it is characterised in that its amino acid sequence is Val-Pro-Ile-Thr-
Pro-Thr-Leu-Asn-Arg-Glu。
2. a kind of biologically active polypeptide VPITPTLNRE according to claim 1, it is characterised in that the bioactivity is more
Peptide is milk-derived.
3. encode the nucleotide fragments of biologically active polypeptide VPITPTLNRE described in claim 1, it is characterised in that the nucleosides
The sequence of acid fragment such as SEQ ID NO：Shown in 2.
4. biologically active polypeptide VPITPTLNRE as claimed in claim 1 preparation method, it is characterised in that pass through genetic engineering
Method it is artificial synthesized, or directly obtained from dairy products by the method isolated and purified, or directly prepared by chemical synthesis.
5. biologically active polypeptide VPITPTLNRE as claimed in claim 1 application, it is characterised in that the biologically active polypeptide
Applications of the VPITPTLNRE in the food with anti-oxidation function, health products, medicine or cosmetics are prepared.
6. biologically active polypeptide VPITPTLNRE as claimed in claim 1 application, it is characterised in that the biologically active polypeptide
Applications of the VPITPTLNRE in the food with anti-senescence function, health products or medicine is prepared.
7. biologically active polypeptide VPITPTLNRE as claimed in claim 1 application, it is characterised in that the biologically active polypeptide
Applications of the VPITPTLNRE in the food with anti-oxidation function and anti-senescence function, health products or medicine is prepared.
8. a kind of oxidation resistant product, it is characterised in that including biologically active polypeptide VPITPTLNRE as claimed in claim 1 or institute
State biologically active polypeptide VPITPTLNRE derivative；Described oxidation resistant product include antioxidant food, antioxidant health-care product,
Anti-oxidation medicine or antioxidation cosmetic product；The derivative of the biologically active polypeptide VPITPTLNRE, refers to more in bioactivity
On peptide VPITPTLNRE amino acid side groups, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate,
Acetylation, phosphorylation, esterification or glycosylation modified, obtained polypeptide derivative.
9. a kind of anti-aging product, it is characterised in that including biologically active polypeptide VPITPTLNRE as claimed in claim 1 or institute
State biologically active polypeptide VPITPTLNRE derivative；Described anti-aging product includes antisenility cistanche food, antisenescence health product
Or antiaging agent；The derivative of the biologically active polypeptide VPITPTLNRE, refers in biologically active polypeptide VPITPTLNRE
Amino acid side groups on, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate, acetylation, phosphoric acid
Change, 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 VPITPTLNRE or described biologically active polypeptides VPITPTLNRE derivative；With anti-oxidation function and anti-ageing
The product of old function includes food, health products or medicine；The derivative of the biologically active polypeptide VPITPTLNRE, refers in life
On thing active peptides VPITPTLNRE amino acid side groups, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonyl
Change, methylate, acetylation, phosphorylation, esterification or glycosylation modified, obtained polypeptide derivative.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1374885A1 (en) * | 2002-06-27 | 2004-01-02 | Ingredia | Use of at least one peptide of alpha-s2 casein with inhibiting activity of ACE for the preparation of medicaments and foodstuffs |
| WO2005081628A2 (en) * | 2004-03-01 | 2005-09-09 | Peptera Pharmaceutical Ltd. | Casein derived peptides and therapeutic uses thereof |
| CN103254278A (en) * | 2005-06-08 | 2013-08-21 | 康斯乔最高科学研究公司 | Bioactive peptides identified in enzymatic hydrolyzates of milk caseins and method of obtaining same |
| CN104710524A (en) * | 2014-12-19 | 2015-06-17 | 上海交通大学 | Bovine alpha s2-casein source bioactive peptides preparation and application thereof |
| CN105254740A (en) * | 2015-10-16 | 2016-01-20 | 上海交通大学 | Bioactive polypeptide NQFYQKF as well as preparation and application thereof |
-
2017
- 2017-12-11 CN CN201711311343.XA patent/CN107880106B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1374885A1 (en) * | 2002-06-27 | 2004-01-02 | Ingredia | Use of at least one peptide of alpha-s2 casein with inhibiting activity of ACE for the preparation of medicaments and foodstuffs |
| WO2005081628A2 (en) * | 2004-03-01 | 2005-09-09 | Peptera Pharmaceutical Ltd. | Casein derived peptides and therapeutic uses thereof |
| CN103254278A (en) * | 2005-06-08 | 2013-08-21 | 康斯乔最高科学研究公司 | Bioactive peptides identified in enzymatic hydrolyzates of milk caseins and method of obtaining same |
| CN104710524A (en) * | 2014-12-19 | 2015-06-17 | 上海交通大学 | Bovine alpha s2-casein source bioactive peptides preparation and application thereof |
| CN105254740A (en) * | 2015-10-16 | 2016-01-20 | 上海交通大学 | Bioactive polypeptide NQFYQKF as well as preparation and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| GEMMA PUJADAS等: "The dipeptidyl peptidase-4 (DPP-4) inhibitor teneligliptin functions as antioxidant on human endothelial cells exposed to chronic hyperglycemia and metabolic high-glucose memory", 《ENDOCRINE》 * |
| HIROSHI UENISHI等: "Isolation and identification of casein-derived dipeptidyl-peptidase 4 (DPP-4)-inhibitory peptide LPQNIPPL from gouda-type cheese and its effect on plasma glucose in rats", 《INTERNATIONAL DAIRY JOURNAL》 * |
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