CN109467589A - Active peptide, recombinant vector, recombinant cell, antioxidant composition and its preparation method and application - Google Patents
Active peptide, recombinant vector, recombinant cell, antioxidant composition and its preparation method and application Download PDFInfo
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- CN109467589A CN109467589A CN201811290354.9A CN201811290354A CN109467589A CN 109467589 A CN109467589 A CN 109467589A CN 201811290354 A CN201811290354 A CN 201811290354A CN 109467589 A CN109467589 A CN 109467589A
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- active peptide
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- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003746 solid phase reaction Methods 0.000 description 1
- 238000010671 solid-state reaction Methods 0.000 description 1
- 229940083466 soybean lecithin Drugs 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000001845 taraxacum officinale leaf extract Substances 0.000 description 1
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 229940035936 ubiquinone Drugs 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/18—Antioxidants, e.g. antiradicals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- 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
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- Zoology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Dermatology (AREA)
- Pharmacology & Pharmacy (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The present invention relates to a kind of active peptides, recombinant vector, recombinant cell, antioxidant composition and its preparation method and application.The active peptide contains amino acid sequence small peptide as shown in SEQ ID No.1.The active peptide with have inoxidizability and safety it is higher.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to a kind of active peptide, recombinant vector, recombinant cell, anti-oxidant
Composition and its preparation method and application.
Background technique
Organism can be constantly be generated free radical in normal living process, and suitable free radical is able to maintain that organism just
Normal physiological metabolism, but excessive free radical will lead to body tissue by oxidative stress, causes the oxidative system of body and resists
Oxidative system is unbalance, and causes the large biological molecules such as cell, albumen and nucleic acid in body tissue that oxidative damage, Jin Eryin occurs
The problems such as sending out cancer, angiocarpy and aging, causes greatly to damage to the health of body.Therefore, effective antioxidant into
Row is anti-oxidant to have extremely important effect for maintenance body health.Currently, medically there are many anti-oxidation medicines, but this
A little drugs have different degrees of side effect mostly, should not take for a long time.
Summary of the invention
Based on this, it is necessary to provide a kind of with inoxidizability and the higher active peptide of safety.
In addition, also providing a kind of recombinant vector, recombinant cell, antioxidant composition and its preparation method and application.
A kind of active peptide, the active peptide contain amino acid sequence small peptide as shown in SEQ ID No.1.
Above-mentioned active peptide contains Gly-Glu-Gly-Val-Phe-Gly-His, which is effectively improved
The activity of antioxidase, and the active peptide has no toxic side effect, safety is higher.Experiment proves that the active peptide is able to extend N2
The service life of Wild-type C. elegans improves the superoxide dismutase of paraquat treated N2 Wild-type C. elegans
The activity of (SOD, Superoxide Dismutase) reduces the activity of paraquat treated N2 Wild-type C. elegans
The content of oxygen cluster (ROS, reactive oxygen species) and malonaldehyde (MDA, Malondialdehyde), reach reparation
With the purpose for the treatment of oxidative damage.
A kind of recombinant vector, the coded sequence containing small peptide described above.
Coded sequence nucleotide sequence as shown in SEQ ID No.2 in one of the embodiments,；Or
The coded sequence is the nucleotide sequence for having 75% homology with nucleotide sequence shown in SEQ ID No.2；
Or
The coded sequence is that one or more bases are lacked, substituted in the nucleotide sequence as shown in SEQ ID No.2
Or increase obtained nucleotide sequence.
A kind of recombinant cell, the recombinant cell contain the nucleotide for encoding active peptide described above；Alternatively,
The recombinant cell contains recombinant vector described above.
A kind of antioxidant composition, including active component, the active component include active peptide described above.
Active peptide described above, recombinant vector described above, recombinant cell described above or described above anti-
Oxidising composition improves the drug of activities of antioxidant enzymes or content, reduces the drug of content of propylene glycol or reduces active oxygen in preparation
Application in the drug of cluster content.
Active peptide described above, recombinant vector described above, recombinant cell described above or described above anti-
Application of the oxidising composition in the drug of preparation prevention or treatment oxidative damage.
Active peptide described above is preparing the application in food, health care product or cosmetics.
The preparation method of active peptide described above includes the following steps: the isolated activity from stone small water turtle
Peptide；Or
The active peptide is synthesized by chemical synthesis process.
The active peptide isolated from stone small water turtle includes the following steps: in one of the embodiments,
Stone small water turtle meat is digested, zymolyte is obtained；And
The zymolyte is extracted using organic solvent, obtains the active peptide.
Detailed description of the invention
Fig. 1 is the mass spectrogram of the active peptide of embodiment 1；
Fig. 2 is the high-efficient liquid phase chromatogram of the active peptide of embodiment 1；
Fig. 3 is the comparison diagram in the service life of the nematode of I group, II group, III group, IV group；
Fig. 4 is the comparison diagram of the survival rate of the nematode of I group, II group, III group, IV group, Par group；
Fig. 5 is the comparison diagram of ROS content in the nematode body of I group, II group, III group, IV group, Par group；
Fig. 6 is the comparison diagram of MDA content in the nematode body of I group, II group, III group, IV group, Par group；
Fig. 7 is the active comparison diagram of the intracorporal superoxide dismutase of nematode of I group, II group, III group, IV group, Par group.
Specific embodiment
To facilitate the understanding of the present invention, a more comprehensive description of the invention is given in the following sections with reference to the relevant attached drawings.In attached drawing
Give preferred embodiment of the invention.But the invention can be realized in many different forms, however it is not limited to herein
Described embodiment.On the contrary, purpose of providing these embodiments is keeps the understanding to the disclosure more saturating
It is thorough comprehensive.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.
The active peptide of one embodiment contains amino acid sequence small peptide as shown in SEQ ID No.1.
Specifically, amino acid sequence shown in SEQ ID No.1 is Gly-Glu-Gly-Val-Phe-Gly-His.Wherein,
Gly indicates glycine, is abbreviated as G.Glu indicates glutamic acid, is also abbreviated as E.Val indicates valine, is abbreviated as V.Phe indicates benzene
Alanine is abbreviated as F.His indicates histidine, is abbreviated as H.
Due to the polymorphism and variation of albumen coded sequence, naturally occurring protein will appear gene mutation, code sequence
Base is by the missing of missing, substitution or increase or amino acid, insertion, substitution or other variations in column, so as to cause protein
There are one or more amino acid by missing, substitution or increase in amino acid sequence.Accordingly, there exist some physiology and bioactivity
On be essentially identical to the albumen of no variant protein matter.These structures are different from corresponding protein, but not bright with the protein
More peptide or proteins of aobvious function difference are known as function and are equal to variant.
The equivalent variant of function is equally applicable to change by artificial means such as missing, insertion and mutation one or more
Codon, to import this kind of variation into a kind of amino acid sequence of protein and manufactured small peptide.Even now can obtain
More various forms of variants, but resulting variant as the premise that function is equal to variant be its physiological activity substantially etc.
It is same as the activity of original no variant protein matter.
In general, function be equal variant coded sequence be it is homologous, therefore, by least one change (such as protein
Coded sequence in have one or more in missing, insertion or the substitution of one or more bases or the amino acid sequence of protein
A amino acid deletions, insertion or substitution) resulting more peptide or proteins generally have the activity for being functionally equivalent to the protein.
Therefore, the small peptide being made of above-mentioned amino acid sequence is also included within if its active peptide constituted does not have apparent function difference
In the range of this research.
In a particular embodiment, small peptide has the following structure formula:
The amino acid sequence of active peptide is as shown in SEQ ID No.1 in one of the embodiments,.The peptide chain of the active peptide
It is shorter, it is easy to absorb and utilize.
Above-mentioned active peptide contains Gly-Glu-Gly-Val-Phe-Gly-His, which is effectively improved
The activity of antioxidase, and the active peptide has no toxic side effect, safety is higher.Experiment proves that the active peptide is able to extend N2
The service life of Wild-type C. elegans improves the superoxide dismutase of paraquat treated N2 Wild-type C. elegans
(SOD) activity reduces the reactive oxygen species (ROS) and malonaldehyde of paraquat treated N2 Wild-type C. elegans
(MDA) content achievees the purpose that reparation and treatment oxidative damage.Meanwhile the active peptide can directly participate in protein
Synthesis improves body to the utilization rate of protein.Further, since the active peptide has the activity that can be improved antioxidase, drop
The content of low ROS and MDA can be used in preparation and improve the drug of activities of antioxidant enzymes or content, reduce the medicine of content of propylene glycol
In the drug of object or reduction reactive oxygen species, and it can be used in the drug of preparation prevention or treatment oxidative damage, additionally it is possible to
Applied in food, health care product or cosmetics.
Wherein, superoxide dismutase is antioxidase important in organism, has special physiological activity, is biology
The primary substance of free radical is removed in vivo.ROS is one of biological aerobic metabolism process byproduct, including oxonium ion, peroxide
Compound and oxygen radical etc..There are excessive ROS can result in cell rapid apoptosis in cell.Malonaldehyde can cause albumen
The cross-linked polymeric of the life macromolecules such as matter, nucleic acid, and have cytotoxicity, malonaldehyde be the most important product of Lipid peroxidation metabolism it
One, the damage of film can be aggravated.
The food of one embodiment includes above-mentioned active peptide.Above-mentioned active peptide is as edible additive, to enhance food
Inoxidizability, to reach healthcare function, meanwhile, above-mentioned active peptide can supplement required albumen and amino acid to body.
Food is tablet, capsule, pulvis, granule, pill, syrup, solution, mixes in one of the embodiments,
Suspension or aerosol.
The mass percentage of active peptide is 5%~10% in food in one of the embodiments,.
The health care product of one embodiment includes above-mentioned active peptide.Above-mentioned active peptide is as active constituent, so that health care product has
There is preferably anti-oxidation efficacy, meanwhile, above-mentioned active peptide can supplement required albumen and amino acid to body.
In one of the embodiments, health care product be tablet, capsule, pulvis, granule, pill, syrup, solution,
Suspension or aerosol.
The mass percentage of active peptide is 3%~5% in health care product in one of the embodiments,.
The cosmetics of one embodiment include above-mentioned active peptide.Above-mentioned active peptide as the performance component in cosmetics,
So that cosmetics have stronger inoxidizability, the aging of delaying skin keeps the elasticity of skin.
Cosmetics are cream shape, emulsion form, gel or aqua shape in one of the embodiments,.
Cosmetics further include excipient matrix in one of the embodiments,.Excipient matrix is selected from ultraviolet absorbing agent, metal
In ion chelating agent, monosaccharide, oligosaccharides, polysaccharide, amino acid, organic amine, preservative, pH adjusting agent, anti-oxidant auxiliary agent and fragrance
It is at least one.Further, the mass ratio of active peptide and excipient matrix is 3%~5% in cosmetics.
The recombinant vector of one embodiment contains the coded sequence of small peptide.
Coded sequence is the nucleotide sequence as shown in SEQ ID No.2 in one of the embodiments,.Specifically, SEQ
Nucleotides sequence shown in ID No.2 is classified as 5 '-GGCGAAGGAGUAUUCGGGCAU-3 '.
Coded sequence is homologous with 75% with nucleotide sequence shown in SEQ ID No.2 in one of the embodiments,
The nucleotide sequence of property.
Coded sequence is one or more in the nucleotide sequence as shown in SEQ ID No.2 in one of the embodiments,
Base is by missing, substitution or increases obtained nucleotide sequence.
Further, coded sequence is that one or more bases are simultaneous by its in the nucleotide sequence as shown in SEQ ID No.2
And the nucleotide sequence that base substitutes.
It should be noted that by above-mentioned nucleotide sequence coded small peptide, if its active peptide constituted is not apparent
Function difference is also included in the range of this research.
Recombinant vector is recombinant expression carrier or recombinant cloning vector in one of the embodiments,.
Recombinant vector contains purification tag in one of the embodiments,.By the way that purification tag is arranged, be conducive to active peptide
Isolate and purify.Further, purification tag is His label, GST label or SUMO label.It should be noted that purification tag
Be not limited to it is above-mentioned point out purification tag, other common purification tags can also act on the purification tag of recombinant vector.
Recombinant vector includes engineering carrier in one of the embodiments,.The nucleotide for encoding above-mentioned active peptide is inserted
Enter in engineering carrier.Further, engineering carrier is pET-32a carrier, pGEX-6P-1 carrier, pPIC-9K carrier
Or pPIC-Z α carrier.It should be noted that engineering carrier be not limited to it is above-mentioned point out engineering carrier, other are common
Engineering carrier can also act on the engineering carrier of recombinant vector.
Above-mentioned recombinant vector can preferably save the nucleotide for encoding above-mentioned active peptide, be conducive to the expression of active peptide,
The drug of activities of antioxidant enzymes or content is improved can be used in preparation, and can be applied to preparation prevention or treatment oxidisability
The drug of damage.
The recombinant cell of one embodiment contains the nucleotide or above-mentioned recombinant vector for encoding above-mentioned active peptide.
Recombinant cell is the cell for the nucleotide that clone encodes above-mentioned active peptide in one of the embodiments,.
Recombinant cell is the cell for the nucleotide that expression encodes above-mentioned active peptide in one of the embodiments,.
Recombinant cell includes recipient cell in one of the embodiments,.Encode the nucleotide or above-mentioned of above-mentioned active peptide
Recombinant vector is located in recipient cell.
Recipient cell is Escherichia coli, saccharomyces cerevisiae, Pichia pastoris, zooblast or plant in one of the embodiments,
Object cell.Further, recipient cell is bacillus coli DH 5 alpha, Escherichia coli Top10, Escherichia coli Orgami (DE3), Bi Chi
Yeast GS115 or Pichia pastoris SMD1168.
It should be noted that recipient cell be not limited to it is above-mentioned point out recipient cell, other common recipient cells can also be with
Act on the recipient cell of recombinant cell.
Above-mentioned active peptide can be cloned or be expressed to above-mentioned recombinant cell, make it possible to prepare the active peptide on a large scale, and
And by the recombinant cell orientation expression active peptide, the higher active peptide of purity can be obtained, and then be conducive to active peptide
Using therefore, above-mentioned recombinant cell can be used in preparation and improve the drug of activities of antioxidant enzymes or content, and can be applied to
The drug of preparation prevention or treatment oxidative damage.
The antioxidant composition of one embodiment, including active component, active component include above-mentioned active peptide.
Antioxidant composition further includes carrier in one of the embodiments,.Carrier is used for load active component.
Carrier includes at least one of solvent, polymer and liposome in one of the embodiments,.
Solvent is for dissolution or suspended active component.Solvent includes in sterile water, physiological saline and common non-aqueous solvent
One kind.Wherein, commonly using non-aqueous solvent for example can be ethyl alcohol.
Polymer is used to improve the water solubility and stability of bioactive peptide.Polymer includes that polylysine, polylysine change
At least one of property object, polyethyleneimine, polyethyleneimine-modified object, chitosan, polylactic acid and gelatin.Wherein, gather bad ammonia
The modifier of acid for example can be the water-soluble modified object of polylysine.The modifier of polyethyleneimine for example can be polyethylene
The water-soluble modified object of imines.
Liposome is for wrapping up bioactive peptide, to obtain the liposoluble substance containing small peptide, to be applied to fat-soluble ring
In border.Liposome includes at least one of cholesterol and lecithin.Wherein, lecithin is selected from beans lecithin and egg yolk lecithin
At least one of.Specifically, beans lecithin for example can be soybean lecithin.
Carrier further includes at least one of diluent and excipient in one of the embodiments,.
Diluent is for diluting active component.Diluent includes at least one of starch, sugar, cellulose and inorganic salts.
Specifically, starch for example can be amylopectin.Sugar for example can be sugarcane polysaccharide.Celluloses such as can be sugarcane fibre
Element.Inorganic salt such as can be sodium chloride.
Excipient is for making antioxidant composition that specific form be presented.Specifically, excipient makes the antioxidant composition be in
The forms such as tablet, semisolid preparation, liquid preparation, capsule, pulvis, granule, pill, syrup, suspension or aerosol.It needs
It is noted that antioxidant composition be not limited to it is above-mentioned point out form, can also be other forms, such as can be paste.
Excipient includes at least one of binder, filler and lubricant in one of the embodiments,.Pass through packet
The excipient is included, so that antioxidant composition is in tablet.
Excipient includes the base portion of ointment or the base portion of creme in one of the embodiments,.Pass through packet
The excipient is included, so that antioxidant composition is in semisolid preparation.
Excipient includes preservative, antioxidant, corrigent, aromatic, cosolvent, emulsification in one of the embodiments,
At least one of agent and colorant.By including the excipient, so that antioxidant composition is in liquid preparation.
In one of the embodiments, in antioxidant composition, the mass ratio of active component and carrier is 5:75~95:4.
Optionally, in antioxidant composition, the mass ratio of active component and carrier is 10:50~90:3.Further, anti-oxidant combination
In object, the mass ratio of active component and carrier is 15:45~85:10.Further, in antioxidant composition, active component
Mass ratio with carrier is 30:60~80:15.
Antioxidant composition further includes helper component in one of the embodiments,.Helper component includes vitamin C, dimension
At least one of raw element E, ubiquinone, glutathione, carrotene and glycine betaine.By adding helper component, to enhance antioxygen
Change the oxidation resistant ability of composition.It should be noted that helper component is not limited to the above-mentioned component pointed out, it can also include tool
There is the substance of other auxiliary effects, such as can be the substance with anti-inflammatory activity.Specifically, with the substance example of anti-inflammatory activity
It such as can be dandelion extract.
Helper component and the mass ratio of active component are 5:85~5:90 in one of the embodiments,.
Above-mentioned antioxidant composition contains active peptide, has and improves activities of antioxidant enzymes and prevention or treatment oxidative damage
Characteristic, can be used in preparation and improve the drug of activities of antioxidant enzymes or content, and can be applied to preparation prevention or treatment
The drug of oxidative damage.
The active peptide of one embodiment can be isolated from stone small water turtle, can also be closed by chemical synthesis process
At.
Isolated active peptide includes following operation S110~S120 in the slave stone small water turtle of one embodiment:
S110, stone small water turtle meat is digested, obtains zymolyte.
Specifically, S110 includes following operation S111~S112:
S111, water mixing is added to stone small water turtle meat gruel, obtains meat gruel water.
Stone small water turtle meat gruel is to obtain after stone small water turtle digested tankage is broken in one of the embodiments,.
Water is deionized water or ultrapure water in one of the embodiments,.
The ratio between the quality of stone small water turtle meat gruel and the volume of water are 1~40 in one of the embodiments,.Further,
The ratio between quality and the volume of water of stone small water turtle meat gruel are 5~15.
S112, into meat gruel water, addition enzyme is digested, and obtains zymolyte.
Enzyme is alkali protease in one of the embodiments,.Further, enzyme is that the big rich biotechnology in Zhengzhou City is limited
The alkali protease of the 2017-JD-0812 of company.Further, final concentration of 200U/mL~800U/mL of enzyme.Meat gruel water
PH with the mixture of enzyme is 8~10.Hydrolysis temperature is 40 DEG C~60 DEG C.Enzymolysis time is 3h~6h.Optionally, the end of enzyme is dense
Degree is 600U/mL~800U/mL.The pH of the mixture of meat gruel water and enzyme is 8~9.Hydrolysis temperature is 40 DEG C~50 DEG C.
It further include operating as follows: to enzyme in one of the embodiments, after enzyme is added into meat gruel water and is digested
Meat gruel water after solution carries out destroy the enzyme treatment；After enzyme deactivation, supernatant is collected by centrifugation；Supernatant is dried, zymolyte is obtained.
Specifically, the operation of destroy the enzyme treatment is carried out to the meat gruel water after enzymatic hydrolysis specifically: be put into the meat gruel water after enzymatic hydrolysis
Enzyme deactivation 10min~30min at 85 DEG C~95 DEG C.
It is collected by centrifugation in the operation of supernatant, centrifugal rotational speed is 6000r/min~12000r/min.Centrifugation time is 10min
~30min.
The mode that supernatant is dried is freeze-drying.It is 0.1%~0.3% by zymolyte drying to water content.Its
In, water content is the mass percentage of water in zymolyte.It should be noted that dry mode is not limited to be freeze-dried,
It can also be other drying modes, such as air-dry.
S120, zymolyte is extracted using organic solvent, obtains active peptide.
Specifically, zymolyte is mixed with organic solvent, extracts, obtains aqueous layer；Aqueous layer is purified, is lived
Property peptide.Wherein, aqueous layer is the solution containing active peptide.It should be noted that if the purity of active peptide can in aqueous layer
If meeting actual demand, the operation purified to aqueous layer be can be omitted.
The volume ratio of zymolyte and organic solvent is 1:1.5~1:7 in one of the embodiments,.
Organic solvent is selected from least one of ethyl acetate and acetone in one of the embodiments,.
The time extracted in one of the embodiments, is 10min~20min.
Zymolyte is mixed with organic solvent in one of the embodiments, is extracted, the operation for obtaining aqueous layer is specific
Are as follows: zymolyte, organic solvent are mixed with water, extracts, obtains aqueous layer.By mixing zymolyte, organic solvent with water, more
Be conducive to the progress of extraction.Wherein, water is deionized water or ultrapure water.
More specifically, by the zymolyte of 450g~550g, the ethyl acetate and 900mL~1100mL of 3150mL~3850mL
Water mixing, extraction, obtain aqueous layer.Further, extraction is repeated at least three times；The water phase being obtained by extraction every time is laminated
And to be purified.Optionally, the water of the zymolyte of 500g, the ethyl acetate of 3500mL and 1000mL is mixed, is extracted, weight
It is five times multiple, obtain aqueous layer.
The operation that aqueous layer is purified in one of the embodiments, specifically: aqueous layer is dried；It will do
Aqueous layer after dry carries out chromatography separation, obtains active peptide.
Specifically, in operation aqueous layer being dried, dry mode is vacuum and low temperature freeze-drying.It needs to illustrate
, it can also be other drying modes, such as air-dry that dry mode, which is not limited to be freeze-dried,.
It is HPLC, i.e. high performance liquid chromatography (High by the method that the aqueous layer after drying carries out chromatography separation
Performance Liquid Chromatography).Specifically, the condition of HPLC are as follows: chromatographic column Cosmosil5C18,
Mobile phase is the aqueous solution for the methanol that volumn concentration is 35%, and flow velocity is 0.5mL/min~1.2mL/min, Detection wavelength
For 220nm, sample volume is 80 μ L.
It should be noted that if the concentration of aqueous layer can satisfy when needing of chromatography separation, aqueous layer is done
Dry operation can be omitted.
By the available natural active peptide of the preparation method of above-mentioned active peptide, and the purity of active peptide is higher.
The preparation method of the active peptide of one embodiment includes the following steps: through chemical synthesis process synthesizing activity peptide.
Chemical synthesis process is polypeptide solid-state reaction method in one of the embodiments,.Further, chemical synthesis process
For Boc solid-phase synthesis or Fmoc solid-phase synthesis.Wherein, Boc is tertbutyloxycarbonyl.With easy acidolysis in Boc solid-phase synthesis
Boc group as N- ɑ-blocking group.Fmoc is 9-fluorenylmethyloxycarbonyl.With the Fmoc base of easy acidolysis in Fmoc solid-phase synthesis
Group is used as N- ɑ-blocking group.
The preparation method of above-mentioned active peptide, it is simple process, easy to operate, and the higher active peptide of purity can be prepared.
The following are specific embodiment parts.
In following embodiment, unless otherwise instructed, test method without specific conditions, usually according to normal condition,
For example, see Pehanorm Brooker, EF, not the written molecular cloning of Ritchie, T Manny A Disi etc. (Jin Dongyan, Li Mengfeng etc. are translated) is real
It tests condition described in guide [M] (Beijing: Science Press, 1992) or method that kit manufacturer is recommended is realized.
Reagent used in embodiment is commercially available.
If not otherwise specified, in following embodiment, Fmoc-Glu-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-
Phe-OH and Fmoc-His-OH are purchased from Aladdin reagent Co., Ltd.N2 Wild-type C. elegans are purchased from Chinese science
Institute's cell bank.NGM culture medium is purchased from Life Technologies, Grand Island, NY, USA company.
Embodiment 1
The preparation process of the active peptide of the present embodiment is as follows:
(1) the ultrapure water mixing of 2500mL is added to the stone small water turtle meat gruel of 300g, obtains meat gruel water.Add into meat gruel water
Enter alkali protease (the as basic protein of the 2017-JD-0812 of Zhengzhou City Wei Feng Biotechnology Co., Ltd of 700U/mL
Enzyme) it descends to digest 4h for 8.0 in 45 DEG C, pH, the meat gruel water after enzymatic hydrolysis is put into enzyme deactivation 10min at 85 DEG C, after enzyme deactivation,
9000r/min is centrifuged 20min and collects supernatant；It is 0.2% that supernatant, which is dried to water content, obtains zymolyte.
(2) ultrapure water of the zymolyte of 500g, the ethyl acetate of 3500mL and 1000mL is mixed, stands extraction 15min,
Aqueous layer is collected, extraction four times is repeated, the aqueous layer that four extractions are collected is mixed and carries out vacuum and low temperature freeze-drying, after being dried
Aqueous layer.
(3) aqueous layer after drying is subjected to chromatography separation, obtains active peptide.Aqueous layer after drying is subjected to chromatography
The condition of method separation are as follows: chromatographic column Cosmosil5C18, mobile phase is the aqueous solution for the methanol that volumn concentration is 35%,
Flow velocity is 0.8mL/min, and Detection wavelength 220nm, sample volume is 80 μ L.
Embodiment 2
The preparation process of the active peptide of the present embodiment is as follows:
(1) take 100mg Fmoc-Gly Wang Resin (glycine pre-install resin, be purchased from Aladdin company and article No. be
It 2018-01-225-N23) is placed in synthesis in solid state pipe, the n,N-Dimethylformamide (DMF) that 25mL is added is stood afterwards to be made to pre-install
Resin is sufficiently swollen, and filters off solvent, the DMF solution of the piperidines for being 20% containing mass percentage of 5mL is added, after oscillation
Solvent is filtered out, the resin that obtains that treated.
(2) by the 1- of the Fmoc-Glu-OH of 4mg (be purchased from Aladdin company and article No. is 2018-01-225-N37), 5mg
Hydroxybenzotriazole, 3mg O- benzotriazole-tetramethylurea hexafluorophosphoric acid ester be dissolved in the DMF of 20mL, 30mg is being added
N,N-diisopropylethylamine is protected from light after mixing, the Fmoc-Glu-OH activated.Step is added in Fmoc-Glu-OH after activation
Suddenly in (1) resin that obtains that treated, nitrogen blows stirring 70min under room temperature, filtering, successively using DMF and methylene chloride
Washing precipitating, removes solvent, obtains the first reactant.
(3) Fmoc-Gly-OH, the Fmoc-Gly-OH activated are activated according to the operation of step (2)；According to step (2)
Operation, by after activation Fmoc-Gly-OH be added the first reactant in, obtain the second reactant.According to the operation of step (2)
Activate Fmoc-Val-OH, the Fmoc-Val-OH activated；According to the operation of step (2), by the Fmoc-Val-OH after activation
It is added in the second reactant, obtains third reactant.Fmoc-Phe-OH is activated according to the operation of step (2), is activated
Fmoc-Phe-OH；According to the operation of step (2), the Fmoc-Phe-OH after activation is added in third reactant, obtains the 4th
Reactant.According to the operation of step (2), the Fmoc-Gly-OH after activation is added in the 4th reactant, obtains the 5th reaction
Object.Fmoc-His-OH, the Fmoc-His-OH activated are activated according to the operation of step (2)；According to the operation of step (2),
Fmoc-His-OH after activation is added in the 5th reactant, the 6th reactant is obtained.
(4) the 6th reactant is washed with dehydrated alcohol, be separated by solid-liquid separation, supernatant is successively carried out to rotary evaporation concentration, cold
It is lyophilized dry, obtains active peptide.
Test:
1, it is measured using purity of the high performance liquid chromatography to the active peptide of Examples 1 to 2, and using mass spectrum to implementation
The active peptide of example 1 is identified.
Wherein, high-efficient liquid phase chromatogram determining condition are as follows: Boston Green ODS-AQ chromatographic column (250*4.6mm)；With body
The aqueous solution for the trifluoroacetic acid that product percentage composition is 0.1% is mobile phase A, the trifluoroacetic acid for being 0.1% with volumn concentration
Acetonitrile solution be Mobile phase B, the volume ratio of mobile phase A and Mobile phase B is 75:25；Flow velocity is 1mL/min；Detection wavelength is
220nm；10 μ L of sample volume；Standard items are histidine；
Mass spectroscopy condition: ESI positive ion mode, capillary voltage 3kV, orifice potential 50V, extraction voltage are
5V, desolventizing temperature are 350 DEG C, atomization air flow 350L/h.
See Table 1 for details and Fig. 1~2 for measurement result.What table 1 indicated is the purity of the active peptide of Examples 1 to 2.Fig. 1 is to implement
The mass spectrogram of the active peptide of example 1.Fig. 2 is the high-efficient liquid phase chromatogram of the active peptide of embodiment 1, wherein arrow (2-1) meaning
Peak is the absorption peak of active peptide.
The purity of the active peptide of 1 Examples 1 to 2 of table
| Embodiment 1 | Embodiment 2 | |
| Purity (%) | 95.5 | 96.2 |
From Fig. 1~2 as can be seen that the amino acid sequence of the active peptide of embodiment 1 is Gly-Glu-Gly-Val-Phe-
Gly-His, structural formula are as follows:
As it can be seen from table 1 the purity for the active peptide that Examples 1 to 2 obtains illustrates above-mentioned embodiment party 95% or more
The preparation method of the active peptide of formula can obtain the active peptide with higher degree.
2, influence of the measurement active peptide to the service life of N2 Wild-type C. elegans (hereinafter referred to as nematode)
(1) experiment is divided into four groups, respectively control group (i.e. I group), experimental group 1 (i.e. II group), 2 (i.e. III of experimental group
Group), experimental group 3 (i.e. IV group), every group of three parallel laboratory tests.
(2) nematode is inoculated in NGM culture medium and is cultivated in 20 DEG C.Wherein, in the NGM culture medium of experimental group 1~3
Middle to add 0.25 μM, 1.25 μM, the active peptide of 7.25 μM of embodiment 1 respectively, addition is isometric in the NGM culture medium of control group
PH7.45 phosphate buffer.
(3) service life of four groups of nematodes is measured.Specifically, by each group nematode culture to the 5th day, using microscope observation method
The nematode of four groups of cultures is once counted daily, until all groups of nematode is dead.Measurement result is detailed in Fig. 3.Fig. 3 is
The comparison diagram in the service life of the nematode of I group, II group, III group, IV group." * " in Fig. 3 indicates significant difference < 0.05, " * * " table
Show significant difference < 0.01.
From figure 3, it can be seen that the addition of active peptide is able to extend the service life of nematode.The nematode longevity of II group, III group, IV group
Life provides 17.7%, 45.6%, 73.7% than I group respectively.With the increase of the additive amount of active peptide, the service life of nematode is longer.
3, repair of the measurement active peptide to body oxidative damage
(1) experiment is divided into five groups, respectively control group (i.e. I group), experimental group 1 (i.e. Par group), 2 (i.e. II of experimental group
Group), experimental group 3 (i.e. III group), experimental group 4 (i.e. IV group), every group of three parallel laboratory tests.
(2) N2 Wild-type C. elegans (hereinafter referred to as nematode) are inoculated in NGM culture medium and are trained in 20 DEG C
It supports.Wherein, 0.25 μM, 1.25 μM, the activity of 7.25 μM of embodiment 1 are added respectively in the NGM culture medium of experimental group 2~4
The phosphate buffer of isometric pH7.45 is added in the NGM culture medium of peptide, control group and experimental group 1.It cultivates to the 4th day,
100 μM of paraquat is added in the culture medium of experimental group 1~4, continues culture 1 day.
(3) it after cultivating, is counted using nematode of the microscope observation method to five groups of cultures, and calculates five tissue cultures
The survival rate of feeding nematode；It is measured in the nematode of five groups of cultures and is surpassed using ELISA method (superoxide dismutase ELISA kit)
The activity (unit U/mL) of Superoxide dismutase (SOD)；Five tissue cultures are measured using ELISA method (malonaldehyde ELISA kit)
The content (unit pg/mL) of malonaldehyde in feeding nematode；Five tissue cultures are measured using ELISA method (ROS ELISA kit)
The content (unit pg/mL) of ROS in feeding nematode.Measurement result is detailed in Fig. 4~7.Wherein, Fig. 4 be I group, II group, III group,
The comparison diagram of the survival rate of the nematode of IV group, Par group, " * " in Fig. 4 indicate significant difference < 0.05.Fig. 5 be I group, II group,
III group, IV group, Par group nematode body in ROS content comparison diagram, " * " in Fig. 5 indicates significant difference < 0.05.Fig. 6 is
I group, II group, III group, IV group, Par group nematode body in MDA content comparison diagram.Fig. 7 be I group, II group, III group, IV group,
The active comparison diagram of the intracorporal superoxide dismutase of the nematode of Par group.
From fig. 4, it can be seen that survival rate of the survival rate of the nematode of Par group lower than the nematode of I group, and be computed, Par group
The survival rate ratio I group of nematode reduce 23.6%, illustrate that the processing of paraquat generates serious damage to nematode, significantly reduce line
The service life of worm.The survival rate of the nematode of II group, III group, IV group is better than Par group, illustrates that the addition of active peptide can be alleviated and repair
Multiple damage of the paraquat to nematode, extends the service life of nematode；And be computed, II group, III group, IV group nematode survival rate point
10.4%, 13.6%, 23.3% is not improved than Par group, illustrates the increase of the concentration with active peptide, active peptide makes paraquat
At damage repair it is stronger.Particularly, the survival rate of the nematode of IV group is substantially suitable with I group, illustrates active peptide energy
It enough treats and is damaged caused by paraquat.
From fig. 5, it can be seen that the intracorporal ROS content of the nematode of Par group is apparently higher than I group, illustrate that the processing of paraquat is led
It causes to generate a large amount of reactive oxygen species in nematode body, and then serious oxidative damage is caused to nematode, it is a large amount of to eventually lead to nematode
It is dead.II group, III group, the intracorporal ROS content of nematode of IV group are below Par group, illustrate that the addition of active peptide is able to suppress
The generation of ROS, and then alleviate and repair oxidative damage caused by paraquat.The intracorporal ROS of nematode of II group, III group, IV group
Content gradually decreases, and illustrates the increase of the concentration with active peptide, and the repair of the oxidative damage caused by paraquat is got over
By force.
From fig. 6, it can be seen that the intracorporal MDA content of the nematode of Par group is apparently higher than I group, illustrate that the processing of paraquat is led
It causes to generate a large amount of MDA in nematode body, causes nematode serious Lipid peroxidation metabolism occur, eventually lead to nematode mortality.II
Group, III group, the intracorporal MDA content of nematode of IV group are below Par group, illustrate that the addition of active peptide is able to suppress the production of MDA
It is raw, and then alleviate and repair Lipid peroxidation metabolism caused by paraquat.II group, III group, the intracorporal MDA content of nematode of IV group by
It gradually reduces, illustrates the increase of the concentration with active peptide, the repair of the Lipid peroxidation metabolism caused by paraquat is stronger.Especially
Ground, the intracorporal MDA of the nematode of IV group is substantially suitable with I group, illustrates that active peptide can treat film rouge peroxide caused by paraquat
Change.
From figure 7 it can be seen that the intracorporal SOD activity of the nematode of Par group is lower than I group, illustrate that the processing of paraquat leads to line
The intracorporal SOD inactivation of worm, reduces the oxidation resistance of nematode.II group, III group, the intracorporal SOD activity of nematode of IV group are excellent
In Par group, illustrate that the addition of active peptide is able to suppress the active reduction of SOD.The intracorporal SOD of nematode of II group, III group, IV group
Activity gradually increases, and illustrates the increase of the concentration with active peptide, and the active reduced effect of SOD is inhibited to be more obvious.Particularly,
The intracorporal SOD activity ratio I group of the nematode of IV group it is strong, illustrate active peptide can be improved nematode SOD activity.
To sum up, above-mentioned active peptide improves the activity of antioxidase, extends the service life of body, prevention or treatment oxidisability damage
Wound, meanwhile, the peptide chain of above-mentioned active peptide is shorter, is easy to absorb, and safety is higher, can be used in preparation and improves activities of antioxidant enzymes
Or in the drug of content, and it can be used in the drug of preparation prevention or treatment oxidative damage.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
<110>the triumphant health Biotechnology Co., Ltd in Shenzhen
<120>active peptide, recombinant vector, recombinant cell, antioxidant composition and its preparation method and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 2
<211> 7
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Gly Glu Gly Val Phe Gly His
1 5
<210> 2
<211> 21
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ggcgaaggag uauucgggca u 21
Claims (10)
1. a kind of active peptide, which is characterized in that the active peptide contains amino acid sequence small peptide as shown in SEQ ID No.1.
2. a kind of recombinant vector, which is characterized in that the recombinant vector contains the coded sequence of small peptide described in claim 1.
3. recombinant vector according to claim 2, which is characterized in that the coded sequence is as shown in SEQ ID No.2
Nucleotide sequence；Or
The coded sequence is the nucleotide sequence for having 75% homology with nucleotide sequence shown in SEQ ID No.2；Or
The coded sequence is bases one or more in the nucleotide sequence as shown in SEQ ID No.2 by missing, substitution or increasing
The nucleotide sequence added.
4. a kind of recombinant cell, which is characterized in that the recombinant cell contains the nucleosides of active peptide described in coding claim 1
Acid；Alternatively,
The recombinant cell contains the described in any item recombinant vectors of claim 2~3.
5. a kind of antioxidant composition, which is characterized in that including active component, the active component includes described in claim 1
Active peptide.
6. the described in any item recombinant vectors of active peptide described in claim 1, claim 2~3, as claimed in claim 4
Antioxidant composition described in recombinant cell or claim 5 improves drug, the reduction of activities of antioxidant enzymes or content in preparation
Application in the drug of content of propylene glycol or the drug of reduction reactive oxygen species content.
7. the described in any item recombinant vectors of active peptide described in claim 1, claim 2~3, as claimed in claim 4
The answering in the drug of preparation prevention or treatment oxidative damage of antioxidant composition described in recombinant cell or claim 5
With.
8. active peptide described in claim 1 is preparing the application in food, health care product or cosmetics.
9. the preparation method of active peptide described in claim 1, which comprises the steps of: divide from stone small water turtle
From obtaining the active peptide；Or
The active peptide is synthesized by chemical synthesis process.
10. the preparation method of active peptide according to claim 9, which is characterized in that described to be separated from stone small water turtle
Include the following steps: to the active peptide
Stone small water turtle meat is digested, zymolyte is obtained；And
The zymolyte is extracted using organic solvent, obtains the active peptide.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630318A (en) * | 2015-01-21 | 2015-05-20 | 华南理工大学 | Preparation method of small water turtle anti-tumor polypeptide |
| CN106399437A (en) * | 2016-09-07 | 2017-02-15 | 深圳凯联龟业有限公司 | Preparation method of mauremys mutica polypeptide and mauremys mutica polypeptide facial mask |
| CN106474453A (en) * | 2016-11-30 | 2017-03-08 | 深圳凯联龟业有限公司 | The application of stone small water turtle mixtures of polypeptides and antineoplastic |
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2018
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630318A (en) * | 2015-01-21 | 2015-05-20 | 华南理工大学 | Preparation method of small water turtle anti-tumor polypeptide |
| CN106399437A (en) * | 2016-09-07 | 2017-02-15 | 深圳凯联龟业有限公司 | Preparation method of mauremys mutica polypeptide and mauremys mutica polypeptide facial mask |
| CN106474453A (en) * | 2016-11-30 | 2017-03-08 | 深圳凯联龟业有限公司 | The application of stone small water turtle mixtures of polypeptides and antineoplastic |
Non-Patent Citations (2)
| Title |
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| HE S等: "Separation and nanoencapsulation of antitumor peptides from Chinese three-striped box turtle (Cuora trifasciata)", 《J MICROENCAPSUL》 * |
| 王艳梅 等: "黄缘盒龟肉的酶解工艺优化及其体外抗氧化活性研究", 《水产学报》 * |
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