CN107325154A - It is a kind of that there is the polypeptide and its method for separating and preparing and purposes for improving memory effect - Google Patents
It is a kind of that there is the polypeptide and its method for separating and preparing and purposes for improving memory effect Download PDFInfo
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- CN107325154A CN107325154A CN201710484352.2A CN201710484352A CN107325154A CN 107325154 A CN107325154 A CN 107325154A CN 201710484352 A CN201710484352 A CN 201710484352A CN 107325154 A CN107325154 A CN 107325154A
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 37
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 22
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 title claims abstract description 16
- 230000003446 memory effect Effects 0.000 title claims abstract description 9
- 230000004083 survival effect Effects 0.000 claims abstract description 24
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- 238000004366 reverse phase liquid chromatography Methods 0.000 claims abstract description 8
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- 238000001514 detection method Methods 0.000 claims description 7
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- 108091005804 Peptidases Proteins 0.000 claims description 5
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 5
- 239000003513 alkali Substances 0.000 claims description 5
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- 239000003643 water by type Substances 0.000 claims description 5
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- 238000005119 centrifugation Methods 0.000 claims description 4
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- 230000036541 health Effects 0.000 claims description 4
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- 238000001914 filtration Methods 0.000 claims description 3
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- 239000012498 ultrapure water Substances 0.000 claims description 3
- 210000001835 viscera Anatomy 0.000 claims description 3
- 239000000376 reactant Substances 0.000 claims description 2
- QEWYKACRFQMRMB-UHFFFAOYSA-N fluoroacetic acid Chemical compound OC(=O)CF QEWYKACRFQMRMB-UHFFFAOYSA-N 0.000 claims 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims 1
- 244000189799 Asimina triloba Species 0.000 claims 1
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- 235000009467 Carica papaya Nutrition 0.000 claims 1
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- 108010000912 Egg Proteins Proteins 0.000 claims 1
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- 108010037850 glycylvaline Proteins 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 31
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 10
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- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
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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
-
- 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
Abstract
The invention discloses a kind of polypeptide and its method for separating and preparing and purposes for having and improving memory effect, the amino acid sequence of the polypeptide is Tyr Ser Gly Val Cys, and its method for separating and preparing comprises the following steps：Enzymolysis liquid will be obtained after long tail anchovy plus mixed enzyme enzymolysis, the gel columns of Sephadex G 25 are eluted on enzymolysis liquid, collect each eluting peak and determine its influence to being damaged PC12 cell survival rates, survival rate highest component is further purified using RPLC, collect each eluting peak and determine its influence to being damaged PC12 cell survival rates, survival rate highest component is the polypeptide of the present invention.The present invention prepares a kind of protein peptides for having and being obviously improved memory effect using biological enzymolysis technology and chromatographic separation technology, and preparation method simple possible, obtained product purity is high.
Description
Technical field
The invention belongs to protein intensive processing field, and in particular to a kind of to have the polypeptide for improving memory effect and its divide
From preparation method and purposes.
Background technology
Research shows that Alzheimer's disease (Alzheimer ' s diseases, AD) is that a kind of age-related nerve is moved back
Row disease, it can cause people during the age increases, gradually lose cognitive function.Up to the present, research work
Persons have found that the pathology of mankind's degenerative disease is extremely complex.In numerous cases, the protective effect for nerve cell is studied
Turn into people to prevent, improve or treat the break-through point of nerve degenerative diseases.
Oxidative stress aggravates or function of human body aging can damage nerve cell in human brain, so as to reduce cell work
Power.Therefore, nutritional ingredient, or addition functional active components are provided for nerve cell, so as to improve the anti-oxidant, anti-of nerve cell
Inflammation ability, strengthens cell survival rate, has very important significance for improving, improving cognitive ability tool.
Prevent and/or the treatment disease such as nerve degenerative diseases and depression in addition, part food source active component has turned into
The potential target of disease.Especially for senile dementia, diet treatment is more avoided that the unfavorable factor of pharmaceutical intervention treatment.
In addition, the peptide matters in food proteins source, because have steady sources, cheap, preparation technology simply, have no side effect etc. it is excellent
Point, is increasingly becoming the focus of people's research.
The content of the invention
The primary and foremost purpose of the present invention, which is that offer is a kind of, has the polypeptide for improving memory effect, and its amino acid sequence is Tyr-
Ser-Gly-Val-Cys。
Another object of the present invention is to provide the method for separating and preparing of aforementioned polypeptides.
It is still another object of the present invention to provide the purposes of aforementioned polypeptides.
The purpose of the present invention is achieved through the following technical solutions：
A kind of polypeptide, its amino acid sequence is Tyr-Ser-Gly-Val-Cys；
Described polypeptide molecular weight is 527.20Da.
The method for separating and preparing of aforementioned polypeptides, comprises the following steps：
(1) internal organ are removed into long tail anchovy decaptitating, is twisted into meat gruel, add water mixing, add and account for the mixed of meat gruel quality 0.5~1.2%
Synthase, is digested after 6~10h, go out enzyme, is cooled to after room temperature at 45~60 DEG C, and centrifugation is collected by filtration filtrate, is long tail anchovy enzyme
Solve liquid；
The mass ratio of the mixing that adds water described in step (1), water and meat gruel is (1~3):1；
Mixed enzyme described in step (1), is made up of papain and alkali protease；Wherein, papain accounts for meat
The 0.2~0.5% of rotten quality, alkali protease accounts for the 0.3~0.7% of meat gruel quality；Described alkali protease is preferred
Alcalase 2.4L；
The enzyme that goes out described in step (1) is that reactant is heated into 15min at 95 DEG C；
Centrifugation described in step (1) preferably centrifuges 10min under 3500r/min rotating speeds；
(2) long tail anchovy enzymolysis liquid is added to Sephadex G-25 gel columns, with deionized water with 0.5~1.5mL/min
Flow velocity eluted, Detection wavelength is 220nm, collects each eluting peak and simultaneously determines its to the shadow that is damaged PC12 cell survival rates
Ring, choose and cause the component corresponding to impaired PC12 cell survival rate highest eluting peaks to carry out next step separation；
(3) target components chosen using RPLC to step (2) are further purified, and collect each eluting peak
And determining its influence to being damaged PC12 cell survival rates so that impaired PC12 cell survival rate highest elution fractions are this
The peptide T yr-Ser-Gly-Val-Cys of invention；
RPLC preferably following parameter described in step (3)：Waters e2695HPLC, 2998PDA inspections
Device is surveyed, chromatographic column is XBridgeTMPrep BEH130 C18 posts (10 × 150mm, 5 μm, Waters, USA), mobile phase is A phases
With B phases；A phases are the trifluoroacetic acid ultra-pure water solutions of mass fraction 0.1%, and B phases are methanol；
Elution program is：A and B volume ratios is (95 in the mobile phase used in 0-1min:5)-(90:10)；In 1-35min
A and B volume ratios is (95 in the mobile phase used:5)-(60:Or (90 40):10)-(70:30)；The stream used in 35-36min
A and B volume ratios are 60 in dynamic phase:40；A and B volume ratios is (60 in the mobile phase used in 36-40min:40)-(95:5) or
(60:40)-(90:10), flow velocity is 1mL/min, and Detection wavelength is 220nm.
The assay method of impaired PC12 cell survival rates described in step (2) and (3) is as follows：
PC12 cells (the thermophilic chromium knurl noble cells strain of rat adrenal medulla), which are cultivated, uses the culture mediums of PRIM 1640, and
5% hyclone and 5% horse serum are added in culture medium and dual anti-, in 37 DEG C and 5%CO2Cultivated under environment；After passing on
Cell be laid on 96 orifice plates, cell density is 1.0 × 105Per hole, after culture 24h, add after 2mL eluent cultures 24h and add
30mM glutamic acid is damaged, and trauma time is 24h；After trauma time terminates, 0.5mg/mL MTT are added, continue to cultivate
4h, removes culture medium, adds 150 μ L DMSO, light absorption value is determined at 550 nm；The calculation formula of cell survival rate is：
Cell survival rate (%)=(Asample-Ablank)/(Acontrol-Ablank) × 100%
Wherein, Asample、Ablank、AcontrolRepresent respectively under 550nm wavelength detectings, sample, PBS blank and normal right
According to the light absorption value of group.
Said determination method is also known as mtt assay, is a kind of method for detecting cell survival and growth.Its Cleaning Principle is living thin
The bluish violet that succinate dehydrogenase in born of the same parents' mitochondria can make exogenous MTT be reduced to water-insoluble crystallizes first a ceremonial jade-ladle, used in libation (Formazan)
And be deposited in cell, and dead cell is without this function.Dimethyl sulfoxide (DMSO) (DMSO) can dissolve the first a ceremonial jade-ladle, used in libation in cell, use enzyme linked immunological
Detector determines its absorbance value, and living cells quantity can be reflected indirectly.In the range of certain cell number, MTT crystallizes the amount to be formed
It is directly proportional to cell number.
The polypeptide of the present invention can be used for preparing medicine, health products or food with improvement memory effect.
The present invention has the following advantages and effect relative to prior art：
(1) raw material used in polypeptide of the present invention is long tail anchovy, and wide material sources are cheap, and system is used as current long tail anchovy more
Make canned fish, now as raw material, develop into improvement memory peptide, be remarkably improved the added value of long tail anchovy.
(2) present invention using biological enzymolysis technology and chromatographic separation technology prepare it is a kind of have be obviously improved memory effect
Protein peptides, preparation method simple possible, obtained product purity is high.
(3) bioactivity for the improvement memory peptide that the present invention is provided is excellent, is acted on good neurocyte protection, can
Using as functional components, in health products.
(4) the improvement memory peptide that the present invention is provided is five peptide products, and its peptide molecular weight is small, can be directly absorbed by the body.
(5) five peptide products that the present invention is obtained, by chemical synthesis and experiments verify that its neurocyte protection effect.
Brief description of the drawings
Fig. 1 is the Sephadex G-25 gel chromatography separation elution curves of long tail anchovy enzymolysis product.
Fig. 2 is survival of the long tail anchovy enzymolysis product Sephadex G-25 gel chromatographies elution fractions to impaired PC12 cells
Rate influences.
Fig. 3 separates elution curve for the RPLC of gel chromatography target collection component.
Fig. 4 RPLCs separate survival rate of the elution fraction to impaired PC12 cells.
Fig. 5 remembers the amino acid sequence mass spectral analysis figure of peptide to improve.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited
In this.
Embodiment
It is a kind of to improve the polypeptide of memory, it is made by following steps：
(1) internal organ are removed into long tail anchovy decaptitating, crosses meat grinder and be twisted into meat gruel, add the water of 2 times of meat gruel quality, add long tail anchovy
The Alcalase 2.4L of the papain 0.25% of meat gruel quality 0.25%, the insulation hydrolysis 7h at 55 DEG C, after enzymolysis terminates
15min is heated at a temperature of 95 DEG C and carries out the enzyme that goes out, is cooled to after room temperature, 10min is centrifuged under 3500r/min rotating speeds, filtering is received
Collect filtrate, obtain long tail anchovy enzymolysis liquid；
(2) long tail anchovy enzymolysis liquid is isolated and purified using Sephadex G-25 gel columns, with deionized water with
0.5mL/min flow velocity is eluted, and Detection wavelength is 220nm, elution curve such as Fig. 1, and 6 eluting peaks are collected into altogether, are passed through
Influence (Fig. 2) of each eluting peak to impaired PC12 cell survival rates is determined, the active highests of component Fr.4 is found, chooses Fr.4 components
It is further purified into RPLC (Waters e2695HPLC, 2998PDA detector), chromatographic column is
XBridgeTMPrep BEH130 C18 posts (10 × 150mm, 5 μm, Waters, USA), mobile phase is A phases (0.1% trifluoro
Acetic acid ultra-pure water solution) and B phases (acetonitrile), elution program is：A in 0-1min:B is 90:10 (volume ratios, similarly hereinafter), 1-35min
Interior A:B is 90:10-70:A in 30,35-36min:B is 70:A in 30,36-40min:B is 70:30-90:10, flow velocity is 1mL/
Min, Detection wavelength is 220nm, and elution curve is shown in Fig. 3, and 10 peaks are collected into altogether, thin to impaired PC12 by determining each eluting peak
The influence (Fig. 4) of born of the same parents' survival rate, finds the active highests of component F5, collects component F5, obtains improving memory peptide.
(3) sequence analysis (Fig. 5) is finally carried out to component F5 using esi-msn, measuring improves the ammonia of memory peptide
Base acid sequence is Tyr-Ser-Gly-Val-Cys.
The neurocyte protection effect experiment of polypeptide of the present invention is as follows：
PC12 cells (the thermophilic chromium knurl noble cells strain of rat adrenal medulla), which are cultivated, uses the culture mediums of PRIM 1640, and
5% hyclone and 5% horse serum are added in culture medium and dual anti-, in 37 DEG C and 5%CO2Cultivated under environment；After passing on
Cell be laid on 96 orifice plates, cell density is 1.0 × 105Per hole, after culture 24h, 2mL eluents are added, continue to cultivate after 24h
Add 30mM glutamic acid to be damaged, trauma time is 24h；After trauma time terminates, 0.5mg/mL MTT are added, continue to train
4h is supported, culture medium is removed, 150 μ L DMSO is added, light absorption value is determined at 550 nm；The calculation formula of cell survival rate is：
Cell survival rate (%)=(Asample-Ablank)/(Acontrol-Ablank) × 100%
Wherein, Asample, Ablank, AcontrolRepresent respectively under 550nm wavelength detectings, sample, PBS blank and normal right
According to the light absorption value of group.
Improvement memory peptide and Cerebrolysin (Cerebrolysin) prepared by the present invention is to impaired PC12 cell survival rates
Influence is shown in Table 1.
Table 1 improves the influence of memory peptide and Cerebrolysin to impaired PC12 cells
Note：It is 1mg/mL to improve memory peptide and Cerebrolysin concentration.
Existing document shows, because memory declines many damages with nerve cell and apoptosis phase caused by function of human body aging
Close.Improvement memory peptide of the invention has outstanding neurocyte protection effect as shown in Table 1, with god in potential improvement brain
Through mediator disturbance state and the effect of nerve cell apoptosis rate is reduced, show that the improvement memory peptide that invention is provided has very strong god
Through cytoprotection function, available in the industries such as medicine, health products and food.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (10)
1. a kind of polypeptide, it is characterised in that amino acid sequence is Tyr-Ser-Gly-Val-Cys.
2. the method for separating and preparing of polypeptide described in claim 1, it is characterised in that comprise the following steps：
(1) internal organ are removed into long tail anchovy decaptitating, are twisted into meat gruel, add water mixing, add the mixed enzyme for accounting for meat gruel quality 0.5~1.2%,
Digested at 45~60 DEG C after 6~10h, go out enzyme, is cooled to after room temperature, centrifugation is collected by filtration filtrate, is long tail anchovy enzymolysis liquid；
(2) long tail anchovy enzymolysis liquid is added to Sephadex G-25 gel columns, with deionized water with 0.5~1.5mL/min stream
Speed is eluted, and Detection wavelength is 220nm, is collected each eluting peak and is determined its influence to being damaged PC12 cell survival rates, choosing
Take so that impaired PC12 cell survival rate highests component carries out next step separation；
(3) target components chosen using RPLC to step (2) are further purified, and are collected each eluting peak and are surveyed
Determine its influence to being damaged PC12 cell survival rates, make the polypeptide that impaired PC12 cell survival rate highest components are the present invention
Tyr-Ser-Gly-Val-Cys。
3. method for separating and preparing according to claim 2, it is characterised in that：Add water mixing, water and meat described in step (1)
Rotten mass ratio is (1~3):1.
4. method for separating and preparing according to claim 2, it is characterised in that：Mixed enzyme described in step (1), by pawpaw egg
White enzyme and alkali protease composition.
5. method for separating and preparing according to claim 4, it is characterised in that：Described papain accounts for meat gruel quality
0.2~0.5%.
6. method for separating and preparing according to claim 4, it is characterised in that：Described alkali protease accounts for meat gruel quality
0.3~0.7%.
7. method for separating and preparing according to claim 2, it is characterised in that：The enzyme that goes out described in step (1) is by reactant
15min is heated at 95 DEG C.
8. method for separating and preparing according to claim 2, it is characterised in that：Centrifugation described in step (1) is in 3500r/
10min is centrifuged under min rotating speeds.
9. method for separating and preparing according to claim 2, it is characterised in that：
RPLC preferably following parameter described in step (3)：Waters e2695 HPLC, 2998 PDA detections
Device, chromatographic column is XBridgeTMPrep BEH130 C18 posts, mobile phase is A phases and B phases；A phases are the three of mass fraction 0.1%
Fluoroacetic acid ultra-pure water solution, B phases are methanol；
Elution program is：A and B volume ratios is (95 in the mobile phase used in 0-1min:5)-(90:10)；Used in 1-35min
Mobile phase in A and B volume ratios be (95:5)-(60:Or (90 40):10)-(70:30)；The mobile phase used in 35-36min
Middle A is 60 with B volume ratios:40；A and B volume ratios is (60 in the mobile phase used in 36-40min:40)-(95:Or (60 5):
40)-(90:10), flow velocity is 1mL/min, and Detection wavelength is 220nm.
10. application of the polypeptide in preparing with the medicine, health products or the food that improve memory effect described in claim 1.
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