CN107325154B - Polypeptide with memory improving effect and separation and preparation method and application thereof - Google Patents
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Images
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 polypeptide with memory improving effect, a separation preparation method and application thereof, wherein the amino acid sequence of the polypeptide is Tyr-Ser-Gly-Val-Cys, and the separation preparation method comprises the following steps: adding mixed enzyme into anchovy for enzymolysis to obtain enzymolysis liquid, eluting the enzymolysis liquid on a Sephadex G-25 gel column, collecting each elution peak and determining the influence of each elution peak on the survival rate of damaged PC12 cells, further purifying the component with the highest survival rate by adopting reverse-phase high performance liquid chromatography, collecting each elution peak and determining the influence of each elution peak on the survival rate of damaged PC12 cells, wherein the component with the highest survival rate is the polypeptide. The invention adopts the biological enzymolysis technology and the chromatographic separation technology to prepare the protein peptide with the function of obviously improving the memory, the preparation method is simple and feasible, and the prepared product has high purity.
Description
Technical Field
The invention belongs to the field of protein deep processing, and particularly relates to a polypeptide with a memory improving effect, and a separation preparation method and application thereof.
Background
Studies have shown that Alzheimer's Disease (AD) is an age-related neurodegenerative disease that leads to the gradual loss of cognitive function in people during their aging. To date, researchers have found that the pathology of human degenerative diseases is very complex. In many cases, the study of the protective effect on nerve cells has been the breakthrough point for people to prevent, improve or treat neurodegenerative diseases.
The nerve cells in the brain of a human body are damaged by the aggravation of oxidative stress or the functional aging of the human body, so that the cell activity is reduced. Therefore, the nutrition component is provided for nerve cells, or functional active components are added, so that the antioxidant and anti-inflammatory capabilities of the nerve cells are improved, the cell survival rate is enhanced, and the method has important significance for improving the cognitive ability.
In addition, some food-derived active ingredients have become potential targets for preventing and/or treating neurodegenerative diseases and depression. Especially for senile dementia, the adverse factors of drug intervention treatment can be avoided by daily diet treatment. In addition, peptide substances derived from food proteins are gradually the hot spots of research because of the advantages of stable sources, low price, simple preparation process, no side effects and the like.
Disclosure of Invention
The invention aims to provide a polypeptide with memory improving effect, wherein the amino acid sequence from C end to N end is Tyr-Ser-Gly-Val-Cys.
Another objective of the invention is to provide a method for separating and preparing the polypeptide.
It is a further object of the present invention to provide uses of the above polypeptides.
The purpose of the invention is realized by the following technical scheme:
the amino acid sequence from the C end to the N end of the polypeptide is Tyr-Ser-Gly-Val-Cys;
the molecular weight of the polypeptide is 527.20 Da.
The method for separating and preparing the polypeptide comprises the following steps:
(1) removing the head and the internal organs of the anchovy, mincing the anchovy into minced meat, adding water for mixing, adding mixed enzyme accounting for 0.5-1.2% of the mass of the minced meat, carrying out enzymolysis at 45-60 ℃ for 6-10 h, then carrying out enzyme deactivation, cooling to room temperature, centrifuging, and filtering to collect filtrate, thus obtaining anchovy enzymolysis liquid;
adding water for mixing in the step (1), wherein the mass ratio of water to meat paste is (1-3) to 1;
the mixed enzyme in the step (1) consists of papain and alkaline protease; wherein, the papain accounts for 0.2-0.5% of the mass of the meat paste, and the alkaline protease accounts for 0.3-0.7% of the mass of the meat paste; the alkaline protease is preferably Alcalase 2.4L;
the enzyme deactivation in the step (1) is to heat reactants for 15min at 95 ℃;
the centrifugation in the step (1) is preferably performed for 10min at the rotating speed of 3500 r/min;
(2) adding the anchovy enzymatic hydrolysate into a Sephadex G-25 gel column, eluting with deionized water at a flow rate of 0.5-1.5 mL/min, detecting the wavelength of 220nm, collecting each elution peak, determining the influence of each elution peak on the survival rate of damaged PC12 cells, and selecting a component corresponding to the elution peak which enables the highest survival rate of the damaged PC12 cells for next separation;
(3) further purifying the target component selected in the step (2) by adopting reverse-phase high performance liquid chromatography, collecting each elution peak and determining the influence of each elution peak on the survival rate of damaged PC12 cells, so that the elution component with the highest survival rate of damaged PC12 cells is the polypeptide of the invention, and the amino acid sequence from the C end to the N end is Tyr-Ser-Gly-Val-Cys;
the reversed phase high performance liquid chromatography in the step (3) preferably has the following parameters: waters e2695HPLC, 2998PDA detector, column XbridgeTMPrep BEH 130C 18 column (10X 150mm,5 μm, Waters, USA), mobile phase A and B; phase A is trifluoroacetic acid ultrapure water solution with mass fraction of 0.1%, phase B is methanol;
the elution procedure was: the volume ratio of A to B in the mobile phase adopted within 0-1min is (95:5) - (90: 10); the volume ratio of A to B in the mobile phase adopted within 1-35min is (95:5) - (60:40) or (90:10) - (70: 30); the volume ratio of A to B in the mobile phase adopted within 35-36min is 60: 40; the volume ratio of A to B in the mobile phase adopted within 36-40min is (60:40) - (95:5) or (60:40) - (90:10), the flow rate is 1mL/min, and the detection wavelength is 220 nm.
The method for determining the survival rate of the damaged PC12 cells in the steps (2) and (3) is as follows:
PC12 cell (rat adrenal medulla chromaffin tumor differentiated cell strain) is cultured by adopting PRIM 1640 culture medium, adding 5% fetal calf serum and 5% horse serum and double antibody into the culture medium, and culturing at 37 deg.C and 5% CO2Culturing under the environment; the cells after passage were plated in 96-well plates at a cell density of 1.0X 105After culturing for 24h, adding 2mL of eluent to culture for 24h, and adding 30mM glutamic acid to damage each well for 24 h; after the injury time is over, adding 0.5mg/mL MTT, continuing to culture for 4h, removing the culture medium, adding 150 mu L DMSO, and measuring the light absorption value at 550 nm; the cell viability was calculated as:
cell survival rate (%) ═ asample－Ablank)/(Acontrol－Ablank)×100％
Wherein A issample、Ablank、AcontrolRespectively represent the absorbance of the sample, the PBS blank and the normal control under the detection of the wavelength of 550 nm.
The above assay method is also called MTT method, and is a method for detecting cell survival and growth. The detection principle is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT into water-insoluble blue-purple crystalline Formazan (Formazan) and deposit the blue-purple crystalline Formazan in the cells, and dead cells do not have the function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and its light absorption value is measured by ELISA, which can indirectly reflect living cell number. Within a certain range of cell number, MTT crystals are formed in an amount proportional to the cell number.
The polypeptide can be used for preparing medicines, health-care products or foods with the effect of improving memory.
Compared with the prior art, the invention has the following advantages and effects:
(1) the raw material used by the polypeptide is anchovy, the source is wide, the price is low, the anchovy is mostly used for manufacturing canned fish at present, and the anchovy is developed into the memory improving peptide by taking the anchovy as the raw material, so that the additional value of the anchovy can be obviously improved.
(2) The invention adopts the biological enzymolysis technology and the chromatographic separation technology to prepare the protein peptide with the function of obviously improving the memory, the preparation method is simple and feasible, and the prepared product has high purity.
(3) The memory improving peptide provided by the invention has excellent bioactivity and good nerve cell protection effect, and can be used as a functional component for a health-care product.
(4) The memory improving peptide provided by the invention is a pentapeptide product, has small peptide molecular weight and can be directly absorbed by a human body.
(5) The pentapeptide product obtained by the invention is chemically synthesized and is verified to have the effect of protecting nerve cells through experiments.
Drawings
FIG. 1 is a Sephadex G-25 gel chromatographic separation elution curve of anchovy enzymolysis products.
FIG. 2 shows the effect of Sephadex G-25 gel chromatography elution fraction on the survival rate of damaged PC12 cells.
FIG. 3 is a reversed phase HPLC separation elution profile of a target collection fraction of gel chromatography.
FIG. 4 reversed phase high performance liquid chromatography elution fraction effects on viability of damaged PC12 cells.
FIG. 5 is a diagram of amino acid sequence mass spectrometry of the improved memory peptide.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Examples
A polypeptide for improving memory is prepared by the following steps:
(1) removing the head and the viscera of the anchovy, mincing the anchovy into minced meat by a meat mincer, adding water which is 2 times of the mass of the minced meat, adding Alcalase 2.4L which is 0.25 percent of papain and 0.25 percent of the mass of the minced anchovy meat, carrying out heat preservation and hydrolysis for 7h at 55 ℃, heating for 15min at the temperature of 95 ℃ after the enzymolysis is finished, carrying out enzyme deactivation, cooling to the room temperature, centrifuging for 10min at the rotating speed of 3500r/min, filtering and collecting filtrate to obtain anchovy enzymatic hydrolysate;
(2) separating and purifying the anchovy enzymolysis liquid by adopting a Sephadex G-25 gel column, eluting with deionized water at the flow rate of 0.5mL/min, detecting the wavelength of 220nm, collecting 6 elution peaks in total as shown in figure 1, determining the influence of each elution peak on the survival rate of damaged PC12 cells (figure 2), finding that the Fr.4 component has the highest activity, selecting the Fr.4 component to enter a reversed phase high performance liquid chromatography (Waters e2695HPLC, 2998PDA detector) for further purification, and adopting an Xbridge chromatographic column as the chromatographic columnTMPrep BEH 130C 18 column (10X 150mm,5 μm, Waters, USA), mobile phase A (0.1% trifluoroacetic acid ultrapure water solution) and B phase (acetonitrile), elution program: the ratio of A to B is 90:10 (volume ratio, the same below) within 0-1min, the ratio of A to B is 90:10-70:30 within 1-35min, the ratio of A to B is 70:30 within 35-36min, the ratio of A to B is 70:30-90:10 within 36-40min, the flow rate is 1mL/min, the detection wavelength is 220nm, the elution curve is shown in figure 3, 10 peaks are collected in total, the activity of the component F5 is the highest by measuring the influence of each elution peak on the survival rate of damaged PC12 cells (figure 4), and the improved memory peptide is obtained by collecting the component F5.
(3) And finally, carrying out sequence analysis on the component F5 by adopting an electrospray tandem mass spectrometry (figure 5), and determining that the amino acid sequence from the C end to the N end of the memory improving peptide is Tyr-Ser-Gly-Val-Cys.
The nerve cell protection effect experiment of the polypeptide of the invention is as follows:
PC12 cell (rat adrenal medulla chromaffin tumor differentiated cell strain) is cultured by adopting PRIM 1640 culture medium, adding 5% fetal calf serum and 5% horse serum and double antibody into the culture medium, and culturing at 37 deg.C and 5% CO2Culturing under the environment; the cells after passage were plated in 96-well plates at a cell density of 1.0X 105After culturing for 24h, adding 2mL of eluent into each hole, continuing culturing for 24h, and adding 30mM glutamic acid for damage for 24 h; after the injury time is over, adding 0.5mg/mL MTT, continuing to culture for 4h, removing the culture medium, adding 150 mu L DMSO, and measuring the light absorption value at 550 nm; the cell viability was calculated as:
cell survival rate (%) ═ asample－Ablank)/(Acontrol－Ablank)×100％
Wherein A issample，Ablank，AcontrolRespectively represent the absorbance of the sample, the PBS blank and the normal control under the detection of wavelength of 550 nm.
The effect of the improved memory peptide and the brain active substance (Cerebrolysin) prepared by the present invention on the survival rate of the damaged PC12 cells is shown in Table 1.
TABLE 1 Effect of improving memory peptide and Naphthokinin on injured PC12 cells
Note: the concentration of the memory improving peptide and the concentration of the brain activity are both 1 mg/mL.
It has been shown in the literature that the memory decline due to human functional aging is mostly related to the damage and apoptosis of nerve cells. As can be seen from Table 1, the memory improving peptide has excellent nerve cell protection effect, has the potential effects of improving the neurotransmitter disorder state in the brain and reducing the apoptosis rate of nerve cells, and can be used in the industries of medicines, health products, foods and the like.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (6)
1. A polypeptide, characterized by: the amino acid sequence from the C end to the N end is Tyr-Ser-Gly-Val-Cys.
2. A method for the isolation of a polypeptide according to claim 1, comprising the steps of:
(1) removing the head and the internal organs of the anchovies, mincing the anchovies into minced meat, adding water which is 2 times of the mass of the minced meat, adding Alcalase 2.4L which is 0.25 percent of papain and 0.25 percent of the mass of the minced anchovies, carrying out heat preservation hydrolysis for 7h at 55 ℃, heating the anchovies at the temperature of 95 ℃ for 15min after the enzymolysis is finished, carrying out enzyme deactivation, cooling the anchovies to room temperature, centrifuging, filtering and collecting filtrate to obtain an anchovies enzymolysis solution;
(2) adding the anchovy enzymatic hydrolysate into a Sephadex G-25 gel column, eluting with deionized water at a flow rate of 0.5mL/min, detecting the wavelength of 220nm, collecting each elution peak, determining the influence of each elution peak on the survival rate of damaged PC12 cells, and selecting the component with the highest survival rate of damaged PC12 cells for next separation;
(3) further purifying the target component selected in the step (2) by adopting reverse-phase high performance liquid chromatography, collecting each elution peak and determining the influence of each elution peak on the survival rate of damaged PC12 cells, so that the component with the highest survival rate of damaged PC12 cells is the polypeptide of claim 1, wherein the amino acid sequence from the C end to the N end is Tyr-Ser-Gly-Val-Cys;
the damaged PC12 cells are glutamic acid damaged PC12 cells.
3. The separation preparation method according to claim 2, characterized in that: the enzyme deactivation in the step (1) is to heat the reactant at 95 ℃ for 15 min.
4. The separation preparation method according to claim 2, characterized in that: the centrifugation in the step (1) is performed for 10min at the rotating speed of 3500 r/min.
5. The separation preparation method according to claim 2, characterized in that:
the reversed phase high performance liquid chromatography in the step (3) preferably has the following parameters: waters e2695HPLC, 2998PDA detector, column XbridgeTMA Prep BEH 130C 18 column, and the mobile phase is A phase and B phase; phase A is trifluoroacetic acid ultrapure water solution with mass fraction of 0.1%, phase B is methanol;
the elution procedure was: the volume ratio of A to B is 90:10 within 0-1min, the volume ratio of A to B is 90:10-70:30 within 1-35min, the volume ratio of A to B is 70:30 within 35-36min, the volume ratio of A to B is 70:30-90:10 within 36-40min, the flow rate is 1mL/min, and the detection wavelength is 220 nm.
6. The use of the polypeptide of claim 1 for the preparation of a medicament, health product or food for improving memory.
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| Publication number | Priority date | Publication date | Assignee | Title |
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