CN105380818A - Application of chick embryo bioactive peptide in preparing product for protecting cell activity - Google Patents
Application of chick embryo bioactive peptide in preparing product for protecting cell activity Download PDFInfo
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Abstract
The invention relates to the technical field of biology, and in particular relates to chick embryo bioactive peptide, as well as a preparation method and application thereof. The chick embryo bioactive peptide provided by the invention comprises the chick embryo bioactive peptide and free amino acid, wherein the molecular weight of the chick embryo bioactive peptide is less than 10kDa. The chick embryo bioactive peptide provided by the invention can protect the activity of human fibroblast, and in comparison with a matched group without adding the chick embryo bioactive peptide provided by the invention, the activity of the human fibroblast of an experimental group adding the chick embryo bioactive peptide is protected. Compared with a blank group without ultraviolet radiation, the activity of the human fibroblast of the experimental group is basically equal to, even slight higher than, that of the blank group. The preparation method of the chick embryo bioactive peptide, provided by the invention is simple to operate, the cytomembrane is crushed to the greatest extent through homogenate, freeze thawing and ultrasonication, so that bioactive peptide in a cell is well dissolved out, and then through the biotechnologies of centrifugation, ultrafiltration and the like, the extracting efficiency is improved.
Description
Technical field
The present invention relates to biological technical field, particularly relate to the application of Embryo Gallus domesticus bioactive peptide in the product preparing Cell protection vigor.
Background technology
Skin covers whole body as the organ that human body is maximum, is the protective layer of health, plays an important role to human body.And skin of face can not only resist extraneous antibacterial intrusion health, be also measurement one personal health looks standards of beauty.Common skin of face problem has: microgroove, red blood streak, cicatrix, the colour of skin are obscure.Cause the main cause of these skin problems to be: skin of face exposure year in year out in atmosphere, ultraviolet irradiation, afloat dirt, dust, the natural harmful substance chafe surface of antibacterial in air.Add the oils and fats, perspiration, dead cell etc. self secreted, these factors can affect the performance of skin normal function, cause the vigor of skin of face cell to decline even dead, thus cause a series of skin problem.
The embryo of Embryo Gallus domesticus and chicken is after fertilized eggs hatching to certain natural law, the life entity that also non-broken shell edible is complete.Research shows, the nutritional labeling comprised in Embryo Gallus domesticus comprises protein, polypeptide, aminoacid, carbohydrate (glucose, fructose, galactose, arabinose, furanose etc.), lipid, fatty acid etc.In addition, infection composition and bioactive substance is also comprised in Embryo Gallus domesticus.Described infection composition has globulin, interferon, fetoprotein, lysozyme and phosphodiesterase; Described bioactive substance has sialic acid, youngster's phenol amine, insulin like growth factor-1, insulin, kininase, histaminase, choline, erythropoietin, deoxyribonuclease and ribonuclease etc.Relative to unhatched egg, the content of Embryo Gallus domesticus Middle nutrition composition, particularly small molecular protein or polypeptide is abundanter, and fat, cholesterol equal size then decline to a great extent.
Embryo Gallus domesticus is nutritious, have that potential skin protection is anti-ageing, quench antiallergic, health care and medical value.But also less at present to the research of peptide in Embryo Gallus domesticus at present, tackle it and carry out further development and utilization in cosmetics, the antianaphylactic research of health product quench.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is to provide the application of Embryo Gallus domesticus bioactive peptide in the product preparing Cell protection vigor.The present invention tests proof, and Embryo Gallus domesticus bioactive peptide can promote the growth of cell, maintains the activity of cell.
The invention provides the application of Embryo Gallus domesticus bioactive peptide in the product preparing Cell protection vigor.
In an embodiment of the present invention, cell is human fibroblasts.
In an embodiment of the present invention, the dosage of Embryo Gallus domesticus bioactive peptide Cell protection vigor protection is 250 μ g/mL ~ 500 μ g/mL.
In embodiments of the present invention, the dosage of Embryo Gallus domesticus bioactive peptide Cell protection vigor protection is 250 μ g/mL.
The present invention also provides, the application of Embryo Gallus domesticus bioactive peptide in the cosmetics of preparation; Cosmetics are the cosmetics with the function of repairing red blood streak, whitening, wrinkle removal, the reparation of skin-active cell recovery or going cicatrix.
A kind of cosmetics, comprise Embryo Gallus domesticus bioactive peptide, hyaluronic acid, xanthan gum, Jojoba oil, arbutin and glycerol.
Wherein, the concentration of Embryo Gallus domesticus bioactive peptide is 0.5mg/mL ~ 3.0mg/mL, and hyaluronic mass fraction is 0.3%, xanthan gum mass fraction 0.3%, Jojoba oil mass fraction 1%, arbutin mass fraction 0.5%, the mass fraction of glycerol is 3%, and surplus is deionized water.
Human fibroblasts is the cell of a kind of mesoderma origin be prevalent in connective tissue, is positioned at skin corium.The extracellular matrix components such as collagen, fibronectin and collagenase can be secreted.Problems such as promoting fibroblastic activity that skin can be made to recover elasticity, improve microgroove, red blood streak, cicatrix, the colour of skin are obscure.
The present invention confirms through test, and Embryo Gallus domesticus bioactive peptide provided by the invention can protect the activity of human fibroblasts, and compared with not adding the matched group of Embryo Gallus domesticus bioactive peptide of the present invention, the fibroblastic activity of experimental group of adding Embryo Gallus domesticus bioactive peptide is protected.Therefore, Embryo Gallus domesticus bioactive peptide can as improving microgroove, the colour of skin is obscure, removes the cosmetics of red blood streak, cicatrix.
In the Embryo Gallus domesticus bioactive peptide that the present invention obtains, bioactive peptide 300 detected altogether, respectively from 69 kinds of different protein.According to PSMs (Peptide-SpectralMatch), these protein are mainly: Ovalbumin (accession number P01012), ApolipoproteinB (accession number Q197X2), Ovotransferrin (accession number E1BQC2), HistoneH3.2 (accession number P84229), Apovitellenin-1 (accession number P02659), OvoglobulinG2typeAB (accession number I0J174), OvoglobulinG2 (accession number I0J170), Vitellogenin-2 (accession number P02845), Alpha-1-acidglycoprotein (accession number A7UEB0) and HistoneH4 (accession number P62801).
In Embryo Gallus domesticus bioactive peptide of the present invention, the concentration of Embryo Gallus domesticus bioactive peptide is 0.1g/ μ L.
The present invention extracts the Embryo Gallus domesticus bioactive peptide obtained and comprises 18 kinds of free amino acids, comprising 7 kinds of essential amino acids, is respectively threonine, valine, isoleucine, leucine, phenylalanine, lysine and tryptophan.Also comprise 11 kinds of non essential amino acid in addition, be respectively aspartic acid, serine, glutamic acid, glycine, alanine, methionine, tyrosine, histidine, arginine, cystine and proline.
In Embryo Gallus domesticus bioactive peptide of the present invention, the concentration of free amino acid is 14.3mg/mL.
The preparation method of Embryo Gallus domesticus bioactive peptide provided by the invention comprises: shelled by Embryo Gallus domesticus, after homogenate, smudge cells; After pancreatin enzymolysis, collected by centrifugation supernatant, ultrafiltration molecular cut off is less than the component of 10kDa.
Embryo Gallus domesticus of the present invention is the Embryo Gallus domesticus of hatching to 14 ~ 19 days.
Concrete, the obtain manner of Embryo Gallus domesticus is, gets SPF level fertilized eggs, puts into incubator and hatch, and incubation temperature controls at 37.5 DEG C, relative humidity 60%, and every 2 hours automatic egg turnings once, treat that hatching was to 14 ~ 19 days,
As preferably, adopt the Embryo Gallus domesticus of hatching to 16 ~ 17 days.
The embryo of the Embryo Gallus domesticus of fertilization hatching to 14 day ~ 19 days is in the stem cell height rise period, and various organ basically forms.In the hatching process of Embryo Gallus domesticus, the metabolism of protein is very violent, and between the incubation period, the content of proteins and peptides is in continuing propradation.Research shows, at the 14th day ~ 19 days of hatching, in Embryo Gallus domesticus, the increasing degree of proteins and peptides was maximum, and the content of free amino acid also reaches the highest during this period.
Before preparing Embryo Gallus domesticus polypeptide, hatching chicken embryo stops hatching.Be specially, hatching chicken embryo be positioned over-20 DEG C and stop hatching.
In the present invention, described homogenate adopts tissue refiner.
Be specially: the egg surface sterilization to 14d ~ 19d will be hatched, with tissue refiner homogenate after removal eggshell.As preferably, the temperature of homogenate is 4 DEG C, and the time is 20min rotating speed is 2500r/min.
Through tissue homogenate, chicken embryo tissue is pulverized, and cell is preliminary broken under the mechanism of refiner.In order to further smudge cells, carry out freeze thawing and ultrasonication.
In embodiments of the present invention, smudge cells comprises the step of freeze thawing and ultrasonication;
The cryogenic temperature of freeze thawing is-20 DEG C ~-40 DEG C, and the time is 60h ~ 80h; Solution temperature is 4 DEG C ~ 8 DEG C;
The temperature of ultrasonication is 4 DEG C ~ 8 DEG C, and power is 800W ~ 1200W, and the time is 15min ~ 25min.
In certain embodiments, smudge cells comprises the step of freeze thawing and ultrasonication;
The cryogenic temperature of freeze thawing is-30 DEG C, and the time is 72h; Solution temperature is 4 DEG C;
The temperature of ultrasonication is 4 DEG C, and power is 1000W, and the time is 20min.
Freeze thawing makes cell in the process of freeze thaw, makes the salinity of residue cytosol increase and cause cell breakage owing to forming ice pellets in cell.In the present invention, the temperature of dissolving is 4 DEG C, is dissolved to cell homogenates and melts.After freeze thawing, cell membrane further breaks, then carries out ultrasonic disruption, thus makes the degree of crushing of cell membrane more complete, improves the extraction efficiency of bioactive peptide.
In the present invention, after the tissue homogenate of ultrasonication mixes with isopyknic water, enzymolysis is carried out.
Described enzymolysis refers to jointly hatch at tissue homogenate and pancreatin, makes protein that the process of decomposing occur.
In the present invention, during enzymolysis, the mass fraction of pancreatin is 2.5%.Adding tissue homogenate to mass fraction by pancreatin is 2.5%.
In the present invention, the enzyme activity of pancreatin is 37000u/g.
In the present invention, the temperature of enzymolysis is 45 DEG C ~ 55 DEG C, and pH value is 7.3 ~ 7.7, and enzymolysis time is 40min ~ 80min.
In certain embodiments, the temperature of enzymolysis is 50 DEG C, and pH value is 7.5, and enzymolysis time is 1h.
After centrifugal, reject fragment of tissue, collects supernatant.Rich in proteins, bioactive peptide and free amino acid in this supernatant.
In an embodiment of the present invention, centrifugal rotating speed is 4500r/min, and the centrifugal time is 20min, and centrifugal temperature is 4 DEG C.
Ultrafiltration retains the filter membrane of specifically instigating supernatant through 10kDa, collects the ultrafiltrate within 10kDa.
Those skilled in the art can also carry out lyophilization according to known method to the component that tissue homogenate, supernatant and ultrafiltration are collected.
As preferably, after retaining, also comprise the step that adjust ph is 6.8 ~ 7.2.
In the scope that pH value is 6.8 ~ 7.2, the stable in properties of Embryo Gallus domesticus bioactive peptide, activity is higher.
Described lyophilization is that Embryo Gallus domesticus bioactive peptide mixes with lyophilized preparation, obtained by freeze drying lyophilized powder.Described lyophilization excipient is mannitol.Concrete, adding mannitol to mass fraction in the component that tissue homogenate, supernatant and ultrafiltration are collected is 8% carry out lyophilization.Preferably, adding mannitol to mass fraction in the component of collecting in ultrafiltration is 8% carry out lyophilization.
The invention provides the application of Embryo Gallus domesticus bioactive peptide in the product preparing Cell protection vigor.Embryo Gallus domesticus bioactive peptide of the present invention comprises Embryo Gallus domesticus bioactive peptide and free amino acid; Wherein, the molecular weight of Embryo Gallus domesticus bioactive peptide is less than 10kDa.The present invention tests confirmation, and Embryo Gallus domesticus bioactive peptide can protect the activity of human fibroblasts, and compared with not adding the matched group of Embryo Gallus domesticus bioactive peptide of the present invention, the activity of adding the experimental group human fibroblasts of Embryo Gallus domesticus bioactive peptide is protected.Therefore, Embryo Gallus domesticus bioactive peptide can as improving microgroove, the colour of skin is obscure, remove red blood streak, whitening, wrinkle removal, the reparation of skin-active cell recovery or remove the cosmetics of function of cicatrix.
Accompanying drawing explanation
Fig. 1 shows that embodiment 1 obtains the protective effect of Embryo Gallus domesticus bioactive peptide to human fibroblasts.
Detailed description of the invention
The invention provides and provide the application of Embryo Gallus domesticus bioactive peptide in the product preparing Cell protection vigor, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can change methods and applications herein or suitably changes and combination not departing from content of the present invention, spirit and scope, realizes and applies the technology of the present invention.
The material that the present invention adopts, instrument or reagent are all common commercially available product, all can buy in market.
Below in conjunction with embodiment, set forth the present invention further:
Embodiment 1
(1) hatching chicken embryo: get SPF level fertilized eggs, put into incubator and hatch, incubation temperature controls at 37.5 DEG C, relative humidity 60%, every 2 hours automatic egg turnings once, treat that hatching was to 16 days, take out fertilized eggs, put in the freezer of-20 DEG C, stop hatching and saving backup.
(2) homogenate and cell breakage: surface sterilization is carried out to the egg of hatching, remove eggshell and take out Embryo Gallus domesticus, at 4 DEG C by Embryo Gallus domesticus with after tissue refiner homogenate, pour in the seal box after sterilizing, to be put in the freezer of-30 DEG C freezing 72 hours, in the environment of 4 DEG C, cell ultrasonication is carried out to it after melting, add the water for injection of equivalent, be positioned in the freezer of 4 DEG C for subsequent use.
(3) enzyme hydrolysis and centrifugal: get the cold preservation liquid heated water bath to 50 DEG C after cell breakage, adjust pH to 7.5, adding pancreatin to concentration is that 2.5% (mass fraction) is hydrolyzed water bath with thermostatic control 1 hour, at 4 DEG C, under 4500r/min condition centrifugal 20 minutes, receive supernatant to be cooled to 4 DEG C of cold preservations for subsequent use.
(4) ultrafiltration and tune pH value: by the filter membrane of Embryo Gallus domesticus serosity ultra filtration 10kDa, collect the ultrafiltrate within 10kDa, adjust pH to 6.8 ~ 7.2, obtained Embryo Gallus domesticus bioactive peptide.
Bioactive peptide is analyzed through shotgun, and the content of bioactive peptide is 0.1 μ g/ μ L; Peptide hop count amount amounts to 300, and respectively from 70 kinds of different albumen, concrete outcome is as table 1:
Table 1shotgun analysis result
The method adopting GB/T5009.124-2003 to provide, utilizes L-8800 type automatic amino acid analyzer, detects Embryo Gallus domesticus bioactive peptide Free Amino Acids.Result is:
Table 2 free amino acid detects
Embodiment 2
Utilize MTT colorimetric method for determining cell survival rate.Its Cleaning Principle is that the succinate dehydrogenase in living cells mitochondrion can make exogenous MTT be reduced to water-insoluble bluish violet crystallization first a ceremonial jade-ladle, used in libation (Formazan) and be deposited in cell, and dead cell is without this function.First a ceremonial jade-ladle, used in libation in dimethyl sulfoxide (DMSO) energy dissolved cell, measures its absorbance value with enzyme-linked immunosorbent assay instrument at 550nm wavelength place, can indirectly reflect living cells quantity.Within the scope of certain cell number, the amount that MTT crystallization is formed is directly proportional to viable count.
Mtt assay measures cell viability:
By human fibroblasts (HDF cell) (the 6th generation) with 1 × 10
4individual/hole is inoculated in 96 orifice plates;
Experiment is divided into experimental group, blank group;
After 24 hours, the Embryo Gallus domesticus bioactive peptide (20,50,100,250,500 μ g/mL) that the embodiment 1 that experimental group gives variable concentrations provides processes cell; Blank group does not add bioactive peptide.Continue cultivation 48 hours.Before cultivation terminates, 4 hours every holes add 20 LMTT20 LMTT (5 μ g/mL).The purple crystal DMSO produced dissolves, under 550nm wavelength condition, measure absorbance value.With the light absorption value of blank for 100%, calculate the activity of the relative blank group cell of each group of cell.Result is as Fig. 1.
Result shows, when the dosage of Embryo Gallus domesticus bioactive peptide is 20 ~ 100 μ g/mL, fibroblastic activity and blank group are without significant difference (p>0.05); And when the dosage of Embryo Gallus domesticus bioactive peptide is 250 ~ 500 μ g/mL, the remarkable rising (p<0.05) compared with blank of fibroblastic activity, increased proportion is respectively 20% and 40%.These results suggest that, Embryo Gallus domesticus bioactive peptide can promote fibroblastic activity, all effective in 250 ~ 500 μ g/mL dosage ranges.
Below be only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. the application of Embryo Gallus domesticus bioactive peptide in the product preparing Cell protection vigor.
2. application according to claim 1, is characterized in that, described cell is human fibroblasts.
3. application according to claim 1, is characterized in that, the dosage of described Embryo Gallus domesticus bioactive peptide Cell protection vigor protection is 250 μ g/mL ~ 500 μ g/mL.
4. application according to claim 1, is characterized in that, described Embryo Gallus domesticus bioactive peptide comprises Embryo Gallus domesticus bioactive peptide and free amino acid; Wherein, the molecular weight of described Embryo Gallus domesticus bioactive peptide is less than 10kDa.
5. application according to claim 1, is characterized in that, the preparation method of described Embryo Gallus domesticus bioactive peptide is: shelled by Embryo Gallus domesticus, after homogenate, smudge cells; After pancreatin enzymolysis, collected by centrifugation supernatant, ultrafiltration molecular cut off is less than the component of 10kDa.
6. application according to claim 5, is characterized in that, described smudge cells comprises the step of freeze thawing and ultrasonication;
The cryogenic temperature of described freeze thawing is-20 DEG C ~-40 DEG C, and the time is 60h ~ 80h; Solution temperature is 4 DEG C ~ 8 DEG C;
The temperature of described ultrasonication is 4 DEG C ~ 8 DEG C, and power is 800W ~ 1200W, and the time is 15min ~ 25min.
7. application according to claim 5, is characterized in that, in the enzymolysis solution of described enzymolysis, the mass fraction of pancreatin is 2.5%; The temperature of described enzymolysis is 45 DEG C ~ 55 DEG C, and pH value is 7.3 ~ 7.7, and enzymolysis time is 40min ~ 80min.
8. application according to claim 5, is characterized in that, described centrifugal rotating speed is 4500r/min, and the centrifugal time is 20min, and centrifugal temperature is 4 DEG C.
9. the application of Embryo Gallus domesticus bioactive peptide in the cosmetics of preparation; Described cosmetics are the cosmetics with the function of repairing red blood streak, whitening, wrinkle removal, the reparation of skin-active cell recovery or going cicatrix.
10. cosmetics, is characterized in that, comprise Embryo Gallus domesticus bioactive peptide, hyaluronic acid, xanthan gum, Jojoba oil, arbutin and glycerol.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109370982A (en) * | 2018-12-11 | 2019-02-22 | 浙江大学 | A kind of chick embryo extract and its preparation and application |
| CN109619245A (en) * | 2018-12-18 | 2019-04-16 | 长春大学 | A kind of method that instant active double protein exempts from digesting protein for raw material preparation solid-state |
| CN115025124A (en) * | 2022-06-17 | 2022-09-09 | 济南万泉生物技术有限公司 | Hematopoietic anti-aging active component extracted from chick embryo tissue and application thereof |
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| CN109370982A (en) * | 2018-12-11 | 2019-02-22 | 浙江大学 | A kind of chick embryo extract and its preparation and application |
| CN109619245A (en) * | 2018-12-18 | 2019-04-16 | 长春大学 | A kind of method that instant active double protein exempts from digesting protein for raw material preparation solid-state |
| CN115025124A (en) * | 2022-06-17 | 2022-09-09 | 济南万泉生物技术有限公司 | Hematopoietic anti-aging active component extracted from chick embryo tissue and application thereof |
| CN115025124B (en) * | 2022-06-17 | 2024-04-30 | 济南万泉生物技术有限公司 | Hematopoietic anti-aging active component extracted from chick embryo tissue and application thereof |
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