CN108251480A - Remove the preparation method of the Functional Polypeptides of melanin in skin - Google Patents
Remove the preparation method of the Functional Polypeptides of melanin in skin Download PDFInfo
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- C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
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Abstract
The invention discloses removal skin in melanin Functional Polypeptides preparation method, the specific steps are：Silkworm cocoon is added in into degumming in sodium carbonate liquor, then salt is molten in addition calcium chloride solution obtains silk fibroin solution, add in alkali protease and papain enzymolysis, centrifugation, it is concentrated to give the thick liquid of Functional Polypeptides, then successively with gel filtration chromatography, ion exchange chromatography, high-efficient liquid phase chromatogram purification to get Functional Polypeptides.It has the beneficial effect that：Preparation method simple possible of the present invention, the yield of Functional Polypeptides is high, purity is high, activity is not destroyed, and separation method is simple, can quickly and efficiently isolate Functional Polypeptides, and obtained Functional Polypeptides de-black pigment effect is good, has water-retaining property.
Description
Technical field
The present invention relates to polypeptide extractive technique field, more particularly, to the preparation side of the Functional Polypeptides of melanin in removal skin
Method.
Background technology
Silk is a kind of long azelon, and soft texture, fabric such as silk is comfortable and easy to wear, and has magnificent gloss,
In thousands of years history of its utilization, silk is mainly used as haberdashery, is favourably welcome, and is constantly in confession for a long time
The state that should not be asked.To the end of the twenties, silk industry first appears global production relative surplus.The Japan of Silk Industry prosperity is
It safeguards the international status of its silk production and promotes the healthy benign development of national Silk Industry, from 1933, agricultural saved silk
Test site will be included in government budget to the research of silk cocoon new application, start the exploitation and research of silk cocoon new application.In silk
Protein content be up to 97.6%-98.5%, structure is mainly by two fibroins（Fibroin）With the silk gum of turnover covering
（Sericin）Two parts form, and silk peptide belongs to the intermediate product of Fibroin Hydrolysis.Since fibroin has good hygroscopicity
There is good compatibility with the angle member albumen in moisture retention, with skin, thus help to adjust skin moisture.Meanwhile silk egg
It is white also to have the function of to absorb ultraviolet light, solar radiation can be prevented.Therefore for these functions of fibroin, Japan is in the seventies
Start to develop and use fibroin powder and for foundation cream material.Related fibroin is used as the optimum weight range of cosmetics, in the eighties
Just, the existing related data of Japan is recorded.After using certain density sulfuric acid by Fibroin Hydrolysis, the molecular weight made from alkali neutralization
It, can by the molecular weight of it and neutral salt preparation when the peptide of 1000-3000kDa is used in mixed way in the silk peptide of 300-800kDa
To increase the natural gloss of hair, the elasticity of hair is improved, and play the role of moisturizing to skin.When fibroin is degraded to molecule
When amount is between 1000-6000kDa ranges, the epidermal cell film that it not only readily penetrates through human body is absorbed by the skin, and to reducing
Local skin fine lines and inhibition melanin also have certain effect.But to melanin in skin can be removed in silk peptide
Functional Polypeptides research is less.
Invention content
The purpose of the present invention is to provide a kind of preparation method simple possible, the yield of Functional Polypeptides is high, purity is high, activity not
It is destroyed, separation method is simple, can quickly and efficiently isolate Functional Polypeptides, and obtained Functional Polypeptides de-black pigment effect is good, has
The preparation method of the Functional Polypeptides of melanin in the removal skin of water-retaining property.
The problem of present invention in above-mentioned technology for mentioning, the technical solution taken is：
The preparation method of the Functional Polypeptides of melanin in skin is removed, including degumming, prepared by silk fibroin solution, fibroin albumen digests, solidifying
Plastic column chromatography, ion exchange chromatography, high-efficient liquid phase chromatogram purification, the specific steps are：
Degumming：It is 1 that silkworm cocoon is pressed solid-liquid ratio:30-40（g/mL）It adds in the sodium carbonate liquor of a concentration of 0.3-0.7%, degumming
Then 30-40min is cleaned up the silkworm cocoon of degumming with distilled water, drying is to get degumming silkworm cocoon, silk at 70-90 DEG C
Plain close structure, crystallinity is high, not soluble in water, but can be by salt solution, and silk gum molecule polar groups are slightly more, amorphous
Fraction is higher, thus hydrophily is stronger, and uniform peptizate can be formed with water, which can will be enclosed in around fibroin
Silk gum remove, so as to obtain more single, high-purity fibroin zymolyte, can increase fibroin directly and enzyme effect contact area；
It is prepared by silk fibroin solution：Degumming silkworm cocoon is added in the calcium chloride solution of a concentration of 8-12%, the molten 2- of salt at 95-100 DEG C
5min, a concentration of 40-60g/L of adjusting silk fibroin solution, spare after cooling；
Fibroin albumen digests：The pH to 8-9 of silk fibroin solution is adjusted, is then added by enzyme concentration for 600-700U/g and 300-400U/g
Enter alkali protease and papain, the L-vitamin C of alkali protease weight 3-5% is added, then at 50-60 DEG C
Then temperature is risen to 90-100 DEG C by lower enzymolysis 3-4h, heat preservation 10-20min carries out enzyme deactivation, after cooling temperature be 1-5 DEG C, turn
Speed is that 8-10min is centrifuged in the centrifuge of 8000-10000rpm, the solution of supernatant concentration to a concentration of 45-55mg/mL, i.e.,
Spare for the thick liquid of Functional Polypeptides, which passes through the collective effect of restriction endonuclease alkali protease and papain, to crystallization
The peptide bond for participating in being formed by alanine, glycine or tyrosine in area has specificity, fibroin albumen can be made to hydrolyze to a greater degree,
The content that the Functional Polypeptides of melanin can be removed in the thick liquid of Functional Polypeptides made is high, while the addition of L-vitamin C contributes to
Alkali protease quickly identifies serine position, by proteolysis into the Functional Polypeptides of small molecule, is dissolved in enzymolysis liquid, while can assign
Give Functional Polypeptides that there is protein or its composition unexistent new function-water-retaining property of amino acid, and then at the first time by percutaneous absorption
It utilizes, ingredient is complex in the thick liquid of polypeptide obtained by the step, has because its molecular weight is different with amino acid sequence
Different physical characteristics and bioactivity, in order to deeper into research, it is necessary to be isolated purifying；
Gel filtration chromatography：Sephadex deionized water in boiling water bath is heated into colloidal sol 1-3h, is then pressed with deionized water
It is 2.5-3.5 according to weight ratio:1 is uniformly mixed, and then pours into pillar, the gel column loaded is thick by Functional Polypeptides after balance
Chromatography is carried out in liquid loading to pillar, is eluted with distilled water, setting detector sensitivity is 0.2A, and detector is adjusted to
It detects at 280nm, sample size 1.8-2.2mL, flow velocity 0.5-0.8mL/min, is washed according to the absorbance curve collection under 280nm
De- component, freeze-drying is spare, and this method is different according to movement speed of the enzymolysis liquid in chromatographic column, and the component of macromolecular is first
It is eluted, is eluted after the component of small molecule, it is easy to operate so as to achieve the purpose that isolate and purify, and do not need to
Functional Polypeptides are combined with other substances, reduce the loss of Functional Polypeptides in purification process, do not influence target components chemical property, can be protected
The original activity of Functional Polypeptides is held not to be damaged；
Ion exchange chromatography：Gel filtration chromatography step is obtained into sample and is made into the molten of a concentration of 45-55mg/mL with distilled water
Liquid, then centrifuges 5-10min in the centrifuge that temperature is 1-5 DEG C, rotating speed is 10000-13000rpm, and removal is insoluble miscellaneous
Matter, supernatant are added to DEAE-52 anion exchange resin chromatographic columns, successively with distilled water, 0.1,0.5 and 1M NaCl solution
The most strong component of de-black pigment, as ion-exchange chromatography zymolyte are collected in elution, and this method is according to target substance institute band net charge
Difference, target substance is separated, there is reproducible, easy to operate, advantage that Elution range is wide, can be according to practical feelings
Condition is amplified condition and realizes industrialized production；
High-efficient liquid phase chromatogram purification：Above-mentioned gel chromatography zymolyte with distilled water is made into the solution of 80-100 μ g/mL, utilizes height
Effect liquid phase chromatogram is purified to get Functional Polypeptides, and chromatographic condition is：Setting sample size is 4-6 μ L, chromatographic column is Agilent C18
（250mm × 4.6mm, 5 μm）, column temperature be 25-35 DEG C, mobile phase is 10-15% acetonitrile solutions, elution speed 0.8-1.2mL/
Min, this method have that analyze speed is fast, advantage of high resolution, high sensitivity, good separating effect, can fast separating and purifying mesh
Substance is marked, while sample size needed for the purification step is few, sample size can detach Multiple components simultaneously using μ L as the order of magnitude, can be anti-
Multiple sample introduction, and sample is not destroyed in separation process, is easily recycled, the compositional purity of acquisition is higher.
Preferably, sephadex is modified glucan gel in gel filtration chromatography step, and method of modifying is：By mole
Than being 1:0.8-1.2:0.013-0.016 will add in NaIO in sephadex4With 2,6- dichlorobenzoic acids, add and NaIO4
Equimolar 25-35%H2O2Solution reacts 2-3h at 55-65 DEG C, cooling, is washed with deionized water net, filters, be dried in vacuo with
Afterwards, modified glucan gel, NaIO are obtained4Synergistic effect can be played with 2,6- dichlorobenzoic acids so that NaIO4It can be quick
Oxidation forms carboxyl so that acid molecule is easier to approach with gel molecular, is easier to make for oxidation reaction, it is easier to modified Portugal
The production of polysaccharide gel improves reaction rate and degree of substitution, increases hydrophily functional group-carboxyl on sephadex,
And then improve the water absorption and swelling amount of sephadex so that target substance enters in the micropore of gel, increases the stream of target substance
It is final so that the purity of target substance is higher, while can avoid carboxyl and hydroxyl that the side reactions such as lactone reaction occur through distance
Occur, avoid the formation of interchain ester, improve the water absorption of modified glucan gel so that sephadex can directly make after being dissolved in water
With, the step of detaching is reduced, sephadex swelling is fast with crimping process in addition, is detached with solution easily, is easy to regenerate,
Intensity is good, and service life is long.
Compared with prior art, the advantage of the invention is that：1）The preparation method simple possible of Functional Polypeptides of the present invention, can be fast
Speed efficiently isolates Functional Polypeptides, and function peptide yield is high, loss is low, purity is high, the active Functional Polypeptides de-black for not being destroyed, obtaining
Pigment effect is good；2）The enzymolysis can have specificity to the peptide bond for participating in being formed by alanine, glycine or tyrosine in crystal region,
Fibroin albumen can be made to hydrolyze to a greater degree, while it is unexistent new with protein or its composition amino acid to assign Functional Polypeptides
Function-water-retaining property；3）Hydrophily functional group-carboxyl of gel column sephadex is more in the preparation method, and water suction is molten
Bulk is higher, and can increase target substance flows through distance, final so that the purity of target substance is higher, and is swollen and shrinkage
Journey is fast, is detached with solution easily, is easy to regenerate, intensity is good, and service life is long.
Specific embodiment
The present invention program is described further below by embodiment：
Embodiment 1：
The preparation method of the Functional Polypeptides of melanin in skin is removed, including degumming, prepared by silk fibroin solution, fibroin albumen digests, solidifying
Plastic column chromatography, ion exchange chromatography, high-efficient liquid phase chromatogram purification, the specific steps are：
1）Degumming：It is 1 that silkworm cocoon is pressed solid-liquid ratio:40（g/mL）It adds in a concentration of 0.3% sodium carbonate liquor, degumming
Then 40min is cleaned up the silkworm cocoon of degumming with distilled water, drying is to get degumming silkworm cocoon, fibroin structure at 70 DEG C
Closely, crystallinity is high, not soluble in water, but can be by salt solution, and silk gum molecule polar groups are slightly more, pars amorpha ratio
Example is higher, thus hydrophily is stronger, and uniform peptizate can be formed with water, which can remove the silk gum being enclosed in around fibroin
Remove, so as to obtain more single, high-purity fibroin zymolyte, can increase fibroin directly and enzyme effect contact area；
2）It is prepared by silk fibroin solution：Degumming silkworm cocoon is added in a concentration of 12% calcium chloride solution, the molten 5min of salt at 95 DEG C,
A concentration of 40g/L of adjusting silk fibroin solution, spare after cooling；
3）Fibroin albumen digests：The pH to 9 of silk fibroin solution is adjusted, then adds in alkaline egg for 600U/g and 400U/g by enzyme concentration
White enzyme and papain, add the L-vitamin C of alkali protease weight 3%, 3h are then digested at 60 DEG C, then
Temperature is risen to 100 DEG C, heat preservation 10min carries out enzyme deactivation, after cooling in the centrifuge that temperature is 5 DEG C, rotating speed is 8000rpm from
Heart 10min, the solution of supernatant concentration to a concentration of 45mg/mL, the as thick liquid of Functional Polypeptides are spare, in enzymolysis step process
The collective effect of enzyme cutting alkali protease and papain, to participating in shape by alanine, glycine or tyrosine in crystal region
Into peptide bond have specificity, fibroin albumen can be made to hydrolyze to a greater degree, so as to get the thick liquid of Functional Polypeptides in can remove melanin
Functional Polypeptides content it is high, while the addition of L-vitamin C contributes to alkali protease quickly to identify serine position, by egg
White enzymolysis is dissolved in enzymolysis liquid, while can assign Functional Polypeptides with protein or its composition amino acid into the Functional Polypeptides of small molecule
Unexistent new function-water-retaining property, and then utilized at the first time by percutaneous absorption, ingredient in the thick liquid of polypeptide obtained by the step
It is complex, there is different physical characteristic and bioactivity because its molecular weight is different with amino acid sequence, in order to deeper
The research entered, it is necessary to be isolated purifying；
4）Gel filtration chromatography：Sephadex deionized water in boiling water bath is heated into colloidal sol 3h, is then pressed with deionized water
It is 2.5 according to weight ratio:1 is uniformly mixed, and then pours into pillar, the gel column loaded, will be on the thick liquid of Functional Polypeptides after balance
Chromatography is carried out in sample to pillar, is eluted with distilled water, setting detector sensitivity is 0.2A, and detector is adjusted to
It is detected at 280nm, sample size 2.2mL, flow velocity 0.5mL/min, elution fraction is collected according to the absorbance curve under 280nm,
Freeze-drying, spare, this method is different according to movement speed of the enzymolysis liquid in chromatographic column, and the component of macromolecular is first eluted down
Come, be eluted after the component of small molecule, it is easy to operate so as to achieve the purpose that isolate and purify, and do not need to Functional Polypeptides with
Other substances combine, and reduce the loss of Functional Polypeptides in purification process, do not influence target components chemical property, can keep Functional Polypeptides
Original activity is not damaged；
5）Ion exchange chromatography：Gel filtration chromatography step is obtained into the solution that sample is made into distilled water a concentration of 55mg/mL,
Then 5min is centrifuged in the centrifuge that temperature is 1 DEG C, rotating speed is 13000rpm, removes insoluble impurities, supernatant is added to
DEAE-52 anion exchange resin chromatographic columns, successively with distilled water, 0.1,0.5 and 1M NaCl solution elute, collect black removal
The most strong component of element, as ion-exchange chromatography zymolyte, this method according to target substance the difference with net charge, by object
Matter is separated, and is had the advantage reproducible, easy to operate, Elution range is wide, can be amplified condition according to actual conditions
Realize industrialized production；
6）High-efficient liquid phase chromatogram purification：Above-mentioned gel chromatography zymolyte with distilled water is made into the solution of 100 μ g/mL, utilizes height
Effect liquid phase chromatogram is purified to get Functional Polypeptides, and chromatographic condition is：Setting sample size is 4 μ L, chromatographic column is Agilent C18
（250mm × 4.6mm, 5 μm）, column temperature be 35 DEG C, mobile phase is 10% acetonitrile solution, elution speed 1.2mL/min, this method
With analyze speed is fast, advantage of high resolution, high sensitivity, good separating effect, can fast separating and purifying target substance, simultaneously
Sample size needed for the purification step is few, and sample size can detach Multiple components simultaneously using μ L as the order of magnitude, can sample introduction, and dividing repeatedly
It is not destroyed from sample in the process, easily recycles, the compositional purity of acquisition is higher.
Sephadex is modified glucan gel in above-mentioned gel filtration chromatography step, and method of modifying is：In molar ratio
It is 1:0.8:0.016 will add in NaIO in sephadex4With 2,6- dichlorobenzoic acids, add and NaIO4Equimolar 25%
H2O2Solution reacts 2h at 65 DEG C, cooling, is washed with deionized water only, filters, after vacuum drying, obtain modified glucan and coagulate
Glue, NaIO4Synergistic effect can be played with 2,6- dichlorobenzoic acids so that NaIO4Can Quick Oxidation formed carboxyl so that acid
Molecule is easier to approach with gel molecular, is easier to make for oxidation reaction, it is easier to which the production of modified glucan gel improves
Reaction rate and degree of substitution increase hydrophily functional group-carboxyl on sephadex, and then improve sephadex
Water absorption and swelling amount so that target substance enters in the micropore of gel, increases the distance that flows through of target substance, finally so that object
The purity of matter is higher, while can avoid carboxyl and hydroxyl that the generation of the side reactions such as lactone reaction occurs, and avoids the formation of interchain ester, carries
The water absorption of high modified glucan gel so that sephadex can be used directly after being dissolved in water, reduce the step of detaching, in addition
The sephadex is swollen with solution will detach easily soon, be easy to regenerate, intensity is good, and service life is long with crimping process.
Embodiment 2：
The preparation method of the Functional Polypeptides of melanin in skin is removed, including degumming, prepared by silk fibroin solution, fibroin albumen digests, solidifying
Plastic column chromatography, ion exchange chromatography, high-efficient liquid phase chromatogram purification, the specific steps are：
1）Degumming：It is 1 that silkworm cocoon is pressed solid-liquid ratio:30（g/mL）It adds in a concentration of 0.7% sodium carbonate liquor, degumming
Then 30min is cleaned up the silkworm cocoon of degumming with distilled water, drying is to get degumming silkworm cocoon at 90 DEG C；
2）It is prepared by silk fibroin solution：Degumming silkworm cocoon is added in a concentration of 8% calcium chloride solution, the molten 2min of salt at 100 DEG C,
A concentration of 60g/L of adjusting silk fibroin solution, spare after cooling；
3）Fibroin albumen digests：The pH to 8 of silk fibroin solution is adjusted, then adds in alkaline egg for 700U/g and 300U/g by enzyme concentration
White enzyme and papain, add the L-vitamin C of alkali protease weight 5%, 4h are then digested at 50 DEG C, then
Temperature is risen to 90 DEG C, heat preservation 20min carries out enzyme deactivation, after cooling in the centrifuge that temperature is 1 DEG C, rotating speed is 10000rpm from
Heart 8min, the solution of supernatant concentration to a concentration of 55mg/mL, the as thick liquid of Functional Polypeptides are spare；
4）Gel filtration chromatography：Sephadex deionized water in boiling water bath is heated into colloidal sol 1h, is then pressed with deionized water
It is 3.5 according to weight ratio:1 is uniformly mixed, and then pours into pillar, the gel column loaded, will be on the thick liquid of Functional Polypeptides after balance
Chromatography is carried out in sample to pillar, is eluted with distilled water, setting detector sensitivity is 0.2A, and detector is adjusted to
It is detected at 280nm, sample size 1.8mL, flow velocity 0.8mL/min, elution fraction is collected according to the absorbance curve under 280nm,
Freeze-drying, it is spare；
5）Ion exchange chromatography：Gel filtration chromatography step is obtained into the solution that sample is made into distilled water a concentration of 45mg/mL,
Then 10min is centrifuged in the centrifuge that temperature is 5 DEG C, rotating speed is 10000rpm, removes insoluble impurities, supernatant is added to
DEAE-52 anion exchange resin chromatographic columns, successively with distilled water, 0.1,0.5 and 1M NaCl solution elute, collect black removal
The most strong component of element, as ion-exchange chromatography zymolyte；
6）High-efficient liquid phase chromatogram purification：Above-mentioned gel chromatography zymolyte is made into the solution of 80 μ g/mL with distilled water, using efficient
Liquid chromatogram is purified to get Functional Polypeptides, and chromatographic condition is：Setting sample size is 6 μ L, chromatographic column is Agilent C18
（250mm × 4.6mm, 5 μm）, column temperature be 25 DEG C, mobile phase is 15% acetonitrile solution, elution speed 0.8mL/min.
Sephadex is modified glucan gel in above-mentioned gel filtration chromatography step, and method of modifying is：In molar ratio
It is 1:1.2:0.013 will add in NaIO in sephadex4With 2,6- dichlorobenzoic acids, add and NaIO4Equimolar 35%
H2O2Solution reacts 3h at 55 DEG C, cooling, is washed with deionized water only, filters, after vacuum drying, obtain modified glucan and coagulate
Glue.
Embodiment 3：
The preparation method of the Functional Polypeptides of melanin in skin is removed, including degumming, prepared by silk fibroin solution, fibroin albumen digests, solidifying
Plastic column chromatography, ion exchange chromatography, high-efficient liquid phase chromatogram purification, the specific steps are：
1）Degumming：It is 1 that silkworm cocoon is pressed solid-liquid ratio:35（g/mL）It adds in a concentration of 0.5% sodium carbonate liquor, degumming
Then 35min is cleaned up the silkworm cocoon of degumming with distilled water, drying is to get degumming silkworm cocoon at 80 DEG C；
2）It is prepared by silk fibroin solution：Degumming silkworm cocoon is added in a concentration of 10% calcium chloride solution, the molten 4min of salt at 98 DEG C,
A concentration of 50g/L of adjusting silk fibroin solution, spare after cooling；
3）Fibroin albumen digests：The pH to 8.5 of silk fibroin solution is adjusted, then adds in alkalinity for 500U/g and 400U/g by enzyme concentration
Protease and papain add the L-vitamin C of alkali protease weight 4%, then digest 3.5h at 55 DEG C,
Then temperature is risen to 100 DEG C, heat preservation 15min carries out enzyme deactivation, in the centrifuge that temperature is 4 DEG C, rotating speed is 9000rpm after cooling
Middle centrifugation 8min, the solution of supernatant concentration to a concentration of 50mg/mL, the as thick liquid of Functional Polypeptides are spare；
4）Gel filtration chromatography：Sephadex deionized water in boiling water bath is heated into colloidal sol 2h, is then pressed with deionized water
It is 3.0 according to weight ratio:1 is uniformly mixed, and then pours into pillar, the gel column loaded, will be on the thick liquid of Functional Polypeptides after balance
Chromatography is carried out in sample to pillar, is eluted with distilled water, setting detector sensitivity is 0.2A, and detector is adjusted to
It is detected at 280nm, sample size 2.0mL, flow velocity 0.6mL/min, elution fraction is collected according to the absorbance curve under 280nm,
Freeze-drying, it is spare；
5）Ion exchange chromatography：Gel filtration chromatography step is obtained into the solution that sample is made into distilled water a concentration of 50mg/mL,
Then 8min is centrifuged in the centrifuge that temperature is 4 DEG C, rotating speed is 12000rpm, removes insoluble impurities, supernatant is added to
DEAE-52 anion exchange resin chromatographic columns, successively with distilled water, 0.1,0.5 and 1M NaCl solution elute, collect black removal
The most strong component of element, as ion-exchange chromatography zymolyte；
6）High-efficient liquid phase chromatogram purification：Above-mentioned gel chromatography zymolyte is made into the solution of 90 μ g/mL with distilled water, using efficient
Liquid chromatogram is purified to get Functional Polypeptides, and chromatographic condition is：Setting sample size is 5 μ L, chromatographic column is Agilent C18
（250mm × 4.6mm, 5 μm）, column temperature be 30 DEG C, mobile phase is 12% acetonitrile solution, elution speed 1.0mL/min.
Sephadex is modified glucan gel in above-mentioned gel filtration chromatography step, and method of modifying is：In molar ratio
It is 1:1.0:0.015 will add in NaIO in sephadex4With 2,6- dichlorobenzoic acids, add and NaIO4Equimolar 30%
H2O2Solution reacts 2.5h at 60 DEG C, cooling, is washed with deionized water only, filters, after vacuum drying, obtain modified glucan
Gel.
Routine operation in the operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme of the present invention is described in detail in embodiment described above, it should be understood that the above is only
For specific embodiments of the present invention, it is not intended to restrict the invention, all any modifications made in the spirit of the present invention,
Supplement or similar fashion replacement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. remove skin in melanin Functional Polypeptides preparation method, including degumming, silk fibroin solution prepare, fibroin albumen enzymolysis,
Gel filtration chromatography, ion exchange chromatography, high-efficient liquid phase chromatogram purification, it is characterised in that：The fibroin albumen enzymolysis step
For：Alkali protease, papain and L-vitamin C, enzymolysis are added in silk fibroin solution, enzyme deactivation centrifuges, supernatant after cooling
Liquid concentrates, as the thick liquid of Functional Polypeptides, spare.
2. the preparation method of the Functional Polypeptides of melanin in removal skin according to claim 1, it is characterised in that：The alkali
Property protease and papain enzyme concentration for 600-700U/g and 300-400U/g, the enzymolysis pH is 8-9, temperature is
50-60 DEG C, time 3-4h.
3. the preparation method of the Functional Polypeptides of melanin in removal skin according to claim 1, it is characterised in that：The left side
Revolve the 3-5% that ascorbic additive amount is alkali protease weight.
4. the preparation method of the Functional Polypeptides of melanin in removal skin according to claim 1, it is characterised in that：It is described de-
Glue step is：It is 1 that silkworm cocoon is pressed solid-liquid ratio:30-40（g/mL）It adds in the sodium carbonate liquor of a concentration of 0.3-0.7%, degumming
Then 30-40min is cleaned up the silkworm cocoon of degumming with distilled water, drying is to get degumming silkworm cocoon at 70-90 DEG C.
5. the preparation method of the Functional Polypeptides of melanin in removal skin according to claim 1, it is characterised in that：The silk
Plain solution preparation step is：Degumming silkworm cocoon is added in the calcium chloride solution of a concentration of 8-12%, the molten 2- of salt at 95-100 DEG C
5min, a concentration of 40-60g/L of adjusting silk fibroin solution, spare after cooling.
6. the preparation method of the Functional Polypeptides of melanin in removal skin according to claim 1, it is characterised in that：It is described solidifying
Plastic column chromatography preparation process is：Sephadex deionized water is heated into colloidal sol 1-3h in boiling water bath, then with deionization
Water is 2.5-3.5 according to weight ratio:1 is uniformly mixed, and then pours into pillar, the gel column loaded is after balance, by function
Carry out chromatography in the thick liquid loading to pillar of peptide, eluted with distilled water, setting detector sensitivity be 0.2A, detector
It is adjusted to detect at 280nm, sample size 1.8-2.2mL, flow velocity 0.5-0.8mL/min are received according to the absorbance curve under 280nm
Collect elution fraction, freeze-drying is spare.
7. the preparation method of the Functional Polypeptides of melanin in removal skin according to claim 6, it is characterised in that：The Portugal
Polysaccharide gel is modified glucan gel, and the method for modifying of the sephadex is：NaIO will be added in sephadex4With
2,6- dichlorobenzoic acids, add H2O2Solution reacts, cooling, and net, suction filtration is washed with deionized water, dry, obtains modified Portugal and gathers
Sugared gel.
8. the preparation method of the Functional Polypeptides of melanin in removal skin according to claim 7, it is characterised in that：The Portugal
Polysaccharide gel, NaIO4Molar ratio with 2,6- dichlorobenzoic acids is 1:0.8-1.2:0.013-0.016.
9. the preparation method of the Functional Polypeptides of melanin in removal skin according to claim 1, it is characterised in that：It is described from
Sub-exchange resin chromatographic step is：Gel filtration chromatography step is obtained into sample and is made into the molten of a concentration of 45-55mg/mL with distilled water
Liquid, then centrifuges 5-10min in the centrifuge that temperature is 1-5 DEG C, rotating speed is 10000-13000rpm, and removal is insoluble miscellaneous
Matter, supernatant are added to DEAE-52 anion exchange resin chromatographic columns, successively with distilled water, 0.1,0.5 and 1M NaCl solution
The most strong component of de-black pigment, as ion-exchange chromatography zymolyte are collected in elution.
10. the preparation method of the Functional Polypeptides of melanin in removal skin according to claim 1, it is characterised in that：It is described
High-efficient liquid phase chromatogram purification step is：Above-mentioned gel chromatography zymolyte with distilled water is made into the solution of 80-100 μ g/mL, is utilized
High performance liquid chromatography is purified to get Functional Polypeptides, and chromatographic condition is：Setting sample size is 4-6 μ L, chromatographic column Agilent
C18（250mm × 4.6mm, 5 μm）, column temperature be 25-35 DEG C, mobile phase is 10-15% acetonitrile solutions, elution speed 0.8-
1.2mL/min。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111214385A (en) * | 2020-03-13 | 2020-06-02 | 珀莱雅化妆品股份有限公司 | Nanoparticle emulsion loaded with skin nutrient and preparation method thereof |
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