CN107523601B - The preparation method and applications of scallop edge whitening peptide - Google Patents
The preparation method and applications of scallop edge whitening peptide Download PDFInfo
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Abstract
The invention discloses a kind of preparation method and applications of scallop edge whitening peptide, the preparation method of the whitening peptide includes: the pretreatment of (1) scallop edge: taking scallop edge, it shreds, the water that scallop edge quality settings multiple is added is homogenized, and scallop edge homogenate is obtained；(2) it digests: sequentially adding pepsin in the scallop edge homogenate in step (1) and trypsase is digested, obtain enzymolysis liquid；(3) pH to 6.8~7.2 of the enzymolysis liquid of regulating step (2), centrifuging and taking supernatant are concentrated, and the dehydrated alcohol of setting ratio is added, and stir alcohol precipitation, supernatant is scallop edge polypeptide crude extract after centrifugation；After purification up to scallop edge whitening peptide.The present invention only needs simple double enzymolysis, alcohol precipitation process is to obtain the scallop edge whitening peptide with tyrosinase activity is significantly inhibited, have many advantages, such as the application potential as skin-lightening cosmetic effective component, while active height, good biocompatibility, safe without toxic side effect.
Description
Technical field
The invention belongs to cosmetic technical fields, and in particular to a kind of preparation method and applications of scallop edge whitening peptide.
Background technique
With the continuous growth of people's consumption demand, cosmetic industry development is also very rapid, a just so-called " white screening three
It is ugly ", bright and clean, pale, ruddy skin is always what Nails were pursued, so skin whitening, moisturizing is gradually at consistent aesthetic
Standard.And the substance for influencing skin color is mainly melanin.Melanin is a kind of biochrome, is tyrosine or 3,4- dihydroxy
Phenylpropyl alcohol ammonia is formed by a succession of chemical reaction, is generated and is stored therein in by melanocyte.In melanocyte
Melanosome has a kind of special enzyme i.e. tyrosinase, can then be oxidized to DOPA quinone, finally by oxidizing tyrosine at DOPA
Generate melanin.Moving layer by layer through cell metabolism, arrival skin epidermis form the shapes such as freckle, sunburn, blackspot to melanin again
Shape.Since the links such as tyrosinase activity and melanocyte conveying are influenced vulnerable to the external world, so that the research and development for skin-lightening cosmetic mention
Having supplied may.The action target spot of common skin-lightening cosmetic is mainly tyrosinase, melanocyte and melanocyte transhipment metabolism at present.It presses
The effective component of skin-lightening cosmetic is divided by different mechanism of action: tyrosinase activity inhibitor, such as quinhydrones, arbutin, song
Acid etc.；Melanocyte toxic agents, such as oil soluble licorice extract, quinhydrones；Influence Melanin Metabolism agent, such as linoleic acid；Opacifier；
Reducing agent, such as vitamin C and its derivative；Chemical peeling agent, such as tartaric acid, linoleic acid, linolenic acid.
These chemical whitening agents such as domestic common arbutin, vitamin C and its derivative, kojic acid all have good beauty
White effect is applied to have some defects in formula: though as kojic acid can inhibit the activity of tyrosinase, but its to light, heat and
Metal ion is unstable, easy to change, and is used for a long time and has cytotoxicity, will increase gonad cell chromosome interchange and dye
The probability of colour solid distortion；Although arbutin safety is good, there is no irritation and anaphylaxis substantially, excessive concentration can allow normal skin
Skin decoloration.In addition there are mainly two ways for vitamin C whitening mechanism: first is that it is black that oxidisability melanocyte is reduced to colourless reproducibility
Element；Second is that inhibiting the activity of tyrosinase.But the possible re-oxidation of the melanin because being restored through vitamin C, so must be long-term
It is just effective to keep enough concentration, but Excess free enthalpy vitamin C can be harmful to the human body, and vitamin C is water-soluble substances, no
Easily penetrate to the skin cuticula, itself is oxidizable unstable, therefore its application is restricted.Cosmetics usually with metal such as sodium,
Calcium is combined to maintain its stability, or is existed in the form of vitamin C derivatives.The defect of this kind of chemical whitening agents is all in a word
Leverage the whitening effect and appearance of product.
The skin-whitening agents of nowadays demand should comply with two standards: first is that having higher inhibition to tyrosinase activity
Rate can reduce the generation of melanin significantly；Second is that have higher safety, it is nontoxic to human skin non-stimulated.Therefore it makes up
Product safely, effectively, mildness become the primary factor that considers when consumer buys cosmetics.Whitening agent is skin-lightening cosmetic
Core functional component, the whitening agent in natural products source due to its good safety and it is both effectiveness and increasingly by market
It welcomes.This just need it is a kind of realized healthy whitening function by means of the whitening active of natural extract, and marine product is developed
It is a promising rising industry, research marine active substance can yet be regarded as one well in improvement people's image direction such as whitening etc.
Application prospect.
Summary of the invention
In view of the above shortcomings of the prior art, inventor gets a foothold through long-term technology and practical exploration, success from scallop
Material --- a kind of polypeptide is extracted in scallop body, evaluates scallop edge polypeptide by measuring the inhibiting rate to tyrosinase activity
Whitening active finds the scallop edge polypeptide whitening active with higher extracted, can be used in cosmetics, can also develop
Provide the marine active substance of whitening active.
Specifically, the present invention relates to following technical schemes:
The first aspect of the invention, provides a kind of preparation method of scallop edge whitening peptide, the preparation method include with
Lower step:
(1) scallop edge pre-processes: taking scallop edge, shreds, the water that scallop edge quality settings multiple is added is homogenized, and must be fanned
Shellfish side homogenate；
(2) it digests: sequentially adding pepsin in the scallop edge homogenate in step (1) and trypsase carries out enzyme
Solution, obtains enzymolysis liquid；
(3) pH to 6.8~7.2 of the enzymolysis liquid of regulating step (2), centrifuging and taking supernatant are concentrated, and setting ratio is added
The dehydrated alcohol of example stirs alcohol precipitation, and supernatant is scallop edge polypeptide crude extract after centrifugation；After purification up to scallop edge whitening peptide；
It in step (1), specifically, scallop edge tap water and distilled water are successively rinsed well scallop edge, shreds, uses
Distilled water impregnates 0.4~1h, removes water, weighing；
It is homogenized using refiner, 6~10 times of water of scallop edge quality, the distilled water of preferably 8 times quality is added.
In step (2), when being digested using pepsin, the addition quality of pepsin is the 0.3~0.5% of scallop edge
(preferably 0.4%), hydrolysis temperature are 45~55 DEG C (preferably 50 DEG C), and enzymatic hydrolysis pH is 2.0~2.5 (preferably 2.3), enzymatic hydrolysis
Time is 4~6h (preferably 5h)；
When using trypsin digestion, the addition quality of trypsase be scallop edge 0.4~0.5% (preferably
0.45%), hydrolysis temperature is 45~55 DEG C (preferably 50 DEG C), and enzymatic hydrolysis pH is 8.0~8.5 (preferably 8.2), and enzymolysis time is
4~6h (preferably 5h)；
Preferably, enzyme digestion reaction is stopped using high-temperature inactivation mode, specifically using after trypsin digestion in step (2)
High-temperature inactivation condition are as follows: 85~90 DEG C of inactivation temperature (preferably 87 DEG C), inactivation time are 3~8 minutes (preferably 5 minutes)；
Then by enzymolysis liquid be quickly cooled to 30 DEG C hereinafter, place 20~40 minutes (preferably 30 minutes) thoroughly to stop reaction into
Row.
In step (3), the pH to 7.0 of the enzymolysis liquid of regulating step (2), centrifugation (10000rpm, 10~30 minutes, preferably
Be 20 minutes) after take supernatant to be concentrated, into concentrate be added dehydrated alcohol to dehydrated alcohol account for the 50 of overall solution volume
~70% (preferably 60%), stirring carries out alcohol precipitation reaction, after centrifugation (10000rpm, 10~20 minutes, preferably 15 minutes)
Supernatant is scallop edge polypeptide crude extract；
Step purifies specific steps described in (3) are as follows: takes scallop edge polypeptide crude extract through 5000Da ceramic membrane ultrafitration, filtration
Liquid is through vacuum-concentrcted, and scallop edge whitening peptide to obtain the final product after spray drying；
The second aspect of the invention discloses the scallop edge whitening peptide being prepared using the above method；
The third aspect of the invention discloses the application of above-mentioned scallop edge whitening peptide in inhibiting tyrosinase activity；Tool
Body application form is that the scallop edge whitening peptide is preparing the application in skin-lightening cosmetic, more specifically, the scallop edge whitening
Application of the peptide in preparation lotion, face cream, surfactant or Essence.
Beneficial effects of the present invention:
The preparation method of scallop edge whitening peptide is simple, easy to operate in the present invention, and temperature is low, and safe preparation process, environmental protection are complete
It is whole to remain active constituent therein, it uses reagent safety high in preparation process, dissolvent residual will not be led to the problem of, simultaneously
Using scallop leftover --- scallop edge is raw material, is also various pharmacological actives so that waste resource be made to be utilized effectively
The exploitation of matter provides new approaches；
The present invention only needs simple double enzymolysis, and alcohol precipitation process is to obtain the scallop edge beauty with tyrosinase activity is significantly inhibited
White peptide, experiment proves that, scallop edge polypeptide is up to 45.3% (scallop edge whitening peptide concentration to the inhibiting rate of tyrosinase activity
0.16mg/ml)；With the application potential as skin-lightening cosmetic effective component, at the same active height, good biocompatibility,
The advantages that safe without toxic side effect.
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to the illustrative embodiments of the application.As used herein, unless the context clearly indicates otherwise, otherwise singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.
As background technique is introduced, in the prior art the generally existing stability of chemical whitening agent, safety, poison is secondary makees
The problems such as using；
In view of this, a kind of preparation method of scallop edge whitening peptide is provided in an exemplary embodiment of the invention, it should
Preparation method the following steps are included:
(1) scallop edge pre-processes: taking scallop edge, shreds, the water that scallop edge quality settings multiple is added is homogenized, and must be fanned
Shellfish side homogenate；
(2) it digests: sequentially adding pepsin in the scallop edge homogenate in step (1) and trypsase carries out enzyme
Solution, obtains enzymolysis liquid；
(3) pH to 6.8~7.2 of the enzymolysis liquid of regulating step (2), centrifuging and taking supernatant are concentrated, and setting ratio is added
The dehydrated alcohol of example stirs alcohol precipitation, and supernatant is scallop edge polypeptide crude extract after centrifugation；After purification up to scallop edge whitening peptide；
It in step (1), specifically, scallop edge tap water and distilled water are successively rinsed well scallop edge, shreds, uses
Distilled water impregnates 0.4~1h, removes water, weighing；
It is homogenized using refiner, 6~10 times of water of scallop edge quality, the distilled water of preferably 8 times quality is added.
In step (2), when being digested using pepsin, the addition quality of pepsin is the 0.3~0.5% of scallop edge
(preferably 0.4%), hydrolysis temperature are 45~55 DEG C (preferably 50 DEG C), and enzymatic hydrolysis pH is 2.0~2.5 (preferably 2.3), enzymatic hydrolysis
Time is 4~6h (preferably 5h)；
When using trypsin digestion, the addition quality of trypsase be scallop edge 0.4~0.5% (preferably
0.45%), hydrolysis temperature is 45~55 DEG C (preferably 50 DEG C), and enzymatic hydrolysis pH is 8.0~8.5 (preferably 8.2), and enzymolysis time is
4~6h (preferably 5h)；
Using after trypsin digestion in step (2), enzyme digestion reaction, specific high-temperature inactivation are stopped using high-temperature inactivation mode
Condition are as follows: 85~90 DEG C of inactivation temperature (preferably 87 DEG C), inactivation time are 3~8 minutes (preferably 5 minutes)；Then by enzyme
Solution liquid is quickly cooled to 30 DEG C hereinafter, placing the progress for coming thoroughly to stop reaction for 20~40 minutes (preferably 30 minutes).
In step (3), the pH to 7.0 of the enzymolysis liquid of regulating step (2), centrifugation (10000rpm, 10~30 minutes, preferably
Be 20 minutes) after take supernatant to be concentrated, into concentrate be added dehydrated alcohol to dehydrated alcohol account for the 50 of overall solution volume
~70% (preferably 60%), stirring carries out alcohol precipitation reaction, after centrifugation (10000rpm, 10~20 minutes, preferably 15 minutes)
Supernatant is scallop edge polypeptide crude extract；
Step purifies specific steps described in (3) are as follows: takes scallop edge polypeptide crude extract through 5000Da ceramic membrane ultrafitration, filtration
Liquid is through vacuum-concentrcted, and scallop edge whitening peptide to obtain the final product after spray drying；
In still another embodiment of the invention, the scallop edge whitening peptide being prepared using the above method is provided；
In still another embodiment of the invention, above-mentioned scallop edge whitening peptide is provided in inhibiting tyrosinase activity
Using；Concrete application form is that the scallop edge whitening peptide is preparing the application in skin-lightening cosmetic, more specifically, the fan
Application of the whitening peptide in shellfish side in preparation lotion, face cream, surfactant or Essence.
Explanation is further explained to the present invention by the following examples, but is not construed as limiting the invention.
Embodiment 1
(1) scallop edge pre-processes: new fresh scallops 2kg is rinsed well with tap water, and gutting, silt, scallop column take fan
Scallop edge is successively rinsed well with tap water and distilled water, is shredded by Bei Bian, impregnates 0.5h, water removal weighing with pure water.It is added 8
The pure water of amount obtains scallop edge homogenate with refiner decentralized processing again.
(2) double enzymolysis: by 50 DEG C of heating water baths of scallop edge homogenate, homogenate pH to 2.0 is adjusted with 6mol/L HCl, is added
The weight for entering pepsin is the 0.4% of scallop edge, and adjusting pH is to digest 5h under 2.3,50 DEG C of waters bath with thermostatic control, is stirred continuously, whole
A process pH is between 2.0~2.5.By 50 DEG C of heating water baths of enzymolysis liquid, is continued to adjust pH to 8.2 with 0.5mol/L NaOH, be added
Enter trypsase, weight is the 0.45% of scallop edge, adjusts pH to 8.2,5h is digested under 50 DEG C of condition of water bath heating, constantly
Stirring, whole process pH is between 8.0~8.5.The temperature of water-bath is set as 87 DEG C, places 5min, cooling rapidly by enzyme-deactivating
To 30 DEG C hereinafter, placing 30min.Enzymolysis liquid pH to 7.0 is adjusted, room temperature is centrifuged (10000rpm, 20min), takes supernatant, and it is heavy to abandon
It forms sediment.
(3) reduced pressure and ethanol precipitation: it will be slowly added into a certain amount of dehydrated alcohol after the liquid concentration after centrifugation, added
Enter process to be stirred continuously, ethyl alcohol accounts for 60%, and 4 DEG C of refrigeration 12h are placed after being sealed with preservative film.By double enzymes of the scallop edge after alcohol precipitation
Liquid room temperature centrifugation (10000rpm, 15min) is solved, supernatant is scallop edge polypeptide crude extract after centrifugation, is precipitated as the polysaccharide of scallop edge
Crude extract.Take the crude extract of scallop edge polypeptide through 5000Da ceramic membrane ultrafitration, filtered solution is spray-dried through vacuum-concentrcted
Obtain scallop edge polypeptide 50g.
Embodiment 2
(1) scallop edge pre-processes: new fresh scallops 2kg is rinsed well with tap water, and gutting, silt, scallop column take fan
Scallop edge is successively rinsed well with tap water and distilled water, is shredded by Bei Bian, impregnates 1h, water removal weighing with pure water.It is added 6 times
The pure water of amount obtains scallop edge homogenate with refiner decentralized processing.
(2) double enzymolysis: by 50 DEG C of heating water baths of scallop edge homogenate, homogenate pH to 2.0 is adjusted with 6mol/L HCl, is added
The weight for entering pepsin is the 0.5% of scallop edge, and adjusting pH is to digest 5h under 2.3,50 DEG C of waters bath with thermostatic control, is stirred continuously, whole
A process pH is between 2.0~2.5.By 50 DEG C of heating water baths of enzymolysis liquid, is continued to adjust pH to 8.0 with 0.5mol/L NaOH, be added
Enter trypsase, weight is the 0.45% of scallop edge, adjusts pH to 8.2,5h is digested under 50 DEG C of condition of water bath heating, constantly
Stirring, whole process pH is between 8.0~8.5.The temperature of water-bath is set as 87 DEG C, places 5min, cooling rapidly by enzyme-deactivating
To 30 DEG C hereinafter, placing 30min.Enzymolysis liquid pH to 7.0 is adjusted, room temperature is centrifuged (10000rpm, 20min), takes supernatant, and it is heavy to abandon
It forms sediment.
(3) reduced pressure and ethanol precipitation: it will be slowly added into a certain amount of dehydrated alcohol after the liquid concentration after centrifugation, added
Enter process to be stirred continuously, ethyl alcohol accounts for 60%, and 4 DEG C of refrigeration 12h are placed after being sealed with preservative film.By double enzymes of the scallop edge after alcohol precipitation
Liquid room temperature centrifugation (10000rpm, 15min) is solved, supernatant is scallop edge polypeptide crude extract after centrifugation, is precipitated as the polysaccharide of scallop edge
Crude extract.Take the crude extract of scallop edge polypeptide through 5000Da ceramic membrane ultrafitration, filtered solution is spray-dried through vacuum-concentrcted
Obtain scallop edge polypeptide 45g.
Embodiment 3
(1) scallop edge pre-processes: new fresh scallops 2kg is rinsed well with tap water, and gutting, silt, scallop column take fan
Scallop edge is successively rinsed well with tap water and distilled water, is shredded by Bei Bian, impregnates 1h, water removal weighing with pure water.It is added 10 times
The pure water of amount obtains scallop edge homogenate with refiner decentralized processing.
(2) double enzymolysis: by 50 DEG C of heating water baths of scallop edge homogenate, homogenate pH to 2.0 is adjusted with 6mol/L HCl, is added
The weight for entering pepsin is the 0.4% of scallop edge, and adjusting pH is to digest 5h under 2.3,45 DEG C of waters bath with thermostatic control, is stirred continuously, whole
A process pH is between 2.0~2.5.By 50 DEG C of heating water baths of enzymolysis liquid, is continued to adjust pH to 8.5 with 0.5mol/L NaOH, be added
Enter trypsase, weight is the 0.5% of scallop edge, adjusts pH to 8.2,5h is digested under 45 DEG C of condition of water bath heating, constantly
Stirring, whole process pH is between 8.0~8.5.The temperature of water-bath is set as 87 DEG C, places 5min, cooling rapidly by enzyme-deactivating
To 30 DEG C hereinafter, placing 30min.Enzymolysis liquid pH to 7.2 is adjusted, room temperature is centrifuged (10000rpm, 20min), takes supernatant, and it is heavy to abandon
It forms sediment.
(3) reduced pressure and ethanol precipitation: it will be slowly added into a certain amount of dehydrated alcohol after the liquid concentration after centrifugation, added
Enter process to be stirred continuously, ethyl alcohol accounts for 70%, and 4 DEG C of refrigeration 12h are placed after being sealed with preservative film.By double enzymes of the scallop edge after alcohol precipitation
Liquid room temperature centrifugation (10000rpm, 15min) is solved, supernatant is scallop edge polypeptide crude extract after centrifugation, is precipitated as the polysaccharide of scallop edge
Crude extract.Take the crude extract of scallop edge polypeptide through 5000Da ceramic membrane ultrafitration, filtered solution is spray-dried through vacuum-concentrcted
Obtain scallop edge polypeptide 47g.
Embodiment 4: the concentration mensuration of scallop edge polypeptide
1, the preparation of coomassie brilliant blue staining liquid
Coomassie brilliant blue G250 0.02g is weighed, is dissolved with 90% ethyl alcohol of 5mL, 10mL85% phosphoric acid is added and is uniformly mixed
Double pure water dilutions are added to be settled to 100ml again afterwards.
2, the preparation of standard protein liquid
0.01g bovine serum globulin is weighed, a certain amount of double pure water dissolutions are added, are settled to 100ml with the dilution of double pure water,
Being configured to concentration is the bovine serum globulin liquid of 0.1mg/mL as standard protein liquid.
3, determination of protein concentration in Specification Curve of Increasing and sample extracting solution
(1) it is prepared according to shown in following table
1 preparation method of table
(2) by prepared test tube mix place 5min after, with glass cuvette at 595nm colorimetric, measure A595nm.With
Protein concentration (C) is abscissa, is that ordinate draws Coomassie brilliant blue standard curve with light absorption value (A).According to measurement gained
Light absorption value calculates the protein concentration of sample liquid after alcohol precipitation revolving.
The data of standard curve are as shown in table 2, absorbance measured at the wavelength of 595nm, using absorbance as ordinate
(A), standard curve is obtained: A=4.92C+0.0486R for abscissa with standard sample concentration (C)2=0.993.
2 Coomassie brilliant blue standard curve tables of data of table
Embodiment 5: scallop edge whitening peptide whitening active measurement experiment:
1, the preparation of PBS
NaCl 8.0g, KCl 0.2g, Na2HPO4 1.44g,KH2PO40.24g, distilled water dilution are settled to 1000ml,
Shake up pH7.2-7.4 PBS, HCl adjust pH to 6.83 after it is spare.
2, the preparation of tyrosinase
Precision weighs the tyrosinase of 1mg, and PBS solution is added and obtains the tyrosinase solution that concentration is 10KU/L.Junket ammonia
The every 1mg of the potency of sour enzyme is not less than 1000U, is calculated with 1mg=1000U, takes the tyrosinase of 1mg to be made into 100ml solution, now match
It is current.
3, the preparation of L-Dopa solution
Precision weighs 25mg L-Dopa, is configured to the L-Dopa solution that mass fraction is 0.05% with PBS (pH6.83):
It weighs 25mg L-Dopa addition water and obtains the solution of 50ml.It is unable to measure because absorbance value is too small, L-Dopa solution is dense
It is to weigh 1.0g L-Dopa addition distilled water to be diluted to 50ml that degree, which improves 40 times,.
4, the preparation of analyte sample fluid
With VC:0.2mg/ml, 0.1mg/ml of the PBS of pH6.83 configuration various concentration, 0.05mg/ml, 0.025mg/ml,
0.0125mg/ml totally five concentration (standard items)；Scallop edge whitening peptide liquid is different dense at five according to certain gradient dilution
Degree.
5, the measurement of tyrosinase activity
(1) it before measuring, is accurately prepared with micropipettor:
A1 test specimens: 2mL PBS+1mL tyrosinase solution；
A2 test specimens: 3mLPBS；
A3 test specimens: 1mL sample solution+1mL PBS+1mL tyrosinase solution；
A4 test specimens: 1mL sample solution+2mL PBS.
(2) parallel laboratory test, places 10min in 25 DEG C of waters bath with thermostatic control, respectively adds prepared L-Dopa solution immediately
1mL surveys rapidly absorbance after reacting 5min in 25 DEG C of water-baths after mixing at 475nm wavelength.Record different samples
The absorbance of solution various concentration gradient simultaneously calculates respective inhibitory activity against tyrosinase.
(3) inhibitory activity against tyrosinase (%)=[(A1- A2)-(A3- A4)]/(A1- A2)×100.According to
The light absorption value A measured at 475nm wavelength calculates different inhibitory activity against tyrosinase.Table 3, table 4 are respectively various concentration
The inhibiting rate of VC and scallop edge whitening peptide of the present invention to tyrosinase activity.
Inhibiting rate of 3 VC of table to tyrosinase activity
Inhibiting rate of the 4 scallop edge whitening peptide of table to tyrosinase activity
Using VC as standard reference material, 98.8% (concentration 0.2mg/ml) is up to the inhibiting rate of tyrosinase activity；
Scallop edge polypeptide is up to 45.3% (concentration 0.16mg/ml), the inhibiting rate of two samples to the inhibiting rate of tyrosinase activity
It all reduces and reduces with concentration.
In conclusion scallop edge polypeptide has a degree of whitening active, scallop edge polypeptide can be used to make up by explanation
In conduct industry, to develop the marine active substance with whitening active.
The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field
For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair
Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.
Claims (7)
1. a kind of preparation method of scallop edge whitening peptide, which is characterized in that the preparation method comprises the following steps:
(1) scallop edge pre-processes: scallop edge successively being rinsed well by scallop edge tap water and distilled water, is shredded, with double steamings
Water impregnates 0.4~1h, removes water, weighing；
It is homogenized using refiner, 6~10 times of water of scallop edge quality is added, obtains scallop edge homogenate；
(2) it digests: sequentially adding pepsin in the scallop edge homogenate in step (1) and trypsase is digested, obtain
Enzymolysis liquid；
When being digested using pepsin, the addition quality of pepsin is the 0.3~0.5% of scallop edge, hydrolysis temperature is 45~
55 DEG C, enzymatic hydrolysis pH is 2.0~2.5, and enzymolysis time is 4~6h；
When using trypsin digestion, the addition quality of trypsase is the 0.4~0.5% of scallop edge, hydrolysis temperature is 45~
55 DEG C, enzymatic hydrolysis pH is 8.0~8.5, and enzymolysis time is 4~6h；
After trypsin digestion, enzyme digestion reaction, specific high-temperature inactivation condition are stopped using high-temperature inactivation mode are as follows: inactivation temperature
85~90 DEG C of degree, inactivation time are 3~8 minutes；Enzymolysis liquid is then quickly cooled to 30 DEG C hereinafter, placing 20~40 minutes
Thoroughly stop the progress of reaction；
(3) it is taken after being centrifuged 10~30 minutes under the conditions of the pH to 6.8~7.2,10000rpm for the enzymolysis liquid that regulating step (2) obtains
Supernatant is concentrated, into concentrate be added dehydrated alcohol to dehydrated alcohol account for the 50~70% of overall solution volume, stir into
The reaction of row alcohol precipitation, centrifugation 10 under the conditions of 10000rpm~after twenty minutes up to scallop edge polypeptide crude extract；
Take scallop edge polypeptide crude extract through 5000Da ceramic membrane ultrafitration, filtered solution is through vacuum-concentrcted, and after being spray-dried
Obtain scallop edge whitening peptide.
2. preparation method as described in claim 1, which is characterized in that in step (2), when being digested using pepsin, stomach egg
The addition quality of white enzyme is the 0.4% of scallop edge, and hydrolysis temperature is 50 DEG C, and enzymatic hydrolysis pH is 2.3, enzymolysis time 5h.
3. preparation method as described in claim 1, which is characterized in that when in step (2) using trypsin digestion, tryptose
The addition quality of enzyme is the 0.45% of scallop edge, and hydrolysis temperature is 50 DEG C, and enzymatic hydrolysis pH is 8.2, enzymolysis time 5h.
4. preparation method as described in claim 1, which is characterized in that step (2) high temperature inactivates condition are as follows: inactivation temperature is
87 DEG C, inactivation time is 5 minutes；Enzymolysis liquid is then quickly cooled to 30 DEG C to come within 30 minutes thoroughly to stop reacting hereinafter, placing
Progress.
5. preparation method as described in claim 1, which is characterized in that in step (3), enzymolysis liquid that regulating step (2) obtains
PH to 7.0, centrifugation take supernatant to be concentrated after twenty minutes, into concentrate be added dehydrated alcohol to dehydrated alcohol account for solution
The 60% of total volume, stirring carry out alcohol precipitation reaction, and supernatant is scallop edge polypeptide crude extract after centrifugation 15 minutes.
6. the scallop edge whitening peptide that any one of the claim 1-5 preparation method is prepared.
7. scallop edge whitening peptide described in claim 6 is preparing the application in skin-lightening cosmetic.
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| CN101701241A (en) * | 2009-11-05 | 2010-05-05 | 山东好当家海洋发展股份有限公司 | Preparation method of scallop active peptide |
| CN105385744A (en) * | 2015-12-29 | 2016-03-09 | 广西钦州市绿源天然食品加工有限公司 | Preparation method of scallop brim polypeptide extractive |
| CN105640862A (en) * | 2016-03-24 | 2016-06-08 | 王伟娜 | Skin beautifying cosmetic for whitening, moisture preserving, anti-wrinkling and pore shrinking |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101701241A (en) * | 2009-11-05 | 2010-05-05 | 山东好当家海洋发展股份有限公司 | Preparation method of scallop active peptide |
| CN105385744A (en) * | 2015-12-29 | 2016-03-09 | 广西钦州市绿源天然食品加工有限公司 | Preparation method of scallop brim polypeptide extractive |
| CN105640862A (en) * | 2016-03-24 | 2016-06-08 | 王伟娜 | Skin beautifying cosmetic for whitening, moisture preserving, anti-wrinkling and pore shrinking |
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