CN106866807A - The preparation method of pearl protein, the pearl protein prepared by the method and its application - Google Patents
The preparation method of pearl protein, the pearl protein prepared by the method and its application Download PDFInfo
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- CN106866807A CN106866807A CN201510929202.9A CN201510929202A CN106866807A CN 106866807 A CN106866807 A CN 106866807A CN 201510929202 A CN201510929202 A CN 201510929202A CN 106866807 A CN106866807 A CN 106866807A
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
-
- 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
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/74—Biological properties of particular ingredients
- A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
- A61K2800/782—Enzyme inhibitors; Enzyme antagonists
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Toxicology (AREA)
- Biophysics (AREA)
- Dermatology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Birds (AREA)
- Cosmetics (AREA)
Abstract
Preparation method the invention discloses pearl protein, the pearl protein by the method preparation and its application.Effective substance it is an object of the invention to determine pearl whitening activity, there is the activity of preferable antityrosinase by an albuminoid in research discovery pearl, medicine and cosmetics can be used for as the raw material of whitening product, preparation method to such pearl protein is investigated, and determines preparation technology.
Description
Technical field
The present invention relates to medicine and cosmetic technical field, specifically, preparation method the present invention relates to pearl protein,
The pearl protein prepared by the method and its application.
Background technology
Pearl is recorded in Chinese Pharmacopoeia (version in 2015), is Pteriidae animal Pinctada martensii Pteria martensii
(Dunker), Unionidae animal hydriopsis cumingii Hyriopsis cumingii (Lea) or cristaria plicata Cristaria plicata
(Leach) the stimulated formation of bivalve such as, its nature and flavor is sweet, salty, cold；Enter the heart, Liver Channel, pearl is China's traditional Chinese medicine,
With arresting convulsion of calming the nerves, improving eyesight disappears screen, the effects such as solve taking poison and growing muscle due, moistening skin and eliminating spot.Modern pharmacology research shows that pearl has calmness
Calm the nerves, anti-oxidant, anti-inflammatory, promote the effect such as wound healing effect, hypotensive.Pearl is shell marine drug, contains 90%
Calcium carbonate above, in addition, also contains a small amount of water-solubility protein, angle glutelin, peptides, trace element etc..
China is pearl big country, with hydriopsis cumingii as Main Resources, main product in China south China, East China and other places, with Jiangsu Weihe
The pool and the main product of Zhuji, zhejiang two ground are representative.Early stage people are most for the use of pearl in ornament, jewel etc., with science and technology
Development and the improvement of people's living standards, many medicinal efficacies of pearl are gradually recognized, such as because of its whitening, the work(of moisturizing
Effect is widely used in the skin type product such as cosmetic additive agent, facial mask.The principle of whitening spot-removing is commonly considered as inhibiting melanin
Generation.Melanin is, with tyrosine as matrix, DOPA, DOPA quinone, istain, DOPA to be converted into the presence of tyrosinase
Pigment etc., various intermediate oxidation material polymerizations, ultimately forms melanin.In this process, tyrosinase is that melanin is closed
Major rate-limiting enzyme during, therefore suppress the activity of tyrosinase, the generation of melanin is reduced, the mesh of whitening can be reached
's.
But, the whitening product with pearl as raw material is mostly used with full powder at present, including ordinary pearl powder, granularity 200
More than mesh, national basic demand can be reached；Superfine pearl powder, more than the mesh of fineness 1000 (or below 15 microns of particle diameter)；Nanometer
Pearl powder, more than the mesh of fineness 150,000 (or less than 100 nanometers).It is generally believed that pearl powder is thinner, its silty is finer and smoother, absorptivity
Higher, effect is more preferable.But the effective substance for pearl whitening activity is still not clear, and lacks system research, on phase
The basic blank of exploitation of product is closed, blindness that pearl uses is result in and active component utilization ratio is low.
The content of the invention
In order to overcome the above not enough, present inventor on the basis of early-stage Study, according to the property of albumen in pearl
Classified, and carried out the screening of antityrosinase activity, to determine the effective substance of pearl whitening activity, and conduct is opened
The raw material of pearl whitening nti-freckle product is sent out, the value of pearl is substantially increased, increases economic benefit.
Therefore, it is an object of the present invention to provide a kind of method for preparing pearl angle glutelin, the method has technique
Simply, low cost, it is reproducible, it is easy to the features such as industrialized production.
It is a further object to provide pearl angle glutelin, its by above-mentioned preparation pearl angle glutelin method system
It is standby.
A further object of the present invention is to provide purposes of the above-mentioned pearl angle glutelin in whitening product is prepared.
It is also another object of the present invention to provide a kind of method for preparing pearl protein, the pearl protein includes water-soluble egg
In vain, sour molten albumen and angle glutelin, the method have process is simple, and low cost is reproducible, it is easy to the spy such as industrialized production
Point.
It is also another object of the present invention to provide pearl aqueous soluble protein, the pearl molten albumen of acid or pearl angle glutelin, its by
It is prepared by the above-mentioned method for preparing pearl protein.
Exist it is also another object of the present invention to provide above-mentioned pearl aqueous soluble protein, the molten albumen of pearl acid or pearl angle glutelin
Prepare the purposes in whitening product.
According to an aspect of the invention, there is provided a kind of method for preparing pearl angle glutelin, it includes：
Added in appropriate Pearl 5-100 times of volume (preferably 10-50 times volume, more preferably 10-20 times volume, most
It is preferred that 10 times of volumes) acid (preferably 5%-30% hydrochloric acid or acetic acid, more preferably 10% hydrochloric acid or acetic acid), under agitation, 0
8-24 hour (preferably 12-20 hours, more preferably 12 hours) is processed under DEG C -8 DEG C (preferably 2 DEG C -6 DEG C, more preferably 4 DEG C), is centrifuged
(preferably 4000rev/min-12000rev/min, more preferably 8000rev/min-12000rev/min, most preferably 12000rev/
Min) it is precipitated, gained precipitation is lyophilized to be angle glutelin.
According to another aspect of the present invention, there is provided a kind of pearl angle glutelin, it is by above-mentioned preparation pearl angle shell egg
It is prepared by white method.
It is very high by the amino acid content in the pearl angle glutelin that the above method is obtained in the present invention, mainly containing sweet ammonia
Acid, alanine, aspartic acid etc., and experiments verify that the activity with suppression tyrosinase very high.
According to a further aspect of the invention, there is provided purposes of the above-mentioned pearl angle glutelin in whitening product is prepared.
In the present invention, it is preferable that above-mentioned whitening product is food, medicine, cosmetics or health products etc..
According to another aspect of the present invention, there is provided a kind of method for preparing pearl aqueous soluble protein, it includes：
Added in appropriate Pearl 5-100 times of volume (preferably 10-50 times volume, more preferably 10-20 times volume, most
It is preferred that 10 times of volumes) water, (preferably 20-48 is small within cold soaking 16-48 hours under 0 DEG C -8 DEG C (preferably 2 DEG C -6 DEG C, more preferably 4 DEG C)
When, more preferably 24 hours) after extract solution is filtered, filtrate is obtained the water-soluble egg of pearl using ammonium sulfate precipitation method or direct desivac
In vain.
According to another aspect of the present invention, there is provided a kind of pearl aqueous soluble protein, it is by the above-mentioned aqueous soluble protein for preparing
It is prepared by method.
According to another aspect of the present invention, there is provided purposes of the above-mentioned pearl aqueous soluble protein in whitening product is prepared.
In the present invention, it is preferable that above-mentioned whitening product is food, medicine, cosmetics or health products etc..
According to another aspect of the present invention, there is provided a kind of side for preparing the molten albumen of pearl acid or pearl angle glutelin
Method, it includes：
After extract solution is filtered in the method for above-mentioned preparation pearl aqueous soluble protein, filter residue is taken, add 5-100 times of volume
Acid (preferably 5%-30% hydrochloric acid or the vinegar of (preferably 10-50 times volume, more preferably 10-20 times volume, most preferably 10 times volumes)
Acid, more preferably 10% hydrochloric acid or acetic acid), under agitation, 8-24 is processed under 0 DEG C -8 DEG C (preferably 2 DEG C -6 DEG C, more preferably 4 DEG C)
Hour (preferably 12-20 hours, more preferably 12 hours), and centrifugation (preferably 4000rev/min-12000rev/min, more preferably
8000rev/min-12000rev/min, most preferably 12000rev/min) supernatant and precipitation are obtained, gained supernatant is used and is cut
Bag filter dialysis 40-80 hour (preferably 40-50 hour, more preferably 48 hour) of the molecular weight less than 3000 is stayed, by bag filter
Gained material is lyophilized to be sour molten albumen；Gained precipitation is freezed and obtains final product pearl angle glutelin.
According to another aspect of the present invention, there is provided a kind of molten albumen of pearl acid or pearl angle glutelin, it is by above-mentioned
It is prepared by the method for preparing sour molten albumen or pearl angle glutelin.
According to another aspect of the present invention, there is provided the molten albumen of above-mentioned pearl acid or pearl angle glutelin are preparing whitening
Purposes in product.
In the present invention, it is preferable that above-mentioned whitening product is food, medicine, cosmetics or health products etc..
According to a further aspect of the invention, there is provided a kind of method for preparing pearl protein, it includes：
Step 1) 5-100 times of volume (preferably 10-50 times volume, more preferably 10-20 times body are added in appropriate Pearl
Product, most preferably 10 times volumes) water, cold soaking 16-48 hours (preferably under 0 DEG C -8 DEG C (preferably 2 DEG C -6 DEG C, more preferably 4 DEG C)
20-48 hours, more preferably 24 hours) after extract solution is filtered, filtrate is obtained treasure using ammonium sulfate precipitation method or direct desivac
Pearl aqueous soluble protein；
Step 2) in step 1) in extract solution is filtered after, take filter residue, add 5-100 times of volume (preferably 10-50 times body
Product, more preferably 10-20 times volume, most preferably 10 times volumes) acid (preferably 5%-30% hydrochloric acid or acetic acid, more preferably 10% salt
Acid or acetic acid), under agitation, (preferably 12-20 is small within 8-24 hours for treatment under 0 DEG C -8 DEG C (preferably 2 DEG C -6 DEG C, more preferably 4 DEG C)
When, more preferably 12 hours), (preferably 4000rev/min-12000rev/min, more preferably 8000rev/min- is centrifuged
12000rev/min, most preferably 12000rev/min) supernatant and precipitation are obtained, gained supernatant is less than with molecular cut off
3000 40-80 hours (preferably 40-50 hours, more preferably 48 hours) of bag filter dialysis, gained material in bag filter is freezed
As sour molten albumen, gained precipitation is lyophilized to be angle glutelin.
According to a further aspect of the invention, there is provided pearl aqueous soluble protein, the molten albumen of pearl acid or pearl angle glutelin,
It is prepared by the above-mentioned method for preparing pearl protein.
Experiments verify that, in the pearl protein by the preparation method preparation of above-mentioned pearl protein, amino in the glutelin of angle
Acid content highest, different parts amino acid content differs greatly, and angle glutelin mainly contains glycine, alanine, aspartic acid, water
Molten albumen mainly includes glycine and alanine, and wherein aspartic acid, leucine, serine content is also of a relatively high, sour molten egg
Amino acid content is less in white.
Still another aspect of the invention, there is provided purposes of the above-mentioned pearl protein in whitening product is prepared.
In the present invention, it is preferable that above-mentioned whitening product is food, medicine, cosmetics or health products etc..
The pearl protein that the present invention is obtained is all-natural product, and the work for suppressing tyrosinase is respectively provided with through screening active ingredients
Property, particularly, angle glutelin has the activity of suppression tyrosinase higher.The extraction separation method technique letter that the present invention is used
Single, low cost is reproducible, it is easy to industrialized production.The product obtained by the method can as the raw material of whitening product,
Can be used for medicine and cosmetics.
Brief description of the drawings
Fig. 1 is the process chart of the extraction process according to the embodiment of the present invention 1.
Fig. 2 is according to content assaying method [the Journal of Separation of amino acid reported in the literature
Science.2015,38:1552-1560.], the ammonia of aqueous soluble protein, sour molten albumen and angle glutelin to being extracted in embodiment 1
The figure of the result that base acid is measured.
Fig. 3 is according to content assaying method [the Journal of Separation of amino acid reported in the literature
Science.2015,38:1552-1560.], the result being measured to the amino acid of the angle glutelin of extraction in embodiment 2
Figure.
Fig. 4 is the junket ammonia of aqueous soluble protein, sour molten albumen and angle glutelin in the pearl extracted according to embodiments of the invention 1
Sour enzyme inhibition rate curve.
Fig. 5 is the tyrosinase inhibition rate curve of the angle glutelin extracted according to embodiments of the invention 2.
Specific embodiment
With reference to embodiment, the invention will be further elaborated, and implementation below only describes this by way of example
Invention.It is obvious that those of ordinary skill in the art can in the scope of the present invention and essence, the present invention is carried out it is various flexible and
Modification.It is to be understood that this invention is intended to cover the accommodation and modification that include in the following claims.
Embodiment
Material, instrument and reagent：
The pearl place of production used is Zhuji, zhejiang in the present embodiment, is studied through Shanghai Pharmaceutical Inst., Chinese Academy of Sciences's fruit Dean
Member identifies source for Unionidae animal hydriopsis cumingii Hyriopsis cumingii (Lea).
Hydrochloric acid, NaOH, methyl alcohol, ethanol, dimethyl sulfoxide (DMSO) (DMSO), bag filter (molecular cut off 3000), ice vinegar
Acid, ammonium sulfate, TYR, disodium hydrogen phosphate, sodium dihydrogen phosphate are purchased from Chemical Reagent Co., Ltd., Sinopharm Group；Tyrosinase
Purchased from Sigma companies；Vitamin C is biological purchased from Shanghai source leaf；Water：It is prepared by Mili-Q ultra-pure waters instrument.
BRANSON B3500S-DTH ultrasonic washing instruments：Holland must believe Co., Ltd；Turbine mixer：
SCIENTIFIC INDUSTTRIES, INC；Freeze drier：German Alpha.ELIASA：BioTek SYNFRGY4
(BioTek, Winooski, USA).
Embodiment 1：The extraction of pearl protein
1st, the extraction of aqueous soluble protein：
The extraction of aqueous soluble protein uses the cold-maceration, specific method to be：Take medicinal powder (according to《Chinese Pharmacopoeia》Version in 2010
It is required that, crushing pearl is into most fine powder) 100g, 10 times of volume of water are added, cold soaking filtered extract solution after 24 hours at 4 DEG C, filtered
Liquid is respectively adopted ammonium sulfate precipitation or directly lyophilized method is processed, and takes product obtained above, is reported according in document
Amino acid content assaying method [Journal of Separation Science.2015,38:1552-1560.], determine
The content of amino acid, the results are shown in Table 1 in two methods gained albumen, it can be seen that amino acid in direct desivac gained albumen
Content is higher than ammonium sulfate precipitation, and operates relative ease.
Amino acid content in the different preparation method gained aqueous soluble proteins of table 1
2nd, sour molten albumen, the extraction of angle glutelin：
The molten albumen of acid and angle glutelin derive from water-insoluble part, and specific preparation method is：It is water-soluble in above-mentioned part 1
After the completion of protein extraction, filtering takes filter residue, 10 times the 10% of volume of acid (hydrochloric acid or acetic acid) is added, under agitation at 4 DEG C
Reason 12h, supernatant and precipitation, the bag filter (retention point of gained supernatant are obtained by the high speed centrifugation under 12000rev/min
Less than the 48h that 3000) dialyses, except deionization, in bag filter, gained material is lyophilized is sour molten albumen to son amount, and gained precipitation is lyophilized i.e.
It is angle glutelin.
10% hydrochloric acid and 10% acetic acid are investigated, sour molten albumen obtained above and angle glutelin has been taken respectively, according to text
Offer content assaying method [the Journal of Separation Science.2015,38 of the amino acid of middle report:1552-
1560.], the content of amino acid in gained albumen is determined respectively, the results are shown in Table 2 and table 3, it is either sour molten as can be seen from the results
Albumen or angle glutelin, the content of amino acid is all higher than acetic acid in gained albumen during using hydrochloric acid.
Amino acid content in the sour molten albumen of different acid treatment gained of table 2
Amino acid content in the different acid treatment gained angle glutelins of table 3
Conclusion
It is final to determine three albuminoids (that is, aqueous soluble protein, sour molten egg in pearl and mother-of-pearl by the optimization of extraction conditions
In vain, angle glutelin) extraction process, technological process is as shown in Figure 1.
3rd, the assay of three albuminoids：
According to content assaying method [the Journal of Separation of amino acid reported in the literature
Science.2015,38:1552-1560.], the amino acid to three albuminoids is determined, as a result as shown in table 4.Extract
Amino acid content highest in the glutelin of angle, different parts amino acid content differs greatly, and angle glutelin mainly contains glycine, the third ammonia
Acid, aspartic acid, aqueous soluble protein mainly include glycine and alanine, wherein aspartic acid, leucine, serine content also phase
To higher, amino acid content is all less in sour molten albumen, and chromatogram is as shown in Figure 2.The yield of three kinds of albumen is respectively in pearl：
Angle glutelin 1.30%, sour molten albumen 0.40%, aqueous soluble protein 0.16%.
In the pearl of table 4 in three albuminoids amino acid comparision contents (%)
Embodiment 2：The preparation of angle glutelin in pearl
The preparation of angle glutelin, specific method is：Take medicinal powder (according to《Chinese Pharmacopoeia》Version requirement in 2010, pearl
It is ground into most fine powder) 100g, 10 times the 10% of volume of acid (hydrochloric acid or acetic acid) is added, 12h is processed in 4 DEG C under agitation, lead to
The high speed centrifugation under 12000rev/min is crossed, gained precipitation is lyophilized to be angle glutelin.
10% hydrochloric acid and 10% acetic acid are investigated, angle glutelin obtained above has been taken, according to amino reported in the literature
Content assaying method [the Journal of Separation Science.2015,38 of acid:1552-1560.], institute is determined respectively
The content of amino acid in albumen, 5 are the results are shown in Table, from the results, it was seen that the content of amino acid is all in albumen during using hydrochloric acid
Higher than acetic acid.
The content of amino acid in the different acid treatment gained angle glutelins of table 5
The assay of angle glutelin
According to content assaying method [the Journal of Separation of amino acid reported in the literature
Science.2015,38:1552-1560.], the amino acid to constituting angle glutelin is measured, as a result as shown in table 6.Composition
The amino acid of albumen mainly has glycine, alanine and aspartic acid, and chromatogram is as shown in Figure 3.Angle glutelin obtains in pearl
Rate is 1.30%.
Angle glutelin amino acid content (%) in the pearl of table 6
Embodiment 3：The measure of the whitening active of pearl protein prepared by embodiment 1
The preparation of phosphate buffer solution：
Disodium hydrogen phosphate 13.40g is taken in 250mL measuring bottles, plus deionized water dissolving, scale is settled to, shake up；Take phosphoric acid
Sodium dihydrogen 6.90g is in 250mL measuring bottles, plus deionized water dissolving, is settled to scale, shakes up；It is each that precision pipettes above-mentioned two solution
50mL is in 250mL measuring bottles, plus deionized water is diluted to scale, obtains final product the phosphate buffer solution of pH 6.8.
The preparation of TYR solution：
Take TYR 3.7mg accurately weighed, in 50mL measuring bottles, plus phosphate buffer solution dissolving, scale is settled to, shake
It is even, obtain final product the TYR solution that concentration is 74 μ g/mL.
The preparation of tyrosinase solution：
This experiment is 5771U/mg with tyrosinase specification, weighs tyrosinase 0.6065mg in 10mL measuring bottles, in ice
Add phosphate buffer solution to dissolve and be settled to scale in water-bath, shake up, obtain 350U/mL tyrosinase solutions.
The preparation of sample and reference substance：
Obtained pearl aqueous soluble protein, sour molten albumen and angle glutelin are appropriate during precision weighs embodiment 1, are configured to respectively
0.2,0.4,0.6,0.8,1.0,1.2mg/ml six solution of concentration, positive control Vc is made into 0.2,0.4,0.6,0.8,1.0,
Six solution of concentration of 1.2mg/ml, wherein water-solubility protein and Vc are dissolved in water, and sour molten albumen and angle glutelin are dissolved in DMSO.
Three kinds of measure of albumen whitening active in pearl：
By each concentration samples solution and positive control respectively according to the volume shown in table 7, by sample, buffer solution and L- junket ammonia
Acid is added in 96 orifice plates, after reaction 10min, adds tyrosinase, continues to react 10min, is put into ELIASA rapidly, is read
The absorbance of each reaction system under 475nm, and the tyrosinase inhibition rate of each position albumen is calculated according to equation below.
Inhibiting rate %=(C-D)/A × 100%
A：Absorbance of the tyrosinase in blank solvent
C：Absorbance of the tyrosinase in sample solution
D：The absorbance of blank solvent
The tyrosinase of table 7 suppresses reaction system institute solubilizer
Experimental result：
Calculated according to the absorbance for determining, the tyrosinase inhibition rate curve of the albuminoid of pearl three is drawn, such as Fig. 4 institutes
Show, it can be seen that sour molten protein concentration be more than 0.8mg/ml when inhibiting rate decline, therefore consider albumen main whitening active into
It is divided into aqueous soluble protein and angle glutelin two parts, but aqueous soluble protein inhibiting rate is relatively low, the two compares and understands, the junket ammonia of angle glutelin
Sour enzyme inhibition activity is higher.
Conclusion：
The measure of tyrosinase activity is carried out to three albuminoids in pearl, to determine the drug effect of whitening active in pearl
Material base, is the main active of whitening by angle glutelin in research discovery pearl.
Embodiment 4：The measure of the whitening active of angle glutelin prepared by embodiment 2
The preparation of phosphate buffer solution：
Disodium hydrogen phosphate 13.40g is taken in 250mL measuring bottles, plus deionized water dissolving, scale is settled to, shake up；Take phosphoric acid
Sodium dihydrogen 6.90g is in 250mL measuring bottles, plus deionized water dissolving, is settled to scale, shakes up；It is each that precision pipettes above-mentioned two solution
50mL is in 250mL measuring bottles, plus deionized water is diluted to scale, obtains final product the phosphate buffer solution of pH 6.8.
The preparation of TYR solution：
Take TYR 3.7mg accurately weighed, in 50mL measuring bottles, plus phosphate buffer solution dissolving, scale is settled to, shake
It is even, obtain final product the TYR solution that concentration is 74 μ g/mL.
The preparation of tyrosinase solution：
This experiment is 5771U/mg with tyrosinase specification, weighs tyrosinase 0.6065mg in 10mL measuring bottles, in ice
Add phosphate buffer solution to dissolve and be settled to scale in water-bath, shake up, obtain 350U/mL tyrosinase solutions.
The preparation of sample and reference substance：
Obtained pearl angle glutelin is appropriate during precision weighs embodiment 2, and 0.2,0.4,0.6 is configured to respectively with DMSO,
0.8,1.0,1.2mg/ml six solution of concentration, positive control Vc water is made into 0.2,0.4,0.6,0.8,1.0,1.2mg/ml
Six solution of concentration.
The measure of angle glutelin antityrosinase activity：
By each concentration samples solution and positive control respectively according to the volume shown in table 8, by sample, buffer solution and L- junket ammonia
Acid is added in 96 orifice plates, after reaction 10min, adds tyrosinase, continues to react 10min, is put into ELIASA rapidly, is read
The absorbance of each reaction system under 475nm, and the tyrosinase inhibition rate of each position albumen is calculated according to equation below.
Inhibiting rate %=(C-D)/A × 100%
A：Absorbance of the tyrosinase in blank solvent
C：Absorbance of the tyrosinase in sample solution
D：The absorbance of blank solvent
The tyrosinase of table 8 suppresses reaction system institute solubilizer
Experimental result：
Calculated according to the absorbance for determining, draw tyrosinase inhibition rate curve, as shown in Figure 5, it can be seen that angle shell
Albumen has the inhibitory activity of moderate strength to tyrosinase.
Claims (8)
1. a kind of method for preparing pearl angle glutelin, it includes：
5-100 times of volume (preferably 10-50 times volume, most preferably more preferably 10-20 times volume, 10 times are added in Pearl
Volume) acid (preferably 5%-30% hydrochloric acid or acetic acid, more preferably 10% hydrochloric acid or acetic acid), it is under agitation, (excellent at 0 DEG C -8 DEG C
Select 2 DEG C -6 DEG C, more preferably 4 DEG C) under process 8-24 hour (preferably 12-20 hours, more preferably 12 hours), be centrifuged (preferably
4000rev/min-12000rev/min, more preferably 8000rev/min-12000rev/min, most preferably 12000rev/min)
To precipitation, gained precipitation is lyophilized to be angle glutelin.
2. a kind of pearl angle glutelin, it is prepared as the method for preparing pearl angle glutelin described in claim 1.
3. purposes of the pearl angle glutelin described in claim 2 in whitening product is prepared.
4. purposes according to claim 3, wherein, the whitening product is food, medicine, cosmetics or health products.
5. a kind of method for preparing pearl protein, it includes：
Step 1) 5-100 times of volume is added in Pearl, and (preferably 10-50 times volume, more preferably 10-20 times volume is optimal
Select 10 times of volumes) water, (preferably 20-48 is small within cold soaking 16-48 hours under 0 DEG C -8 DEG C (preferably 2 DEG C -6 DEG C, more preferably 4 DEG C)
When, more preferably 24 hours) after extract solution is filtered, filtrate is obtained the water-soluble egg of pearl using ammonium sulfate precipitation method or direct desivac
In vain；
Step 2) in step 1) in extract solution is filtered after, take filter residue, add 5-100 times of volume (preferably 10-50 times volume, more
It is preferred that 10-20 times of volume, most preferably 10 times volumes) acid (preferably 5%-30% hydrochloric acid or acetic acid, more preferably 10% hydrochloric acid or vinegar
Acid), under agitation, under 0 DEG C -8 DEG C (preferably 2 DEG C -6 DEG C, more preferably 4 DEG C) process 8-24 hours (preferably 12-20 hours, more
It is preferred that 12 hours), centrifugation (preferably 4000rev/min-12000rev/min, more preferably 8000rev/min-12000rev/min,
Most preferably 12000rev/min) supernatant and precipitation are obtained, the bag filter by gained supernatant with molecular cut off less than 3000
40-80 hours (preferably 40-50 hours, more preferably 48 hours) of dialysis, by the lyophilized as sour molten albumen of gained material in bag filter,
Gained precipitation is lyophilized to be angle glutelin.
6. pearl aqueous soluble protein, the pearl molten albumen of acid or pearl angle glutelin, it is respectively as the preparation pearl described in claim 5
The step of described in the method for albumen 1) and/or step 2) prepare.
7. the sour molten albumen of pearl aqueous soluble protein described in claim 6, pearl or pearl angle glutelin are in whitening product is prepared
Purposes.
8. purposes according to claim 7, wherein, the whitening product is food, medicine, cosmetics or health products.
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