Patent Application
20180258147
The present invention relates to the field of pearl extract production, in particular, relates to a pearl protein preparation method and a water-soluble pearl protein and acid-soluble pearl protein obtained by adopting this method.
Currently, in the application of pearl powder, numerous problems such as grain size, potential safety hazard, poor liquidity, water insolubility and difficulty in the release of the functional components have been encountered, which directly affect the application fields of pearl powder. To solve the problems above, water-soluble pearl powder and pearl hydrolysate have been developed. However, during these processes, we only conduct simple compounding of the original components or extensive hydrolysis, while no research was performed on the functions of each component therein, and fine separation and purification were rarely conducted.
The existing pearl extracts are generally produced by adopting enzymolysis method. For example, the Chinese invention patent (application number: 01139157.X; application date: Dec. 19, 2001) discloses a pearl powder preparation method by biological enzymolysis. The key lies in the use of lactic acid solution as a solvent for dissolving the pearl, adjusting the pH of the solution to near neutral, readjusting it to a meta-acid by using acid, and then adding appropriate amounts of biological active enzyme to realize catalytic decomposition of the protein to obtain an enzymatic pearl glue solution, adopting spray drying to realize rapid drying of the enzymatic pearl glue solution, curing and pelletizing, and obtaining the superfine enzymatic pearl powder. The pearl powder prepared reserves effective components such as various amino acids and microelements of the pearl, and the calcium carbonate of the pearl are translated into active calcium that is easily absorbed by the human body, and the keratin protein insoluble in gastric acid are converted to free amino acids that are easily absorbed by enzymolysis.
The Chinese invention patent (application number: 200710141569.X; application date: Aug. 7, 2007) discloses a pearl bioactive peptide preparation method as follows: taking out pearl from fresh pearl shell, mixing pearl with deionized water, adjusting PH of the mixed solution to a meta-acid with phosphate buffer solution, and then using serrapeptass to realize an enzymatic hydrolysis reaction with pearl; obtaining pearl and a clear solution by filtering separation with filters, boiling the reaction solution for enzyme deactivation; and then performing spray drying of the obtained solution to obtain a pearl bioactive peptide.
The Chinese invention patent (application number: 200910114409.5; application date: Sep. 18, 2009) discloses a pearl hydrolysate preparation method, with steps as follows: (1) putting pearl powder in hydrochloric acid water solution, stirring it until full reaction, adjusting pH of this mixture to neutral with ammonium hydroxide, and performing centrifugation to obtain sediment A; (2) dissolving the sediment A in purified water, adjusting the pH value to 7.5˜8.5 with ammonium hydroxide, and performing centrifugation to obtain sediment B; (3) dissolving the sediment B in purified water, realizing enzymolysis by adding in an enzyme, performing centrifugation to the enzymatic hydrolysate, and obtaining a supernatant and sediment C; wherein the named enzyme is a mixture of snail protease and papain, with an addition quantity of 0.3˜1.05% based on a weight of sediment B; (4) dissolving the sediment C in purified water, adding in an enzyme to perform secondary enzymolysis, performing centrifugation, and collecting the supernatant; wherein the named enzyme is a mixture of trypsin, papain and neutral protease, with an addition quantity of 0.2˜0.45% based on a weight of sediment B; (5) combining the two supernatants, boiling it, filtering by using a nanofiltration membrane with a pore diameter 1˜3 nm, and obtaining the pearl hydrolysate.
The aforementioned methods adopt enzymolysis, which can be used to obtain amino acids and polypeptide with low molecular weight, however, enzymolysis can destroy some activated protein substances and simultaneously cause loss of the active substances.
The Chinese invention patent (application number: 201410055730.1; application date: Feb. 19, 2014) discloses a nanoscale pearl protein preparation method as follows: grinding pearl powder into micro pearl powder having mean grain size of 1˜4 micrometers, performing acidolysis and filtering, washing the filtered residue with purified water to obtain coarse pearl protein, adding a dispersing agent to the coarse pearl protein and grinding it, and performing ultrasonic oscillation during the process; concentrating the solution obtained after grinding, heating it for sterilization, and saving it to obtain a nanoscale pearl protein. In this method, a dispersing agent is added, which is hard to be eliminated in subsequent processing steps, hence limiting the application of the product.
To solve the aforementioned technical problem, this invention aims to provide a pearl protein preparation method as follows: a nanoscale pearl powder is used, desalination is performed after hydrolysis and acidolysis, a water-soluble pearl protein and an acid-soluble pearl protein are obtained respectively, which provides raw materials for pearl skin care products and health care products.
To realize the purpose above, this invention adopts the technical solution below:
A pearl protein preparation method, the method includes the following steps:
Preferably, a grain size of the described nanoscale pearl powder is 10˜100 nm.
Preferably, the described weak acid adopts edible acetic acid, citric acid or carbonic acid.
Preferably, the described desalination adopts dialysis desalination: the desalination is performed by using a dialysis bag with molecular weight of 3000˜5000 Da at a 2˜10° C. environment for 3-5 days, wherein 1 volume of sample corresponds with 10-20 volumes of distilled water, and water needs to be changed 1-2 times each day.
Preferably, the described desalination adopts chromatography desalination: using Sephadex G-15 for column packing, start to collect after first desalination and sample adding, collect once every 1-2 minutes, gather several samples, add in little amounts of ammonium sulfate respectively, determine if sediments are present, verify which of the samples have salt out, and collect solutions before this time point which is to be treated as a protein solution after desalination.
As to a water-soluble pearl protein prepared by adopting the method stated in this invention, the water-soluble pearl protein has a main molecular weight distribution of less than 15 kd. “Main” of this invention refers to over 90% of the substances in this protein.
As to an acid-soluble pearl protein prepared by adopting the method stated in this invention, the acid-soluble pearl protein has a main molecular weight distribution of 14 kd˜50 kd. “Main” of this invention refers to over 90% of the substances in this protein.
As this invention adopts the aforementioned technical solution, which uses nanoscale pearl powder, performs desalination after hydrolysis and acidolysis to obtain water-soluble pearl protein and acid-soluble pearl protein respectively, raw materials for pearl skin care products and health care products can be provided. Specifically, acid-soluble pearl protein possesses strong antioxidant capacity, can promote fibroblast growth, simultaneously endorse the secretion of collagen, is favorable for wound healing, and has powerful potential in the field of cosmetic treatment. On the other hand, water-soluble pearl protein can obtain favorable effect in aspects of skin whitening and moisturizing.
A pearl protein preparation method, the method includes the following steps:
{circle around (1)} Dialysis desalination: the desalination is performed by using a dialysis bag with molecular weight of 3500 Da at a 4˜7° C. environment for 4 days, wherein 1 volume of sample corresponds with 10-15 volumes of distilled water, and water needs to be changed 1 time each day.
Or {circle around (2)} chromatography desalination: Sephadex G-15 was used for column packing, start to collect after first desalination and sample adding, collect once every 1 minute, gather 15 samples, add in little amounts of ammonium sulfate respectively, determine whether sediments are present, verify which of the samples have salt out in this step, and collect solutions before this time point which is to be treated as a protein solution after desalination; finally, a water-soluble pearl protein a and an acid-soluble pearl protein b can be obtained correspondingly;
Molecular weight detection of different pearl proteins, using test method: SDS-PAGE detection method
a) Take 20 μl of a 1 times diluted water-soluble pearl protein and acid-soluble pearl protein respectively, add in 5 μl of a 5-times concentrated sample buffer solution respectively, and boil at an 80° C. water bath kettle for 5 min;
b) Prepare SDS-PAGE gel according to the recipe above, install an electrophoresis apparatus, and add electrode buffer solution in the trough;
c) Add samples in sample adding holes in a particular sequence, wherein an amount of added sample is selected based on a difference between a gap thickness of the SDS-PAGE electrophoresis glass plates. In general, for 0.75 mm gap with 15 holes, a sample adding amount is less than 10 μL/hole, for 1 mm gap with 10 holes, a sample adding amount is less than 20 μL/hole. The sample adding amount may be determined according to practical experience;
d) Open power supply, adjust voltage to 80v (generally takes about 15 min), and adjust voltage to 120v after the sample passes through the compressed gel, maintain the voltage until the electrophoresis of the sample reaches the bottom of the gel;
e) Carefully remove the gel and placing it into a container, add in staining fluid, and shake for 2-3 h with a shaker;
f) Pour out the staining fluid after the stripe becomes clear, then add in a destaining solution, and leave overnight;
g) Pour away the destaining solution, use Quality One, or a corresponding imaging system to take a picture, then analyze and estimate the protein concentration.
Test Results
1. Research on the Influence of Different Pearl Proteins on Fibroblast Growth
a) Test Method
Cell Growth Curve Diagram Test
Influence of different pearl proteins on fibroblast proliferation can be reflected by detecting e number of cells in an 8-day cultivation process. The acid-soluble pearl protein and the water-soluble pearl protein were each prepared into a series of 15.6 μg/ml medium, and 1% calf serum was added; take 1% of calf serum culture medium as blank contrast. The number of cells were counted using cell counting plates each day.
b) Test Results
As shown in
2. Research on DDPH Free Radical Scavenging Abilities of Different Pearl Proteins
a) Test Method
DPPH Free Radical Inhibiting Ability
Test steps: preparing DPPH95% ethanol solution with a concentration of 2×10−4 mol/L, and protecting it from light. Taking 2 mlDPPH solution, and adding in 2 ml of acid-soluble pearl protein solution or water-soluble pearl protein solution with different concentrations respectively, mixing it well, reacting it for 30 min at 25° C., measuring the absorption value at 517 nm (the maximum absorption wavelength), calculating the scavenging rate, and taking it as reference.
Calculation formula of DPPH free radical scavenging activity:
P=[(1−(Ai−Aj)/Ac)]×100%
In the formula: Ai=absorbance of 2 mlDPPH solution+2 ml sample solution.
Aj=absorbance of 2 ml sample solution+2 ml solvent.
Ac=absorbance of 2 mlDPPH solution+2 ml solvent.
b) Test Results
As shown in
3. Influence of Different Pearl Proteins on Collagen Generation of Cells
a) Test Method
Hydroxyproline Test
The collagen products of fibroblast can be evaluated by quantifying the amount of hydroxyproline. Fibroblasts are inoculated in a 24-hole plate, and treated in the presence of an acid-soluble pearl protein with a concentration of 15.6 μg/ml for 72 hours. The 1% calf serum culture medium and 10% calf serum culture medium are used as blank contrast and positive contrast respectively. Chloramine T method is used for detecting the content of hydroxyproline. The data refers to microgram of the collagen in 106 cells, assuming that there are 13.5% hydroxyprolines contained in collagen.
b) Test Result
As shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 201510020372.5 | Jan 2015 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2015/093020 | 10/28/2015 | WO | 00 |
