WO2020221145A1

WO2020221145A1 - Pearl shell whitening factor and preparation method therefor and use thereof

Info

Publication number: WO2020221145A1
Application number: PCT/CN2020/086910
Authority: WO
Inventors: 王菁; 杨安全; 张丽华; 莫家欢; 谢敏
Original assignee: 欧诗漫生物股份有限公司
Priority date: 2019-04-29
Filing date: 2020-04-26
Publication date: 2020-11-05
Also published as: CN111850073B; CN111850073A

Abstract

Disclosed is a method for preparing a pearl shell whitening factor, comprising the following steps of: clam shell crushing and sieving; performing mixing with a weak acid aqueous solution, stirring for decomposition, and a solid being separated, the solid being a crude protein; cleaning the crude protein using deionized water, performing mixing with a buffer solution and performing treatment by ultra-high pressure, and then reacting with a protease under ultrasound condition; inactivating enzyme and then filter pressing to obtain an aseptic filtered fluid; freezing and drying the filtered fluid; finally, obtaining freeze-dried powder. Further disclosed is use of the pearl shell whitening factor obtained by the method in the preparation of an endothelin antagonist.

Description

Pearl oyster whitening factor, its preparation method and application

Technical field

The application relates to a pearl oyster whitening factor and a preparation method thereof, and uses of the pearl oyster whitening factor, in particular to a freeze-dried powder of the pearl oyster whitening factor and a preparation method thereof, and the antagonistic effect of the pearl oyster whitening factor on endothelin.

Background technique

Whitening is the skin care demand most concerned by Asians. The research on the mechanism of skin whitening effect, in which the inhibition of endothelin to stimulate the secretion of melanin and the inhibition of tyrosinase activity are two key links in the whitening effect. Therefore, many research units at home and abroad have carried out the search for endothelin antagonists. For example, the patent (application number: 200610030123.5, title of invention: enzyme-mediated mixed peptide of natural protein with endothelin antagonism) lists the preparation of many related protein polypeptides. However, the patent ignores many low-value hard protein products, especially the clam shell of Hyriopsis cumingii, which is currently produced in large quantities. There is a clear difference between the mussel shell and the pearl. For this reason, existing national standards provide a near-infrared identification method for identifying pearl powder and mussel shell powder. In addition, there are many differences between the two functions in Chinese medicine. There are few reports on the research of mussel shells at home and abroad, and even reports mainly focus on the research of water purification by Hyriopsis mussel. Since it was discovered last year that the fired clam shell powder has a super-strong absorption of heavy metals, we have also begun to pay more attention to the research on the clam shell, so we began to explore the ingredients in the Hyriopsis mussel to test whether it has a whitening effect and the mechanism of action how is it.

The purpose of this work is to enhance the added value of mussel shells and open up their application fields in skin care products. On the basis of the patent application of the existing pearl compound whitening factor, we propose an innovation for its preparation method. On the basis of mechanochemistry, part of the calcium in the clam shell is retained, and the protein in the clam shell is selectively Enzyme digestion, freeze-drying after the planned enzymolysis, to obtain a whitening freeze-dried powder with endothelin antagonism. This technology overcomes the instability of pearl protein hydrolysate, strict requirements on the molecular weight of active ingredients, and the exploration of optimizing the ratio of calcium ions to active peptides. These are problems that have not been considered or overcome in the prior art. Moreover, the whitening effect is obvious, meeting expectations, and the inexpensive clam shell powder can obtain significant added value, and is widely used in whitening skin care products and calcium supplement health foods.

Summary of the invention

The problem to be solved by this application is how to produce products with endothelin inhibition through low cost and large amount of by-products. Specifically: First, pretreatment: how to deal with the calcium in the clam shell, how to deal with the powder particle size of the clam shell powder; second, how to increase the enzymatic hydrolysis rate of the semi-finished clam shell; third, how to determine the type of enzyme and Degree of enzymolysis. Fourth, how to simulate the model of skin melanin production and the research of whitening mechanism.

Based on the above problems, the invention points of the present invention are: 1. Retaining part of the calcium in the clam shell is beneficial to improve skin calcium ion channels, and is beneficial to the clam shell polypeptide to cross the skin barrier and act on the melanocyte membrane. 2. The inventors of the present invention found in the research that the endothelin inhibitory effect does not need to be enzymatically hydrolyzed to polypeptides below 2000 Da, which will greatly improve the utilization rate, and the use of the cheap and large by-product of Hyriopsis mussel shell The ability to produce products with endothelin inhibition is also a discovery of waste recycling. 3. Based on the mechanochemical method to promote the opening of the clam shell protein spatial structure and promote the enzymatic hydrolysis of protease; 4. Establish a 3D endothelin melanocyte model, and scientific simulation to determine that the clam shell polypeptide components are beneficial to the antagonism of endothelin and the inhibition of melanin Transfer.

The present invention provides a method for preparing pearl oyster whitening factor. The method includes the following steps:

Step 1) After crushing the clam shells, sieving to obtain small particles of clam shell powder;

Step 2) After mixing the clam shell powder with a 10-20% weak acid aqueous solution in a mass ratio of 1:5-20, stir at 30-70°C at 100-200rpm and fully decompose for 1-4 hours. The separated solid is crude protein;

Step 3) Wash the crude protein with deionized water until the TDS value in the cleaning solution is 100-500ppm, and then mix it with the pH 6.0-7.5 buffer solution. The mass ratio of crude protein to buffer solution is 1:5-15. After the mixed solution is treated by ultra-high pressure 100-200Mpa at 40-80°C for 20-40 minutes, it is mixed and reacted with protease under ultrasonic conditions at 40-60°C for 2-7h;

Step 4) After stirring at 85-100°C for 20-50min to inactivate the enzyme, press filtration through a filter membrane to obtain a sterile filtrate;

Step 5) The freeze-dried protective agent and the filtrate are mixed in a mass-volume ratio of 0.5～4:100g/mL, pre-freeze under liquid nitrogen or -70～-85℃, and then put into a vacuum freeze-drying bin. Lyophilize for 12-20h; finally harvest the lyophilized powder with a moisture content of 2-5%.

Preferably, in step 1), the clam shell is pretreated before being crushed, that is, the clam shell is cleaned to remove skin dirt and algae, and dried; the sieving in step 1) is preferably a 200-mesh sieve.

Preferably, the weak acid in step 2) is selected from one or more of acetic acid, lactic acid, citric acid, carbonic acid, malic acid, etc.; more preferably, it is one or more of acetic acid, lactic acid or citric acid.

Preferably, the pH 6.0-7.5 buffer solution in step 3) is PBS, Tris-HCl or citrate buffer.

Preferably, the protease in step 3) is one or more of neutral protease, papain, trypsin, subtilisin, pepsin, etc.; more preferably, neutral protease, papain and trypsin One or more of.

Preferably, the mass-volume ratio of the protease and the mixed solution in step 3) is 1:100-1:10 g/mL.

Preferably, the filter membrane in step 4) is a 0.22 μm filter membrane.

Preferably, the freeze-drying protective agent in step 5) is one or more of sucrose, glucose, glutamic acid, albumin, chitosan, etc.; more preferably, sucrose, glucose or glutamic acid.

Preferably, the freeze-dried powder obtained by the preparation method of the present invention can be canned in a brown vial and stored at 1-10°C.

The invention also provides a pearl oyster whitening factor, which is prepared by the aforementioned method.

The present invention also provides the use of the pearl oyster whitening factor in the preparation of an endothelin antagonist; the endothelin antagonist can be health care products or medicines.

The pearl oyster whitening factor obtained by the method of the present invention is different from the full-component enzymatic hydrolysis and the extraction method of quantitatively selecting the molecular weight range of the proteolytic solution. The above two methods are the more common methods for extracting the pearl oyster whitening factor. Advantage of:

1. The preparation time is short. Compared with the previous extraction time, the extraction time is generally 3-7 days. The extraction time of the present invention can be controlled within 2-4 days, and the time efficiency is reduced by half.

2. The process is simple and close to non-destructive extraction. The decalcification of the present invention adopts weak acid extraction to retain acid-insoluble organic matter, and then carries out enzymatic extraction. There is no need to screen proteins for separation. If it involves extracting all the mussel shell proteins, there will be many methods involved, and other organic substances in the mussel shell, such as polysaccharides and lipids, must be removed.

3. In the similar prior art, it is necessary to screen the extract in molecular weight segment to obtain protein polypeptides within 20kda, which will cause the waste of protein above 20kda. The present invention utilizes technological improvement to increase the degree of enzymatic hydrolysis without the need for molecular weight segment screening.

4. The whitening effect is more obvious. The freeze-dried powder obtained by the present invention has more obvious whitening effect and effective concentration than the pearl endothelin antagonist sold in the market.

Description of the drawings

Figure 1 is a photo of the electrophoresis results of different samples, which can show the molecular weight distribution of their composition; among them: 1 and 6 are macromolecular standards; 5 is small molecule protein standards, from top to bottom are 12kd and 7kd; 2 is pearl As the endothelin antagonist, 3 is the pearl oyster whitening factor of the present invention, and 4 is the pearl endothelin antagonist.

Figure 2 shows the apparent results of different samples on the seventh day of gas-liquid culture after acting on the melanin model;

Figure 3 is a silver stained photo of histomorphology on the model after sampling.

Detailed ways

Example 1

A method for preparing pearl oyster whitening factor, the method comprising the following steps:

Step 1) Clean the clam shells, remove the epidermal dirt and algae, dry and pulverize, pass through a 200-mesh sieve to obtain small particles of clam shell powder;

Step 2) Mix the clam shell powder with 15% acetic acid solution in a mass ratio of 1:20, stir and fully decompose at 60°C at 200 rpm for 3 hours, and separate the solid to obtain crude protein;

Step 3) After the crude protein is washed with deionized water (the TDS value in the cleaning solution is 150ppm), it is mixed with the pH 7.5 buffer solution PBS at a mass ratio of 1:5, and the mixed solution is treated by ultra-high pressure 100Mpa at 40°C for 30min Then, it was mixed and reacted with trypsin under ultrasound at 60°C for 3 hours, and the mass-volume ratio of trypsin to the mixed solution was 1:100g/mL;

Step 4) After stirring at 85°C for 30 minutes to inactivate the enzyme, obtain a sterile filtrate by pressure filtration through a 0.22μm filter membrane, and add sucrose and albumin to the filtrate to mix; the mass-volume ratio of sucrose and filtrate is 2:100g/ mL, the mass-volume ratio of albumin to filtrate is 2:100g/mL;

Step 5) Pre-freeze the mixture at -85°C, and then freeze-dry to obtain a freeze-dried powder, which is the pearl oyster whitening factor freeze-dried powder, which has endothelin antagonism.

Example 2

Step 2) Mix the clam shell powder and the 10% lactic acid solution with a mass fraction of 1:20 at a mass ratio of 1:20, stir and fully decompose at 60°C at 200 rpm for 4 hours, and separate the solid to obtain crude protein;

Step 3) After cleaning the crude protein with deionized water (the TDS value in the cleaning solution is 100ppm), mix it with pH 7.5 buffer solution Tris-HCl at a mass ratio of 1:10, and pass the mixture through ultra-high pressure 100Mpa, 40°C After 30 minutes of treatment, it was mixed and reacted with neutral protease under ultrasound at 60°C for 3 hours. The mass-volume ratio of neutral protease and the mixed solution was 1:100g/mL;

Step 4) Stir at 85°C for 35 minutes to inactivate the enzyme and obtain the supernatant. Pass through a 0.22μm membrane filter to obtain a sterile filtrate. Add sucrose and glutamic acid to the filtrate to mix; the mass-volume ratio of sucrose and filtrate 2:100g/mL, the mass-volume ratio of glutamic acid to the filtrate is 2:100g/mL;

Example 3

Step 2) Mix the clam shell powder and the 20% citric acid solution with a mass fraction of 1:5 at a mass ratio of 1:5, stir and fully decompose at 50°C and 100 rpm for 3 hours, and separate the solid to obtain crude protein;

Step 3) Wash the crude protein with deionized water until the TDS value in the cleaning solution is 120ppm, then mix it with the pH 7.0 citric acid buffer solution at a mass ratio of 1:15, and treat the mixed solution with ultra-high pressure 200Mpa and 45°C After 40 minutes, it was mixed and reacted with papain under ultrasound at 40°C for 7 hours. The mass-volume ratio of papain to the mixed solution was 1:100g/mL.

Step 4) Obtain the supernatant after stirring at 100°C for 20 minutes to inactivate the enzyme, and obtain a sterile filtrate by pressure filtration through a 0.22μm filter membrane. Chitosan and glutamic acid are added to the filtrate to mix; chitosan and filtrate The mass-volume ratio of glutamic acid is 2:100g/mL, and the mass-volume ratio of glutamic acid to the filtrate is 2:100g/mL;

Step 5) Pre-freeze the mixed solution under liquid nitrogen conditions, and then freeze-dry to obtain a freeze-dried powder, which is the pearl oyster whitening factor freeze-dried powder, which has endothelin antagonism.

Example 4

Step 2) Mix the clam shell powder and the 15% lactic acid solution with a mass fraction of 1:5 in a mass ratio of 1:5, stir and fully decompose at 70°C and 100 rpm for 2 hours, and separate the solid to obtain crude protein;

Step 3) Wash the crude protein with deionized water until the TDS value in the cleaning solution is 500ppm, then mix it with pH 7.5Tris-HCl buffer solution at a mass ratio of 1:5, and pass the mixture through ultra-high pressure 200Mpa, 800°C After being treated for 20 minutes, it was mixed and reacted with papain under ultrasound at 55°C for 5 hours. The mass-volume ratio of papain to the mixed solution was 1:100g/mL.

Step 4) Obtain the supernatant after stirring at 90°C for 20 minutes to inactivate the enzyme, and obtain a sterile filtrate by pressure filtration through a 0.22μm filter membrane. Chitosan and albumin are added to the filtrate to mix; The mass-volume ratio is 2:100g/mL, and the mass-volume ratio of albumin to filtrate is 2:100g/mL;

Test Case 1-Ingredient Matching

The quality verification of pearl oyster whitening factor components ensures that it has the corresponding components of endothelin antagonists. The electrophoresis molecular weight test is performed to verify that the extract of the present invention is consistent with the existing pearl endothelin antagonist.

As shown in Figure 1, the experiment proved that the present invention can use low-cost shells as biomass raw materials to prepare products consistent with the molecular weight distribution of endothelin antagonists in pearls.

Test case 2-strong efficacy

In order to fully prove whether the pearl oyster whitening factor prepared in the present invention has endothelin antagonism, in view of the fact that endothelin stimulates melanocytes to secrete melanin through the secretion of keratinocytes, the present invention uses a 3-dimensional model of melanocytes for the first time, using endothelin (ET -1) Inducing the melanin model to form melanin deposition, so as to better mimic the pigment deposition during skin stress. Using the 3D melanin skin model (purchased from Guangzhou Boxi Biotechnology Co., Ltd.) as the test tool, the sample is used to simulate the use of the human body by the method of surface administration, and the sample is evenly coated on the 3D melanin skin model in vitro to simulate the surface, and the appearance of the color changes , Melanin content, melanin distribution and other indicators to evaluate the inhibitory effect and whitening effect of the sample on the melanin synthesis caused by endothelin.

1. Experimental protocol

Table 1 In vitro evaluation scheme of active substance whitening based on ET-1 stimulated melanin model

Refer to the experimental groupings and corresponding treatment conditions in Table 1. After the establishment of the 3D cell model, 5nM ET-1 submerged stimulation treatment was performed every day. After 3 consecutive days of stimulation, a negative control group was established, followed by pearl oyster whitening factor and endothelin antagonist Spread on the surface of the negative control group model, the administration volume is 10 μL/each time, once every 2 days, the operation is finished for 4 consecutive days of stimulation, and it is reserved.

1.1 Grouping and administration

The 3D melanin skin model was divided into 8 groups, namely the blank group (Control), the negative control group (ET-1), SPE-0.01% group, SPE-0.1% group, SPE-1% group, OSPE-0.01% group, OSPE-0.1% group, OSPE-1% group. Test the appearance, L* value and melanin content of the blank group.

Except for the blank group, the 3D melanin skin models of each group were subjected to subliquid stimulation treatment with endothelin (ET-1) 5nM. The volume of endothelin administration was 10μL/each time for 3 consecutive days. The blank group and the negative control group were tested. Appearance, L* value and melanin content.

Then, in addition to the blank group and the negative control group, the pearl oyster whitening factor (SPE) or the commercially available endothelin antagonist (OSPE) of the present invention was used to evenly smear and administer, and the administration volume was 10 μL/each time, every 2 days Drug once; after each administration of endothelin antagonist, use endothelin (ET-1) 5nM for one administration stimulation, the administration volume is 10μL/each time, 4 days after administration (ie twice administration) End the operation and test the appearance, L* value and melanin content of each group.

Wherein, the administration mode of the endothelin antagonist is:

For the SPE-0.01% group, the lyophilized pearl oyster whitening factor powder prepared in Example 1 is formulated into a solution with a mass fraction of 0.01% with a diluent solvent for administration;

In the SPE-0.1% group, the lyophilized pearl oyster whitening factor powder prepared in Example 1 was formulated into a solution with a mass fraction of 0.1% with a diluent solvent for administration;

In the SPE-1% group, the lyophilized pearl oyster whitening factor powder prepared in Example 1 was formulated into a solution with a mass fraction of 1% with a dilution solvent for administration;

In the OSPE-0.01% group, the control pearl endothelin antagonist was formulated into a solution with a mass fraction of 0.01% with a dilution solvent for administration;

In the OSPE-0.1% group, the control pearl endothelin antagonist was formulated into a solution with a mass fraction of 0.1% with a diluent solvent for administration;

In the OSPE-1% group, the commercially available endothelin antagonist was formulated into a solution with a mass fraction of 1% with a dilution solvent for administration;

The dilution solvent is a mixed solvent with a volume ratio of propylene glycol: ethanol of 7:3.

1.2 Take pictures and observe the model's appearance

The models of different experimental groups were taken to observe the appearance by taking photos. If there is a small amount of water or sample on the tissue surface, use a sterile cotton swab to carefully dry the tissue surface. The photos were taken in a studio with a fixed light intensity. The specific photographing operation standards are as follows: (1) Camera mode: manual; photographing parameter settings: focal length=5.8mm, aperture=f/8, aperture F22, shutter speed -1/80s, ISO=1600. (2) Put the melanin model Take a photo at the center of the color chart. The results are shown in Figure 2.

1.3 L* value detection

Perform L* value detection on models of different experimental groups. The specific detection standard operations are as follows: (1) Use a surgical blade to cut and remove the model along the edge of the chamber; (2) Place the model on a flat and hard white surface, and follow the colorimeter operating instructions to align the colorimeter detection hole vertically to the model The surface is tested, each model is read three times repeatedly, and the data is recorded. The results are shown in Table 2.

Table 2 Determination of L* value of different sample cell models

1.4 Model melanin distribution detection

The models of different experimental groups were tested for the distribution of melanin, and the model used for the detection of melanin distribution was the corresponding grouped model after L* value detection. The detection method is as follows: the model to be tested is fixed with 4% paraformaldehyde, the tissue is embedded, and the slice is subjected to Fontana-Masson staining, the result of the slice is recovered, and the result is photographed with a microscope, and two parallel experiments are performed. The results are shown in Figure 3.

1.5 Model melanin content detection

After treatment with reference to 4.2.1, the models of different experimental groups were tested for melanin content, and the model used for melanin content testing was the corresponding grouped model after L* value detection. The detection method is as follows: the model to be tested uses 0.6mL 0.25% pancreatin, and stands at 37°C for 2 hours; after the end, pipette the tissue repeatedly with an elbow pipette for about 1 min; after pipetting, use an elbow forceps to take out the detached PC membrane; Termination: add 0.6mL PBS containing 10% serum, 2000r/min, centrifuge for 10min, discard the supernatant; add 1N NaOH (containing 10% DMSO) to the pellet, pipette several times, react in a water bath at 80°C for 30min, to make the melanin particles complete Dissolve, then centrifuge at 1200r/min for 5min, add the supernatant to a 96-well plate, 200μL per well, and detect the OD value at the A405nm of the microplate reader. The results are shown in Table 3.

Table 3 The effect of pearl oyster whitening factor on melanin content

The statistical method in the present invention:

Use Graph Pad Prism to plot, and the result is expressed as Mean±SD. One-way ANOVA one-way statistical analysis of variance was used for multiple comparisons between groups. All statistical analyses are two-tailed. p<0.05 is considered to have significant difference, where *p<0.05, 0.005<**p<0.01, ***p<0.001, the smaller the p value, the more significant.

The above experimental results show that the pearl oyster whitening factor of the present invention has a very significant ability to inhibit melanin at a concentration of 1/10,000. Compared with the endothelin antagonist extracted from hundreds of thousands of kilograms of pearls purchased on the market, it has a more obvious ability to inhibit melanin. Regardless of the cost, the development of by-products, or the whitening effect, the pearl oyster whitening factor of the present invention has a very obvious improvement effect.

Test case 3—quality is more stable

The stability test of the extract prepared from the clam shell as the raw material and the endothelin antagonist in the pearl was carried out, and it was found that the pearl oyster whitening factor of the present invention has stronger stability.

Table 4 Comparison of physical and chemical indicators in the stability of two endothelin antagonists

Claims

A method for preparing pearl oyster whitening factor is characterized in that it comprises the following steps:

Step 1) After crushing the clam shells, sieving to obtain small particles of clam shell powder;

Step 2) After mixing the clam shell powder with a 10-20% weak acid aqueous solution in a mass ratio of 1:5-20, stir at 30-70°C at 100-200rpm and fully decompose for 1-4 hours. The separated solid is crude protein;

Step 3) Wash the crude protein with deionized water until the TDS value in the cleaning solution is 100-500ppm, and then mix it with the pH 6.0-7.5 buffer solution. The mass ratio of crude protein to buffer solution is 1:5-15. After the mixed solution is treated by ultra-high pressure 100-200Mpa at 40-80°C for 20-40 minutes, it is mixed and reacted with protease under ultrasonic conditions at 40-60°C for 2-7h;

Step 4) After stirring at 85-100°C for 20-50min to inactivate the enzyme, press filtration through a filter membrane to obtain a sterile filtrate;

Step 5) The freeze-dried protective agent and the filtrate are mixed in a mass-volume ratio of 0.5～4:100g/mL, pre-freeze under liquid nitrogen or -70～-85℃, and then put into a vacuum freeze-drying bin. Lyophilize for 12-20h; finally harvest the lyophilized powder with a moisture content of 2-5%.
The preparation method according to claim 1, wherein the weak acid in step 2) is selected from one or more of acetic acid, lactic acid, citric acid, carbonic acid, and malic acid.
The preparation method of claim 1, wherein the buffer solution with a pH value of 6.0-7.5 in step 3) is PBS, Tris-HCl or citric acid buffer.
The preparation method according to claim 1, wherein the protease in step 3) is one or more of neutral protease, papain, trypsin, subtilisin, and pepsin.
The preparation method according to claim 1, characterized in that: in step 3), the mass-volume ratio of the protease and the mixed solution is 1:100-1:10 g/mL.
The preparation method according to claim 1, wherein the filter membrane in step 4) is a 0.22 μm filter membrane.
The preparation method of claim 1, wherein the freeze-dried protective agent in step 5) is one or more of sucrose, glucose, glutamic acid, albumin, and chitosan.
A pearl oyster whitening factor, which is prepared by the method according to any one of claims 1-7.
The use of the pearl oyster whitening factor of claim 8 in the preparation of an endothelin antagonist.