CN112125967A - Method for separating jellyfish mucin and application of jellyfish mucin in repairing cosmetics - Google Patents
Method for separating jellyfish mucin and application of jellyfish mucin in repairing cosmetics Download PDFInfo
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Abstract
A separation method of jellyfish mucin and its application in repairing cosmetics utilizes the difference of isoelectric points of mucin and collagen, combines with ethanol fractional precipitation, and separates high-purity and high-viscosity mucin from jellyfish mesoglea; through human clinical tests, the mucin can repair the damaged skin barrier and reduce the sensitivity of the skin; the method comprises the following steps: cutting the jellyfish from the top end of the umbrella part after rinsing, and digging out the gel substance of the mesoglea layer; homogenizing the jelly, and putting the homogenate into an ultrasonic extraction kettle to melt to obtain ablation mucus; adjusting the pH value of the ablated mucus to 5-8, and adding ethanol to make the ethanol reach 10% -40%; centrifuging for the first time, and collecting supernatant of the first centrifugation; adjusting pH of the supernatant to 2-4, and adding ethanol to make ethanol reach 40-70%; centrifuging for the second time, collecting precipitate, and rinsing the precipitate with acidic ethanol; re-dissolving the precipitate with buffer solution, and lyophilizing to obtain white cotton-like mucin.
Description
The technical field is as follows:
the invention belongs to the technical field of development and utilization of marine active ingredients and the field of cosmetic development, and particularly relates to a separation method of jellyfish mucin and application of the jellyfish mucin in repairing cosmetics.
Background art:
mucins (mucins), also known as mucins, are a class of highly glycosylated macromolecular proteins that are secreted primarily by epithelial cells, have gel-like properties of mucosal barriers, and play important roles in the immune defense of the respiratory and digestive tracts. Mucin products are derived from secretions of the mandible of cattle, pigs and sheep or mucus of the gastric mucosa. Mucin secreted from the bovine jawbone gland is applied to cosmetics by Japanese Shandong Xixiong et al, and the mucin has moisturizing and wrinkle removing effects as proved by experiments. In recent years, domestic enterprises have made certain progress in extracting and separating mucin from other animals and plants. The phyllosticta has been industrialized to extract snail mucin, lysozyme and other bioactive substances from snail secretion. Nevertheless, the market for high quality mucins is still limited and expensive.
Jellyfish (Jellyfish) belongs to lower zooplankton of coelenterate (coelenteras), 3 class and 5 order are provided, more than 400 jellyfishes are discovered in China probably, and most of jellyfishes are toxic. Jellyfish is a general name of a large edible jellyfish and has high nutritional value. The quantity of the jellyfishes cultured or caught in China is more than 30 ten thousand tons every year. Cyanea nozakii (Cyanea nozakii) is a representative of Cyanea nozakii and is also the jellyfish with the most abundant coastal resources in Fujian, but the jellyfish has deviation in taste and low market price.
More than 96% of fresh jellyfish is water, but has many bioactive substances, such as collagen, mucin, and jellyfish toxin. Aequorin (Qniumucin) was first isolated from the mesoderm of aequorea by Kiminori Ushida, has properties very close to those of mammalian gastric mucus, and is expected to be used in adjuvant therapy of gastric ulcer. When the experiment is carried out on the rabbit suffering from the osteoarthritis by the Zodiac Zhengren and the like at the university of east China sea, the rabbit receiving the injection of the hyaluronic acid and aequorin mixed medicament is found to have the joint recovery condition far better than the rabbit receiving the injection of the hyaluronic acid only.
Related studies have been made on methods for extracting and separating jellyfish mucin. Patent 2007101711890 discloses a glycoprotein derived from Cyanea nozakii and having anti-fatigue activity and a preparation process thereof, wherein the preparation process mainly comprises the following steps: desalting, heat preservation and melting, enzymolysis, centrifugation, concentration and spray drying, and the inventor proves that the glycoprotein has the anti-fatigue effect through a mouse test. Patent 2013800318499 provides a method for fractional extraction of mucin and collagen, which mainly comprises washing with water, crushing, water extraction, alcohol fractionation, centrifugation, etc.
Many beauty-conscious people wash their faces many times a day or over-use some exfoliating products for beauty pursuit, resulting in thinning of the cutin, impaired skin barrier and very sensitive skin. Therefore, repair-type cosmetics have always had a large market.
The sample obtained in patent 2007101711890 has a protein content of 75%, i.e. a polysaccharide fraction of at most 25%. The sugar content of the common cyanea nozakii mucin can reach more than 60 percent. This difference was probably due to the fact that the patent did not further purify the crude extract, resulting in mucopolysaccharides containing large amounts of collagen.
The process of patent 2013800318499 is complicated and requires a large amount of organic solvent. In addition, a large amount of electric energy is consumed in the whole production process to maintain the low temperature of materials in the production process.
The invention content is as follows:
in view of the above problems of the prior art, the present invention aims to provide a method for separating jellyfish mucin and application thereof in a cosmetic preparation of a restorative type.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for separating aequorin, comprising the following steps:
(1) cutting the jellyfish from the top end of the umbrella part after rinsing, and digging out the gel substance of the mesoglea layer;
(2) homogenizing the jelly, and putting the homogenate into an ultrasonic extraction kettle to melt to obtain ablation mucus;
(3) adjusting the pH value of the melt mucus to 5-8, adding ethanol to enable the ethanol to reach 10-40%, and standing for 1-4 h;
(4) centrifuging for the first time, and collecting supernatant of the first centrifugation;
(5) adjusting the pH of the supernatant liquid of the first time to 2-4, adding ethanol to make the ethanol reach 40-70%, and standing for 1-4 h;
(6) centrifuging for the second time, collecting precipitate, and rinsing the precipitate with acidic ethanol;
(8) re-dissolving the precipitate with buffer solution, and lyophilizing to obtain white cotton-like mucin.
Further, the jellyfish is the jellyfish which is not pickled, namely the frozen jellyfish or the running jellyfish, and the extraction part is the mesoglea of the jellyfish.
Further, in the step (2), the homogenate is put into an ultrasonic extraction kettle, and a trisodium citrate solution and a preservative are added, wherein the concentration of the trisodium citrate solution is 0.1-5%, and the feeding amount is 1-5 times of that of the homogenate; the melting temperature is 5-60 ℃, the time is 0.5-24 h, and the ultrasonic pulse power is 0.5-5W/cm3。
Further, in the step (3), the pH of the ablated mucus is firstly adjusted to 6.5 by citric acid; the ethanol concentration was about 30%.
Further, the centrifugation conditions in the steps (4) and (6) are centrifugal force of 3000-10000 g, and the centrifugation time is 15-120 min.
Further, the pH in the above step (5) is 2.5; the concentration of the acidic ethanol is 50%; the acidic ethanol is a mixture of 0.5% citric acid aqueous solution and ethanol according to the volume ratio of 1: 1.
Further, the concentration of the trisodium citrate solution is 0.5 percent, and the feeding amount is 2 times of the weight of the homogenate; the preservative is sodium dehydroacetate, and the addition amount is 0.05 percent; the ablation temperature is 50 ℃, and the time is 2 h.
The application of jellyfish mucin in a repairing cosmetic is characterized in that jellyfish mucin is added into a skin care product in the cosmetic, wherein the product is freeze-dried powder, cream, emulsion, essence or a facial mask, and the addition amount of the product is 0.01% -10%.
Further, the jellyfish mucin is added into the essence to prepare the essence containing the mucin, and the formula of the essence is as follows:
name of raw materials | Ratio (%) |
Pure water | 83 |
|
5 |
Butanediol | 5 |
Alpha- |
5 |
Vitamin C ethyl ether | 0.5 |
Phenoxyethanol | 0.5 |
Jellyfish mucin | 0.5 |
Citric acid sodium salt | 0.2 |
Xanthan gum | 0.2 |
Zinc pyrrolidone carboxylate | 0.1 |
The technical scheme has the advantages that: the invention only takes the middle adhesive layer rich in mucin as the raw material, which is beneficial to the extraction of the mucin and can make full use of other materials; the extraction is assisted by high-intensity ultrasonic waves, so that the dissolution of mucin can be improved, and the sterilization effect is achieved. Sodium dehydroacetate is used as a preservative, so that the material is effectively prevented from being corrupted, low-temperature processing is avoided, and the energy consumption is reduced. The combination of pH (controlling the isoelectric point of protein) and ethanol fractional precipitation is utilized to improve the purity of mucin and reduce the usage amount of ethanol. By using acid alcohol precipitation and rinsing, the fishy smell in the mucin can be effectively reduced, and the residual preservative is also removed.
The invention has the following beneficial effects: the invention discloses a method for obtaining high-purity mucin from jellyfishes, which promotes the comprehensive utilization of the jellyfish resource and improves the economic value of the jellyfishes. Cyanea nozakii is a common large jellyfish in Fujian coastal areas, and is influenced by marine environmental pollution in recent years, so that many times of Cyanea nozakii outbreaks occur, and marine ecological balance is seriously influenced. The jellyfish can be processed into edible jellyfish, but the jellyfish has relatively rotten meat and is not popular in the market. The jellyfish mucin prepared by the method has high purity and large molecular weight, can be used as an efficacy raw material of cosmetics, and can effectively repair skin barriers and reduce skin allergy. In addition, the aequorin has high sugar ratio, can avoid being hydrolyzed by protease, has certain viscosity and lubricating effect, and is expected to be used for protecting gastrointestinal mucosa and developing biological medicine preparations such as joint lubricating liquid.
Description of the drawings:
the invention is described in detail below with reference to the accompanying drawings:
FIG. 1 is a process for preparing jellyfish mucin according to the present invention;
FIG. 2 is a gel chromatogram of jellyfish mucin according to the present invention;
fig. 3 shows the skin barrier function repairing effect of the present invention (n-10).
The specific implementation mode is as follows:
the invention is described in detail below with reference to the following figures and examples:
fig. 1-3 illustrate embodiments of the present invention.
Example 1:
(1) rinsing several fresh white cyanea nozakii (total weight about 10kg) with clear water, cutting from the top of umbrella part, and digging out the mesoglea to obtain gel about 1.2 kg;
(2) homogenizing the jelly, putting the homogenate into an ultrasonic extraction kettle, adding 2L of 0.5% trisodium citrate solution and 1.6g of sodium dehydroacetate, and setting the ablation conditions to be ultrasonic pulse power of 2000W, heating temperature of 45 ℃ and time of 2 h;
(3) adding 10% citric acid solution about 20mL into the ablated mucus, mixing, adding 1.6L 95% ethanol, mixing, and standing for 2 hr;
(4) centrifuging at 5000g for 30min, and collecting supernatant liquid 4.5L per month;
(5) adding 250mL of 10% citric acid into the supernatant, adding 2.5L of 95% ethanol, uniformly mixing, and standing;
(6) centrifuging at 5000g for 30min, collecting precipitate, and rinsing the precipitate with 50% ethanol containing 0.5% citric acid for 2 times;
(7) the precipitate was redissolved in 0.2M phosphate buffer pH7.2 and dried under vacuum to give 17.31g of white cotton.
Example 2:
a serum containing mucin has a formula shown in Table X.
Table 1 formula of essence having skin barrier repair effect
To demonstrate the effectiveness of the present invention, the inventors conducted the following tests:
1) GPC measurement of molecular weight and purity
The experimental method comprises the following steps:
a lyophilized mucin sample (25 mg) was dissolved in 10mL of mobile phase, filtered through a 0.45 μm filter head, and then 25 μ L of the solution was injected into a high performance gel chromatograph. Dextran (580,000Da) was used as a control and the samples were analyzed for molecular weight and molecular weight distribution using Shimadzu GPC software.
The instrument conditions were as follows:
the chromatographic column is TSK-gel; column temperature: 40 ℃; mobile phase: 0.05mol/L sodium nitrate; the flow rate is 0.6 mL/min; shimadzu RID-20 shows a differential refractive detector (40 ℃ C.).
The experimental results are as follows:
the gel chromatogram of the jellyfish mucin is shown in FIG. 2 and calculated by Shimadzu GPC software using dextran (10.9min) as a control. The analytical results were as follows: peak 1(Rt 11.3min) had a peak area ratio of 94% and a number average molecular weight of 570000 Da; peak 2(Rt 16.1min) has a peak area ratio of 6% and a number average molecular weight of 1700Da, i.e., the prepared aequorin has a high purity (94%) and a high molecular weight.
2) Analysis of sugar or protein content
The test method comprises the following steps:
1) and (3) rinsing the mouth and wrist with the jelly layer removed and the umbrella cloth respectively by pure water as in example 1, homogenizing, and carrying out vacuum freeze drying to obtain the sample to be tested.
2) Measuring the protein content in the sample by using a BCA method (with bovine serum albumin as a control), and measuring the sugar content in the sample by using a DNS method (firstly hydrolyzing the sample by using 2M trifluoroacetic acid at 105 ℃ for 5 hours, then removing the trifluoroacetic acid by rotary evaporation, and finally fixing the volume; glucose as control).
And (3) detection results: as shown in the table below. In each part of jellyfish body, the sugar content of the middle glue layer is 34%, which is far higher than 4.2% of the mouth and wrist and 7.9% of the umbrella part; combined with the experience in jellyfish processing, the viscous liquid is mainly present in the mesogen layer. Therefore, it is suitable to extract mucopolysaccharide from the mesoglea, and other parts are more suitable for extracting collagen or other purposes. The cotton package of the invention has the sugar content as high as 78 percent and the protein content as high as 13 percent, and can be preliminarily judged to be mucin by combining a GPC spectrogram and physicochemical properties (high viscosity and smoothness, high wiredrawing property and isoelectric point).
Results of sugar and protein detection
Sample name | Sugar content (%) | Protein content (%) |
Example 1 | 78% | 13% |
Middle glue layer | 34% | 29% |
Mouth and wrist | 4.2% | 51% |
Umbrella part (removing middle glue layer) | 7.9% | 46% |
3) Skin repair effect after SLS (sodium lauryl sulfate) treatment
The test results are shown in fig. 3, and it can be seen from the control group (water) that the human skin barrier is destroyed after 3% SLS treatment, the transdermal water loss rate is increased from 6.73mg/(cm2 × h) of normal skin to 13.51mg/(cm2 × h), and after 4h natural recovery of the human body, it is decreased to 9.12mg/(cm2 × h), but it fails to reach the level before destruction. Relative to the blank group, the initial transdermal water loss after application of 0.5% mucin dropped to 8.34mg/(cm2 × h), and the transdermal water loss at 2h was 6.90mg/(cm2 × h), roughly equivalent to that before failure. In addition, it can be seen from the figure that 0.5% HA (hyaluronic acid) HAs a weak skin barrier repair effect. In total, 0.5% mucin was effective in repairing skin barrier breakdown caused by SLS.
4) Lactic acid sting test:
test protocol:
collecting 10 volunteers in the experiment, drawing two 2 cm-4 cm areas at corresponding positions on the inner side of the right front arm of each volunteer, and randomly marking A or B; group A is smeared with 0.1mL of 0.5% of the mucin every morning and evening; group B is smeared with 0.1mL of distilled water every morning and evening; the application is continued for 1 week.
The specific operation method comprises the following steps:
a1 cm by 2cm piece of filter paper was cut and soaked in 10% lactic acid solution, and the filter paper was applied to the A or B mark of the forearm of the volunteer, and the degree of sting at the test site of the subject was recorded. (0 for no sensation; 1 for slight sting; 2 for moderate sting; 3 for intense sting.)
Test results
The following table shows the results of the lactic acid stinging test. The average pricking pain score of the experimental group is 0.6, the average pricking pain score of the control group is 1.1, and the average pricking pain score is 4.2 through SPSS statistical software F test; looking up a table, when F1 is 9, F2 is 9, and α is 0.05, the F threshold is 3.18; f is more than F0.05, and the data of the two groups have significant difference (P is less than 0.05), namely 0.5 percent of mucin is smeared for 1 week, so that the skin barrier can be effectively improved, and the skin sensitivity is reduced.
Statistics of lactic acid sting test results
The method utilizes the difference of isoelectric points of the mucin and the collagen, combines ethanol fractional precipitation, and separates the mucin with high purity and high viscosity from the jellyfish middle glue layer; the mucin can repair damaged skin barrier and reduce skin sensitivity.
While the foregoing description shows and describes the preferred embodiments of the present invention, it is to be understood that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as described herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (9)
1. A method for separating aequorin, which is characterized by comprising the following steps:
(1) cutting the jellyfish from the top end of the umbrella part after rinsing, and digging out the gel substance of the mesoglea layer;
(2) homogenizing the jelly, and putting the homogenate into an ultrasonic extraction kettle to melt to obtain ablation mucus;
(3) adjusting the pH value of the melt mucus to 5-8, adding ethanol to enable the ethanol to reach 10-40%, and standing for 1-4 h;
(4) centrifuging for the first time, and collecting supernatant of the first centrifugation;
(5) adjusting the pH of the supernatant liquid of the first time to 2-4, adding ethanol to make the ethanol reach 40-70%, and standing for 1-4 h;
(6) centrifuging for the second time, collecting precipitate, and rinsing the precipitate with acidic ethanol;
(7) re-dissolving the precipitate with buffer solution, and lyophilizing to obtain white cotton-like mucin.
2. The method for separating jellyfish mucin according to claim 1, wherein the jellyfish is not salted jellyfish, i.e., frozen jellyfish or live jellyfish, and the extraction site is the mesoglea of jellyfish.
3. The method for separating aequorin according to claim 1, wherein in the step (2), the homogenate is put into an ultrasonic extraction kettle, and a trisodium citrate solution and a preservative are added, wherein the concentration of the trisodium citrate solution is 0.1-5%, and the adding amount is 1-5 times of that of the homogenate; the melting temperature is 5-60 ℃, the time is 0.5-24 h, and the ultrasonic pulse power is 0.5-5W/cm3。
4. The method for separating aequorin according to claim 1, wherein in the step (3), the pH of the ablated mucus is adjusted to 6.5 by citric acid; the ethanol concentration was about 30%.
5. The method for separating aequorin according to claim 1, wherein the centrifugation conditions in the steps (4) and (6) are centrifugal force of 3000-10000 g for 15-120 min.
6. The method for separating aequorin according to claim 1, wherein the pH in the step (5) is 2.5; the concentration of the acidic ethanol is 50%; the acidic ethanol is a mixture of 0.5% citric acid aqueous solution and ethanol according to the volume ratio of 1: 1.
7. The method for separating aequorin according to claim 3, wherein the concentration of the trisodium citrate solution is 0.5%, and the feeding amount is 2 times of the weight of the homogenate; the preservative is sodium dehydroacetate, and the addition amount is 0.05 percent; the ablation temperature is 50 ℃, and the time is 2 h.
8. The application of the aequorin in the repair cosmetics is characterized in that the aequorin extracted by the aequorin separation method according to any one of claims 1 to 7 is added into skin care products in the cosmetics, wherein the products are freeze-dried powder, cream, emulsion, essence or facial mask, and the addition amount of the products is 0.01% -10%.
9. The use of jellyfish mucin for a restorative cosmetic as claimed in claim 8, wherein the jellyfish mucin is added to an essence to prepare an essence containing mucin, and the formula thereof is as follows:
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Citations (5)
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CN102516378A (en) * | 2011-12-07 | 2012-06-27 | 河南科技大学 | Method for extracting jellyfish glycoprotein by ultrahigh-pressure combined ultrasonic technology |
CN104379593A (en) * | 2012-08-23 | 2015-02-25 | 株式会社海月研究所 | Method for fractionally extracting mucin and collagen |
JP2017145200A (en) * | 2016-02-15 | 2017-08-24 | 学校法人北里研究所 | Method for producing mucin |
JP2018145106A (en) * | 2017-03-01 | 2018-09-20 | エーザイ・アール・アンド・ディー・マネジメント株式会社 | Skin external compositions |
CN111393521A (en) * | 2020-03-20 | 2020-07-10 | 张培华 | Extraction method of jellyfish collagen |
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2020
- 2020-09-28 CN CN202011041642.8A patent/CN112125967A/en active Pending
Patent Citations (5)
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CN102516378A (en) * | 2011-12-07 | 2012-06-27 | 河南科技大学 | Method for extracting jellyfish glycoprotein by ultrahigh-pressure combined ultrasonic technology |
CN104379593A (en) * | 2012-08-23 | 2015-02-25 | 株式会社海月研究所 | Method for fractionally extracting mucin and collagen |
JP2017145200A (en) * | 2016-02-15 | 2017-08-24 | 学校法人北里研究所 | Method for producing mucin |
JP2018145106A (en) * | 2017-03-01 | 2018-09-20 | エーザイ・アール・アンド・ディー・マネジメント株式会社 | Skin external compositions |
CN111393521A (en) * | 2020-03-20 | 2020-07-10 | 张培华 | Extraction method of jellyfish collagen |
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Title |
---|
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ZOHA BARZIDEH等: "Isolation and characterisation of collagen from the ribbon jellyfish (Chrysaora sp.)", 《INTERNATIONAL JOURNAL OF FOOD SCIENCE AND TECHNOLOGY》 * |
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王倩等: "糖蛋白的制备、结构分析及生理功能研究现状", 《福建水产》 * |
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Application publication date: 20201225 |
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