CN104725466A - Method for extracting earthworm active protein for cosmetics - Google Patents
Method for extracting earthworm active protein for cosmetics Download PDFInfo
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- CN104725466A CN104725466A CN201510088980.XA CN201510088980A CN104725466A CN 104725466 A CN104725466 A CN 104725466A CN 201510088980 A CN201510088980 A CN 201510088980A CN 104725466 A CN104725466 A CN 104725466A
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Abstract
The invention discloses a method for extracting earthworm active protein for cosmetics. The method comprises the following steps of taking cleaned fresh earthworms and adding the earthworms into a phosphate buffer, and performing high speed homogenization under an ice-bath condition to obtain an earthworm homogenate; adding the earthworm homogenate into a centrifuge tube, performing high-speed centrifugation and getting supernate; adding comminuted ammonium sulfate to perform salting-out, and separating out impurity protein; putting salting-out liquid into a centrifugation tube, getting supernate to pass through a membrane of 0.45 micrometers, and getting filter liquid; removing macromolecular matters by adopting an ultrafiltration method, then desalting in a mode of ultrafiltration utilizing a hollow fiber membrane component with a molecular cut-off of 20k-50k, and finally performing freeze drying to obtain the earthworm active protein. According to the method, fresh eisenia fetida is taken as the raw material, and active protein in the eisenia fetida is separated and purified by adopting the technologies of homogenization, centrifugation, salting-out, microfiltration, ultrafiltration, freeze drying and the like so as to obtain the earthworm active protein with higher proliferative activity, no micromolecular impurities are contained, the purity is high, and the use effect is good.
Description
Technical field
The invention belongs to biological technical field, particularly a kind of extracting method of earthworm active protein used for cosmetic.
Background technology
Along with the sustainable development of medicine cosmetic industry, research and development strategy will turn to the product of medical-type more, utilize the medicine cosmetic of advanced medical technology to be developed rapidly, and this trend also will be more powerful.At present, cosmetic surgery comprises microdermabrasion and chemistry mill skin art, novain nursing and laser upgrade skin etc., these methods can eliminate rough skin surface, senile plaque and tiny wrinkle, but most of human consumer favors more in makeup such as use astringent and anti-wrinkle elite etc. to beautify skin.
The earthworm kind that this institute uses is Eisenia foetida, and this kind belongs to Lumbricidae and likes that victory belongs on taxonomy.Eisenia foetida breeding potential is high, strong adaptability, is well suited for propagating artificially, is to cultivate a most general kind in the world at present, and the medicinal ingredients in its body is also very abundant, so preparation is clinically raw material mainly with Eisenia foetida.Along with the development of biotechnology, the pharmaceutical use of Eisenia foetida is more and more studied by people and pays close attention to.
Active substance in the pharmaceutical use of earthworm and its body is in close relations; Find that earthworm active protein extract can promote the healing of scald wound in animal level in the research in earlier stage of laboratory, fibroblastic propagation and migration can be promoted on a cellular level; But yet there are no the data extracting crease-resistant composition from earthworm, in prior art, earth-worm extractive as raw material extraction process is not also very perfect, and the mixture, the purity that often comprise small molecules composition in extract are low; The main task of research of the present invention is that science extracts in Eisenia foetida the activated protein promoting fibroblast proliferation, and with it for the high quality cosmetic with antiwrinkle active developed by raw material.
Summary of the invention
The object of the present invention is to provide a kind of extracting method of earthworm active protein used for cosmetic, the earthworm active protein extracted by the method is not containing small molecular weight impurity, and purity is high, and result of use is good.
The extracting method of earthworm active protein used for cosmetic of the present invention, comprises the following steps:
A, get the fresh earthworm cleaned up, add the phosphate buffered saline buffer that 20mmol/L, pH are 6.8 ~ 7.2, high-speed homogenization under condition of ice bath, obtain earthworm homogenate, and leave standstill 2 ~ 3h at 4 DEG C, make the abundant stripping of albumen;
B, be sub-packed in centrifuge tube by earthworm homogenate, at 4 DEG C, high speed centrifugation 20 ~ 30min, gets supernatant liquor; At ambient temperature, the ammonium sulfate adding porphyrize is saltoutd, and reaches 50% saturation ratio, makes foreign protein Precipitation, liquid of must saltouing;
C, liquid of saltouing are sub-packed in centrifuge tube, and at 4 DEG C, high speed centrifugation 20 ~ 30min, gets the film that supernatant liquor crosses 0.45 μm, get filtrate;
D, described filtrate of saltouing adopt the mode of the hollow fiber film assembly ultrafiltration of 0.2 μm to remove macromolecular substance, then be the mode desalination of 20k-50k hollow fiber film assembly ultrafiltration with molecular weight cut-off, namely last lyophilize obtains described earthworm active protein used for cosmetic.
The invention has the beneficial effects as follows: present method with fresh Eisenia foetida for raw material, adopt homogenate, centrifugal, saltout, micro-filtration, ultrafiltration, the technology such as lyophilize carry out separation and purification to activated protein wherein, adopt NIH3T3 inoblast to carry out activity tracking, obtains the earthworm active protein had compared with high proliferation activity; On this basis, orthonormal design of experiments is adopted to investigate solvent, solid-liquid ratio, temperature, the extraction time four factor impact of extracting activated protein, saltout on the impact of earthworm active protein extraction by the study tour of different salting point, by investigating the effect of PSPP hollow-fibre membrane desalination, the earthworm active protein obtained is not containing small molecular weight impurity, purity is high, and result of use is good; Experimental basis is provided for extracting the albumen with short proliferative activity of fibroblasts from earthworm; Earthworm active protein the inventive method prepared is applied to makeup, has the effect making skin slip, replenishing water and preserving moisture and delay senility, can be used as skin care product, be directly used in skin.
Embodiment
Carry out clear, complete description by the scheme in the embodiment of the present invention below, based on embodiments of the invention, those of ordinary skill in the art, not making other embodiments all obtained under creative work prerequisite, belong to the scope of protection of the invention.
embodiment 1:
Get the fresh earthworm 500g cleaned up, add the phosphate buffered saline buffer of 1000ml 20mmol/L, pH7.2, with high-speed homogenization machine with 22000rpm, homogenate 5min under condition of ice bath; By earthworm homogenate 4 DEG C of standing 2h, make the abundant stripping of albumen.
Homogenate is sub-packed in centrifuge tube, at 4 DEG C, with 15000 rpm high speed centrifugation 25min, gets supernatant liquor.At ambient temperature, the ammonium sulfate adding porphyrize is saltoutd, and reaches 50% saturation ratio, makes foreign protein Precipitation, will constantly stir in this process; Ammonium sulfate must add in 30min, and add rear continuation stirring 30min and ammonium sulfate is fully dissolved, 4 DEG C of standing 1h, make foreign protein fully precipitate.
Liquid of saltouing is sub-packed in centrifuge tube, at 4 DEG C, with the centrifugal 28min of 16500 rpm, gets the film that supernatant liquor crosses 0.45 μm, gets filtrate.
Supernatant liquor of saltouing adopts the mode of the hollow fiber film assembly ultrafiltration of 0.2 μm to remove macromolecular substance, then be the mode desalination of 20k-50k hollow fiber film assembly ultrafiltration with molecular weight cut-off, namely last lyophilize obtains earthworm active protein used for cosmetic of the present invention.
embodiment 2:
Get the fresh earthworm 500g cleaned up, add the phosphate buffered saline buffer of 800ml 20mmol/L, pH6.8, with high-speed homogenization machine with 18000rpm, homogenate 5min under condition of ice bath.By earthworm homogenate 4 DEG C of standing 2.5h, make the abundant stripping of albumen.
Homogenate is sub-packed in centrifuge tube, at 4 DEG C, with 15000 rpm high speed centrifugation 25min, gets supernatant liquor.At ambient temperature, the ammonium sulfate adding porphyrize is saltoutd, and reaches 50% saturation ratio, makes foreign protein Precipitation, will constantly stir in this process.Ammonium sulfate must add in 30min, and add rear continuation stirring 30min and ammonium sulfate is fully dissolved, 4 DEG C of standing 1h, make foreign protein fully precipitate.
Liquid of saltouing is sub-packed in centrifuge tube, at 4 DEG C, with the centrifugal 30min of 12000 rpm, gets the film that supernatant liquor crosses 0.45 μm, gets filtrate.
Supernatant liquor of saltouing adopts the mode of the hollow fiber film assembly ultrafiltration of 0.2 μm to remove macromolecular substance, then be the mode desalination of 20k-50k hollow fiber film assembly ultrafiltration with molecular weight cut-off, namely last lyophilize obtains earthworm active protein used for cosmetic of the present invention.
embodiment 3:
Earthworm active protein is adopted to prepare the detection of astringent and physico-chemical property thereof:
(1) formula of astringent: glycerine 1g; 1% hyaluronic acid 10g; Reed fragrant plant glue 1g; Trehalose 2g; Watermiscible vitamin E 0.2g; Outstanding horse 0.4g; Earthworm active protein 5g; Radix Ginseng extractive solution 5g; Essence 0.01g; Pure water supplies 100g.
(2) preparation method of astringent is: according to above formula accurate weighing glycerine, 1% hyaluronic acid solution, trehalose, and watermiscible vitamin E and outstanding horse are put in beaker, after glass stick stirs evenly, add 5g Radix Ginseng extractive solution and 5g earthworm active protein extracting solution; Continue to be stirred to evenly and add distilled water to 100g, add the odor type of 1 essence to makeup after all dissolving and regulate.
(3) detected result is as shown in table 1: the pH of astringent is 6.8, observes the color and luster of astringent under non-direct sunlight condition, light yellow in homogeneous transparent, inclusion-free; Wiped by astringent on skin, observe under indoor and non-direct sunlight condition, structure is fine and smooth, and affinity is better; The odor type of osmesthesia astringent, taste conforms with the regulations odor type.In heat-resistant experiment, after 40 DEG C of maintenance 24h, front without obvious Traits change with test after recovering room temperature.In cold-resistant experiment, after 4 DEG C of maintenance 24h, front without obvious Traits change with test after recovering room temperature.
embodiment 4
Earthworm active protein is adopted to prepare the detection of anti-wrinkle elite and physico-chemical property thereof:
(1) formula of anti-wrinkle elite: glycerine 3g; 1% hyaluronic acid 25g; Reed fragrant plant glue 1.5g; Water-soluble Jojoba oil 2g; Trehalose 3g; Watermiscible vitamin E 0.2g; Outstanding horse 0.4g; Earthworm active protein 15g; Radix Ginseng extractive solution 15m; Essence 0.02g; Pure water supplies 100g.
(2) anti-wrinkle elite method is: according to above formula accurate weighing glycerine, 1% hyaluronic acid solution, reed fragrant plant glue, water-soluble Jojoba oil, trehalose, watermiscible vitamin E and outstanding horse are put in beaker, after glass stick stirs evenly, add 15g Radix Ginseng extractive solution and 15g earthworm active protein extracting solution.Continue to be stirred to evenly and add distilled water to 100g, add the odor type of 1 essence to makeup after all dissolving and regulate.
(3) detected result is as shown in table 2: the pH of anti-wrinkle elite is 7.0.The color and luster of anti-wrinkle elite is observed, the light brown in homogeneous transparent, inclusion-free under non-direct sunlight condition.Wiped by astringent on skin, observe under indoor and non-direct sunlight condition, structure is fine and smooth, and affinity is better.The odor type of osmesthesia anti-wrinkle elite, taste conforms with the regulations odor type.In heat-resistant experiment, after 40 DEG C of maintenance 24h, front without obvious Traits change with test after recovering room temperature.In cold-resistant experiment, after 4 DEG C of maintenance 24h, front without obvious Traits change with test after recovering room temperature.
Claims (1)
1. the extracting method of earthworm active protein used for cosmetic, is characterized in that comprising the steps:
A, get the fresh earthworm cleaned up, add the phosphate buffered saline buffer that 20mmol/L, pH are 6.8 ~ 7.2, high-speed homogenization under condition of ice bath, obtain earthworm homogenate, and leave standstill 2 ~ 3h at 4 DEG C, make the abundant stripping of albumen;
B, be sub-packed in centrifuge tube by earthworm homogenate, at 4 DEG C, high speed centrifugation 20 ~ 30min, gets supernatant liquor; At ambient temperature, the ammonium sulfate adding porphyrize is saltoutd, and reaches 50% saturation ratio, makes foreign protein Precipitation, liquid of must saltouing;
C, liquid of saltouing are sub-packed in centrifuge tube, and at 4 DEG C, high speed centrifugation 20 ~ 30min, gets the film that supernatant liquor crosses 0.45 μm, get filtrate;
D, described filtrate of saltouing adopt the mode of the hollow fiber film assembly ultrafiltration of 0.2 μm to remove macromolecular substance, then be the mode desalination of 20k-50k hollow fiber film assembly ultrafiltration with molecular weight cut-off, namely last lyophilize obtains described earthworm active protein used for cosmetic.
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| CN201510088980.XA CN104725466A (en) | 2015-02-27 | 2015-02-27 | Method for extracting earthworm active protein for cosmetics |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106137910A (en) * | 2016-07-27 | 2016-11-23 | 杨凌三度蚯蚓科技有限公司 | A kind of preparation method of Lumbricus nutrition anti-wrinkle cream |
| CN109260231A (en) * | 2017-07-18 | 2019-01-25 | 首都儿科研究所 | The preparation method of the anti-inflammatory antimicrobial extract of cough-relieving apophlegmatic in a kind of earthworm |
| CN112205622A (en) * | 2020-10-10 | 2021-01-12 | 广东丽姿生物科技有限公司 | Antioxidant and anti-aging composition, preparation method and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102525866A (en) * | 2012-01-11 | 2012-07-04 | 山东大学威海分校 | Cosmetic additive, and preparation method and application thereof |
| CN103845272A (en) * | 2014-03-14 | 2014-06-11 | 江苏隆力奇生物科技股份有限公司 | Earthworm extract having skin aging resisting effect and preparation method and application thereof |
| CN104042548A (en) * | 2014-06-26 | 2014-09-17 | 广东省保化检测中心有限公司 | Snail grume extract as well as preparation method and application thereof in cosmetics |
-
2015
- 2015-02-27 CN CN201510088980.XA patent/CN104725466A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102525866A (en) * | 2012-01-11 | 2012-07-04 | 山东大学威海分校 | Cosmetic additive, and preparation method and application thereof |
| CN103845272A (en) * | 2014-03-14 | 2014-06-11 | 江苏隆力奇生物科技股份有限公司 | Earthworm extract having skin aging resisting effect and preparation method and application thereof |
| CN104042548A (en) * | 2014-06-26 | 2014-09-17 | 广东省保化检测中心有限公司 | Snail grume extract as well as preparation method and application thereof in cosmetics |
Non-Patent Citations (2)
| Title |
|---|
| 彭芙: "蚯蚓（地龙）综合利用的现状与展望", 《中药与临床》 * |
| 肖连冬等: "《酶工程》", 30 September 2008, 化学工业出版社 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106137910A (en) * | 2016-07-27 | 2016-11-23 | 杨凌三度蚯蚓科技有限公司 | A kind of preparation method of Lumbricus nutrition anti-wrinkle cream |
| CN109260231A (en) * | 2017-07-18 | 2019-01-25 | 首都儿科研究所 | The preparation method of the anti-inflammatory antimicrobial extract of cough-relieving apophlegmatic in a kind of earthworm |
| CN109260231B (en) * | 2017-07-18 | 2021-09-03 | 首都儿科研究所 | Preparation method of earthworm extract with cough stopping, phlegm eliminating, anti-inflammatory and antimicrobial functions |
| CN112205622A (en) * | 2020-10-10 | 2021-01-12 | 广东丽姿生物科技有限公司 | Antioxidant and anti-aging composition, preparation method and application |
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