KR20150024628A - Method for preparing fermented vegitable mucin from yam and cosmetic compositions with it having superior collagen synthetic abilities, moisturizing effect, skin protection and wound healing - Google Patents

Abstract

The present invention relates to a method for producing vegetable mucin from horse mackerel and a cosmetic composition comprising the same, which comprises the steps of (a) pulverizing hemp, (b) filtering the pulverized hemp of step (a) (D) precipitating the filtrate of step (b) to obtain a supernatant, (d) filtering the product of step (c), and (e) fermenting the filtrate of step , And to provide a cosmetic composition comprising such a vegetable mucin as an active ingredient.
INDUSTRIAL APPLICABILITY According to the present invention, it is possible to produce mucins having high immunogenicity and high plant density, which are superior in moisture retention and collagen synthesis ability to conventional mucins derived from animals. In addition, there is an advantage that a cosmetic composition which is safe to the skin because of no cytotoxicity, and which is effective for skin protection and cell regeneration can be mass-produced.

Description

FIELD OF THE INVENTION The present invention relates to a method for producing a vegetable mucin fermented from horse mackerel, and a cosmetic composition having excellent collagen composition performance, moisturizing, skin protection and wound regeneration, EFFECT, SKIN PROTECTION AND WOUND HEALING}

The present invention relates to a method for producing vegetable mucin from horse mackerel and a cosmetic composition containing the same, and more particularly, to a method for producing vegetable mucin by fermenting horse extract and a cosmetic composition containing the same.

Mucin (mucin) is a mucus substance secreted from the mucous membrane and is sometimes referred to as mucin or mucin. It is difficult to dissolve in water and neutral salt solution, it does not dissolve in acid but precipitates, but in alkaline it becomes viscous liquid. The chemical body is a type of glycoprotein, but it contains several glycoproteins depending on the origin of the mucus, and more than two kinds of glycoproteins are often contained depending on the kind of the mucus. There is a typical submandibular gland mucin, but its structure differs greatly in sheep, pigs, and cows. In addition, there are gastric mucin, small intestine mucin, trachomycin, and cervical mucin. Among these, the mucins of digestive organs serve as lubricants for the protection and digestion of organs, and gastric mucin is used for the treatment of gastric hyperplasia and gastric ulcers.

In addition, mucin contains chondroitin, a natural complex substance such as collagen, elastin, and protein, thus preventing moisture loss from the harsh environment from the outside, preventing skin dryness and quickly regenerating scratched skin. Therefore, mucin is a new substance that regenerates and gives elasticity to acne scars and sensitive skin.

Cosmetics including snail mucus filtrate have been used, such as "Manufacturing Method of Cosmetic Products Using Mucin Components Extracted from Snails" of Korean Patent Laid-Open No. 1995-002741, which is now filed as cosmetics containing mucin components. However, since it is an animal-derived moisturizing agent, it can not exclude infections caused by skin irritation or contamination when it is used over a proper amount, and since the cost is high, only a very small amount of mucin is contained as an effective ingredient in the cosmetic itself, In fact.

However, vegetable mucins derived from plant extracts have the advantage of significantly less cytotoxicity and skin irritation than animal mucins having strong skin irritation. Above all, in the case of plants containing vegetable mucin, unlike snails containing animal mucin, it is easy to cultivate and harvest, so that it can be produced in a large capacity, is much less costly, and is environmentally friendly. Therefore, it can be contained in a large amount in cosmetics, so that it is possible to obtain a more excellent effect when actually using cosmetics.

For these reasons, it can overcome the disadvantages of animal mucin which has disadvantages such as adverse effects on skin, cost and environment, but it also has excellent antioxidative effect. It also increases skin elasticity, improves skin moisturizing effect and wrinkle, There has been a demand for the development of a cosmetic composition containing a plant mucin.

In accordance with this demand, in the 'composition for external application for skin containing vegetable mucin extract' of Korean Patent Laid-Open Publication No. 2013-0047806, a skin external preparation composition containing mucin extract extracted from Applema, Paekyuncho, However, it was not possible to produce mushins of high concentration by extracting mucins by merely stirring apple mash, pseudomonas sp., Doduck mushroom, and licorice root with a magnetic stirrer using pure water as a solvent. Which contains the heavy metals and residual pesticides contained in the carrageenan, lotus root, and the like, which leads to cytotoxicity.

KR 10-1995-0002741 A KR 10-2013-0047806 A

The present invention has been proposed in order to solve the above-mentioned problems. It is an object of the present invention to provide a plant mucin which is obtained by pulverizing and filtering marl to obtain an extract and fermenting the extract into Lactobacillus, It is an object of the present invention to produce a high concentration mucin having excellent synthetic ability.

It is also intended to provide a cosmetic composition which is safe to the skin because of no cytotoxicity, and which is effective for skin protection and cell regeneration.

In order to achieve the above object, the present invention provides a process for preparing vegetable mucin from the step of: (a) crushing hemp, (b) filtering the crushed hemp of step (a) (d) precipitating the filtrate of step (b) to obtain a supernatant, (d) filtering the product of step (c), and (e) fermenting the filtrate of step .

In the step (e), the filtrate of step (d) is fermented by inoculating lactobacillus of the genus Lactobacillus.

The step (d) is carried out at 25 to 50 ° C for 20 to 100 hours.

Further comprising filtering the fermented product of step (e).

In addition, the cosmetic composition of the present invention contains vegetable mucin which is obtained by fermenting a horse extract as an active ingredient.

The vegetable mucin is contained in an amount of 0.0001 to 10% by weight based on the total weight of the cosmetic composition.

The plant mucin is fermented through lactobacillus of the genus Lactobacillus.

According to the method for producing vegetative mucin from the strain of the present invention, it has an advantage of being able to produce a plant-derived mucin at a high concentration which is superior in moisture retention and collagen synthesis ability as compared with conventional mucins derived from animals and has immunity-enhancing action .

In addition, there is an advantage that a cosmetic composition which is safe to the skin because of no cytotoxicity, and which is effective for skin protection and cell regeneration can be mass-produced.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a process diagram of a method for producing vegetative mucin according to the present invention.
Fig. 2 is a photograph of a cell after 24 hours of treatment of the material in the skin cell safety test according to the present invention. Fig.
FIG. 3 is a graph showing the cell viability of the examples in the skin cell safety test according to the present invention. FIG.
Fig. 4 is a photograph of cells after 24 hours of treatment of the material of the comparative example in the skin cell safety test according to the present invention. Fig.
5 is a graph showing the cell viability of Comparative Example in the skin cell safety test according to the present invention.
6 is a graph showing the results of the collagen synthesis performance test according to the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be described in detail with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a process diagram showing a method for producing vegetative mucin fermented from horse mackerel according to the present invention. FIG.

The method for producing vegetable mucin of the present invention is characterized in that vegetable mucin having no cytotoxicity and excellent collagen synthesis ability can be produced by pulverizing and filtering the margarine, precipitating and filtering it again, and fermenting it.

That is, as shown in FIG. 1, the method for producing a plant mucin of the present invention comprises the steps of (a) crushing hemp, (b) filtering the crushed hemp of step (a) (d) precipitating the precipitate of step (b), and (e) fermenting the filtrate of step (d). The method may further include, after step (e), filtering the fermentation product of step (e).

As in the prior art, when crushing hemp and adding distilled water to centrifuge the hemp to obtain vegetative mucin from hemp, there is a disadvantage that heavy metals or residual pesticides harmful to the human body are contained in plant mucin as it is and can not be used as a raw material for cosmetics.

However, the method for producing vegetable mucin according to the present invention is advantageous in that not only the heavy metals and residual pesticides are decomposed by fermenting the extract of the horse to obtain the vegetable mucin, but also the skin absorption rate of the plant mucin, which is an effective substance through fermentation, have.

Hereinafter, the present invention will be described step by step.

(a) a step of crushing hemp

Clean the hemp and remove the bark. And crushes the skin with the skin removed. The pulverization method is not limited and can be carried out using a pulverizer. In addition, water may be added for pulverization and pulverized while stirring with a stirrer. The amount of water to be added may be 2 to 10 times based on the weight of the pulp, and pulverized by stirring at 50 to 1,000 rpm for 30 to 180 minutes .

The root used in the present invention is yam (Dioscorea japonica Thunb.) Because it contains the highest amount of vegetable mushin in the roots of yams.

(b) filtering the crushed hemp of step (a)

The crushed marl is filtered through a filter paper or a filter to remove impurities. The filtration method can use a filter or a filter. Since there is a filtration step again in the subsequent step, rough filtration is performed at this stage to remove only the residue and some impurities. For example, the filtration is performed using a filter paper or a standard of about 20 to 400 mesh. In addition, the crushed form of the hemp is in the form of a sticky mucilage, which is not easy to filtrate, so that the coarse filtration is performed first.

(c) precipitating the filtrate of step (b) to obtain a supernatant

After the primary filtration step (b), the obtained filtrate is precipitated to obtain only the mucus component which is the supernatant after the unnecessary sludge and starch components are submerged. The precipitation method can be performed by centrifugation at about 20 to 30 ° C. for about 10 to 15 minutes at about 1,000 to 8,000 rpm.

That is, only the mucus component containing the mucin component as the supernatant is obtained through the precipitation step.

Prior to precipitation, the filtrate of step (b) may be treated with a preservative. Any kind of beverages used in the production of general foods may be used.

(d) filtering the product of step (c)

After obtaining the supernatant of step (c), it is filtered again. Filtering at this stage can also be done with filter paper or filters. The secondary filtration is preferably performed using a filter paper or a small hole of a standard body rather than the advanced primary filtration, but the size thereof is not limited. For example, 400 to 600 mesh can be used.

(e) fermenting the filtrate of step (d)

Next, the filtrate of step (d) is fermented. The fermentation step (e) is a step of decomposing the filtrate of step (d) using microorganisms. That is, since the substance is naturally decomposed by the enzyme secreted by the microorganism and the microorganism, the molecular size of the substance becomes small, so that the skin is easily absorbed, and the existing components following the fermentation are strengthened. In addition, toxic substances such as heavy metals and residual pesticides harmful to the human body are completely decomposed by the microorganisms, and the cytotoxicity is lost. The fermentation products are resistant to heat and carbon dioxide and do not change well, so that the preservability of the active ingredient increases.

The microorganism used in the fermentation of the present invention is not particularly limited, but is preferably fermented by inoculation with lactic acid bacteria in Lactobacillus. The Lactobacillus genus is a bacterium that produces a large amount of lactic acid by fermenting sugars and acquiring energy, and is morphologically polymorphic in gram (Gram) positive spore bacillus. The size of the lactobacillus is 0.5 ~ 0.9 × 1 ~ 11μm, In various forms of enterobacteria. This genus develops in an environment of low oxygen and produces lactic acid in various sugars. It is also known that they are resistant to acidity (pH 3 to 4), nutritional requirements are very complicated, and many amino acids and vitamins other than saccharides are required, so that they can not be developed unless microorganisms or strains are added with micronutrients.

It is effective that the fermentation is carried out at 25 to 50 DEG C for 20 to 100 hours. If the fermentation temperature is lower than 25 ° C. or higher than 50 ° C., the activity of the lactic acid bacterium is not properly maintained and fermentation is not smoothly performed. If the fermentation time is shorter than 20 hours, And if it exceeds 100 hours, since sufficient fermentation has already been performed, it is meaningless to maintain the fermentation period longer. The humidity condition at the time of fermentation is preferably 73 to 75%.

The inoculum amount of the microorganism is preferably 0.01 to 0.10% by weight of lactobacillus of the genus Lactobacillus, relative to 99.90 to 99.99% by weight of the filtrate. This is because if the inoculation amount of the lactic acid bacteria is too small, the fermentation does not sufficiently proceed and the functionality of the moisturizing cosmetic can be degraded. If the lactic acid bacteria use amount exceeds 0.10 wt%, the excessive amount becomes unnecessary.

When the fermentation is completed as described above, the fermentation product of step (e) is filtered again. This is the final filtration, and filtration can proceed from one to many times. At this time, a filter having a pore size of 0.1 to 10 μm can be used, and the filtration is performed a plurality of times.

When the fermentation broth is filtered, sterilized preservatives and excipients are added as needed. The preservatives and excipients used are not limited. The preservatives include parabens most commonly used as preservatives for general cosmetics and dextrin as excipient. .

When the vegetable mucin is obtained from horse mackerel using the above-mentioned method, it can be used as an effective ingredient in the cosmetic composition. That is, unlike the conventional vegetable mucin, when the vegetable mucin as described above is used in a cosmetic composition, there is no heavy metal and pesticide ingredient in the fermentation, and the skin absorption rate is increased. In addition, it has excellent collagen synthesis ability than the conventional snail mucilage extract, and is effective for improving wrinkles, and is effective for skin protection and wound regeneration.

The cosmetic composition of the present invention preferably contains the vegetable mucin in an amount of 0.0001 to 10% by weight based on the total weight of the cosmetic composition. When the content is less than 0.0001% by weight, the effect of the vegetable mucin becomes insignificant, If it exceeds, it is excessive and it is inefficient because the effect is less than the input.

The cosmetic composition of the present invention may contain conventional auxiliary agents and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances in addition to the vegetable mucins mentioned above, It does not limit its kind.

In addition, the cosmetic composition of the present invention is not limited to be produced in any formulations conventionally manufactured in the art, and examples thereof include solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, , Surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation and spray, and the type is not limited thereto.

Hereinafter, the present invention will be described in more detail by way of specific examples and test examples.

(Example)

The yams were prepared, cleaned, and skin removed. 2.5 L of water was added to 500 g of the yam removed from the skin, and the mixture was pulverized with a stirrer at 800 rpm for 30 minutes. Thereafter, primary filtration was performed using a 300 mesh filter paper, and the filtrate was centrifuged (5,000 rpm, 15 minutes, 25 DEG C) to obtain a supernatant. The filtrate was subjected to secondary filtration with 500 mesh filter paper. Next, the lactic acid bacteria of the genus Lactobacillus was inoculated (0.01 wt% with respect to the total weight of the secondary filtrate) to the secondary filtrate, and then fermented (temperature 35 ° C, humidity 74%, 50 hours). The final fermentation product was filtered three times using a filter of 5 mu m. The final filtrate was lyophilized at -50 ° C for 20 hours to obtain final mucin.

Using the vegetable mucin thus obtained, cosmetic compositions were prepared at concentrations of 0.1%, 0.5%, 1% and 5% as shown in Table 1 below.

(Unit:% by weight) of the cosmetic composition according to the concentration of botanical mucin according to the embodiment, division Concentration 0.1% Concentration 0.5% Concentration 1% Concentration 5% Vegetable mushine 0.1 0.5 1.0 5.0 glycerin 3.0 3.0 3.0 3.0 Butylene glycol 3.0 3.0 3.0 3.0 Propylene glycol 1.0 1.0 1.0 1.0 -26-piperazine-26 / phage-40
Dragenated castor oil 1.1 1.1 1.1 1.1 ethanol 8.0 8.0 8.0 8.0 Triethanolamine 0.2 0.2 0.2 0.2 Purified water to 100 to 100 to 100 to 100

(Comparative Example)

The snails, 5 to 8 cm in size, were washed in clean running water and cleaned with 70% ethanol. After disinfected snails were placed in a black mesh, the mucus secreted for 6 hours was collected. After feeding again, the mucilage was recovered by the same method as that of above.

The cosmetic composition was prepared in the same manner as in the above examples except that the mucilage collected instead of the vegetative mucin was used.

Skin cell  safety in vitro  exam( MTT )

① Cell culture

Human skin cell lines (keratinocyte HaCaT) were cultured in 100 mm / 60.1 cm² culture dishes with Dulbecco's Modified Eagle's Medium (DMEM), 10% fetal bovine serum (FBS) and antibiotic-antimycotic (GIBCO, Cat No. 15240-062) And cultured at 37 ° C and 5% CO ₂ . When these cells were 80%, they were subcultured and used in the experiment.

② Test method

The first step in the search for functionality of various functional cosmetic raw materials is to select a range of concentrations that do not exhibit cytotoxicity in the cell types (keratinocyte, melanocyte, fibroblast, etc.) that make up the skin. To dissolve or dilute the test substance, DMEM medium without FBS was used. In order to investigate these concentrations, the MTT test is used to investigate the effect of various concentrations of the test substance on the cell growth and proliferation or toxicity after culturing the cells.

This is an assay using MTT (3- (4,5-dimethylthiazol-2yl) -2,5 diphenyl-2H-tetrazolium bromide) which specifically reacts with the mitochondrial dehydrogenase of cells. The influence can be tested. As a specific test method, cells are seeded at 1 × 10 4 cells or 1 × 10 5 cells / well in 24 wells and cultured under cell culture conditions until cell number reaches 50 ~ 60%. Discard the medium, wash with 10% PBS (phosphate buffered saline), add a new medium not containing 10% FBS, treat with a certain concentration of test substance (AP1 ~ 20) and incubate for 48 hours. 50 μl of MTT solution (6.6 mg / ml, 3- (4,5-dimethylthiazol-2yl) -2,5 diphenyl-2H-tetrazolium bromide solution) is added to each well and further incubated for 3 hours. After removing the culture medium, add 200 μl of dimethylsulfoxide (DMSO, Amresco, 0231-500ML) and shake for 10 minutes. Then, take 100 μl of 96-wells and measure the absorbance at 540 nm with an ELISA (Enzyme-Linked Immunosorbent Assay) reader. The degree of cytotoxicity (cell viability) was expressed as a percentage based on the absorbance of the control using pure water.

Cell viability (%) = (absorbance of test group / absorbance of control group) x 100

③ Test result

Cell safety in Examples and Comparative Examples in HaCaT, a human keratinocyte cell line, was evaluated. The test was conducted in the concentration range of 0.1%, 0.5%, 1% and 5% for the example and the comparative example.

FIGS. 2 and 3 are photographs of cells after 24 hours of material treatment. In FIGS. 4 and 5, cells having cell viability of 80% or more were regarded as not cytotoxic.

As a result, as shown in FIG. 2 to FIG. 5, it was confirmed that both of the examples and the comparative examples did not have cytotoxicity even though the concentration was increased.

Collagen( Collagen ) Sum performance  exam

① Test method

The major component of the skin is collagen. Collagen accounts for 70-80% of the dermal dry weight. Type I and III collagen are the major components of human dermis. Type I is 80% of total collagen and type III is 15% of total collagen. Skin wrinkles are caused by an imbalance in the synthesis and degradation of collagen. In normal skin, the synthesis of type I collagen and its degrading enzyme, MMP-1, are in balance. However, it has been reported that the synthesis of type I and III collagen degrades and the activity of MMP-1 increases in photoaged skin. In addition, collagen is synthesized in the cell as procollagen, then secreted outside the cell and polymerized into collagen fiber.

In this test method, the degree of increase in the expression level of intracellular procollagen in a sample of human fibroblast (CCD986-sk) was compared with that of the untreated group at the level of mRNA. To measure the amount of collagen, real-time PCR Respectively. Human fibroblasts (CCD986-sk) were cultured in 100 mm / 60.1 cm2 culture dish (DMEM) supplemented with Dulbecco's Modified Eagle's Medium (DMEM), 10% Fatal bovine serum (FBS), 1% Antibiotic- Antimycotic (GIBCO, Cat No. 15240-062) At 37 ° C and 5% CO ₂ . When human fibroblasts were confluent at a concentration of 100% or more, cells were seeded at 1 × 10 4 cells / well in a 96-well plate and cultured until confluence reached 80% or higher in cell culture conditions. After that, the test substances (Examples and Comparative Examples: 0.1%, 0.5%, 1%, 5%) were treated with a certain concentration of the test substance after the medium was replaced with the DMEM free medium (without FBS). Thereafter, the cells were cultured for 24 hours under the cell culture conditions to examine the effect of each substance on the expression of the pleocyte-related gene, procollagen. The test method was real-time PCR, and the test procedure was as follows.

Ⓐ RNA Isolation and cDNA Synthesis

RNA isolation and cDNA synthesis were performed using FastLane Cell one-step buffer set (QIAGEN 216213). Cells from which the medium had been removed were washed with FCW (cell wash buffer), added with 50 μl cell processing mixture (Buffer FCPW supplemented with Buffer FCPW and gDAN Wipeout buffer), and incubated at room temperature for 5 minutes. Then, they were transferred to a new E-tube and reacted at 75 ° C for 5 minutes.

Ⓑ Real-time PCR analysis

To analyze the procollagen gene, the cDNA synthesized above was used as a template and subjected to a Rotor Gene Q Real-time PCR Machine (Qiagen) according to the manual of 2X QuantiTect SYBR Green RT-PCR Master Mix. The primers used in the experiments were Qiagen's QuantiTect® primer assays (18S rRNA; QT00199367, procollagen C-endopeptidase enhancer protein (PCOLCE); QT01005725). Expression rates were normalized to 18S rRNA. Real-time PCR conditions are shown in Table 2 below.

Real-time PCR conditions step time temp. 역류 전류 30min 50 ℃ PCR initial activation step 15 min - 3-step cycling - 95 ℃ Denaturation 15 s 94 ° C Annealing 30 s 50-60 ° C Extension 30 s 72 ℃ Number of cycles 35 35-45 -

② Test results

In the fibroblasts (CCD986sk), real-time PCR methods were used to examine the influence of the concentrations of 0.1%, 0.5%, 1% and 5% on the expression of pro-collagen in the Examples and Comparative Examples. The results are shown in the graph of FIG.

The expression of procollagen can be said to be a substance that increases the expression of procollagen when a value higher than 100, which is a positive control value, is compared with the negative control group (untreated group). Especially, the expression level of negative control (TGFβ-1) As shown in FIG. 6, when compared to the untreated group, the comparative example increased from about 0.1% to about 1.3-fold and from 1% to about 1.18-fold , And the concentrations of the samples were increased by about 1.3 times, 1.9 times, and 1.19 times at 0.5%, 1%, and 3% concentrations except 0.1%, respectively. Thus, it can be seen that both Examples and Comparative Examples have excellent collagen synthesis ability, but the Examples of the present invention have more excellent effects on collagen synthesis than the Comparative Examples.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the scope of the present invention is not limited to the disclosed exemplary embodiments. It will also be appreciated that many modifications and variations will be apparent to those skilled in the art without departing from the scope of the present invention.

Claims (7)

(a) crushing hemp,
(b) filtering the crushed hemp of step (a); and
(c) precipitating the filtrate of step (b) to obtain a supernatant; and
(d) filtering the product of step (c)
(e) fermenting the filtrate of step (d)
≪ RTI ID = 0.0 > 1, < / RTI > wherein the plant mucin is fermented.
The method according to claim 1,
The step (e)
Wherein the fermented product is fermented by inoculating lactobacillus of the genus Lactobacillus into the filtrate of step (d).
3. The method of claim 2,
Wherein the step (d) is carried out at 25 to 50 ° C for 20 to 100 hours. The method of claim 3,
Wherein the step (e) further comprises filtering the fermented product of step (e).
The cosmetic composition according to any one of claims 1 to 3, wherein the cosmetic composition contains vegetable mucin, which is obtained by fermenting hemp extract.
6. The method of claim 5,
Wherein the vegetable mucin is contained in an amount of 0.0001 to 10% by weight based on the total weight of the cosmetic composition.
The method according to claim 6,
Wherein the plant mucin is fermented through a lactobacillus of the genus Lactobacillus.

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