JP2003277225A - Cosmetic composition containing levan having cell- proliferation, skin-moisturizing and irritation- alleviating effects - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cosmetic composition containing a polysaccharide (levan) which exerts an excellent cell-proliferating, skin moisturizing and skin irritation- alleviating effects and is produced by microbes free of stability- and safety- related problems. <P>SOLUTION: The cosmetic composition contains the polysaccharide (levan) which is isolated and purified from microbes known to produce levan using sucrose as a substrate, such as Rahnella aquatilis, Zymomonas mobilis and Pseudomonas aurantiaca. Levan is isolated and purified from media in which the microbes have been cultured, and dried. The levan extracted and purified from the microbes is shown to have an excellent skin-moisturizing effect through a measurement of its moisturizing effect, no toxicity through a cytotoxicity test and a skin irritation-alleviating effect through a skin irritation test employing a three-dimensional bioartificial skin and a human patch test. The composition is also shown to exert an excellent elasticity-enhancing effect via moisturization by directly applying it onto a human skin at a concentration equal to or higher than a predetermined level. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cosmetic composition containing a polysaccharide levan produced by using a microorganism and having cell proliferation action, skin moisturizing and stimulating effect.

[0002]

BACKGROUND OF THE INVENTION The stratum corneum, which is the outermost layer of the skin, has a function of regulating skin water evaporation and absorption, and a barrier function for permeation of external substances such as chemical substances, toxic substances and bacteria. The stratum corneum consists of flat keratinocytes and intercellular lipids between them, and has a lamellar structure. The stratum corneum of the skin contains about 20% water,
It is well known that the water content is an important factor in determining the flexibility and smoothness of skin.

The moisturizing ability of the stratum corneum is the natural moisturizing factor (N) composed of sebum components and amino acids, lactic acid, urea and inorganic salts.
It is one of the important research subjects in cosmetics to develop a substance that is controlled by MF), is a skin external preparation, is safe and has excellent usability like natural moisturizing factors, and has a high moisturizing effect. Recently, it has been clarified that the factors that affect the functions of the stratum corneum (percutaneous absorption, moisturizing, exfoliation) are keratin lipids, and research on this has been actively conducted.

With age, the skin gradually deteriorates in its inherent function and undergoes an aging phenomenon, of which typical examples are wrinkle formation and melanin production. The main cause of such aging is photoaging by sunlight. The free radicals generated by the ultraviolet rays of sunlight promote the oxidative cleavage of collagen and elastin, which are connective tissues of the skin, and as a result, wrinkles are formed by abnormal cross-connecting of connective tissues. In addition, it promotes the cleavage and lower molecular weight of hyaluronic acid, which plays the most important role in the moisturizing ability of the skin, which is one of the causes for reducing the moisturizing ability of the stratum corneum. Generally, when the water content of the stratum corneum is 10% or less, the physiological action of the skin decreases, the skin loses its original function, and the texture becomes rough, causing various skin problems. Therefore, the use of moisturizers to prevent skin dryness is essential in cosmetics. First of all, the moisturizer required for cosmetics has an appropriate moisturizing power, secondly, the moisturizing power is not easily affected by changes in ambient environmental conditions, and third, affinity for the skin. The fourth is that it is safe for the skin and the living body, and the fifth is that it is highly compatible with other ingredients used in cosmetics. Moisturizers that are currently widely used include polyols such as glycerin, propylene glycol and butylene glycol, natural moisturizing factors such as amino acids, pyrrolidone, carboxylates and lactates, and biological agents such as hyaluronate, chondroitin sulfate and chitosan. There are polymeric substances. Some of these have excellent moisturizing power, but since they are synthetic compounds or animal-origin moisturizers, when used in an appropriate amount, they are humectants that cannot avoid infection due to skin irritation or contamination. .

Levan, which is mainly found in microbial products (and recently also found in various plants and tree trunks and plants such as wheat and wheat), is a fructose linked by β (2 → 6). It has hundreds of thousands of fructose residues in the polymer and is used in various microorganisms [Bacillus subti
lis), Bacillus Polymyxa, Eerobacter levanicum, Streptococcus sp., Pseudomonas sp., Corynebacterium revaniformans laevaniformans),
Zymomonas mobilis] is produced using sucrose as a substrate. This polysaccharide has an excellent moisturizing effect on the skin, has no toxicity to cells, and is a material suitable for use as a cosmetic ingredient.

[0006]

The object of the present invention is to include a polysaccharide (levan) produced by the action of a microorganism-derived enzyme, and has excellent cell growth action and moisturizing and skin irritation alleviation effect, stability and safety. The present invention provides a cosmetic composition free from problems.

[0007]

The above-mentioned object of the present invention is achieved by a polysaccharide (levan) produced by the action of a microorganism-derived enzyme and a cosmetic composition containing the polysaccharide.

The inventors of the present invention measured the moisturizing power of levan extracted and purified from microorganisms in order to find a substance having an excellent skin moisturizing power. It was confirmed that there was no toxicity in the experiment. The irritation test using the three-dimensional bioartificial skin and human patch test also showed the skin irritation alleviation effect. In addition, it was found that the cosmetic composition was blended at a certain concentration or more and directly applied to human skin, and thus the effect of enhancing elasticity by moisturizing was excellent.

The type of levan used in the present invention is Rahnella aquatili, which is known to be a microorganism that produces levan when cultured with sucrose as a substrate.
s), Zymomonas mobilis, Pseudomonas aurantiac
a), Pseudomonas syringa
e), Gluconobacter oxidans
xydans), Aerobacter levan
icum), Bacillus amyloliquefaciens (Bacillus
amyloliquifaciens), Bacillus polymixa (Bacillus)
polymyxa), Bacillus subtilis
s), Corynebacterium revaniformans (Coryneb
acterium laevaniformans), Erwinia amylovora, and Streptococcus salivarius.

The method for producing the polysaccharide (levan) from the above-mentioned microorganism is as follows.

Levan is a polysaccharide (levan) obtained by culturing levan-producing microorganisms in a medium containing sucrose and yeast extract as main components and then centrifuging or removing the bacterial cells using a membrane. Alcohol is added to the solution containing
(Levan) is precipitated and then dried, or Levan separated and purified using a membrane is obtained by drying.

As another method, a culture solution containing levan sucrase is used as an enzyme solution, and alcohol is added to a solution containing the polysaccharide (levan) obtained by reacting sucrose to add the polysaccharide (levan). It is obtained by drying after precipitation, or by drying the levane separated and purified using a membrane.

As another method, the levansucrase gene isolated from the microorganism is cloned to obtain E. coli.
After insertion into JM109 and culturing, the expressed levansucrase was precipitated in (NH ₄ ) ₂ SO ₄ aqueous solution and purified by dialysis, mixed with sucrose aqueous solution, and then obtained by enzymatic reaction. The product is obtained by adding 3 times the amount of alcohol to precipitate and then filtering and drying.

More detailed culture and separation conditions are as follows.

Levan-producing microorganisms were used at a temperature of 30-37 ° C. and pH 5.0 at 30-37 ° C. in a medium containing 5-25% sucrose and 1-2% yeast extract as main components.
-Incubate at 7.0 for 20-50 hours, then centrifuge or 0.2
-To the polysaccharide-containing (levan) solution obtained after removing the cells using a 0.4-micron membrane, add 2-3 times the amount of alcohol to precipitate the polysaccharide (levan) and then dry it, or 0.03 -
Levan separated and purified from the polysaccharide-containing (levan) solution using a 0.1 micron membrane can be dried to obtain levan powder. As another method, using a culture solution containing levansucrase obtained by culturing as described above containing levansucrase as an enzyme solution, 5-50% sucrose solution (pH 5.0-7.0) -35 ° C
To the polysaccharide-containing (levan) solution obtained by reacting for 20-50 hours at the temperature of 2-3 times the amount of alcohol is added to precipitate the polysaccharide (levan) and then dried, or 0.03- Levan separated and purified from the polysaccharide-containing (levan) solution using a 0.1 micron membrane can be dried to obtain levan powder. Alternatively, as another method, the Levansucrase gene isolated from the above-mentioned microorganism is cloned to obtain Escherichia coli JM10.
After inserting into 9, culture (temperature 30 ℃, medium LB 30hr).
Expressed levansucrase is obtained by precipitation into (NH ₄ ) ₂ SO ₄ and purification by dialysis. The purified levansucrase was mixed with sucrose (pH 4.8, 0-30 ° C), and the product obtained by enzymatic reaction for 1 day was precipitated by adding 3-fold amount of alcohol, followed by filtration and drying. Then, levan powder can be obtained.

The following examples and experiments illustrate the invention in more detail, but the invention is not limited to these examples.

[0017]

[Examples] Production Example 1 Whether Ranella aquatilis was cultured at 37 ° C and pH 5.5 for 24 hours in a medium containing 10% sucrose and 1% yeast extract as main components, and then centrifuged. Alternatively, the levan-containing solution obtained after removing the cells using a 0.2 micron membrane,
Levan powder is obtained by adding twice the amount of alcohol to precipitate levan and then drying it or by drying the levan separated and purified from the levan-containing solution using a 0.03 micron membrane. Another method is to clone the Levan sucrase gene of Ranella aquatilis and clone it into E. coli JM.
After inserting into 109, culture (temperature 35 ℃, medium LB 35hr). The expressed sucrase is obtained by precipitation in 30% (NH ₄ ) ₂ SO ₄ and purified by dialysis. After mixing purified levansucrase with 40% sucrose (pH 4.8, 20 ° C), the product obtained by enzymatic reaction for 3 days was precipitated by adding 3 volumes of alcohol, followed by filtration and drying. And get levan powder. Production Example 2 Pseudomonas aurantiaca was used at 37 ° C. and pH 5.5 for 24 hours using a medium containing 10% sucrose and 1% yeast extract as main components.
After culturing for a period of time, centrifuge or removing the cells using a 0.2-micron membrane, the levan-containing solution obtained was mixed with 3 times the amount of alcohol to precipitate levan, and then dried, or 0.03 The levan separated and purified from the levan-containing solution is dried using a micron membrane to obtain levan powder. As another method, the Pseudomonas aurantiacae levansucrase gene is cloned and inserted into Escherichia coli JM109, and then cultured (temperature 35 ° C, medium
LB 35hr). Expressed sucrase in 30% (NH ₄ ) ₂ SO
Obtained by precipitation in ₄ and purification by dialysis. Purified levansucrase was added to 40% sucrose (pH 4.8, 2
After mixing at 0 ° C.) for 3 days, the product obtained by enzymatic reaction is added with 3 times volume of alcohol to precipitate, and then filtered and dried to obtain levan powder. Production Example 3 Zymomonas mobilis was cultivated at 37 ° C. and pH 5.5 for 24 hours in a medium containing 10% sucrose and 1% yeast extract as main components, and then centrifuged or 0.2 micron membrane. After removing the bacterial cells using, the levan-containing solution obtained was added with 3 times the amount of alcohol to precipitate levan and then dried, or separated and purified from the levan-containing solution using a 0.03 micron membrane. The levan is dried to obtain levan powder.
As another method, the Levan sucrase gene of Zymomonas mobilis is cloned, inserted into Escherichia coli JM109, and then cultured (temperature 35 ° C., medium LB 35 hr). The expressed sucrase is obtained by precipitation in 30% (NH ₄ ) ₂ SO ₄ and purified by dialysis. After mixing the purified levansucrase with 40% sucrose (pH 4.8, 20 ° C),
The product obtained by enzymatic reaction for 3 days is precipitated by adding 3 volumes of alcohol, and then filtered and dried to obtain levan powder. Production Example 4 Pseudomonas syringae was cultivated at 37 ° C. and pH 5.5 for 24 hours in a medium containing 10% sucrose and 1% yeast extract as a main component, followed by centrifugation or a 0.2 micron membrane. Levan-containing solution obtained after removing the bacterial cells by using 3 times amount of alcohol to precipitate Levan and then dried, or separated and purified from the Levan-containing solution using 0.03 micron membrane. To obtain levan powder. Production Example 5 Gluconobacter oxidans was used at 37 ° C. and pH 5.5 for 24 times in a medium containing 10% sucrose and 1% yeast extract as main components.
After culturing for a period of time, centrifuge or removing the cells using a 0.2-micron membrane, the levan-containing solution obtained was mixed with 3 times the amount of alcohol to precipitate levan, and then dried, or 0.03 The levan separated and purified from the levan-containing solution is dried using a micron membrane to obtain levan powder. Production Example 6 After culturing Erobacter revanicum at 37 ° C. and pH 5.5 for 24 hours in a medium containing 10% sucrose and 1% yeast extract as the main components, centrifugation or 0.2-micron membrane is performed. After removing the bacterial cells using, the levan-containing solution obtained was added with 3 times the amount of alcohol to precipitate levan and then dried, or separated and purified from the levan-containing solution using a 0.03 micron membrane. The levan is dried to obtain levan powder. Production Example 7 Bacillus amyloliquefaciens was mixed with 10% sucrose and 1%
Use a yeast extract-based medium at 37 ° C, pH 5.5
After culturing for 24 hours at 37 ° C, centrifuge or removing the cells using a 0.2-micron membrane, the resulting solution containing levan is mixed with 3 times the amount of alcohol to precipitate levan and then dried, Alternatively, the levan separated and purified from the levan-containing solution is dried using a 0.03 micron membrane to obtain levan powder. Production Example 8 Bacillus polymixa was cultivated at 37 ° C. and pH 5.5 for 24 hours using a medium containing 10% sucrose and 1% yeast extract as a main component, followed by centrifugation or a 0.2-micron membrane. To the levanin-containing solution obtained after removing the bacterial cells using 3 times the amount of alcohol to precipitate levan and then dry it, or
Alternatively, the levan separated and purified from the levan-containing solution is dried using a 0.03 micron membrane to obtain levan powder. Production Example 9 Bacillus subtilis was cultivated in a medium containing 10% sucrose and 1% yeast extract as main components at 37 ° C. and pH 5.5 for 24 hours and then centrifuged or a 0.2 micron membrane was used. Levan-containing solution obtained after removing the bacterial cells by using 3 times amount of alcohol to precipitate Levan and then dried, or separated and purified from the Levan-containing solution using 0.03 micron membrane. To obtain levan powder. Production Example 10 Erwinia amylovora was cultivated in a medium containing 10% sucrose and 1% yeast extract as main components at 37 ° C. and pH 5.5 for 24 hours, followed by centrifugation or a 0.2 micron membrane. After removing the bacterial cells using the solution containing levan,
Levan powder is obtained by adding twice the amount of alcohol to precipitate levan and then drying it, or by drying the levan separated and purified from the levan-containing solution using a 0.03 micron membrane. Production Example 11 Corynebacterium revaniformans was cultured at 37 ° C. and pH at 37 ° C. using a medium containing 10% sucrose and 1% yeast extract as main components.
After culturing at 5.5 for 24 hours, centrifuge or removing the cells using a 0.2-micron membrane, and then adding 3 times the amount of alcohol to the resulting levan-containing solution to precipitate levan and then dry it. Alternatively, a levan powder is obtained by drying levan separated and purified from the levan-containing solution using a 0.03 micron membrane. Production Example 12 Streptococcus salivarius was cultivated at 37 ° C. and pH 5.5 using a medium containing 10% sucrose and 1% yeast extract as main components.
After culturing for 4 hours, centrifuge or removing the cells using a 0.2-micron membrane, the solution containing levan obtained was mixed with 3 times the amount of alcohol to precipitate levan, and then dried, or Levan separated and purified from the levan-containing solution using a 0.03 micron membrane is dried to obtain levan powder. In the following Experimental Examples and Formulation Examples, in the above Production Examples, after culturing the microorganisms, alcohol was added to the solution containing levan obtained after removing the cells using the membrane to precipitate levan, and then, The levan powder obtained by the method of the drying mode was used. Experimental Example 1. Cell protection effect experiment from ultraviolet rays (1) The cell protection effect against ultraviolet rays was measured for the levan powder obtained in the above-mentioned levan production Examples 1 to 12. Experimental method Human fibroblasts were placed in a well plate at 2 × 10 ⁵ cells / well, 0.2 ml of DMEM medium containing 10% FBS was supplied, and then cultured in a CO ₂ incubator for 24 hours. This is treated with levane dissolved in DMSO, then irradiated with 2 MED of UV light for 5 minutes, and then cultured for 24 hours. MT for investigating cell viability
Perform T assay. In the MTT assay, MT the cultured cells
After adding 0.1 ml of T solution and culturing for 4 hours, the medium was removed, 0.52 ml of DMSO was added, and the formazan formed was measured for absorbance at 570 nm using an ELISA reader. The results are shown in Table 1. In addition, efficacy tests at different concentrations were carried out using the levan produced in Production Example 2. The results are shown in Fig. 1.

[0018]

[Table 1]

Experimental Example 2. Cytoprotective Effect Experiment of Levan (2) The cytoprotective effect of the levan powder obtained in Levan Production Examples 1 to 12 against superoxide radicals generated by ultraviolet irradiation was measured. Experimental Method Human fibroblasts were placed in a well plate at 2 × 10 ⁵ cells / well, 0.2 ml of DMEM medium containing 10% FBS was supplied, and then cultured in a CO ₂ incubator for 24 hours. This is treated with levan dissolved in DMSO and incubated for 30 minutes. After 10 μl of 1.5 mM xanthine and 10 μl of 0.25 U xanthine oxidase was added to the cultured cells to generate superoxide radicals, the cells were cultured in a CO ₂ incubator for 24 hours. Perform MTT assay to check cell viability. In the MTT assay, the formazan formed by adding 0.1 ml of the MTT solution to the cultured cells was measured for absorbance at 570 nm using an Elisa reader. The results are shown in Table 2. In addition, efficacy tests at different concentrations were conducted using the levan produced in Production Example 2. The result is shown in FIG.

[0020]

[Table 2]

Experimental Example 3 Cell Proliferation Effect Experiment of Levan The cell proliferation effect of the levan powder obtained in the above-mentioned levan production Examples 1 to 7 was measured. Experimental method 5% fetal bovine serum (FBS) in 96 microwell plates
After adding 100 μl of each solution, the sample was dispersed in DMEM medium containing 2% FBS at a constant concentration to make it thin, and 100 μl was added again.
Fibroblasts obtained from neonatal foreskin tissue were dispersed in 100 μl medium at 4000 cells per well using a hemacytometer, and then cultured for 7 days. After culturing,
Remove the supernatant and re-apply 5% PBS (phosphate buffered saline) 200
μL was added to each well for washing, 150 μl of DMSO was added to each well, and the mixture was shaken for 10 minutes to dissolve it, and then the absorbance was measured at 570 nm. At this time, the experiment conducted by the same method without addition of levan was used as a control group, and the cell proliferation effect was compared from the relative difference in absorbance. The results are shown in Table 3. In addition, efficacy tests at different concentrations were conducted using the levan produced in Production Example 2. The result is shown in FIG.

[0022]

[Table 3]

Experimental Example 4. Moisturizing test of levan The moisturizing ability of the levan powder obtained in the above-mentioned levan production Examples 1 to 12 was measured. Experimental Method 5 g of levane was placed in a dish and left in a desiccator containing CaCl ₂ at a relative humidity of 41% at 25 ° C., and the weight change was measured with time. The results are shown in Table 4. In addition, efficacy tests at different concentrations were conducted using the levan produced in Production Example 2.
The result is shown in FIG.

[0024]

[Table 4]

Experimental Example 5. Utilizing a three-dimensional bioartificial skin
Skin irritation alleviation effect test of levan The skin irritation alleviation effect was measured for the levan powder obtained in the above-mentioned levan production Examples 1 to 12. Experimental method The MTT assay in the three-dimensional bioartificial skin was performed based on the monolayer culture system method with modification according to this experiment. The culture medium was replaced with new medium and each insert (inser
After 10 hours, 10 μl of skin stimulant (SLS: sodium lauryl sulfate) is administered, and after 4 hours, 10 μl of levan at each concentration is added, and then the mixture is cultured at 37 ° C. under 5% CO ₂ for 24 hours. After washing with PBS, MTT solution (0.33 mg / ml) is administered to each well. After leaving it in the incubator for 4 hours, it is extracted with isopropanol for 24 hours at room temperature. For the interleukin-1α assay, absorbance was measured at 450 nm using an ELISA kit (manufactured by Endogen Corporation, Boston, Mass., USA). The results are shown in Table 5.
Shown in. In addition, the efficacy test at different concentrations was manufactured in Example 2
It was carried out using the levan manufactured in. The result is shown in FIG.

[0026]

[Table 5]

Experimental Example 6. Human patch test of Levan
Skin irritation alleviation effect The skin irritation alleviation effect was measured for the levan powders obtained in Levan Production Examples 1 to 12 described above. Experimental method A fixed amount of Levan 5.0% aqueous solution and sodium lauryl sulfate 0.08% aqueous solution for each sample for 50 healthy men and women
(0.2 g) was applied for 24 hours, the fin chamber was removed, and after 4 hours, the change in skin condition was visually read and the result was shown. The results are shown in Table 6. In addition, efficacy tests at different concentrations were conducted using the levan produced in Production Example 2. The result is shown in FIG.

[0028]

[Table 6]

Prescription Example 1. Among cosmetics containing levan, prescription examples of lotion (skin lotion) are as follows. The levan here is that of manufacturing example 2.

[0030]

[Table 7]

<Preparation Method> Formulation Example 1: Nos. 11, 2, 3, 4 and 8 are sequentially added to No. 11 and stirred to dissolve. On the other hand, after heating No. 5 to about 60 ℃ to melt it, add No. 10 to this and stir to add to No. 11 above.

Finally, after adding Nos. 1, 6, 7, and 9 and stirring sufficiently, they are passed through a Microfluidizer and then aged.

Comparative Prescription Example 1: Nos. 11, 2, 3, 4, and 8 were sequentially added and dissolved by stirring. On the other hand, after heating No. 5 to about 60 ° C. to melt it, add No. 10 to this and stir to add to No. 11 above.

Finally, after adding Nos. 6, 7, and 9 and thoroughly stirring, they are passed through a microfluidizer and then aged. Prescription example 2. Among cosmetics containing levan, the prescription example of nutrition lotion is as follows. Levan here is manufacturing example 2
belongs to.

[0035]

[Table 8]

<Preparation Method> Formulation Example 2: Raw materials 2, 3, 4, 5 and 6 are homogenized at 70 ° C to 80 ° C. This is called a nonionic amphipathic lipid. The above-mentioned nonionic amphipathic lipid and the raw materials 1, 7, 8 and 14 are mixed, homogenized at 70 ° C to 80 ° C, passed through a microfluidizer, and then the raw material 9 is heated at 70 ° C to 80 ° C. After gradually adding and homogenizing, the mixture is passed through the microfluidizer again. Then add 10, 11, 12, 13 and disperse to stabilize and age.

Comparative Formulation Example 2: 70% of raw materials 2, 3, 4, 5 and 6
Homogenize at -80 ° C. This is called a nonionic amphipathic lipid. The above-mentioned nonionic amphiphilic lipid and the raw materials 7, 8 and 14 are mixed, homogenized at 70 ° C to 80 ° C, passed through a microfluidizer, and then the raw material 9 is gradually heated at 70 ° C to 80 ° C. After addition and homogenization, it is passed through the microfluidizer again. Then add 10, 11, 12, 13 and disperse to stabilize and age. Prescription example 3 The following is a prescription example of nutrition cream among cosmetics containing levan. The levan here is that of the second embodiment.

[0038]

[Table 9]

<Preparation Method> Formulation Example 3: Raw materials 2, 3, 4, 5 and 6 are homogenized at 70 ° C to 80 ° C. This is called a nonionic amphipathic lipid. The above-mentioned nonionic amphipathic lipid and the raw materials 1, 7, 8 and 14 are mixed, homogenized at 70 ° C to 80 ° C, passed through a microfluidizer, and then the raw material 9 is heated at 70 ° C to 80 ° C. After gradually adding and homogenizing, the mixture is passed through the microfluidizer again. Then add 10, 11, 12, 13 and disperse to stabilize and age. Comparative formulation example 3: Raw materials 2, 3, 4, 5 and 6 are homogenized at 70 ° C to 80 ° C. This is called a nonionic amphipathic lipid. The above-mentioned nonionic amphipathic lipid and the raw materials 7, 8 and 14 are mixed, homogenized at 70 ° C to 80 ° C, passed through a microfluidizer, and then the raw material 9 is gradually heated at 70 ° C to 80 ° C. In addition to homogenizing, the mixture is passed through the microfluidizer again. Then add 10, 11, 12, 13 and disperse to stabilize and age. Prescription example 4. Among the cosmetics containing levan, the prescription example of massage cream is as follows. Levan here is Example 2
belongs to.

[0040]

[Table 10]

<Preparation Method> Formulation Example 4: 8, 9, 10, 11, 13, 16 are mixed and stirred at 80
After heating at ~ 85 ° C and putting it into the production department, operate the emulsifier to heat 2,3,4,5,6,7,12 to 80 ~ 85 ° C and put in it, then emulsify. After the emulsification is complete, cool to 50 ° C with stirring using a stirrer, then add No. 14 and
After cooling to ℃, add No. 15, then cool to 35 ℃, then add No. 1. Cool to 25 ° C and age. Comparative Prescription Example 4: 8, 9, 10, 11, 13, 16 were heated to 80 to 85 ° C. while being mixed and stirred and charged into the production section, and then the emulsifying machine was operated to operate 2, 3, 4, 5, 6 , 7 and 12 are heated at 80-85 ℃ and added, then emulsified. After completion of the emulsification, cool to 50 ° C with stirring using a stirrer, add No. 14, cool to 45 ° C, add No. 15, cool to 35 ° C, and then age. Prescription example 5. Among the cosmetics containing levan, the prescription example of nutrition essence is as follows. The levan here is that of the second embodiment.

[0042]

[Table 11]

<Preparation Method> Formulation Example 5: Raw materials 2, 3, 4, 5 and 6 are homogenized at 70 ° C to 80 ° C. This is called a nonionic amphipathic lipid. The above-mentioned nonionic amphipathic lipid and the raw materials 1, 7, 8 and 14 are mixed, homogenized at 70 ° C to 80 ° C, passed through a microfluidizer, and then the raw material 9 is heated at 70 ° C to 80 ° C. After gradually adding and homogenizing, the mixture is passed through the microfluidizer again. Then add 10, 11, 12, 13 and disperse to stabilize and age. Comparative formulation example 5: Raw materials 2, 3, 4, 5 and 6 are homogenized at 70 ° C to 80 ° C. This is called a nonionic amphipathic lipid. The above-mentioned nonionic amphiphilic lipid and the raw materials 7, 8 and 14 are mixed, homogenized at 70 ° C to 80 ° C, passed through a microfluidizer, and then the raw material 9 is gradually heated at 70 ° C to 80 ° C. After addition and homogenization, it is passed through the microfluidizer again. Then add 10, 11, 12, 13 and disperse to stabilize and age. Test Example 1. Skin elasticity improvement test In order to measure the skin elasticity improvement effect when the nutritional creams of Prescription Example 3 and Comparative Prescription Example 3 were applied to the skin, normal skin was applied to women aged 20 to 55 years, Have oily, dry or mixed skin
For 20 people, the nutritional cream of Prescription Example 3 on the left side of the face and the nutritional cream of Comparative Prescription Example 3 on the right side of the face
The composition was applied twice a day and used, and after 1 week, 2 weeks, 3 weeks and 4 weeks, the elasticity of the skin was measured using a Ballistometer. The measurement results are shown in Table 12 and FIG.

[0044]

[Table 12]

As can be seen from the above results, the nutritional cream of Formulation Example 3 containing levan according to the present invention has the effect of improving the skin condition of levan, as compared with the nutritional cream of Comparative Formulation Example 3 prepared without adding levan. Shows that it has a skin elasticity effect. Test Example 2. In order to test the effect of Levan on skin elasticity, 20 people were applied with Formulation Example 5 twice a day on the left outer corner of the eye. Comparative formulation example 5 was applied to the right outer corner of the eye. 45 days after each treatment, the skin elasticity of the skin surface was measured by a Cutometer SEM 474. Skin elasticity measurement using a ctometer SEM 474 measures the depth of wrinkles, and the lower the value, the better the elasticity. The elasticity value shows the value decreased as compared with the control group in%, and
The average value of persons is shown in the results. The control group is the measured value before treatment with the sample.

[0046]

[Table 13]

Test Example 3. To test the effect of Levan on skin elasticity, a prescription example was given to the inside of the left arm for 20 people.
2 and Comparative Prescription Example 2 were applied. After each application, the skin moisturizing power of the skin surface is measured by Corneometer CM.
It was measured by the 825 every 5 minutes. Corneometer CM
The skin moisturization measurement by 825 measures the water content of the skin, and the higher the value, the better the moisturizing power. The average value of 20 subjects is shown in the results. The experiment was performed at 20 ° C and 20% relative humidity.

[0048]

[Table 14]

[0049]

As can be seen from the above experimental examples, the cosmetic composition containing the levan of the present invention is excellent in improving skin elasticity, reducing wrinkle depth of skin, reducing skin irritation and moisturizing effect.

[Brief description of drawings]

FIG. 1 is a graph showing the cytoprotective effect of levan.

FIG. 2 is a graph showing the cytoprotective effect of levan.

FIG. 3 is a graph showing the cell proliferation effect of levan.

FIG. 4 is a graph showing the moisturizing effect of Levan.

FIG. 5 is a graph showing the skin irritation alleviating effect of levan using three-dimensional bioartificial skin.

FIG. 6 is a graph showing the skin irritation alleviating effect of levan using a human patch test.

FIG. 7 is a graph showing the skin elasticity improving effect of a cosmetic containing Levan.

FIG. 8 is a graph showing a skin wrinkle depth reducing effect of a cosmetic containing Levan.

FIG. 9 is a graph showing the moisturizing effect of a cosmetic containing Levan.

   ─────────────────────────────────────────────────── ─── Continued front page    (71) Applicant 501 255 239             Toa Kasei Co., Ltd.             1-4-4 Awaji-cho, Chuo-ku, Osaka-shi, Osaka (72) Inventor Kim Hoho             South Korea, Chungcheongnam-do, Cheonan, Sinbang             Dong, 869 101 Shin-Dong apartments             No. 402 (72) Inventor Kim Ki-soo             South Korea, Chungcheongnam-do, Cheonan, Achecheon, Seo             Nzonri 19-4 (72) Inventor Liang             South Korea, Chungcheongnam-do, Cheonan, Shinbu-dong,             Delim apartment 301 building No. 704 (72) Inventor Lee Daiwa             Republic of Korea, Chungcheongbuk-do, Cheongju-shi, Shangli-gu, Yo             Seonghan Alm Apartment, Nam-dong             108 building 1301 (72) Inventor Kim Tengho             Republic of Korea, Daejeon, Dong-gu, Yongda-dong, Sindh             N'a Apartment, 2 Buildings No. 1101 F term (reference) 4B064 AF11 CA21 CB30 CD09 CE03                       CE16 DA20                 4C083 AA031 AA032 AA082 AA122                       AC102 AC122 AC422 AC432                       AC442 AC542 AC642 AC902                       AD092 AD211 AD212 AD352                       AD492 BB21 BB48 CC02                       CC04 CC05 EE12 EE17 FF01

Claims (7)

[Claims] 1. A cosmetic composition comprising levan produced by the action of a microorganism-derived enzyme. 2. The microorganisms are Ranella aquatilis, Zymomonas mobilis, Pseudomonas aurantiaca, Pseudomonas syringe, Gluconobacter oxidans, Erobacter revanicum, Bacillus amyloliquefaciens, Bacillus polymyxa. , Bacillus subtilis, Corynebacterium revaniformans, Erwinia amylovora, and one or more microorganisms selected from the group consisting of Spreptococcus salivarius, characterized in that
The cosmetic composition according to claim 1. 3. The levan is subjected to the following steps: 1) culturing the levan-producing microorganism using a medium containing sucrose and a yeast extract as a main component, and 2) centrifuging or culturing the membrane using a membrane. Remove the bacterial cells from the liquid, and 3) 3-1) Add alcohol to the polysaccharide-containing (levan) solution thus obtained to precipitate the polysaccharide (levan) and dry it, or 3-2 The cosmetic composition according to claim 1, which is obtained through a step of drying levan separated and purified from the polysaccharide-containing (levan) solution using a membrane. 4. The polysaccharide containing levan obtained in the following step, that is, 1) reacting sucrose with a culture solution containing 1) levansucrase as an enzyme solution, 2) 2-1) It is obtained through the steps of adding alcohol to the solution to precipitate the polysaccharide (levan) and then drying it, or 2-2) drying the levan separated and purified from the polysaccharide-containing (levan) solution using a membrane. The cosmetic composition according to claim 1 or 2, wherein the cosmetic composition is prepared. 5. The levan is subjected to the next step, 1) cloning of the levansucrase gene isolated from the microorganism, inserting into E. coli JM109 and culturing, and 2) expressing the levansucrase (NH ₄ ) ₂ It is precipitated in SO ₄ aqueous solution and purified by dialysis, 3) the purified Levansucrase is mixed with sucrose aqueous solution, enzymatically reacted to obtain a product, and 4) alcohol is added to the product. After precipitating,
The cosmetic composition according to claim 1 or 2, wherein the cosmetic composition is obtained through a step of filtering and drying. 6. Levan is 0.1-10% by dry weight,
Desirably characterized in that it contains in an amount of 0.5-5% (w / v),
The cosmetic composition according to any one of claims 1 to 5. 7. The formulation according to claim 1, which has a dosage form of a lotion (skin lotion), nutritional lotion, nutritional cream, massage cream or nutritional essence.
Any one of the cosmetic compositions.