JPH0710734A - Cosmetic - Google Patents

Abstract

PURPOSE:To prepare a cosmetic compounded with an ingredient having effects on the prevention of the production of melamine causing stains, freckles, dark skin due to sunburn, etc., and on the lightening of deposited pigments when applied to skins, and capable of making white and beautiful skins. CONSTITUTION:The characteristic of this cosmetic comprises compounding a fermentation solution prepared by inoculating and culturing the genus Saccharomyces yeast separated from kefir particles in a medium containing milk ingredients as main ingredients and subsequently removing the cells of the yeast from the obtained culture solution.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention has an excellent skin whitening effect,
The present invention relates to a cosmetic having an active oxygen scavenging effect and a cell activating effect, and more specifically to a cosmetic containing a fermentation broth obtained from a Saccharomyces yeast culture broth as an active ingredient.・

[0002]

2. Description of the Related Art Generally, it is considered that dark skin, spots, freckles, etc. due to sunburn are caused by the production of melanin which is a blackish brown pigment. , Tyrosinase (tyrosine oxidase) present in melanocytes of the skin is activated, and tyrosine, which is one of the protein-constituting amino acids, is oxidized and produced. From this, since it is expected that the effect of whitening the skin by suppressing the activity of tyrosinase involved in melanin production is expected, the incorporation of a tyrosinase activity suppressing component into cosmetics has been proposed.

[0003] However, many cosmetics have an effect of suppressing tyrosinase activity, but have a weak effect of suppressing melanin production in cultured cells of B16 melanoma, or have an insufficient effect of lightening the deposited pigment when actually applied to the skin. .

In order to actually exert the whitening effect when the cosmetic is applied to the skin, the above-mentioned tyrosinase activity suppressing effect and melanin production suppressing effect alone are insufficient,
In addition to these effects, effects such as suppression of pigmentation by eliminating active oxygen and promotion of melanin excretion by activation of epidermal cells are also required.

On the other hand, as a cosmetic using yeast, JP-A-61-53208, "Skin cosmetic", JP-A-61-53208
260009, "Yeast Extract", JP-A-62-23
No. 4007, "Skin cosmetics containing antibacterial substances produced by yeast", JP-A-63-277605, "Cosmetics", JP-A-64-63505, "Cosmetics" and JP-A-3-163168. Red dye and its manufacturing method and rice food or cosmetics "and the like,
Most of these conventional cosmetics using yeasts use extracts of brewer's yeast and baker's yeast, and most of them do not have a whitening effect.

[0006]

DISCLOSURE OF THE INVENTION The present invention solves the problems of the above-mentioned conventional techniques and has an excellent skin whitening effect based on tyrosinase activity inhibition, melanin production inhibition, active oxygen elimination and cell activation, In addition, the purpose is to provide a safe cosmetic product.

[0007]

Means for Solving the Problems The inventors of the present invention have conducted diligent research to solve such problems, and found that a fermentation broth obtained from a Saccharomyces yeast culture broth has the above-mentioned various effects. The present invention can be provided.

That is, according to the present invention, a medium containing milk components as a main component is inoculated with a yeast of the genus Saccharomyces separated from Kefir grains and cultivated, and yeast cells are obtained from the obtained culture solution. The present invention relates to a cosmetic comprising a fermentation liquor prepared by removing it.

[0009]

The yeast used in the present invention is a yeast separated from kefir grains, which is a starter of traditional fermented milk kefir, which is considered to originate in the Caucasus region, and is similar to brewing yeast and baker's yeast. It is a highly safe yeast that has been used by humankind for a long period of time.

In the present invention, a lactose-utilizing yeast of the genus Saccharomyces isolated from commercial kefir grains manufactured by Christian Hansen Co., Ltd. is used.

In this case, cow's milk or 5 to 20% reduced skim milk is used as a medium for culturing the yeast, and the yeast is then inoculated at a temperature of 20 to 30 ° C.
Stir culture or aeration stir culture for 2 to 7 days.

Then, the obtained culture broth is immediately removed by centrifugation or filtration to remove insoluble components such as yeast cells and casein to obtain a desired fermentation broth. In this case, 1 volume part of the obtained culture broth is used. On the other hand, after adding a solvent containing 0.5 to 2 parts by volume of methanol or ethanol alone or a mixed solution and stirring at a temperature of 20 to 40 ° C. for 2 to 24 hours,
The target fermentation broth may be obtained by removing insoluble components such as yeast cells and casein by a centrifuge or filtration.

The fermented liquor thus obtained has an effect of suppressing tyrosinase activity, an effect of suppressing melanin production in B16 melanoma cells, an effect of scavenging active oxygen, a cell activating effect and a lightening effect of a deposition pigment on the skin of brown guinea pigs. This has been confirmed by the inventors' tests.

This fermented liquid was used as a lotion, cream, emulsion,
By blending 0.05 to 10% in a pack or the like, a cosmetic having a skin whitening effect, an active oxygen scavenging effect and a cell activating effect can be obtained.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited thereto.

[0016]

Example 1 Separation of Saccharomyces yeast from kefir grains.

105 kefir grains obtained from Christian Hansen (Copenhagen, Denmark)
Inoculate 10% reduced skim milk sterilized at ℃ for 20 minutes,
Kefir grains were activated by subculture daily at -22 ° C.
Next, 2 g of the activated kefir grains were collected in a sterilized mortar, and 2 ml of sterilized physiological saline was added to sufficiently crush the kefir grains.

Next, the suspension containing the finely divided kefir grains was diluted with sterile physiological saline in 10 steps, and 0.1 ml of a diluting solution at an appropriate dilution step was prepared in advance on a YM agar medium (physical and chemical). Laboratory-published microbial strain catalog 5th edition, 19
1992 (see page 396), and cultured at 25 ° C. for 4 days.

Using an optical microscope, 20 yeast strains showing an elliptic shape were isolated from the resulting colonies, and each was purified by repeating single colony isolation three times on a new YM agar medium. Of the isolates thus obtained, any one strain was used as the representative strain Saccharomyces yeast strain Y14 (Institute of Biotechnology, National Institute of Biotechnology, FERMP-13627).

The mycological properties of this Saccharomyces sp. Strain Y14 were as follows.

・ Shape Ellipse ・ Proliferation pattern Sprouting ・ YM agar-like colony properties Round, white ・ Lactose assimilating

[0022]

Example 2 Preparation of yeast fermentation broth.

A medium was prepared by autoclaving 2 liters of 10% reduced skim milk at 110 ° C. for 20 minutes. Then, the medium was inoculated with a culture solution of the Saccharomyces yeast Y14 strain that had been precultured in advance using 50 ml of the same medium.
Culture was performed with aeration and agitation for 5 days (aeration rate of 2 liters / minute,
Rotation speed 200 rotations / minute).

After the completion of the culture, 3 liters of ethanol was added to the culture medium, and the mixture was stirred at 25 ° C. for 18 hours. Then, this solution was centrifuged (8,000 × g, 15 minutes) to obtain about 4.4 liters of a supernatant, and 1N hydrochloric acid was added to this supernatant in an appropriate amount to adjust the pH to 4.6. Then
After standing at -20 ° C for 18 hours, the precipitate was removed by filtration to obtain a filtrate.

Then, the pH of the filtrate was adjusted to 6.8 by adding an appropriate amount of 1N sodium hydroxide aqueous solution, the mixture was allowed to stand at -20 ° C. for 18 hours, the precipitate was removed by filtration, and the mixture was pale yellow and transparent. 4.2 liters of yeast fermented liquid was obtained.

[0026]

Example 3 A test for measuring the tyrosinase activity inhibition rate.

First, 1 m of the yeast fermentation broth obtained in Example 2
l, 2 ml, 3 ml, 4 ml, and 5 ml were dried under reduced pressure, and 0.1 ml of distilled water was added to each to dissolve, and these solutions were used as test solutions in the following tests.

In a test tube, 1.8 ml of 100 mM sodium succinate buffer (pH 5.5), 0.1 ml of 270 units / ml mushroom tyrosinase (manufactured by Sigma) and 0.1 ml of the above sample solution were separately charged. The mixture was mixed and incubated in a constant temperature water bath at 30 ° C. for 15 minutes.

Then, 6 mM L- was added to each of the individual test tubes.
After adding 1 ml of DOPA solution (manufactured by Wako Pure Chemical Industries: dissolved in the above 100 mM sodium succinate buffer) and stirring, each test tube was placed in a constant temperature chamber at 30 ° C. for about 45 minutes on a reciprocal shaker. The sample was tilted and set, and shaken for 40 minutes (reciprocating frequency 150 times / minute). After that, the absorbance at 475 nm was measured using a spectrophotometer, and the measured value was (A).
And

On the other hand, as a control liquid, the absorbance at 475 nm was measured in the same manner as above except that distilled water was added instead of the sample solution, and the measured value was designated as (B). Also, L
The absorbance at 475 nm was measured in the same manner as above except that the sodium succinate was added instead of the DOPA solution, and the measured value was designated as (C).

The tyrosinase activity inhibition rate of the sample solution was calculated using the following formula, and the results are shown in Table 1. Tyrosinase activity inhibition rate (%) = {1- (AC) /
B} × 100

[0032]

[Table 1]

[0033]

Example 4 Measurement test of melanin production suppression rate in B16 melanoma cells.

First, 1 ml of the yeast fermentation broth obtained in Example 2
ml, 3 ml, 4 ml, and 5 ml were dried under reduced pressure, and then each of the
Dissolved in 1 ml of distilled water, these solutions were used as sample solutions in the following tests.

Then, in an Eagle MEM medium containing 10% fetal bovine serum, B16 melanoma cells (B16-FO.A), which are melanin-producing malignant melanoma cells of mouse origin.
TCC No. CRU-6322) at a final concentration of 3 × 10 ^3.
After inoculating so that the amount of the culture medium becomes / ml, 6 ml of this medium is put into each well of a 6-well plate (FALCON) and CO
_The cells were cultured in a ₂ incubator (5% CO ₂ , 37 ° C) for 5 days.

Then, this medium was added to the novel Eagle ME containing 0.03% theophylline (manufactured by Sigma) and having the above composition.
The medium was replaced with M medium (6 ml), and the sample solution (0.1 ml) or the solution of the substance used as a control was added to each well, followed by culturing for 3 days.

After culturing, the medium is discarded and 1 well is added to each well.
ml of physiological saline is added, and the cells attached to the bottom of the well are suspended using a scraper so that the cells are marked down. Then, the cell suspension is pipetted into a microcentrifuge tube (1.5 ml volume, Eppendorf). (Made by the company) and centrifuged (1,000 xg, 15 minutes).

After removing the supernatant after centrifugation, physiological saline was added so that the final volume was 1 ml, and the pelleted cells were suspended to form a cell suspension. 1
20 μl of 0% SDS solution was added and vigorously stirred to lyse the cells.

Then, using a spectrophotometer, the absorbance at 260 nm (A) and the absorbance at 475 nm (B) of the above cell lysate.
And were measured respectively. On the other hand, as a control, sterilized water was added instead of the sample solution and the same test as above was performed.
The absorbance (C) at nm and the absorbance (D) at 475 nm were measured, and the melanin inhibition rate was calculated using the following formula, and the results are shown in Table 2.

Melanin production inhibition rate (%) = {1- (B /
A) / (D / C)} × 100

[0041]

[Table 2]

[0042]

[Example 5] A test for measuring the lightening effect of a deposited pigment in a brown guinea pig.

A-1 brown guinea pig (male, body weight 400 g at the start of the experiment) was shaved on the back to expose the skin, and irradiated with ultraviolet rays (wavelength 312 nm, intensity 420 mJ) once a day,
This was repeated for 5 days. After that, the animal was kept without treatment, and after 7 days, skin pigmentation was obtained.

Next, the pigmentation site was divided into 2 cm × 2 cm squares, and 50 μl of the yeast fermentation broth obtained in Example 2 was applied to one section once a day, and this was repeated for 20 days. . As a control, a 60% ethanol solution and distilled water were also applied in the same manner to determine the pigmentation lightening effect.

In this case, the lightening effect of pigmentation was determined by applying the above-mentioned coated sample for 20 days and visually determining it according to the following criteria, and the results are shown in Table 3.

Criteria 0: No difference in pigmentation is recognized as compared with the non-coated area. 1: A slight lightening of the deposited pigment is observed as compared with the uncoated area. 2: Moderate lightening of the deposited pigment is recognized as compared with the uncoated area. 3: Significant lightening of the deposited pigment is recognized as compared with the uncoated area.

[0047]

[Table 3]

[0048]

[Example 6] A test for measuring the effect of eliminating active oxygen.

First, 1 ml of the yeast fermentation broth obtained in Example 2
ml, 3 ml, 4 ml, and 5 ml were dried under reduced pressure, and then each of the
Dissolved in 1 ml of distilled water, these solutions were used as sample solutions for the following tests.

The active oxygen scavenging effect was measured in the following manner according to the method using cytochrome C in the superoxide dismutase (SOD) activity measuring method.

Spectrophotometer cuvette (4 ml capacity, light path 1
cm), 2.6 ml of 100 mM Tris-HCl buffer (pH 7.8), 0.1 ml of 1 mM Cytochrome C solution (dissolved in the above Tris-HCl buffer), xanthine oxidase solution (Boehringer Mannheim, product number) 11
0.1 ml of 0434 diluted with the above Tris-HCl buffer), 0.1 ml of sample solution, and 0.1 ml of 15 mM xanthine solution (dissolved in 0.025N NaOH solution) were added and stirred. Then, using a spectrophotometer with a 30 ° C constant temperature cuvette holder set,
The change (increase) in the absorbance at nm was measured, and the initial speed of the increase rate was V.

On the other hand, as a control, when 0.1 ml of distilled water was added instead of the sample solution, the change in absorbance was measured in the same manner, and the initial rate of increase in absorbance at that time was V _0.
As a result, the active oxygen scavenging rate was calculated using the following formula, and the results are shown in Table 4.

Active oxygen scavenging rate (%) = (1−V / V ₀ ) × 100

[0054]

[Table 4]

[0055]

Example 7: Test for measuring cell activation effect.

First, 1 ml of the yeast fermentation broth obtained in Example 2 and 2
ml, 3 ml, 4 ml, and 5 ml were dried under reduced pressure, and then each of the
Dissolved in 1 ml of distilled water, these solutions were used as sample solutions for the following tests.

Eagle MEM with 10% fetal bovine serum
Human-derived normal fiber cells (CCD-45SK.A) were added to the medium.
TCC No. CRL-1506) at a final concentration of 1 × 10 ⁴
After inoculating so that the amount of the culture medium becomes / ml, 5 ml of this medium is put into each well of a 6-well plate (FALCON) and CO
₂ incubator _{(5% CO 2, 37 ℃} ) 24 hours and cultured in, then medium was changed to Eagles MEM medium containing 1% fetal calf serum, the sample solution (0.1 ml) to each well, or control Then, 0.1 ml of distilled water was added, and the cells were further cultured for 5 days.

After culturing, the medium is discarded and 0.25 is added to each well.
% Trypsin solution (manufactured by Kojin Bio Co., Ltd.) was added to wash the cell surface. Discard the trypsin solution, add 1 ml of fresh trypsin solution again, perform the same operation, discard the trypsin solution, put the plate in a CO ₂ incubator, keep warm for 20 minutes, put 5 ml of physiological saline in each well, and gently After pipetting to suspend the cells,
Cell concentration was measured.

The cell activation effect was represented by the cell concentration of the sample solution-added experiment group, where the cell concentration of the control experiment group was 100, and the results are shown in Table 5.

[0060]

[Table 5]

[0061]

Example 8 Composition of lotion.

Using the yeast fermented liquor obtained in Example 2, a lotion having the following formulation can be produced.

(Wt%) ・ Yeast fermentation liquor 10 ・ Glycerin 5 ・ Polyoxyethylene sorbitan monolaurate 1.5 ・ Perfume proper amount ・ Preservative proper amount ・ Dye proper amount ・ Purified water balance

[0064]

As described above, the yeast fermented liquor according to the present invention has a tyrosinase activity inhibitory effect, a melanin production inhibitory effect, an active oxygen scavenging effect, a cell activating effect and a lightening effect of a deposition pigment in brown guinea pigs. The cosmetic containing the yeast fermentation liquid exhibits an excellent skin whitening effect.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location A61K 35/72 ADS 7431-4C AED 7431-4C C12P 1/02 Z 7417-4B // (C12P 1 / 02 C12R 1:85)

Claims (1)

[Claims] 1. Saccharomyces (Saccha) separated from Kefir grains in a medium containing milk as a main component.
romyces) genus yeast is inoculated and cultivated, and a fermentation liquor prepared by removing yeast cells from the obtained culture broth is added to the cosmetic.