JPH05163134A - Cosmetic - Google Patents

Abstract

PURPOSE:To obtain a cosmetic having excellently beautifying effects on skin, sufficient shelf stability and high safety. CONSTITUTION:First, kefia granules are inoculated into 10% reduced defatted milk, subjected to subculture every day at 20 deg.C and activated. The activated kefia granules are cultured by using a medium for separating cells of lactic acid bacteria of the genus Lactobacillus and the cells of lactic acid bacteria of the genus Lactobacillus are separated. Then, the cells are inoculated into a medium, subjected to stationary culture or mild spinner culture at 20-35 deg.C and the culture solution is centrifuged to collect cells. 1 pt.wt. of the cells is extracted with 2-10 pts.wt. solvent at 0-40 deg.C for 1-24 hours, the obtained filtrate is concentrated to give a cell extract (extracted solution). A cosmetic is blended with 0.01-0.5% of the extract as an active ingredient.

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 an extract of cultured lactic acid bacteria having excellent skin whitening effect and active oxygen scavenging effect.

[0002]

2. Description of the Related Art It is believed that dark skin, spots, freckles, etc. due to sunburn are caused by the production of melanin, which is a dark brown amorphous pigment, and this melanin is produced when the skin receives external stimuli such as ultraviolet rays. , 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. Therefore, since the effect of whitening the skin is expected by suppressing the activity of tyrosinase involved in melanin production, the incorporation of a tyrosinase activity suppressing component into cosmetics has been proposed.

Conventionally, as cosmetics having a whitening effect, JP-B-55-43443, "Whitening cosmetics" and JP-B-54-
As disclosed in Japanese Patent No. 974, "Composition containing crude drug extract," a composition containing ascorbic acid or a derivative thereof is known. In addition, external skin preparations containing arbutin (JP-A-60-16906, etc.), bleaching cosmetics containing kojic acid (JP-B-32-8100), plant components (JP-A-63-2913, etc.) ) Or an animal ingredient (Japanese Patent Laid-Open No. 63-8312, etc.) is known to have a whitening effect.

However, many of the above-mentioned conventional cosmetics do not have a sufficient whitening effect, and also have some problems such as insufficient storage stability and irritation, which are problematic to the skin. There were many.

[0005]

DISCLOSURE OF THE INVENTION The present invention solves the above-mentioned problems of the prior art and provides a cosmetic having an excellent skin whitening effect, sufficient storage stability and high safety. The purpose is to

[0006]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to solve such problems, and as a result, a lactic acid bacterium cell extract isolated from Kefir grains belonging to the genus Lactobacillus has an inhibitory effect on tyrosinase activity, melanin. It has been found that it has an effect of suppressing generation and an effect of eliminating active oxygen, and exhibits an excellent whitening effect, and was able to provide the present invention.

[0007] That is, the present invention relates to a cosmetic composition containing a lactic acid bacterium extract of Lactobacillus separated from kefir grains.

[0008]

The cosmetic of the present invention can be manufactured by the following method.

First, 10% reconstituted skim milk is inoculated with kefir grains and subcultured at 20 ° C. every day for activation. Then, the activated kefir grains are cultured using a medium for separating lactic acid bacterium of the genus Lactobacillus to separate the lactic acid bacterium of the genus Lactobacillus (arbitrary bacterium of homofermentative lactic acid bacterium that does not produce gas when cultured). As a medium for separating Lactobacillus cells from Lactobacillus, for example, Rogosa's medium (Eftymiou, C, and Hansen,
PA 1962 Journal of Infectious Disease 110, p. 258
-267), a medium in which the carbon source is changed from glucose to lactose (hereinafter abbreviated as L-Rogosa medium) can be used.

Next, the cells separated from the kefir grains as described above are inoculated into the medium, and static culture or gentle stirring culture is carried out at a temperature of 20 ° C to 35 ° C. After culturing, cells are collected by centrifuging the obtained culture solution. The collected cultured bacterial cells may be directly subjected to the extraction operation described below as they are, or may be stored after being frozen or lyophilized. As a medium used for culturing Lactobacillus of genus Lactobacillus, for example, a medium capable of growing the used bacterium, such as Rogosa's medium, can be used.

Then, a solvent is added to 1 part by weight or 1 part by volume of live cells, cells that have been thawed after freezing, or dried cells.
Add 2 to 10 parts by weight or 2 to 10 parts by volume, and add at 0 to 40 ° C 1
Extraction is performed for -24 hours, and the obtained filtrate is concentrated to obtain the target cell extract (extract). As the solvent, a lower alkyl alcohol (methanol, nitanol, inpropanol) and water alone or a mixture of two or more of these can be used. In addition, the above-mentioned bacterial cell extract (extract) may be further concentrated and dried, if necessary, to give a solid extract.

It has been confirmed by the present inventors that the bacterial cell extract thus obtained has an effect of suppressing tyrosinase activity, an effect of suppressing melanin production in melanoma cells, and an effect of eliminating active oxygen. By blending 0.01 to 5% as an active ingredient, a cosmetic having a whitening effect and an active oxygen scavenging effect can be obtained.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the scope of the present invention is not limited by the following examples.

[0014]

【Example】

 (1) Isolation of lactic acid bacteria of the genus Lactobacillus from kefir grains.

First, 1 kefir grain obtained from Christian Hansen (Copenhagen, Denmark)
Inoculate 10% reduced skim milk sterilized at 05 ℃ for 20 minutes at 20-22 ℃
Each day, the seeds were subcultured to activate the kefir grains. Then
Collect about 2 g of activated kefir grains in a sterilized mortar,
Ro which used lactose as a carbon source instead of glucose
2 ml of gosa medium (hereinafter abbreviated as L-Rogosa medium) was added, and kefir grains were thoroughly ground.

Next, the suspension containing the finely divided kefir grains was diluted with L-Rogosa medium in 10 steps, and 0.1 ml of a diluent at an appropriate dilution step was prepared in advance in Rogosa agar medium ( Agar concentration of 1.5%) was applied to the agar using a conradi stick. Then, this agar medium was anaerobically cultured at 30 ° C. for 4 days using a gas pack jar. After culturing, from the colonies generated by using an optical microscope, about
Twenty cells were separated, and each of these bacteria was purified by repeating single colony isolation three times using a new L-Rogosa agar medium.

After purification, each strain was tested in a test tube with a screw stopper in order to investigate the properties of the obtained isolate during liquid culture.
L-Rogosa liquid medium was inoculated, and after hermetically sealing, static culture was performed at 30 ° C for 24 to 48 hours, and a strain that did not produce gas at the end of the culture was selected. In this example, any one of several strains thus obtained was used as the representative strain R202.

The bacteriological properties of the R202 strain were as follows. Morphology / Cell size ・ ・ ・ 1 〜 1.2 × 5 〜 20 μm ・ Shape ‥ Rod-like ・ Motility ‥‥‥ None ・ Presence or absence of spores ‥ None ・ Gram stain ‥‥ Growth state in positive medium (L by gas pack method) -30 on Rogosa agar plate surface
Properties of colonies after culturing at 4 ° C for 4 days) ・ Shape ・ ・ ・ Circle ・ Size ・ ・ ・ diameter 0.5 to 3 mm ・ Color tone ・ ・ ・ Physical properties・ Reduction of nitrate ・ ・ ・ None ・ Dye formation ・ ・ ・ None ・Catalase: Negative ・ Growth range: Temperature: 20-35 ℃, pH: 5.0-
6.5 ・ Attitude toward oxygen: facultative anaerobic Note: The strain (R202 strain) that exhibits the above-mentioned mycological properties was sent to the Institute of Microbiology, Institute of Industrial Science and Technology (Microtechnical Laboratory).
Deposited as No. 24.

(2) Preparation of Lactobacillus sp. Lactobacillus Extract First, a basal medium having the composition (A) and an aqueous lactose solution having the composition (B) shown below are prepared, and at 120 ° C for 20 minutes.
Separately autoclaved. (A) Peptone (Kyokuto Pharmaceutical) 200 (g) Powdered yeast extract (Kyokuto Pharmaceutical) 100 (g) 1 potassium phosphate 5 (g) 2 potassium phosphate 5 (g) 3 ammonium citrate 10 (g) acetic acid sodium (anhydrous) 10 (g) sodium succinate 5 (g) Tween _{80 5 (g) MgSO 4 ·} 7H 2 O 2.3 (g) MnSO 4 · (4~6) H 2 O 0.48 (g) FeSO 4 · 7H ₂ O 0.14 (g) Distilled water 4 (l) (B) Lactose 500 (g) Distilled water 6 (l)

After sterilization, the above basal medium and lactose aqueous solution were mixed, and 100 ml of the culture solution of the R202 strain pre-cultured in L-Rogosa liquid medium was inoculated and gently stirred at 30 ° C. for 5 days (100 r
pm) while culturing. From 24 hours after the start of the culture, neutralization culture of pH 5.6 was performed using a 20% sodium carbonate aqueous solution (high pressure sterilized). Centrifuge after culturing
(5,000 xg for 15 minutes), collect the cells, wash the collected cells with physiological saline, centrifuge again to collect the cells, suspend them in 500 ml of distilled water, and use a freezer at -20 ° C. It was stored frozen overnight.

Next, after the frozen cell suspension was thawed at room temperature, ethanol and distilled water were added so that the total amount was 1 liter and the final ethanol concentration was 20%, and the mixture was gently stirred (1
The extraction was performed for 24 hours while (00 rpm). After the extraction, the obtained extract was centrifuged to remove most of the cells, and the supernatant was filtered once to obtain about 900 ml of filtrate. Then, this filtrate was concentrated under reduced pressure by an evaporator to obtain a concentrated liquid of about 50 ml. The concentrate thus obtained was placed in a dialysis tube and dialyzed overnight (4 ° C.) in about 1 liter of distilled water. After dialysis, the external dialysate was concentrated under reduced pressure using an evaporator, and
Thus, a concentrated solution (ml extract of R202 strain) was obtained in ml.

(3) Measurement test of tyrosinase activity inhibitory rate of Lactobacillus lactic acid bacterium extract First, 100
mM sodium succinate buffer (pH 5.5) 1.8 ml, 270 uni
ts / ml mushroom tyrosinase solution (manufactured by Sigma)
0.1 ml, and sample solution (

0.1 ml of the R202 strain cell extract obtained in (2)
Were mixed with each other and incubated in a constant temperature water layer at 30 ° C. for 15 minutes. Then, 1 ml of a 6 mM L-DOPA solution (manufactured by Wako Pure Chemical Industries: dissolved in the above 100 mM sodium succinate buffer) was added to this test tube and stirred. After stirring, the test tube was set on a reciprocal shaker installed in a thermostatic chamber at 30 ° C. with a tilt of about 45 °, and shaken for 40 minutes (reciprocating frequency 150 / min).
After shaking, the absorbance at 475 nm was measured using a spectrophotometer, and the measured value was designated as A.

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

The tyrosinase activity inhibition rate of the sample solution was calculated from the measured value of the absorbance at 475 nm. The calculation of the tyrosinase activity suppression rate is performed using the following calculation formula,
The results are shown in Table 1. Tyrosinase activity inhibition rate (%) = {1- (AC) /
B} × 100

[Table 1]

(4) Lactobacillus lactic acid bacterium extract
B16 Melanoma Cell Inhibitory Effect Measurement Test First, B16 melanoma cells (B16-F0, ATCC No. CRL-632), which are mouse-derived malignant melanoma cells that produce melanin.
2) was cultured in Eagle's MEM medium supplemented with fetal bovine serum to a final concentration of 10%, and then cultured in a 6-well plate (FALC
6 ml of the above medium containing the cells at a concentration of 3 × 10 ³ / ml was put into each well of (ON), and CO ₂ incubator (5% C
The cells were cultured in O ₂ at 37 ° C. for 5 days.

Then, this medium was replaced with a new Eagle MEM medium (6%) containing 0.03% of theophylline (manufactured by Sigma).
ml) and replace each well with an appropriate amount of sample solution ((2)
The cell extract of the R202 strain obtained in step 1) was added, and the mixture was further cultured for 3 days. After completion of the culture, the medium was discarded, 1 ml of physiological saline was added to each well, and the cells attached to the bottom of the well were suspended by scraping with a scraper. Next, the cell suspension was transferred to a microcentrifuge tube (1.5 ml volume, manufactured by Eppendorf) using a pipette and centrifuged (10,000 xg, 15 minutes).

On the other hand, as a control, sterilized water was added instead of the sample solution, and the same test as above was conducted. In addition, as an experimental section for comparing whitening of cells, 2% L-ascorbic acid aqueous solution (a) 60 μl, (b) 15 was used instead of the sample solution.
0 μl and (c) 300 μl were added, and the same test as above was conducted.

Next, the degree of whitening of the pelletized cells was visually compared to determine the melanin production inhibitory effect. At that time, the degree of whiteness of cells in the control experiment group (sterile water addition group) was “−”, and the degree of whiteness of cells in the comparative experiment group to which L-ascorbic acid was added was (a): “+”,
(b): "++", (c): "++++", the degree of whiteness of the cells when the sample solution was added was-, +, ++, ++
It was visually judged which of + was applied, and the strength of the melanin production inhibitory effect of the sample solution was evaluated in four stages. The results are shown in Table 2.

[Table 2]

(5) Measurement test of active oxygen scavenging effect of Lactobacillus sp. Lactic acid bacterium extract The active oxygen scavenging effect is measured according to the method using cytochrome C in the superoxide dismutase (SOD) activity measuring method. went.

First, the sample solution (the cell extract of the R202 strain obtained in (2)) was put in a cuvette for spectrophotometer (4 ml volume, optical path 1 cm) in the range of 0.01 ml to 1 ml. Was designated as Aml. Next, 100 mM Tris-HCl buffer (pH 7.8) (2.7-A) ml, 1 mM Cytochrome C solution (dissolved in the above Tris-HCl buffer) 0.1 ml,
Add 0.1 ml of xanthine oxidase solution (Boehringer Mannheim, product number 110434 diluted to 80 media with Tris-HCl buffer) and 0.1 ml of 15 mM xanthine solution (dissolved in 0.025N NaOH solution) and stir. did. After stirring, the change (increase) in the absorbance at 550 nm was measured with a spectrophotometer equipped with a 30 ° C. constant temperature cuvette holder, and the initial rate of the increase in absorbance was V.

Further, as an experiment for comparing the active oxygen scavenging action, superoxide.
Dismutase (SOD) solution (manufactured by Sigma, product number
S-2515 was diluted with the above-mentioned Tris-HCl buffer to a final concentration of 10 activity units / ml, and the change (increase) in absorbance at 550 nm was measured in the same manner as above, and the rate of increase in absorbance was measured. The initial speed V was calculated.

On the other hand, as a comparison, instead of the sample solution, a superoxide dismutase (SOD) solution (manufactured by Sigma: product number S-2515 was diluted with the above-mentioned Tris-HCl buffer solution to give a final concentration of 10 activity units / ml. Was prepared in the same manner as above), the change (increase) in the absorbance at 550 nm was measured in the same manner as above, and the initial rate of increase in the absorbance was Vo.

The active oxygen scavenging rate of the sample solution was calculated from the above measured V and Vo. The active oxygen scavenging rate was calculated using the following formula, and the results are shown in Table 3. Active oxygen scavenging rate (%) = (1-V / Vo) × 100

[Table 3] From the above results, it was determined that 25 μl of the sample solution contained 4 activity units or more as SOD.

(6) Composition of lotion Using the Lactobacillus lactic acid bacterium extract obtained in (2),
It is possible to produce a cosmetic having the following composition. (Wt%) ・ Cell extract 1 ・ Glycerin 5 ・ Polyoxyethylenesorbitan monolaurate 1.5 ・ Ethanol 10 ・ Perfume proper amount ・ Preservative proper amount ・ Dye proper amount ・ Purified water proper amount

[0036]

EFFECT OF THE INVENTION An extract extracted from a lactic acid bacterium belonging to the genus Lactobacillus separated from kefir grains with a solvent has an excellent tyrosinase activity inhibitory effect, and an excellent melanin production inhibitory effect in melanoma cells,
And has an active oxygen scavenging effect, and a cosmetic containing this extract exhibits an excellent skin whitening effect and an active oxygen scavenging effect.

Claims (1)

[Claims] 1. A cosmetic comprising a lactobacillus lactic acid bacterium bacterial cell extract separated from kefir grains.