WO2017113263A1

WO2017113263A1 - Novel use of gluconacetobacter xylinus fermentation broth as cosmetic composition

Info

Publication number: WO2017113263A1
Application number: PCT/CN2015/100043
Authority: WO
Inventors: 林宜全; 林圣凯; 李亚洁
Original assignee: 奈菲儿生医股份有限公司
Priority date: 2015-12-31
Filing date: 2015-12-31
Publication date: 2017-07-06

Abstract

Provided is a cosmetic composition, with gluconacetobacter xylinus fermentation broth as an active ingredient thereof, which has effects such as anti-aging, whitening and moisture retention, and has the property of biostability.

Description

Novel use of xylobacter acetate fermentation broth as a cosmetic composition

Technical field

The present invention relates to the use of a microbial fermentation broth as a cosmetic composition, in particular, to the use of a xyloacetic acid fermentation broth as a cosmetic composition.

Background technique

In recent years, a variety of microbial fermentation broth products have been introduced. The microbial fermentation broth is known to contain vitamins, minerals, peptides, amino acids or natural moisturizing factors, and has the effects of promoting metabolism, strengthening moisturizing, anti-oxidation, soothing inflammation, regulating sebum and the like on the skin.

For example, the trade name SKII (Procter &Gamble;P&G) uses Pirate ^TM, a fermentation broth produced by yeast (Saccharomycopsis), as an active ingredient in the winemaking process to achieve skin care effects such as moisturizing (SKII Official Website; www.sk-ii.com ). Tsai et al. have also published that the fermentation broth of Saccharomycopsis has anti-inflammatory and cell repair effects (Journal of Dermatological Science, 2006, 42, pp. 249-257).

Based on the biosafety and efficacy of natural fermentation products, further development of cosmetic compositions using natural fermented materials as active ingredients has market demand.

Summary of the invention

The present invention provides a cosmetic composition comprising a fermentation broth of Gluconacetobacter xylinus as an active ingredient.

The present invention further provides a use of a fermentation broth of Gluconacetobacter xylinus as a cosmetic composition.

The above xyloacetic acid fermentation broth is produced by the following method:

(i) blending a medium containing a carbon source, a nitrogen source, a phosphorus source, an inorganic salt, and an organic acid;

(ii) inoculating the lignin acetic acid bacteria (Gluconacetobacter xylinus);

(iii) allowing the culture medium containing the xylobacteria to be cultured; and

(iv) The cellulose film formed in the medium was removed, and the remaining culture liquid was collected.

The inoculum amount of the xylobacteria is preferably from 10 ² to 10 ⁵ cells/mL.

The medium containing the xylobacteria is preferably cultured at 25 to 32 ° C, more preferably in two steps. Stage culture, which comprises culturing at a temperature of 25 to 28 ° C in the first stage and 29 to 32 ° C in the second stage after the first stage.

Further, the method for producing the xylosonic acid fermentation broth may further include a purification step comprising at least one of adsorbent addition, ultrafiltration, or filtration.

The adsorbent may include a food adsorbent, activated carbon or a product having an adsorption effect.

The above method for producing the xylosonic acid fermentation broth may further comprise the step of drying the culture liquid collected in the step (iv).

Further, the concentration of the xylosidum fermentation broth is 5% by weight or less based on the weight of the cosmetic composition.

DRAWINGS

Figure 1 is a graph showing the cytotoxicity of the fermentation broth of the xylobacteria in human skin keratinocytes (HaCaT cells) in an embodiment of the present invention.

Fig. 2 is a view showing the effect of the fermentation broth of the xylosylobacter oxysporum on the cell cycle distribution of human skin keratinocytes (HaCaT cells) in an embodiment of the present invention.

Fig. 3 is a view showing the ability of the acetic acid bacteria fermentation broth to remove DPPH (di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium) free radicals in an embodiment of the present invention.

Fig. 4 is a view showing the ability of the xyloacetic acid fermentation broth to remove ABTS (2, 2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) radicals in an embodiment of the present invention.

Fig. 5 is a view showing the ability of the fermentation broth of the xylobacteria bacterium to inhibit the production of intracellular reactive oxygen species (ROS) in an embodiment of the present invention.

Fig. 6 is a view showing the ability of the fermentation broth of the xylobacteria bacterium to inhibit the formation of lipoxygenase in an embodiment of the present invention.

Fig. 7 is a view showing the ability of the fermentation broth of the xylobacteria bacterium to inhibit the production of nitric oxide (NO) in an embodiment of the present invention.

Figure 8 is a graph showing the ability of the fermentation broth of the xylobacteria bacterium to inhibit the activity of mushroom tyrosinase in an embodiment of the present invention.

detailed description

The object of the present invention is to provide a novel use of a fermentation broth of Gluconacetobacter xylinus as a cosmetic composition. Traditional acetic acid bacteria are often used in the production of vinegar, such as cereal vinegar Or fruit vinegar and so on. On the other hand, acetic acid bacteria are also known to be metabolized to produce biocellulose, which is formed into a film shape, and can be applied to foods, industrial materials, biomedical materials, cosmetic compositions and the like. In terms of biomedical materials, it is known as, for example, a burn dressing, artificial skin, or the like. In the case of a cosmetic composition, a microbial fiber membrane is also commercially available as a mask or the like. However, prior to the present invention, there has not been a technique in which a fermentation broth of xyloacetic acid bacteria is used as a cosmetic composition.

Specifically, the fermentation broth of Gluconacetobacter xylinus described in the present invention is a fermentation broth left by removing the microbial cellulose membrane produced by fermentation of xylobacteria. Although the fermentation broth described in the present case may contain partially residual cellulose, based on the aerobic nature of the xyloacetic acid bacterium, the microbial cellulose is formed at a liquid surface in contact with the liquid medium to form a film, and thus the removal is performed. After the membrane, there is almost no microbial cellulose in the fermentation broth.

The xylobacteria fermentation broth of the present invention is produced by the following methods:

(ii) inoculating the lignin acetic acid bacteria (Gluconacetobacter xylinus);

(iv) The cellulose film in the medium was removed, and the remaining culture liquid was collected.

The medium prepared in the above step (i) contains a carbon source, a nitrogen source, a phosphorus source, an inorganic salt, and an organic acid. The carbon source may be derived from a five-carbon sugar, a six-carbon sugar, a hydrolyzed sugar liquid, a sugar-containing wastewater, etc., and the nitrogen source may be derived from a compound such as yeast extract or peptone. The phosphorus source can be derived from Na ₂ HPO ₄ , NaH ₂ PO ₄ , K ₂ HPO _{4 ,} and the like. The inorganic salts may be derived from minerals, vitamins such as NaCl, CaCO ₃ , C ₂ H ₃ O ₂ Na, and the like. The organic acid can be derived from citric acid, acetic acid or the like. In one embodiment of the present invention, glucose having a concentration of 10 to 30 g/L is used as a carbon source, and 2 to 10 g/L of Na ₂ HPO _{4 is} used as a phosphorus source. However, the composition of the medium formulated in this case can be appropriately adjusted according to the conditions of culture.

Further, in the above method for producing a xyloacetic acid fermentation broth, the bacterial inoculation amount of Gluconacetobacter xylinus may be 10 ² to 10 ⁵ cells/mL, but is not limited thereto, and the culture conditions and desired fermentation broth may be used. Adjust the concentration appropriately.

Further, in the above method for producing a xyloacetic acid fermentation broth, the step (iii) of the culture medium containing the xylosylic acid bacterium may be carried out at 25 to 32 °C. In one embodiment of the present invention, the above static culture is carried out at room temperature. In another embodiment of the present invention, the static culture is a two-stage culture comprising culturing at a temperature of 25 to 28 ° C in the first stage and 29 to 32 ° C in the second stage after the first stage.

Furthermore, in the above method for producing a xyloacetic acid fermentation broth, in addition to the above steps (i) to (iv), a purification step may be further included, including the addition of an adsorbent, ultrafiltration, or Filter at least one of them. The adsorbent may include a food adsorbent, activated carbon or a product having an adsorption effect. In one embodiment, the adsorbent may be activated carbon, which adsorbs odor and decolorizes by the porous structure of activated carbon, and then removes activated carbon by filtration or the like. Ultrafiltration uses a nanoporous ultrafiltration membrane to filter out impurities in the fermentation broth.

In the method for producing a xyloacetic acid fermentation broth of the present invention, in addition to the above steps (i) to (iv), the step of drying the culture liquid collected in the step (iv) may be further included. According to the invention of the present invention, the dried lactic acid bacteria fermentation liquid can be stored for a long period of time, and only needs to be added with water to dissolve, and the activity can be reproduced.

According to the invention of the present invention, the xyloglucanase fermentation liquid preferably contains a concentration of 5% by weight or less, more preferably 0.1% by weight to 5% by weight, even more preferably 2% by weight to 5%, based on the total weight of the cosmetic composition to be prepared. weight%. The cosmetic composition containing the lactic acid bacteria fermentation liquid having a concentration of 5 wt% or less has an effective DPPH (di(phenyl)-(2,4,6-trinitrophenyl) iminoazanium) free radical scavenging effect, ABTS (2, 2'-azino- Bis (3-ethylbenzthiazoline-6-sulfonic acid) free radical scavenging effect, inhibitory effect of reactive oxygen species (ROS) production, inhibition of nitric oxide (NO) production, inhibition of lipoxygenase activity, and The inhibitory effect of mushroom tyrosinase activity can be seen that the cosmetic composition described in the present invention has anti-oxidation, anti-aging, moisturizing, whitening and the like, and in the cytotoxicity test, the concentration is 5 weight. The cell viability of the xylobacteria fermentation broth of less than % is more than 50%, and the biosafety of the cosmetic composition described in the present invention can be apparent.

The type of the cosmetic composition described in the present invention may be selected from the group consisting of a lotion, a gel, a frozen film, a mud film, an emulsion, a cream, a lipstick, a foundation, a powder, a powder, and a makeup cosmetic. , but not limited to, cleansing oil, cleansing milk, facial cleanser, shower gel, shampoo, hair lotion, sunscreen lotion, hand cream, nail polish and perfume.

The cosmetic compositions of the present invention may optionally contain cosmetically acceptable ingredients such as emulsifiers, penetration enhancers, softeners, solvents, excipients, antioxidants, or combinations of these.

The cosmetic composition described in the present invention may further comprise a second active ingredient such as hyaluronic acid, salicylic acid, ursolic acid, kojic acid, a polypeptide, an oligopeptide, a growth factor, an enzyme, or the like, or a combination thereof.

The specific embodiments of the present invention are described in detail below, but the following embodiments are only used to further disclose the technical content of the present invention, and should not limit the scope of the invention.

Example

[Example 1] Preparation of xyloacetic acid fermentation broth (1)

Prepare a liquid medium of 10~30g/L of glucose, 5~10g/L of yeast extract powder (Qixin Biotechnology Co., Ltd.), and after sterilization, access to Gluconacetobacter xylinus (which is obtained) From the Bioresource Conservation and Research Center-Food Industry Development Research Institute, after the strain has been activated and isolated, the strains with better performance are selected for use in the process), aerated at 30 °C, and cultured for 3-7 days as an inoculum. . On the other hand, a culture solution of 10 to 30 g/L glucose, 5 to 10 g/L yeast extract powder, 2 to 10 g/L Na ₂ HPO ₄ , and 1 to 5 g/L citric acid (sterilization at 121 ° C for 30 minutes) is prepared. The previously cultured inoculum was added to control the initial concentration of xyloacetic acid bacteria at 10 ² to 10 ⁵ cells/mL, and the culture was carried out in an A4-sized culture pot. The initial culture temperature was 25-28 ° C. After 2-4 days, it is raised to 29 to 32 ° C, and further cultured for 3 to 10 days. Thereafter, the crude fermentation broth was collected, and the fermentation broth was brown in color and was a flowable liquid. The obtained filtrate was allowed to stand for 10 hours by adding 10 g of activated carbon to decolorize and deodorize. Then, suction filtration was performed using Whatman filter paper, and the collected filtrate was 90 mL of the purified fermentation liquid. The purified fermentation broth was dried under vacuum to obtain 2.25 g of a dried fermented product, which was stored at 25 °C. The dried fermented product was added with water and dissolved back before use to obtain a 100%-concentrated fermentation broth of xylobacter aceti to carry out the following tests.

[Example 2] Preparation of xyloacetic acid fermentation broth (2)

Prepare liquid culture medium for pre-culture of glucose 10～30g/L and yeast extract powder 5～10g/L. After sterilization, connect to Gluconacetobacter xylinus, aerate culture at 30°C, and culture for 3-7 days. Used as an inoculum. On the other hand, a culture solution of 10 to 30 g/L glucose, 5 to 10 g/L yeast extract powder, 2 to 10 g/L Na ₂ HPO ₄ , and 1 to 5 g/L citric acid (sterilization at 121 ° C for 30 minutes) is prepared. The previously cultured inoculum was added to control the initial concentration of xyloacetic acid bacteria at 10 ² to 10 ⁵ cells/mL, and the culture was carried out in an A4-sized culture pot. The initial culture temperature was 25-28 ° C. After 2-4 days, it is raised to 29 to 32 ° C, and further cultured for 3 to 10 days. Thereafter, the crude fermentation broth was collected, and the fermentation broth was brown in color and was a flowable liquid. The collected fermentation broth will further add 1-10% by weight of adsorbent for pre-decolorization and deodorization treatment, the treatment condition is stirring at normal temperature for 10 minutes, and then the adsorbent is removed by suction filtration, and the obtained product is obtained. It can be used directly after sterilization without any processing.

[Example 3] Cell survival test

step

Human skin keratinocytes (HaCaT cells obtained from the Center for Bioresource Conservation and Research - Food Industry Development Institute, BCRC 60038) were cultured in a 96-well plate (NEST 701001) at 1 × 10 ⁵ cells/mL. Incubate for 24 hours at 37 ° C in a 5% CO ₂ incubator. On the other hand, the above-mentioned xylosonic acid fermentation broth was diluted with water to prepare a fermentation broth of xylobacter xylinum at concentrations of 1 wt%, 2 wt%, 5 wt% and 10 wt%, respectively. Further, 1 μL of the above-mentioned various concentrations of the xylosylobacter fermentation broth was added to the 96-well plate and cultured in a 37 ° C, 5% CO ₂ incubator for 24 hours. The above fermentation broth was not added to the control group. Then remove the culture solution, wash it with PBS (phosphate buffered saline), replace the fresh medium, and add 10 μL of MTT (3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (5 mg). /mL) The solution was reacted for 4 hours. The supernatant was then removed, and after adding 100 μL of DMSO, the absorbance was measured at a wavelength of 570 nm (BioTek, Synergy ^TM 2, USA).

The human skin keratinocyte culture medium was composed of Dube's improved Ig's culture medium, and 10% (v/v) fetal bovine serum and 1 mM sodium pyruvate were additionally added.

result

The results are shown in Fig. 1. The cell viability of the control group to which no fermentation broth was added (100%), human skin keratinocytes (HaCaT cells) and the concentrations of 1 wt%, 2 wt%, 5 wt%, and 10 wt% above. After 24 hours of co-cultivation of the xylobacter acetate fermentation broth, the cell viability was 97.5%, 95.4%, 70.0% and 50.5%, respectively. This result indicates that the xylobacteria fermentation broth having a concentration of 5% or less is not cytotoxic.

[Example 4] Cell cycle test

step

1×10 ⁵ cells/mL of human skin keratinocytes (HaCaT cells) were cultured in a 24-well dish for at least 24 hours, after which 2% of the above-mentioned xyloacetic acid fermentation broth was added and reacted in an incubator for 24 hours. . The supernatant was collected into a 15 mL centrifuge tube, and the cells were removed into a centrifuge tube with trypsin-EDTA solution, and centrifuged at 1200 rpm for 5 minutes with the supernatant. The supernatant was removed, 300 μL of PBS was added, and the mixture was slowly shaken, and 700 μL of absolute alcohol was added dropwise to fix the cells, and then transferred to a microcentrifuge tube.

The above microcentrifuge tube was centrifuged at 1200 rpm for 5 minutes at 4 ° C, and the supernatant was removed. Then, 445 μL of PBS, 5 μL of RNase (10 mg/mL), and 50 μL of 10% Triton X-100 were sequentially added to disrupt the RNA of the cells, and then reacted at 37 ° C for 30 minutes, centrifuged at 1200 rpm for 5 minutes, and the supernatant was removed. . Thereafter, 400 μL of PBS was added and mixed uniformly, and 5 μL of PI (5 mg/mL) was further added thereto, and the reaction was carried out in the dark at 4 ° C for 5 minutes, and filtered through a filtration membrane. Cell cycle distribution ratio (%) was analyzed using a flow cytometer (FACScan) in conjunction with Winmdi computer software.

result

The results are shown in Figure 2, human skin keratinocytes (HaCaT cells) with a concentration of 2wt% After the above-mentioned xyloacetic acid fermentation broth was co-cultured for 24 hours, the sub-G1 in the cell cycle was 1.9% and less than 10%. The subG1 phase is one of the characteristics of apoptosis. If it is a normal cell, there will be no subG1 phase. According to the induced death factor and time, the proportion of this phase in the stained cells will also be different.

[Example 5] Scavenging DPPH free radical evaluation

step

1 μL of the above-mentioned xyloacetic acid fermentation broth at a concentration of 1 wt%, 2 wt%, 5 wt%, and 10 wt%, and 1000 μg/mL of vitamin C as a positive control group, respectively, and 99 μL of freshly prepared 100 μM DPPH (di(phenyl)- A solution of (2,4,6-trinitrophenyl)iminoazanium). Each of the above solutions was shaken and mixed uniformly, and allowed to stand at room temperature for 30 minutes, and then the absorbance at 517 nm was measured using a spectrophotometer. The control is a solution of DPPH to which no fermentation broth or vitamin C is added.

result

The content of DPPH radical detected by the positive control group to which 1000 μg/mL of vitamin C was added was used as a reference (100%), and as shown in Fig. 3, the concentrations were 1 wt%, 2 wt%, 5 wt%, and 10 wt% as described above. The DPPH clearance rates of the xylobacteria fermentation broth were 37.8%, 54%, 77.2% and 84.0%, respectively, and the IC _{50 of the} DPPH free radical scavenging was <10%. The higher the value of DPPH free radical scavenging, the stronger the antioxidant capacity of the fermentation broth.

[Example 6] Inhibition of ABTS free radical evaluation

step

Formulation of ABTS+ stock solution: 7 mM ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (FW=548.68) and 4.9 mM K ₂ S ₂ O ₈ (potassium persulfate, FW=270.32), After dissolving in ddH ₂ O, the reaction was allowed to stand at room temperature for 16 hours in the dark.

ABTS analysis solution: The ABTS+ stock solution prepared above was diluted 40 times with secondary ddH ₂ O, and the absorbance was measured at a light absorption value of 734 nm at about 0.7. Thereafter, 2.45 mM K ₂ S ₂ O ₈ and 7 mM ABTS solution were mixed, and after standing for 16-18 hours in the dark, the reaction generated a radical. Thereafter, 1.5 μL of a concentration of 1 wt%, 2 wt%, 5 wt%, and 10 wt% of the above-mentioned fermentation liquid of xylobacteria and 1000 μg/ml of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were respectively taken. As a positive control group, 3.5 μL of ddH ₂ O and 145 mL of a 7 mM ABTS solution were mixed and allowed to stand for 20 minutes, and the absorbance at 734 nm was measured by a spectrophotometer. The lower the absorbance value, the better the ability of the sample to scavenge ABTS free radicals. The control group was a solution in which the above fermentation broth or Trolox was not added.

result

The ABTS free radical is a cationic free radical. This test can evaluate whether the above-mentioned xyloacetic acid fermentation broth has the effect of scavenging cationic radicals. As a result, as shown in FIG. 4, the above-mentioned xylobacteria were added at a concentration of 1 wt%, 2 wt%, 5 wt%, and 10 wt% based on the concentration of ABTS radical detected by adding a 1000 μg/ml Trolox positive control group. The ABTS free radical scavenging rates of the fermentation broth were 33.0%, 76.6%, 77.2% and 84.2%, respectively, and the IC _{50 of the} ABTS free radicals was eliminated by <10%.

[Example 7] Evaluation of inhibition of intracellular production of reactive oxygen species (ROS)

step

The number of cells of 1×10 ⁵ cells/mL was cultured in a 96-well plate, and grown in a 37° C., 5% CO 2 incubator for 24 hours or more, and then added at a concentration of 0 wt%, 1 wt%, 2 wt%, and 5 wt%, respectively. % and 10% by weight of the above-mentioned xylosonic acid fermentation broth were co-cultured. After the reaction time, the supernatant was removed, washed with PBS, and freshly cultured with 0.1 mM H ₂ O ₂ was added thereto for further 1 hour. Then, the culture solution was removed, and a fresh culture solution containing 10 μm of DCFH ₂ DA (2',7'-dichlorofluorescin diacetate) was added, and after 1 hour from the light, the culture solution was removed. After the addition amount of PBS, the excitation light 502nm / 524nm scattered light measured at the absorbance value ^{(BioTek, Synergy TM 2, USA} ). The control group was a cell to which no fermentation broth or H ₂ O ₂ was added.

result

DCFH ₂ DA is a fluorescent agent that emits fluorescence when combined with reactive oxygen species, and uses this property to detect intracellular reactive oxygen species. As a result, as shown in FIG. 5, the ROS concentration generated by the cells to which the fermentation broth or H ₂ O ₂ was not added was used as a reference (100%), and the cells in which the fermentation broth was not added were subjected to H ₂ O ₂ treatment, and the intracellular activity was observed. The amount of oxygen production (121%) increased by 21%. However, in the cells co-cultured with the above-mentioned xyloglucanase fermentation broth at concentrations of 1%, 2%, 5% and 10%, the amount of intracellular ROS production was significantly decreased, indicating that the above-mentioned xyloacetate fermentation broth can be effectively reduced by H. ₂ O ₂ induces the production of ROS. The ROS production of the cells was lower than 110.5% (50% of active oxygen production) after the action of the above-mentioned 1%, 2%, 5% and 10% concentrations of the xylosonic acid fermentation broth, indicating that the above-mentioned xylobacteria fermentation broth inhibited intracellular cells. Active oxygen (ROS) production has an IC ₅₀ <10%.

[Example 8] Inhibition of lipoxygenase activity test

step

1 μL of 0.1 wt%, 0.2 wt% of the above-mentioned xyloacetic acid fermentation broth and 1000 μg/ml caffeic acid were used as positive control groups, and 2 μL of LOX-1 (135 units) were added. It Thereafter, 1.5 μL of 10 mM linoleic acid was added as a substrate for causing the reaction, and 95.5 μL of Tris-HCl buffer (pH 9.0) was added. The control group was completely uniformly mixed with an equal dose of DMSO, and its absorbance was measured at 234 nm with a spectrophotometer.

result

The LOX-1 enzyme is one of the important enzymes in the inflammatory reaction. This test can be used to evaluate whether the above-mentioned xyloacetate fermentation broth has anti-inflammatory efficacy. Since this test has a wavelength limitation, since the above-mentioned xyloacetic acid fermentation broth is dark brown at a high concentration, only the inhibition rate of the lipoxygenase in the low concentration of the xylobacteria fermentation broth can be measured. The results are shown in Fig. 6. Based on the LOX-1 concentration measured by the positive control group to which 1000 μg/ml of caffeic acid was added (100%), the concentration of 0.1% and 0.2% of the above-mentioned lignin fermentation broth was inhibited in LOX. The ability of -1 activity was 58.7% and 65.2%, respectively, and its IC _{50 of} inhibition of LOX-1 activity was <10%.

[Example 9] Nitric oxide (NO) removal test

step

98 μL of sodium nitroprusside 5 mM was added to 2 μL of 1%, 2%, 5% and 10% of the above-mentioned xyloacetic acid fermentation broth, and 1000 μg/ml rutin was used as a positive control group at 25 ° C. Cultured for 150 minutes. Thereafter, 100 μL of Griess Reagent (0.1% naphthylenediamine dihydrochloride, 5% phosphoric acid and 1% sulfanilamine) was added, and the absorbance at 560 nm was measured. The control group was a solution in which the above fermentation broth or rutin was not added.

result

The concentrations of 1% by weight, 2% by weight, 5% by weight and 10% by weight of the above-mentioned xylosidum fermentation broth to remove NO radicals were about 34.0%, 38.9%, 47.1% and 51.2%, respectively, and the ability of the rutin to remove NO radicals was about 84%, which inhibits ₅₀ <10% of the enzyme tyrosinase IC.

[Example 10] Determination of inhibition of mushroom tyrosinase activity

step

Using 1000 μg/ml of vitamin C as a standard, the test group was 2 μL of the concentration of 1 wt%, 2 wt%, 5 wt%, and 10 wt% of the above-mentioned xyloacetic acid fermentation broth mixed with 18 μL of DMSO, and added to a 96-well plate, followed by 25 μL. A 100 unit solution of mushroom tyrosinase was reacted at room temperature for 10 minutes. Thereafter, the background value of the mushroom tyrosinase was measured at a wavelength of 475 nm. Subsequently, 155 μL of 2.5 mM L-DOPA solution was added in the dark to mix at room temperature, and the absorbance of mushroom tyrosinase (BioTek, Synergy ^TM 2, USA) was measured at a wavelength of 475 nm. The inhibitory activities of different concentrations of Phytohemagglutinin fermentation broth on mushroom tyrosinase were calculated.

result

As shown in FIG. 8 , the above-mentioned xyloacetate fermentation broths having a concentration of 1% by weight, 2% by weight, 5% by weight, and 10% by weight, respectively, based on the concentration of the mushroom tyrosinase measured by adding 1000 μg/mL of vitamin C, respectively. It has a tyrosinase inhibition rate of 30.4%, 55.8%, 93.8% and 94.3%, indicating that the above-mentioned xyloacetic acid fermentation broth has a good whitening effect, and its inhibitory tyrosinase has an IC ₅₀ <10%.

Although the present invention has been described above in terms of the preferred embodiments, it is not intended to limit the present invention, and it is to be understood that those skilled in the art can make some modifications and refinements without departing from the spirit and scope of the invention. The scope of the invention is defined by the scope of the appended claims.

Claims

A cosmetic composition comprising a fermentation broth of Gluconacetobacter xylinus as an active ingredient.
The cosmetic composition according to claim 1, wherein the xylobacteria fermentation broth is produced by the following method:

(i) blending a medium containing a carbon source, a nitrogen source, a phosphorus source, an inorganic salt, and an organic acid;

(ii) inoculating the lignin acetic acid bacteria (Gluconacetobacter xylinus);

(iii) allowing the culture medium containing the xylobacteria to be cultured; and

(iv) The cellulose film formed in the medium was removed, and the remaining culture liquid was collected.
The cosmetic composition according to claim 2, wherein the xylobacteria is inoculated in an amount of from 10 2 to 10 5 cells/mL.
The cosmetic composition according to claim 2, wherein the static culture of the medium containing the xylobacteria is carried out at 25 to 32 °C.
The cosmetic composition according to claim 2, wherein the static culture of the culture medium containing the xylobacteria is a two-stage culture comprising the first stage of culturing at a temperature of 25 to 28 ° C and at the same The second stage after one stage is cultured at 29 to 32 °C.
The cosmetic composition according to claim 2, wherein the method for producing the xylosonic acid fermentation broth further comprises a purification step comprising at least one of addition of an adsorbent, ultrafiltration, or filtration.
The cosmetic composition according to claim 6, wherein the adsorbent comprises a food adsorbent, activated carbon or a product having an adsorption effect.
The cosmetic composition according to claim 2, wherein the method for producing the xylemacetic acid fermentation broth further comprises the step of drying the culture solution collected in the step (iv).
The cosmetic composition according to claim 1, wherein the concentration of the xylosonic acid fermentation broth is 5% by weight or less based on the weight of the cosmetic composition.
A use of a fermentation broth of Gluconacetobacter xylinus as a cosmetic composition.
The use according to claim 10, wherein the xylobacteria fermentation broth is produced by the following method:

(i) blending a medium containing a carbon source, a nitrogen source, a phosphorus source, an inorganic salt, and an organic acid;

(ii) inoculating the lignin acetic acid bacteria (Gluconacetobacter xylinus);

(iii) allowing the culture medium containing the xylobacteria to be cultured; and

(iv) The cellulose film formed in the medium was removed, and the remaining culture liquid was collected.
The use according to claim 11, wherein the xylobacteria is inoculated in an amount of from 10 2 to 10 5 cells/mL.
The use according to claim 11, wherein the static culture of the medium containing the xylobacteria is carried out at 25 to 32 °C.
The use according to claim 11, wherein the static culture of the medium containing the xylobacteria is a two-stage culture comprising the first stage of culturing at a temperature of 25 to 28 ° C and at the first stage The second stage after the incubation was carried out at 29 to 32 °C.
The use according to claim 11, wherein the method for producing the xylosonic acid fermentation broth further comprises a purification step comprising at least one of addition of an adsorbent, ultrafiltration, or filtration.
The use according to claim 15, wherein the adsorbent comprises a food adsorbent, activated carbon or a product having an adsorption effect.
The use according to claim 11, wherein the method for producing the xylosonic acid fermentation broth further comprises the step of drying the culture liquid collected in the step (iv).
The use according to claim 11, wherein the concentration of the xylosonicum fermentation broth is 5% by weight or less based on the weight of the cosmetic composition.