WO2022050559A1 - Method for preparing deinoxanthin fermented extract and cosmetic composition comprising deinoxanthin fermented extract - Google Patents

Abstract

The present invention relates to a method for preparing a deinoxanthin fermented extract, the method enabling deinoxanthin production efficiency to be maximized by optimizing deinoxanthin production conditions. In addition, antioxidant, anti-inflammatory and anti-wrinkle effects may be enhanced by providing a cosmetic composition comprising the deinoxanthin fermented extract. Research related to the present invention was carried out with support from the SME Technology Innovation Development Program of The Ministry of SMEs and Startups (microbial fermentation-based mass production of a novel carotenoid deinoxanthin containing a biocompatible biodegradable polymer, and development of a cosmeceutical cosmetic material; project no. S2954110).

Description

Manufacturing method of deinoxanthin fermented extract and cosmetic composition comprising deinoxanthin fermented extract

The present invention relates to a method for producing a deinoxanthin (deinoxanthin) fermentation extract.

In addition, the present invention relates to a cosmetic composition comprising the fermented deinoxanthin extract.

Deinococcus radiodurans ( Deinococcus radiodurans ) is an organism that is highly resistant to radiation as a microorganism that survives in an extreme environment.

This microorganism produces carotenoid-based pigments, and among them, deinoxanthin is known to have high antioxidant capacity.

Conventionally, by utilizing the genetic resources of Deinococcus radiodurans, there has been an improved deinoxanthin biosynthetic strain with an improved yield.

However, since this method for producing the improved strain involves the overexpression of a foreign gene by a plasmid, an expensive inducer is required, and the administration of antibiotics as a selective marker during culture and fermentation of the improved strain is essential

In addition, there is a risk that the productivity of the final product may be lowered because the plasmid is not stable in the cell.

Accordingly, research on strains for improving the production of deinoxanthin is being actively conducted, and a method for further improving the production efficiency of deinoxanthin by utilizing the strain is required.

It is an object of the present invention to provide a method for producing deinoxanthin with improved production efficiency.

Another object of the present invention is to provide a cosmetic composition comprising the deinoxanthin.

According to one aspect of the present invention, there is provided a cosmetic composition comprising a Deinococcus sp. strain fermentation broth, culture broth, or extract thereof as an active ingredient.

In one embodiment, the fermentation broth or culture broth may be obtained by culturing the Deinococcus sp. strain at 30°C.

In one embodiment, the fermentation broth or culture broth may be obtained by primary and secondary culture of the Deinococcus sp. strain at 30 °C.

In one embodiment, the Deinococcus sp. strain may be Deinococcus sp. KACC 81132BP.

In one embodiment, the cosmetic composition may be for antioxidant or anti-inflammatory.

In one embodiment, the cosmetic composition may be for anti-aging.

According to another aspect of the present invention, the step of inoculating the strain KACC 81132BP (Agricultural Biotechnology Research Institute) of the deinococcus genus into the primary culture medium;

30° C., with stirring at 200 rpm and culturing for the first time;

Inoculating the primary cultured strain into the secondary culture medium;

Secondary incubation at 30 ℃;

Recovering cells by centrifuging the secondary culture medium at 4000 rpm for 20 minutes at room temperature;

Including; suspending the recovered cells, homogenizing for 1 hour and filtering;

The primary culture medium and the secondary culture medium contain 5 g of tryptone, 5 g of yeast extract, 10 g of glucose, 0.5 g of MgSO ₄ ·7H ₂ O and 1 mg of MnCl ₂ per 1 L of water,

The primary culture comprises the step of culturing with stirring for 24 hours,

The secondary culture is provided with a method for producing a deinoxanthin fermentation extract comprising the step of culturing with stirring for 48 hours.

The specific details of other embodiments according to the present invention are included in the detailed description below.

According to the present invention, it is possible to maximize the production efficiency of deinoxanthin by optimizing the production conditions of deinoxanthin. In addition, it is possible to improve the antioxidant, anti-inflammatory, anti-wrinkle effect by providing a cosmetic composition containing the deinoxanthin.

1 is a graph showing the results of strain growth according to the difference in the composition of the medium.

Figure 2 is a graph showing the results of strain growth and deinoxanthin production over time.

3 and 4 are graphs showing the DPPH scavenging ability according to the concentration of deinoxanthin ferment extract or astaxanthin.

5 and 6 are graphs showing the change in the amount of active oxygen (ROS) production according to the concentration of deinoxanthin fermentation extract or astaxanthin.

7 is a graph showing the change in the amount of nitric oxide (NO) production according to the concentration of deinoxanthin fermentation extract or astaxanthin.

8 is a graph showing the change in the amount of MMP-1 production according to the concentration of deinoxanthin fermentation extract.

Since the present invention can apply various transformations and can have various embodiments, specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

Unless otherwise specified in the present specification, the expression "between" is used as an expression including the corresponding numerical value. Specifically, for example, the expression "1 to 2" is meant to include all numbers between 1 and 2 as well as 1 and 2.

Hereinafter, a method for producing a deinoxanthin ferment extract according to an embodiment of the present invention and a cosmetic composition comprising the same will be described in more detail.

According to one aspect of the present invention, there is provided a cosmetic composition comprising a Deinococcus sp. strain fermentation broth, culture broth, or extract thereof as an active ingredient.

The Deinococcus genus ( Deinococcus sp. ) The strain is known to have resistance to ionizing radiation that can directly damage the DNA or protein of the organism, specifically Deinococcus radiodurans ( Deinococcus radiodurans ) It may be .

The resistance of the Deinococcus sp. strain can quickly repair DNA damage caused by ionizing radiation through an enzymatic or non-enzymatic system, and effectively remove Reactive Oxygen Species (ROS) generated in cells. It can be attributed to the ability

The deinococcus sp. strain produces a large amount of deinoxanthin, a carotenoid, in the culture and fermentation process, and can be used as a use for sulfuration and skin improvement.

The culture medium may be the culture medium itself obtained by culturing the deinococcus sp. strain, or the culture supernatant obtained by removing the strain therefrom, or the concentrate or freeze-dried product of the culture supernatant.

The culture medium is Deinococcus sp. strain glucose, fructose, mannose, galactose, xylose, ribose, maltose, sucrose, dextrin, glycerin, and at least one carbon source selected from the group consisting of inulin; And potassium nitrate, ammonium nitrate, ammonium sulfate, ammonium oxalate, and may be obtained by culturing the strain in a medium containing at least one nitrogen source selected from the group consisting of diammonium phosphate inulin.

The carbon source or nitrogen source used in the culture medium is, for example, about 0.1 to 30 w / v%, 1 to 25 w / v%, 1 to 20 w / v%, 1 to 10 w / v%, 1 to 5 w / v %, or 1 to 1.5 w/v%.

The culture medium may include salts commonly used in the culture medium of the strain, and the salts include, for example, at least one salt selected from the group consisting of potassium nitrate, potassium sulfate, magnesium carbonate, magnesium sulfate, and magnesium nitrate. and the salt may be included in, for example, about 0.01 to 10 w/v%, 0.1 to 7 w/v%, or 0.5 to 5 w/v%.

The extract may be obtained by extracting the separated culture solution of the deinococcus sp. strain with various organic solvents.

The Deinococcus sp. strain may be Deinococcus sp. KACC 81132BP.

The present inventors have found that the separately isolated Deinococcus sp. strain (KACC 81132BP) not only has excellent deinoxanthin production capacity compared to the Deinococcus sp. strain, but also produces a variety of active ingredients to exhibit more excellent skin improvement activity. Confirmed.

The cosmetic composition may be for antioxidant or anti-inflammatory.

The “antioxidation” refers to the action of inhibiting oxidation, and the human body has a balance between an oxidation promoting substance and an oxidation inhibitory substance. , oxidative stress is induced in the living body, which may cause cell damage and pathological diseases.

Reactive oxygen species (ROS), the direct cause of oxidative stress, are chemically unstable and highly reactive, so they can easily react with various biological materials such as DNA, proteins, lipids and carbohydrates, and attack macromolecules in the living body. It causes irreversible damage to cells and tissues, mutation, cytotoxicity, cancer, etc., and is also a direct cause of aging. By removing or reducing the reactive oxygen species, it is possible to obtain an antioxidant effect to prevent aging and maintain health.

The “anti-inflammatory” refers to the action of inhibiting inflammation, and the regulation of the inflammatory response is known to be very complicated, which reduces the enhancement and damage of the repair system in vivo.

If the inflammatory response continues due to repeated tissue damage or regeneration, ROS and RNS are overproduced in inflammation-related cells, and permanent gene modification can be caused. That is, ROS and RNS are deeply related to the inflammatory response that regulates the actions of various cells in vivo.

The cosmetic composition may be for anti-aging.

The “anti-aging” may refer to acting on the skin to inhibit or reverse aging. As the aging progresses, symptoms in which the content and arrangement of collagen, elastin, hyaluronic acid, and glycoproteins, which are substances constituting the skin, change or decrease, appear, and oxidative stress caused by free radicals and free radicals is received. In addition, as aging progresses, changes such as wrinkles of the skin are reduced and elasticity is reduced may appear.

In addition, the present invention,

Inoculating the deinococcus genus KACC 81132BP (Agricultural Biotechnology Research Institute) strain into the primary culture medium;

First incubation at 30 ℃, 200rpm;

Inoculating the primary cultured strain into the secondary culture medium;

Secondary incubation at 30 ℃;

Centrifuging the culture solution after the secondary culture at 4000 rpm for 20 minutes at room temperature to recover the cells, and washing with distilled water;

It provides a method for producing a deinoxanthin fermentation extract comprising a; suspending the recovered cells, homogenizing for 1 hour and filtering.

The primary culture medium and the secondary culture medium are tryptone 5 g, yeast extract 5 g, glucose 10 g, MgSO ₄ ·7H ₂ O 0.5g and MnCl ₂ Preparation of deinoxanthin fermentation extract containing 1 mg per 1L of water provide a way

Compared to the case of using the general microbial culture medium TGY as the primary medium or the secondary medium, when the medium according to the present invention is used, the growth rate of microorganisms is improved and the initial inoculation amount is increased, so that the growth state of high OD is rapidly achieved. can be introduced

According to one embodiment, the glucose of the primary culture medium or the secondary culture medium may be included as a glucose solution prepared by dissolving 500 g of glucose in 1 L of water.

In addition, MnCl ₂ of the secondary culture medium may be included as a MnCl ₂ solution prepared by dissolving MnCl ₂ 1g in 1L of water.

When the content of tryptone in the composition of the culture medium increases, it is preferable not to add more than 8 g because it may inhibit the production of deinoxanthin, and when sodium glutamate or HEPES is added, the growth of microorganisms increases, but deinoxanthin It is preferable not to include it because it may inhibit the production of

In addition, the addition of MnCl ₂ of 0.5 mg or more is effective for the production of deinoxanthin.

According to one embodiment, the step of recovering the cells by centrifugation may be carried out at room temperature. In general, the cell recovery step is carried out at a low temperature to prevent damage to the cells, but in the present invention, the final product can be effectively obtained even when the cells are recovered at room temperature.

According to one embodiment, after the centrifugation to recover the cells, the method may further include lyophilizing the recovered cells.

In addition, the step of suspending the cells may include suspending by adding 10 to 30 mL of ethanol at a concentration of 90 to 100% (v/v) per 1 g of dried cells, for example, 15 to 25 mL of ethanol. .

Alternatively, it may include adding ethanol at a concentration of 90 to 100% (v/v) in a volume of 10 to 50 times, for example 30 to 50 times, with respect to the dry cells. When suspending cells, using ethanol rather than methanol is suitable for use as a cosmetic composition.

According to one embodiment, the step of homogenizing the cells may use an ultrasonicator (ultrasonicator), but is not limited as long as it is generally used. The homogenization treatment time may be, for example, from 10 minutes to 120 minutes, for example from 30 minutes to 90 minutes, for example from 40 minutes to 80 minutes. If the homogenization treatment time is shorter than the above range, the target product may not be sufficiently eluted, and if it is longer than the above range, the target product may be damaged.

According to one embodiment, in the filtering step, for example, 0.25um, for example, a filter paper having a pore size of 0.45um may be used, but if it corresponds to this, it may be replaced without limitation.

According to one embodiment, the primary culturing may include culturing for 24 hours. In addition, the secondary culture may include culturing for 48 hours.

According to another embodiment of the present invention, there is provided a cosmetic composition comprising a deinoxanthin ferment extract prepared by the method as described above. For example, the cosmetic composition can be applied for the purpose of antioxidant, anti-inflammatory or anti-wrinkle.

According to one embodiment, the cosmetic composition according to the present invention may be prepared in any formulation conventionally prepared in the art, for example, a solution, suspension, emulsion, emulsion, paste, gel, pack, cream, It may be formulated as lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, spray, and hair cosmetics, but is not limited thereto.

Specifically, the cosmetic composition includes skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, foundation, essence, nourishing essence, pack, It can be prepared in the formulation of soap, hair shampoo, foot shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion and body cleanser.

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily carry out the present invention. However, the present invention may be embodied in several different forms and is not limited to the embodiments described herein.

Experimental Example 1: Selection of optimal medium

In order to efficiently obtain a deinoxanthin fermentation extract by optimizing the medium, the deinococcus genus KACC 81132BP (Research Institute of Agriculture and Biotechnology) strain was cultured in the medium of the composition as shown in Table 1.

At this time, 1/50 of glucose was prepared at 500 g/L, and 1/1000 of MnCl ₂ was prepared at 1 g/L and then added.

division		Badge 1	Badge 2	Badge 3	Badge 4
tryptone	5g	5g	5g	10g
Na 2 HPO 4		6g
KH 2 PO 4		3g
yeast extract	5g	5g	5g	5g
glucose	10g	10g	10g	10g
sodium glutamate				10g
HEPES				12g
MgSO 4 7H 2 O		0.5g	0.5g	0.5g
MnCl 2		1mg	1mg	1mg
Distilled water	1L	1L	1L	1L

Incubated at 30 ° C. for 66 hours, OD measurement results are shown in Table 2 and FIG. 1 .

0.128 = media 2 blank OD
time (hr)	Badge 1	Badge 2	Badge 3	Badge 4
0	0.396	0.396	0.396	0.396
3.8	0.94	0.8	0.73	0.69
19	2.83	1.74	3.22	3.05
25	4.7	5.69	9.19	7.73
43	5.46	9.2	13.94	12.48
49	5.14	10.46	16.64	14.4
66	4.98	10.8	17.02	14.58

As shown in Table 2 and Figure 1, it was confirmed that the strain grows best in the composition of medium 3.

Example 1

Deinococcus genus KACC 81132BP (agricultural biotechnology) in a medium (medium 3 in Table 1) containing 5 g of tryptone, 5 g of yeast extract, 10 g of glucose, 0.5 g of MgSO ₄ 7H ₂ O and 1 mg of MnCl ₂ per 1 L of water Researcher) The strain was inoculated, and the primary culture was performed while stirring at 30°C and 200rpm for 24 hours.

The primary cultured strain was inoculated into the same medium, and the secondary culture was performed while stirring at 30° C. at 200 rpm for 48 hours.

Cells were recovered by centrifugation for 20 minutes at 4000 rpm at room temperature from the secondary culture medium.

The recovered cells were freeze-dried at -80°C for 72 hours. To 1 g of dry cells, 20 mL of 99% (v/v) concentration of ethanol was added and suspended, followed by pulverization for 1 hour using an ultrasonicator to obtain an extract from the cells using a Rotery Vaccum Evaporator.

The obtained extract was filtered with a filter paper of 0.25um pore size to obtain a deinoxanthin fermentation extract, a final product from which cell residues were removed.

Example 2

Primary and secondary culture was performed with the same strain and method as in Example 1. At this time, the culture time was 0 to 140 hours to confirm the strain growth and deinoxanthin content.

After the secondary culture, cells were recovered in the same manner as in Example 1, and the recovered cells were freeze-dried.

To dry cells, 99% ethanol was added in a volume of 40 times, extracted for 2 hours in a water bath at 70° C., and filtered with 0.45 μm pore filter paper to obtain a final product.

comparative example

Deinococcus genus KACC 81132BP (Agricultural Biotechnology Research Institute) strain was inoculated into a medium containing 5 g of tryptone, 3 g of yeast extract and 1 g of glucose with respect to 1 L of water, and the seed culture was cultured with stirring at 30 ° C., 200 rpm for 24 hours.

The seed cultured strain was cultured in a medium containing 10 g of tryptone, 5 g of yeast extract, 10 g of glucose, 10 g of sodium glutamate, 12 g of HEPES, 0.5 g of MgSO ₄ 7H ₂ O and 1 mg of MnCl ₂ per 1 L of water (medium 4 in Table 1). ) and incubated with stirring at 200rpm for 48 hours at 37°C.

Cells were recovered by centrifugation of the cultured strain culture solution at 4000 rpm at 4° C. for 20 minutes and washed twice with sterile distilled water. The recovered cells were dried with a freeze dryer for 12 hours.

The dried cells were suspended by adding 1 mL of methanol, and homogenized for 1 minute using a sonicator.

The homogenized extract was centrifuged at 4,000 rpm and 4° C. for 20 minutes to separate from the cell lysate, and then the methanol extract containing deinoxanthin was filtered through a 0.2 μm PTPE injection filter.

Experimental Example 2: Deinoxanthin content analysis

The products according to Example 1 and Comparative Example were placed in a 2 mL brown HPLC tube and subjected to HPLC analysis under the following conditions.

Equipment: Waters HPLC system 2969

Column: Zorbax Eclipse XDB-C18 5um (4.6 x 150mm)

Temperature: 30℃

Flow rate: 0.5ml/min

UV Wavelength: 480nm

Analysis time: 20 minutes

Injection volume: 10uL

Mobile phase: acetonitrile, methanol and isopropanol 40:50:10 (v/v)

Since there is no commercial quantitative standard product of deinoxanthin, astaxanthin was used as a standard material.

5 mg of astaxanthin powder was completely dissolved in 50 mL of methanol to prepare a standard solution with a final concentration of 100 mg/L.

A calibration curve was prepared for each standard diluted stepwise with methanol.

The R2 value of the prepared calibration curve was 0.9976, and the concentration of astaxanthin versus deinoxanthin was calculated using the formula Y=[42079 X deinoxanthin concentration (mg/L)] + 30691].

The results are shown in Table 3.

division	Migration Time	Area	Concentration (ppm)
comparative example	6.280	168950	15.2
Example 1	6.275	371425	33.4
Astaxanthin 10ppm	4.458	111342	10

Experimental Example 3: Confirmation of strain amount and deinoxanthin production

For the product according to Example 2, strain amount and deinoxanthin production were confirmed.

The amount of strain was confirmed by the OD measurement value, and the deinoxanthin production was analyzed in the same manner as in Experimental Example 2. The results are shown in FIG. 2 .

When the strain was cultured for 72 hours, it showed the optimal growth rate, and it was confirmed that the content of deinoxanthin was maximized. The content of deinoxanthin is 55.2ug/g based on wet cell and 387.7ug/g based on dry cell.

After the maximum growth point of the strain, it can be seen that the content of deinoxanthin increases at the time passing through the stationary phase, and deinoxanthin is decomposed after 72 hours.

Example 3: Deinoxanthin Solubilization

In order to apply the non-polar deinoxanthin fermentation extract to the cosmetic composition, water-phase processing is required.

For the aqueous phase of deinoxanthin, the product according to Example 1 was dissolved in hexanediol and solubilized at 65° C. with polyglyceryl 10 stearate, a natural solubilizer.

The composition of specific soluble deinoxanthin is shown in Table 4.

division	content(%)
Example 1 Product	10
hexanediol	2
Polyglyceryl 10 Stearate	2
Distilled water	86

Experimental Example 4: Confirmation of antioxidant effect

Experimental Example 4-1: DPPH

With respect to the extraction product according to Example 1, the antioxidant effect of the fermented deinoxanthin extract was confirmed using 2,2-Diphenyl-1-picrylhydrazyl (DPPH).

Astaxanthin (AX) was used as a control, and 1000 ppm of the extract contains 5 ppm of deinoxanthin.

A 1 mM DPPH solution was dissolved in methanol and used immediately. The sample solution and the DPPH solution were mixed at a ratio of 1:1 and left in a dark place at room temperature for 20 minutes.

In this case, ascorbic acid was used as a positive control. After 20 minutes of incubation, absorbance was measured at 540 nm using an absorbance measuring instrument Multiskan GO (Thermo Fisher Scientific).

The radical rate (%) compared to the control group was calculated and shown in Table 5 and FIGS. 3 and 4.

division	Antioxidant Activity (%)
	1ppm	2ppm	3ppm	4ppm	5ppm	10ppm	20ppm
Deinoxanthin (DX)	-0.6	11.4	25.1	32.5	41.4	89.3	123.9
Astaxanthin (AX)	-0.1	-	-	-	1.5	1.7	1.5
DX/AX	-	-	-	-	27.6	52.5	82.6

It was confirmed that deinoxanthin had about 50 times more DPPH antioxidant effect than astaxanthin at 10ppm concentration.

Experimental Example 4-2: ROS

The antioxidant effect of the deinoxanthin fermented extract was confirmed by measuring the intracellular active oxygen (ROS) for each concentration of the extract according to Example 1.

HaCaT Keratinocyte cell line, a skin keratinocyte, was cultured in a thermostat at 37°C, 5% CO ₂ using DMEM medium containing 10% FBS, inoculated in a 96 well plate, and after 24 hours, samples were treated by concentration and incubated for 24 hours. did

DCFDA (dichlorofluorescein diacetate) 20uM, a dye for measuring intracellular reactive oxygen species (ROS), was treated for 20 minutes, incubated at RT, and tBHP (55uM) was treated for 1 hour, followed by measurement using a fluorescence microplate reader.

The average value for each sample group was obtained, and the ROS antioxidant capacity was analyzed compared with the value of the control group.

Figure 5 shows the change in active oxygen (ROS) according to the product concentration of Example 1, Figure 6 shows the change in active oxygen according to the concentration of astaxanthin.

Both the deinoxanthin fermented extract and astaxanthin reduced the ROS increased by H2O2, but it was confirmed that the ROS antioxidant efficacy for the deinoxanthin fermented extract was more excellent than that of the astaxanthin compound at the same concentration.

Experimental Example 5: Confirmation of anti-inflammatory effect

The anti-inflammatory effect was confirmed for the final product according to Example 1.

After counting RAW264.7 cells, diluted to 2.5 x 10 ⁵ cells/well, 2.5 x 10 ⁵ cells/well cells were seeded in a 48-well plate, and cultured at 5% CO ₂ , 37° C. in an incubator for 24 hours.

After incubation, the medium was replaced with a serum-free medium, followed by starvation, and each sample was diluted and processed by concentration.

After treatment with dexamethasone used as a positive control, 5% CO ₂ , incubated at 37° C. for 1 hour, and LPS was treated to 1 μg/ml, followed by 5% CO ₂ , incubated at 37° C. for 24 hours.

100 μl of a medium containing synthesized NO was transferred to a 96-well plate, and treated in a 96-well plate at each concentration using 500 μM NaNO 2 as a reference, followed by reaction at room temperature for 20 minutes.

After executing the multiskan GO program for absorbance measurement, the absorbance was set to 540 nm, a measurement area was selected on the plate, and the absorbance was measured.

The value of each synthesized Nitric Oxide was calculated using a standard curve, and the result value was derived from the average of the values, and the anti-inflammatory effect was confirmed and shown by measuring the amount of NO production.

From the results of Figure 7, it was confirmed that the anti-inflammatory effect of the deinoxanthin ferment extract was superior to that of the astaxanthin compound at the same concentration.

Experimental Example 6: Confirmation of anti-aging effect

MMP-1 was analyzed to confirm the anti-aging effect on the final product according to Example 1.

HaCat cell line was cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1X penicillin/streptomycin (P/S), counted, and diluted to 2 x 10 ⁵ cells/well in a 6 cm2 plate. was seeded and incubated for 24 hours at 5% CO ₂ , 37° C. in an incubator.

Another cell, a fibroblast cell line, was cultured in FBM medium containing 10% FBS and 1X penicillin/streptomycin (P/S), counted, and diluted to 3 x 10 ⁴ cells/well in a 24-well plate. was seeded and incubated for 24 hours at 5% CO ₂ , 37° C. in an incubator.

The supernatant of the cultured HaCaT cells was removed, and after washing with PBS, 1 ml of PBS was added and UV irradiation was performed at 15 mJ/cm ² .

After removing the PBS and replacing the DMEM culture medium containing 1% FBS for the starvation step, 5% CO ₂ , incubated for 24 hours in a 37° C. incubator.

The supernatant of HaCaT cells irradiated with UV and the supernatant of HaCaT cells that were not irradiated were each transferred to a 50ml tube, set to a non-toxic concentration range, and diluted with the supernatant of HaCat cells.

The supernatant of the cultured cell fibroblasts was removed. Samples made using the HaCaT supernatant were treated with each concentration in the cultured cell fibroblasts, and then incubated for 24 hours at 5% CO ₂ , 37° C. in an incubator.

150 μL of the supernatant of the cultured cell fibroblasts was placed in a 96 well plate. The amount of MMP-1 present in the fibroblast supernatant was quantified using the Human Total MMP-1 ELISA kit (DY901).

In order to confirm the wrinkle improvement efficacy of the final product according to Example 1, it was checked whether the expression level of MMP-1, a collagen degrading enzyme, decreased by treating the samples by concentration, and this is shown in FIG. 8 .

As shown in FIG. 8, compared to the UV-treated cells, MMP-1 was reduced by about 2.6 times or more in cells treated with 1% (v/v) of the deinoxanthin ferment extract according to the present invention, and 10% (v/v) When treated with v), it was confirmed that the amount of MMP-1 almost similar to that of the control group not treated with UV was shown, so that the deinoxanthin fermentation extract was effective for anti-aging.

Experimental Example 9: Deinoxanthin production according to temperature

In order to confirm the deinoxanthin content according to the temperature, the KACC 81132BP (Agricultural Biotechnology Research Institute) strain of Deinococcus genus was cultured in the same manner as in Example 1, except that the secondary culture temperature condition was applied differently, and in a 5L fermenter 3L culture.

Specifically, the secondary culture temperature was respectively applied to 37 ℃ or 30 ℃ to obtain a deinoxanthin fermentation extract. The results are shown in Table 6.

division	Comparison of deinoxanthin production according to temperature
	30°C incubation	37°C incubation
48hr O.D.	26.3	12.2
Dry cell weight (recovery amount)	13.98g	5.12g
Deinoxanthin production (3L culture)	5.402mg	2.24mg

As can be seen from the results, it can be confirmed that the production of deinoxanthin is about 2.4 times higher when the strain is cultured at 30°C than when the strain is cultured at 37°C.

As described above, it was confirmed that deinoxanthin can be efficiently extracted and obtained from a specific strain by the method according to the present invention.

Although the embodiments of the present invention have been described above, the present invention is not limited to the above embodiments, but may be manufactured in a variety of different forms, and those of ordinary skill in the art to which the present invention pertains will appreciate the technical spirit of the present invention. However, it will be understood that the invention may be embodied in other specific forms without changing essential features. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

[Accession number]

Name of deposit institution: Agricultural Biotechnology Research Institute

Accession number: KACC81132BP

Deposit date: 20200821

Claims (7)

1. A cosmetic composition comprising a deinococcus sp. strain fermentation broth, culture broth, or extract thereof as an active ingredient.
2. According to claim 1,

   The fermentation broth or culture broth is one obtained by culturing the Deinococcus sp. strain at 30° C., a cosmetic composition.
3. 3. The method of claim 2,

   The fermentation broth or culture broth is one obtained by primary and secondary culturing of the Deinococcus sp. strain at 30° C., a cosmetic composition.
4. The method of claim 1,

   Deinococcus sp. strain is Deinococcus genus KACC 81132BP, the cosmetic composition.
5. 5. The method according to any one of claims 1 to 4,

   An antioxidant or anti-inflammatory, cosmetic composition.
6. 5. The method according to any one of claims 1 to 4,

   A cosmetic composition for anti-aging.
7. Inoculating the deinococcus genus KACC 81132BP (Agricultural Biotechnology Research Institute) strain into the primary culture medium;

   30° C., with stirring at 200 rpm and culturing for the first time;

   Inoculating the primary cultured strain into the secondary culture medium;

   Secondary incubation at 30 ℃;

   Recovering cells by centrifuging the secondary culture medium at 4000 rpm for 20 minutes at room temperature;

   Including; suspending the recovered cells, homogenizing for 1 hour and filtering;

   The primary culture medium and the secondary culture medium contain 5 g of tryptone, 5 g of yeast extract, 10 g of glucose, 0.5 g of MgSO ₄ ·7H ₂ O and 1 mg of MnCl ₂ per 1 L of water,

   The primary culture comprises the step of culturing with stirring for 24 hours,

   The secondary culture is a method for producing a deinoxanthin fermentation extract comprising the step of culturing with stirring for 48 hours.

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