KR102214150B1 - Preparation method for deinoxanthin fermented extract and cosmetic composition comprising deinoxanthin fermented extract - Google Patents

Abstract

The present invention relates to a method for producing a deinoxanthin fermented extract, and by optimizing production conditions of deinoxanthin, it is possible to maximize the production efficiency of deinoxanthin. In addition, it is possible to improve antioxidant, anti-inflammatory, anti-wrinkle effects by providing a cosmetic composition comprising the deinoxanthin fermented extract.

Description

The manufacturing method of the deinoxanthine fermented extract and the cosmetic composition containing the deinoxanthine fermented extract {PREPARATION METHOD FOR DEINOXANTHIN FERMENTED EXTRACT AND COSMETIC COMPOSITION COMPRISING DEINOXANTHIN FERMENTED EXTRACT}

The present invention relates to a method for preparing a deinoxanthin ferment extract.

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

Deinococcus radiodurans is a microorganism that survives in extreme environments and is highly resistant to radiation. These microorganisms produce carotenoid pigments, and in particular, deinoxanthin is known to have high antioxidant capacity.

Conventionally, the biosynthetic strain of deinoxanthin with improved yield has been improved using the genetic resources of Deinococcus radiodurans.

However, the manufacturing method of these improved strains involves the process of overexpression of foreign genes by plasmids, so an expensive inducer is required. Is essential. In addition, there is a concern that the productivity of the final product may be lowered because the plasmid is not stable in cells.

Accordingly, studies on strains for improving the production amount of deinoxanthin are actively in progress, and a method for further improving the production efficiency of deinoxanthin using the strain is required.

An object of the present invention is to provide a method for producing deinoxanthine with improved production efficiency.

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

In order to solve the above problems, the present invention

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

1st culturing at 30° C. and 200 rpm;

Inoculating the primary cultured strain into the secondary culture medium;

Secondary culture at 30°C;

Recovering the cells by centrifuging the culture solution after the secondary culture 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 are 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 with respect to 1 L of water. to provide.

According to one embodiment, the primary culture may include the step of culturing while stirring for 24 hours.

In addition, the secondary culture may include, for example, culturing while stirring for 48 hours.

According to another embodiment of the present invention, it provides a cosmetic composition comprising a deinoxanthine fermented extract prepared by the method as described above.

According to one embodiment, the composition may be used for antioxidant, anti-inflammatory or anti-wrinkle applications.

Other details of the embodiments according to the present invention are included in the detailed description below.

According to the present invention, the production efficiency of deinoxanthin can be maximized by optimizing the production conditions for deinoxanthin. In addition, antioxidant, anti-inflammatory, and anti-wrinkle effects can be improved by providing a cosmetic composition containing the deinoxanthine.

1 is a graph showing the strain growth results according to the medium composition difference.
2 is a graph showing the strain growth and deinoxanthin production results over time.
3 and 4 are graphs showing DPPH scavenging ability according to the concentration of deinoxanthine fermented extract or astaxanthin.
5 and 6 are graphs showing changes in the amount of active oxygen (ROS) produced according to the concentration of deinoxanthine fermented extract or astaxanthin.
7 is a graph showing changes in the amount of nitric oxide (NO) produced according to the concentration of deinoxanthine fermented extract or astaxanthin.
8 is a graph showing the change in the amount of MMP-1 produced according to the concentration of the deinoxanthine fermented extract.

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

Unless otherwise specified in the specification, the expression “to” is used as an expression including the corresponding numerical value. Specifically, for example, the expression "1 to 2" means not only including 1 and 2, but also including all numerical values between 1 and 2.

Hereinafter, a method for preparing a deinoxanthine fermented extract according to an embodiment of the present invention and a cosmetic composition including the deinoxanthine fermented extract will be described in more detail.

Specifically, the present invention,

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

1st culturing at 30° C. and 200 rpm;

Inoculating the primary cultured strain into the secondary culture medium;

Secondary culture at 30°C;

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 preparing a deinoxanthine fermented extract comprising; suspending the recovered cells, homogenizing for 1 hour, and filtering.

Preparation of a deinoxanthine fermented extract 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 with respect to 1 L of water for the primary culture medium and the secondary culture medium Provides a way. Compared to the case of using TGY, which is a general microorganism culture medium as the primary or 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, resulting in a rapid growth of high OD. Can be introduced.

According to an 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 1 g of MnCl _{2 in} 1 L of water.

When the content of tryptone in the culture medium composition is increased, it is preferable not to add more than 8g because it may inhibit the production of deinoxanthin. 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, adding 0.5 mg or more of MnCl ₂ is effective in the production of deinoxanthine.

According to an embodiment, the step of recovering the cells by centrifugation may be performed at room temperature. In general, the cell recovery step is performed at a low temperature to prevent damage to the cells, whereas the present invention can effectively obtain the final product even when the cells are recovered at room temperature.

According to an embodiment, after the step of recovering the cells by centrifugation, the step of lyophilizing the recovered cells may be further included.

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 dried cells. When suspending cells, ethanol is more suitable for use in cosmetic compositions than methanol.

According to one embodiment, the step of homogenizing the cells may use an ultrasonicator, but is not limited as long as it is generally used. The homogenization treatment time may be, for example, 10 to 120 minutes, for example 30 to 90 minutes, for example 40 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 an embodiment, in the filtering step, for example, filter paper having a pore size of 0.25 um, for example 0.45 um may be used, but any corresponding one may be replaced without limitation.

According to one embodiment, the primary culture 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 deinoxanthine fermented extract prepared by the method as described above. For example, the cosmetic composition can be applied for antioxidant, anti-inflammatory or anti-wrinkle applications.

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, solutions, suspensions, emulsions, emulsions, pastes, gels, packs, creams, It may be formulated as a lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, spray, hair cosmetic, etc., but is not limited thereto.

Specifically, the cosmetic composition is a skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, foundation, essence, nutrition essence, pack, It can be prepared in the form 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 skilled in the art can easily implement the present invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

Experimental Example 1: Optimal medium selection

In order to optimize the medium to efficiently obtain the deinoxanthine fermented extract, a strain of deinococcus genus KACC 81132BP (Agricultural Biotechnology Research Institute) was cultured in the medium of the composition as shown in Table 1. At this time, glucose was prepared at 500 g/L and then 1/50 was added, and MnCl ₂ was prepared at 1 g/L, and then 1/1000 was added.

division Badge 1 Badge 2 Badge 3 Badge 4 Trypton 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, and OD measurement results are shown in Table 2 and FIG. 1.

0.128 = medium 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 FIG. 1, it was confirmed that the strain grows best in the composition of medium 3.

Example 1

In a medium 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, Dinococcus KACC 81132BP (Agricultural Biotechnology) Researcher) The strain was inoculated, and the first culture was performed while stirring at 30°C and 200 rpm for 24 hours. The primary cultured strain was inoculated into the same medium, stirred at 30° C. for 48 hours at 200 rpm, followed by secondary culture.

The cells were recovered by centrifugation for 20 minutes at 4000 rpm at room temperature from the culture solution after the secondary culture. The recovered cells were lyophilized at -80°C for 72 hours. To 1 g of dry cells, 20 mL of ethanol at a concentration of 99% (v/v) was added and suspended, and pulverized for 1 hour using an ultrasonicator to obtain an extract from the cells with a Rotery Vaccum Evaporator. The obtained extract was filtered through a filter paper having a pore size of 0.25 μm to obtain a deinoxanthine fermented extract, a final product from which cell residues were removed.

Example 2

Primary and secondary cultures were performed in the same strain and method as in Example 1. At this time, strain growth and deinoxanthine content were confirmed by setting the culture time to 0 to 140 hours. After the secondary culture, cells were recovered in the same manner as in Example 1, and the recovered cells were lyophilized.

To the dried cells, 99% ethanol was added in a volume of 40 times, extracted for 2 hours in a 70° C. waterbath, and filtered through a filter paper having a pore size of 0.45 μm to obtain a final product.

Comparative example

In a medium containing 5 g of tryptone, 3 g of yeast extract, and 1 g of glucose with respect to 1 L of water, a strain of Dinococus genus KACC 81132BP (Agricultural Biotechnology Research Institute) was inoculated and seed cultured with stirring at 30° C. and 200 rpm for 24 hours. 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 at 37° C. for 48 hours while stirring at 200 rpm.

After the culture was completed, the culture solution of the strain was centrifuged at 4000 rpm at 4° C. for 20 minutes to recover the cells and washed twice with sterile distilled water. The recovered cells were dried for 12 hours with a lyophilizer. The dried cells were suspended by adding 1 mL of methanol, and homogenized for 1 minute with an ultrasonic grinder. The homogenized extract was separated from the cell lysate by centrifugation at 4,000 rpm for 20 minutes at 4° C., and the methanol extract containing deinoxanthine was filtered through a 0.2 μm PTPE injection filter.

Experimental Example 2: Deinoxanthine content analysis

The products according to Example 1 and Comparative Example were put in a 2 ml brown HPLC tube, and HPLC analysis was performed 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)

As 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 having a final concentration of 100 mg/L. A calibration curve was prepared for each standard diluted step by step using methanol. The R2 value of the prepared calibration curve was 0.9976, and the concentration of deinoxanthin compared to astaxanthin was calculated using the formula of Y=[42079 X deinoxanthin concentration (mg/L)] + 30691].

The results are shown in Table 3.

division Migration Time Area density
(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 amount

With respect to the product according to Example 2, the amount of strain and the amount of deinoxanthin production were confirmed.

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

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

It can be seen that after the highest growth point of the strain, the content of deinoxanthin increases when the stationary phase passes, and the deinoxanthin is decomposed after 72 hours.

Example 3: Deinoxanthine solubilization

In order to apply the non-polar deinoxanthine fermentation extract product to a cosmetic composition, water-phase processing is required. For the aqueous phase of deinoxanthine, the product according to Example 1 was dissolved in hexanediol and solubilized with polyglyceryl 10 stearate, a naturally derived solubilizing agent at 65°C. The composition of specific soluble deinoxanthine 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

For the extracted product according to Example 1, the antioxidant effect of the deinoxanthine fermented extract was confirmed using 2,2-Diphenyl-1-picrylhydrazyl (DPPH). Astaxanthin (AX) was used as a control, and 1000 ppm of the extracted product contains 5 ppm of deinoxanthin.

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 the dark at room temperature for 20 minutes. At this time, ascorbic acid was used as a positive control. After 20 minutes of prevention, absorbance was measured at 540 nm using an absorbance measuring instrument Multiskan GO (Thermo Fisher Scientific). The radical ratio (%) compared to the control group was calculated and shown in Table 5, FIGS. 3 and 4.

Antioxidant Activity(%) 1ppm 2ppm 3ppm 4ppm 5ppm 10ppm 20ppm Deinoxanthine (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 at a concentration of 10 ppm, deinoxanthin had about 50 times more DPPH antioxidant efficacy than astaxanthin.

Experimental Example 4-2: ROS

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

HaCaT Keratinocyte cell line, a skin keratinocyte, was cultured in a DMEM medium containing 10% FBS in a 37°C, 5% CO ₂ incubator and inoculated into a 96-well plate. After 24 hours, samples were treated by concentration and incubated for 24 hours. I did. DCFDA (dichlorofluorescein diacetate) 20uM, a dye for measuring intracellular active oxygen (ROS), was treated for 20 minutes, incubated at RT, and tBHP (55uM) was treated for 1 hour, and then measured using a Fluorescence microplate reader. The average value for each sample group was calculated, and ROS antioxidant activity was analyzed by comparing it with the value of the control group.

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

Both the deinoxanthine fermented extract and astaxanthin reduced ROS increased by H ₂ O ₂ , but it was confirmed that the ROS antioxidant efficacy for the deinoxanthine fermented extract was more excellent compared to 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 the RAW264.7 cells were cultured at 2.5 x 10 ⁵ cell / well after diluted to this, a 2.5 x 48-well plate at 10 ⁵ cells / well for cell seeding and 5% CO _2, 37 ℃ incubator for 24 hours. After incubation, it was replaced with a serum-free medium, followed by starvation, and the samples were diluted and treated for each concentration. Dexamethasone, used as a positive control, was treated with 5% CO ₂ , incubated for 1 hour in a 37°C incubator, and treated with LPS to 1 μg/ml, followed by 5% CO ₂ incubating for 24 hours in a 37°C incubator. 100 μl of the synthesized NO-containing medium was transferred to a 96-well plate, and 500 μM NaNO ₂ was used as a reference to treat the 96-well plate for each concentration and reacted at room temperature for 20 minutes. To measure absorbance, after executing the multiskan GO program, 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 resulting value was derived from the average of the values, and the anti-inflammatory effect was confirmed and indicated by measuring the amount of NO produced.

From the results of FIG. 7, it was confirmed that the deinoxanthine fermented extract was more excellent in anti-inflammatory efficacy than 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% FBS (fetal bovine serum) and 1X penicillin/streptomycin (P/S), counted, and then 2 x 10 ⁵ cells/well in a 6 cm ² plate. Diluted and seeded, and incubated for 24 hours in a 5% CO ₂ , 37°C incubator. Another cell, 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. It was seeded and incubated for 24 hours in 5% CO ₂ , 37°C incubator.

The supernatant of the cultured HaCaT cells was removed, washed with PBS, 1 ml of PBS was added, and UV was irradiated with 15 mJ/cm ² . The PBS was removed, and the DMEM culture medium containing 1% FBS was replaced for the starvation step, followed by incubation in 5% CO ₂ , 37° C. incubator for 24 hours. The supernatant of UV-irradiated HaCaT cells and the supernatant of non-irradiated HaCaT cells were transferred to a 50 ml tube, respectively, set to a non-toxic concentration range, and diluted using the supernatant of HaCat cells.

The supernatant of the cultured cell fibroblasts was removed. Samples made using the HaCaT supernatant were treated by concentration on the cultured cell fibroblasts, and then cultured in an incubator at 5% CO ₂ , 37°C for 24 hours. The supernatant of cultured fibroblast cells was added to a 96 well plate by 150 µl. Human Total MMP-1 ELISA kit (DY901) was used to quantify the amount of MMP-1 present in the fibroblast supernatant.

In order to check the wrinkle improvement effect of the final product according to Example 1, it was confirmed whether the expression level of MMP-1, a collagen-degrading enzyme, decreased by treating the sample by concentration, which is shown in FIG. 8.

As shown in Figure 8, compared to the UV-treated cells, MMP-1 decreased by about 2.6 times or more in cells treated with 1% (v/v) of the deinoxanthine ferment extract according to the present invention, and 10% (v/ When treated with v), it was confirmed that the deinoxanthine fermented extract was effective in anti-aging, because it showed an amount of MMP-1 almost similar to that of the control group not treated with UV.

Experimental Example 9: Confirmation of deinoxanthine production according to temperature

In order to check the deinoxanthin content according to the temperature, the strain of KACC 81132BP (Agricultural Biotechnology Research Institute) 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 fermentor Incubated 3L. Specifically, a deinoxanthine fermentation extract was obtained by applying the secondary culture temperature to 37°C or 30°C, respectively. The results are shown in Table 6.

Comparison of deinoxanthine production according to temperature 30℃ culture 37℃ culture 48hr O.D. 26.3 12.2 Dry cell weight
(Recovery amount) 13.98g 5.12g Deinoxanthine production
(3L culture) 5.402mg 2.24mg

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

As described above, it was confirmed that deinoxanthine 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 various different forms, and those of ordinary skill in the technical field to which the present invention pertains are It will be appreciated that it can be implemented in other specific forms without changing any or essential features. Therefore, the embodiments described above are illustrative in all respects, and should be understood as non-limiting.

Institute of Agricultural Biotechnology KACC81132BP 20200821

Claims (5)

Inoculating the strain of the genus Deinococcus KACC 81132BP (Institute of Agricultural Biotechnology) into the primary culture medium;
Stirring at 30° C. and 200 rpm and first culturing;
Inoculating the primary cultured strain into the secondary culture medium;
Secondary culture at 30°C;
Recovering the cells by centrifuging the culture solution after the secondary culture 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 includes the step of culturing while stirring for 24 hours,
A method for producing a deinoxanthine fermented extract comprising the step of culturing the secondary culture while stirring for 48 hours. A cosmetic composition comprising a deinoxanthine fermented extract prepared by the method according to claim 1. The method of claim 2,
The composition is for antioxidant, anti-inflammatory or anti-wrinkle, cosmetic composition. delete delete

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