WO2024167084A1 - Emulsion using rhodococcus koreensis strain and composition for improving skin condition comprising fermented product thereof - Google Patents

Abstract

The present invention relates to a cosmetic composition for improving skin condition, wherein a multiple emulsion composition is prepared during microbial fermentation so that there is no multi-step purification process, and thus has excellent usability. The multiple emulsion cosmetic composition is in the form of a bio-liquid crystal and exhibits excellent skin barrier strengthening and moisturizing effects without irritating the skin, and thus can be used for skin improvement.

Description

Composition for improving skin condition comprising emulsion using Rhodococcus coriensis strain and fermented product thereof

The present invention relates to a cosmetic composition for improving skin condition.

In general, a multiple emulsion is a system composed of smaller, different-phase emulsions within an emulsion. It refers to a multi-phase emulsion created when the emulsion is added to another continuous phase and stirred, and is mainly used in fields such as cosmetics, drug delivery, and food. Multiple emulsions can be largely divided into W/O/W (water in oil in water) emulsions and O/W/O (oil in water in oil) emulsions. A W/O/W multiple emulsion is a form in which a W/O emulsion of the inner phase is redispersed in an outer aqueous phase, and the dispersed phase itself has a unique overlapping structure that contains different small dispersed particles to become a continuous phase.

W/O/W multiple emulsions can secure the moisturizing effect, which is the advantage of W/O emulsions, and the refreshing feeling, which is the advantage of O/W emulsions, at the same time, and can be used by adding active substances that react with each other to the inner and outer phases, respectively. However, W/O/W multiple emulsions have larger droplets and are not uniform in size compared to simple emulsions, which makes it easy for droplets to coalesce or separate into phases, and when the energy balance and osmotic pressure between the inner and outer phases are not balanced, the innermost phase and the outermost phase may coalesce and eventually turn into an O/W emulsion, etc., have problems.

Meanwhile, in order to solve these stability problems of multiple emulsions, if high-grade alcohol and glucoside series surfactants are used to increase the viscosity of the emulsion, it may result in a sticky feeling characteristic of high-grade alcohol-based liquid crystal emulsions, and if a polymer such as polyethylene glycol-diacrylate is introduced into the innermost phase, a W/O/W multiple emulsion with a solid core-shell structure can be produced, but it may result in a negative feeling and cause skin irritation.

Against this backdrop, the inventors of the present invention sought to develop a composition that has the advantages of a W/O/W multiple emulsion, while simplifying the process and improving stability by utilizing the emulsification phenomenon using microorganisms rather than the complicated manufacturing process of the existing W/O/W multiple emulsion, and developed a multiple emulsion composition with excellent skin usability and skin improvement effect from a fermented product of microorganisms using non-animal raw materials.

One aspect provides a method for producing an emulsion composition, comprising the steps of: producing a seed culture by culturing a Rhodococcus koreensis strain; producing a pre-culture by pre-cultivating the seed culture; and inoculating the pre-culture into a culture medium containing oil and performing fermentation.

Another aspect provides a cosmetic composition for improving skin, comprising a culture or fermented product of Rhodococcus koreensis.

Another aspect provides a medium composition for culturing a Rhodococcus koreensis strain.

Another aspect provides a Rhodococcus koreensis strain BT1 deposited under the accession number KCCM12825P, comprising 16S rRNA of sequence number 1, and a culture medium thereof.

One aspect is to provide a method for producing an emulsion composition, comprising the steps of: producing a seed culture by culturing a Rhodococcus koreensis strain; producing a pre-culture by pre-cultivating the seed culture; and inoculating the pre-culture into a culture medium containing oil and fermenting it. Specifically, the emulsion composition may be characterized in that it is a multiple emulsion composition.

The term "multiple emulsion" in this specification refers to a system in which smaller emulsions of different phases are composed within an emulsion, and may mean a multi-phase emulsion generated when the emulsion is stirred while being added to another continuous phase. Multiple emulsions can be largely divided into W/O/W (water in oil in water) emulsions and O/W/O (oil in water in oil) emulsions. The W/O/W multiple emulsion is a form in which a W/O emulsion of an inner phase is redispersed in an outer water phase, and may have an overlapping structure in which the dispersed phase itself contains different small dispersed particles to become a continuous phase.

The above multiple emulsion composition may be a W/O/W multiple emulsion composition. Specifically, in one embodiment, the aqueous phase of an emulsion containing a Rhodococcus koreensis BT1 (KCCM 13266P) culture was stained with 5-carboxyfluorescein and observed under a fluorescence microscope, and it was confirmed that the multiphase structure had small emulsions in the aqueous phase within a relatively large emulsion (Fig. 5).

In addition, the multi-emulsion composition may be characterized by having a form of a bio-liquid crystal. The term "liquid crystal" as used herein refers to a system that is created when organic molecules having hydrophilicity and lipophilicity as an intermediate phase between a solid and a liquid are arranged in a lamellar structure in which the hydrophilic parts are arranged hydrophilically and the hydrophobic parts are arranged hydrophobically to form multiple layers. The term "bio-liquid crystal" as used herein may refer to a liquid crystal manufactured using a fermentation process of a microorganism, and may include a culture solution or fermented product of the Rhodococcus coriensis strain.

The above liquid crystal has a structure similar to the intercellular lipid of the stratum corneum of the skin, so it has excellent affinity with skin lipids and can form a skin protective film, so it can be used in cosmetic compositions, etc.

In addition, the bio-liquid crystal is characterized by being a lamellar-type spherical multiple liquid crystal having a hexagonal (Maltese cross) multi-layer particle shape, and the bio-liquid crystal particle may have a particle size of 10 to 50 ㎛. In one embodiment, as a result of microscopically photographing multiple emulsions (Example 1 and Comparative Example 1) before and after fermentation, it was confirmed that in the case of Example 1, an emulsion with a uniform particle size was formed after fermentation compared to before fermentation in Comparative Example 1 (FIGS. 1 and 2).

In the present invention, the microorganism that produces a multiple emulsion capable of taking on a bio-liquid crystal form through a fermentation process may be " Rhodococcus koreensis", and more specifically, may be the Rhodococcus koreensis BT1 strain deposited under the deposit number KCCM 13266P. The Rhodococcus koreensis BT1 strain may include 16s rRNA of sequence number 1.

In the step of producing a culture by culturing the above strain as a spawn, and in the step of producing a culture by pre-culturing the above strain as a pre-culture, shaking culture may be performed using an LB medium, but there is no particular limitation to this as long as it is a medium or culture method that contains components necessary to activate the above strain.

In one embodiment, the step of preparing the seed culture or pre-culture may be performed under conditions of 10 to 30° C. and 100 to 300 rpm for 24 to 72 hours. In addition, the step of preparing a fermented product by inoculating the pre-culture into a culture medium containing oil may be performed under aerobic conditions of 10 to 30° C., 100 to 300 rpm, 0.5 to 2.0 vvm, and pH 5.5 to 6.5 for 24 to 120 hours.

The above culture medium is for performing the main culture of the activated preculture, and may contain at least one selected from the group consisting of _{KH2PO4 , K2HPO4} , ( _{NH4 ) 2SO4} , _MgSO4 · _{7H2O , H3BO4 , CaCl2 ,} NaMoO4· _2H2O , FeNaEDTA _{, FeSO4} · _{7H2O , ZnSO4} ·H2O _, MnSO4· _H2O , _NiCl2 · _H2O , CuSO4· _5H2O , _MnCl2 , and _CoCl2 · _6H2O , but is not particularly limited thereto. Specifically, the culture medium contains _KH2PO4 , _0.5 to 10 g/L, _{K2HPO4 ,} 5 to 20 g/L, ( _NH4 ) _{2SO4 ,} 10 to 30 g/L, _MgSO4 · _7H2O , 0.001 to 2 g/L, _{H3BO4 ,} 1 to 10 mg/L, _CaCl2, 1 to 50 mg/L, _NaMoO4 · _2H2O , 1 to 10 mg/L, FeNaEDTA, 0.1 to 1 mg/L, _{ZnSO4·H2O ,} 0.1 to 1 mg/L, _MnSO4 · _H2O , 0.01 to 0.1 mg/L, _NiCl2 · _H2O , 0.01 to 0.1 mg/L, _CuSO4 · _5H2O , 0.01 to 0.1 mg/L, MnCl ₂ 0.01 to 0.1 mg/L, CoCl ₂ ·6H ₂ O 0.01 to 0.1 mg/L, and vegetable oil 1 to 100 g/L. In addition, the culture medium may additionally contain water (purified water).

The above oil is for supplying a carbon source to the above microorganism or strain and may be a vegetable oil. The vegetable oil may be extracted from at least one selected from the group consisting of, for example, canola, grape seed, sunflower seed, olive, soybean, argan, rice bran, perilla seed, sesame seed, almond, macadamia, rosehip, coconut, peanut, corn, tiger nut, shea fruit, oil palm, bergamot fruit, vitamin tree seed, camellia seed, safflower seed, apricot seed, poppy seed, evening primrose seed, castor seed, green tea seed, meadowfoam seed, flax seed, hemp seed and rapeseed, and may be specifically, rapeseed oil, but is not particularly limited thereto.

In one embodiment, the method for producing the multi-emulsion composition may further include a step of heat treating at 10 to 100° C. for 5 to 200 minutes after the step of producing the fermented product. In addition, a step of removing remaining microbial fragments using a centrifuge may be further included after the step of heat treating.

Another aspect is to provide a cosmetic composition for improving skin, comprising a culture or fermented product of Rhodococcus koreensis . The same parts as described above are also applied to the cosmetic composition.

The above culture solution or fermented product may be produced through a step of producing a culture by culturing a Rhodococcus coriensis strain as a culture medium; a step of producing a pre-culture by pre-cultivating the culture medium; and a step of producing a fermented product by inoculating the pre-culture into a culture medium containing oil.

The above skin improvement may be, but is not particularly limited to, skin barrier strengthening or skin moisturizing. In this specification, the term "skin barrier strengthening" may mean any action that enhances the function of the skin barrier, which is located at the outermost part of the skin and prevents moisture loss, and the term "skin moisturizing" may mean any action that maintains moisture in the skin or prevents moisture loss.

In addition, the cosmetic composition may be characterized as being a W/O/W multiple emulsion composition, and the multiple emulsion composition may be characterized as having a form of bio-liquid crystal.

Another aspect is to provide a medium composition for culturing a Rhodococcus koreensis strain. The same parts as described above are also applied to the medium composition. Specifically, the medium composition may include at least one selected from the group consisting of oil; and KH ₂ PO ₄ , K ₂ HPO ₄ , (NH ₄ ) ₂ SO ₄ , MgSO ₄ ·7H ₂ O, H ₃ BO ₄ , CaCl ₂ , NaMoO ₄ ·2H ₂ O, FeNaEDTA, FeSO ₄ ·7H ₂ O, ZnSO ₄ ·H ₂ O, MnSO ₄ ·H ₂ O, NiCl ₂ ·H ₂ O, CuSO ₄ ·5H ₂ O, MnCl ₂ and CoCl ₂ ·6H ₂ O, but is not particularly limited thereto.

Another aspect provides a Rhodococcus koreensis BT1 strain deposited under the accession number KCCM12825P, comprising 16S rRNA of sequence number 1, and a culture medium thereof.

The present invention relates to a method for producing a multiple emulsion composition during fermentation of microorganisms, which has excellent usability without the complicated process of existing multiple emulsions, and a multiple emulsion cosmetic composition produced by this method can take the form of a bio-liquid crystal, and has excellent effects of strengthening the skin barrier and moisturizing without irritating the skin, so that it can be used for skin improvement purposes.

Figure 1 shows microscopic photographs of the multiple emulsions of the present invention (Example 1 and Comparative Example 1) before and after fermentation.

Figure 2 shows the particle size distribution of multiple emulsions (Example 1 and Comparative Example 1) before and after fermentation.

Figure 3 shows photographs comparing the degree of emulsification by strain in Example 1, Comparative Example 1, and Comparative Examples 3 to 6.

Figure 4 shows a photograph comparing the formation of multiple emulsions in fermented products (Example 1, Comparative Examples 4 and 5) that exhibit emulsification.

Figure 5 shows the results of confirming whether multiple emulsions were formed according to water dyeing in Example 1.

Figure 6 shows a photograph confirming the formation of bio-liquid crystals in each multiple emulsion.

Figure 7 shows the results of comparing the thermal stability of a multiple emulsion (Example 1) having a bio-liquid crystal form (micrographs taken before and after heat treatment).

Figure 8 shows the results of comparing the effects of the multiple emulsions of the present invention (Example 1, Comparative Example 1, and Comparative Examples 3 to 6) on the expression of the skin barrier factor filaggrin (FLG).

Figure 9 shows the results of comparing the effects of the multiple emulsions of the present invention (Example 1, Comparative Example 1) on the expression of the skin barrier factor caspase-14 (CASP-14).

Figure 10 shows the results of comparing the effects of the multiple emulsions of the present invention (Example 1, Comparative Example 1) on the expression of the moisture-retaining factor hyaluronan synthase-2 (Hyaluronic acid synthase-2; HAS-2).

The present invention will be described in more detail through the following examples. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1. Preparation of an emulsion containing a culture of Rhodococcus coriensis strain

Rhodococcus koreensis BT1 (KCCM 13266P, 2022.11.11) was inoculated into LB medium and shaken at 30°C and 200 rpm for 48 hours to culture the spawn. Preculture was performed by inoculating 1% of the spawn culture into sterilized LB medium and shaking at 30°C and 200 rpm for 24 hours to culture the spawn. The goal was to induce maximum bacterial activation up to preculture to induce multiple emulsion formation in the main culture.

For the main culture, the medium was _{composed of KH2PO4 5} g/L, _K2HPO4 10 g/L, ( _NH4 ) _2SO4 20 g/L, _MgSO4 ·7H2O _0.2 g/L, _{H3BO4 5} mg/L, _CaCl2 20 mg/L, _NaMoO4 · _2H2O 5 mg/L, FeNaEDTA 0.5 mg/L, _ZnSO4 · _H2O 0.5 mg/L, _MnSO4 · _H2O 0.04 mg/L, _NiCl2 · _H2O 0.02 mg/L, CuSO4·5H2O 0.04 mg/L, MnCl2 0.02 mg/L, _CoCl2 · _{6H2O 0.04} mg/L, and rapeseed oil ₂₅ g/ _L . 3.5 L was prepared in a 5 L fermenter. The medium was inoculated with 1% of the pre-culture fermentation product and cultured for 96 hours under the conditions of 30°C, 100 rpm, pH 6.0 correction, and 0.5 vvm.

Comparative Example 1. Emulsion at 0 hour of culture

The manufacturing method up to the main culture of the above Example 1 was the same, and the time at which the culture was 0 hours was set as Comparative Example 1.

Comparative Example 2. Preparation of general liquid crystal emulsion

This is a liquid crystal manufacturing method widely used in the cosmetics field, and a liquid crystal emulsion was manufactured using a homomixer. Table 1 below shows representative composition ratios for making a liquid crystal emulsion. Specifically, the water phase was accurately weighed so that the total amount was 100 g, heated to 75 to 80°C to completely dissolve, and stored separately. In addition, the oil phase was accurately weighed, heated to 75 to 80°C to completely dissolve, and stored separately. Afterwards, the oil phase was slowly added to the water phase using a homomixer to sufficiently disperse it, and an emulsification reaction was performed at about 3,000 rpm for 5 minutes. Afterwards, it was cooled to room temperature to complete the defoaming process.

Distinction between the top Full ingredient list Comparative Example 2 (wt%) premier
(Water phase) Purified water To 100 glycerin 2 Butylene glycol 3 Hydrogenated Lecithin 3 EDTA-2Na 0.01 Paid
(Oil phase) Stearyl alcohol 3 Rapeseed oil 25 Ceramide 3 Cholesterol 1 Phospholipid 1 Total 100

Comparative examples 3 to 6. Emulsion formation conditions by strain

A comparative experiment on emulsion formation by strain was conducted with a total of four strains: Rhodococcus erythropolis KCCM40161 (Comparative Example 3), Rhodococcus ruber KCCM41053 (Comparative Example 4), Rhodococcus opacus KCCM41722 (Comparative Example 5), and Candia bombiocola KCCM11858 (Comparative Example 6).

Rhodococcus erythropolis KCCM40161 was inoculated into LB medium and cultured with shaking at 26°C and 200 rpm for 24 hours to culture the spawn. Preculture was performed by inoculating 1% of the above spawn culture into sterilized LB medium and cultured with shaking at 26°C and 200 rpm for 24 hours to culture the spawn. The aim was to induce the emulsification phenomenon in the main culture by maximally inducing the activation of the bacteria up to the preculture.

For the main culture, the medium was _{composed of KH2PO4 5} g/L, _K2HPO4 10 g/L, ( _NH4 ) _2SO4 20 g/L, _MgSO4 ·7H2O _0.2 g/L, _{H3BO4 5} mg/L, _CaCl2 20 mg/L, _NaMoO4 · _2H2O 5 mg/L, FeNaEDTA 0.5 mg/L, _ZnSO4 · _H2O 0.5 mg/L, _MnSO4 · _H2O 0.04 mg/L, _NiCl2 · _H2O 0.02 mg/L, CuSO4·5H2O 0.04 mg/L, MnCl2 0.02 mg/L, _CoCl2 · _{6H2O 0.04} mg/L, and rapeseed oil ₂₅ g/ _L . 3.5 L was prepared in a 5 L fermenter. The medium was inoculated with 1% of the pre-culture fermentation product and cultured for 96 hours under the conditions of 26°C, 100 rpm, pH 6.0 correction, and 0.5 vvm.

Rhodococcus ruber KCCM41053 was inoculated into LB medium and cultured with shaking at 28°C and 200 rpm for 48 hours to culture the spawn. Preculture was performed by inoculating 1% of the above spawn culture into sterilized LB medium and cultured with shaking at 28°C and 250 rpm for 24 hours to culture the spawn. The aim was to induce maximum activation of the bacteria up to the preculture to induce the emulsification phenomenon in the main culture, and the conditions for the main culture were the same as those of Example 1.

Rhodococcus opacus KCCM41722 was inoculated into LB medium and cultured with shaking at 30°C and 200 rpm for 48 hours to culture the spawn. Preculture was performed by inoculating 1% of the above spawn culture into sterilized LB medium and cultured with shaking at 30°C and 200 rpm for 24 hours to culture the spawn. The preculture was performed to induce the emulsification phenomenon in the main culture by inducing the maximum activation of the bacteria up to the preculture, and the main culture conditions were the same as those of Example 1.

Candia bombiocola KCCM11858 was inoculated into YM medium and cultured with shaking at 30°C and 200 rpm for 48 hours to culture the spawn. Preculture was performed by inoculating 1% of the above spawn culture into sterilized YM medium and cultured with shaking at 30°C and 200 rpm for 24 hours to culture the spawn. The preculture was performed to induce the emulsification phenomenon in the main culture by inducing the maximum activation of the fungus up to the preculture, and the main culture conditions were the same as those of Example 1.

Experimental Example 1. Observation of the formation of multiple emulsions of W/O/W

As a result of taking microscopic photographs of multiple emulsions (Example 1 and Comparative Example 1) before and after fermentation, it was confirmed that in the case of Example 1, an emulsion with uniform particle size was formed after fermentation compared to before fermentation in Comparative Example 1 (Figs. 1 and 2).

In addition, for Example 1, the aqueous phase was stained with 5-carboxyfluorescein and observed under a fluorescence microscope, and it was confirmed that small emulsions in the aqueous phase existed inside a relatively large emulsion (Fig. 5), and from this, it was confirmed that the structure of the emulsion including the culture of Example 1 was a multiple emulsion of W/O/W.

Experimental Example 2. Comparison of emulsifying power and multiple emulsion formation by strain

The emulsifying power of each strain was compared and evaluated for Example 1 and Comparative Examples 3 to 6. As a result, Example 1 using the strain of Rhodococcus koreensis BT1 of the present invention showed the highest emulsifying power. It was confirmed that Comparative Examples 3 and 6 were not emulsified, and Comparative Examples 4 and 5 showed an emulsifying appearance compared to Comparative Examples 3 and 6, but showed a large difference in the degree of emulsification and stability compared to Example 1 (Fig. 3).

In addition, as a result of comparing whether multiple emulsions were formed for the fermented products exhibiting emulsification (Example 1, Comparative Examples 4 and 5), it was observed that Example 1 formed a multiple emulsion by observing small emulsions inside a large emulsion, but it was confirmed that such a multiple emulsion structure was not observed for Comparative Examples 4 and 5 (Fig. 4).

Experimental Example 4. Observation of the formation of bio-liquid crystals in multiple emulsions

The results of determining whether a liquid crystal with a lamellar structure was formed using a polarizing microscope (BX53T-32F01, Olympus, Japan) for Example 1 and Comparative Examples 1 and 2 are shown in Fig. 6.

As a result, in the case of Example 1, it was confirmed that a Maltese cross shape was observed similarly to Comparative Example 2, which is known to form a liquid crystal. On the other hand, in the case of Comparative Example 1, which was composed of materials unrelated to liquid crystal formation and did not use a surfactant, the Maltese cross shape was not observed at all (Fig. 6).

Experimental Example 5. Evaluation of thermal stability of multiple emulsions with bio-liquid crystal form according to heat treatment

For the above Example 1, the stability of the multiple emulsion according to the heat treatment was evaluated. Specifically, the emulsion of Example 1 was heat treated at 70℃ for 30 minutes and then the state was observed using a polarizing microscope. The particle size analysis was performed using SLS (LA-960, Horiba, Japan). As a result, in the case of Example 1, it was confirmed that the emulsion state and particle size exhibited thermal stability by comparing before and after the heat treatment (Fig. 7).

Experimental Example 6. Sensory Evaluation

A sensory evaluation was conducted on the usability of the above Example 1 and Comparative Examples 2 to 5. Ten evaluators applied the compositions of Example 1 and Comparative Examples 2 to 5, and then conducted a comparative test on the items of spreadability, moisturizing power, stickiness, and fermentation odor, and evaluated them on a 10-point scale. The results are shown in Table 2 below. ([Evaluation score] 1 point/---: worst, with unpleasant odor, 10 points/+++: best, without unpleasant odor). According to Table 2 below, Example 1, which is a multiple emulsion according to the present invention, has excellent spreadability compared to Comparative Examples 3 to 5, and also does not significantly generate the unique fermentation odor generated during fermentation. In addition, it was confirmed that Example 1 had less stickiness and the highest moisturizing power compared to Comparative Examples 2 to 5. Through this, it was confirmed that the multi-emulsion composition of the present invention has almost no fermentation odor characteristic of fermented compositions, is environmentally friendly, and has excellent usability.

Usability Evaluation Items Spreadability Moisturizing power Stickiness Fermented flavor Total usability Example 1 7 7.5 8 7 ++ Comparative Example 2 7 6 5.5 8 + Comparative Example 3 2 4 4 4 -- Comparative Example 4 5 5.5 6 5 - Comparative Example 5 5 6.5 6.5 5.5 - Comparative Example 6 5 5.5 6 4 -

Experimental Example 7. Confirmation of the skin barrier strengthening and moisturizing effects of multiple emulsions

The effects of the multiple emulsion of Example 1 on the skin barrier and moisturizing-related factors were analyzed. Specifically, human epidermal cells (HaCaT) and human dermal fibroblasts were cultured using DMEM (Dulbecco's Modified Eagle's Medium) medium containing 10% fetal bovine serum (FBS) and 1% penicillin, respectively, at 37°C in a 5% _CO2 incubator. 3.0 x 10 ⁴ cells were dispensed into a 96-well plate and cultured in an incubator for 24 hours. After 24 hours, the medium was replaced with new one, and the multiple emulsion sample of Example 1 was treated at the desired concentration and cultured for 24 hours. After culture, the cells were sufficiently washed with saline solution, cDNA was synthesized, and quantified at 100 ng/mL, and then Real-time Quantitative Polymerase Chain Reaction (RT-qPCR) was performed. The expression of Filaggrin (FLG) and Caspase-14 (CASP-14), which are skin barrier factors, was confirmed using epidermal cells, and the expression of Hyaluronic acid synthase-2 (HAS-2), which is a moisturizing factor, was confirmed using dermal fibroblasts. The relative expression rate was expressed by normalizing the expression amount of each gene to the expression amount of β-actin, which is a housekeeping gene. The effects of Example 1 and Comparative Example 1 and Comparative Examples 3 to 6 on the expression of FLG, a skin barrier factor, are shown in Fig. 8, the effects on CASP-14 expression are shown in Fig. 9, and the effects on HAS-2 expression are shown in Fig. 10.

gene Primer Direction order Sequence number FLG Forward 5'-TCCCCTACGCTTTCTTGTCCT-3' 2 Reverse 5'-TGCTTGAGCCAACTTGAATACC-3' 3 CASP-14 Forward 5'-GCTGGCGACAATGGAGTCAA-3' 4 Reverse 5'-TGGATCTTGTCAGCCCAACC-3' 5 HAS-2 Forward 5'-TGGGCGAGAAATTGAGTGTT-3' 6 Reverse 5'-GGTCTCCACATTCCTGCCA-3' 7 β-actin Forward 5'-GGCCATCTCTTGCTCGAAGT-3' 8 Reverse 5'-GAGACCTTCAACACCCCAGC-3' 9

As a result, the expression of Filaggrin, a skin barrier factor, was most significantly increased by Example 1, and it was confirmed that the skin barrier effect was the highest. In other comparative examples, no significant increase in FLG expression was observed compared to the untreated group (Control) (Fig. 8). In addition, it was confirmed that Example 1 increased the expression of CASP-14, another skin barrier factor, and HAS-2, a moisture retention factor, compared to the untreated group and Comparative Example 1 (Figs. 9 and 10).

The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

[Acceptance number]

Name of depositor: Korea Center for Microbiological Conservation (KCCM)

Accession number: KCCM13266P

Date of Consignment: 20221111

Claims (19)

A step of producing a spawn culture by culturing a Rhodococcus koreensis strain; A step of producing a pre-culture by pre-cultivating the above-mentioned inoculum culture; and A method for producing an emulsion composition, comprising the step of inoculating the pre-culture into a culture medium containing oil to produce a fermented product. A manufacturing method in claim 1, wherein the Rhodococcus coriensis strain is deposited under the deposit number KCCM 13266P. A manufacturing method according to claim 1, characterized in that the emulsion composition has a form of a bio-liquid crystal. A manufacturing method in claim 1, wherein the oil is a vegetable oil. A manufacturing method in claim 1, wherein the culture medium includes at least one selected from the group consisting of KH ₂ PO ₄ , K ₂ HPO ₄ , (NH ₄ ) ₂ SO ₄ , MgSO ₄ ·7H ₂ O, H ₃ BO ₄ , CaCl ₂ , NaMoO ₄ ·2H ₂ O, FeNaEDTA, FeSO ₄ ·7H ₂ O, ZnSO ₄ ·H ₂ O, MnSO ₄ ·H ₂ O, NiCl ₂ ·H ₂ O, CuSO ₄ ·5H ₂ O, MnCl ₂ and CoCl ₂ ·6H ₂ O. A manufacturing method in claim 1, wherein the step of manufacturing the seed culture and the pre-culture is performed under conditions of 10 to 30°C and 100 to 300 rpm for 24 to 72 hours. A manufacturing method in claim 1, wherein the step of manufacturing the fermented product is performed under aerobic conditions of 10 to 30°C, 100 to 300 rpm, 0.5 to 2.0 vvm, and pH 5.5 to 6.5 for 24 to 120 hours. A manufacturing method in claim 3, characterized in that the bio-liquid crystal is a lamellar-type spherical multiple liquid crystal having a hexagonal (Maltese cross) multi-layer particle shape. A manufacturing method according to claim 1, characterized in that the composition is a multiple emulsion composition. A cosmetic composition for improving skin, comprising a culture or fermented product of Rhodococcus koreensis. A cosmetic composition in claim 10, wherein the Rhodococcus coriensis strain is deposited under the deposit number KCCM 13266P. A cosmetic composition, characterized in that in claim 10, the culture solution or fermented product is prepared by a method according to any one of claims 1 to 8. A cosmetic composition according to claim 10, characterized in that the composition is a multiple emulsion composition. A cosmetic composition according to claim 13, characterized in that the multi-emulsion composition has a form of a bio-liquid crystal. _{A medium composition for culturing a Rhodococcus koreensis strain, comprising at least one selected from the group consisting of oil; and KH 2 PO 4 , K 2 HPO 4 , (NH 4 ) 2 SO 4 , MgSO 4 ·7H 2 O, H 3 BO 4 , CaCl 2 , NaMoO 4 ·2H 2 O , FeNaEDTA , FeSO 4 · 7H 2 O , ZnSO 4 · H 2 O} , MnSO ₄ ·H ₂ O, NiCl ₂ ·H ₂ O, CuSO 4 ·5H ₂ O, MnCl ₂ and CoCl ₂ ·6H ₂ O. A medium composition in claim 15, wherein the oil is a vegetable oil. A medium composition in claim 15, wherein the Rhodococcus coriensis strain is deposited under the deposit number KCCM 13266P. Rhodococcus koreensis BT1 strain, deposited under the accession number KCCM12825P, containing 16S rRNA of sequence number 1. Culture solution of Rhodococcus coriensis strain of Article 18.

Images