KR102412689B1 - Functional fermented oil comprising medicinal herbs and preparation thereof - Google Patents

Abstract

According to the present invention, by fermenting vegetable oil containing herbal medicines using microorganisms, it relates to a method for producing a functional fermented oil containing a large amount of active ingredients of herbal medicines. In addition, by optimizing the fermentation process of functional fermented oil including active ingredients of herbal medicines, it is possible to improve emulsification power, skin absorption, and formulation stability. In addition, by including the active ingredients of herbal medicines, cell proliferation can be promoted, and skin elasticity and whitening effects can be added. In addition, it is safe with little skin irritation, and can exhibit anti-inflammatory and antibacterial effects.

Description

Functional fermented oil containing active ingredients of herbal medicines and manufacturing method {FUNCTIONAL FERMENTED OIL COMPRISING MEDICINAL HERBS AND PREPARATION THEREOF}

The present invention relates to a functional fermented oil containing an active ingredient of a herbal medicine and a manufacturing method, and a fermented oil having excellent emulsification activity, antioxidant, cell proliferation, anti-aging, whitening, anti-inflammatory, antibacterial, etc. and skin safety and skin safety. It relates to a manufacturing method.

Conventional herbal extracts or fermented herbal extracts were prepared in a highly polar aqueous phase. However, actually extracting the active ingredient from the aqueous phase may not have high productivity and practicality. Since most of the active ingredients of herbal medicines are aromatic compounds and have a low polarity structure, hot water extraction is not a suitable manufacturing method in terms of extraction efficiency and stability. It is logical to extract with low polarity organic solvents, oils or supercritical would. However, this method is difficult to use industrially because the production yield is substantially lowered and the production cost is high.

In addition, most active ingredients extracted by organic solvents are substances with low polarity and have low solubility, so when applied to cosmetic formulations in general, stability and skin absorption are poor.

Therefore, in order to compensate for the shortcomings of this extraction method, cold or warm acupuncture was used as a method for extracting fat-soluble active ingredients using oil. However, this method has the disadvantages of a long extraction time and a low extraction yield of active ingredients.

Therefore, the existing methods for extracting the active ingredients of herbal medicines are practically inferior to industrial practicality.

Accordingly, the present invention devised a method for increasing the content of active ingredients by studying the optimal timing of inputting herbal medicines in the process of fermenting vegetable oils and herbal medicines using microorganisms.

In addition, the active ingredients of the fermented oriental medicinal oil were analyzed, and the fermented herbal medicinal oil contained active ingredients to improve skin affinity, efficacy, safety, and stability.

Prior art literature

(Patent Document 1) Republic of Korea Patent Publication No. 10-2016-0125784, November 01, 2016

What the present invention is trying to solve is by setting the time point at which herbal medicines are effectively added in the fermentation process of vegetable oils using Candida bombicola UA-06 (KCTC 18592P), functional fermented oil that can efficiently obtain the active ingredients of herbal medicines will provide

In addition, the present invention is to provide a functional fermented oil having excellent emulsification activity, antioxidant, skin elasticity, whitening, anti-inflammatory, antibacterial, etc. skin efficacy and skin safety as a vegetable fermented oil containing oriental medicinal active ingredients.

In order to solve the above problems, the present invention

Oil fermented by microorganisms,

It provides a fermented oil containing herbal medicines in which the microorganism is Candida bombicola UA-06 (KCTC 18592P).

According to one embodiment, the fermented oil containing the herbal medicine may have 5 to 20 times higher emulsification activity than before fermentation.

In addition, the fermented oil containing the herbal medicine may have a free radical scavenging ability of 10 to 80% than before fermentation.

In addition, the fermented oil containing the herbal medicine may have an increase in collagen production by 10 to 50% compared to before fermentation.

In addition, the fermented oil containing the herbal medicine may have a melanin formation inhibition rate of 5 to 60% than before fermentation.

In addition, the fermented oil containing the herbal medicine may have reduced nitric oxide production by 5 to 50% compared to before fermentation.

According to another embodiment of the present invention,

Candida bombicola UA-06 (KCTC 18592P) First seed fermentation step of fermenting microorganisms and oil in a medium;

A secondary seed fermentation step of sub-culturing the first seed fermented product, and

It provides a method for producing fermented oil containing herbal medicines, which includes the main fermentation step of sub-culturing the secondary seed fermented product and fermenting it together with herbal medicines.

According to one embodiment, the medium used in the first seed fermentation step includes yeast extract, malt extract, peptone, glucose, urea, and vegetable oil. may be doing

According to one embodiment, the medium used in the secondary seed fermentation step may include glycerin.

According to one embodiment, the medium used in the present fermentation step may include glycerin, K₂HPO₄, KH₂PO₄, NH₄NO₃, MgSO₄, yeast extract, vegetable oil, and herbal medicines.

According to one embodiment, the first and second seed fermentation step is to ferment for 10 to 30 hours in aerobic conditions of 15 to 25 ℃, the main fermentation step is fermentation for 90 to 100 hours in aerobic conditions of 15 to 25 ℃ it may be to do

According to one embodiment, the input time of the herbal medicine may be 30 to 50 hours after the start of the main fermentation.

According to one embodiment, the fermentation product is left standing after the main fermentation step to separate the upper layer and the lower layer, the lower layer is discarded, MgSO ₄ is added, stirred and centrifuged to remove microorganisms and moisture, and affinity chromatography column It may include the step of recovering the oil from which fatty acids and impurities are removed by using magnesium silicate as a filler.

According to another embodiment of the present invention, there may be provided a cosmetic composition comprising the fermented oil.

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

The present invention can provide a method for producing a functional fermented oil that can obtain an environmentally friendly active ingredient by fermenting herbal medicines and vegetable oils by microorganisms and can effectively increase the content of active ingredients contained in herbal medicines.

In addition, according to the method for producing a functional fermented oil including an active ingredient of an herbal medicine according to the present invention, by performing a simple process, it is possible to improve the emulsification activity by offsetting the hydrophobic interaction characteristics of the oil.

In addition, the functional fermented oil containing the active ingredient of the herbal medicine according to the present invention has excellent antioxidant, cell proliferation, skin elasticity, whitening, anti-inflammatory, and antibacterial effects, and has little irritation to the skin, so it can be used as an external preparation for skin or a cosmetic composition. It can be useful.

1 is a graph showing the results of measuring the amount of microorganisms.
2 is a photograph in which the oil was observed with the naked eye.
3 is a photograph showing the degree of emulsification of oil after 1 hour.
4 is a photograph showing the degree of emulsification of oil after 48 hours.
5 is a graph showing the measurement results of fibroblast viability.
6 is a graph showing the measurement result of the proliferation rate of keratinocytes.
7 is a graph showing the measurement result of melanin content.
8 is a graph showing the amount of nitrogen oxide produced after LPS treatment.

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.

Hereinafter, a fermented oil containing a herbal medicine according to an embodiment of the present invention and a method for manufacturing the same will be described in more detail.

Fermented oil containing herbal medicine according to the present invention is fermented by microorganisms, specifically, Candida bombicola UA-06 (KCTC 18592P) is fermented by microorganisms.

Candida bombicola is a microorganism that can produce emulsified substances, and it plays an important role in improving the emulsification rate and miscibility with oil without the use of additives such as surfactants, thereby improving skin absorption and formulation stability. etc. can be improved.

According to one embodiment, the fermented oil containing the herbal medicine may have an emulsification activity of 5 to 20 times, for example, 8 to 10 times, than before fermentation. In addition, by increasing the free radical scavenging ability by 10 to 80%, for example, by 20 to 70%, it is possible to improve the antioxidant ability. In addition, the fermented oil containing herbal medicines can improve skin elasticity by increasing the collagen production rate by 10 to 50%, for example by 15 to 40%, compared to before fermentation. In addition, the skin whitening effect can be improved by increasing the inhibition rate of melanin formation by 5 to 60%, for example, by 20 to 40%. In addition, the fermented oil containing herbal medicines can improve the anti-inflammatory effect by reducing the amount of nitric oxide production by 5 to 50% compared to before fermentation.

The method for producing fermented oil containing herbal medicine according to the present invention includes a primary seed fermentation step, a secondary seed fermentation step, and the main fermentation step.

Specifically, the present invention

Candida bombicola UA-06 (KCTC 18592P) First seed fermentation step of fermenting microorganisms and oil in a medium;

A secondary seed fermentation step of sub-culturing the first seed fermented product, and

It provides a method for producing fermented oil containing herbal medicines, which includes the main fermentation step of sub-culturing the secondary seed fermented product and fermenting it together with herbal medicines.

According to one embodiment, the primary seed fermentation is Candida bombicola in a medium containing yeast extract, malt extract, peptone, glucose, urea and vegetable oil. UA-06 (KCTC 18592P) strain may be inoculated and fermented for 10 to 30 hours under aerobic conditions of 15 to 25 °C.

According to one embodiment, the secondary seed fermentation is performed by using a culture medium in which glucose is replaced with glycerin in the primary seed fermentation medium composition, and the primary fermentation product is subjected to aerobic conditions at 15 to 25 ° C. It may be subcultured for 10 to 30 hours.

According to one embodiment, the present fermentation is carried out using a culture medium containing glycerin, K₂HPO₄, KH₂PO₄, NH₄NO₃, MgSO₄, yeast extract, vegetable oil, and herbal medicines at aerobic conditions at 15 to 25 ° C. It may be subcultured for up to 100 hours.

According to one embodiment, in order to prevent inhibition of growth and metabolism of cells due to changes in the environment in the culture medium, herbal medicines may be added just before transition from the logarithmic growth phase of the microorganism to the stationary phase. Specifically, the herbal medicine may be added 30 to 50 hours after the start of the main fermentation, for example, 36 to 42 hours later.

According to one embodiment, it may further include a purification process step after the main fermentation step. Specifically, in order to remove moisture and herbal medicines, the fermented product is left standing after main fermentation to separate the oil layer located on the upper layer and the medium and impurity layer located on the lower layer to discard the lower layer, and MgSO ₄ is added and stirred. Oil from which residual impurities and herbal medicines are removed can be obtained by using a centrifugal separator.

In addition, for purification of fatty acids and impurities in the fermented product after the main fermentation, a chromatography method may be used. Specifically, by using magnesium silicate as a filler in the affinity chromatography column, fatty acids and impurities can be adsorbed and filtered.

According to the present invention, it is possible to reduce the surface tension by canceling the hydrophobic interaction of oil by using metabolites by microorganisms to improve the affinity of oil and highly polar substances, so surfactants, emulsifiers, etc. It is possible to easily improve emulsification power, absorption power, formulation stability, acid odor, etc. without additives. In addition, by including the active ingredient obtained from herbal medicine, it is possible to add functions such as antioxidant power, skin elasticity, whitening, anti-inflammatory, antibacterial.

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.

Manufacturing example: selection of time to inject herbal medicines

In order to prevent the growth and metabolic process of cells from being disturbed due to environmental changes in the culture medium that may occur due to the input of herbal medicines, the input time was set just before the transition from the logarithmic growth phase to the stationary phase. Thus, the growth rate of microorganisms was confirmed. The medium was the same composition as the secondary seed fermentation medium described below, and the microorganism was Candida bombicola UA-06 (KCTC 18592P) strain.

After the strain was inoculated into the medium, 100 ml of the culture solution was taken at 6 hour intervals for incubation time, and the result of measuring the amount of microorganisms by the weight loss method is shown in FIG. 1 below. As shown in FIG. 1 , it can be confirmed that the microorganisms enter the stationary phase at 48 hours, so the time of injecting oriental medicine can be set to 36 to 42 hours.

Example

Examples 1 to 3: Preparation of fermented oil containing herbal medicines

The oils of Examples were prepared with the medium composition according to Table 1 below, and a photograph of each oil is shown in FIG. 2 .

division Example 1 Example 2 Example 3 Seed fermentation Glycerin 20g/L Glycerin 20g/L Glycerin 20g/L Rice oil 50g/L Rice oil 50g/L Rice oil 50g/L Yeast extract 3g/L Yeast extract 3g/L Yeast extract 3g/L Malt extract 3g/L Malt extract 3g/L Malt extract 3g/L Peptone 5g/L Peptone 5g/L Peptone 5g/L Glucose 10g/L Glucose 10g/L Glucose 10g/L Urea 1g/L Urea 1g/L Urea 1g/L Distilled water 928g/L Distilled water 928g/L Distilled water 928g/L main fermentation Glycerin 20g/L Glycerin 20g/L Glycerin 20g/L Rice Oil 500g/L Rice Oil 500g/L Rice Oil 500g/L Yeast extract 10g/L Yeast extract 10g/L Yeast extract 10g/L Schisandra 20g/L Turmeric 20g/L Green tea 20g/L Gardenia 20g/L - - Turmeric 20g/L - - K ₂ HPO ₄ 25g/L K ₂ HPO ₄ 25g/L K ₂ HPO ₄ 25g/L KH ₂ PO ₄ 7g/L KH ₂ PO ₄ 7g/L KH ₂ PO ₄ 7g/L NH ₄ NO _{3 3} g/L NH ₄ NO _{3 3} g/L NH ₄ NO _{3 3} g/L MgSO ₄ 2 g/L MgSO ₄ 2 g/L MgSO ₄ 2 g/L Distilled water 433g/L Distilled water 433g/L Distilled water 433g/L

1st seed fermentation

Candida bombicola UA-06 (KCTC 18592P) strain was inoculated into 1 L of medium, and primary seed fermentation was performed for 24 hours under aerobic conditions of 20° C., 300 rpm, and 1 NL/min (1 vvm). The medium used was Yeast extract 3g/L, Malt extract 3g/L, Peptone 5g/L, Glucose 10g/L, Urea 1g/L, Oil 50g/L, Distilled water 928g/L to optimize the growth and metabolism of microorganisms. It was set as the composition.

Secondary seed fermentation

Except that Glucose 10g/L was replaced with Glycerin 20g/L, 10L of the culture medium of the same composition as the primary seed fermentation medium was placed under aerobic conditions of 20℃, 300rpm, 10NL/min (1vvm) for 2 hours for 24 hours. Tea seed fermentation was performed.

main fermentation

The secondary seed fermentation culture medium was glycerin 20g/L, K ₂ HPO ₄ 25g/L, KH ₂ PO ₄ 7g/L, NH ₄ NO ₃ 3g/L, MgSO ₄ 2g/L, Yeast extract 10g/L, distilled water 433g The main fermentation was carried out for 96 hours under aerobic conditions of 20 °C, 300 rpm, and 100 NL/min (1 vvm) by subculture with 100 L of a culture medium having a composition of /L and 500 g/L of oil. In addition, herbal medicines were added 36 to 42 hours after the start of the main fermentation. The amount of herbal medicine input may be added in an amount of 0.2 to 20 parts by weight, for example, 0.5 to 15 parts by weight, for example, 1 to 10 parts by weight, based on 100 parts by weight of the total culture solution.

refine

In order to remove microorganisms, etc., after the main fermentation, the fermented product was allowed to stand and separated into an upper layer (oil) and a lower layer (medium and impurities), all of the lower layers were discarded, and MgSO ₄ 20g/L was added to remove moisture. and stirred. After 2 hours of stirring, using a continuous centrifuge equipment, microbial cells, MgSO ₄ , and oil from which impurities and herbal medicines remaining in the oil layer were removed was obtained.

In addition, in order to remove fatty acids and impurities, magnesium silicate was used as a filler inside an affinity chromatography column with a diameter of 45 pie and a length of 2M, which was made to order, and fatty acids and impurities were adsorbed and filtered at a flow rate of 30L/hour. did

Examples 4 and 5: Formulations comprising fermented herbal medicine oil

Example 4: Preparation of lotion

An external lotion was prepared according to the composition of Table 2 below. Specifically, the raw materials 2 to 4 are sequentially added to the raw material No. 1 in Table 2 below, stirred and heated to 60-70 ° C. to dissolve, and the raw materials No. 5 to 7 are put in a separate container and heated to 60-70 ° C. After dissolution by heating, it was put into the raw material reaction vessel Nos. 1 to 4 and emulsified. After the emulsification was completed, the mixture was cooled to 40°C, and then the 8th raw material was added and stirred, cooled to 30°C, and aged. Example 1 was used as the herbal medicine fermented oil.

division Raw material weight (%) One Purified water 55.79 2 glycerin 10.00 3 Butylene Glycol 20.00 4 antiseptic 0.40 5 Example 1 8.00 6 PEG-8 Caprylic/Capric Glyceride 5.00 7 PEG-40 Hydrogenated Castor Oil 0.80 8 fare 0.01 Sum 100

Example 5: Cream preparation

Creams for external use were prepared according to the composition of Table 3 below. Specifically, the raw materials 2 to 6 are sequentially added to No. 1 in Table 3 below, stirred, heated to 60 to 70 ° C. to dissolve, and 7 to 10 raw materials are put in a separate container, and 60 to 70 ° C. After dissolution by heating with a furnace, it was put into the raw material reaction vessel Nos. 1 to 6 and emulsified. When the emulsification is finished, raw materials No. 11 and 12 are put in, dissolved and mixed uniformly, cooled to 40° C., and raw materials No. 13 are added and stirred, cooled to 30° C., and aged. Example 1 was used as the herbal medicine fermented oil.

division Raw material weight (%) One Purified water 68.94 2 1% carboxyl vinyl polymer aqueous solution 0.20 3 glycerin 10.00 4 Butylene Glycol 8.00 5 antiseptic 0.40 6 xanthan gum 0.05 7 Example 1 8.00 8 cetearyl alcohol 1.00 9 Glyceryl Stearate 1.50 10 PEG-8 Beeswax 1.50 11 Polyacrylate-13/polyisobutene/polysorbate 20 0.20 12 triethanolamine 0.20 13 Spices 0.01 Sum 100

Experimental example

Experimental Example 1: Emulsifying activity

Experimental Example 1-1: Absorbance Measurement

In order to analyze the emulsification activity before and after fermentation of Examples 1 to 3, fermented oil and distilled water were mixed at a ratio of 1:9, stirred vigorously for 1 minute to emulsify, and then left still for 10 minutes to collect the solution of the lower layer and measure the absorbance did Emulsification activity was confirmed by measuring the absorbance at 660 nm using a spectrophotometer (Microplate, BIOTECK, USA), and the results are shown in Table 4 below.

division Absorbance before fermentation (O.D/660nm) Absorbance after fermentation (O.D/660nm) Example 1 0.133 1.154 Example 2 0.133 1.281 Example 3 0.133 1.107

As shown in Table 4, it can be confirmed that the emulsification activity of the oil after fermentation is 8 to 10 times higher than before fermentation.

Experimental Example 2-2: Visual evaluation

Visual evaluation was performed to confirm the emulsifying activity of the oil. Specifically, for each of the oils before and after fermentation according to Examples 1 to 3 and Comparative Example 1, the emulsified state after 1 hour by mixing with distilled water 1:1 and stirring for 5 minutes is shown in FIG. 3 below, 48 The emulsified state after standing for time is shown in FIG. 4 below. As Comparative Example 1, unfermented rice seed oil was used.

As shown in FIGS. 3 and 4, when the emulsified state with distilled water was observed for each time for Comparative Example 1 and Examples 1 to 3, Examples 1 to 3 showed dispersion results in a stable form, whereas , Comparative Example 1 can be confirmed that the separation phenomenon of distilled water and oil appears distinctly over time.

Experimental Example 3: Oxidative stability

In order to evaluate the oxidation stability with respect to Examples 1 to 3, the acid value was measured. Acid value was measured using the Acid Value Test Method of the Food Code. After taking 5 g of oil in an Erlenmeyer flask, add 100 ml of an ethanol-ether mixture (1:2, v/v) to dissolve the oil well, add 100 μl of 1% phenolphthalein solution, mix well, and 0.1N ethanol until it turns pale red Titrated with aqueous potassium hydroxide solution. A blank test was performed using distilled water instead of oil.

The calculation formula is as follows.

Acid value = (5.611×a×f)/s

s = amount of sample taken

a=0.1N Consumption of KOH solution (ml)

f=0.1N KOH solution titer

The results are shown in Table 5 below.

division before fermentation
(mg KOH/g) after fermentation
(mg KOH/g) Example 1 0.84 1.12 Example 2 0.84 1.11 Example 3 0.84 1.12

As shown in Table 5, it can be confirmed that the oil according to Examples 1 to 3 has an acid value of about 1 to 2 times higher than that before fermentation.

Experimental Example 4: Antioxidant activity

In order to evaluate the antioxidant efficacy of Comparative Examples and Examples, the DPPH test method was used. As Comparative Example 1, unfermented rice seed oil was used. Each oil was dissolved in n-hexane for each concentration, mixed with 0.1 mM DPPH test solution, and absorbance was measured at 517 nm with a microplate reader. The free radical scavenging ability (%) was expressed as "{1-(sample OD/blank OD)}x100", and the results are shown in Table 6 below.

division density(%) Scavenging ability (%) Tocopherol acetate 10 5.71 Comparative Example 1 One 10.44 5 25.63 10 58.98 Example 1 One 20.86 5 46.00 10 77.71 Example 2 One 33.14 5 53.43 10 74.00 Example 3 One 30.86 5 50.57 10 70.57

As shown in Table 6, it can be confirmed that Examples 1 to 3 have improved radical scavenging ability than Comparative Example 1.

Experimental Example 5: Cell proliferation and toxicity

Experimental Example 5-1: Human fibroblast cell proliferation rate

In order to verify the human fibroblast proliferation effect and toxicity of the fermented oil according to Examples 1 to 3, an MTT test was performed. Human fibroblasts, Hs68 cells, were cultured in a 24-well culture plate at a concentration of 1x10 ⁵ cells/well for 24 hours. After serum starvation of the cells for 2 hours, each oil was treated with different concentrations (0-1%) and cultured for 24 hours. In order to measure the proliferation rate of cells, all of the culture medium was removed, treated with MTT reagent, and cultured for 2 hours. The formed formazan was dissolved in DMSO and absorbance was measured at 570 nm with a microplate reader, and the results are shown in FIG. 5 below. 5, Examples 1 to 3 promoted cell proliferation up to 128% at a concentration of 0.125%, and it can be confirmed that cytotoxicity does not appear up to a concentration of 1%.

Experimental Example 5-2: Human Keratinocyte Proliferation Rate

In order to verify the keratinocyte proliferation effect of the fermented oil according to Examples 1 to 3, an MTT test was performed. HaCaT cells, which are human keratinocytes, were cultured in a 24-well culture plate at a concentration of 1x105 cells/well for 24 hours. After serum starvation for 2 hours, the cells were treated with each oil concentration (0-1%) and cultured for 24 hours. In order to measure the proliferation rate of cells, all of the culture medium was removed, treated with MTT reagent, and cultured for 2 hours. The formed formazan was dissolved in DMSO and absorbance was measured at 570 nm with a microplate reader, and the results are shown in FIG. 6 below. As shown in Figure 6, Examples 1 to 3 promoted cell proliferation from 0.125% to a maximum of 125%, and it can be confirmed that cytotoxicity does not appear up to a concentration of 1%.

Experimental Example 6: Evaluation of Collagen Synthesis Effect

In order to compare the collagen synthesis effect of Examples 1 and 2, the human fibroblast culture medium was treated so that the concentration of each oil was 0.125%, 0.0625%, and after culturing the medium for 1 day, the culture medium was taken and measured was used for A PICP EIA kit (procollagen type I c-peptide enzyme immunoassay kit) was used, and the amount of synthesized collagen was measured at 450 nm using a spectrophotometer. The degree of collagen biosynthesis was calculated relative to that of the control (serum-free medium), and the results are shown in Table 7 below.

sample Collagen production increase rate (%) Example 1 20 Example 2 35

As shown in Table 7, it can be confirmed that Examples 1 and 2 exhibit a collagen synthesis increase rate of 20 to 35%.

Experimental Example 7: Evaluation of melanin production inhibition

In order to measure the inhibition rate of melanin production of Example 1, a melanin content measurement test was performed. Mouse-derived melanocytes, B16F10 cells, were cultured in a 6 well culture plate at a concentration of 2x105 cells/well for 24 hours, and the oil according to Example 1 was treated for 2 hours at different concentrations (0-0.5%), and then α- Melanin Stimulating Hormone (α-MSH) was treated and cultured for 48 hours. Cells were obtained by washing once with PBS, treatment with 0.25% Trypsin-EDTA, and centrifugation (3,000 rpm, 5 minutes). After that, the cells were washed once again with PBS and centrifuged (3,000 rpm, 5 minutes), dried at 80° C. for 5 minutes, and then melanin was dissolved at 80° C. for 1 hour with 1N NaOH (containing 1% DMSO). did The content of melanin was indicated by measuring the absorbance at 490 nm using a microplate reader. In addition, the amount of protein was corrected by the Bradford test method, and the results are shown in FIG. 7 . As shown in FIG. 7 , the group treated with α-MSH cells increased the amount of melanin production by 32% compared to the control group, and it can be confirmed that Example 1 inhibits melanin production by 30% at a concentration of 0.5%.

Experimental Example 8: Anti-inflammatory evaluation

In order to evaluate the anti-inflammatory efficacy of the oils according to Examples 2 and 3, the ability to inhibit nitric oxide production was measured. Nitric oxide is known as a chemical molecule that is produced in immune cells and regulates the immune system against external or internal harmful substances. Raw264.7 cells, a mouse-derived macrophage cell line, were cultured in a 12 well culture plate at a concentration of 5x104 cells/well for 24 hours and each oil was treated with each concentration (0-0.5%) for 2 hours, followed by Lipopolysaccharide (0.1 μg /ml) and cultured for 24 hours. Obtain a culture solution, centrifuge (3,000 rpm, 5 minutes), and mix 100 μl of the supernatant with 100 μl of griess reagent (mixed solution of 1% sulfanilamide and 0.1% N-(1-naphthyl)ethylene diamine dihydrochloride) to block light was reacted for 15 minutes. The resulting reactant was measured for absorbance at 450 nm with a microplate reader. In addition, the amount of nitric oxide produced was quantified using NaNO ₂ , and the results are shown in FIG. 8 . As shown in FIG. 8 , after the LPS treatment, nitric oxide was generated about 10 times higher, and it was confirmed that the nitric oxide production amount was inhibited according to the concentration of Examples 2 and 3.

Experimental Example 9: Evaluation of antibacterial activity

Propionibacterium acnes, a bacterium that forms acne, is known to cause inflammation by excessively proliferating in hair follicles and sebaceous glands under anaerobic conditions.

In order to evaluate the antibacterial efficacy of Example 3, a disk diffusion method was used. Before the experiment, the Propionibacterium acnes strain was cultured in liquid RC medium (reinforced clostridium medium) to make a bacterial culture solution with a concentration of 1x10 ⁵ CFU/ml. 1 ml of the culture medium was smeared on the solid RC medium and dried. After soaking Example 3 in a circular filter paper with a diameter of 6 mm at each concentration (0-0.5%), it was put on the medium and incubated in anaerobic conditions (5% H2, 95% N2; 37°C) for 48 hours. Growth inhibition (diameter of clear zone) was measured using a micrometer (Mitutoyo, Kawasaki, Japan), and the results are shown in Table 8 below.

sample Concentration (%) Diameter of clear zone (mm) Example 3 0 0.1 0.125 1.3 0.25 3.6 0.5 7.2

As shown in Table 8, the growth inhibitory ring increased in size according to the concentration, and it was confirmed that the antibacterial activity against acne bacteria was increased by forming a growth inhibitory ring of about 7.2 mm at 0.5%.

Experimental Example 10: skin irritation

In order to evaluate whether or not skin irritation of Example 1 according to the present invention was performed, a skin primary irritation evaluation was performed. The evaluation institution was commissioned by Korea Dermatological Research Institute. Thirty-one subjects were selected and applied on the back for 24 hours, and then at 30 minutes, 24 hours, and 48 hours after the patch was removed, the dermatologist judged the presence or absence of primary skin irritation, respectively. Skin reaction determination was based on the International Contact Dermatitis Association (ICDRG) standards and the American Cosmetics Association (PCPC) standards, and the results are shown in Table 9.

As shown in Table 9, as a result of calculating the irritation index using the skin reaction scores of each subject, 0.027 points were obtained, and Example 1 was judged to be a hypoallergenic substance.

Experimental Example 11: Active ingredient analysis

The active ingredient analysis of Examples 1 to 3 was performed. First, Examples 1 to 3 were used in the test by extracting the active ingredient using ethanol. Gomisin N, Curcumin, Catechin, and (-) epigallocatechin-3-gallate (EGCG) were purchased from Sigma and used as standard products.

Table 10 shows the HPLC analysis conditions of Gomisin N. A Waters Alliance2690 liquid chromatograph system (Waters, USA) and a Waters 2420 detector were used. As a column, Zorbax eclipse XDB C18 1.8 μm, 250×4.6 mm was used.

Curcumin was measured at absorbance at 428 nm using a spectrophotometer (UV-spectrophotometer), and curcumin was prepared by concentration and quantified by preparing a standard quantitation curve.

Table 11 shows the conditions for HPLC analysis of catechin and (-) epigallocatechin-3-gallate (EGCG). A Waters Alliance2690 liquid chromatograph system (Waters, USA) and a Waters 2420 detector were used. As a column, Ascentis RP-amide C18 5㎛, 150 × 4.6mm was used. The results are shown in Table 12.

Item Condition Eluent A Water Eluent B Acetonitrile gradient A:B =50:50 in 10min flow rate 1.5ml/min Injection volume 5ul Detection UV at 254nm

Item Condition Eluent A 10mM ammonium formate, pH3.0 with conc, formic acid Eluent B Methanol gradient Time A (%) B (%) 0 100 0 One 55 45 30 55 45 45 25 75 flow rate 0.5ml/min Injection volume 10ul Detection UV at 273nm

sample Ingredient content (%) Example 1 Gomisin N: 0.1%
Curcumin : 0.2% Example 2 Curcumin: 0.2% Example 3 Catchin: 40%
(-)epigallocatechin-3-gallate (EGCG): 20%

As can be seen from the above results, according to the present invention, metabolites of vegetable oil can improve affinity with other highly polar substances by offsetting the hydrophobic interaction of the oil, thereby improving emulsification power, skin absorption, and formulation stability. It can be confirmed that there is In addition, it was confirmed that it can promote cell proliferation and add elasticity and whitening effect to the skin while minimizing toxicity, and can exhibit antibacterial and anti-inflammatory effects as a hypoallergenic substance for the skin. In addition, the active ingredients included in Examples 1 to 3 were also confirmed.

The above description is merely illustrative of the technical idea of the present invention, and various modifications and variations are possible for those of ordinary skill in the art to which the present invention pertains without departing from the essential characteristics of the present invention. In addition, the embodiments disclosed in the present invention are not intended to limit the technical spirit of the present invention, but to explain, and the scope of the technical spirit of the present invention is not limited by these embodiments. The protection scope of the present invention should be construed by the following claims, and all technical ideas within the scope equivalent thereto should be construed as being included in the scope of the present invention.

Claims (14)

Candida bombicola UA-06 (KCTC 18592P) First seed fermentation step of fermenting microorganisms and oil in a medium;
A second seed fermentation step of sub-culturing the first seed fermented product, and
It includes the main fermentation step of sub-culturing the second seed fermented product and fermenting it with herbal medicines,
A method for producing fermented oil containing herbal medicines, wherein the medium used in the secondary seed fermentation step contains glycerin. The method of claim 1,
A method for producing fermented oil, wherein the fermented oil containing the herbal medicine has an emulsification activity 5 to 20 times higher than before fermentation. The method of claim 1,
A method for producing a fermented oil, wherein the fermented oil containing the herbal medicine has a free radical scavenging activity of 10 to 80% compared to before fermentation. The method of claim 1,
A method for producing a fermented oil, wherein the fermented oil containing the herbal medicine has an increase in collagen production by 10 to 50% compared to before fermentation. The method of claim 1,
The method for producing a fermented oil comprising the herbal medicine has a melanin formation inhibition rate of 5 to 60% than before fermentation. The method of claim 1,
The fermented oil production method of the fermented oil containing the herbal medicine, the amount of nitric oxide production is reduced by 5 to 50% compared to before fermentation. A fermented oil prepared by the manufacturing method according to claim 1 . The method of claim 1,
The medium used in the primary seed fermentation step is yeast extract, malt extract, peptone (peptone), glucose (glucose), urea (urea) and fermented oil production comprising vegetable oil Way. delete The method of claim 1,
The medium used in the main fermentation step is glycerin (glycerin), K₂HPO₄, KH₂PO₄, NH₄NO₃, MgSO₄, yeast extract (yeast extract), vegetable oil and a method for producing a fermented oil comprising herbal medicines. The method of claim 1,
The first and the second seed fermentation step is to ferment for 10 to 30 hours under aerobic conditions of 15 to 25 ℃,
The method for producing a fermented oil wherein the main fermentation step is fermented for 90 to 100 hours under aerobic conditions of 15 to 25 ℃. The method of claim 1,
The method for producing fermented oil is that the time of input of the herbal medicine is 30 to 50 hours after the start of the main fermentation. The method of claim 1,
After the main fermentation step, the fermentation product is left standing to separate the upper layer and the lower layer, the lower layer is discarded, and MgSO ₄ is added thereto, followed by centrifugation to remove microorganisms and moisture;
A method for producing fermented vegetable oil comprising the step of recovering the oil from which fatty acids and impurities have been removed by using magnesium silicate as a filler in the affinity chromatography column. A cosmetic composition comprising the fermented oil according to claim 7.

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