KR20210068811A - Antiaging composition using fermented emulsifier forming reverse micell - Google Patents

Abstract

The present invention relates to a composition in which an antioxidant effective substance is captured using a fermented emulsifier that forms reverse micelles. An active substance can pass through the skin efficiently and can be effectively delivered to the lipid layer. In addition, solubility, emulsification, oxidative stability, and the efficacy of the active material can be improved. In addition, the composition is eco-friendly as a fermented product rather than a compound, and is human-friendly because there is no toxicity to skin cells, thereby being easily applied to various fields.

Description

Anti-aging composition using a fermented emulsifier that forms reverse micelles {ANTIAGING COMPOSITION USING FERMENTED EMULSIFIER FORMING REVERSE MICELL}

The present invention relates to a composition in which antioxidant-based anti-aging substances are captured using a fermented emulsifier that forms reverse micelles.

Human skin is largely divided into epidermis, dermis, and subcutaneous fat, and the epidermis is further subdivided into stratum corneum, transparent layer, granular layer, stratum corneum, and basal layer. The stratum corneum, which is the outermost layer of the skin, is composed of a mass of dead keratinocytes, and is mainly composed of keratinocytes composed of protein, and has a structure in which lipids such as ceramide, cholesterol, and free fatty acid fill the spaces between the keratinocytes. This lipid layer prevents the evaporation of moisture from the body and has a health function to protect the body against external invasion, and an aesthetic function to make the skin look shiny and smooth.

As the skin ages, it is exposed to the external environment and stress, and aging occurs due to an inflammatory reaction. Inflammaging is a compound word of inflammation and aging, and as humans age, they are easily exposed to chronic inflammatory responses, which can be related to aging, and are susceptible to immune aging appearing in the biological aging process.

On the other hand, there are intrapathway, interpathway, and transcelluar pathways for permeating an active substance into the skin. Among them, the interpathway is high for the efficiency of material permeation into the skin. On the other hand, the stratum corneum of the skin is easier to absorb and permeate the hydrophobic material than the hydrophilic material.

Factors affecting the skin absorption of drugs are size, shape, surface charge, lipophilicity, penetration enhancer, form of formulation, and the state of the stratum corneum. In order to allow the active ingredient to pass through the skin naturally, hydrophobicity or a carrier combining hydrophilicity and hydrophobicity should be used. In particular, a carrier in the form of micelles or liposomes is used to deliver water-soluble active substances to the skin. it is common

However, these micelles or liposomes have a limit in absorbing active substances through the lipid layer of the skin. This is because the stratum corneum, which is the outermost layer of the skin, is stacked with keratinocytes and the lipid component between them is structurally easier for hydrophobic substances to permeate between keratinocytes rather than hydrophilic substances.

Reverse micelles mean that nanometer-sized (1-10 nm) round particles are dispersed in an organic solvent by the activity of a surfactant.

The reverse micelle system is an aggregate that spontaneously arranges so that the hydrophobic part (tail) of the surfactant faces outward and the hydrophilic part (head) faces inward in the environment of a non-polar solvent. It can be dispersed into nano-sized particles.

Therefore, there is a need for a method that can effectively penetrate active ingredients from the skin by applying reverse micelles. In addition, it was attempted to improve stability by capturing active substances that are vulnerable to oxidation and decomposition.

It is an object of the present invention to provide a composition in which an anti-aging active material is captured using a fermentation emulsifier that forms reverse micelles.

The present invention in order to solve the above problems,

Fermented product formed from Candida bombicola UA-06 (Accession No.: KCTC13855BP, Korea Research Institute of Bioscience and Biotechnology); and

Crosine, ergothioneine, glutathione, retinol, fullerene, resveratrol, including an active substance consisting of sulforaphane and caustic acid,

The fermented product forms a reverse micelle structure, and the active material is entrapped in the reverse micelle structure.

According to one embodiment, the fermented product is a seed culture step of activating Candida bombicola UA-06 microorganisms; and

It may be prepared by a method comprising a fermentation step of fermenting the seed cultured microorganism and vegetable oil.

In addition, the medium used in the seed culture step may include yeast extract, glycerin, K ₂ HPO ₄ , KH ₂ PO ₄ , NH ₄ NO ₃ and MgSO ₄ .

In addition, the medium used in the fermentation step is yeast extract, glycerin, K ₂ HPO ₄ , KH ₂ PO ₄ , NH ₄ NO ₃ , MgSO ₄ , glucuronolactone and vegetable oil may be included.

Specifically, the medium used in the seed culture step is yeast extract 1g/L, glycerin 20g/L, K ₂ HPO ₄ 25g/L, KH ₂ PO ₄ 7g/L, NH ₄ NO ₃ 3 g/L and MgSO ₄ 2 g/L;

The medium used in the fermentation step is yeast extract 1g/L, 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, glucuronolactone 100g/L and vegetable oil 500g/L.

According to one embodiment, the seed culture step is carried out at 20 ℃, 300rpm, 1vvm, aerobic conditions,

The fermentation step may be performed at a temperature of 25° C. and mixing conditions of 100 rpm.

In addition, the seed culture step may be terminated at a time point of 60 hours from the start of the culture.

According to one embodiment, the vegetable oil may be sunflower seed oil.

According to one embodiment, it may further include a purification process step after the fermentation step,

The purification step is a step of separating the upper and lower layers by allowing the fermented product to stand, discarding the lower layer _{, adding MgSO 4} , stirring and centrifuging to remove microorganisms and moisture;

Using magnesium silicate as a filler in the affinity chromatography column may include recovering the oil from which fatty acids and impurities are removed.

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

The present invention uses a reverse micelle to efficiently pass an anti-aging active material that is functionally excellent but has low stability in aqueous solution through the skin to effectively deliver it to the lipid layer.

By trapping anti-aging active substances in reverse micelles, solubility, emulsification, oxidative stability, efficacy of the active substances, etc. can be improved. In addition, as it is a fermented product rather than a synthetic product, it is eco-friendly and has no toxicity to skin cells, so it is human-friendly, so it can be easily applied to various fields.

1 shows a schematic diagram of reverse micelle formation.
Figure 2 shows the collection process of the water-soluble material.
3 is a graph confirming the cell proliferation rate.

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 it 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.

Naturally derived surfactants contain a large amount of ingredients useful for the skin, and among them, bio-surfactants using microorganisms have many advantages.

On the other hand, when the surfactant is at a certain concentration or more, it is common to form a micelle structure in which hydrophobic groups are concentrated in the center of the aqueous phase. Conversely, in the oil phase, a reverse micelle structure is formed in which low molecular weight hydrophilic groups are concentrated in the center. In relation to the structure of the reverse micelle, a schematic diagram of the formation of the reverse micelle is shown in FIG. 1 .

Since reverse micelles are more efficient at passing through the lipid layer of the skin than micelles, they can be applied as a system that stably delivers water-soluble active substances to skin cells through the intercellular space.

Anti-aging active substances based on antioxidant action are functionally excellent, but have low stability in aqueous solution. According to the present invention, it is possible to enhance the skin absorption rate and permeation rate through the skin by collecting such an active material with reverse micelles, and it is possible to maximize the function of the active material. In addition, since the active material has a structure in a state of being trapped in the hydrophobic oil, the stability of the active material can be improved, and it can be grafted into various formulations. The process of collecting a water-soluble substance into reverse micelles according to an embodiment of the present invention is shown in FIG. 2 .

Hereinafter, an anti-aging composition according to an embodiment of the present invention will be described in more detail.

As used herein, the term "reverse micelle" refers to a group of molecules in which polar groups are oriented inward and hydrophobic groups are oriented toward the outside non-polar solvent side in the presence of a hydrophilic lysate, forming a nearly spherical oriented population.

As used herein, the term “collection” refers to a form in which a water-soluble substance is encapsulated in the reverse micelle.

Specifically, the present invention

Fermented product formed from Candida bombicola UA-06 (Accession No.: KCTC13855BP, Korea Research Institute of Bioscience and Biotechnology); and

Crosine, ergothioneine, glutathione, retinol, fullerene, resveratrol, including an active substance consisting of sulforaphane and caustic acid,

The fermented product forms a reverse micelle structure, and the active material is entrapped in the reverse micelle structure.

Crocin (crocin) is the main component of the saffron pigment extracted from Crocus sativus L,flowers and Irisaceae, and has a yellow color. In addition, it is easily soluble in water and is widely used as a food material and a cosmetic material. However, crosine has low stability when exposed to environments such as heat, oxygen, light and acid. Crocin has also been reported to have antioxidant and anti-inflammatory effects (ref. Crocin, a natural molecule with potentially beneficial effects against skin aging, International Journal of Cosmetic Science [09 Aug 2018, 40(4):388-400]).

Ergothioneine (L-Ergothioneine) is a powerful natural antioxidant, protects cells from oxidative stress, and has anti-aging action against light, so it has high utility value as a cosmetic material (Reference: L-Ergothioneine Protects Skin Cells against UV-Induced Damage??A Preliminary Study, Cosmetics 2014, 1, 51-60; doi:10.3390/cosmetics1010051).

Glutathione is a tripeptide composed of three amino acids: cysteine, glycine, and glutamate, and its main function is reported to have antioxidant and whitening effects. However, due to high polarity, skin penetration is difficult (Partition Coefficient and Glutathione Penetration of Topical Antiaging: Preformulation Study, International Journal of Drug Delivery Technology 2018; 8(2); 39-43). In addition, since heat or photostability is very low, there is a problem in that it is difficult to express an efficacy effect even when applied to a formulation.

Retinol is the chemical name for vitamin A1, also known as pure vitamin A. It is present in the intestinal mucosa cells of animals and is abundant in green-yellow plants. It is also transformed into the active form of retinoic acid, and is known to play an important role in maintaining the original function of epidermal cells. That is, it is known to have the effect of reducing wrinkles and increasing skin elasticity, and has been developed as a cosmetic for removing wrinkles or preventing skin aging.

Fullerenes are carbon allotropes such as graphite and diamond. It has carbon rings in which carbon atoms are arranged in a pentagonal or hexagonal arrangement, and has a shape similar to that of a soccer ball. In addition, it has strong antioxidant and bactericidal properties.

Resveratrol (Resveratrol), a kind of polyphenol, is contained in many berry plants, secretes antibacterial substances, and has antioxidant, anti-inflammatory and anti-aging effects.

Sulforaphane is a substance contained in cruciferous vegetables such as broccoli, Brussels sprouts, and cabbage, and has a strong antioxidant effect. However, there is a disadvantage in that the stability against conditions such as photostability, temperature, pH, and oxygen is very low.

Chebulic acid is a phenolic compound isolated from ripened fruits.

According to one embodiment, the fermented product is a seed culture step of activating Candida bombicola UA-06 microorganisms; and

It may be prepared by a method comprising a fermentation step of fermenting the seed cultured microorganism and vegetable oil.

The medium used in the seed culture step may include yeast extract, glycerin, K ₂ HPO ₄ , KH ₂ PO ₄ , NH ₄ NO ₃ and MgSO ₄ . Specifically, for example, yeast extract 1.0g/L, glycerin 20g/L, K ₂ HPO ₄ 25g/L, KH ₂ PO ₄ 7 g/L, NH ₄ NO ₃ 3 g/L and MgSO ₄ 2g/L. In addition, it may further contain water, specifically, purified water is preferable, for example, purified water using an ion exchange resin may be used.

The medium used in the fermentation step is yeast extract, glycerin, K ₂ HPO ₄ , KH ₂ PO ₄ , NH ₄ NO ₃ , MgSO ₄ , glucuronolactone and vegetable oil. may include Specifically, for example, yeast extract 1g/L, glycerin 20g/L, K ₂ HPO ₄ 25g/L, KH ₂ PO ₄ 7g/L, NH ₄ NO ₃ 3g/L, MgSO ₄ 2 g/L, glucuronolactone 100 g/L and vegetable oil 500 g/L. In addition, it may further contain water, specifically, purified water is preferable, for example, purified water using an ion exchange resin may be used.

By including glucuronolactone in the fermentation step, it is possible to produce an emulsifier with increased hydrophilicity in the form of glucuronolactone-fatty acid, thereby increasing the emulsifying power of the fermentation emulsifier or improving the capturing power of hydrophilic active substances. can

According to one embodiment, the seed culture step may be performed at 20° C., 300 rpm, 1 vvm, aerobic conditions. In addition, the fermentation step may be carried out for 3 days at a temperature of 25 ℃ and 100 rpm.

According to one embodiment, the seed fermentation step may be terminated at the endpoint of exponential growth of the bacteria. Specifically, for example, the seed fermentation may be terminated at a time point of, for example, 60 hours as an exponential growth endpoint.

According to one embodiment, the vegetable oil may be sunflower seed oil.

According to one embodiment, it may further include a purification process step after the fermentation step. Specifically, for the removal of moisture and microorganisms, the fermented product is left standing after oil fermentation to separate the hydrophobic portion (fermented product) located in the upper layer and the hydrophilic portion (medium and impurities) located in the lower layer to discard the lower layer, and MgSO ₄ can be added and stirred to remove residual impurities and microorganisms using a centrifuge.

In addition, in order to purify the fatty acids and impurities of the fermented product after the 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 one embodiment, the composition of the present invention may be prepared in any formulation that can be applied dermatologically, such as a conventional cosmetic composition, a pharmaceutical composition for external application to the skin, and the like. Dermatologically applicable is a composition that can have a relatively non-toxic and harmless effective action to the subject, and may include an external application that can be applied to the skin, and the side effects resulting from the composition can affect the efficacy of the active ingredient. It may mean that the activity and physical properties of the active ingredients are not impaired while not lowering the application target and causing serious irritation.

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 many different forms and is not limited to the embodiments described herein.

manufacturing example

Obtained Candida bombicola UA 06 (Accession No.: KCTC13855BP, Korea Research Institute of Bioscience and Biotechnology).

Candida bombicola UA 06 (Accession No.: KCTC13855BP, Korea Research Institute of Bioscience and Biotechnology), yeast extract 1.0 g/L, glycerin 20 g/L, K ₂ HPO ₄ 25 g/L, KH ₂ PO ₄ 7 Inoculated into a medium composed of g/L, NH ₄ NO ₃ 3 g/L, MgSO ₄ 2 g/L and distilled water 933 g/L, and seed culture was performed under aerobic conditions at 20°C, 300rpm, 1NL/min (1vvm). proceeded. The culture was terminated at 60 hours, the end point of exponential growth, by measuring absorbance at 600 nm with a spectrophotometer (Epoch2, BioTek). The culture medium was centrifuged (16,000 g, 30 minutes) to precipitate the cells and washed several times with a phosphate buffer to obtain the cells.

Fermentation of oil

For oil fermentation, yeast extract 1.0 g/L, glycerin 20 g/L, K ₂ HPO ₄ 25 g/L, KH ₂ PO ₄ 7 g/L, NH ₄ NO ₃ 3 g/L , MgSO ₄ 2 g/L, glucuronolactone 100 g/L, vegetable oil 500 g/L, and distilled water 333 g/L. In a 5 L fermenter, the fermentation broth is mixed at 25°C and 100 rpm. prepared. When the temperature of the fermentation broth was 25° C., 2.5 g/L of the cells was added, and fermentation was performed for 3 days. As vegetable oil, sunflower seed oil was used.

refine

After 3 days of fermentation, the remaining enzyme activity with respect to the fermented product was lost at a high temperature of 90° C., the pH was adjusted to 4 to 5 with 1N HCl, and the fermented product was left to stand for 6 to 12 hours to obtain an aqueous layer (microorganism and medium) The water layer was removed by separating the oil layer (reverse micellar formation ferment emulsion). To remove moisture remaining in the fermented product, _{20 g/L of MgSO 4} was added, and the mixture was stirred at 50° C. and 300 rpm for 2 hours to obtain a transparent oil layer (reverse micelle-forming fermentation emulsion) after centrifugation. In addition, after purifying the fatty acids and impurities of the fermentation emulsion with a column filled with magnesium silicate, the fermented product (U-able HBG) is filtered through a 0.45 μm filter to completely remove the _{remaining small particles of MgSO 4 to form reverse micelles (U-able HBG).} ) was obtained.

Example 1 and Comparative Example 1: Preparation of anti-aging composition

A composition was prepared with the composition shown in Table 1, and Comparative Example 1 was obtained by simply dissolving the active material. Specifically, raw materials 1 to 8 in Table 1 below are water-soluble substances, transparently dissolved in raw materials 9 to 11, and then added to raw material 12 heated to 50° C., and raw materials 1 to 11 are used as raw materials 12 was collected in The collection was carried out by using an agi-mixer for collection at 500 ~ 1000 rpm, 30 minutes ~ 1 hour. Thereafter, it was cooled to 25° C. to observe the transparency and long-term stability of each composition. However, the concentration of the active material dissolved in the solvent in Example 1 was prepared in the same manner.

Example 2: Preparation of Essence

Essence was prepared according to the composition of Table 2. Specifically, the raw materials of the A phase and B phase of Table 2 were sequentially added, mixed and dissolved. Thereafter, phase B was added to phase A and mixed at 70° C. with a homomixer at 3000 rpm for 10 minutes. After dissolving phase C, it was put into a container in which phases A to B were mixed, and mixed at 3000 rpm for 3 minutes. Phase D was put into a container in which phases A to C were mixed, and mixed with a homomixer at 3500 rpm for 3 minutes. Thereafter, it was defoamed and cooled to 30°C.

Experimental Example 1: Stability evaluation by long-term storage temperature

In order to evaluate the stability according to the storage temperature conditions, the composition according to Example 1, which is a stable composition, for 1 month at room temperature and 25° C., low temperature (-5° C.), high temperature (50° C.), circulation (-5 to 40 ℃ / 12 hr) condition was observed for storage for 3 months. The evaluation results are shown in Tables 3 and 4.

From the results of Tables 3 and 4, it was confirmed that Example 1 was excellent in transparency according to storage conditions and stability according to precipitation, and from this, it can be seen that the composition of Example 1 is a condition for stably collecting active substances.

Experimental Example 2: Antioxidative power test (DPPH radical scavenging ability measurement)

DPPH assay was performed to compare the antioxidant activity over time at room temperature of Example 1.

Example 1 and Comparative Example 1 were compared. Each sample was stored at room temperature for one month.

DPPH (2,2-diphenyl-1-picrylhydrazyl) was prepared by dissolving in methanol at a concentration of 0.2 mM. After adding 500ul of DPPH reagent to 500ul of sample and reacting for 15 minutes, absorbance was measured at 517nm. The results are shown in Table 5.

From the results of Table 5, Example 1 confirmed that the radical scavenging ability was significantly higher than that of Comparative Example 1, and in Example 1, it was confirmed that the stability of the component was high even with the passage of time of the collected composition.

Experimental Example 3: Human fibroblast cell proliferation rate

In order to verify the human fibroblast proliferation effect and toxicity of Example 1 and Comparative Example 1 stored for one month at room temperature, 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 sample was treated with different concentrations (0 to 1%) and cultured for 24 hours. 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. 3 . As shown in Figure 3, Example 1 promoted cell proliferation up to 120% at a concentration of 0.05%, it can be confirmed that the cytotoxicity does not appear up to a concentration of 0.5%. However, the sample used for the cell experiment was dissolved in 50% DMSO and then diluted with PBS.

Experimental Example 4: Collagen Synthesis

In order to compare the collagen synthesis effect of the human fibroblasts of Example 1 and Comparative Example 1 stored at room temperature for a month, the human fibroblast culture medium was treated so that the concentrations of each sample were 0.05% and 0.01%, and the medium was After culturing for 1 day, the culture solution was taken and used for measurement.

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 6.

As shown in Table 6, Example 1 shows a collagen synthesis increase rate of 30%, which can be confirmed that it is increased by 20 times compared to Comparative Example 1.

Experimental Example 5: Comparison of skin efficacy of the compositions according to Example 2 and Comparative Example 2

In order to investigate the skin efficacy effects of the compositions of Example 2 and Comparative Example 2, an experiment was performed on human skin.

Specifically, after dividing into a test group and a comparative group by 10 subjects or subjects with wrinkle or already generated wrinkles, Example 2 as a test group and Comparative Example 2 as a comparison group were administered in the morning and evening on the first day, respectively. It was applied twice for 4 weeks. The skin condition was precisely measured using a facial analyzer (Janus) with normal light, polarized light, and ultraviolet light. Normal light analyzes skin texture, pores, wrinkles, etc., polarized light analyzes pigmentation and inflammatory expression such as acne, and ultraviolet light can analyze sebum, pigmentation, and dead skin cells. This was comprehensively evaluated to derive the effect of the skin, and the results are shown in Tables 7 and 8.

As shown in Tables 7 and 8, when Example 2 and Comparative Example 2 were applied to human facial skin, improvement in skin tone, pigmentation, wrinkles, and skin texture were efficaciously compared. In Example 2, a composition in which three types of effective substances were captured by reverse micelles was added to the formulation, and Comparative Example 2 is a formulation in which the reverse micelles were not collected but dissolved in a solvent and directly added to the formulation. As a result, it can be confirmed that the formulation of Example 2 exhibits a superior effect on skin aging and skin tone improvement than Comparative Example 2. Therefore, when water-soluble effective substances based on antioxidant function were applied to the formulation in the same concentration as reverse micelles in hydrophobic oil and non-collected ones, the skin efficacy effect showed a marked difference. Therefore, since the water-soluble substances applied to the cosmetic formulation are difficult to penetrate into the skin, the fusion and absorption rate in the lipid layer of the skin can be increased by using the method of collecting with reverse micelles, thereby maximizing the skin effect.

As can be seen from the above results, the present invention uses a fermented emulsifier (U-ableㄾ HBG) to form reverse micelles, and Crocin, Ergothioneine, Glutathione ), Retinol, Fullerene, Resveratrol, Sulforaphane, and Chebulic acid are captured in the oil phase to limit the absorption and penetration of antioxidants into the skin. This can be overcome with emulsifiers. In addition, since it is possible to stably prepare an unstable substance in the aqueous phase, dermatological efficacy can be maximized.

Therefore, skin permeability can be improved by inclusion of easily oxidized, heat-denatured substances, poorly soluble substances, etc. according to the present invention, and can be applied stably and easily to cosmetic formulations.

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 equivalent range should be construed as being included in the scope of the present invention.

Claims (9)

Fermented product formed from Candida bombicola UA-06 (Accession No.: KCTC13855BP, Korea Research Institute of Bioscience and Biotechnology); and
Crosine, ergothioneine, glutathione, retinol, fullerene, resveratrol, including an active substance consisting of sulforaphane and caustic acid,
The anti-aging composition, wherein the fermented product forms a reverse micelle structure, and the active material is entrapped in the reverse micelle structure. According to claim 1,
Seed culture step in which the fermentation product activates Candida bombicola UA-06 microorganism; and
The anti-aging composition, which is prepared by a method comprising a fermentation step of fermenting the seed cultured microorganism and vegetable oil. 3. The method of claim 2,
The medium used in the seed culture step is yeast extract (yeast extract), glycerin (glycerin), K ₂ HPO ₄ , KH ₂ PO ₄ , NH ₄ NO ₃ And MgSO _{4 The} anti-aging composition comprising. In the second,
The medium used in the fermentation step is yeast extract, glycerin, K ₂ HPO ₄ , KH ₂ PO ₄ , NH ₄ NO ₃ , MgSO ₄ , glucuronolactone and vegetable oil. It comprises, the anti-aging composition. 3. The method of claim 2,
The medium used in the seed culture step is yeast extract 1g/L, glycerin 20g/L, K ₂ HPO ₄ 25g/L, KH ₂ PO ₄ 7g/L, NH ₄ NO ₃ 3g /L and MgSO ₄ 2 g/L,
The medium used in the fermentation step is yeast extract 1g/L, 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, glucuronolactone (glucuronolactone) 100g / L, and vegetable oil 500g / L, the anti-aging composition comprising a. 3. The method of claim 2,
The seed culture step is carried out at 20 ° C., 300 rpm, 1 vvm, aerobic conditions,
The anti-aging composition, wherein the fermentation step is performed at a temperature of 25° C. and a mixing condition of 100 rpm. 3. The method of claim 2,
The seed culture step is to end at 60 hours from the start of the culture, anti-aging composition. 3. The method of claim 2,
The anti-aging composition, wherein the vegetable oil is sunflower seed oil. 3. The method of claim 2,
It may further include a purification process step after the fermentation step,
The purification step is a step of separating the upper and lower layers by allowing the fermented product to stand, discarding the lower layer _{, adding MgSO 4} , stirring and centrifuging to remove microorganisms and moisture;
Using magnesium silicate as a filler in the affinity chromatography column, the anti-aging composition comprising the step of recovering the oil from which fatty acids and impurities are removed.

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