KR102459312B1 - Fermentation method for the preparation of hyaluronic acid with hydrophobic properties - Google Patents

Abstract

The present invention provides an eco-friendly composition in which hydrophobic properties are imparted to hyaluronic acid using microorganisms. In addition, by giving hyaluronic acid hydrophobicity, it is easy to be applied as an emulsion stabilizer or dispersion stabilizer with oil dispersion stabilization properties, and the feeling of use and skin intimacy is improved to be easy to be applied as cosmetics or external skin preparations and the like.

Description

Fermentation method for producing hydrophobic hyaluronic acid

The present invention relates to a fermentation method for producing hyaluronic acid having hydrophobic properties.

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, mainly composed of keratinocytes composed of proteins, and has a structure in which lipids such as ceramide, cholesterol, and free fatty acids 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. In order to allow effective functional substances to pass through the skin naturally, hydrophobic or hydrophilic and hydrophobic carriers should be used. It is common to use

However, these micelles or liposomes have a limit in allowing the active material to be absorbed by passing 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 structure between them is composed of lipid components.

On the other hand, hyaluronic acid is a linear polysaccharide composed of repeating disaccharide units in which N-acetyl-glucosamine and glucuronic acid are bonded by β-1,3-glucosidic bonds. It has high hydrophilicity and is easily combined with moisture and has excellent moisturizing effect, but has a limit in penetrating the skin. In addition, since hyaluronic acid can act as a lubricant in the body, such as joints and eyes, and can serve as a part of the extracellular matrix, it is necessary to control the degree of dissolution for various applications.

Conventionally, in order to impart hydrophobic properties to hyaluronic acid, a hydrophobic group was combined by a chemical synthesis method. For example, a carboxyl group is bonded to hyaluronic acid through an esterification reaction, and organic compounds such as 2,4,6-trichlorobenzoic acid (TCBA) and alcohol are used.

Therefore, the present invention intends to provide a method for producing hydrophobic hyaluronic acid using a microorganism.

It is an object of the present invention to provide a method for imparting hydrophobic properties to hyaluronic acid using microorganisms.

In order to solve the above problems, the present invention

Candida bombicola UA-06 (Accession No.: KCTC13855BP, Korea Research Institute of Bioscience and Biotechnology) Seed culture step to activate microorganisms;

A fermentation step of fermenting the seed cultured microorganisms and vegetable oil to produce a fermented emulsion;

separating the fermented product from the fermented emulsion; and

purifying the fermented product,

It provides a method for producing hydrophobic hyaluronic acid, wherein the medium of the fermentation step includes hyaluronic acid.

According to one embodiment, the separating may include adjusting the pH of the fermented emulsion to 1 to 5 and separating the aqueous layer, the intermediate layer, and the oil layer by standing for 1 to 20 hours, respectively.

According to one embodiment, the purifying step includes adding 80 to 100% (w/w) ethanol in 1 to 10 weight multiples to the fermented product and standing at 1 to 10° C. for 1 to 20 hours; and

It may include the steps of filtration, concentration and drying.

Alternatively, the purifying may include suspending the fermented product in water and adjusting the pH to 4 to 8; and

It may include a step of filtration and drying.

According to one embodiment, 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 .

According to one embodiment, the medium used in the fermentation step may include yeast extract, glycerin, K ₂ HPO ₄ , KH ₂ PO ₄ , NH ₄ NO ₃ , MgSO ₄ , hyaluronic acid and vegetable oil.

According to one embodiment, the medium used in the seed culture step is 5 to 30 g/L of yeast extract, 10 to 40 g/L of glycerin, K ₂ HPO ₄ 10 to 50 g/L, KH ₂ PO ₄ 3 to 15 g/L, NH ₄ NO ₃ 1 to 10 g/L and MgSO ₄ 0.5 to 5 g/L may be included.

According to one embodiment, the fermentation step is yeast extract 0.1 to 10 g / L, glycerin 1 to 40 g / L, K ₂ HPO ₄ 10 to 50 g / L, KH ₂ PO ₄ 3 to 15 g / L, NH ₄ NO ₃ 1 to 10 g/L, MgSO ₄ 0.5 to 5 g/L, hyaluronic acid 0.01 to 20 g/L and vegetable oil 50 to 1000 g/L 0.1 to 10 g/L of the seed cultured microorganism to a medium containing 50 to 1000 g/L can

According to one embodiment, the seed culture step may include the step of culturing for 40 to 100 hours at 15 to 30 ℃ 100 to 500 rpm and 0.5 to 2 vvm, aerobic conditions.

According to one embodiment, the fermentation step may include mixing at a temperature of 20 to 35 ° C. at 50 to 200 rpm, and fermenting for 1 to 10 days.

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

The present invention can provide a method for preparing a composition with improved feeling of use by imparting hydrophobic properties to hyaluronic acid in a nature-friendly method to improve moisturizing power and reduce stickiness.

1 is a photograph after completion of fermentation.
2 is a photograph of visually observing the hydrophobic hyaluronic acid composition.
3 is a photograph of a hydrophobic hyaluronic acid composition observed under a microscope.

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.

Hyaluronic acid acts as a lubricant in the body and has potential effects in various fields, such as being used for moisturizing the skin. However, there is a disadvantage in that its utility is limited due to its high hydrophilic properties. Conventionally, there is a method for producing hyaluronic acid having fat-soluble properties by inducing an esterification reaction by a chemical synthesis method, for example, in order to overcome these disadvantages.

The present invention provides a method for producing hydrophobic hyaluronic acid by a nature-friendly method rather than a chemical synthesis method.

Specifically, the present invention

Candida bombicola UA-06 (Accession No.: KCTC13855BP, Korea Research Institute of Bioscience and Biotechnology) Seed culture step to activate microorganisms;

A fermentation step of fermenting the seed cultured microorganism and vegetable oil to produce a fermented emulsion;

separating the fermented product from the fermented emulsion; and

purifying the fermented product,

It provides a method for producing hydrophobic hyaluronic acid, wherein the medium of the fermentation step includes hyaluronic acid.

According to one embodiment, in the separating step, the fermented emulsion is adjusted to pH 1 to 5, for example, pH 1 to 4, for example, pH 2 to 3, and 1 to 20 hours, for example 3 to 15 It may include the step of separating the aqueous layer, the intermediate layer, and the oil layer by standing for a period of time, for example, 6 to 12 hours. In this case, the aqueous layer contains microorganisms and a medium, the intermediate layer contains a fermented product, and the oil layer contains a fermented oil.

According to one embodiment, the purifying step is 80 to 100% (w / w), for example 90 to 98 (w / w) in 1 to 10 weight multiples, for example 1 to 5 weight times, in the fermented product. adding ethanol and standing at 1 to 10° C., for example, 2 to 8° C., for 1 to 20 hours, for example, 3 to 15 hours, for example, 5 to 10 hours; and

It may include the steps of filtration, concentration and drying.

Alternatively, the purifying may include suspending the fermented product in water and adjusting the pH to 4 to 8, for example, to pH 5 to 7, for example, to pH 6 to 8; and

It may include a step of filtration and drying.

Water that can be used in the present invention may include distilled water, purified water, ultrapure water, and the like, but is not particularly limited to the above description.

The filtration method applied to the purification step is not particularly limited as long as it is a general one, but may include, for example, filtration with 200 to 400 mesh.

In addition, the concentration step is not particularly limited as long as it is a general method of removing and drying ethanol, but, for example, a reduced pressure concentration method may be applied. For example, ethanol may be used by substituting one or more of the substances having a polarity of 5 to 6 or adding one or more of the substances, such as methanol and acetonitrile.

The drying step may be, for example, a freeze-drying method, but is not particularly limited as long as it is a conventional one.

According to one embodiment, 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 may include yeast extract, glycerin, K ₂ HPO ₄ , KH ₂ PO ₄ , NH ₄ NO ₃ , MgSO ₄ , hyaluronic acid and vegetable oil.

Specifically, for example, the medium used in the seed culture step is yeast extract 5 to 30 g / L, for example 5 to 20 g / L, for example 5 to 15 g / L, glycerin 10 to 40 g / L, for example For example, 10 to 30 g/L, for example 15 to 25 g/L, K ₂ HPO ₄ 10 to 50 g/L, for example 15 to 40 g/L, for example 20 to 30 g/L, KH ₂ PO ₄ 3 to 15 g/L, for example 5-10 g/L, NH ₄ NO ₃ 1-10 g/L, for example 1-5 g/L and MgSO ₄ 0.5-5 g/L, for example 1-3 g/L can do.

In addition, the medium used in the fermentation step is yeast extract 0.5 to 3 g / L, for example 0.5 to 2 g / L, for example 0.5 to 1.5 g / L, glycerin 10 to 40 g / L, for example 10 to 30 g /L, for example 15 to 25 g/L, K ₂ HPO ₄ 10 to 50 g/L, for example 15 to 40 g/L, for example 20 to 30 g/L, KH ₂ PO ₄ 3 to 15 g/L, For example, 5 to 10 g/L, NH ₄ NO ₃ 1 to 10 g/L, for example 1 to 5 g/L, MgSO ₄ 0.5 to 5 g/L, for example 1 to 3 g/L, hyaluronic acid 0.5 to 20 g /L, for example from 1 to 10 g/L, for example from 1 to 5 g/L and vegetable oil from 50 to 1000 g/L, for example from 100 to 800 g/L, for example from 300 to 700 g/L. 0.1 to 10 g/L, for example, 1 to 5 g/L, for example, 2 to 3 g/L of the seed cultured microorganism to the .

According to one embodiment, the hyaluronic acid may include sodium hyaluronate.

According to one embodiment, the seed culture step is 20 to 40 ℃, for example, 23 to 30 ℃ 100 to 500rpm, for example 200 to 400rpm and 0.5 to 2vvm, for example 0.5 to 1.5vvm, aerobic conditions It may include culturing for 40 to 100 hours, for example 50 to 80 hours, for example 50 to 70 hours.

In addition, the seed culture step may be terminated at the end point of exponential growth of the bacteria. Specifically, the exponential growth endpoint may be 40 to 120 hours, for example, 50 to 100 hours, for example, the seed culture may be terminated at a time point of 60 hours after culture.

According to one embodiment, in the seed culture step, after completion of the culture, 10,000 to 20,000 g, for example, 13,000 to 18,000 g, and 10 minutes to 1 hour, for example, centrifugation for 20 minutes to 40 minutes of the culture medium Separation may include the step of precipitating the cells.

In addition, after precipitation of the cells, for example, the step of washing several times with a phosphate buffer to obtain the cells may be further included.

According to one embodiment, the fermentation step is 20 to 35 ℃, for example, at a temperature of 20 to 30 ℃ 50 to 200 rpm, for example, 80 to 150 rpm mixing, 1 to 10 days, for example 1 to 5 It may include a step of fermenting for days.

According to one embodiment, the vegetable oil is, for example, sunflower seed, grape seed, canola, rice germ, olive, soybean, argan, brown rice, perilla, sesame, almond, peanut, corn, red ginseng, avocado, macadamia, coconut, rosehip , vitamin tree seed, shea fruit, oil palm, bergamot fruit, camellia seed, safflower seed, apricot seed, poppy seed, evening primrose seed, castor seed, green tea seed, meadowfoam seed, flax seed and hemp seed, etc. It may include one or more, but is not limited to the above description, and if it is an edible or human-friendly oil, it may be included in the scope of the present invention.

According to one embodiment, the hydrophobic hyaluronic acid formed according to the method as described above may have a hydrophobicity characteristic as a form in which a fat-soluble substance is bound to hyaluronic acid.

The predicted structure of the hydrophobic hyaluronic acid according to the present invention is as shown in Formula 1.

R may include various fatty acids which may be derived from vegetable oils. For example, it may include unsaturated and saturated fatty acids such as oleic acid, linoleic acid, eiconenoic acid, palmitic acid, stearic acid, and the like.

In addition, R may be a monoglyceride or diglyceride in which the fatty acids are randomly composed.

In addition, R may be a fat-soluble substance derived from vegetable oil in addition to the substances described above.

The hydrophobic hyaluronic acid to which the fat-soluble material is bound as in the above structure may have improved hydrophobic properties regardless of the molecular weight of the hyaluronic acid.

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.

Examples 1 to 3 and Comparative Example 1

seed culture

In order to obtain the cells, Candida bombicola UA 06 (Accession No.: KCTC13855BP, Korea Research Institute of Bioscience and Biotechnology) was mixed with yeast extract 10 g/L, glycerin 20 g/L, K ₂ HPO ₄ 25 g/L, Inoculated into a medium composed of KH ₂ PO ₄ 7 g/L, NH ₄ NO ₃ 3 g/L, MgSO ₄ 2 g/L and distilled water 933 g/L, aerobic at 25°C, 300rpm, 1NL/min (1vvm) Cultivation was carried out under the conditions. 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

Each medium composed of the composition shown in Table 1 was mixed in a 5 L fermenter at 25° C. and 100 rpm. When the temperature of the medium was 25° C., 2.5 g/L of the cells was added, and fermentation was performed for 3 days to prepare a fermented emulsion. A photograph after completion of fermentation for Example 1 is shown in FIG. 1 .

Comparative Example 1 Example 1 Example 2 Example 3 yeast extract
1 g/L yeast extract
1 g/L yeast extract
1 g/L yeast extract
1 g/L Glycerin 20 g/L Glycerin 20 g/L Glycerin 20 g/L Glycerin 20 g/L K ₂ HPO ₄ 25 g/L K ₂ HPO ₄ 25 g/L K ₂ HPO ₄ 25 g/L K ₂ HPO ₄ 25 g/L KH ₂ PO ₄ 7 g/L KH ₂ PO ₄ 7 g/L KH ₂ PO ₄ 7 g/L KH ₂ PO ₄ 7 g/L NH ₄ NO ₃ 3 g/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 MgSO ₄ 2 g/L - Sodium Hyaluronate 3 g/L Sodium Hyaluronate 3 g/L Sodium Hyaluronate 3 g/L Distilled water 442 g/L Distilled water 439 g/L Distilled water 439 g/L Distilled water 439 g/L High Oleic Acid Sunflower Seed Oil 500 g/L High Oleic Acid Sunflower Seed Oil 500 g/L Grape Seed Oil 500 g/L 500 g/L canola oil

separation

After losing the activity of the cells remaining in the fermented emulsion at a high temperature of 90° C., the pH is adjusted to 2-3 with 1N HCl, and the fermented product is left for 8 hours to form an aqueous layer (microorganisms and medium) and an intermediate layer (fermented product) and The oil layer (oil) was separated to remove the water layer and the oil layer.

Fermentation Purification

In order to obtain hydrophobic hyaluronic acid from the fermented product of the intermediate layer, the fermented product was suspended in distilled water. Thereafter, the pH was adjusted to 6 to 7 with 1N NaOH, and 95% (w/w) ethanol in 2 weights was added. After standing in refrigeration (4°C) for 8 hours to precipitate the fermented product, the precipitate was filtered through a 300 mesh and turbid in purified water. All ethanol was evaporated using a rotary vacuum concentrator (EV-1001V, SciLab), and the powder was prepared by freeze-drying for 3-5 days.

Experimental Example 1: Detection of hydrophobic hyaluronic acid

In order to confirm the hydrophobic hyaluronic acid of the fermented product, a uronic acid detection test was performed using carbazole. Carbazole reacts with uronic acid at high temperatures to form a reddish-purple compound. In a glass test tube, 200 mg of the powder prepared according to Example 1 and 800 uL of 25 mM sodium tetraborate dissolved in concentrated sulfuric acid were mixed, heated at 100° C. for 10 minutes, and cooled to room temperature. 200 uL of 0.125% carbazole dissolved in ethanol was added and reacted by heating at 100° C. for 10 minutes. After cooling to room temperature, uronic acid was confirmed by measuring absorbance at 550 nm with a microplate reader (Epoch2, BioTek). The uronic acid detection results are shown in Table 2 below.

division Comparative Example 1 Example 1 detection - ++

As a result of the measurement, uronic acid was detected in the fermented product separated in Example 1. Hyaluronic acid has a structure of a high molecular sugar chain in which glucuronic acid and N-acetylglucosamine are bonded, and carbazole reacts with this glucuronic acid to show a reddish purple color. Therefore, glucuronic acid constituting hyaluronic acid was detected in the fermented product that was not mixed with the aqueous layer, and finally hyaluronic acid with increased hydrophobicity was confirmed.

Experimental Example 2: Observation of physical properties

To observe the change in the physical properties of the composition, the composition was prepared as shown in Table 3 below and observed on the same day and the next day.

The preparation methods of Comparative Examples 3 and 6 are as follows. Raw materials 1 to 4 or 5 were mixed at 70° C. at 2,000 rpm using a homomixer in the main container. After mixing, the raw material 6 heated to 70° C. was added and homogenized at 2,000 rpm for 5 minutes.

division Raw material Comparative Example 3 (%) Example 6 (%) One Distilled water 82 82 2 Butylene Glycol 10 10 3 hexanediol 2 2 4 Example 1 - One 5 Sodium Hyaluronate One - 6 Macadamia Seed Oil 5 5 Sum 100 100

As a result of observing the physical properties of the composition, as shown in FIG. 2, Example 6 had higher turbidity than Comparative Example 3 on the same day, and on the next day, although the oil layer and the aqueous layer were separated in Comparative Example 3, Example 6 was observed to be stable.

In addition, as shown in FIG. 3 , it was confirmed that the density of micelles in Example 6 was higher than that of Comparative Example 3 and thus turbidity was increased.

The fermented product according to the present invention is a form in which hyaluronic acid and a fat-soluble substance are combined, and exhibits amphipathic properties due to increased hydrophobicity compared to conventional hyaluronic acid. Therefore, it can be understood that the amphiphilic properties of the fermented product in Example 6 increase the stability of the formulation by maintaining the micelles in the formulation at a stable and high density level.

Examples 7 to 9: Preparation of hydrophobic hyaluronic acid by molecular weight

seed culture

Microorganisms were obtained by activating microorganisms in the same manner as in Example 1.

fermentation

In order to bind fat-soluble substances to hyaluronic acid by molecular weight, each medium having the composition shown in Table 4 was mixed in a 5 L fermenter at 25° C. and 100 rpm. When the temperature of the medium was 25° C., 2.5 g/L of the cells was added, and fermentation was performed for 3 days to prepare a fermented emulsion. The molecular weight range of low molecular weight hyaluronic acid is 10-100KDa, the molecular weight range of medium molecular weight hyaluronic acid is 800-1200kDa, and the molecular weight range of high molecular weight hyaluronic acid is 1500-2000kDa.

Example 7 Example 8 Example 9 Yeast extract 1 g/L Yeast extract 1 g/L Yeast extract 1 g/L Glycerin 20 g/L Glycerin 20 g/L Glycerin 20 g/L K ₂ HPO ₄ 25 g/L K ₂ HPO ₄ 25 g/L K ₂ HPO ₄ 25 g/L KH ₂ PO ₄ 7 g/L KH ₂ PO ₄ 7 g/L KH ₂ PO ₄ 7 g/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 Low molecular weight sodium hyaluronate 3 g/L Medium molecular weight sodium hyaluronate 3 g/L Polymer Sodium Hyaluronate 3 g/L Distilled water 439 g/L Distilled water 439 g/L Distilled water 439 g/L High Oleic Acid Sunflower Seed Oil 500 g/L High Oleic Acid Sunflower Seed Oil 500 g/L High Oleic Acid Sunflower Seed Oil 500 g/L

separation

The intermediate layer (fermented product) was separated in the same manner as in Example 1.

Fermentation Purification

1) Example 7

The intermediate layer was suspended in distilled water to neutralize and the pH was adjusted to 6-7 with 1N NaOH. This was ultra-filtered (MWCO 10kDa) and freeze-dried for 3-5 days to prepare a powder.

2) Examples 8 and 9

The intermediate layer was suspended in distilled water to neutralize and the pH was adjusted to 6-7 with 1N NaOH. In Example 8, 4 times volume of 95% ethanol, Example 9, 2 times volume of 95% ethanol was added and refrigerated (4°C) for 8 hours to precipitate the fermented product. The precipitate was filtered through a 300 mesh ethanol and turbid in purified water, and then all of the ethanol was evaporated using a rotary vacuum concentrator (EV-1001V, SciLab). The fermented turbidity was freeze-dried for 3-5 days to prepare a powder.

Examples 10 to 12 Preparation

In order to evaluate the formulation stability by molecular weight of the fermented product, Examples were prepared as shown in Table 5 below. The preparation methods of Examples 10 to 12 are as follows. Raw materials No. 1 to 3 and hydrophobic hyaluronic acid for each molecular weight were mixed at 70° C. at 2,000 rpm using a homomixer in the main container. After mixing, raw material No. 7 heated to 70° C. was added, and homogenized at 2,000 rpm for 5 minutes.

division Raw material Example 10 (%) Example 11 (%) Example 12 (%) One Distilled water 82 82 82 2 Butylene Glycol 10 10 10 3 hexanediol 2 2 2 4 Example 7 One - - 5 Example 8 - One - 6 Example 9 - - One 7 Macadamia Seed Oil 5 5 5 Sum 100 100 100

Experimental Example 3: Stability evaluation

In order to evaluate the stability of the formulation according to Examples 10 to 12, after storing the formulation for a period of 1 day, 1 month, and 3 months at a temperature condition of -5 ° C., circulation (-5 to 50 ° C), and 50 ° C. Visual observation was made. The cycle (-5 to 50°C) temperature condition was set to change for 12 hours as one cycle. The stability results are shown in Table 8 below.

temperature hour division -5℃ -5~50℃ 50℃ Example 10 1 day separation doesn't exist doesn't exist doesn't exist sedimentation doesn't exist doesn't exist doesn't exist 1 month separation doesn't exist doesn't exist doesn't exist sedimentation doesn't exist doesn't exist doesn't exist 3 months separation doesn't exist doesn't exist doesn't exist sedimentation doesn't exist doesn't exist doesn't exist Example 11 1 day separation doesn't exist doesn't exist doesn't exist sedimentation doesn't exist doesn't exist doesn't exist 1 month separation doesn't exist doesn't exist doesn't exist sedimentation doesn't exist doesn't exist doesn't exist 3 months separation doesn't exist doesn't exist doesn't exist sedimentation doesn't exist doesn't exist doesn't exist Example 12 1 day separation doesn't exist doesn't exist doesn't exist sedimentation doesn't exist doesn't exist doesn't exist 1 month separation doesn't exist doesn't exist doesn't exist sedimentation doesn't exist doesn't exist doesn't exist 3 months separation doesn't exist doesn't exist doesn't exist sedimentation doesn't exist doesn't exist doesn't exist

As a result of observing the stability of Examples 10 to 12 for 3 months, no deformation of the formulation was observed under all temperature conditions.

As can be seen from the above results, according to the present invention, it is possible to prepare an oil emulsion stabilizer or dispersion stabilizer having thickening by preparing hydrophobic hyaluronic acid. In addition, according to the present invention, by imparting hydrophobic properties to hyaluronic acid, it is possible to improve skin penetration and emulsion stability, and to maximize skin moisturizing, elasticity, and skin barrier improvement effects. In addition, the method for producing hydrophobic hyaluronic acid according to the present invention is a method using a natural product, not a chemical synthesis method, and is environmentally friendly and has low human toxicity.

The above description is merely illustrative of the technical idea of the present invention, and various modifications and variations are possible without departing from the essential characteristics of the present invention by those skilled in the art to which the present invention pertains. 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.

Korea Institute of Bioscience and Biotechnology KCTC13855BP 20190604

Claims (10)

Candida bombicola UA-06 (Accession No.: KCTC13855BP, Korea Research Institute of Bioscience and Biotechnology) Seed culture step to activate microorganisms;
A fermentation step of fermenting the seed cultured microorganisms and vegetable oil to produce a fermented emulsion;
separating the fermented product from the fermented emulsion; and
purifying the fermented product,
A method for producing hydrophobic hyaluronic acid, wherein the medium of the fermentation step includes hyaluronic acid. According to claim 1,
The method for producing hydrophobic hyaluronic acid, wherein the separating step comprises the step of adjusting the fermented emulsion to pH 1 to 5, and separating the aqueous layer, the intermediate layer and the oil layer by standing for 1 to 20 hours. According to claim 1,
The purifying step is a step of adding 80 to 100% (w/w) ethanol to the fermented product in 1 to 10 weight multiples and standing at 1 to 10° C. for 1 to 20 hours; and
A method for producing hydrophobic hyaluronic acid, comprising the steps of filtration, concentration and drying. According to claim 1,
The purifying step includes suspending the fermented product in water and adjusting the pH to 4 to 8; and
A method for producing hydrophobic hyaluronic acid, comprising the steps of filtration and drying. According to claim 1,
The medium used in the seed culture step is yeast extract, glycerin, K ₂ HPO ₄ , KH ₂ PO ₄ , NH ₄ NO ₃ And MgSO ₄ The method for producing a hydrophobic hyaluronic acid comprising. According to claim 1,
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 , hyaluronic acid and a method for producing a hydrophobic hyaluronic acid comprising vegetable oil. The method of claim 1,
The medium used in the seed culture step is yeast extract 5 to 30 g/L, glycerin 10 to 40 g/L, K ₂ HPO ₄ 10 to 50 g/L, KH ₂ PO ₄ 3 to 15 g/L, NH ₄ NO ₃ 1 to 10 g/L and MgSO ₄ A method for producing a hydrophobic hyaluronic acid comprising 0.5 to 5 g/L. The method of claim 1,
The fermentation step is yeast extract 0.1 to 10 g / L, glycerin 1 to 40 g / L, K ₂ HPO ₄ 10 to 50 g / L, KH ₂ PO ₄ 3 to 15 g / L, NH ₄ NO ₃ 1 to 10 g / L, MgSO ₄ 0.5 to 5 g / L, 0.01 to 20 g / L of hyaluronic acid, and the hydrophobic hyaluronic acid comprising the step of adding 0.1 to 10 g / L of the seed cultured microorganism to a medium containing 50 to 1000 g / L of vegetable oil manufacturing method. According to claim 1,
The method for producing hydrophobic hyaluronic acid, wherein the seed culture step comprises culturing at 15 to 30° C. at 100 to 500 rpm and 0.5 to 2 vvm, aerobic conditions for 40 to 100 hours. According to claim 1,
The method for producing hydrophobic hyaluronic acid, wherein the fermentation step comprises the step of mixing at 50 to 200 rpm at a temperature of 20 to 35 ° C, and fermenting for 1 to 10 days.

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