KR101715652B1 - Fermented oil comprising nitro fatty acid and producing method thereof - Google Patents

Abstract

The present invention relates to a fermented oil containing a nitro fatty acid through fermentation of a vegetable oil using microorganisms and a fermented vegetable oil through a mixed fermentation using nitric acid bacteria and a method for producing the fermented oil, And is not cytotoxic. Therefore, it can be applied to various fields such as cosmetics, medicines, and foods.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a fermented oil containing a nitric acid and a method for producing the fermented oil,

The present invention relates to a fermented oil containing a nitro fatty acid and a process for producing the same.

The cause of the formation of wrinkles, which is one form of skin aging, can be broadly classified into the generation of wrinkles by natural aging and the generation of wrinkles by exposure to ultraviolet rays (photoaging). However, the mechanism of wrinkling due to two causes has not yet been clarified . A common phenomenon found in association with skin changes due to natural aging and photoaging is the reduction and modification of collagen and elastin, the main constituent proteins in the dermis, and the reduction of skin elasticity due to the reduction of the hyaluronic acid, the substrate of the dermis. Particularly, according to the results thus far, reduction and modification of collagen and elastin, which are main proteins in the dermis, are directly related to the generation of wrinkles and the reduction of elasticity of skin.

Collagen is a major substrate protein produced in fibroblasts of the skin and exists in extracellular epilepsy. Its important functions are mechanical rigidity of skin, resistance of connective tissues and binding force of tissues, support of cell adhesion, division of cells and differentiation And the induction of wound healing). Such collagen is reduced by aging and photoaging by ultraviolet irradiation, which is known to be closely related to the wrinkling of the skin. In recent years, as a result of extensive research on skin aging, important functions of collagen in the skin have been revealed.

It is necessary to protect the cell membrane by destroying free radicals mediated by free radicals, ultraviolet rays, and inflammation that occur during the metabolism of the body, and to proliferate cells by regenerating already damaged cells by active metabolism. And maintain healthy skin. Aging involves not only free radicals but also matrix metalloprotease (MMP) enzymes. In vivo, the synthesis and degradation of extracellular matrix such as collagen are properly regulated, but their synthesis decreases as aging progresses. Collagen Expression of substrate metalloproteinase (MMP), which is an enzyme to degrade, promotes skin elasticity and wrinkles. These degrading enzymes are also activated by ultraviolet irradiation. Therefore, it is required to develop a substance capable of controlling the activation of MMP expression in cells or inhibiting its activity. Until now, the raw materials used as cosmetic materials mostly inhibited only the enzyme activity. Therefore, we tried to control the amount and activity of MMP expression induced by intracellular natural aging and photoaging.

The inventors of the present invention have sought to develop a material capable of promoting collagen synthesis of skin damaged by ultraviolet rays and improving the skin aging phenomenon harmless to human body through a method using natural materials.

Accordingly, there is provided a fermented oil containing a nitro fatty acid using a microorganism and a method for producing the fermented oil.

In order to solve the above problems, the present invention provides a method for producing a microorganism, comprising the steps of: (A) pre-culturing a microorganism in a suitable medium (a);

A first fermentation step of adding a vegetable oil to the microorganism culture solution to ferment;

(B) a pre-culturing step of nitric acid bacteria for culturing nitrate bacteria in an appropriate medium (b);

A secondary fermentation step of adding the culture broth of the nitrifying bacteria to the primary fermentation broth to mix and ferment; And

And recovering the vegetable oil containing the nitro fatty acid after the second fermentation. The present invention also provides a method for producing a fermented oil containing a nitro fatty acid.

The microorganism may be preferably Pseudozyma sp.

It is more preferable that the microorganism is Pseudozyma sp. SY16 (KCTC 8950P).

The appropriate medium (a) of the microorganism is glucose, yeast extract, malt extract, peptone, soya bean meal, (NH ₄ ) ₂ SO ₄ , KH ₂ PO ₄ , MgSO ₄ , CaCl ₂ , and NaCl.

The pre-cultivation step (A) of the microorganism may be preferably carried out at 22 to 28 DEG C under aerobic conditions for 16 to 72 hours.

The vegetable oil may be selected from the group consisting of olive oil, palm oil, argan oil, green tea seed oil, grape seed oil, meadowfoam oil, soybean oil, sunflower seed oil, grape seed oil, sesame oil, perilla oil, flaxseed oil, Rapeseed oil, canola oil, and the like.

The acid value of the vegetable oil is preferably in the range of 0.1 to 2, more preferably 0.1 to 1 or 0.1 to 0.5.

It is preferable that the vegetable oil is 100 parts by weight or less based on 100 parts by weight of the microorganism culture solution.

The primary fermentation may be preferably fermented at 22 to 28 DEG C under aerobic conditions for 70 to 120 hours.

The nitrate bacteria are preferably at least one selected from the group consisting of Nitrosomonas sp. DM-0521, Nitrobacter sp. DM-0514 and Nitrobacter sp. DM-2309 can do.

It is further preferred that the nitric acid bacteria are Nitrosomonas sp. DM-0521.

Titration medium of the jilsangyun (b) is (NH ₄₎ from _{2 SO 4, KH 2 PO 4} , MgSO 4, CaCl 2, FeSO 4, CuSO 4, NaH 2 PO 4, Na 2 CO 3, the group comprising NaCl It may be desirable to have at least one selected.

The pre-cultivation step (B) of the nitrifying bacteria may be preferably carried out at 22 to 28 DEG C for 6 to 15 days under aerobic conditions.

The secondary fermentation step may be preferably carried out at 22 to 28 ° C under aerobic conditions for 96 to 120 hours.

The present invention also provides a fermented oil containing the nitro fatty acid produced by the above-mentioned method.

The present invention also provides a cosmetic composition comprising a fermented oil containing a nitro fatty acid produced by the above-described method.

The cosmetic composition is at least one cosmetic composition selected from the group consisting of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing oils, powder foundations, emulsion foundations, wax foundations and sprays Composition.

The vegetable oil containing the Nitro fatty acid according to the present invention has an anti-aging effect and can be applied to various fields such as cosmetics, medicines and foods.

FIG. 1 shows the results of cytotoxicity measurement of fibroblasts of nitro fatty acid through MTT-assay.
FIG. 2 shows the results of cytotoxicity measurement of UV-irradiated fibroblasts of nitro-fatty acids through MTT-assay.
FIG. 3 shows the results of measurement of the collagen (COL1A1) aggregation performance by irradiating ultraviolet light to the fibroblasts of nitro fatty acid.
Fig. 4 shows the results of measurement of inhibitory activity of collagenase (MMP-1) upon irradiation of ultraviolet light to the fibroblasts of nitro fatty acid.
FIG. 5 shows the results of cytotoxicity measurement of keratinocytes of nitro fatty acid through MTT-assay.
FIG. 6 shows cytotoxicity measurement results of UV-irradiated keratinocytes of nitro fatty acid through MTT-assay.
FIG. 7 shows the results of measuring the inhibitory activity of collagenase (MMP-1) upon keratinocytes of nitro fatty acid upon irradiation with ultraviolet light.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a method for producing a microorganism, which comprises (A) a step of pre-culturing a microorganism in which the microorganism is cultured in an appropriate medium (a);

A first fermentation step of adding a vegetable oil to the microorganism culture solution to ferment;

(B) a pre-culturing step of nitric acid bacteria for culturing nitrate bacteria in an appropriate medium (b);

A secondary fermentation step of adding the culture broth of the nitrifying bacteria to the primary fermentation broth to mix and ferment; And

And recovering the vegetable oil containing the nitro fatty acid after the second fermentation. The present invention also provides a method for producing a fermented oil containing a nitro fatty acid.

The microorganism is preferably Pseudozyma sp., And more preferably Pseudozyma sp. SY16 (KCTC 8950P).

The Pseudozyma sp. SY16 (KCTC 8950P) grows in a cylindrical shape, a polar germination type, and forms endogenous spores distinct from mycelia and ascospores. The Pseudozyma sp. Is known to produce a biosurfactant (MEL-SY16) upon fermentation using an oil or the like as a carbon source (Korean Patent Registration No. 10-0532690).

It is important that the microorganism culture solution contains no vegetable oil yet, and it is important to blend various culture materials so that the vegetable oil is fermented well.

The appropriate medium (a) of the microorganism is glucose, yeast extract, malt extract, peptone, soya bean meal, (NH ₄ ) ₂ SO ₄ , KH ₂ PO ₄ , MgSO ₄ , CaCl ₂ , and NaCl.

In addition, the step (A) for pre-culturing the microorganisms is preferably carried out at 22 to 28 ° C under aerobic conditions for 16 to 72 hours, more preferably 30 to 60 hours.

In order to optimize the fermentation of the vegetable oil in the primary fermentation step, it is important to use vegetable oil having a low acid value. That is, the optimum fermentation condition is determined by the acid value of the vegetable oil, and the lower the acid value, the higher the fermentation efficiency. The acid value of the vegetable oil is preferably in the range of 0.1 to 2, and more preferably the acid value is in the range of 0.1 to 1 or 0.1 to 0.5. Vegetable oils having an acid value of less than 0.1 are scarcely present, and when the acid value is more than 2, the fermentation efficiency is lowered.

The vegetable oil may be selected from the group consisting of olive oil, coconut oil, argan oil, green tea seed oil, grape seed oil, meadowfoam oil, soybean oil, sunflower seed oil, grape seed oil, sesame oil, perilla oil, flaxseed oil, Rapeseed oil, canola oil, and the like.

The vegetable oil is preferably added in an amount of 100 parts by weight or less based on 100 parts by weight of the microorganism culture solution.

The amount of the vegetable oil to be added can be appropriately adjusted according to the amount of the desired fermented vegetable oil. However, when the vegetable oil is added in an amount exceeding 100 parts by weight based on 100 parts by weight of the microorganism culture solution, the fermentation efficiency of the oil is lowered.

Preferably, the primary fermentation is carried out at 22 to 28 ° C under aerobic conditions for 70 to 120 hours. As the primary fermentation progresses, free fatty acid is produced. Since the efficiency of the secondary fermentation can be changed according to the content of the free fatty acid, it must be fermented for a time such that the free fatty acid can be maximally produced. However, since the amount of free fatty acid production reaches a maximum value depending on the kind of the vegetable oil, it is preferable to measure the increase amount of the free fatty acid with time and to perform the primary fermentation until the free fatty acid is not further increased.

The nitrate bacteria are preferably at least one selected from the group consisting of Nitrosomonas sp. DM-0521, Nitrobacter sp. DM-0514 and Nitrobacter sp. DM-2309 , And more preferably Nitrosomonas sp. DM-0521.

Titration medium of the jilsangyun (b) is (NH ₄₎ from _{2 SO 4, KH 2 PO 4} , MgSO 4, CaCl 2, FeSO 4, CuSO 4, NaH 2 PO 4, Na 2 CO 3, the group comprising NaCl It is preferable that at least one is selected.

Preferably, the pre-cultivation step (B) of culturing the nitrifying bacteria is carried out at 22 to 28 DEG C for 6 to 15 days under aerobic conditions.

In the secondary fermentation in which the culture solution after the primary fermentation has been mixed and inoculated with the culture broth of the nitric acid bacteria through the pre-cultivation step, nitrite (nitrite) or nitrate (nitrate) produced through nitrification of nitrifying bacteria It is important that active nitrogen species such as nitrate be added well to the double bond of the free fatty acid and be well-induced.

Therefore, it is preferable that the content of the fatty acid liberated by the fermentation of the vegetable oil in the primary fermentation is high and the content of the unsaturated fatty acid is high.

The secondary fermentation step is preferably carried out at 22 to 28 ° C under an aerobic condition for 96 to 120 hours, more preferably until the amount of nitrite (NO ₂ ^- ) is no longer increased.

As the secondary fermentation proceeds, the amount of nitrite (NO ₂ ^- ) increases, but reaches a maximum value at 96 hours, and there is no significant difference thereafter.

The fermented oil recovered in the step of recovering the vegetable oil containing the nitro fatty acid after the secondary fermentation is in the form of free fatty acid oxidized by nitrification and contained in the fermented vegetable oil, and there is no particular limitation on the recovery method.

The present invention also provides a fermented oil containing the nitro fatty acid produced by the above-mentioned method.

The present invention also provides a cosmetic composition comprising a fermented oil containing a nitro fatty acid produced by the above-described method.

The cosmetic composition may be prepared in any conventional formulation, for example, as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant- Oils, powder foundations, emulsion foundations, wax foundations and sprays, but are not limited thereto. More specifically, it can be manufactured in the form of softening agent, convergent lotion, nutritional lotion, nutritional cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.

Hereinafter, the present invention will be described in more detail with reference to examples. It will be apparent, however, to one skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the following claims. It is also within the scope of the appended claims.

≪ Examples 1 to 8 > Selection of an optimum oil for producing fermented oil by microorganisms

(Pseudozyma sp.) SY16 [KCTC 8950P] was inoculated in 1 L culture medium containing the different plant oils as shown in Table 1 at 25 ° C and 300 rpm in order to determine the oil suitable for fermented oil production by microorganisms , And cultured under aerobic conditions.

Fermentation oil medium composition of each vegetable oil by microorganism Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Example 8 vegetable oil Olive oil
100 g / L Argan oil
100 g / L Green tea seed oil
100 g / L Grape seed oil
100 g / L Meadow Foam Seeds
100 g / L Macadamia yu
100 g / L Camellia seed oil
100 g / L Jojoba seed oil
100 g / L Glucose 10 g / L 10 g / L 10 g / L 10 g / L 10 g / L 10 g / L 10 g / L 10 g / L yeast extract 3 g / L 3 g / L 3 g / L 3 g / L 3 g / L 3 g / L 3 g / L 3 g / L malt extract 3 g / L 3 g / L 3 g / L 3 g / L 3 g / L 3 g / L 3 g / L 3 g / L peptone 3 g / L 3 g / L 3 g / L 3 g / L 3 g / L 3 g / L 3 g / L 3 g / L soya bean meal 3 g / L 3 g / L 3 g / L 3 g / L 3 g / L 3 g / L 3 g / L 3 g / L (NH _{4) 2} SO ₄ 2 g / L 2 g / L 2 g / L 2 g / L 2 g / L 2 g / L 2 g / L 2 g / L KH ₂ PO ₄ 1 g / L 1 g / L 1 g / L 1 g / L 1 g / L 1 g / L 1 g / L 1 g / L MgSO ₄ 0.5 g / L 0.5 g / L 0.5 g / L 0.5 g / L 0.5 g / L 0.5 g / L 0.5 g / L 0.5 g / L CaCl ₂ 0.1 g / L 0.1 g / L 0.1 g / L 0.1 g / L 0.1 g / L 0.1 g / L 0.1 g / L 0.1 g / L NaCl 0.1 g / L 0.1 g / L 0.1 g / L 0.1 g / L 0.1 g / L 0.1 g / L 0.1 g / L 0.1 g / L Distilled water Up to 1L Up to 1L Up to 1L Up to 1L Up to 1L Up to 1L Up to 1L Up to 1L

<Experimental Examples 1 to 3> Microbial growth rate, lipase activity and acid value analysis of the fermented oil culture solution

1 ml of the fermented oil culture solution cultured for 24 hours in Examples 1 to 8 was sampled and the growth and lipase activity of the microorganism were measured by using a spectrophotometer. Then, acid value analysis was performed to determine the amount of free fatty acid.

1. Growth of microorganisms

When a culture of microorganisms passes light of a certain wavelength, the solution can absorb or scatter the light, and the amount of absorbed light is proportional to the concentration of the microorganism in the solution.

Using the above-described principle, the culture medium cultured under the conditions of Examples 1 to 8 was centrifuged to remove the oil layer, mixed strongly for 20 seconds, and diluted 10 times with distilled water to prepare a sample. The optical density (OD) of the microorganism was measured at 660 nm using a spectrophotometer. The results are shown in Table 2.

2. Lipase activity of microorganisms

Lipase, an enzyme that is a biological catalyst, is involved in various chemical catalytic reactions such as hydrolysis, alcoholysis, acidolysis, esterification, and aminolysis, Isomers that exhibit substrate specificity for a variety of substrates and which do not require a separate cofactor in the catalytic reaction process, maintain activity in an organic solvent as well as in an aqueous solution state, and are active only in one of the optical isomers It is produced in the microbial process and is involved in the production of free fatty acid which is important in the fermentation of vegetable oil.

In order to measure the lipase activity, the cultures cultured under the conditions of Examples 1 to 8 were centrifuged at 12,000 rpm for 3 minutes, and the supernatant was used as the coenzyme solution. The amount of p-nitrophenol (pNP) bound to the substrate by the enzyme was measured by a microplate analysis method in which the optical density (OD) was measured at 405 nm using a spectrophotometer to measure the absorbance, The results are shown in Table 2.

Lipase activity was defined as one unit in which 100 μl of coenzyme produced 1 μg of tyrosine per minute.

3. Acid value measurement of fermented oil

In order to compare the free fatty acid content of the cultured oil under the respective conditions of the above Examples 1 to 8, the acid value was measured.

The acid value represents the number of mg of potassium hydroxide necessary to neutralize 1 g of lipid, and the acid value is determined by the acid value test of the food. Specifically, 5 to 10 g of oil is precisely weighed and placed in a stoppered Erlenmeyer flask, and 100 mL of a neutral ethanol-ether mixture (1: 2) is added to dissolve it. The phenolphthalein reagent is used as an indicator until the pale red color persists for 30 seconds The product was titrated with 0.1 N ethanolic potassium hydroxide solution, and the acid value was determined by the following formula (1).

S: Amount of sample (g)

a: consumption of 0.1N ethanolic potassium hydroxide solution (mL)

f: Potency of 0.1N ethanolic potassium hydroxide solution

Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Example 8 Absorbance (OD at 660 nm) 1.892 1.375 1.652 1.305 1.702 1.475 1.213 1.008 Lipase activity (U / mL) 3.6 2.5 3.1 2.3 3.2 2.8 2.1 1.8 Acid value (ml / g) 36.45 24.51 31.21 22.67 32.08 23.75 15.22 10.10

Experimental Examples 1 to 3 showed the highest microbial growth and lipase activity in Example 1 using olive oil and the highest content of free fatty acid was found to be the optimum composition in the production of fermented oil by microorganisms Example 1 was selected.

&Lt; Preparation Examples 1 to 2 and Experimental Examples 4 to 5 > Preparation of fermentation oils by microorganisms

1. Pre-culture of Pseudozyma sp. SY16 [KCTC 8950P]

Glucos 10 g / L, yeast extract 3 g / L, malt extract 3 g / L, peptone 3 g / L, soya bean meal 3 g / L, (NH ₄ ) ₂ SO ₄ 2 g / L, KH ₂ PO ₄ (Pseudozyma sp.) SY16 [KCTC 8950P] was inoculated in 1 L of culture medium consisting of 1 g / L, MgSO ₄ 0.5 g / L, CaCl ₂ 0.1 g / L and NaCl 0.1 g / , And cultured under aerobic conditions.

The cultured microorganisms were sampled at 1, 3, 7, 12, 24, 48, 72, and 96 hours for each hour, and the absorbance was measured using a spectrophotometer to measure the growth rate and lipase activity of the microorganism Respectively. As a result of the measurement, it was confirmed that the time of the highest lipase activity was 48 hours.

As a result, it was confirmed that 48 hours was most suitable for pre-culture.

2. Pseudozyma sp. SY16 [KCTC 8950P] Primary fermentation by adding vegetable oil to fermentation broth

100 L of olive oil was added to 100 L of Pseudozyma sp. SY16 [KCTC 8950P] cultured medium, which had been pre-cultured, and then primary fermentation was carried out at 25 ° C and 500 rpm under aerobic conditions.

 0.3 to 0.5 L of the primary fermented oil was sampled at time intervals (3, 7, 12, 24, 48, 72, 96 hours) to collect only the oil portion. The degree of fermentation of the oil was measured by measuring the free fatty acid of the oil recovered at each time period. As a result of measurement of the amount of free fatty acid, it was confirmed that the amount of free fatty acid showed the maximum value at 96 hours after the oil addition, and thereafter did not increase.

As a result, it was confirmed that 96 hours was most suitable for primary fermentation.

PREPARATION EXAMPLE 3 Preparation of Fermentation Oils Containing Nitrogen Fatty Acids

In order to prepare a fermented oil containing a nitro fatty acid, nitrate acid bacteria pre-cultured in a culture solution after primary fermentation were mixed and fermented.

1. Nitrosomonas sp. DM-0521 pre-culture of Nitrosomonas sp.

Nitrosomonas sp. DM-0521 was inoculated into a culture solution having the composition shown in Table 3 below and cultured under aerobic conditions at 25 캜.

Nitrosomonas sp. DM-0521 Culture medium composition sample Volume The first solution (NH ₄ ) ₂ SO ₄ (for 50 mM NH ₄ ⁺ ) 4.95 g KH ₂ PO ₄ 0.62 g MgSO _4. 7H ₂ O 0.27 g CaCl ₂ . 2H ₂ O 0.04 g FeSO ₄ (30 mM in 50 mM EDTA at pH 7.0) 0.5 ml CuSO ₄ . 5H ₂ O 0.2 mg Sterile distilled water 1.2 L The second solution KH ₂ PO ₄ 8.2 g NaH ₂ PO ₄ 0.7 g Sterile distilled water 300ml The third solution Na ₂ CO ₃ anhydrous 0.6 g Sterile distilled water 12 ml

2. Co-fermentation by adding the Nitrosomonas sp. DM-0521 medium to the primary fermentation broth,

The primary fermented culture was inoculated with the Nitrosomonas sp. DM-0521 culture medium and subjected to mixed fermentation (secondary fermentation) at 25 ° C under aerobic conditions.

<Experimental Example 6> Measurement of microbial growth rate of the second fermentation oil (mixed fermentation oil)

1 ml of each of the mixed fermented oils of Preparation Example 3 was taken at time intervals (0, 6, 12, 24, 48, 72, 96, 120, and 150 hours), and the absorbance was measured using a spectrophotometer, And the results are shown in Table 4. &lt; tb &gt;&lt; TABLE &gt;

Absorption of mixed fermented oils by time Time (h) 0 6 12 24 48 72 96 120 150 Absorbance
(OD at 660 nm) 0.026 0.048 0.081 0.120 0.298 0.567 0.765 0.833 0.874

<Experimental Example 7> The nitrite of the second fermentation oil (mixed fermentation oil) by time (NO ₂ ^- ) Production amount

The Griess Reagent Reaction Assay was used for the determination of nitrite (NO ₂ ^- ) in the mixed fermented oil culture solution of Production Example 3. Nitrite (NO ₂ ^- ) accumulated in the culture solution develops a purplish color by reacting with N- (1-naphthyl) -ethylenediamidedihydrochloride (NEDHC) of Griess reagent and sulfanilamide. At this time, the maximum absorbance value at a wavelength of 540 nm was used, and the principle of indirectly determining the amount of NO produced by measuring this value was used.

The amount of nitrite (NO ₂ ^- ) produced in the culture was measured in the same manner as described above. The results are shown in Table 5.

Production of nitrite (NO ₂ ^- ) in the culture of mixed fermented oil Time (h) 0 6 12 24 48 72 96 120 150 Absorbance
(OD at 540 nm) 0.014 0.016 0.031 0.078 0.154 0.242 0.287 0.290 0.287

As a result of measurement of the amount of nitrite produced in the mixed fermentation oil culture medium, it was confirmed that nitrite was produced in proportion to the microbial growth of Experimental Example 6, and the maximum yield was obtained at about 96 hours after the culturing and there was no significant difference thereafter.

As a result, it was confirmed that 96 hours was most suitable for the secondary fermentation.

<Experimental Example 8> Anti-aging effect on human fibroblasts: Evaluation of cytotoxicity

The effect of nitrofatty acid on cell viability was confirmed by MTT assay using human dermal fibroblast.

Human dermal fibroblasts were cultured in FGM-2 kit medium at 37 ° C in a 5% CO ₂ incubator (Thermo Fisher scientific, USA).

The cells were cultured in FGM-2 kit medium at a concentration of 3 × 10 ⁴ cells / well in a 24-well plate. After 18 hours, Nitro fatty acid was treated by FGM-2 kit medium Lt; 0 &gt; C, 5% CO ₂ incubator for 48 hours. Cells were washed once with PBS (Phosphate Buffer Saline) solution, and 50 μl of 5 mg / ml MTT reagent (Sigma, USA) and 450 μl of FGM-2 kit medium were added for 3 to 4 hours The supernatant was removed from each well. The formazan crystals produced in each well were identified and DMSO (Dimethylsulfoxide) was added in the same amount as the medium. After shaking for 30 minutes in the shade state, the absorbance was measured at 570 nm.

The results of the measurement of the cytotoxicity of nitrofatty acid to fibroblasts are shown in FIG. 1. As a result, the nitro fatty acid showed a cell growth rate of 80% or more up to a concentration of 0.7 μg / ml.

<Experimental Example 9> Anti-aging effect on human fibroblasts: Evaluation of cytotoxicity by ultraviolet irradiation

The effect of nitrofatty acid on cell viability was confirmed by MTT assay using human dermal fibroblast.

Human dermal fibroblasts were cultured in FGM-2 kit medium at 37 ° C in a 5% CO ₂ incubator (Thermo Fisher scientific, USA).

The cells cultured in FGM-2 kit medium in ^{24 well plate 3 × 10 4 cells} / a well divided by the concentration of the counter after 18 hours UV B (ultraviolet B: UVB) 37 ℃ after a review of 25mJ / cm ^2, 5% CO ₂ incubator for 48 hours. At this time, transforming growth factor-β (TGF-β) was treated as a positive control, and cultured in a 5% CO ₂ incubator at 37 ° C. for 48 hours. Cells were washed once with PBS (Phosphate Buffer Saline) solution, and 50 μl of 5 mg / ml MTT reagent (Sigma, USA) and 450 μl of FGM-2 kit medium were added for 3 to 4 hours The supernatant was removed from each well. The formazan crystals produced in each well were identified and DMSO (Dimethylsulfoxide) was added in the same amount as the medium. After shaking for 30 minutes in the shade state, the absorbance was measured at 570 nm.

The result of the cytotoxicity test by ultraviolet ray based on the result of the measurement of the cytotoxicity of nitro fatty acid to fibroblasts is shown in FIG. 2. As a result, it was confirmed that cytotoxicity by ultraviolet light was reduced by 60% when irradiated with ultraviolet rays. On the other hand, it was confirmed that nitro-fatty acid and TGF-β did not significantly affect cell growth rate.

<Experimental Example 10> Anti-aging effect on human fibroblasts: Measurement of collagen (COL1A1) aggregation performance by ultraviolet irradiation

Human dermal fibroblasts were cultured in FGM-2 kit medium at 37 ° C in a 5% CO ₂ incubator (Thermo Fisher scientific, USA).

Cells were cultured in FGM-2 kit medium at a concentration of 3 × 10 ⁵ cells / well in a 6-well plate. After 18 hours, nitro-fatty acids were transfected with 0.05, 0.1 and 0.5 μg / The transforming growth factor-β (TGF-β) was treated at 5 ng / ml and incubated at 37 ° C in a 5% CO ₂ incubator for 6 hours. After incubation for 6 hours, each well was washed once with phosphate buffered saline (PBS), 1 mL of PBS was added to each well, and ultraviolet B (UVB) 25 mJ / cm ² was irradiated. To the FGM-2 kit medium, nitro-fatty acids were added at concentrations of 0.05, 0.1, 0.5 ㎍ / ml and TGF-β 5 ng / ml. Ultraviolet B was irradiated to remove PBS from each well, and the prepared nitro-fatty acid mixed medium and TGF-β mixed medium were added and cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours.

After 24 hours, separate the RNA from the cultured cells, followed by synthesizing cDNA using a PCR (Applied Biosystems ^®. Synthesized cDNA using collagen gene (COL1A1) TaqMan to verify the expression ^® assay (Life technologies) Real time PCR (Applied Biosystems ^® collagen gene (COL1A1) amount was measured.

The effect of nitric acid on the synthesis of collagen-associated gene (COL1A1) by UV irradiation is shown in Fig. As a result, it was confirmed that the nitro fatty acid increased the expression of COL1A1 gene by about 25%, and increased the expression of collagen gene (COL1A1) to that of TGF-β used as a positive control.

<Experimental Example 11> Anti-aging effect on human fibroblasts: Measurement of inhibitory activity of collagenase (MMP-1) by ultraviolet irradiation

Human dermal fibroblasts were cultured in FGM-2 kit medium at 37 ° C in a 5% CO ₂ incubator (Thermo Fisher scientific, USA).

Cells were cultured in FGM-2 kit medium at a concentration of 3 × 10 ⁵ cells / well in a 6-well plate. After 18 hours, nitro-fatty acids were transfected with 0.05, 0.1 and 0.5 μg / The transforming growth factor-β (TGF-β) was treated at 5 ng / ml and incubated at 37 ° C in a 5% CO ₂ incubator for 6 hours. After incubation for 6 hours, each well was washed once with phosphate buffered saline (PBS), 1 mL of PBS was added to each well, and ultraviolet B (UVB) 25 mJ / cm ² was irradiated. To the FGM-2 kit medium, nitro-fatty acids were added at concentrations of 0.05, 0.1, 0.5 ㎍ / ml and TGF-β 5 ng / ml. Ultraviolet B was irradiated to remove PBS from each well, and the prepared nitro-fatty acid mixed medium and TGF-β mixed medium were added and cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours.

After 24 hours, separate the RNA from the cultured cells, followed by synthesizing cDNA using a PCR (Applied Biosystems ^®. A synthetic cDNA was TaqMan ^® to verify the collagen-decomposing enzyme gene (MMP-1) expression assay (Life technologies ) Was used to measure changes in the amount of real-time PCR (Applied Biosystems ^® collagenase gene (MMP-1)).

The effect of nitric acid on the synthesis of collagenase gene (MMP-1) by ultraviolet irradiation is shown in FIG. As a result, it was confirmed that the nitro fatty acid inhibited the expression of the MMP-1 gene by about 21% as compared with the negative control group irradiated only with ultraviolet light.

In the Experimental Examples 8 to 11, the nitro fatty acid did not inhibit cell growth, increased the expression of the collagen gene (COL1A1), and the collagenase gene (MMP-1) in human dermal fibroblast, 1) expression was not cytotoxic, and it was confirmed that it has anti - aging effect by helping collagen synthesis.

<Experimental Example 12> Anti-aging effect on keratinocytes: evaluation of cytotoxicity

The effect of nitric acid on cell survival rate was confirmed by MTT assay using human epidermal keratinocyte.

Human epidermal keratinocytes were cultured in EpiLife ^® Medium containing HKGS (Human Keratinocyte Growth Supplement) at 37 ° C in a 5% CO ₂ incubator (Thermo Fisher scientific, USA).

The cultured cells were seeded at a concentration of 3 × 10 ⁴ cells / well in a 24-well plate. After 18 hours, the nitric acid was treated at different concentrations and cultured at 37 ° C. in a 5% CO ₂ incubator for 48 hours . After 48 hours of incubation, cells were washed once with phosphate buffered saline (PBS), and 50 μl of 5 mg / ml MTT reagent (Sigma, USA) and 450 μl of culture medium were added and incubated for 3-4 hours. Lt; / RTI &gt; The formazan crystals produced in each well were identified and DMSO (Dimethylsulfoxide) was added in the same amount as the medium. After shaking for 30 minutes in the shade state, the absorbance was measured at 570 nm.

The results of the measurement of the cytotoxicity of nitrofatty acid to fibroblasts are shown in FIG. 5. As a result, the nitro fatty acid showed a cell growth rate of 80% or more up to a concentration of 0.5 μg / ml.

<Experimental Example 13> Anti-aging effect on keratinocytes: evaluation of cytotoxicity by ultraviolet irradiation

The effect of nitric acid on cell survival rate was confirmed by MTT assay using human epidermal keratinocyte.

Human epidermal keratinocytes were cultured in EpiLife ^® Medium containing HKGS (Human Keratinocyte Growth Supplement) at 37 ° C in a 5% CO ₂ incubator (Thermo Fisher scientific, USA).

Cells cultured in the culture medium were dispensed at a concentration of 3 × 10 ⁴ cells / well in a 24-well plate and irradiated with 25 mJ / cm ² of ultraviolet B (UVB) 18 hours later. And then cultured in a 5% CO ₂ incubator at 37 ° C for 48 hours. At this time, transforming growth factor-β (TGF-β) was treated as a positive control, and cultured in a 5% CO ₂ incubator at 37 ° C. for 48 hours. After 48 hours of incubation, cells were washed once with phosphate buffered saline (PBS), and 50 μl of 5 mg / ml MTT reagent (Sigma, USA) and 450 μl of culture medium were added and incubated for 3-4 hours. Of the supernatant was removed. The formazan crystals produced in each well were identified and DMSO (Dimethylsulfoxide) was added in the same amount as the medium. After shaking for 30 minutes in the shade state, the absorbance was measured at 570 nm.

The cytotoxicity test result of ultraviolet ray based on the result of the measurement of the cytotoxicity of nitric acid on keratinocytes is shown in FIG. 6. As a result, it was confirmed that cytotoxicity by ultraviolet light was reduced by 70% . On the other hand, it was confirmed that nitro-fatty acid and TGF-β did not significantly affect cell growth rate.

<Experimental Example 14> Anti-aging effect on keratinocytes: Measurement of inhibitory activity of collagenase (MMP-1) by ultraviolet irradiation

Human epidermal keratinocytes were cultured in EpiLife ^® Medium containing HKGS (Human Keratinocyte Growth Supplement) at 37 ° C in a 5% CO ₂ incubator (Thermo Fisher scientific, USA).

The cultured keratinocytes were seeded at a density of 3 × 10 ⁵ cells / well in a 6-well plate. After 18 hours, nitro-fatty acids were added at a concentration of 0.05, 0.1, 0.5 μg / ml and a positive control TGF-β) was treated at 5 ng / ml and incubated in a 5% CO ₂ incubator at 37 ° C for 6 hours. After incubation for 6 hours, each well was washed once with phosphate buffered saline (PBS), 1 mL of PBS was added to each well, and ultraviolet B (UVB) 25 mJ / cm ² was irradiated. To the FGM-2 kit medium, nitro-fatty acids were added at concentrations of 0.05, 0.1, 0.5 ㎍ / ml and TGF-β 5 ng / ml. Ultraviolet B was irradiated to remove PBS from each well, and the prepared nitro-fatty acid mixed medium and TGF-β mixed medium were added and cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours.

After 24 hours, RNA was isolated from the cultured cells and cDNA was synthesized using PCR (Applied Biosystems). Synthesized cDNA was determined the collagen-decomposing enzyme gene (MMP-1) to confirm the expression ^{TaqMan ® assay (Life technologies) Real} -time PCR (Applied Biosystems) using the collagen-decomposing enzyme gene (MMP-1) Amount of change .

The effect of nitric acid on the synthesis of collagenase gene (MMP-1) by UV irradiation is shown in FIG. As a result, it was confirmed that the nitro fatty acid inhibited the expression of the MMP-1 gene by about 25% as compared with the negative control group irradiated only with ultraviolet light.

In Experimental Examples 12 to 14, it was found that nitro fatty acid does not inhibit cell growth on human epidermal keratinocytes and inhibits the expression of collagenase gene (MMP-1) It has no toxicity, and it has been confirmed that it has anti-aging effect by helping collagen synthesis.

Claims (16)

(A) a step of pre-culturing the microorganism in a suitable medium (a);
A first fermentation step of adding a vegetable oil to the microorganism culture solution to ferment;
(B) a pre-culturing step of nitric acid bacteria for culturing nitrate bacteria in an appropriate medium (b);
A secondary fermentation step of adding the culture broth of the nitrifying bacteria to the primary fermentation broth to mix and ferment; And
Recovering the vegetable oil comprising the nitro fatty acid after the secondary fermentation,
Wherein the microorganism is Pseudozyma sp.
Wherein said primary fermentation is fermentation at 22 to 28 DEG C under aerobic conditions for 70 to 120 hours,
Wherein said secondary fermentation step is carried out at 22 to 28 DEG C under aerobic conditions for 96 to 120 hours.
delete The method according to claim 1,
Wherein the microorganism is Pseudozyma sp. SY16 (KCTC 8950P).
The method according to claim 1,
The appropriate medium (a) of the microorganism is glucose, yeast extract, malt extract, peptone, soya bean meal, (NH ₄ ) ₂ SO ₄ , KH ₂ PO ₄ , MgSO ₄ , CaCl ₂ , and NaCl. The method of producing a fermented oil containing a nitro fatty acid according to claim 1,
The method according to claim 1,
Wherein the pre-cultivation step (A) of the microorganism is cultivated at 22 to 28 DEG C under aerobic conditions for 16 to 72 hours.
The method according to claim 1,
Wherein the acid value of the vegetable oil is 0.1 to 2. 12. The fermented oil according to claim 1,
The method according to claim 1,
The vegetable oil may be selected from the group consisting of olive oil, coconut oil, argan oil, green tea seed oil, grape seed oil, meadowfoam oil, soybean oil, sunflower seed oil, grape seed oil, sesame oil, perilla oil, flaxseed oil, Rapeseed oil, rapeseed oil, rapeseed oil, canola oil, and the like.
The method according to claim 1,
Wherein the vegetable oil is 100 parts by weight or less based on 100 parts by weight of the microorganism culture solution.
delete The method according to claim 1,
Characterized in that said nitric acid bacteria are at least one selected from the group consisting of Nitrosomonas sp. DM-0521, Nitrobacter sp. DM-0514 and Nitrobacter sp. DM-2309 By weight based on the total weight of the fermentation oil.
The method according to claim 1,
Titration medium of the jilsangyun (b) is (NH ₄₎ from _{2 SO 4, KH 2 PO 4} , MgSO 4, CaCl 2, FeSO 4, CuSO 4, NaH 2 PO 4, Na 2 CO 3, the group comprising NaCl Wherein the nitric acid is at least one selected from the group consisting of nitric acid and nitric acid.
The method according to claim 1,
Wherein the pre-culturing step (B) of culturing the nitric acid bacteria is carried out at 22 to 28 DEG C under aerobic conditions for 6 to 15 days.
delete delete delete delete

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