KR102588606B1 - Method for the production of cosmetic composition and γ-PGA by mixed culture of Saccharomyces cerevisiae J2K-23(KCTC 13695BP) and Bacillus subtilis J2K-51(KCTC 14053BP) - Google Patents

Abstract

The present invention is a mixed culture of Saccharomyces cerevisiae J2K-23 (KCTC 13695BP) isolated from green tea flowers and Bacillus subtilis J2K-51 (KCTC 14053BP) isolated from yeast, showing excellent moisturizing, inflammation alleviating, erythema improvement and skin soothing effects. A cosmetic composition can be manufactured. In addition, the present invention purifies γ-PGA with significantly reduced odor through mixed culture of Saccharomyces cerevisiae J2K-23 (KCTC 13695BP) isolated from green tea flowers and Bacillus subtilis J2K-51 (KCTC 14053BP) isolated from yeast. It can be produced with high yield.

Description

Preparation of cosmetic compositions and production of poly-gamma-glutamic acid through mixed culture of Saccharomyces cerevisiae J2K-23 (KCTC 13695BP) isolated from green tea flowers and Bacillus subtilis J2K-51 (KCTC 14053BP) isolated from yeast {Method for the production of cosmetic composition and γ-PGA by mixed culture of Saccharomyces cerevisiae J2K-23(KCTC 13695BP) and Bacillus subtilis J2K-51(KCTC 14053BP)}

The present invention relates to the preparation of a cosmetic composition through mixed culture of Saccharomyces cerevisiae J2K-23 (KCTC 13695BP) isolated from green tea flowers and Bacillus subtilis J2K-51 (KCTC 14053BP) isolated from yeast and polygamma Regarding the production of glutamic acid, it is possible to manufacture cosmetic compositions that exhibit excellent moisturizing, inflammation relief, erythema improvement, and skin soothing effects, and to produce γ-PGA that can be used in cosmetics in high yield with significantly reduced odor generation. there is.

Recently, due to changes in the environment or lifestyle, environmental pollution due to various stresses and pollutants, frequent washing of the face due to makeup habits, and natural skin deterioration due to aging, the skin becomes dry, the surface becomes rough, and the skin loses its luster and becomes dull. As phenomena such as looking hazy occur frequently, the importance of skin health issues is increasing.

As a way to maintain skin health from skin stress, much effort has been put into developing moisturizing materials to prevent drying of skin moisture and improve moisture retention in the stratum corneum.

When dry skin is formed, the barrier function of the skin surface is lost and external irritants or allergens not only easily invade the skin, but also cause a rejection reaction of the skin against these invaders. The rejection reaction of the skin is caused by keratinocytes called keratinocytes, which make up the majority of the surface layer components, activating a small number of cells such as Langerhans and melanocytes, and cytokines produced and released by these cells. It appears as an inflammatory phenomenon caused by cytokines.

The cytokine acts to recruit inflammatory cells, and in particular stimulates the skin's constituent cells, causing an inflammatory reaction centered on the upper layer of the skin and forming an eczema reaction, which is called non-allergic skin disease.

Therefore, substances with strong skin soothing and moisturizing effects have the potential to extend to anti-inflammatory effects, and conversely, substances with anti-inflammatory effects can be predicted to be effective in skin soothing and skin moisturizing, but in reality, such natural materials There was a problem in that it had to be a very difficult process to find.

Meanwhile, poly-gamma-glutamic acid (γ-PGA) is a biopolymer derived from Bacillus subtilis , which is present in Cheonggukjang. Poly-gamma-glutamic acid, which has been traditionally consumed through Cheonggukjang, is a substance that has been proven to be safe, water-soluble, non-toxic, and biodegradable. It promotes the secretion of pro-inflammatory cytokines and promotes antigen presenting cells through TLR4 (toll-like receptor 4). It is known to induce innate immune responses, such as activation of APC). In addition, it is known to promote Th1 immune response and induce cellular immune responses such as cytotoxic T lymphocyte (CTL) activity. Poly-gamma-glutamic acid (γ-PGA), which has such excellent functions, is widely used in cosmetics for the purpose of moisturizing the skin and improving elasticity.

Republic of Korea Patent Registration No. 10-1824179 (2018.01.25.) Republic of Korea Patent Registration No. 10-2293081 (2021.08.18.) Republic of Korea Patent Registration No. 10-0517114 (2005.09.16)

The purpose of the present invention is to produce a cosmetic composition using Saccharomyces cerevisiae J2K-23 (KCTC 13695BP) isolated from green tea flowers and Bacillus subtilis J2K-51 (KCTC 14053BP) isolated from yeast and a method for producing γ-PGA that can be used as a cosmetic raw material. We would like to develop and provide.

The present invention is a mixture of Saccharomyces cerevisiae J2K-23 (KCTC 13695BP) strain isolated from green tea flowers and Bacillus subtilis J2K-51 (KCTC 14053BP) strain isolated from yeast. Provided is a cosmetic composition for alleviating skin irritation, characterized in that it contains a culture solution obtained by culturing. At this time, the cosmetic composition may preferably have any one effect selected from skin moisturizing effect, inflammation alleviating effect, erythema improving effect, and skin soothing effect.

Meanwhile, the present invention relates to the Saccharomyces cerevisiae J2K-23 (KCTC 13695BP) strain isolated from green tea flowers and the Bacillus subtilis J2K-51 (KCTC 14053BP) strain isolated from yeast. Provides a method for producing γ-PGA, characterized by mixed culture. At this time, the method for producing γ-PGA may preferably have reduced culture odor.

In addition, the present invention relates to the Saccharomyces cerevisiae J2K-23 (KCTC 13695BP) strain isolated from green tea flowers and the Bacillus subtilis J2K-51 (KCTC 14053BP) strain isolated from yeast. Provides a method for producing a culture medium containing γ-PGA, characterized in that mixed culture. At this time, the method for producing the γ-PGA-containing culture medium may preferably have reduced culture odor.

The present invention is a mixed culture of Saccharomyces cerevisiae J2K-23 (KCTC 13695BP) isolated from green tea flowers and Bacillus subtilis J2K-51 (KCTC 14053BP) isolated from yeast, showing excellent moisturizing, inflammation alleviating, erythema improvement and skin soothing effects. A cosmetic composition can be manufactured.

In addition, the present invention was able to produce γ-PGA in high yield through mixed culture of Saccharomyces cerevisiae J2K-23 (KCTC 13695BP) isolated from green tea flowers and Bacillus subtilis J2K-51 (KCTC 14053BP) isolated from yeast. The odor is reduced and the purification process can be simplified. γ-PGA produced in this way can be used in cosmetics for the purpose of moisturizing the skin and improving elasticity.

Figure 1 shows the measured effect of Aquaporin-3 expression in the culture prepared through Preparation Example 3 of the present invention.
Figure 2 shows the measured effect of inhibiting COX-2 production in the culture prepared through Preparation Example 3 of the present invention.
Figure 3 shows an evaluation of the skin soothing (relieving redness, alleviating irritation) effect caused by external stimulation (SLS) of the cosmetic composition prepared through Example 1 of the present invention.
Figure 4 is a report confirming the identification results of Saccharomyces cerevisiae J2K-23.
Figure 5 is a report confirming the identification results of Bacillus subtilis J2K-51.

The present invention provides Bacillus subtilis J2K-51 (KCTC 14053BP) strain isolated from yeast. Additionally, Saccharomyces cerevisiae J2K-23 (KCTC 13695BP) strain isolated from green tea flowers is provided. When cultivating the strain isolated in the present invention, a culture medium with skin irritation alleviating effect can be obtained.

Additionally, the present invention provides a cosmetic composition containing a culture medium of the Bacillus subtilis J2K-51 (KCTC 14053BP) strain. Additionally, a cosmetic composition containing a culture medium of Saccharomyces cerevisiae J2K-23 (KCTC 13695BP) strain isolated from green tea flowers is provided. The cosmetic composition of the present invention has an irritation alleviating effect on skin irritation. In addition, one or more effects selected from skin moisturizing effect, inflammation alleviating effect, erythema improving effect, and skin soothing effect may be further exerted.

In the present invention , Bacillus subtilis J2K-51 (KCTC 14053BP) strain isolated from yeast and Saccharomyces cerevisiae J2K-23 (KCTC 13695BP) strain isolated from green tea flowers. It is recommended to use a culture medium obtained by mixed culture. At this time, it is preferable that the culture medium obtained through the mixed culture is mixed cultured by simultaneously culturing the two strains.

However, the Saccharomyces cerevisiae J2K-23 (KCTC 13695BP) strain isolated from green tea flowers was inoculated into the medium and cultured for the first time, and then the medium was sterilized, and Bacillus subtilis isolated from yeast ( Mixed culture can also be performed by additionally inoculating Bacillus subtilis J2K-51 (KCTC 14053BP) strain and performing secondary culture. In this way, when primary and secondary cultures are performed separately from each other within one batch, problems such as inhibition of growth of some bacteria due to mixed culture of the above strains can be resolved, and one batch is used. It has the advantage of lowering production costs.

The cosmetic composition according to the present invention exhibits the effect of relieving skin irritation, and as similar effects, it can be interpreted as showing the effect of alleviating skin inflammation, improving skin erythema, and soothing the skin.

In the cosmetic composition of the present invention, the mixed culture of Saccharomyces cerevisiae J2K-23 (KCTC 13695BP) isolated from green tea flowers and Bacillus subtilis J2K-51 (KCTC 14053BP) isolated from yeast is preferably used as a cosmetic at a ratio of 0.0005 to 30% by weight. may be added to the composition.

Meanwhile, the cosmetic composition of the present invention is not limited to a specific one, and examples include solutions, external ointments, creams, foams, nourishing lotions, softening lotions, packs, softening water, emulsions, makeup bases, essences, liquid cleansers, bath additives, Can be a formulation selected from the group consisting of sunscreen creams, sun oils, suspensions, emulsions, pastes, gels, lotions, powders, surfactant-containing cleansing oils, powder foundations, emulsion foundations, wax foundations, patches and sprays. there is. In addition, to achieve each formulation, additives commonly used in the relevant field may be further included.

In addition, the cosmetic composition of the present invention may additionally contain one or more cosmetically acceptable carriers that are blended with general skin cosmetics, and common ingredients include, for example, oil, water, surfactant, moisturizer, lower alcohol, Thickeners, chelating agents, pigments, preservatives, fragrances, etc. can be appropriately mixed, but are not limited thereto, and the cosmetically acceptable carrier included in the cosmetic composition of the present invention can be applied in various ways depending on the formulation.

Meanwhile, the present invention relates to the Saccharomyces cerevisiae J2K-23 (KCTC 13695BP) strain isolated from green tea flowers and the Bacillus subtilis J2K-51 (KCTC 14053BP) strain isolated from yeast. Provides a method for producing γ-PGA, characterized by mixed culture.

In addition, the present invention relates to the Saccharomyces cerevisiae J2K-23 (KCTC 13695BP) strain isolated from green tea flowers and the Bacillus subtilis J2K-51 (KCTC 14053BP) strain isolated from yeast. Provides a method for producing a culture medium containing γ-PGA, characterized in that mixed culture.

The method for producing γ-PGA and the method for producing a culture medium containing γ-PGA of the present invention uses Malt Extract (M.E) instead of Yeast Extract (Y.E) as a medium component and mixes J2K-23 strain and J2K-51 strain. Through culture, γ-PGA could be produced in high yield, and the purification process could be simplified by reducing the unique odor. In order to remove odor, an additional process of recovering and re-dissolving the precipitate after solvent precipitation is generally required, but in the present invention, this additional process is unnecessary, making it economical and environmentally friendly.

In the method for producing γ-PGA and the method for producing a γ-PGA-containing culture medium of the present invention, the culture medium preferably contains 10 to 30 g/L of glucose, 1 to 9 g/L of Malt Extract, and 25 to 75 g/L of sodium glutamate. It is good to include, more preferably, 0.1 to 0.9 g/L of potassium phosphate and 0.05 to 0.15 g/L of magnesium sulfate heptahydrate.

Hereinafter, the contents of the present invention will be described in more detail through the following production examples, examples, and experimental examples. However, the scope of the present invention is not limited to the following manufacturing examples, examples, and experimental examples, and includes modifications of the technical idea equivalent thereto.

<Production Example 1> From green tea flowers Saccharomyces cerevisiae Isolation of J2K-23 (KCTC 13695BP)

Green tea flowers collected from green tea fields in the Oltis area of Jeju Island were used as strain isolation samples. The sample was suspended and diluted in sterile saline solution, and 0.1 mL of it was taken and spread on Difco Yeast Extract Peptone Dextrose (YPD) agar medium. Afterwards, the plate was cultured in a constant temperature incubator at 30°C for 3 days. Each resulting colony was inoculated onto a YPD plate, cultured at 30°C for 3 days, and colonies showing off-white colonies were harvested to inoculate Saccharomyces cerevisiae from green tea flowers in the Oltis region of Jeju. ) J2K-23 was isolated.

18S rRNA nucleic acid sequence analysis was performed on the isolated strain, and as a result, the same nucleic acid sequence as described in SEQ ID NO: 1 was confirmed. Through this, the isolated strain could be identified as Saccharomyces cerevisiae .

Figure 4 is a report confirming the identification results of Saccharomyces cerevisiae J2K-23.

<Production Example 2> From yeast Bacillus subtilis Isolation of J2K-51 (KCTC 14053BP)

Nuruk from the Jeju Island region was used as a strain isolation sample. The sample was suspended and diluted in sterile saline solution, and 0.1 mL of it was taken and spread on Millipore Tryptic Soy Agar (TSA) medium. Afterwards, the plate was cultured in an incubator at 35°C for 1 day. Each resulting colony was inoculated onto a TSA plate, and Bacillus subtilis J2K-51 was isolated from Jeju Island yeast by culturing at 35°C for 2 days and collecting off-white colonies.

16S rRNA nucleic acid sequence analysis was performed on the isolated strain, and as a result, the same nucleic acid sequence as described in SEQ ID NO: 2 was confirmed. Through this, the isolated strain could be identified as Bacillus subtilis .

Figure 5 is a report confirming the identification results of Bacillus subtilis J2K-51.

<Production Example 3> Saccharomyces cerevisiae J2K-23 (KCTC 13695BP) and Bacillus subtilis Preparation of mixed cultures with J2K-51 (KCTC 14053BP)

The medium contains 2% (w/v) glucose, 0.5% (w/v) malt extract, 5.0% (w/v) sodium glutamate, and potassium phosphate (K ₂ HPO ₄ ). Use a medium containing 0.05% (w/v) and 0.01% (w/v) of magnesium sulfate (MgSO ₄ ). After dissolving the above medium components, sterilize at 121°C for 20 minutes, cool, and cool. The strains isolated in Preparation Examples 1 and 2 were sequentially and simultaneously inoculated into the medium and cultured for 50 hours at a temperature of 33°C and a rotation speed of 120 rpm to inoculate Saccharomyces cerevisiae J2K-23 (KCTC 13695BP) and Bacillus subtilis J2K-51 ( A mixed culture was prepared with KCTC 14053BP).

<Example 1> Preparation of cosmetic composition using strains of the present invention

A cosmetic composition (Metabiome HK-503) was prepared by containing 3% by weight of a mixed culture of Saccharomyces cerevisiae J2K-23 (KCTC 13695BP) and Bacillus subtilis J2K-51 (KCTC 14053BP) prepared in Preparation Example 3 in purified water. was manufactured.

<Experimental Example 1> Confirmation of cell viability

Cell viability was confirmed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphinyltetrazolium bromide) assay. Cells of 5×10 ⁴ cells/ml (HaCaT cells) were dispensed into a 96-well plate, samples were processed, and cultured in a CO ₂ incubator at 37°C for an incubation time corresponding to each immunoexperiment condition. Afterwards, 10 ㎕ MTT solution (stock concentration: 5 mg/ml) was added and further reaction was induced for 3 hours. To terminate the reaction and dissolve the formazan crystals, 100㎕ MTT stopping solution (10% Sodium dodecyl sulfate in 0.01M HCl) was additionally added to each well. Cell viability was calculated through the OD value obtained by measuring the absorbance at 570 nm of the amount of MTT reduced to formazan.

As a result of checking the survival rate of HaCaT cells, which are human keratinocytes, and mouse-derived macrophages (RAW264.7) of the culture prepared through Preparation Example 3, it was confirmed that cytotoxicity did not appear up to a concentration of 5%. .

<Experimental Example 2> Confirmation of increased expression of Aquaporin-3

In this experiment, we attempted to confirm the expression of Aquaporin-3, a skin moisturizing factor, in the culture prepared through Preparation Example 3. Using HaCaT cells, which are human keratinocytes, ³ The cells were pre-cultured for 24 hours in a 60 mm cell culture dish at a concentration of cells/ml. After replacing with DMEM medium that does not contain FBS and treating the samples for 24 hours, cells were collected, lysis buffer was added to lyse the cells to obtain Western samples, and the protein concentration of each sample was measured using BSA as the standard. Western blotting assay was performed with each sample amount corresponding to protein concentration based on the value obtained in this way. First, SDS-PAGE was performed, proteins were transferred to a PVDF membrane, and the membrane was blocked using 5% skim milk. After primary treatment with a target protein antibody solution and washing, secondary treatment with an antibody solution and washing again. Color was developed using ECL solution and analyzed using Chemi-doc equipment.

As shown in Figure 1, it was found that Aquaporin-3 expression was increased in the culture prepared through Preparation Example 3 of the present invention in proportion to the concentration.

<Experimental Example 3> Confirmation of COX-2 production inhibition effect

In this experiment, we sought to confirm the effect of inhibiting the production of COX-2, an inflammation-related factor, in the culture prepared through Preparation Example 3 above. To inhibit COX-2 production, mouse-derived macrophages (RAW264.7) were dispensed in 3 ml each at a concentration of ^4.0 After pretreatment of the sample for 2 hours, lipopolysaccharide (LPS), known as an inflammation-inducing substance, was treated to a final concentration of 1 μg/ml and cultured for 4 hours. After removing the medium, the cells were removed from the culture vessel and homogenized using a protein elution solution (CelLyticTM-MT Tissue Lysis Reagent, Sigma, USA) containing a protease inhibitor cocktail (Roche, USA). The extract was centrifuged at 14000 rpm for 20 minutes, and then the supernatant and insoluble aggregates were separated. The protein concentration of the separated supernatant was measured using a Bio-Rad protein assay kit (Bio-Rad, USA). Additionally, the supernatant was mixed 1:4 with 5×SDS (0.156M Tris-HCl, pH 6.8, 2.5% SDS, 37.5% glycerol, 37.5 mM DTT) and boiled at 100°C for 10 minutes. 40 ug of protein from the boiled sample was separated on an SDS 4-12% SDS-PAGE gel and transferred to a PVDF membrane. The protein-free portion of the membrane to which the protein has been transferred is blocked with skim milk powder, followed by sequential binding of primary and secondary antibodies, color development using ECL solution, and analysis using Chemi-doc equipment. did.

As shown in Figure 2, it was confirmed that the culture prepared through Preparation Example 3 of the present invention inhibited the production of COX-2.

<Experimental Example 4> Clinical test for skin soothing effect

We attempted to conduct a clinical test on the skin soothing effect of the cosmetic composition prepared in Example 1 above against external stimulation. The clinical test was performed by applying an appropriate amount of the cosmetic composition prepared in Example 1 to the forearm in the morning and evening (twice a day) after washing it, and applying it twice a day for 3 days. The test subjects were selected according to the exclusion criteria one day before the start of the test. They were asked to clean the test area, then their skin was stabilized for 30 minutes, and photography and equipment evaluation (Spectrophotometer) were performed. After taking pictures and evaluating the device, a chamber impregnated with 1.0% SLS (Sodium Lauryl Sulfate) was attached to the test area for 24 hours.

On the day of the test, the chamber attached to the test area was removed and the condition of the test area was checked. If the erythema induction was successful, photography and device evaluation were performed. If it was determined that erythema induction was insufficient, a new chamber was additionally attached, and the test area was later rechecked and photography and device evaluation were performed. Test subjects who completed the measurement We explained to them how to use the test product and distributed the test product. 24 to 72 hours after the test, safety evaluation, photography, and device evaluation were conducted on test subjects who used the distributed test samples according to the usage method to evaluate efficacy.

For the evaluation, the test subject rested for 30 minutes in a waiting room with constant temperature and humidity (22±2℃, RH40~60%) to adapt the skin surface temperature and humidity to the environment of the measurement space. For objective measurement, the device was used. For the evaluation, one researcher measured the same area for each measurement. The test area was photographed with Digital Cemera (SONY ILCE-5100, THAILAND). For standardization, the photography was conducted by the same researcher, and the photography conditions (setting values and camera height) were fixed. Instrumental measurements (dimensionless units) were applied using a colorimeter (Spectrophotometer CM-2600D, Minolta, Japan), and the aperture was 8 mm. *a value (Redness), a measurement variable, means that the skin color becomes redder as the measurement value increases. Therefore, in this test, the lower the *a value measurement value, the more effective the test product is in soothing (relieving redness). .

Skin irritation evaluation includes adverse reactions to test products such as erythema, edema, scaling, itching, stinging, burning, stiffness, and tingling. ) or other adverse reactions were closely observed, and when adverse skin reactions occurred, they were graded according to severity and test results were written. If you are no longer able to participate in the test even if it is not the day of your visit, you are asked to write a "Consent to Give Up Test Participation" with your signature attached.

Adverse reactions and classification table are shown in Table 1 below.

As shown in Figure 3, as a result of evaluating the skin soothing (redness alleviation) effect caused by external stimulation (SLS) of the cosmetic composition prepared through Example 1 of the present invention, 11.3% was relieved after 72 hours (3.2% in the control group). % relief) was observed.

Therefore, the cosmetic composition according to the present invention contains cultures of Saccharomyces cerevisiae J2K-23 (KCTC 13695BP) isolated from green tea flowers and Bacillus subtilis J2K-51 (KCTC 14053BP) isolated from yeast, and provides excellent moisturizing, inflammation relief, and erythema. It was evaluated as showing improvement and skin soothing effect.

<Example 2> Production of poly gamma glutamic acid (γ-PGA) using strains of the present invention

In this experiment, we attempted to produce γ-PGA using Bacillus subtilis and yeast, which are strains of the present invention. However, when cultivating Bacillus subtilis, that is, Bacillus subtilis, there is a problem in that a unique odor is generated. In this experiment, we attempted to develop a method that can produce γ-PGA in high yield without producing bad odor.

For the experiment, Malt Extract (M.E) was added to the medium instead of Yeast Extract (Y.E), and while mixed culture of the strains of the present invention was performed, γ-PGA production and odor reduction were observed. For mixed culture, a medium composed of 10 g/L of glucose, 10 g/L of nitrogen source (Y.E or M.E), 10 g/L of sodium glutamate, 0.5 g/L of potassium phosphate, and 0.1 g/L of magnesium sulfate heptahydrate was used. It was performed according to the method described in Preparation Example 3.

division strain Yeast Extract Malt Extract Unusual taste ¹⁾ γ-PGA production amount (%) Control group 1 J2K-23 O X One - Control group 2 J2K-23 X O One - Control group 3 J2K-51 O X 4 3.2 Control group 4 J2K-51 X O 3 2.4 Experimental group 1 J2K-23
J2K-51 O X 4 2.7 Experimental group 2 J2K-23
J2K-51 X O 2 2.9

Note 1) Specific odor: 4 (very strong), 3 (strong), 2 (average), 1 (weak), 0 (odorless).

In the case of control groups 1 and 2, that is, when only S. cerevisiae J2K-23 was cultured, γ-PGA was not produced. In addition, in the case of control groups 3 and 4, when only B. subtilis J2K-51 was cultured, γ-PGA was produced and viscosity was formed in the culture medium, but a strong specific odor occurred in the culture medium.

In addition, when culturing using Malt Extract (M.E) (Control Group 4), the odor was found to be reduced compared to culturing using Yeast Extract (Control Group 3), but the viscosity of the culture medium and the yield of γ-PGA were lower than those of Yeast Extract (Control Group 4). It was confirmed that it was significantly lower (3.2% -> 2.4%) compared to when Extract was used (Control Group 3).

Meanwhile, in the case of experimental group 1, where mixed fermentation was performed using Y.E (Yeast Extract), the odor was not significantly different from the control group, and the yield of γ-PGA was lower than that of the control group in which J2K-51 was cultured alone. Confirmed.

However, in the case of experimental group 2, where mixed fermentation was performed using M.E., slightly less γ-PGA was produced than the control group in which J2K-51 was cultured alone, but the unique odor was confirmed to be significantly improved. Through this, it was confirmed that the mixed culture of yeast strains is excellent for removing bad odors.

<Example 3> Development of a medium composition that can increase the production yield of γ-PGA

Through Example 2, it was confirmed that when mixed culture was performed using Malt Extract (M.E), γ-PGA could be produced at a relatively high yield while eliminating bad odor.

However, in this experiment, we attempted to explore the optimal medium composition that can produce γ-PGA with higher yield. Table 3 below shows the medium composition designed to confirm the optimal medium composition. However, all trace elements (0.5 g/L of potassium phosphate, 0.1 g/L of magnesium sulfate heptahydrate) were added in the same way. In Table 3 below, the control group was inoculated with only Bacillus subtilis J2K-51, and the experimental group was a mixed culture of Bacillus subtilis J2K-51 and yeast J2K-23.

division Glucose Malt Extract Sodium
glutamate Unusual taste ¹⁾ r-PGA production amount (%) Control group 4 10 10 10 3 2.4 Experimental group 2 10 10 10 2 2.9 Experimental group 3 20 10 10 2 3.6 Experimental group 4 40 10 10 3 3.5 Experimental group 5 10 5 10 One 2.4 Experimental group 6 10 20 10 3 3.4 Experimental group 7 10 40 10 4 3.8 Experimental group 8 10 10 30 2 4.4 Experimental group 9 10 10 50 3 5.4 Experimental group 10 10 10 70 4 5.0 Control group 5 20 5 50 4 5.7 Experimental group 11 20 5 50 2 5.6

Note 1) Specific odor: 4 (very strong), 3 (strong), 2 (average), 1 (weak), 0 (odorless)

As a result of the experiment, under medium conditions (experimental group 11) of glucose 20g/L, malt extract 5g/L, sodium glutamate 50g/L, dipotassium phosphate 0.5g/L, and magnesium sulfate heptahydrate 0.1g/L, yeast J2K-23 and Through mixed culture of Bacillus subtilis J2K-51, a γ-PGA-containing culture with less specific odor than Bacillus subtilis J2K-51 single culture could be obtained in high yield.

As above, γ-PGA could be produced in high yield by using Malt Extract (M.E) instead of Yeast Extract (Y.E) and mixed culture of J2K-23 and J2K-51, and the unique odor was reduced, improving the purification process. It was confirmed that it can be simplified. In order to remove odor, an additional process of recovering and re-dissolving the precipitate after solvent precipitation is generally required, but in the present invention, this additional process is unnecessary, making it economical and environmentally friendly.

Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC13695BP 20181031 Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC14053BP 20191202

Claims (6)

Obtained by mixed culture of Saccharomyces cerevisiae J2K-23 (KCTC 13695BP) strain isolated from green tea flowers and Bacillus subtilis J2K-51 (KCTC 14053BP) strain isolated from yeast. Contains a culture medium that
The culture medium was mixed and cultured in a culture medium containing 10 to 30 g/L of glucose, 1 to 9 g/L of malt extract, and 25 to 75 g/L of sodium glutamate to produce poly-gamma-glutamic acid (γ). -PGA) and a cosmetic composition for alleviating skin irritation, characterized by reduced culture odor.
According to paragraph 1 above,
The cosmetic composition is,
A composition for relieving skin irritation, characterized in that it further has any one effect selected from skin moisturizing effect, inflammation alleviating effect, erythema improving effect, and skin soothing effect.
Saccharomyces cerevisiae J2K-23 (KCTC 13695BP) strain isolated from green tea flowers and Bacillus subtilis J2K-51 (KCTC 14053BP) strain isolated from yeast with 10 to 30 g of glucose. /L, mixed culture in a culture medium containing 1 to 9 g/L of malt extract and 25 to 75 g/L of sodium glutamate, and poly-gamma-glutamic acid characterized by reduced culture odor. Method for producing acid; γ-PGA).
delete Saccharomyces cerevisiae J2K-23 (KCTC 13695BP) strain isolated from green tea flowers and Bacillus subtilis J2K-51 (KCTC 14053BP) strain isolated from yeast with 10 to 30 g of glucose. /L, mixed culture in a culture medium containing 1 to 9 g/L of malt extract and 25 to 75 g/L of sodium glutamate, and poly-gamma-glutamic acid characterized by reduced culture odor. Method for producing culture medium containing acid; γ-PGA). delete