KR102265388B1 - Fermented product of Glehnia littoralis extract, preparation method and use thereof - Google Patents

Abstract

The present invention provides a lactic acid bacterium fermented product of a liberated wind extract, a method for producing the same, and a use thereof. The lactic acid bacteria fermented product of the liberated wind extract of the present invention has a high GABA content compared to the extract before fermentation, and has anti-melanin activity, excellent antioxidant activity, antibacterial activity and biosafety, and is useful as a cosmetic and food composition.

Description

Fermented product of lactic acid bacteria of Haebangpung extract, manufacturing method and use thereof {Fermented product of Glehnia littoralis extract, preparation method and use thereof}

The present invention relates to a fermented product of lactic acid bacteria of liberated wind extract, a cosmetic or food composition containing the same, and a method for manufacturing the fermented product, and more particularly, to a solvent extract of liberated wind fermented using lactic acid bacteria, anti-melanin, antioxidant, GABA production It relates to a lactic acid bacteria fermented product of a liberated wind extract having an increasing and antibacterial effect and a product using the same as an active ingredient.

Glehnia littoralis is a perennial grass belonging to the Umbrella family that grows wild on sandy soils along the coast of Korea and is a medicinal plant distributed in Japan, Manchuria, and China. is increasing It is a distinct plant from Saposhnikovia divaricate , and in Oriental medicine, it is known that its medicinal effect is similar to that of Sasam (Zandae).

On the other hand, GABA (r-aminobutyric acid, GABA) is a non-protein constituent amino acid widely distributed in nature, and GABA is known as an inhibitory neurotransmitter in the central nervous system of higher organisms. In humans, it is contained in the nervous system and blood, but most of it is present in the bone marrow of the brain and increases the neurotransmitter called acetylcholine and promotes brain function. It is known to be a substance involved in the (Chang et al., 1992; Shelp et al., 1999; Park et al., 2002).

Brown rice, germinated brown rice, green tea, and mulberry leaves are known as natural products with a high GABA content. However, there is a limit to exerting pharmacological action with the low GABA content of the natural product itself, and the need for research and development to produce GABA at a high concentration is recognized. The conventional method of increasing GABA production is to produce GABA through decarbonization of glutamine possessed by most natural products, but in this case, there is a limit in which GABA production is determined by the amount of glutamine possessed by the natural product. Therefore, in order to overcome these limitations, GABA production studies by microorganisms were conducted. Research on industrial production of GABA has been a method for high-concentration production, but the development of a material that maximizes the efficacy of GABA is still insufficient.

Under this background, the present inventors have found that the fermented product obtained by culturing lactic acid bacteria in the extract of liberated wind can increase the r-aminobutyric acid (GABA) content while exhibiting an antibacterial effect against dandruff, acne, caries, etc. It was confirmed that it can be used as a composition for treating diseases.

The present invention relates to a lactic acid bacteria fermented product of a liberated wind extract.

The present invention also provides a method for producing a lactic acid bacteria fermented product of liberated wind extract, comprising the steps of obtaining a solvent extract of liberation wind, inoculating the solvent extract with lactic acid bacteria and fermenting the extract.

In addition, the present invention provides a composition for skin whitening comprising the lactic acid bacteria fermented product of the liberated wind extract.

In addition, the present invention provides an antibacterial composition comprising the lactic acid bacteria fermented extract of the liberated wind.

In addition, the present invention provides an antioxidant composition comprising the lactic acid bacteria fermented product of the extract of liberated wind.

In addition, the present invention is one or more types of skin whitening, antioxidant, acne prevention, improvement and treatment, tooth decay prevention, improvement and treatment, and dandruff prevention, improvement and treatment, including the lactic acid bacteria fermented product of the liberated wind extract. A pharmaceutical, food or cosmetic composition having a use is provided.

The present invention relates to a lactic acid bacteria fermented product of liberated wind solvent extract, a preparation method thereof, and skin whitening, antioxidant, acne prevention, improvement and treatment, tooth decay prevention, improvement and treatment, and dandruff prevention, improvement and treatment. It relates to the above uses.

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

An example of the present invention relates to a lactic acid bacterium fermented product of a liberation pung extract and a method for preparing the same. The fermented lactic acid bacteria of the liberated wind extract is obtained by inoculating the solvent extract of liberated wind with lactic acid bacteria and culturing or fermenting it.

In the present invention, the lactic acid bacteria fermented product of the liberation wind extract is (i) a liquid fermented product obtained by culturing and fermenting lactic acid bacteria in a solvent extract of the liberation wind, (ii) a powder fermented product obtained by freeze-drying and powdering the liquid fermented product of the liberation wind extract, and (iii) After filtering the liquid fermented product and separating the supernatant, it may include all of the supernatant powder of the freeze-dried and powdered fermented product, and the active ingredients included in (i) to (iii) above include all

The fermented lactic acid bacteria of the liberation wind extract according to the present invention can be prepared by a method for producing a lactic acid bacteria fermented product of the liberation wind extract, comprising the steps of obtaining a solvent extract of liberation wind, inoculating the solvent extract with lactic acid bacteria and fermenting the extract have. The manufacturing method may further perform the step of drying the liquid fermented product fermented by culturing lactic acid bacteria, and the drying may be preferably freeze-drying.

The lactic acid bacteria fermented product of Haebangpung extract provided by the present invention may have one or more of the following four characteristics:

i) 1.01 times to 10 times higher GABA content than the unfermented product of the liberated wind solvent extract,

ii) 1.3 times to 3 times higher antioxidant activity than the unfermented product of Haebangpung solvent extract,

iii) high anti-melanin activity compared to the unfermented product of the liberated wind solvent extract, and

iv) antibacterial activity against one or more bacteria selected from the group consisting of Acne, dandruff and caries.

An example of the present invention relates to a method for producing a lactic acid bacteria fermented product of a liberation wind extract, comprising the steps of obtaining a solvent extract of liberation wind, inoculating and fermenting the solvent extract with lactic acid bacteria, it's about how Hereinafter, the method for preparing the lactic acid bacteria fermented product of the liberated wind extract will be described in detail.

The method for producing a lactic acid bacteria fermented product of the liberated wind extract provided by the present invention may further include drying the fermented product after the lactic acid bacteria fermentation step, preferably freeze-drying. The freeze-dried fermented product may be stored in a range in which its activity is maintained, and may be used by mixing with an appropriate solvent if necessary. By further including the freeze-drying step, it is possible to increase the preservation of the lactic acid bacteria fermented product of the liberated wind extract provided by the present invention.

In the method for producing a lactic acid bacterium fermented product of the liberated wind extract according to the present invention, the step of obtaining the solvent extract of the liberated wind may be performed using a conventional method for obtaining a solvent extract from a plant.

In the present invention, "liberation wind" includes one or more parts selected from the group consisting of leaves, roots, petals, calyxes, fruits and stems of liberation winds.

The solvent extract of the liberation wind is prepared by extracting at least one selected from the group consisting of liberation wind, a dried product of liberation wind, and a pulverized product of liberation wind or dry liberation wind with an extraction solvent.

The liberated air used in the preparation of the solvent extract may be a powder obtained by drying the liberated air by a method such as hot air drying, microwave drying, or freeze-drying, and then pulverizing it with a pulverizer. At this time, there is no limitation on the particle size of the powder, and it may be a powder prepared using a general grinder or a food mixer. Alternatively, after freezing the release wind, a powder obtained by grinding together with a buffer may be used. The powder prepared above can be used directly for preparing the extract, and it can be stored at -20 to -70 °C and used as needed. Alternatively, the liberated air may be stored frozen at -70° C. without drying, and then added and pulverized with a solvent.

When using the powder of the liberation wind, a mixing step for mixing the powder and the solvent before solvent extraction, for example, an ultrasonic wave or a vortex chamber may be used. The mixing step using ultrasonic waves serves to dissolve the active material in the cell membrane of the liberation wind well, and can be freely used in a range that does not cause denaturation of proteins or other materials while achieving the purpose of cell disruption. For example, the liberation wind powder and the extraction solvent can be mixed by irradiating at 750 watt, 10 kHz at 10 second intervals for 5 minutes.

As the solvent extraction method, a conventional extraction method may be used, and methods such as extraction with an extraction solvent, supercritical fluid extraction, ultrasonic extraction, and hot water extraction may be used. Preferably, a hot water extraction method may be used. The solvent extraction time may be 4 to 6 hours, or 5 to 6 hours.

The extraction solvent may be at least one selected from the group consisting of water, alcohol, C1 to C3 alcohol, deep sea water, and saltwater. For example, it may be water or ethanol (EtOH). The deep sea water means seawater at a depth of 200 m or more from the surface of the sea water. The saltwater is mineral-containing water, and may include both low-salt groundwater and saltwater groundwater, and may be groundwater having a salt concentration of 0.3‰ (permilliage) or more.

When using the hot water extraction method as the solvent extraction method, 3 to 10 hours, 4 to 8 hours, 4 to 6 hours in hot water of 60 to 100 ° C, 70 to 90 ° C, or 80 to 90 ° C, preferably 85 ° C. , or 5 to 6 hours extraction may be performed.

For example, to obtain a liberated wind hot water extract, 50 to 100 g of liberated wind powder is mixed with 900 to 1 L of water and added so that the liberated wind: water ratio is 0.5:9 to 1:9 by weight, 750 watt, 10 kHz for 10 seconds The powder and water were mixed by irradiating ultrasonic waves at intervals of 5 minutes, and the mixture was cultured with shaking in hot water at 85° C. for 4 to 6 hours, and then the cultured extract was centrifuged at 7000 rpm for 10 minutes to 0.8um coring bottle-top. about 800 to 900 mL (80 to 90%) of liberated wind hot water extract can be prepared by vacuum filtration using

The liberated wind hot water extract of the present invention may be used in a liquid form, or may be used in a powder form obtained by freeze-drying the liquid liberated wind hot water extract. The powdered liberated wind hot water extract can be mixed with water as needed to form a liquid form and used in the fermented product manufacturing process.

The liberated wind solvent extract used for the fermentation of the lactic acid bacteria may or may not have a post-treatment process. The post-treatment process may be selected by a person skilled in the art without limitation within the scope for achieving the object of the present invention, for example, it may be one or more processes selected from the group consisting of centrifugation, filtration, purification and sterilization. . The sterilization may be high-temperature and high-pressure sterilization, and preferably may be sterilized at 121° C. and 0.12 MPa for 20 minutes.

In the method for producing a fermented lactic acid bacteria of the liberated wind extract according to the present invention, the step of inoculating and fermenting the lactic acid bacteria in the solvent extract of the liberated wind is performed.

The liberated wind extract may be used as a fermentation raw material by itself without any additives, or may be used as a fermentation raw material after an additive for fermentation is included.

The additives are the same as described above. The lactic acid bacteria fermented product of the liberation wind solvent extract according to the present invention can be prepared by using the liberated wind solvent extract as it is as a fermentation raw material, or by treating it with a post-treatment process and using it as a fermentation raw material. The post-treatment process may be, for example, one or more processes selected from the group consisting of centrifugation, filtration, purification, and sterilization, as necessary, and may be appropriately selected and used, and the type of the process is not limited.

The liberated wind extract may be used as a raw material for fermentation by itself without any additives, or may be used as a raw material for fermentation of lactic acid bacteria by additionally including an additive for fermentation.

The additive may be a carbon source or a nitrogen source, but is not limited thereto. The type of the carbon source is not particularly limited, but may be at least one selected from the group consisting of dextrose, glucose, sucrose, lactose, and galactose, As the nitrogen source, one or more additives selected from the group consisting of beef extract, peptone, tryptone, and skim milk may be used.

In one embodiment, the liberated wind extract used as the fermentation raw material may have an excellent lactic acid bacteria growth effect without the addition of the additive. Specifically, the liberated wind extract provided by the present invention is 2 times to 300 times, 2 times to 200 times, 2 times to 150 times, 2 times compared to MRS medium (de Man Rogasa and Sharpe) under the condition that no additives are added. to 100 times, 2 to 50 times, 2 to 30 times, 2 to 20 times, 2 to 15 times, 2 to 10 times, 4 to 300 times, 4 to 200 times, 4 to 150 times. Times, 4 times to 100 times, 4 times to 50 times, 4 times to 30 times, 4 times to 20 times, 4 times to 15 times, or may have a lactic acid bacteria proliferation effect of 4 times to 10 times.

The lactic acid bacteria used in the preparation of the lactic acid bacteria fermented product of the liberated wind extract of the present invention may be used without limitation, and preferably may be lactic acid bacteria having a characteristic of producing GABA. For example, Lactobacillus brevis (Lactobacillus brevis), Lactobacillus para casei (Lactobacillus paracasei), Lactobacillus Buh Neri (Lactobacillus buchneri), Lactobacillus four K (Lactobacillus sakei), Lactobacillus flange teorum (Lactobacillus plantarum), Lactobacillus Bacillus pentosus ( Lactobacillus pentosus ) , Lactoccocus lactics ( Lactoccocus lactics ), Lactobacillus delbrueckii ( Lactobacillus delbrueckii ), It may be one or more lactic acid bacteria selected from the group consisting of Lactobacillus acidophilus , Lactobacillus helveticus , and Pediococcus acidilactici. In a preferred embodiment, the fermented product is prepared using Lactobacillus planterum, Pediococcus acidi lactisi, or a combination of Lactobacillus planterum and Pediococcus acidilactisi.

The Lactobacillus planterum strain may be a strain comprising a 16S rRNA gene nucleotide sequence having 97% or more, 98% or more, 99% or more, or 99.9% or more sequence homology with the nucleotide sequence of SEQ ID NO: 5, preferably Preferably, it may be a strain comprising a 16S rRNA sequence consisting of the nucleotide sequence of SEQ ID NO: 3, and more preferably, the accession number KCCM12299P strain deposited with the Korea Microorganism Conservation Center (KCCM) on August 10, 2018. It may be.

The Pediococcus esidilactici strain may be a strain comprising a 16S rRNA gene nucleotide sequence having sequence homology of 97% or more, 98% or more, 99% or more, or 99.9% or more with the nucleotide sequence of SEQ ID NO: 6 And, it may be a strain comprising a 16S rRNA sequence consisting of the nucleotide sequence of SEQ ID NO: 4, preferably, the accession number KCCM12472P strain deposited on March 29, 2019 at the Korea Center for Microorganisms (KCCM).

The Lactobacillus planterum and Pediococcus esidilactici strains may be cultured at 30 to 37° C. using MRS medium. The Lactobacillus planterum and Pediococcus acidi lactisi strains may be cultured in a temperature range of 25 to 40° C. using a liberated wind extract or a liberated wind hot water extract that is not added with a carbon source or nitrogen source as a medium.

In the preparation of the fermented product of the extract according to the present invention, the inoculum of the lactic acid bacteria may be 1x10 ⁸ to 1x10 ¹⁵ cfu/ml, 1x10 ⁸ to 1x10 ¹² cfu/ml, or 1x10 ⁸ to 1x10 ¹⁰ cfu/ml.

In the production of lactic acid bacteria fermented product of liberated wind extract according to the present invention, the fermentation temperature may be carried out at 25 to 40 ℃, 25 to 35 ℃, or 30 ℃, and the fermentation time is 12 to 60 hours, 18 to 53 hours or 24 hours to 48 hours. When the temperature and culture time are outside the above ranges, the number of viable lactic acid bacteria sufficient to maintain the functionality of the lactic acid bacteria is not maintained, and there is a risk of dead cells.

The lactic acid bacteria fermented product of Haebangpung extract provided by the present invention may have one or more of the following four characteristics:

i) GABA content 1.01 to 10 times higher than that of the unfermented extract of Haebangpung solvent extract,

ii) 1.3 times to 3 times higher antioxidant activity than the unfermented product of Haebangpung solvent extract,

iii) high anti-melanin activity compared to the unfermented product of the liberated wind solvent extract, and

iv) antibacterial activity against one or more bacteria selected from the group consisting of Acne acnes, dandruff, dental caries and caries.

The lactic acid bacteria fermented product of the liberated wind extract provided by the present invention may have a higher GABA content than the liberated wind extract or unfermented liberated wind extract before fermentation. Specifically, 1.01 to 10 times compared to the unfermented liberated wind extract. 1.01 to 5 times, 1.01 to 4 times, 1.01 to 3.7 times, 1.05 to 10 times, 1.05 to 5 times, 1.05 to 4 times, 1.05 to 3.7 times, 1.1 to 10 times, 1.1 to 5 times, 1.1 to 0.1% The GABA content was increased to 0.43 ng/ml and 0.87 ng/ml, respectively, at the 1% concentration. In the case of Haebangpung root extract, the GABA content was 0.38ng/ml and 0.49ng/ml, respectively, at 0.1% and 1% concentrations of Haebangpung root extract before fermentation, but increased to 0.46ng/ml and 1.31ng/ml after fermentation, The GABA content was increased in all fermented products in which lactic acid bacteria were cultured in the liberation wind extract.

Therefore, the lactic acid bacteria fermented product of the liberation wind extract provided by the present invention has a high GABA content, so high functionality can be expected when applied to food and cosmetics. In particular, the liberated wind extract fermented product with increased GABA component has a soothing effect, anti-stress effect, and antioxidant , it can help to activate cells in a short period of time by regulating the action of inflammation and prevention, blood circulation regulation, and cell activity.

The lactic acid bacteria fermented product of the haebangpung extract provided by the present invention may have superior antioxidant activity compared to the unfermented haebangpung extract. In the present invention, the antioxidant activity means free radical scavenging ability. In one embodiment, the lactic acid bacteria fermented product of the liberation leaf extract may have a free radical scavenging activity that is 1.3 to 3 times, 1.3 to 2 times, or 1.3 to 1.5 times higher than the free radical scavenging ability of the unfermented product.

The free radical scavenging ability may be expressed as a DPPH inhibition rate, and the DPPH inhibition rate may be calculated by the method of Equation 1 below.

[Equation 1]

DPPH scavenging activity (%) = (1-OD sample/OD blind) x 100

In Equation 1, OD sample is the absorbance of the sample to be measured, and OD blind is the absorbance of the DPPH solution to which no sample is added.

The DPPH inhibition rate (%) of the lactic acid bacteria fermented product of the liberated wind extract provided by the present invention is 60 to 90%, 60 to 85%, 60 to 80%, 70 to 90%, 70 to 85%, 70 to 80%, or 75 to 90%, 75 to 85% or 75 to 80%.

The lactic acid bacteria fermented product of the liberated wind extract of the present invention may have a DPPH inhibition rate of 1-1.4 times, 1-1.3 times, 1-1.2 times, or 1-1.1 times compared to the DPPH inhibition ratio of butyl hydroxytoluene (BHT).

In an embodiment of the present invention, DPPH free radical scavenging activity analysis was performed between the fermented product obtained by culturing L. plantarum and P. acidilactici in the hot water extract of Haebangpung leaf and the unfermented hot water extract, respectively. The DPPH inhibition rate of each extract or fermented product was calculated by the method of Equation 1 above.

As a result of the analysis, the DPPH inhibition rate of the unfermented liberation leaf hot water extract was 56.5%, while the DPPH inhibition rate of the L. plantarum fermented product was 75.6% and the DPH inhibition rate of the P. acidilactici fermented product was 79.2%, respectively, 1.3 to 1.5 in each ferment. It was confirmed that the free radical scavenging ability was twice as high, and it was confirmed that both types of the fermented products had superior free radical scavenging ability compared to the control butyl dihydroxytoluene (BHT). Therefore, the lactic acid bacteria fermented product of Haebangpung leaf extract has superior antioxidant activity compared to the unfermented hot water extract.

In one embodiment of the present invention, as a result of calculating _{the EC 50} value for the DPPH inhibition rate of the fermented product obtained by culturing L. plantarum and P. acidilactici in the hot water extract of Haebangpung leaf, respectively, butyl dihydroxytoluene ( BHT) was found to be 0.063%, lower than the EC50 value of 0.066%, confirming that the lactic acid bacteria fermented product of the Haebangpung leaf ?x water extract was excellent as an antioxidant.

The lactic acid bacteria fermented broth of the liberated peony extract of the present invention has superior anti-melanin activity compared to the unfermented liberated wind extract, and can be utilized for skin whitening purposes. Specifically, when the melanin content in B16F10 cells of untreated mice was set to 100%, the intracellular melanin content was 50 to 99%, 55 to 95% when the lactic acid bacteria fermented extract of the liberated wind extract of the present invention was treated. , may be 60 to 90%, or 60 to 85%, preferably 60 to 82%.

When the lactic acid bacteria fermented liquid of the liberation wind extract of the present invention is treated with the liberation wind extract,

In one embodiment of the present invention, in order to analyze the effect of the lactic acid bacteria fermentation broth of the present invention on the amount of melanin production in the skin, a quantitative analysis of melanin was performed using a mouse B16F10 cell line. Specifically, after the B16F10 cells of the mice were cultured in DMEM medium containing 10% FBS, haebangpung leaf hot water extract (not fermented), haebangpung root hot-water extract (not fermented), haebangpung L.plantarum fermented leaf hot water extract , haebangpung P. acidilactici leaf hot water extract of the fermented product, the hot-water extract of the roots haebangpung L.plantarum during fermentation, and haebangpung roots 48 hours, respectively 0.1% of the processing P. acidilactici fermentation of the hot-water extract after incubation, the absorbance at 475nm was measured. As a result, when the melanin content of the cells of the untreated group that was not treated with the liberated wind extract or the lactic acid bacteria fermented product of the liberated wind extract was set at 100%, the melanin content of the unfermented liberated wind leaf extract in the treated group was 90.5%, and the unfermented liberated wind melanin content of the root extract or've found in 89.2%, L. plantarum in the fermented product of the US haebangpung leaf extract fermented L. plantarum fermented and non-fermented extract of the roots haebangpung show each melanin content of 71.3% and 68.6% non-fermented Compared to the extract, the fermented product of Haebangpung leaf extract showed 19.2%p lower melanin content than the unfermented product, and the fermented product of Haebangpung root extract showed a melanin content that was 20.6%p lower than that of the unfermented product. It was confirmed that both the fermented products of both extracts had an anti-melanin effect. P. acidilactici fermented products of melanin leaf extract and root extract showed melanin content of 81.5% and 77.1%, respectively, and both about 10%p lower melanin content compared to unfermented product, specifically 9% each compared to unfermented product p, 12.1%p was low. Therefore, the lactic acid bacteria fermented product of the liberated wind extract of the present application has an activity of reducing the melanin concentration of the skin, and can be used for skin whitening, and thus has a high utilization potential.

The lactic acid bacteria fermented broth of the liberated wind extract of the present invention has excellent antibacterial activity compared to the unfermented extract. The antibacterial target bacteria may be one or more uniform selected from the group consisting of Listeria, Staphylococcus, Escherichia coli, Bacillus, Acne, dandruff, caries, and caries, but is not limited thereto.

Therefore, the lactic acid bacteria fermented liquid of the liberated peony extract of the present invention can be used for the prevention, improvement or treatment of acne, the prevention, improvement or treatment of dandruff, the prevention, improvement or treatment of tooth decay, pharmaceutical composition, cosmetic composition, oral product Or it can be used as a hair product. Accordingly, the present invention also provides a composition for antibacterial, comprising the lactic acid bacteria fermented extract of the liberation wind. The fermented product includes at least one formulation selected from the group consisting of a fermentation stock solution, a concentrated fermentation broth of the fermentation stock solution, and a fermentation powder prepared from the fermentation stock solution.

The acne bacteria may be, for example , Propionibacterium acnes (ATCC 6919), the dandruff bacteria may be Malassezia furfur (ATCC 14521), and the dental caries or caries bacteria are It may be Streptococcus mutans (ATCC3065) or Streptococcus sobrinus (ATCC33478), but is not limited thereto.

More specifically, in an embodiment of the present invention, L. plantarum ( KCCM12299P ) and P. acidilactici (KCCM12300P) were co-inoculated with Haebangpung leaf hot water extract and Haebangpung root hot water extract to prepare lactic acid bacteria fermented products of each extract, Propionibacterium acnes (ATCC 6919), Malassezia furfur (ATCC 14521), Streptococcus sobrinus (ATCC33478) and Streptococcus mutans (Streptococcus mutans, ATCC3065), respectively After injecting the fermented product or unfermented hot water extract into the sterilized punch hole of the smeared plate medium, the diameter of the inhibition clear zone was measured to analyze the antibacterial properties against each bacteria. As a result, it was confirmed that the antibacterial effect of the Haebangpung root extract was shown to be effective against tooth decay bacteria and acne bacteria before fermentation, but the lactic acid bacteria fermented product of the Haebangpung root extract showed a strong antibacterial ring against all three bacteria, thus improving the antimicrobial activity. In the case of the liberated leaf extract, the hot water extract did not generate any antibacterial rings before fermentation, but the lactic acid bacteria fermented product showed an antibacterial ring in acne, confirming that the antibacterial effect of the fermented product was improved compared to the extract before fermentation.

The present invention also provides a pharmaceutical composition for the prevention or treatment of one or more diseases selected from the group consisting of acne, dandruff and tooth decay, comprising the lactic acid bacteria fermented product of the liberation pung extract.

The pharmaceutical composition of the present invention may be prepared by including one or more additives selected from the group consisting of a dispersing agent, a carrier, and other antibacterial agents, as long as the antimicrobial activity of the lactic acid bacteria fermented product of the lactic acid bacteria fermented product is not inhibited as an active ingredient.

As the dispersant, water, methanol, ethanol, ethylene glycol, propylene glycol, diethylene glycol, glycerin, ketones, acetone, methyl ethyl ketone, ethers, dioxane, tetrahydrofuran, cellosolve, diethylene glycol dimethyl ether, hexane , kerosene, benzene, toluene, xylene, naphthalene, methyl naphthalene, chloroform, carbon tetrachloride, dimethyl formadide, methyl acetate ester, ethyl acetate ester, butyl acetate ester, fatty acid glycelniester, acetonitrile, higher alcohol sulfate At least one selected from the group consisting of esters, alkyl sulfonic acids, alkyl allyl sulfonic acids, quaternary ammonium salts, oxyalkyl amines, fatty acid esters, polyalkylene oxide compounds, and anhydrosorbitol compounds may be included.

The carrier may be, for example, at least one selected from the group consisting of kaolin, bentonite, acid clay, talc powder, pyrophyllite powder, diatomaceous earth, silicic anhydride, mica powder, alumina, sulfur powder, and activated carbon.

The other antibacterial agents are, for example, carvacrol, thymol, citral, isoeugenol, methyleugenol, bamboo extract, lychee extract, rice bran fermented product, isodiazolone compound and aminocarboxylic acid selected from the group consisting of It may be one or more antibacterial substances, but is not limited thereto.

The pharmaceutical composition of the present invention may be prepared in any of liquid, solid and gaseous phases. The pharmaceutical composition of the present invention may be administered in any manner, such as oral or parenteral, and preferably may be administered in a topical manner. Formulations of the oral administration method include tablets, pills, powders, liquids, food forms, and the like, and parenteral formulations include injections, topical administration agents (creams, ointments, etc.), suppositories, sprays, and the like.

The content of the lactic acid bacteria fermented product of the liberated wind extract may be 0.001 to 99.99% by weight, 0.1 to 99% by weight, or 0.1 to 50% by weight in the antibacterial composition of the present invention, but an appropriate content according to the method and purpose of use of the antibacterial composition can be adjusted to

The lactic acid bacteria fermented product of the liberated wind extract is excellent in safety for the living body and/or the human body. In one embodiment of the present invention, a fermented product prepared by culturing a leaf or root extract of liberated punggi in a mouse melanoma cell line (B16F10) culture and L. plantarum ( KCCM12299P ), or P. acidilactici (KCCM12300P) in the extract. was treated by concentrations of 1 mg, 100 ug, 10 ug and 1 ug and cultured for 24 hours, 48 hours or 72 hours, and then reacted by adding CCK-8, and then absorbance (OD 540 nm) was measured. As a result, when the cell viability in the untreated group, which was not treated with either the liberation wind extract or the fermented product at all, was 100%, the fermented products of the liberation wind leaf extract all showed higher cell viability than the control group, regardless of the lactic acid bacteria strain. (FIG. 4), the fermented product of Haebangpung root extract also showed 100% or more viability regardless of the strain (FIG. 5). Therefore, the lactic acid bacteria fermented product of the liberated wind extract of the present invention is not toxic to cells and has excellent safety when taken or applied to a living body or human body.

The present invention also provides a food composition for the prevention or improvement of one or more diseases selected from the group consisting of acne, dandruff and tooth decay, comprising the lactic acid bacteria fermented product of the liberation pung extract.

The formulation of the lactic acid bacteria fermented product of the liberated wind extract contained in the food composition of the present invention is not limited as long as it is a formulation that can be used in a food composition in principle, and may be liquid or solid, filtrate, extract, suspension, concentrate, dry powder, freeze-dried. It may be included in the form of powder, spray-dried powder, or the like.

The lactic acid bacteria fermented product of the Haebangpung extract can prevent or improve acne, dandruff or caries disease by having antibacterial activity against acne bacteria, dandruff bacteria and tooth decay. In the lactic acid bacteria fermented product of the liberated wind extract of the present invention, the antibacterial activity is as described above.

Since the lactic acid bacteria fermented product of the liberated wind extract has no cytotoxicity and is excellent in safety, it can be applied to food. In addition, both liberation wind and lactic acid bacteria, which are raw materials of the fermented lactic acid bacteria, are food ingredients that have been eaten for a long time, and can be used as food.

The lactic acid bacteria fermented product of the liberated wind extract may be included in an amount of 0.001 to 99% by weight or 0.01 to 50% by weight relative to the total weight of the food composition. The content of the lactic acid bacteria fermented product of the liberated wind extract is not particularly limited, but is included so as to exhibit the desired activity of the present invention for preventing or improving acne, dandruff and/or tooth decay.

For the formulation of the food composition of the present invention, various formulations known to those skilled in the art can be applied, for example, conventional health functional food formulations such as beverages, pills, powders, etc. including the lactic acid bacteria fermented product of the liberated wind extract, or It may be in the form of a composition, such as a liquid, pill, suspension, or the like.

The food composition provided by the present invention may further include excipients or adjuvants conventionally used in the food field. The preparation of the food composition of the present invention may be prepared in the form of tablets, capsules, granules, powders, extracts, solutions, syrups, suspensions, or emulsions, but is not limited thereto.

The present invention also provides a cosmetic composition for the prevention or improvement of one or more diseases selected from the group consisting of acne, dandruff and tooth decay, comprising a lactic acid bacterium fermented product of the liberated fermented extract. The antibacterial activity of the lactic acid bacteria fermented product of the liberated wind extract against acne bacteria, dandruff bacteria and tooth decay bacteria is as described above.

The present invention also provides a cosmetic composition for skin whitening, comprising a lactic acid bacterium fermented product of the liberated wind extract. Since the liberated wind extract has an activity of lowering the melanin content in skin cells, it can be included in a cosmetic composition and used for skin whitening. The effect of reducing the melanin content in skin cells of the liberated wind extract is as described above.

The cosmetic composition for preventing or improving the disease or the cosmetic composition for skin whitening may be applied to one or more sites selected from the group consisting of hair, scalp, skin, oral cavity, teeth and periodontal period.

When the cosmetic composition of the present invention is applied to the scalp or hair, it may be prepared in any conventionally prepared formulation, preferably as various hair products such as emulsions, creams, pastes, gels, lotions, packs, lotions, powders, It may be formulated as a cosmetic for hair treatment, such as a spray or soap. Examples of hair products include, but are not limited to, formulations such as hair toner, hair lotion, hair cream, hair spray, hair mousse, hair gel, hair soap, hair shampoo, hair conditioner, hair pack and hair treatment. .

In the present invention, the cosmetic composition is at least one selected from the group consisting of flexible lotion, nutrient lotion, nutrient essence, nutrient oil, moisturizing oil, nutrient cream, powder, pack, foundation, makeup base, cleansing, shampoo, lotion and ointment. It may be in the form

The composition in powder form can be used as it is when it is applied to cosmetics, oral products, or hair products, and can be used by dissolving it in a solvent, and the solvent may be, for example, water or ethanol, but is not limited thereto.

The lactic acid bacteria fermented product of the liberated wind extract may be included in 0.001 to 30% by weight of the total weight of the cosmetic composition, but is not limited thereto.

The present invention also provides an oral composition comprising a lactic acid bacterium fermented product of the liberated wind extract. The composition for oral use is a composition for oral use that can prevent, improve, or treat periodontal disease or tooth decay caused by oral diseases, for example, Streptococcus mutans, and may be a pharmaceutical composition or a composition for oral hygiene.

The oral composition of the present invention may further include a material used in a conventional toothpaste composition, a gargle composition, or a composition for inhibiting bad breath and preventing tooth decay, and using the composition for oral cleaning as an additive, a toothpaste, gargle, or bad breath inhibitor and caries prevention agent can be included in

The formulation of the oral care composition of the present invention may be a warning agent, granule, powder, syrup, liquid, aerosol, spray, ointment, liquid extract, emulsion, suspension, tablet, capsule, cream, or pill. have.

The fermented product of the liberated wind extract provided by the present invention has an increased GABA content than before fermentation, has high biosafety, and has excellent cosmetic and pharmacological effects such as antibacterial, antioxidant, and anti-melanin.

1 is a diagram showing the location of the SNP of Pediococcus acidi lactisi used in the fermentation of the liberated wind extract.
2 is a graph showing the DDPH inhibition rate as a result of the antioxidant activity analysis of each extract or fermented product according to Example 4;
3 is a graph showing _{the IC 50} value of the antioxidant activity analysis result of each extract or fermented extract according to Example 4.
FIG. 4 is a graph showing the results of a toxicity test according to the concentration of an extract or fermented product of Haebangpung leaf on mouse B16F10 cells according to Example 5; FIG.
5 is a graph showing the results of a toxicity test according to the concentration of an extract or a fermented product of a liberated wind root for mouse B16F10 cells according to Example 5;
6 is a graph of the experimental results evaluating the effect of reducing melanin pigment in mouse B16F10 cells of each liberated wind extract or fermented product according to Example 6.
7 is a photograph of the TLC test results for GABA production of L. plantarum and P.acidilactici lactic acid bacteria in the hot water extract of Haebangpung leaves and roots according to Example 7. FIG.
Figure 8 is a graph of GABA production quantitative analysis ELISA test results of each extract or fermented product sample according to Example 8.
9 is a diagram showing the results of analysis of antibacterial properties against dandruff bacteria, acne bacteria and tooth decay bacteria of each extract or fermented product according to Example 9;

The present invention will be described in more detail with reference to the following examples. However, the following examples are intended to illustrate the present invention, and do not limit the scope of the present invention.

Example 1. Preparation of liberated wind extract

A hot water extract was prepared using the liberated wind leaf powder and the liberated wind root powder, which were prepared by separating the leaves and roots of Haebangpung, respectively, and pulverizing them by freeze-drying.

After mixing 50 g of the prepared liberated wind leaf powder or liberated wind root powder with 1000 mL of distilled water, respectively, the ultrasonic wave was applied at 750 watt, 10 kHz at 10 kHz intervals for 5 minutes to dissolve the active substance in the cell membrane well. Cells were disrupted and mixed by irradiation (Sonics Vibra-Cell, USA). Sonication was performed in ice to prevent denaturation of proteins or other substances. Next, the mixture was shaken in hot water at 85° C. for 4 hours, and the extract was centrifuged (7,000 rpm, 10 minutes) and vacuum filtered using a 0.8 um corning bottle-top (Sigma, USA). About 800~900ml (80~90%) of Hotwater Extract of Glehnia leaf (GLE) and Hotwater Extract of Glehnia root (GRE) liquid hot water extract were prepared.

The liquid hot water extract was freeze-dried at -70°C and then freeze-dried for 3 days to be powdered and then used for subsequent experiments.

Example 2. Preparation of fermented product of liberated wind extract

2-1. Isolation of lactic acid bacteria

Lactobacilli for fermenting the liberated peony leaf or root extract prepared in Example 1 were respectively isolated from fermented sawdust or deep-sea water for feeding of white spot flower radish.

First, Pediococcus acidilactici ( P. acidilactici ) was isolated and identified from fermented sawdust, food for edible insects. The fermented sawdust for feeding the edible insects was purchased from Yeongcheon Insect Industry (Korea). Fermented sawdust stored at 25° C. was added to PBS at a concentration of 1% (w/v) and mixed by vortexing. The fermented sawdust mixture was smeared on MRS (Difco, USA) agar medium to which 0.1% (v/v) BCP (Bromocresol purple, Sigma, USA) was added, and then cultured at 37° C. for 1 day. The BCP is acidified by the lactic acid of lactic acid bacteria to perform a function to form yellow colonies.

The process of reselection by selecting only yellow colonies formed by reaction with BCP and smearing them on MRS agar medium. It was repeated to isolate a single species.

Lactobacillus plantarum ( L. plantarum ) was isolated from deep sea water. The deep-sea water was transported in a refrigerated state by taking seawater 200m deep from the surface at Taeha, Jeo-dong, and Hyeonpo in Ulleungdo from November to December 2015. The deep sea water was smeared on MRS agar medium supplemented with 0.1% (v/v) BCP and cultured at 37° C. for 1 day. Thereafter, the selection of yellow colonies formed by the reaction with BCP was repeated to isolate a single species.

The two isolated strains were cultured in MRS liquid medium and mixed with 50% (v/v) glycerol so that the final glycerol concentration was 25%, and then stored at -70°C.

2-2. Identification of lactic acid bacteria

The two types of lactic acid bacteria isolated in Example 2-1 were identified using nucleotide sequence analysis of 16S rRNA. To identify two types of lactic acid bacteria, genomic DNA of each strain was first isolated using a mini genomic DNA extraction kit (Promega).

Using the isolated genomic DNA as a template, the following 27F and 1492R primer sets and AccPower PCR premix kit (Bioneer, Korea) were used to amplify the 16S rRNA gene regions of the two strains using TaKaRa PCR Thermal Cycler (Japan).

- 27F primer (SEQ ID NO: 1): 5'-AGAGTTTGATCMTGGCTCAG -3'

- 1492R primer (SEQ ID NO: 2): 5'-GGTTACCTTGTTACGACTT-3'

The PCR product was purified by PCR clean-up Gel extraction (Macherey Nagel, USA) and the gene sequence was analyzed with an ABI3730 DNA analyzer (Applied Bioxyxtems, USA). The 16S rRNA sequence of the Lactobacillus planterum strain isolated in the present invention is shown in SEQ ID NO: 3, and the 16S rRNA sequence of the Pediococcus esidilactici strain is shown in SEQ ID NO: 4.

16S rRNA sequence similarity analysis was performed using the 16s rRNA sequence information of the isolated Lactobacillus planterum strain and Pediococcus esidilactici strain, and the results are shown in Table 1 below. As shown in Table 1, the lactic acid bacteria isolated from deep sea water showed 99% sequence similarity with Lactobacillus planterum as a result of 16S rRNA analysis, and the lactic acid bacteria isolated from fermented sawdust was 99% with Pediococcus acidi lactisi. Showing the sequence similarity of, was named Lactobacillus planterum and Pediococcus acidi lactisi, respectively.

lactic acid bacteria 16S rRNA inoculation Proximal 16S rRNA sequence sequence similarity deep sea water Lactobacillus plantarum MK311261.1 (SEQ ID NO: 5) 99% Fermented sawdust Pediococcus acidilactici LC311071.1 (SEQ ID NO: 6) 99%

The isolated Lactobacillus planterum strain was deposited with the Korea Center for Microorganisms (KCCM) on August 10, 2018, and the accession number is KCCM12299P. The Pediococcus acidi lactisi strain was deposited with the Korea Microorganism Conservation Center (KCCM) on March 29, 2019, and the accession number is KCCM12472P.

2-3. Preparation of fermented products of liberated wind extract

After preparing a lactic acid bacteria culture medium using the liberated wind leaf extract or liberated wind root extract prepared in Example 1, sterilized by high-temperature and high-pressure sterilization at 121° C. and 0.12 MPa for 20 minutes, cooled at room temperature, inoculated with lactic acid bacteria, and cultured with liberated wind solvent A fermented product of the extract was prepared.

Specifically, in 1 L (pH 6.5 to 7.5) of the liquid hot water extract of liberated wind prepared in Example 1, a medium was prepared and used without addition of a nitrogen source, a carbon source, and inorganic salts. As the liberated wind solvent extract, two types of media were prepared using a liquid extract of liberated wind leaf (GLE) and a liquid hot water extract of liberated wind root (GRE), respectively.

Each of the prepared medium was ^{inoculated with 1 mL of 1×10 8} cfu/ml lactic acid bacteria and cultured at 30° C. for 24 hours to prepare a fermented product of the liberated wind extract. The lactic acid bacteria is Lactobacillus plantarum (KCCM12299P), Pediococcus acidilactici, KCCM12472P) isolated in Example 2-1, or the two strains to a final concentration of 1x10 ⁸ cfu/ml Each of the lactic acid bacteria mixture mixed at 1:1 was inoculated.

After each lactic acid bacteria inoculation, cultured at 30° C. for 24 hours to prepare a fermented product of each liberated wind extract. The lactic acid bacteria fermentation broth was freeze-dried and then pulverized to be prepared in powder form and used for subsequent experiments.

In this example, lactic acid bacteria fermented samples 1 to 6 were prepared with different types of liberated wind extract and lactic acid bacteria, respectively, specific types of inoculated lactic acid bacteria, and fermented products of liberated wind extract prepared by inoculating lactic acid bacteria are shown in Table 2 below.

Specifically, Comparative Sample 1 is a liberated pneumophila leaf hot-water extract (GLE) without lactic acid bacteria inoculation and culture, Comparative Sample 2 is a liberated pneumophila root hot-water extract (GRE) without lactic acid bacteria inoculation and culture, and Sample 1 is liberated wind inoculated with L. plantarum. Fermented product powder of leaf extract (GLELp), Sample 2 is a fermented product powder of Haebangpung root extract inoculated with L. plantarum (GRELp), Sample 3 is a fermented product powder of Haebangpung leaf hot water extract inoculated with P. acidilactici (GLEPa) , Sample 4 is the fermented product powder (GREPa) of the balsam root extract inoculated with P. acidilactici (GREPa), and Samples 5 and 6 are the fermented product powder (GLELpPa) of L. plantarum and P. acidilactici mixture inoculated leaves and root extracts. , GRELpPa).

division denomination Lactobacillus type Comparative sample 1 GLE non-fermented Comparative sample 2 GRE non-fermented sample 1 GLELp Lactobacillus plantarum ( Lactobacillus plantarum ) sample 2 GRELp Lactobacillus plantarum ( Lactobacillus plantarum ) sample 3 GLEPA Pediococcus acidilactici sample 4 GREP Pediococcus acidilactici sample 5 GLELpPa Lactobacillus plantarum ( Lactobacillus plantarum ) Pediococcus acidilactici sample 6 GRELpPa Lactobacillus plantarum ( Lactobacillus plantarum ) Pediococcus acidilactici

Example 3. Confirmation of the necessity of adding a carbon source and a nitrogen source

In the preparation of the fermented product of the liberated wind extract of Example 2, an experiment was performed to determine whether it was necessary to add a carbon source or a nitrogen source capable of effectively performing lactic acid bacteria fermentation.

In a medium containing 1 L of the liberated leaves of the leaf hot water extract prepared in Example 1 or the Haebangpung root hot water extract (GLE, GRE), specifically, 1 L of the liberated wind hot water extract of the liquid extract prepared in Example 1 (pH 6.5 to 7.5) A medium was prepared and used without the addition of a nitrogen source, a carbon source, or inorganic salts. As a control, MRS medium, which is a commonly used lactic acid bacteria medium, was prepared.

Were mixed in a 1: The prepared three kinds of the culture medium 1x10 ⁸ cfu / ml Lactic acid concentration L.plantarum (KCCM12299P), P. acidilactici ( KCCM12300P) or the two strains 1 final concentration of 1x10 ⁸ cfu / ml After inoculating each of the mixed strains and incubating at 30 ° C. for 24 hours, LAB Counts (log cfu/ml) were measured. The LAB Counts measurement results are shown in Table 3 below. In the table below, Lp denotes L. plantarum , Pa denotes P.acidilactici , and LaPa denotes a sample inoculated by mixing both strains.

LAB Counts is a solid medium prepared by adding agarose (BD Difco, USA) to each liquid medium, diluted with 1% (v/v) of the culture, spread 1 ml on the LAB plate, and then cultured at 30°C for 24 hours. This was performed by measuring the number of colonies formed.

badge Lp (cfu/ml) Pa (cfu/ml) LpPa (cfu/ml) MRS 1.00 X 10 ⁹ 1.00 X 10 ⁸ 1.00 X 10 ¹⁰ Liberation wind leaf extract (GLE) 4.00 X 10 ⁹ 6.00 X 10 ⁹ 1.50 X 10 ¹² Liberation Wind Root Extract (GRE) 4.00 X 10 ¹⁰ 5.00 X 10 ⁸ 1.00 X 10 ¹¹

As can be seen in Table 3, when L. plantarum was inoculated in MRS medium, LAB Counts was measured as ^{1.00x10 9} cfu/ml after culturing for 24 hours, but in the case of P. acidilactici ^{1.00x10 8} cfu/ml was measured with L. plantarum and P. acidilactici were inoculated into a liberated leaf extract (GLE) medium and the number of lactic acid bacteria in the culture cultured for 24 hours was 4.00x10 ⁹ cfu/ml and 6.00x10 ⁹ cfu/ml, respectively, and L. plantarum and P. In the case of co-inoculation with acidilactici, the number of lactic acid bacteria was increased to about ^{1.50x10 12 cfu/ml.} L. plantarum and P. acidilactici were inoculated into a liberated root extract (GRE) medium and the number of lactic acid bacteria in the culture cultured for 24 hours was 4.00x10 ¹⁰ cfu/ml, 5.00x10 ⁸ cfu/ml, L. plantarum and P. acidilactici In the case of co-inoculation, the number of lactic acid bacteria ^{proliferated to 1.00x10 11} cfu/ml.

As can be seen from the above experimental results, the lactic acid bacteria culture performed without the addition of a nitrogen source or a carbon source to the liberated wind leaf or root extract proliferated better compared to the general lactic acid bacteria MRS medium. Specifically, compared to MRS medium, L. plantarum showed 4 times and P. acidilactici 60 times in liberation leaf extract (GLE), and when the two strains were inoculated together, 150 times of growth was shown, resulting in very excellent proliferation. showed effect. In the case of liberated wind root extract (GRE), compared to the MRS medium, L. plantarum showed 40-fold and P. acidilactici 5-fold, and when both strains were co-inoculated, the growth was 10-fold.

In both the Haebangpung leaf extract or Haebangpung root extract, superior lactic acid bacteria growth effect was confirmed compared to commercial media without the addition of a carbon source or nitrogen source, so a fermented product was prepared without adding a separate nitrogen or carbon source to the Haebangpung leaf extract or root extract in a later experiment. .

Example 4. Confirmation of antioxidant functionality of fermented extracts from Haebangpung

As shown in Table 1 of Example 2, the L. plantarum fermented product of the Haebangpung leaf extract (Comparative 1), the L. plantarum fermented product of the Haebangpung leaf extract (Sample 1), and the P. acidilactici fermented product of the Haebangpung leaf extract (Sample 3) of the present invention. To confirm the antioxidant effect, free radical scavenging activity analysis was performed using DPPH (2,2-diphenyl-1-picrylhydrazyl).

The fermented product prepared by the method of Example 2 was collected by 10 mg/ml, added to 1 mL of sterile water, and diluted to prepare a 0.1% (w/v) sample solution. A stock solution (100 mM Tri-HCl, pH 7.4) was prepared by dissolving DPPH in 1 mM methanol, and in the analysis experiment, it was diluted to a concentration of 100 μM.

Then, 10 μL of each sample solution prepared before the 96-well plate was dispensed at a concentration of 0.1% (w/v), and the change in the antioxidant effect over time was measured as the DPPH inhibition rate (%). BHT (butyl hidroxytoluene) was used as a control.

The antioxidant effect was confirmed by adding 100 μL of 100 μM DPPH to the well plate, blocking the light, and leaving it at room temperature for 10-30 minutes, then irradiating light with a wavelength of 517 nm to measure the absorbance. As a negative control, 100 uL DPPH was treated in methanol or water to measure the absorbance, and zero-point treatment was performed. After measuring the absorbance, the DPPH inhibition rate was calculated by Equation 1, and the experimental results are shown in FIG. 2 and Table 4 below. 2 is a graph of the measured DPPH inhibition rate (%).

[Equation 1]

DPPH scavenging activity (%) = (1-OD sample/OD blind) x 100

division medium ingredients DPPH inhibition rate (%) Comparative sample 1 Liberation wind leaf extract 56.5 sample 1 Haebangpung leaf extract + L. plantarum fermentation 75.6 sample 3 Haebangpung leaf extract + P. acidilactici fermentation 79.2 control sample Butyl Hydroxytoluene (BHT) 73.5

As shown in FIG. 2 , the DPPH inhibition rate of the unfermented liberation leaf extract was 56.5%, and the Fermented L. plantarum fermented product (Sample 1), P. acidilactici In the case of the fermented product (Sample 3), 75.6 and 79.2%, respectively, showed a significantly high DPPH inhibition rate. Each of the fermented products exhibited a higher DPPH inhibition rate by 20%p compared to the unfermented extract. The DPPH inhibition rate of the fermented product was higher than that of the control group, BHT. Therefore, in the case of the lactic acid bacteria culture of the leaf extract of Haebangpung, it was confirmed that the antioxidant effect was significantly increased compared to the unfermented extract. Therefore, it was confirmed that the fermented product of the liberated peony leaf extract of the present invention increases the antioxidant effect according to the culture of lactic acid bacteria.

In addition, an EC ₅₀ (half maximal effective concentration) value was obtained based on the analysis result and shown in FIG. 3 . _{3 shows the EC 50} of the antioxidant effect shown in FIG. 2 .

As shown in the EC ₅₀ value of 3, the non-fermented haebangpung leaf extract (Comparative Sample 1), the sample 1 and 3 of haebangpung leaf extract fermentation water EC ₅₀ value of each as 0.088, 0.063 and 0.063%, the antioxidant is generally known It was confirmed that high antioxidant effect can be expected with a smaller amount than phosphorus BHT (control sample).

Example 5: Confirmation of cell line toxicity

The cell line toxicity test of Haebangpung extract or fermented product was measured using Cell counting kit-8 (CCK-8, Dojindo). The cell line used in the experiment was a mouse melanoma cell line, B16F10 (ATCC CRL-6475). Cells were prepared by adding 10% (v/v) fetal bovine serum (FBS) and 1% (w/v) penicillin-streptomycin to Dulbecco's modified Eagle's Medium (DMEM) medium at 37°C. , 5% CO ₂ Culturing in an incubator was used for the experiment.

1 mg, 100 ug, 10 ug, 1 ug of unfermented liberated peony leaf extract and root extract (Comparative Samples 1 and 2) and the fermented product (Sample 1 to 4) of the liberated balsam leaf and root extract using lactic acid bacteria prepared by the method of Example 2, respectively A sample solution was prepared at a concentration of /ml.

The melanoma cell line was seeded with 5 x 10 ³ cells per well in a 24-well plate and cultured for 24 hours, and each extract sample was dispensed by concentration and cultured for 24, 48 or 72 hours. After adding CCK-8 to the culture and reacting for 2-4 hours, absorbance was measured at 540 nm, and cell viability was measured using the liberated wind untreated group as a control (Cont, 100%), and the cell line The toxicity test results are shown in FIGS. 4 and 5 .

4 is a graph showing the results of toxicity experiments on mouse skin cells B16F10 with an unfermented liberated leaf extract (Comparative 1), a fermented leaf extract of L. plantarum (Sample 1), and a fermented product of P. acidilactici (Sample 3). , Figure 5 shows the results of a toxicity test on mouse skin cells B16F10 with an unfermented liberated peony root extract (Comparative 2), a fermented root extract of L. plantarum (Sample 2), and a fermented product of P. acidilactici (Sample 4). .

In the toxicity experiment of B16F10 cells, which are mouse skin cells shown in FIGS. 4 and 5 , it was confirmed that all of them were non-toxic in the sample solution at a concentration of 1 mg/ml up to 72 hours.

Through the above results, it is possible to confirm the safety of the liberated wind extract and its fermented product provided by the present invention, and the toxicity of the extract or fermented product to skin cells is not confirmed, so that it can be used in cosmetics, household products, and food.

Example 6: Quantitative analysis of melanin in cell lines

A quantitative analysis of melanin was performed to analyze the effect of the composition of the present invention on the amount of melanin production in skin cells. For quantitative analysis of melanin, the rat B16F10 cells used in Example 5 were cultured in DMEM medium containing 10% FBS. Culture conditions are the same as in Example 5.

B16F10 cells are cells derived from the murine melanin-producing epithelium. As the cells grow, melanin is produced and browned. Therefore, when the cells are treated with the whitening function, the presence or absence of the whitening effect or the level of the whitening effect can be determined through the change in the melanin content measured through the absorbance (OD value) compared to the untreated group.

^{3 x 10 5} cells of each of the B16F10 cells were dispensed in a 6 well plate and incubated for 24 hours at 37° C. and _{5% CO 2 ,} Unfermented leaves or root extracts (Comparative Samples 1 and 2), fermented products of Haemophilus leaf extract using L. plantarum and fermented products of root extracts (Samples 1 and 2), P. acidilactici liberation wind Fermented product of leaf extract and fermented product of root extract (samples 3 and 4) were treated with Arbutin 1% (w/v) solution as an experimental group and negative control treated with 0.1% (v/v), respectively, and cultured for 48 hours. The cultured cells were collected and dissolved in 1 N NaOH with 10% (v/v) DMSO added, followed by incubation at 80° C. for 1 hour to measure the absorbance at 475 nm, and the melanin content calculated from the absorbance measurement results in FIG. 6 . graph was shown.

Analysis of the whitening effect of B16F10 cells in mice confirmed that there was an anti-melanin effect in the fermented product (samples 1 and 2), which is a leaf or root extract culture of 0.1% L. plantarum (FIG. 6). Specifically, when the melanin content of the B16F10 cells of the untreated group that was not treated with the liberated wind extract or the lactic acid bacteria fermented product of the liberated wind extract or the lactic acid bacteria fermented product of the liberated wind extract was set to 100%, the melanin content of the unfermented liberated wind leaf extract in the treated group was 90.5%, represented by fermenting haebangpung melanin content of 89.2% of the root extract nateu or, in the L. plantarum in the fermented leaf extract non-fermented haebangpung L. plantarum fermented and non-fermented extract of the roots haebangpung show respectively 71.3% and melanin content of 68.6% Compared to the unfermented extract, the level was about 20%p lower. Specifically, the fermented product of Haebangpung leaf extract was 19.2%p lower than that of the unfermented product, and the fermented product of Haebangpung root extract had 20.6%p lower melanin than the unfermented product. It was confirmed that both the fermented products of both extracts had an anti-melanin effect. P. acidilactici fermented products of melanin leaf extract and root extract showed melanin content of 81.5% and 77.1%, respectively, and both about 10%p lower melanin content compared to unfermented product, specifically 9% each compared to unfermented product p, 12.1%p appeared as a low value, confirming the anti-melanin activity.

Therefore, the lactic acid bacteria fermented product of the liberation wind extract of the present application can be used for skin whitening by reducing the melanin concentration of the skin, and thus the utilization potential is high.

Example 7. GABA TLC Analysis of Fermented Products of Haebangpung Extract

The presence or absence of gaba was confirmed in the liberated wind extract and the fermented product provided by the present invention.

As shown in Table 1 of Example 2, the hot water extracts of liberated leaves or roots (GLE, GRE; Comparative Sample 1 and Comparative Sample 2 of Example 2), Lactobacillus L. plantarum ( KCCM12299P ) and P. acidilactici (KCCM12300P) Co-inoculated samples 5 and 6 were prepared.

In addition, Comparative Samples 1 and 2 were treated and sterilized at 121° C. for 20 minutes, respectively, to prepare Comparative Samples 3 and 4, respectively. In Samples 5 and 6, the lactic acid bacteria incubation time was 48 hours. Two types of samples were prepared, respectively. Table 5 shows the information of samples comparing the GABA content.

division Contents Comparative sample 1 liberation leaf extract Comparative sample 2 liberation wind root extract Comparative sample 3 Liberation wind leaf extract + sterilization Comparative sample 4 Liberation wind root extract + sterilization sample 5 Haebangpung leaf extract 24h fermented product sample 6 Haebangpung Root Extract 24h Fermented Product sample 7 Haebangpung leaf extract 48h fermented product sample 8 Haebangpung Root Extract 48h Fermented Product

Each sample and GABA 1% (w/v) and MSG 0.2% (w/v) standard solutions were added dropwise at 3 ul each to Merck TLC silica GEL and separated with a solvent to check the GABA content in each sample. The composition of the solvent used for the separation was used by mixing n-butanol:acetic acid:water in a volume ratio of 5:2:2. After solvent separation of each sample, 1% (v/v) ninhydrin was sprayed, and then heat was applied to develop color.

7 shows the TLC results. From the left of FIG. 7, 0.2% MSG, 1% GABA, and the unfermented liberation leaf extract of Comparative Sample 1 and the unfermented liberated leaf extract of Comparative Sample 3, which were sterilized at 121° C. for 20 minutes, L. plantarum (KCCM12299P) , P. acidilactici (KCCM12300P) was co-inoculated and the GABA concentration was measured after incubation for 24 hours (Sample 5) or 48 hours (Sample 7), followed by 0.2% MSG, 1% GABA, and non-fermentation of Comparative Sample 2 Comparative sample 4, which was sterilized at 121 °C for 20 minutes with haebangpung root extract and haebangpung root extract, and L. plantarum (KCCM12299P ) and P. acidilactici (KCCM12300P) from the haebangpung root extract were co-inoculated for 24 hours (Sample 6) and 48 hours, respectively. (Sample 8) The result of measuring GABA after incubation is shown. As can be seen in Figure 7, the presence of GABA was confirmed in Comparative Samples 1 and 2 of the extracts of liberated leaves and roots, and even after sterilization, GABA was confirmed in both the sterilized products of the leaf and root extracts, so that GABA in the liberated leaves or root extracts was exposed to heat. stability was confirmed. In addition, it was qualitatively confirmed that L. plantarum (KCCM12299P ) and P. acidilactici (KCCM12300P) co-cultured samples 5 and 6 produced more GABA than before culturing of lactic acid bacteria in the extracts of liberated leaves and roots (FIG. 7).

Example 8. Quantitative analysis of GABA in Haebangpung extract and its fermented product

GABA ELISA kit (AVIVA Systems biology, USA) was used to measure the amount of GABA in the liberated wind extract and its fermented product. First, GABA standard solutions 50, 25, 12.5 6.25, 3.13, 1.56, and 0 ng/ml were prepared using Lyophilized GABA (AVIVA) 100 ng/ml GABA standard to obtain a GABA calibration curve. In addition, as an experimental group, samples 5 and 6 in which L. plantarum and P. acidilactici were co-cultured in the haebangpung leaf extract (comparative sample 1) and haebangpung root extract (comparative sample 2) obtained by the method of Example 2, and the haebangpung leaf and root extracts, respectively. (Table 2) was prepared at a concentration of 1% (v/v) and 0.1% (v/v), respectively. Each 50 ul of the above solution was dispensed in a 96-well plate. 50 ul of 1xGABA complex to which biotin was bound was added to each well, sealed, and left at 37° C. for 60 minutes. After the binding reaction between GABA and biotin in each sample, the reaction solution was removed. 300 ul of 1 x wash buffer was treated for 2 minutes and discarded, and the process was repeated 3 times. 100 ul of 1x avidin-HRP solution was treated in a 96-well plate, sealed, left at 37° C. for 45 minutes, the buffer was removed, and the process of processing and discarding 300 ul of 1x wash buffer for 2 minutes was repeated 2-3 times. For the color development process, 90 ul of TMB solution was treated, placed in a dark room at 37°C for 15-30 minutes, and the reaction was stopped with 50 ul stop solution, and absorbance was measured by irradiating light with a wavelength of 450 nm, and a calibration curve was drawn up and expressed in ng/ml. It was. A graph of the results of the experiment is shown in FIG. 8, and the GABA content for each condition is shown in Table 6 below.

division denomination Medium composition and content GABA concentration (ng/ml) Comparative sample 5 GLE 0.1% Haebangpung leaf extract (0.1% content) - non-fermented product 0.37 Comparative sample 6 GLE 1% Haebangpung leaf extract (1.0% content) - non-fermented 0.75 Comparative sample 7 GRE 0.1% Haebangpung root extract (0.1% content) - non-fermented product 0.38 Comparative sample 8 GRE 1% Haebangpung root extract (1.0% content) - non-fermented product 0.49 sample 9 GLELpPa 0.1% Haebangpung leaf extract fermented product (0.1% content) 0.43 sample 10 GLELpPa 1% Haebangpung leaf extract fermented product (1.0% content) 0.87 sample 11 GRELpPa 0.1% Haebangpung root extract fermented product (0.1% content) 0.46 sample 12 GRELpPa 1% Haebangpung root extract fermented product (1.0% content) 1.31

Haebangpung leaf extract (comparative samples 5 and 6) had a GABA content of 0.37 and 0.75 ng/ml at 0.1 and 1%, respectively, which was higher than the GABA content of 0.38 and 0.49 ng/ml in 0.1 and 1% of the liberation pung root extract. However, when compared at 1% in the fermented product of Haebangpung leaf extract and Haebangpung root extract, the fermented root extract was 1.32 ng/ml, which was higher. In the case of the extract culture using lactic acid bacteria than in the case of simple extraction, the GABA content was higher, and in particular, the fermented root extract showed the highest GABA content, confirming that the fermented product of the root extract of liberation wind was effective in the production of GABA (Fig. 8).

Example 9. Antibacterial effect of fermented extract of liberated wind

An experiment was performed to confirm the antibacterial activity of each fermented product of each hot water extract of Haebangpung leaf and root of the present invention and of Haebangpung leaf and root extract.

9-1: Antibacterial activity against acne bacteria

Acne bacteria Propionibacterium acnes (Propionibacterium acnes, ATCC 6919) was prepared. The acne bacteria were inoculated in RCM medium and pre-cultured at 37° C. for 24 hours. Then, 20 mL of 1% (w/v) LB agar was dispensed into a Petri dish by 20 mL and coagulated to prepare a plate medium. Then, 200 μL of the pre-cultured test strain was added at a time and evenly applied on the medium with a sterilized glass rod. After that, it was dried for 30 minutes. After high-temperature and high-pressure sterilization, a cavity for sample injection was prepared by punching using a 1ml blue tip prepared in parallel with alcohol lamp sterilization.

Comparison 1 and Comparison 2, L. plantarum ( KCCM12299P ), and P. acidilactici (KCCM12300P) prepared by the method of Example 2, which are hot water extracts (GLE, GRE), prepared by the method of Example 2, sample 5 and sample 5, each co-inoculated with 1 mL of P. acidilactici (KCCM12300P) After preparing a 1% (w/v) homogenous solution sample by diluting the freeze-dried powder of Sample 6 in water by 0.01 g/ml each, 30 μL of each 1% (w/v) homogeneous solution sample was sterilized. It was injected into one hole punched.

After injection of the sample, incubated at 37 ° C. for 24-72 hours, and the diameter of the inhibition ring (inhibition clear zone) around the punch was analyzed to analyze the antibacterial properties. Figure 9 shows the results of the antimicrobial analysis.

9-2: Antibacterial activity against dandruff bacteria

In order to confirm the antibacterial effect on dandruff, dandruff bacterium Malassezia furfur (Malassezia furfur, ATCC 14521) was prepared. The dandruff bacteria were inoculated into LNA medium (addition of 1% (v/v) olive oil) and pre-cultured at 37° C. for 24 hours. Then, 20 mL of 1% (w/v) LB agar was dispensed into a Petri dish by 20 mL and coagulated to prepare a plate medium, 200 μL of the previously pre-cultured dandruff bacteria was added, and evenly applied on the medium with a sterilized glass rod. , and dried for 30 min.

Antimicrobial analysis was performed by measuring the diameter of the inhibition ring in substantially the same manner as in Example 9-1. The experimental results for the antibacterial activity are shown in FIG. 9 .

9-3: Antibacterial activity against tooth decay bacteria

In order to confirm the antibacterial effect against tooth decay bacteria, Streptococcus mutans (ATCC3065) was prepared. The tooth decay bacteria were inoculated in BHI medium and pre-cultured at 37° C. for 24 hours. Then, 20 mL of 1% (w/v) LB agar was dispensed in a Petri dish and coagulated to prepare a plate medium, 200 μL of the previously cultured tooth decay bacteria were added each, and evenly applied on the medium with a sterilized glass rod. , and dried for 30 min.

Antimicrobial analysis was performed by measuring the diameter of the inhibition ring in substantially the same manner as in Example 9-1. The experimental results for the antibacterial activity are shown in FIG. 9 .

9-4: Antibacterial activity against dental caries

In order to confirm the antibacterial effect on dental caries, the dental caries bacterium Streptococcus sobrinus ( ATCC33478) was prepared. The dental caries was inoculated in BHI medium and pre-cultured for 24 hours at 37°C. Then, 20 mL of 1% (w/v) LB agar was dispensed in a Petri dish and coagulated to prepare a plate medium. Then, 200 μL of the previously cultured dental caries was added and evenly applied on the medium with a sterilized glass rod. After that, it was dried for 30 minutes.

Antimicrobial analysis was performed by measuring the diameter of the inhibition ring in substantially the same manner as in Example 9-1. The experimental results for the antibacterial activity are shown in FIG. 9 and Table 7 below.

Inhibition ring diameter for each sample Liberation Wind Leaf Extract (GLE) Liberation Wind Root Extract (GRE) unfermented
(Comparative sample 1) Fermentation
(Sample 5) unfermented
(Comparative sample 2) Fermentation
(Sample 6) dental caries
( S. sobrinus ) - - 0.4mm 0.9mm tooth decay
( S. mutans ) - - 0.2mm 0.5mm acne bacillus
( P.acne ) - 0.4mm 0.4mm 0.5mm dandruff
( M. furfur ) - - 0.1mm 1.2mm

As shown in FIG. 9 , when the liberation pung leaf extract (comparative sample 1) or the liberated pneumophila root extract (comparative sample 2) was inoculated, the liberated pung root extract showed an antibacterial effect on caries bacteria and acne bacteria, and the test strain did not grow, resulting in a circular ring. However, in the case of liberated pung leaf extract, a ring indicating an antibacterial effect was not generated. However, in samples 5 and 6 co-inoculated with L. plantarum (KCCM12299P) and P. acidilactici (KCCM12300P), in the case of the fermented product of the leaf extract (Sample 5), antibacterial ring was observed in acnes bacteria, and in the case of the fermented product of the root extract, antibacterial ring was found. A strong antibacterial ring was confirmed in Acne and dandruff. Therefore, it was confirmed that the antibacterial effect of the fermented product of the present invention was superior to that of the unfermented extract. In particular, the root extract of Haebangpung showed excellent antibacterial activity against all three strains.

Therefore, the haebangpung extract has little antibacterial activity, but in the case of the fermented product of the haebangpung extract of the present invention, it was confirmed that the antibacterial activity against acne bacteria, dandruff bacteria, and tooth decay bacteria was excellent by going through the fermentation step after inoculation with lactic acid bacteria. Accordingly, it was confirmed that the extract fermented product of Haebangpung of the present invention can be used as a substance for improving or treating acne, dandruff and periodontal disease.

Korea Microorganism Conservation Center KCCM12299P 20180810 Korea Microorganism Conservation Center KCCM12472P 20190329

<110> Gyeongbuk Institute for Marine Bioindustry <120> Fermented product of Glehnia littoralis extract, preparation method and use thereof <130> DPP20184164KR <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 27F primer <400> 1 agagtttgat cmtggctca 19 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 1492R primer <400> 2 ggttaccttg ttacgactt 19 <210> 3 <211> 1472 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus plantarum KCCM12299P 16SrRNA <400> 3 gaagcagtgg cgggtgctat acatgcagtc gaacgaactc tggtattgat tggtgcttgc 60 atcatgattt acatttgagt gagtggcgaa ctggtgagta acacgtggga aacctgccca 120 gaagcggggg ataacacctg gaaacagatg ctaataccgc ataacaactt ggaccgcatg 180 gtccgagctt gaaagatggc ttcggctatc acttttggat ggtcccgcgg cgtattagct 240 agatggtggg gtaacggctc accatggcaa tgatacgtag ccgacctgag agggtaatcg 300 gccacattgg gactgagaca cggcccaaac tcctacggga ggcagcagta gggaatcttc 360 cacaatggac gaaagtctga tggagcaacg ccgcgtgagt gaagaaaggt ttcggctcgt 420 aaaactctgt tgttaaagaa gaacatatct gagagtaact gttcaggtat tgacggtatt 480 taaccagaaa gccacggcta actacgtgcc agcagccgcg gtaatacgta cgtggcaagc 540 gttgtccgga tttattgggc gtaaagcgag cgcaggcggt tttttaagtc tgatgtgaaa 600 gccttcggct caaccgaaga agtgcatcgg aaactgggaa acttgagtgc agaagaggac 660 agtggaactc catgtgtagc ggtgaaatgc gtagatatat ggaagaacac cagtggcgaa 720 ggcggctgtc tggtctgtaa ctgacgctga ggctcgaaag tatgggtagc aaacaggatt 780 agataccctg gtagtccata ccgtaaacga tgaatgctaa gtgttggagg gtttccgccc 840 ttcagtgctg cagctaacgc attaagcatt ccgcctgggg agtacggccg caaggctgaa 900 actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagct 960 acgcgaagaa ccttaccagg tcttgacata ctatgcaaat ctaagagatt agacgttccc 1020 ttcggggaca tggatacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg 1080 gttaagtccc gcaacgagcg caacccttat tatcagttgc cagcattaag ttgggcactc 1140 tggtgagact gccggtgaca aaccggagga aggtggggat gacgtcaaat catcatgccc 1200 cttatgacct gggctacaca cgtgctacaa tggatggtac aacgagttgc gaactcgcga 1260 gagtaagcta atctcttaaa gccattctca gttcggattg taggctgcaa ctcgcctaca 1320 tgaagtcgga atcgctagta atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc 1380 ttgtacacac cgcccgtcac accatgagag tttgtaacac ccaaagtcgg tggggtaacc 1440 ttttaggaac cagccgctaa gtgacagaat gg 1472 <210> 4 <211> 1350 <212> DNA <213> Artificial Sequence <220> <223> Pediococcus acidilactici KCCM12472P 16SrRNA <400> 4 cgctggcggc gtgcctaata catgcaagtc gaacgaactt ccgttaattg attatgacgt 60 gcttgcactg aatgagattt taacacgaag tgagtggcgg acgggtgagt aacacgtggg 120 taacctgccc agaagcaggg gataacacct ggaaacagat gctaataccg tataacagag 180 aaaaccgcct ggttttcttt taaaagatgg ctctgctatc acttctggat ggacccgcgg 240 cgcattagct agttggtgag gtaacggctc accaaggcga tgatgcgtag ccgacctgag 300 agggtaatcg gccacattgg gactgagaca cggcccagac tcctacggga ggcagcagta 360 gggaatcttc cacaatggac gcaagtctga tggagcaacg ccgcgtgagt gaagaagggt 420 ttcggctcgt aaagctctgt tgttaaagaa gaacgtgggt gagagtaact gttcacccag 480 tgacggtatt taaccagaaa gccacggcta actacgtgcc agcagccgcg gtaatacgta 540 ggtggcaagc gttatccgga tttattgggc gtaaagcgag cgcaggcggt cttttaagtc 600 taatgtgaaa gccttcggct caaccgaaga agtgcattgg aaactgggag acttgagtgc 660 agaagaggac agtggaactc catgtgtagc ggtgaaatgc gtagatatat ggaagaacac 720 cagtggcgaa ggcggctgtc tggtctgtaa ctgacgctga ggctcgaaag catgggtagc 780 gaacaggatt agataccctg gtagtccatg ccgtaaacga tgattactaa gtgttggagg 840 gtttccgccc ttcagtgctg cagctaacgc attaagtaat ccgcctgggg agtacgaccg 900 caaggttgaa actcaaaaga attgacgggg gcccgcacaa gcggtggagc atgtggttta 960 attcgaagct acgcgaagaa ccttaccagg tcttgacatc ttctgccaac ctaagagatt 1020 aggcgttccc ttcggggaca gaatgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt 1080 gagatgttgg gttaagtccc gcaacgagcg caacccttat tactagttgc cagcattcag 1140 ttgggcactc tagtgagact gccggtgaca aaccggagga aggtggggga cgacgtcaaa 1200 tcatcatgcc ccttatgacc tgggctacac acgtgctaca aatggatggt acaacgagtc 1260 gcgaaaccgc gaggtttagc taatctctta aaaccattct cagttcggga ctgtaggctg 1320 caactcgcct acacgaaagt cggaatcgct 1350 <210> 5 <211> 1470 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus plantarum reference 16SrRNA <400> 5 cgcagggcgg gtgctataca tgcagtcgaa cgaactctgg tattgattgg tgcttgcatc 60 atgatttaca tttgagtgag tggcgaactg gtgagtaaca cgtgggaaac ctgcccagaa 120 gcgggggata acacctggaa acagatgcta ataccgcata acaacttgga ccgcatggtc 180 cgagtttgaa agatggcttc ggctatcact tttggatggt cccgcggcgt attagctaga 240 tggtggggta acggctcacc atggcaatga tacgtagccg acctgagagg gtaatcggcc 300 acattgggac tgagacacgg cccaaactcc tacgggaggc agcagtaggg aatcttccac 360 aatggacgaa agtctgatgg agcaacgccg cgtgagtgaa gaagggtttc ggctcgtaaa 420 actctgttgt taaagaagaa catatctgag agtaactgtt caggtattga cggtatttaa 480 ccagaaagcc acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt 540 gtccggattt attgggcgta aagcgagcgc aggcggtttt ttaagtctga tgtgaaagcc 600 ttcggctcaa ccgaagaagt gcatcggaaa ctgggaaact tgagtgcaga agaggacagt 660 ggaactccat gtgtagcggt gaaatgcgta gatatatgga agaacaccag tggcgaaggc 720 ggctgtctgg tctgtaactg acgctgaggc tcgaaagtat gggtagcaaa caggattaga 780 taccctggta gtccataccg taaacgatga atgctaagtg ttggagggtt tccgcccttc 840 agtgctgcag ctaacgcatt aagcattccg cctggggagt acggccgcaa ggctgaaact 900 caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagctacg 960 cgaagaacct taccaggtct tgacatacta tgcaaatcta agagattaga cgttcccttc 1020 ggggacatgg atacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt 1080 aagtcccgca acgagcgcaa cccttattat cagttgccag cattaagttg ggcactctgg 1140 tgagactgcc ggtgacaaac cggaggaagg tggggatgac gtcaaatcat catgcccctt 1200 atgacctggg ctacacacgt gctacaatgg atggtacaac gagttgcgaa ctcgcgagag 1260 taagctaatc tcttaaagcc attctcagtt cggattgtag gctgcaactc gcctacatga 1320 agtcggaatc gctagtaatc gcggatcagc atgccgcggt gaatacgttc ccgggccttg 1380 tacacaccgc ccgtcacacc atgagagttt gtaacaccca aagtcggtgg ggtaaccttt 1440 taggaaccag ccgcctaagt gacagaatgg 1470 <210> 6 <211> 1516 <212> DNA <213> Artificial Sequence <220> <223> Pediococcus acidilactici reference 16SrRNA <400> 6 gctcaggatg aacgctggcg gcgtgcctaa tacatgcaag tcgaacgaac ttccgttaat 60 tgattatgac gtgcttgcac tgaatgagat tttaacacga agtgagtggc ggacgggtga 120 gtaacacgtg ggtaacctgc ccagaagcag gggataacac ctggaaacag atgctaatac 180 cgtataacag agaaaaccgc ctggttttct tttaaaagat ggctctgcta tcacttctgg 240 atggacccgc ggcgcattag ctagttggtg aggtaacggc tcaccaaggc gatgatgcgt 300 agccgacctg agagggtaat cggccacatt gggactgaga cacggcccag actcctacgg 360 gaggcagcag tagggaatct tccacaatgg acgcaagtct gatggagcaa cgccgcgtga 420 gtgaagaagg gtttcggctc gtaaagctct gttgttaaag aagaacgtgg gtgagagtaa 480 ctgttcaccc agtgacggta tttaaccaga aagccacggc taactacgtg ccagcagccg 540 cggtaatacg taggtggcaa gcgttatccg gatttattgg gcgtaaagcg agcgcaggcg 600 gtcttttaag tctaatgtga aagccttcgg ctcaaccgaa gaagtgcatt ggaaactggg 660 agacttgagt gcagaagagg acagtggaac tccatgtgta gcggtgaaat gcgtagatat 720 atggaagaac accagtggcg aaggcggctg tctggtctgt aactgacgct gaggctcgaa 780 agcatgggta gcgaacagga tagataccc tggtagtcca tgccgtaaac gatgattact 840 aagtgttgga gggtttccgc ccttcagtgc tgcagctaac gcattaagta atccgcctgg 900 gggatacgac cgcaaggttg aaactcaaaa gaattgacgg gggcccgcac aagcggtgga 960 gcatgtggtt taattcgaag ctacgcgaag aaccttacca ggtcttgaca tcttctgcca 1020 acctaagaga ttaggcgttc ccttcgggga cagaatgaca ggtggtgcat ggttgtcgtc 1080 agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt attactagtt 1140 gccagcattc agttgggcac tctagtgaga ctgccggtga caaaccggag gaaggtgggg 1200 acgacgtcaa atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggatggt 1260 acaacgagtc gcgaaaccgc gaggtttagc taatctctta aaaccattct cagttcggac 1320 tgtaggctgc aactcgccta cacgaagtcg gaatcgctag taatcgcgga tcagcatgcc 1380 gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccatgag agtttgtaac 1440 acccaaagcc ggtggggtaa ccttttagga gctagccgtc taaggtggga cagatgatta 1500 gggtgaagtc gtaaca 1516

Claims (21)

As a fermented product of lactic acid bacteria of liberated wind solvent extract obtained by inoculating and fermenting lactic acid bacteria in a solvent extract of liberation wind ( Glehnia littoralis ),
The liberation wind is at least one selected from the group consisting of leaves and roots of the liberation wind,
The solvent extract is obtained using water at 60 to 100 °C,
The lactic acid bacteria is one or more lactic acid bacteria selected from the group consisting of Lactobacillus plantarum , accession number KCCM12299P, and Pediococcus acidilactici, accession number KCCM12472P. The fermentation product according to claim 1, wherein the lactic acid bacteria inoculation and fermentation is performed by inoculating lactic acid bacteria 1x10 ⁸ to 1x10 ¹⁵ cfu/ml, and fermentation at a temperature of 25 to 40°C. The fermented product according to claim 1, wherein the solvent extract of liberated wind does not contain additives that are carbon or nitrogen sources. delete The fermented product according to claim 1, wherein the fermented product has a GABA content of 1.01 to 10 times higher than that of the extract before fermentation. The fermented product of claim 1, wherein the fermented product has antibacterial activity against acnes acne, dandruff, dental caries, and caries. The method according to claim 1, wherein the fermented product has an activity of inhibiting melanogenesis of the skin,
The melanin content of the cells treated with the fermented product is 60 to 85% when the melanin content in the untreated cells is set to 100%, the fermented product. obtaining a solvent extract of liberation wind;
Inoculating and fermenting lactic acid bacteria in the solvent extract of the liberation wind;
As a method for producing a lactic acid bacteria fermented product of liberated wind solvent extract,
The liberation wind is at least one selected from the group consisting of leaves and roots of the liberation wind,
The step of obtaining the solvent extract is carried out using water at 60 to 100 °C,
The lactic acid bacteria is one or more lactic acid bacteria selected from the group consisting of Lactobacillus plantarum , accession number KCCM12299P, and Pediococcus acidilactici, accession number KCCM12472P. The method according to claim 8, wherein the solvent extract of the liberation wind does not contain an additive that is a carbon source or a nitrogen source. The method of claim 8, wherein the solvent in the step of obtaining the solvent extract is at least one selected from the group consisting of water, alcohol, alcohols having 1 to 3 carbon atoms, deep sea water, and brine sewage. delete delete delete According to claim 8, wherein the step of inoculating and fermenting the lactic acid bacteria, by inoculating the lactic acid bacteria 1x10 ⁸ to 1x10 ¹⁵ cfu / ml, to perform the fermentation at a temperature of 25 to 40 ℃, the method. The method of claim 8, further comprising drying the fermented product after inoculating and fermenting the lactic acid bacteria. An antibacterial pharmaceutical composition comprising the lactic acid bacteria fermented product of the solvent extract of liberated wind according to any one of claims 1 to 3. A food composition for preventing or improving one or more diseases selected from the group consisting of acne, dandruff, and tooth decay, comprising the lactic acid bacteria fermented product of the solvent extract of liberated wind according to any one of claims 1 to 3. A pharmaceutical composition for the prevention or treatment of one or more diseases selected from the group consisting of acne, dandruff, and tooth decay, comprising the lactic acid bacteria fermented product of the solvent extract of liberated wind according to any one of claims 1 to 3. A quasi-drug composition for oral hygiene, comprising the lactic acid bacteria fermented product of the solvent extract of liberated wind according to any one of claims 1 to 3. A cosmetic composition for preventing dandruff comprising a lactic acid bacterium fermented product of the solvent extract of liberated wind according to any one of claims 1 to 3. A cosmetic composition for skin whitening, comprising a lactic acid bacterium fermented product of the solvent extract of liberated wind according to any one of claims 1 to 3.

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