KR102350437B1 - Cosmetic compositions containing Hwangryunhaedoktang fermented with Lactobacillus plantarum WiKim0111 for anti-acne - Google Patents

Abstract

The present invention relates to a novel Lactobacillus plantarum WiKim0111 strain, a fermented product of Hwangryeonhaedoktang lactic acid bacteria using the same, a method for preparing the same, and a cosmetic composition for preventing or improving acne comprising the fermented product of Hwangryeonhaedoktang lactic acid bacteria. The Lactobacillus plantarum WiKim0111 strain has superior antibacterial and antioxidant activity than the existing Lactobacillus plantarum, and in the case of Hwangryeon Haedoktang lactic acid bacteria fermented product using it, unlike other herbal medicines, it has excellent antibacterial activity and anti-inflammatory effect, resulting in inflammatory skin diseases such as acne. It can be used as a useful material to improve

Description

Cosmetic compositions containing Hwangryunhaedoktang fermented with Lactobacillus plantarum WiKim0111 for anti-acne}

The present invention relates to a fermented product of Hwangnyeonhaedoktang lactic acid bacteria fermented with Lactobacillus plantarum WiKim0111 strain having excellent antibacterial and antioxidant activity.

Hwangryeon Haedok-tang is a prescription that enhances efficacy while removing toxicity from medicinal herbs by brewing four natural herbal remedies: golden, yellow lily, yellow lily, and gardenia. It is known to be effective against sexual dermatitis, eczema, seborrheic dermatitis, contact dermatitis, and pollen disease.

Yellow lotus contains alkaloids such as berberine and worenine, which are known to have antipyretic, analgesic, sedative, local paralysis, blood pressure lowering and biliary effects. Gold has the effect of removing wet and moist heat and hemostasis and detoxification. known to relieve Hwangbaek contains alkaloids such as phellodendrine and berberine, and substances such as obaculactone and obacunone, and antipyretic and analgesic inhibitory effects have been reported. As such, yellow lily, gold, and yellow lily have a function of detoxifying heat and heat in common, so mixing them increases the effect and is known to have a natural antiseptic effect.

Gardenia has been used as an anti-inflammatory, antipyretic, diuretic, hemostasis, sedative, edema, and blood pressure lowering agent. Gardenia is composed of a glycoside form in which various sugars are bound to crocetin among carotenoids, and crocetin, a crocetin digentiobioside, has been reported as a main component. Gardenia extract has been reported to have skin whitening, monoamine oxidase (MAO) inhibitory and anti-inflammatory effects.

Acne is the proliferation of chronic inflammatory skin disease in adolescence through to adults since hormones, due to environmental pollution, stress, eating excessive sebum production and sebum oxidation, acne bacteria which Kutty tumefaciens Acne (Cutibacterium acne) that occurs in the hair follicle sebaceous Hyperkeratinization and inflammation occur, and the present invention confirmed that the fermented product of Hwangryeonhaedoktang fermented with Lactobacillus plantarum kimchi lactic acid bacteria is effective in antioxidant activity, antibacterial activity and anti-inflammatory activity as a cosmetic material for inflammatory skin diseases such as acne. was completed.

Accordingly, it is an object of the present invention to provide a fermented product of Hwangryeonhaedoktang lactic acid bacteria fermented with Lactobacillus plantarum WiKim0111 strain of accession number KACC 92265P, a method for preparing the same, and a cosmetic composition comprising the fermented product of Hwangryeonhaedoktang lactic acid bacteria is to do

In order to achieve the above object, the present invention provides a fermented product of Hwangryeonhaedoktang lactic acid bacteria fermented with the Lactobacillus plantarum WiKim0111 strain of accession number KACC 92265P.

In addition, the present invention provides a fermented product of Hwangryeonhaedoktang lactic acid bacteria fermented with Lactobacillus plantarum WiKim0111 (KACC 92265P) strain, and a cosmetic composition for preventing or improving acne comprising the fermented product of Hwangryeonhaedoktang lactic acid bacteria as an active ingredient.

In addition, the present invention, after sterilizing Hwangryeonhaedok-tang, Lactobacillus plantarum WiKim0111 (KACC 92265P), a lysate thereof, a culture thereof, a concentrate of the culture, and a dried product of the culture are added and fermented, including the step of fermentation. Provided is a method for producing a fermented product of Dok-tang lactic acid bacteria.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention relates to Lactobacillus plantarum WiKim0111 (KACC 92265P) strain.

Lactobacillus plantarum WiKim0111 (KACC 92265P) strain according to the present invention is a new strain of Lactobacillus plantarum derived from kimchi. Although the Lactobacillus plantarum WiKim0111 strain in the present invention was isolated and identified from kimchi, the acquisition route is not limited thereto.

As a result of 16S rRNA sequencing analysis for microbial identification and classification of a lactic acid bacteria strain with excellent antioxidant activity isolated from kimchi through the following examples, it was found to have the nucleic acid sequence of SEQ ID NO: 1. As a result of confirming the homology of the nucleic acid sequence through the blast search program provided by NCBI GenBank (http://www.ncbi.nin.gov ), the Lactobacillus plantarum standard strain JCM1149 and It was confirmed that they were 98% identical.

Therefore, the microorganism of the present invention having a 16S rRNA base sequence of SEQ ID NO: 1 was named Lactobacillus plantarum WiKim0111 (Lactobacillus plantarum WiKim0111), and was deposited at the National Academy of Agricultural Sciences Agricultural Genetic Resources Center on June 13, 2019. (Accession No. KACC 92265P).

Lactobacillus plantarum WiKim0111 of the present invention is a Gram-positive bacterium, a facultive anaerobe capable of growth under both aerobic and anaerobic conditions, and takes the form of a bacillus. In addition, it was confirmed that the antioxidant and antibacterial activity were excellent at the same time compared to the kimchi lactic acid bacteria, which had excellent antibacterial activity against Cutibacterium acne.

The present invention also provides a fermented product of Hwangryeonhaedoktang lactic acid bacteria fermented with Lactobacillus plantarum WiKim0111 (KACC 92265P) strain and a cosmetic composition for preventing or improving acne comprising the fermented product of Hwangryeonhaedoktang lactic acid bacteria as an active ingredient.

The Lactobacillus plantarum WiKim0111 (KACC 92265P) strain may be derived from kimchi.

In addition, the Lactobacillus plantarum WiKim0111 (KACC 92265P) strain may include 16S rRNA of SEQ ID NO: 1.

The fermentation refers to a process of decomposing organic matter using the efficacy of microorganisms (eg, lactic acid bacteria). Fermentation and decay proceed by a similar process, but when a substance useful in our daily life is produced as a result of decomposition, it is called fermentation. Therefore, when herbal medicines are fermented using microorganisms, the toxicity of herbal medicines is reduced, the molecular weight is reduced to facilitate digestion, or when applied to the skin, transdermal absorption is facilitated, thereby improving bioavailability. In addition, although natural ingredients can exhibit pharmacological action by themselves, the pharmacological effect can be further strengthened through fermentation or can exert a pharmacological action that was not present in traditional herbal medicines prior to fermentation by the metabolism of microorganisms.

The fermented Hwangryeonhaedoktang lactic acid bacteria according to the present invention has excellent antioxidant activity and antibacterial activity, and also nitric oxide (NO), cytokines, which are mediators of inflammation increased by LPS stimulation in RAW264.7 cells compared to the control group. was confirmed to have a significant inhibitory effect.

In particular, antibacterial activity could not be confirmed in the case of Hwangryeonhaedoktang before fermentation, but the antibacterial activity was confirmed in the fermented product of Hwangnyeonhaedoktang lactic acid bacteria fermented with Kimchi Lactobacillus WiKim0111 (KACC 92265P). became In addition, it has excellent antioxidant and anti-inflammatory effects, so it can be used as a cosmetic material useful for improving inflammatory skin diseases such as acne.

In addition, the present invention includes a method for producing a fermented product of lactic acid bacteria in Hwangnyeonhaedoktang, which includes the step of fermenting Hwangryeonhaedoktang by adding Lactobacillus plantarum WiKim0111 (KACC 92265P) after sterilization.

The temperature during fermentation by inoculating lactic acid bacteria in Hwangryeonhaedoktang is not particularly limited as long as it is an appropriate temperature for the growth of lactic acid bacteria, but it is carried out at 40° C. or less, preferably at 10 to 35° C. In addition, it is carried out for 36 hours to 48 hours.

In addition, the hwangryeonhaedoktang can be sterilized before inoculation with lactic acid bacteria.

Lactobacillus plantarum WiKim0111 (KACC 92265P) derived from kimchi, which is excellent in antioxidant activity and antibacterial activity, is used for the lactic acid bacteria used for fermentation of Hwangryeonhaedoktang of the present invention, thereby exhibiting antioxidant and antibacterial activity, so it can be used as a cosmetic material for preventing or improving acne. . The lactic acid bacteria are inoculated so that the initial lactic acid bacteria concentration is 10 ⁵ to 10 ⁷ CFU/ml.

The fermented Hwangryeonhaedoktang lactic acid bacteria produced through the above fermentation process may be the culture itself or the culture supernatant, and when used as a cosmetic material, it may be refrigerated at 4°C after undergoing a sterilization process for long-term preservation.

By confirming that the Lactobacillus plantarum WiKim0111 strain according to the present invention has a superior antibacterial effect compared to the existing kimchi lactic acid bacteria, in particular, it exhibits excellent antibacterial, antioxidant and anti-inflammatory effects when used in the fermentation of poisonous Hwangryeonhaedoktang unlike other herbal medicines. It can be usefully used as a cosmetic material having an excellent effect in preventing or improving acne.

1A is a result of investigating the antibacterial activity of several kimchi isolated lactic acid bacteria for primary selection.
Figure 1b is a graph showing the antioxidant activity of the lactic acid bacteria isolated from kimchi, and an excellent antioxidative strain was finally selected.
2 is a result of sequencing microorganisms for 16S rRNA gene sequencing of isolated lactic acid bacteria.
3 is a result of antibacterial activity of Kimchi Lactobacillus WiKim0111 fermented Hwangnyeonhaedoktang.
Figure 4a is a result of ABTS antioxidant measurement method among the antioxidant activity measurement method of fermented Hwangnyeonhaedoktang fermented with kimchi lactic acid bacteria WiKim0111.
Figure 4b shows the results of the DPPH antioxidant measurement method among the antioxidant activity measurement methods of fermented Hwangnyeonhaedoktang, WiKim0111 fermented kimchi lactic acid bacteria.
Figure 5a shows the results of the nitric oxide anti-inflammatory method among the anti-inflammatory activity measurement method of fermented Hwangnyeonhaedoktang, WiKim0111 fermented kimchi lactic acid bacteria.
Figure 5b is a result of IL-1β anti-inflammatory measurement method among the anti-inflammatory activity measurement method of Kimchi lactic acid bacteria WiKim0111 fermented Hwangnyeonhaedoktang.
Figure 5c is the result of the IL-6 anti-inflammatory measurement method among the anti-inflammatory activity measurement method of kimchi lactic acid bacteria WiKim0111 fermented Hwangnyeonhaedoktang.

Hereinafter, the present invention will be described in detail through examples. The following examples only illustrate the present invention, but the scope of the present invention is not limited to the following examples.

Example 1: Lactobacillus selection

Stock the flow Pocono isolated from a variety of sauerkraut (Leuconostoc), and this cellar (Weissella), Lactobacillus (Lactobacillus) in 300 Kutty bacterium in the culture supernatant of the species Solarium acne (Cutibacterium acne) As a result of measuring the antimicrobial activity of the antimicrobial activity These excellent 30 strains were first selected, and a second repeated experiment was performed in the same manner to select 9 strains (FIG. 1a).

As a result of measuring the antioxidant activity (superoxide dismutase) of the culture supernatant of 9 types of kimchi lactic acid bacteria with excellent antibacterial activity against Cutibacterium acne, 4 strains with excellent antioxidant activity (WiKim0111, J7, PK2, CM2) were selected. Kimchi lactic acid bacterium WiKim0111, which has excellent antibacterial and antioxidant activity at the same time, was finally selected for fermentation of Hwangnyeonhaedoktang (Fig. 1b).

The microorganisms used in this experiment were identified through 16S rRNA sequencing of WiKim0111 Kimchi Lactobacillus with excellent antibacterial and antioxidant activity isolated from Chinese cabbage kimchi, 98% identical to Lactobacillus plantarum standard strain JCM1149. was confirmed and named Lactobacillus plantarum WiKim0111 (Lactobacillus plantarum WiKim0111) (FIG. 2).

Example 2: Measurement of antioxidant activity

Fermentation of Hwangryeonhaedoktang is performed by mixing crushed golden, yellow, yellowish white, gardenia mixture (2%) and yeast extract (0.5%) and heat-treating at 121℃ for 2 hours, and then Lactobacillus plantarum WiKim0111 ( Lactobacillus plantarum WiKim0111) 10 ⁷ CFU/ml was inoculated (0.5%) and fermented at 30°C for 36 hours. After taking the culture supernatant and freeze-drying, antibacterial activity, antioxidant activity and anti-inflammatory activity were investigated.

Antibacterial about to enter into force as a skin disease that Lactobacillus Plan tarum WiKim0111 (Lactobacillus plantarum WiKim0111) barberry root to the antioxidant and antimicrobial activity with excellent kimchi lactic acid bacteria herbal natural products such as doktang that effective acne causing bacteria which Kutty tumefaciens Acne (Cutibacterium acne) As a result of comparing and confirming the activity, growth inhibition was confirmed at a concentration of 100 μg/ml or higher in fermented Hwangnyeonhaedoktang. Since antibacterial activity could not be confirmed in Hwangnyeon Haedok-tang, it was considered that antibacterial substances were produced by fermentation of kimchi lactic acid bacteria (FIG. 3).

The antioxidant activity of Hwangryeonhaedok-tang was 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); ABTS assay, 1,1-Diphenyl-2-picryhydrazyl; As a result of measuring the antioxidant activity of ABTS and DPPH at low concentration (6.25-25 μg/ml) of Hwangnyeon Haedok-tang, the antioxidant activity of Kimchi Lactobacillus WiKim0111 was further increased by more than 5-15%, 50-400 μg/ml), there was almost no significant difference between fermented Hwangnyeonhaedok-tang and non-fermented Hwangnyeonhaedok-tang. 4a, 4b).

Example 3: Measurement of anti-inflammatory activity

In a 96-well plate in which the macrophage line RAW264.7 cell (Murine macrophage cell line) acquired from the Korea Cell Line Bank was cultured, 100 μl of DMEM medium containing 10% MTS solution was put into each well and incubated for 3 hours. As a result of measuring cell viability by measuring absorbance at 490 nm using an ELISA microplate reader, 89.2-99.9% cell viability was shown at the concentration of Kimchi Lactobacillus WiKim0111 fermented Hwangnyeonhaedoktang (25-400 μg/ml), and non-fermented Hwangryeonhae Dok-tang (25-400 μg/ml) also showed 88.7-101.9% cell viability, confirming that all of them did not show a significant toxic reaction against RAW264.7 cells.

For anti-inflammatory activity, the concentration of nitrite in the RAW264.7 cell culture was measured using the Griess reagent system, and the nitric oxide scavenging ability contained in the culture was measured using an ELISA kit. As a result of measuring the effects of fermented Hwangryeonhaedok-tang and non-fermented Hwangryeonhaedok-tang (control group) on NO production in RAW264.7 cells using LPS (10 μg/mL), NO concentration in the LPS-treated group was concentration-dependent. It was confirmed that NO production was inhibited by , and it was confirmed that the fermented Hwangryeonhaedoktang had better anti-inflammatory effect (5.7 μM) than the control at a concentration of 400 μg/ml ( FIG. 5a ).

In order to measure the expression level of inflammatory cytokines secreted into the culture medium, it was measured using the Quantikine® ELISA kit. 6) was confirmed, and 10 μl of the sample was treated in RAW264.7 cell line, followed by _{incubation at 37° C., 5% CO 2 in an} incubator for 48 hours.

In the case of IL-1β, as a result of confirming the effect of reducing IL-1β expression when treated at a concentration of 200 μg/ml, it was confirmed that fermented Hwangnyeonhaedok-tang had a further reduced effect on IL-1β expression level (17.2pg/ml) compared to the control. In the case of IL-6, when treated at a concentration of 400 μg/ml, a further decreased effect (2.4 times) in the IL-6 expression level (623.4 pg/ml) of fermented Hwangryeonhaedok-tang was confirmed, indicating that the anti-inflammatory effect was greater than that of the control group. It was confirmed that it was excellent (FIGS. 5b, 5c).

The present invention, Lactobacillus plantarum WiKim0111 (Lactobacillus plantarum WiKim0111), a new strain with excellent antibacterial activity isolated from kimchi, produced an antibacterial effect on acne-causing bacteria when fermented with Hwangnyeonhaedoktang, and was better than that of the non-fermented Hwangryeonhaedoktang control group. Excellent results were confirmed in that the antioxidant and antibacterial activity and the effect of significantly inhibiting inflammatory mediators such as NO increased by LPS stimulation in RAW264.7 cells were increased.

National Academy of Agricultural Sciences Agricultural Genetic Resource Center (Korea) KACC92265P 20190613

<110> KOREA FOOD RESEARCH INSTITUTE <120> Lactobacillus plantarum WiKim0111 <130> P20R10C2009 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1435 <212> DNA <213> Unknown <220> <223> Lactobacillus plantarum WiKim0111 <220> <221> gene <222> (1)..(1435) <400> 1 gcagtcgaac gaactctggt attgattggt gcttgcatca tgatttacat ttgagtgagt 60 ggcgaactgg tgagtaacac gtgggaaacc tgcccagaag cgggggataa cacctggaaa 120 cagatgctaa taccgcataa caacttggac cgcatggtcc gagcttgaaa gatggcttcg 180 gctatcactt ttggatggtc ccgcggcgta ttagctagat ggtggggtaa cggctcacca 240 tggcaatgat acgtagccga cctgagaggg taatcggcca cattgggact gagacacggc 300 ccaaactcct acgggaggca gcagtaggga atcttccaca atggacgaaa gtctgatgga 360 gcaacgccgc gtgagtgaag aagggtttcg gctcgtaaaa ctctgttgtt aaagaagaac 420 atatctgaga gtaactgttc aggtattgac ggtatttaac cagaaagcca cggctaacta 480 cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggattta ttgggcgtaa 540 agcgagcgca ggcggttttt taagtctgat gtgaaagcct tcggctcaac cgaagaagtg 600 catcggaaac tgggaaactt gagtgcagaa gaggacagtg gaactccatg tgtagcggtg 660 aaatgcgtag atatatggaa gaacaccagt ggcgaaggcg gctgtctggt ctgtaactga 720 cgctgaggct cgaaagtatg ggtagcaaac aggattagat accctggtag tccataccgt 780 aaacgatgaa tgctaagtgt tggagggttt ccgcccttca gtgctgcagc taacgcatta 840 agcattccgc ctggggagta cggccgcaag gctgaaactc aaaggaattg acgggggccc 900 gcacaagcgg tggagcatgt ggtttaattc gaagctacgc gaagaacctt accaggtctt 960 gacatactat gcaaatctaa gagattagac gttcccttcg gggacatgga tacaggtggt 1020 gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1080 ccttattatc agttgccagc attaagttgg gcactctggt gagactgccg gtgacaaacc 1140 ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg 1200 ctacaatgga tggtacaacg agttgcgaac tcgcgagagt aagctaatct cttaaagcca 1260 ttctcagttc ggattgtagg ctgcaactcg cctacatgaa gtcggaatcg ctagtaatcg 1320 cggatcagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca 1380 tgagagtttg taacacccaa agtcggtggg gtaacctttt aggaaccagc cgcct 1435

Claims (1)

Lactobacillus plantarum WiKim0111 { Lactobacillus plantarum WiKim0111} with accession number KACC 92265P.

Images