KR102058622B1 - Lactobacillus curvatus having antimicrobial activity against skin phathgens - Google Patents

Abstract

The present invention relates to a Lactobacillus curvatus AMI-1214 KCCM12569P strain, and a cosmetic composition comprising the same.

Description

LACTOBACILLUS CURVATUS HAVING ANTIMICROBIAL ACTIVITY AGAINST SKIN PHATHGENS}

The present invention relates to a Lactobacillus curvatus AMI-1214 KCCM12569P strain, a cosmetic composition and a food composition comprising the same.

Human skin is an organ that is in direct contact with the surrounding environment, and infections of bacteria, fungi, etc., which are involved in various skin diseases, easily occur. For example, the stapling Philo Staphylococcus aureus causing maturation skin nose kusu aureus (Staphylococcus aureus) the person, including the skin flora of Stability Philo nose kusu epidermidis (Staphlyococcus epidermidis), acne bacteria Propionibacterium sludge tumefaciens arc Ness (Propionibacterium acnes ) are present on the skin, and these bacteria break down sebum and sweat secreted on the skin to produce odors. In particular, Propionibacterium acnes ( Proteibacterium acnes ) is a fatty acid degrading enzyme using lipase (lipase) to break down the sebum to produce free fatty acids. These free fatty acids not only irritate the skin but also cause inflammatory lesions such as papules, pustules and nodules, which are red rashes found in acne.

Antimicrobial compounds such as triclocarban and triclosan have been used as antimicrobial agents for these bacteria, but they are known to cause skin side effects such as skin burning, burning, and redness of the skin after long-term use.

As described above, the conventional antimicrobial synthetic material and the cosmetic composition including the same may cause skin irritation and may cause skin trouble.

Korean Patent Registration No. 10-1906309

It is an object of the present invention to provide a Lactobacillus curvatus AMI-1214 KCCM12569P strain.

It is another object of the present invention to provide a cosmetic composition comprising the strain, the living organism thereof, the mycelium thereof, the culture thereof, the lysate thereof or an extract thereof.

One aspect of the present invention for achieving the above object relates to a Lactobacillus curvatus AMI-1214 KCCM12569P strain.

16S rDNA nucleotide sequence for identification and classification of Lactobacillus curbatus AMI-1214 of the present invention is the same as SEQ ID NO: 1 attached to the present specification. Therefore, Lactobacillus curvatus AMI-1214 of the present invention may comprise 16S rDNA of SEQ ID NO: 1.

As a result of analysis of the 16S rDNA sequence of SEQ ID NO: 1, it showed 99% homology with known Lactobacillus curbatus strains, and showed the highest molecular systemic relationship with Lactobacillus curbatus. Therefore, the lactobacillus was identified as Lactobacillus curvatus ( Lactobacillus curvatus ), was named as Lactobacillus curbatus AMI-1214, and deposited on August 02, 2019 at the Korea Microorganism Conservation Center (Accession No. KCCM12569P).

Lactobacillus curbatus AMI-1214 of the present invention is characterized by a novel lactic acid bacterium of Lactobacillus curbatus isolated and identified from Chinese cabbage kimchi.

Another aspect of the present invention for achieving the above object relates to a cosmetic composition comprising the strain, its living organism, its microorganism, its culture, its lysate or its extract. Specifically, the cosmetic composition may be characterized by exhibiting antimicrobial activity, but is not limited thereto, and may also be used for acne improvement.

In addition, the present invention is not limited thereto, and may be used without limitation as long as it is a form of Lactobacillus curbatus AMI-1214 capable of achieving an antibacterial effect or an acne improving effect.

In the present invention, "culture" or "culture medium" means a thing obtained by culturing the novel strain of the present invention in a known liquid medium or a solid medium, and in the present invention, a novel Lactobacillus curbatus AMI-1214 strain It is a concept to include.

In the present invention, "antibacterial" refers to all actions to inhibit bacteria or fungi, etc., in particular, may mean the suppression or removal of bacteria or fungi harmful to the human body.

In one embodiment of the present invention, Lactobacillus curvatus AMI-1214 (Lactobacillus curvatus AMI-1214) When applying the composition containing the KCCM12569P strain culture medium, it was confirmed that the antibacterial activity that inhibits the bacteria residing on the skin (Table 3), it was confirmed that the acne improvement effect when applied to the skin (Table 5).

Specifically, the antimicrobial activity may be antimicrobial activity against Propionibacterium acnes ( Protonibacterium acnes ), the bacteria are known as acne causing bacteria, the strain of the present invention, its live cells, its microorganisms, Compositions comprising their cultures, their lysates or extracts thereof can be used for anti-acne as well as for acne improvement.

In the present invention, "acne" is an acne, which is caused by an increase in the production of sebum in the pores and abnormally keratinized skin epithelial cells to block the openings of the pores, where acne-causing bacteria proliferate and cause inflammation. By suppressing the acne-causing bacteria, it is possible to improve acne by inhibiting the closure of pores and excessive secretion of sebum.

The cosmetic composition of the present invention is a solution, an external ointment, a cream, a foam, a nourishing lotion, a flexible lotion, a pack, a flexible water, an emulsion, a makeup base, an essence, a soap, a liquid detergent, a bath, a sunscreen cream, a sun oil, a suspension, an emulsion It may be prepared in a formulation selected from the group consisting of liquids, pastes, gels, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations, patches and sprays, but is not limited thereto. no.

The cosmetic composition of the present invention may further include one or more cosmetically acceptable carriers formulated in general skin cosmetics, and as conventional components, for example, oil, water, surfactants, moisturizers, lower alcohols, thickeners , Chelating agents, pigments, preservatives, fragrances and the like may be appropriately blended, but is not limited thereto.

The cosmetically acceptable carrier included in the cosmetic composition of the present invention varies depending on the formulation of the cosmetic composition.

When the formulation of the present invention is an ointment, paste, cream or gel, the carrier component is animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide and the like. May be used, but is not limited thereto. These may be used alone or in combination of two or more thereof.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder and the like can be used, and especially in the case of a spray, additionally chlorofluorohards. Propellants such as carboxylic, propane / butane or dimethyl ether may be included, but are not limited thereto. These may be used alone or in combination of two or more thereof.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsion may be used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Propylene glycol, 1,3-butylglycol oil, and the like can be used, and in particular, cottonseed oil, peanut oil, corn seed oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid ester May be used, but is not limited thereto. These may be used alone or in combination of two or more thereof.

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, micro Crystalline cellulose, aluminum metahydroxyde, bentonite, agar or tracant may be used, but is not limited thereto. These may be used alone or in combination of two or more thereof.

Specifically, in the cosmetic composition as described above, the strain, its living cells, its microorganisms, its culture, its crush or its extract may be included in the 0.005% to 30.0% by weight relative to the total weight of the cosmetic composition. More specifically, it may be included in 0.01% by weight to 20% by weight. Within the above range, there is an advantage in that not only excellent antibacterial activity but also an acne improving effect is present, and the formulation of the composition has an advantage of stabilization.

In addition, the composition of the present invention can be used as a method of transdermal administration, such as direct application or spraying on the skin, the route of administration of the composition of the present invention can be administered through any general route as long as it can reach the target tissue.

The amount of the composition of the present invention can be appropriately adjusted according to individual differences or formulations such as age, degree of lesion, etc., and can be used once a day or several times a day by applying an amount to the skin.

Lactobacillus curvatus AMI-1214 ( Lactobacillus curvatus AMI-1214) KCCM12569P strain or a composition comprising the same of the present invention is excellent in antibacterial activity, in particular excellent antibacterial effect against acne causing bacteria.

In addition, the composition of the present invention is not toxic and does not cause side effects even when applied to the human body has high utility as a cosmetic composition.

It is to be understood that the effects of the present invention are not limited to the above effects, but include all effects deduced from the configuration of the invention described in the detailed description or claims of the present invention.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, the present invention is not limited by the following examples.

Example 1 Preparation and Identification of Lactobacillus Curbatus AMI-1214 Strains

1-1. Separation from Chinese Cabbage Kimchi

After cabbage kimchi was crushed and homogenized, 1.0 g of this was suspended, suspended in 9.0 ml of sterile saline solution (0.9% NaCl), and serially diluted to plate the MRS growth medium and the corresponding selective medium of Table 1, respectively. After culturing at 37 ° C. for 24 to 48 hours, strains having different colony types were selected and purified through several pure cultures. Purely isolated strains were plated in MRS solid medium and incubated for 24 hours at 37 ° C. Purely isolated colonies were cultured in MRS liquid medium and mixed with 20% (v / v) glycerol (l) at a ratio of 1: 1. And stored in -80 ℃ cryogenic freezer.

1-2. Identification of Lactobacillus Curbatus AMI-1214 Strains

Isolation strains were identified by 16S rDNA sequence analysis. Genomic DNA of the isolated strain was extracted using DNA using ZR Bacterial DNA MiniPrepTM kit (ZYMORESEARCH) as a template. PCR primers for amplifying 16S rDNA were used as universal primers 27F (5'- AGAGTTTGATCCTGGCTCAG-3 ') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). And 35 cycles of denaturation for 45 seconds at 94 ℃, annealing for 60 seconds at 55 ℃ and 60 seconds at 72 ℃. 16S rDNA sequence was confirmed by Solgent Co., Ltd.

The 16S rDNA sequence of the isolated Lactobacillus curbatus AMI-1214 strain is shown in SEQ ID NO: 1.

SEQ ID NO: primer order 2 27F 5'-AGAGTTTGATCCTGGCTCAG-3 ' 3 1492R 5'-GGTTACCTTGTTACGACTT-3 '

As a result of comparing the nucleotide sequences of the strains obtained above with those found in the GenBank database, Lactobacillus genus showed 90-99.86% nucleotide sequence similarity, indicating that the strains belong to the genus Lactobacillus . In addition, Lactobacillus curvatus KCCM 40715 was compared to the nucleotide sequence of the strain showed a 99% similarity, based on the 16S rDNA gene sequence is commonly accepted as a standard for classifying the species (species) (Stackebrandt & Goebel, 1994) ; Stackebrandt & Ebers, 2006), which has been identified as belonging to the Lactobacillus curvatus species.

The identified strain was named Lactobacillus curvatus AMI-1214 ( Lactobacillus curvatus AMI-1214), and on August 02, 2019, Korea Microbiological Preservation Center (address: 45, 2ga - gil, Hongjenae , Seodaemun-gu, Seoul, Korea) And a patent number of KCCM12569P.

Single colony of Lactobacillus curvatus AMI-1214 ( Lactobacillus curvatus AMI-1214), the strain isolated in the above example, was added to the flask and incubated in MRS medium for 37 hours and 48 hours. Suspension cultured cells were used after centrifugation to obtain supernatant, filtered through a 0.22um filter to remove the cells. Using the strain culture solution prepared above, the growth test and the acne improvement effect was confirmed along with the cytotoxicity test.

As a control, a culture solution of Lactobacillus curbatus KCCM 40715 strain, which showed 99% homology on the 16S rDNA gene sequence, was used, and the method of preparing strain culture solution of the control group was also performed.

Experimental Example 1. Cytotoxicity Test

To verify skin safety, cytotoxicity tests on human fibroblasts were performed. Fibroblasts were plated at 5 x 10 ³ cells / well in 96 well plates, and then incubated for 24 hours. Thereafter, the prepared strain culture solution was treated for each concentration, and after 48 hours, the medium containing the strain culture solution was removed. Thereafter, 10% EZ-Cytox reagent was mixed in DMEM media, and 100ul of each well was dispensed. After 2 hours of incubation, the absorbance was measured at 450 nm with a Microplate reader (Biotek, Synergy HT). Cell viability was then calculated according to Equation 1 below.

[Equation 1]

Cell viability (%) = (absorbance of strain culture treated group / absorbance of strain culture untreated group) X 100

Human fibroblast cell survival rate (%) Concentration (ug / ml) 0 0.5 One 3 5 Lactobacillus curbatus AMI-1214 strain 100.00 101.08 102.10 98.25 85.31 Lactobacillus curbatus KCCM 40715 strain 100.00 100.50 97.53 95.41 77.83

As shown in Table 2, it was confirmed that the strain of the present invention can be used as a safe material does not appear cytotoxicity.

Experimental Example 2. Confirmation of antimicrobial activity

The antibacterial activity of the strain of the present invention against skin flora was confirmed by using a paper disc diffusion assay.

Specifically, Propionibacterium acnes ( Propionibacterium acnes ATCC 6919), incubated for 48 hours in an RCM liquid medium and then diluted to 1.0 X 10 ⁶ CFU / ml level in sterile saline (0.9% Nacl). After diluting the diluted culture medium with 100 µl of RCM solid medium (Agar 1.8%) (final 1.0 X 10 ⁵ CFU / ml), sterilized tongs are made with the convex part of the paper disc facing up, and then Lactobacillus Curbatus AMI. -1214 strain culture solution, Lactobacillus Curbatus KCCM 40715 strain culture solution and positive control group erythromycin 0.01mg / ml were injected with 40 μl, the paper disc was left for a while, and then inverted and incubated for 48 hours under anaerobic conditions. The antimicrobial activity was confirmed by confirming the formation.

Inhibitory ring (mm) Lactobacillus
Curbatus AMI-1214 Lactobacillus
Curbatus
KCCM 40715 Positive control
(Erythromycin 0.01 mg / ml) Probionibacterium Acne 13 8 17

As shown in Table 3, it was confirmed that the Lactobacillus Curbatus AMI-1214 strain culture solution of the present invention exhibited excellent antibacterial activity. In particular, the Propionibacterium acnes ATCC 6919 strain was acne or atopy Known as a trigger strain suggests that the strain of the present invention can exhibit an improvement effect on acne.

Preparation Example 1 Preparation of Essence Containing Lactobacillus Curbatus AMI-1214 Strain Culture Solution

A cosmetic composition of the essence formulation was prepared as shown in Table 4 below (unit: wt%).

ingredient Content (% by weight) Cetearyl Alcohol 2 Cetearylglucoside One Jojoba Seed Oil 5 Silica 0.5 glycerin 10 Lactobacillus curbatus AMI-1214 strain culture 5 Purified water To 100

Preparation Example 2 Preparation of Cream Containing Lactobacillus Curbatus AMI-1214 Strain Culture Solution A cosmetic composition of a cream formulation was prepared as shown in Table 5 below (unit: wt%).

ingredient Content (% by weight) Lactobacillus curbatus AMI-1214 strain culture 5 Stearic acid 15 ethanol One Potassium hydroxide 0.7 glycerin 5 Propylene glycol 3 antiseptic Quantity incense Quantity Purified water to 100

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form. The scope of the present invention is defined in the following claims. It is to be construed that all changes or modifications represented by the meaning and scope of claims and equivalents thereof are included in the scope of the present invention.

Korea Microorganism Conservation Center (overseas) KCCM12569P 20190802

<110> AMI COSMETIC CO., LTD. <120> LACTOBACILLUS CURVATUS HAVING ANTIMICROBIAL ACTIVITY AGAINST SKIN          PHATHGENS <130> 19PP30865 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 1434 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus curvatus AMI-1214 KCCM12569P 16S rDNA <400> 1 gcagtcgaac gcactctcgt tagattgaag aagcttgctt ctgattgata acatttgagt 60 gagtggcgga cgggtgagta acacgtgggt aacctgccct aaagtggggg ataacatttg 120 gaaacagatg ctaataccgc ataaaaccta gcaccgcatg gtgcaaggtt gaaagatggt 180 ttcggctatc actttaggat ggacccgcgg tgcattagtt agttggtgag gtaaaggctc 240 accaagaccg tgatgcatag ccgacctgag agggtaatcg gccacactgg gactgagaca 300 cggcccagac tcctacggga ggcagcagta gggaatcttc cacaatggac gaaagtctga 360 tggagcaacg ccgcgtgagt gaagaaggtt ttcggatcgt aaaactctgt tgttggagaa 420 gaacgtattt gatagtaact gatcaggtag tgacggtatc caaccagaaa gccacggcta 480 actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga tttattgggc 540 gtaaagcgag cgcaggcggt ttcttaagtc tgatgtgaaa gccttcggct caaccgaaga 600 agtgcatcgg aaactgggaa acttgagtgc agaagaggac agtggaactc catgtgtagc 660 ggtgaaatgc gtagatatat ggaagaacac cagtggcgaa ggcggctgtc tggtctgtaa 720 ctgacgctga ggctcgaaag catgggtagc aaacaggatt agataccctg gtagtccatg 780 ccgtaaacga tgagtgctag gtgttggagg gtttccgccc ttcagtgccg cagctaacgc 840 attaagcact ccgcctgggg agtacgaccg caaggttgaa actcaaagga attgacgggg 900 gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttaccagg 960 tcttgacatc ctttgaccac tctagagata gagctttccc ttcggggaca aagtgacagg 1020 tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1080 caacccttat tactagttgc cagcatttag ttgggcactc tagtgagact gccggtgaca 1140 aaccggagga aggtggggac gacgtcaaat catcatgccc cttatgacct gggctacaca 1200 cgtgctacaa tggatggtac aacgagtcgc gagaccgcga ggtttagcta atctcttaaa 1260 accattctca gttcggattg taggctgcaa ctcgcctaca tgaagccgga atcgctagta 1320 atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac 1380 accatgagag tttgtaacac ccaaagccgg tgaggtaacc ttcgggagcc agcc 1434 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27F primer <400> 2 agagtttgat cctggctcag 20 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 1492R primer <400> 3 ggttaccttg ttacgactt 19

Claims (8)

As Lactobacillus curvatus AMI-1214 KCCM12569P strain,
Wherein the strain exhibits antimicrobial activity against Propionibacterium acnes , Lactobacillus curvatus AMI-1214 KCCM12569P strain. The method of claim 1,
The strain comprises a 16S rDNA sequence of SEQ ID NO: 1, Lactobacillus curvatus AMI-1214 ( Lactobacillus curvatus AMI-1214) KCCM12569P strain. delete delete A cosmetic composition comprising the strain of claim 1 or 2, a living organism thereof, a microorganism thereof, a culture thereof, a lysate thereof or an extract thereof. delete delete The cosmetic composition according to claim 5, wherein the cosmetic composition is for acne improvement.