KR102058623B1 - Leuconostoc citreum having antimicrobial activity against skin phathgens - Google Patents

Abstract

The present invention relates to a Leuconostoc citreum AMI-1227 KCCM12570P strain, and a cosmetic composition comprising the same.

Description

LEUCONOSTOC CITREUM HAVING ANTIMICROBIAL ACTIVITY AGAINST SKIN PHATHGENS}

The present invention relates to a Leuconostoc citreum AMI-1227 KCCM12570P strain, and a cosmetic 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. 10-1992331

It is an object of the present invention to provide a Leuconostoc citreum AMI-1227 KCCM12570P 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 strain of Leuconostoc citreum AMI-1227 KCCM12570P.

16S rDNA nucleotide sequence for the identification and classification of leukonostock citrium AMI-1227 of the present invention is the same as SEQ ID NO: 1 attached to the present specification. Accordingly, the Leuconostoc citreum AMI-1227 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 leuconosstock citrium strains, showing the highest molecular systemic relationship with leuconosstock citrium. Therefore, the lactic acid bacteria were identified as Leuconostoc citreum , was named as Leukonostock citrium AMI-1227, and was deposited with the Korea Microorganism Conservation Center on August 02, 2019 (Accession No. KCCM12570P).

Leukonostock citrium AMI-1227 of the present invention is characterized by a novel lactic acid bacterium of leukonostock citrium isolated and identified from Chinese cabbage kimchi (adaptation).

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 to the above form, and may be used without limitation as long as it is in the form of leukonostock citrium AMI-1227 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 leukonostock citrium AMI-1227 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, Leukonostocitium AMI-1227 ( (Leuconostoc citreum AMI-1227) When applying the composition containing the KCCM12570P strain culture solution, the antibacterial activity of inhibiting the bacteria residing on the skin appears (Table 3), it was confirmed that the acne improvement effect when applied to the skin (Table 5).

Specifically, it may be an antimicrobial activity against Propionibacterium acnes ( Protonibacterium acnes ), the bacteria are known as acne-causing bacteria, the strain of the present invention, its living organism, its microorganism, its culture, its A composition comprising a lysate or extract thereof may be utilized for acne as well as for antibacterial purposes.

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.

Leuconostoc citreum AMI-1227 ( Leuconostoc citreum AMI-1227) KCCM12570P 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 Leukonostock Citrium AMI-1227 Strains

1-1. Separation from Chinese Cabbage Kimchi

After crushing and homogenizing cabbage kimchi (adult), 1.0 g of this was taken and suspended in 9.0 ml of sterile saline solution (0.9% NaCl) and diluted stepwise to smear the MRS growth medium in Table 1 and each corresponding selection medium. It was. 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 Leukonostock Citrium AMI-1227 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 Leukonostock Citrium AMI-1227 strain is shown as 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, it was analyzed that the similarity was 99% compared to the nucleotide sequence of the Leuconostoc citreum KCCM 12030 strain, which is generally accepted as a standard for classifying species based on the 16S rDNA gene sequence (Stackebrandt & Goebel, 1994). (Stackebrandt & Ebers, 2006), which has been identified as belonging to the species Leuconostoc citreum .

The identified strain was named Leuconostoc citreum AMI-1227 ( Leuconostoc citreum AMI-1227), the Korea Microbiological Conservation Center, an official depository institution on August 02, 2019, 45, Yugaenae 2ga-gil, Seodaemun-gu, Seoul, Korea And a patent number of KCCM12570P.

Single colony of Leuconostoc citreum AMI-1227 ( Leuconostoc citreum AMI-1227), 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 medium of the leukonostock citrium KCCM 12030 strain, which showed 99% homology on the 16S rDNA gene sequence, was used, and the method of preparing the strain culture solution of the control group was also performed in the same manner.

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 Leukonostock Citrium AMI-1227 Strain 100.00 102.4 99.7 95.4 90.1 Leukonostock Citrium KCCM 12030 Strain 100.00 101.1 95.8 91.7 88.4

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 ATCC 6919, Propionibacterium acnes ATCC 6919, was incubated for 48 hours in RCM liquid medium and 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 level), sterilized tongs are made with the convex part of the paper disc facing up, and then leuconosstock citrium AMI. -1227 strain culture solution, leukonostock citrium KCCM 12030 strain culture solution and 0.01 mg / ml of positive control erythromycin (erythromycin) inject 40 ㎕, leave the paper disc for a while, and inverted for 48 hours in anaerobic conditions The antimicrobial activity was confirmed by confirming the formation.

Inhibitory ring (mm) Leukonostock Citrium
AMI-1227 Leukonostock Citrium
KCCM 12030 Positive control
(Erythromycin 0.01mg / ml) Probionibacterium Acne 14.5 8 17

As shown in Table 3 above, it was confirmed that the antimicrobial activity of the present invention was applied to the culture medium of the leukonostock citrium AMI-1227 strain. In particular, the Propionibacterium acnes ATCC 6919, a strain induced acne As the strain is known, it suggests that the strain of the present invention may exhibit an improvement effect on acne.

Preparation Example 1 Preparation of Essence Containing Leukonostock Citrium AMI-1227 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 Leukonostock Citrium AMI-1227 Strain Culture 5 Purified water To 100

Preparation Example 2 Preparation of Cream Containing Leukonostock Citrium AMI-1227 Strain Culture Solution A cosmetic composition of a cream formulation was prepared as shown in Table 5 below (unit: wt%).

ingredient Content (% by weight) Leukonostock Citrium AMI-1227 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) KCCM12570P 20190802

<110> AMI COSMETIC CO., LTD. <120> LEUCONOSTOC CITREUM HAVING ANTIMICROBIAL ACTIVITY AGAINST SKIN          PHATHGENS <130> 19PP30866 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 1412 <212> DNA <213> Artificial Sequence <220> <223> Leuconostoc citreum AMI-1227 KCCM12570P 16S rDNA <400> 1 cagtcgaacg cgcagcgaga ggtgcttgca cctttcaagc gagtggcgaa cgggtgagta 60 acacgtggat aacctgcctc aaggctgggg ataacatttg gaaacagatg ctaataccga 120 ataaaactta gtatcgcatg atatcaagtt aaaaggcgct acggcgtcac ctagagatgg 180 atccgcggtg cattagttag ttggtggggt aaaggcttac caagacgatg atgcatagcc 240 gagttgagag actgatcggc cacattggga ctgagacacg gcccaaactc ctacgggagg 300 ctgcagtagg gaatcttcca caatgggcgc aagcctgatg gagcaacgcc gcgtgtgtga 360 tgaaggcttt cgggtcgtaa agcactgttg tatgggaaga aatgctaaaa tagggaatga 420 ttttagtttg acggtaccat accagaaagg gacggctaaa tacgtgccag cagccgcggt 480 aatacgtatg tcccgagcgt tatccggatt tattgggcgt aaagcgagcg cagacggttg 540 attaagtctg atgtgaaagc ccggagctca actccggaat ggcattggaa actggttaac 600 ttgagtgttg tagaggtaag tggaactcca tgtgtagcgg tggaatgcgt agatatatgg 660 aagaacacca gtggcgaagg cggcttactg gacaacaact gacgttgagg ctcgaaagtg 720 tgggtagcaa acaggattag ataccctggt agtccacacc gtaaacgatg aatactaggt 780 gttaggaggt ttccgcctct tagtgccgaa gctaacgcat taagtattcc gcctggggag 840 tacgaccgca aggttgaaac tcaaaggaat tgacggggac ccgcacaagc ggtggagcat 900 gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcct ttgaagcttt 960 tagagataga agtgttctct tcggagacaa agtgacaggt ggtgcatggt cgtcgtcagc 1020 tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttatt gttagttgcc 1080 agcattcagt tgggcactct agcgagactg ccggtgacaa accggaggaa ggcggggacg 1140 acgtcagatc atcatgcccc ttatgacctg ggctacacac gtgctacaat ggcgtataca 1200 acgagttgcc aacctgcgaa ggtgagctaa tctcttaaag tacgtctcag ttcggactgc 1260 agtctgcaac tcgactgcac gaagtcggaa tcgctagtaa tcgcggatca gcacgccgcg 1320 gtgaatacgt tcccgggtct tgtacacacc gcccgtcaca ccatgggagt ttgtaatgcc 1380 caaagccggt ggcctaacct tcgggaggga gc 1412 <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)

Leuconostoc citreum AMI-1227 KCCM12570P strain,
Said strain will exhibit antimicrobial activity against Propionibacterium acnes , Leuconostoc citreum AMI-1227 ( Leuconostoc citreum AMI-1227) KCCM12570P strain. The method of claim 1,
The strain comprises the 16S rDNA sequence of SEQ ID NO: 1, Leuconostoc citreum AMI-1227 ( Leuconostoc citreum AMI-1227) KCCM12570P 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.