KR102349372B1 - Cosmetic composition for promoting the ceramide formation - Google Patents

Abstract

The present invention relates to a cosmetic composition which includes a lactic acid bacteria strain containing a glucosylceramase (gba) gene, and improves ceramide formation in the skin. According to the present invention, the cosmetic composition for improving ceramide formation is provided.

Description

Cosmetic composition for promoting the ceramide formation {Cosmetic composition for promoting the ceramide formation}

The present invention relates to a cosmetic composition that improves ceramide formation.

Recently, as environmental stress increases, interest in skin health is increasing. In particular, the outermost layer of the skin (stratum corneum) is the forefront of skin protection and is mainly composed of keratinocytes and lipids.

Lipids mainly include several types of cholesterol, fatty acids and ceramides. Ceramide, one of the sphingolipids, plays an important role in skin barrier function (skin homeostasis).

In general, ceramides are converted by two hydrolases named glucosylceramides (GCase) and sphingomyelinase (SMase).

Numerous clinical studies have shown that ceramide deficiency is directly or indirectly associated with atopic dermatitis (AD) and psoriasis.

Therefore, in order to maintain a healthy skin condition, it is important to strengthen the skin barrier by increasing the level of ceramide.

Korea Patent Publication No. 10-2018-0046831 (published on May 09, 2018)

An object of the present invention is to provide a cosmetic composition that improves ceramide formation.

The present invention relates to a cosmetic composition for improving ceramide formation in the skin containing a Lactobacillus rhamnosus-based lactic acid bacteria strain as an active ingredient.

The Lactobacillus rhamnosus-based lactic acid bacteria strain may be characterized in that it is Lactobacillus rhamnosus IDCC 3201 deposited as ATCC BAA-2836 by the American Society for Conservation of Microorganisms.

The Lactobacillus rhamnosus IDCC 3201 may be characterized in that it is isolated from the feces of a breast-fed infant.

The Lactobacillus rhamnosus IDCC 3201 may be characterized in the form of a lysate.

The cosmetic composition may be characterized in that it improves ceramide formation by increasing sphingomyelin degrading enzyme (SMase) activity.

The cosmetic composition may be characterized in that it has an effect of improving the skin barrier.

The cosmetic composition is an aqueous solution, suspension, emulsion, paste, gel. It may be characterized as having a formulation selected from the group consisting of creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations, and sprays.

The present invention includes a lactic acid bacteria strain containing a gba (Glucosylceramase) gene, and relates to a cosmetic composition that improves ceramide formation in the skin.

The lactic acid bacteria strain may include Lactobacillus rhamnosus IDCC 3201 deposited with the ATCC BAA-2836 accession number.

According to the present invention, a cosmetic composition for improving ceramide formation is provided.

Figure 1 shows the increase in sphingomyelinase (SMase) activity by the treatment of Lactobacillus rhamnosus IDCC 3201,
Figure 2 shows the increase in sphingomyelinase (SMase) activity according to the cell density after Lactobacillus rhamnosus IDCC 3201 treatment,
3 shows the absence of cytotoxicity of Lactobacillus rhamnosus IDCC 3201 to keratinocytes;
Figure 4 shows that ceramide formation is improved by Lactobacillus rhamnosus IDCC 3201 treatment,
Figure 5 shows the mechanism of improving ceramide formation by Lactobacillus rhamnosus IDCC 3201 and lysate treatment containing it as an active ingredient,
Figure 6 shows a circular genetic map showing the gene location that enhances ceramide formation in Lactobacillus rhamnosus IDCC 3201.

Hereinafter, the present invention will be described in more detail with reference to the drawings.

Since the accompanying drawings are only an example shown in order to explain the technical idea of the present invention in more detail, the spirit of the present invention is not limited to the accompanying drawings. In addition, in the accompanying drawings, the size and spacing may be exaggerated differently from reality in order to explain the relationship between each component.

The present invention relates to a cosmetic composition for improving ceramide formation in the skin containing a Lactobacillus rhamnosus-based lactic acid bacteria strain as an active ingredient.

Lactobacillus rhamnosus is preferably a lactobacillus strain classified as Lactobacillus rhamnosus phylogenetically, and more preferably a Lactobacillus rhamnosus lactic acid bacteria strain derived from human and dairy products such as Breast-fed infant's faces and breast milk mouth.

In the experimental example of the present invention, an extract from the feces of infants fed human milk was used, but the present invention is not limited thereto.

The lactic acid bacteria strain may be included in the cosmetic composition in the form of a lysate. The lysate may be prepared through physical contact, but is not limited thereto. Before dissolution, the lactic acid bacteria strain may undergo a culture process, and may be cultured in any one selected from the group consisting of MRS medium, BL agar medium, and BCP-added plate measurement medium. The form of the lysate is not limited, and may be, for example, a liquid, an emulsion, or a solid.

The cosmetic composition according to the present invention may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing It may be formulated as cleansing, oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto.

More specifically, when the composition of the present invention is used as a cosmetic composition, a cosmetic composition selected from a lotion, an emulsion, a cream, an essence, a gel, a pack, and a cleansing cream; It may be prepared in a formulation selected from the group consisting of makeup cosmetics such as foundation.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component. can

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additional chlorofluorohydrocarbon, propane /may contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, and microcrystals Adult cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used.

When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide as carrier components Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester and the like may be used.

In addition, the cosmetic composition of the present invention requires oil, water, surfactant, moisturizer, lower alcohol, thickener, chelating agent, pigment, preservative, or fragrance, etc. to be formulated in general skin cosmetics in addition to the ingredients described above in each formulation. It can be mixed and used as appropriate.

For example, the softening lotion contains 1 to 10% by weight of polyhydric alcohols (propylene glycol, glycerin, etc.) in addition to the lactic acid bacteria strain according to the present invention and 0.05 to 2% by weight of surfactants (polyethylene oleyl ether, polyoxyethylene hydrogenated castor oil, etc.) It can be prepared to contain.

Nutrient lotion and nourishing cream contain 5 to 20% by weight of oils (squalane, petrolatum, octyldodecanol, etc.) and 3 to 15% by weight of wax components (cetanol, stearyl alcohol, beeswax, etc.) in addition to the lactic acid bacteria strain according to the present invention. It can be prepared to contain, and the essence can be prepared by containing 5 to 30% by weight of polyhydric alcohols such as glycerin and propylene glycol in addition to the strain culture solution according to the present invention.

Massage cream can be prepared by containing 30 to 70% by weight of oil, such as liquid paraffin, petrolatum, isononylisononanoate, in addition to the lactic acid bacteria strain according to the present invention.

In addition to the lactic acid bacteria strain according to the present invention, the pack contains kaolin, talc, zinc oxide, titanium dioxide, etc. It can be prepared as a wash-off pack containing 5 to 30% by weight of the pigment.

The content in the cosmetic composition of the Lactobacillus rhamnosus-based lactic acid bacteria strain according to the present invention is 0.01 to 20% by weight, 0.01 to 10% by weight, 1 to 10% by weight, 0.5 to 5% by weight or 0.1 to 1% by weight % can be

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for illustrating the present invention and should not be construed as limiting the scope of the present invention to these examples.

[Experimental example]

1. Preparation of Samples

1) Preparation of lactic acid bacteria strains and lactic acid bacteria lysates

Lactobacillus rhamnosus IDCC 3201 extracted from the feces of breastfed infants and Lactobacillus thermophilus IDCC 2201 extracted from home-made yogurt were isolated from industrial lactic acid bacteria strains, and other lactic acid bacteria strains from kimchi (Korean fermented food).

All lactic acid bacteria strains were cultured in MRS medium, centrifuged, and freeze-dried to obtain a cell density of ^{about > 10 10 (CFU)/g.}

Thereafter, the freeze-dried lactic acid bacteria strain was suspended in 500 ml of phosphate buffered solution (PBS) and physically dissolved using a microemulsion device (PicoMax MN400BF; Micronox, Seongnam, Korea) to prepare a lactic acid bacteria strain lysate.

2) cell culture

Human epidermal keratinocytes (HEK) were purchased from PromoCell (Heidelberg, Germany). Cells were grown in growth medium 2 containing bovine pituitary extract, epidermal growth factor, insulin, hydrocortisone, epinephrine, transferrin and 0.06 nM CaCl _{2 (Promocell).}

After incubation at 37°C with 5% CO ₂ for 3-5 days, cells were separated using 0.25% trypsin-EDTA and subcultured and maintained.

2. Analysis method

1) Measurement of Sphingomyelinase (SMase) Activity

SMase activity was measured using a SMase assay kit (Abcam, Cambridge, UK). The principle of the assay kit is to quantify phosphocholine produced by hydrolysis of sphingomyeline using colorimetric analysis at 655 nm.

50 μL of sphingomyeline was incubated with various lactic acid bacteria lysates at 37° C. for 1 hour.

Thereafter, color change was measured using a microplate reader (SpectraMax iD3; Molecular Devices, CA, USA).

After the measurement, Smase activity was measured according to standard curves (Caribration curves) using the absorbance value compared to the standard amount of SMase.

2) Cell viability assay

The cell viability of human epidermal keratinocytes (HEK) by the lactic acid bacteria strain lysate according to the experiment was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).

Human epidermal keratinocytes (NHEK) were cultured in 96 well plates and Lactobacillus rhamnosus treated with lysates of IDCC 3201 at cell densities ranging from ^{1 x 10 9} - 5 x 10 ^{10 CFU for 24 hours.} After that, MTT was added to each 96-well plate and incubated at 37° C. for 90 minutes.

After removal of MTT, 100uL of dimethyl sulfoxide (DMSO) was added to dissolve formazan.

The color developed by the above process was measured at 570-630 nm using a microplate (SpectraMax iD3).

3) Measurement of ceramide content

Ceramide content in human epidermal keratinocyte (NHEK) suspension was measured using an ELISA kit (Human Ceramide ELISA Kit; MyBioSource, CA).

A human epidermal keratinocyte (NHEK) culture suspension containing SMase was treated in a 96-well plate coated with human monoclonal antibody.

Cells were incubated with avidin-peroxidase-conjugated biotin-labeled polyclonal antibody. Thereafter, a chromogen solution was added for ELISA chromaticity measurement capable of measuring 450 nm.

Ceramide content was measured according to standard curves (Caribration curves) versus the standard amount of ceramide.

4) Lactobacillus strain gene search for ceramide biosynthesis

Whole genome sequencing of Lactobacillus rhamnosus IDCC 3201 was performed using the PacBio RSII instrument including the Illumina platform (Macrogen, Seoul, Korea).

Nucleotide sequences were formed by a single molecule real-time (SMRT) sequencing system.

For the small strand sequence fragments formed in this way, each small consensus sequence was obtained through sequence correction, sequence error filter, and overlapping gene sequences, and after that, contigs were finally generated.

Finally, higher quality sequences were obtained after error correction of Contig constructed by Pilon (version 1.21).

To find bacterial enzymes involved in ceramide biosynthesis from probiotics, amino acid sequence enzymes were collected.

Table 1 below shows the enzyme sphingomyelinase (EC 3.1.4.12), which is provided by the UniProtKB database and shows a conserved amino acid sequence pattern in the enzyme by constructing HMMs; glucosylceramidase (EC 3.2.1.45); galactosylceramidase (EC 3.2.1.46), ceramide synthase (EC 2.3.1.24), and sphingolipid-4-desaturase (EC 1.14.19.17) are shown.

The genome of Lactobacillus rhamnosus IDCC 3201 was targeted to be searched using the HMM search tool of the HMMER package in which the profile of HMM was built.

Then, candidate enzymes were validated using the BLASTP tool of NCBI BLAST+ and the HMM scan tool of the HMMER package against the SWISS PROT database and the PFAM database.

5) Safety evaluation of Lactobacillus rhamnosus IDCC 3201

(genomic analysis)

Toxicity genes were searched in the VFDB database, and antibiotic resistance genes were searched in ResFider software (ver. 3.2) with CARD database.

The search parameters were set within 80% or greater identity and 80% or greater range for gene identification.

Possibility of transferring genes to other strains or hosts depending on conditions. These transposases or transferases were identified using the protein-protein basic local search program (BLASTP) based on the NCBI GenBank database.

The Prophage area was identified using PHASTER, a PHASTER web-based program.

(in vitro analysis)

Lactobacillus rhamnosus IDCC 3201 is ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloroamphenicol (Sigma-Aldrich, St. Louis , MO) was evaluated for susceptibility to antimicrobial agents, including

A single colony of the plate was inoculated into MRS medium and pre-incubated for 18 hours. The cultured cells and the antibiotic solution were mixed in a 96-well plate ^{to obtain a cell density of 5 x 10 5} CFU/m, and then the plate was incubated anaerobically at 37°C for 18 hours.

The optical density was observed using a microplate reader (BioTek, Winooski, VT), and the minimum inhibitory concentration (MIC) was determined compared to the cut-off value suggested by the European Food Safety Authority (EFSA).

3. Experimental results

1) Screening of lactic acid bacteria with high activity of Sphingomyelinase (SMase)

SMase is a key enzyme in sphingomyelin metabolism, which is converted to ceramide and phosphorylcholine.

Therefore, when SMase activity is reduced, ceramide levels often decrease, leading to skin troubles (eg, atopic dermatitis).

In this experiment, lysates of various lactic acid bacteria strains were prepared in order to screen lactic acid bacteria that produce higher concentrations of ceramide, and the increase in SMase was observed.

In particular, the lysate of the test lactic acid bacteria strain used in the experiment was prepared to have a cell density ^{of 5 x 10 7 CFU/mL.}

Figure 1 shows the activity of SMase by Lactobacillus rhamnosus IDCC 3201 treatment, and showed the highest activity at 1.85±0.03 mU/mL, and in the case of other lactic acid bacteria strains, the activity was shown only at 0.28-0.43 mU/mL.

The activity value of Lactobacillus rhamnosus IDCC 3201 was about 3.4 times higher than that of L. mesenteroides, L. plantarum, L. casei, and S thermophilus.

As a result, Lactobacillus rhamnosus IDCC 3201 lactic acid bacteria strain was selected as the lactic acid bacteria strain showing the highest performance in relation to SMase activity.

The optimal cell density of Lactobacillus rhamnosus IDCC 3201 was evaluated to determine economically feasible concentrations.

Lactobacillus rhamnosus cell lysate was tested at a cell concentration ^{of 1 x 10 7} to 5 x 10 ^{10 CFU/mL.}

As a result, as the concentration of Jepo increased, SMase activity increased, and specifically increased to 1.65 ± 0.09 mU/mL.

In contrast, in terms of efficiency of enzymatic activity (unit/cell), the enzyme efficiency of SMase was measured to be the highest when the ^{cell density was lowest at 1 x 10 7 CFU.}

^{Therefore, a cell amount equivalent to 1 x 10 7} CFU of Lactobacillus rhamnosus IDCC 3201, which can exhibit the maximum SMase activity efficiency, was selected as the optimal cell density for further study.

2) Enhanced ceramide formation by Lactobacillus rhamnosus IDCC 3201

Ceramide, a major lipid component of the epithelial layer, is responsible for maintaining skin barrier homeostasis.

This layer prevents excessive water loss of the skin and protects the skin from a variety of stimuli, including bacterial or microbial infections.

Ceramiad production was investigated whether enhanced SMase activation by Lactobacillus rhamnosus IDCC 3201 affects ceramide production in HEK cells.

Before examining the effect of the lysate of Lactobacillus rhamnosus IDCC 3201, the cytotoxicity of HEK by the lysate was evaluated.

As shown in FIG. 2 , no significant decrease in cell proliferation was observed at cell densities ranging from ^{1×10 7} to 5×10 ^{8 CFU/mL for 24 hours.}

In order to prove that increased SMase activity increases ceramide production, lysates of Lactobacillus rhamnosus and lactic acid bacteria tested in FIG. 1 were treated in HEK cells.

Referring to FIG. 3 , it can be confirmed that the overall increased ceramide formation is correlated to some extent with the ^{increased SMase activity (R 2 =0.77).}

Ceramide concentration by Lactobacillus rhamnosus is 1.7 to 5.2 times higher than that of other lactic acid bacteria. More specifically, 1153.5 ± 334.5 pg / mL of ceramide was measured in HEK treated with Lactobacillus rhamnosus, and 651.1 ± 72.9 and 417.9 in HEK treated with L. mesenteroides, L. plantarum, L. casei, and S. thermophilus, respectively. Ceramides of ± 55.8, 216.8 ± 69.3 and 626.2 ± 216.5 pg/mL were measured.

In a previous study, keratinocytes were treated with a lysate of S. thermophilus S244 to form ceramides in proportion to cell density, and through this, the skin barrier improvement effect of AD patients was shown.

As a result, the lysate of Lactobacillus rhamnosus IDCC 3201 is expected to contribute to improving the skin barrier through the effect of increasing ceramide synthesis.

3) Suggested mechanism through in silico analysis of Lactobacillus rhamnosus IDCC 3201 genome

Information on enzymes capable of synthesizing ceramides in microorganisms to confirm the mechanism of enhancing ceramide production by Lactobacillus rhamnosus IDCC 3201 is shown in [Table 1].

Among the 17 genes, Lactobacillus rhamnosus IDCC 3201 contains galc and gba, which encode galactosylceramidase and glucosylceramidase, respectively.

By genetic analysis using homology search, it was confirmed that the gba gene is responsible for the production of ceramide from glucosylceramide, which is abundant in human keratinocytes.

Therefore, the result of increasing ceramide formation in HEK cells is considered to be due to glucosylceramidase of Lactobacillus rhamnosus IDCC 3201.

As shown in Figure 4, when considering the evidence for the presence of glucosylceramidase and the increased SMase activity in Lactobacillus rhamnosus IDCC 3201, the reason for the enhanced ceramide production is 1) the indirect effect of the lysate component and 2) glucosylceramidase is the ceramide It is summarized as a direct effect on the formation.

4) Safety evaluation of Lactobacillus rhamnosus IDCC 3201

Genome analysis of Lactobacillus rhamnosus IDCC 3201 indicated that the genome size was about 3.03Mbps, the GC content was 46.71%, and the number of open reading flame (ORF) genes was 2,821. In addition, Lactobacillus rhamnosus IDCC 3201 has no virulence genes or virulence factors.

As shown in [Table 2], in the MIC test, Lactobacillus rhamnosus IDCC 3201 was found to be sensitive to all antibiotics except gentamycin and kanamycin.

This resistance is intrinsic and cannot be transferred to the skin microbes because there is no antibiotic resistance gene.

In general, many Lactobacillus species have been shown to be inherently resistant to aminoglycoside antibiotics due to the absence of cytochrome-mediated transport.

Consequently, Lactobacillus rhamnosus IDCC 3201 is considered safe for use as a cosmetic material.

Claims (9)

delete delete delete delete delete delete delete A cosmetic composition that includes a lactic acid bacteria strain containing a gba (Glucosylceramase) gene, and improves ceramide formation in the skin. In claim 8,
The lactic acid bacteria strain is a cosmetic composition comprising Lactobacillus rhamnosus IDCC 3201 deposited with the ATCC BAA-2836 accession number.

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