KR101426191B1 - Cosmetic Composition Having Culture Fluid of Lactobacillus sp. and Stem cells - Google Patents

Abstract

The present invention relates to a cosmetic composition comprising a 16S ribosomal DNA comprising the nucleotide sequence of the first sequence of Sequence Listing and a culture medium of Lactobacillus fermentum strain having an ability to inhibit collagenase activity and a stem cell culture solution .
The present invention can provide a cosmetic composition for promoting epidermal ceramide synthesis, promoting skin collagen synthesis, inhibiting collagenase activity, inhibiting elastase activity, improving skin wrinkles, promoting skin antioxidation, promoting skin irritation, improving skin elasticity, A cosmetic composition which is excellent but has a small side effect and a method for producing the same.

Description

[0001] The present invention relates to a cosmetic composition comprising a lactobacillus culture medium and a stem cell culture liquid. and Stem cells}

The present invention relates to a cosmetic composition comprising a culture medium of a lactobacillus strain and a stem cell culture. More particularly, the present invention relates to a cosmetic composition comprising a 16S ribosomal DNA containing a nucleotide sequence of SEQ ID No. 1 and a lactobacillus Lactobacillus fermentum culture broth and stem cell culture broth.

With the recent improvements in medical technology and public health benefits, an aging society is rapidly emerging. Accordingly, the value and desire for human beauty are increased with the advancement of society, and the cosmetics industry is continuously developing due to the delay in aging and the interest in maintaining beauty. There are many studies in various fields in order to delay and prevent appearance, especially the aging that first appears in the skin, and thus the demand for cosmetic products and antioxidant products will increase steadily in the future.

The skin consists of the epidermis, the dermis and the subcutaneous tissue. The outer skin of the skin consists of a stratified squamous epithelium, which acts as a protective barrier against the external environment. The epidermis is divided from the outside into stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum and stratum basale. The dermis is a layer between the epidermis and the subcutaneous tissue. It is divided into papillary dermis and reticular dermis. The dermis is composed of blood vessels, collagen, elastin fibers, pores, lipids, sebaceous glands, Sensory nerves, fibroblasts, and macrophages, and they occupy the largest portion of the skin.

The dermis consists mainly of collagen and elastin fibers, which play a role in supporting the skin. Therefore, when a problem occurs in the dermis, wrinkles occur and skin elasticity disappears, and skin aging proceeds. Collagen is known to play the most important role in skin regeneration, skin moisture content, wound healing and wrinkle improvement, and is produced from fibroblasts. Collagen can contain a large amount of water, which serves to supply water to the dermis. When aging occurs, the water-containing function of the collagen is lowered and the wrinkles are increased. In addition, when a wound is formed, fibroblast is continuously produced collagen to fill the wound area and heal the wound.

As research on biochemistry and molecular biology of skin aging continues, a small amount of signaling substances such as growth factors in the body are found, and these substances are found to be closely related to aging. Therefore, research on stem cells capable of producing such a large amount of growth factors has attracted attention.

Stem cells are cells that have the ability to differentiate into all kinds of cells that make up the body, such as nerves, blood, There are two ways to obtain such stem cells. First, stem cells from embryos derived from embryos (embryonic stem cells), and stem cells from adult parts of the body (adult stem cells ). Although there are functional differences, embryonic stem cells and adult stem cells all have the ability to differentiate into different types of cells. Although embryonic stem cells have excellent differentiation ability and long telomeres, they have ethical problems and it is difficult to acquire a large number of cells. However, adult stem cells can obtain a large number of cells, There is a drawback that the risk of salt or the ability of differentiation is relatively low.

Adult stem cells are characterized as being very safe for medical applications. Specifically, cancer does not occur even when transplanted into the body for long-term regeneration. Since the immune rejection does not occur because it is in the adult body, autologous transplantation using its own cells is possible. In addition, there is site-specific differentiation that differentiates itself according to the characteristics of surrounding tissues. Since it does not cause cancer even if it is injected in undifferentiated state, it can produce the necessary cells immediately after transplantation, and has the advantage of self-renewal ability to regenerate and store necessary undifferentiated stem cells later. Recently, adult stem cells have been on the rise, and various studies are under way to clarify its usefulness.

Korean Patent Publication No. 2010-0114729 discloses a composition for regenerating skin using cord blood stem cell-derived angiogenic progenitor cells, Korean Patent Publication No. 2010-0032099 discloses a composition for improving skin wrinkles or inhibiting skin aging using mammalian stem cell culture media. Korean Patent Publication No. 2010-0036389 and Korean Patent Publication No. 2011-0097064 disclose a cosmetic composition using a human stem cell culture liquid cultured in a serum-free medium.

1. Korean Patent Publication No. 2010-0114729 2. Korean Patent Publication No. 2010-0032099 3. Korean Patent Publication No. 2010-0036389 4. Korean Patent Publication No. 2011-0097064

The present invention relates to a composition for promoting epidermal ceramide synthesis, promoting skin collagen synthesis, inhibiting collagenase activity, inhibiting elastase activity, improving skin wrinkles, skin antioxidation, promoting skin regeneration, alleviating skin irritation, improving skin elasticity, And to provide a cosmetic composition having a reduced side effect.

In order to accomplish the above object, the present invention provides a recombinant vector comprising 16S ribosomal DNA comprising the nucleotide sequence of SEQ ID No. 1 and having Lactobacillus < RTI ID = 0.0 > fermentum ) culture broth and a stem cell culture broth.

According to one aspect of the present invention, the lactobacillus fermentum strain is characterized by being Lactobacillus fermentum Miev L1106 (KCTC 12082BP).

According to one aspect of the present invention, the stem cells are characterized by being at least one selected from human-derived adult stem cells and embryonic stem cells.

According to one aspect of the present invention, the stem cell is characterized in that the stem cell is derived from any one or more selected from the group consisting of human bone marrow, umbilical cord blood, blood, liver, skin, gastrointestinal tract, placenta, nerve, adrenal gland, epithelium and fat.

According to one aspect of the present invention, there is provided a skin cosmetic composition for promoting epidermal ceramide synthesis, promoting skin collagen synthesis, inhibiting collagenase activity, inhibiting elastase activity, improving skin wrinkles, skin antioxidation, promoting skin regeneration, And the skin condition improving effect selected from the moisturizing improvement is obtained.

According to one aspect of the present invention, the cosmetic composition of the present invention can be used in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, , A spray, and a mixture thereof.

The cosmetic composition of the present invention is effective for promoting epidermal ceramide synthesis, promoting skin collagen synthesis, inhibiting collagenase activity, inhibiting elastase activity, improving skin wrinkles, skin antioxidation, promoting skin regeneration, alleviating skin irritation, The effect is excellent and the side effect is small.

FIG. 1 shows the result of signal analysis of the stem cell culture used in the present invention.
FIG. 2 shows electrophoresis results of the stem cell culture liquid and fractions thereof used in the present invention.
Fig. 3 shows the results of evaluating collagenase inhibitory activity of the example according to the present invention.
Figure 4 shows the results of evaluating the elastase inhibitory activity of the example according to the present invention.
Fig. 5 shows the results of evaluating hyaluronidase inhibitory activity of the example according to the present invention.
Figure 6 shows the result of evaluating cytotoxicity. FIG. 6A shows the result of treating the stem cell culture solution, and FIG. 6B shows the result of treating the culture solution of the lactic acid bacteria.
Fig. 7 shows the result of evaluating the degree of recovery from ultraviolet damage in the example according to the present invention.
Fig. 8 shows the results of evaluation of the degree of regeneration from the cell damage in the example according to the present invention.

In order to accomplish the above object, the present invention provides a recombinant vector comprising 16S ribosomal DNA comprising the nucleotide sequence of SEQ ID NO: 1 and having Lactobacillus < RTI ID = 0.0 > fermentum culture broth and stem cell culture broth. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the drawings.

The Lactobacillus < RTI ID = 0.0 > fermentum strain is a strain producing collagenase activity inhibitory substance isolated from kimchi. It was isolated by pure segregation and identified by 16S rDNA sequence analysis and biochemical characterization. As a result, it belongs to Bacillus sp. It was named as Bacillus Permeumt Miev L1106 and, according to the provisions of the Budapest Treaty on the International Recognition of Microorganism Deposit under the Patent Procedure, on November 22, 2011 at the KCTC (Korean Collection for Type Culture) Deposited under accession number KTCT12081BP.

Lactobacillus fermentum strains according to the present invention may have one or more of the following characteristics: (i) separation from kimchi, (ii) optimal incubation time from 12 hours to 48 hours, (iii) , (Iv) optimal initial pH is 3.5 to 4.5, (v) sucrose is 0.5 to 3% (W / V) of the total volume of medium as the optimum carbon source of the culture medium, (vi) (W / v) beef extracts in a volume of 3 to 5% (w / v), and (iii) 0.15 to 0.5% (w / v) sodium chloride relative to the total volume of the medium as an optimal trace element source of the culture medium.

The lactobacillus fermentum strain according to the present invention may be a lactobacillus fermentum Miev L1106 (KCTC 12082BP).

The lactobacillus fermentum strain according to the present invention may have one or more of the following characteristics: (i) lactobacillus fermentum Miev L1106 (KCTC 12082BP), (ii) optimal incubation time of 24 hours, (iii) optimal culture temperature (Iv) optimal initial pH of 4.5, (v) sucrose of 0.5% (W / V) relative to the total volume of the medium as the optimum carbon source of the culture medium, (vi) optimum nitrogen source of the culture medium Containing 3% (W / V) beef extracts compared to (ⅶ) Includes 0.3% (W / V) sodium chloride as the optimal source of trace elements in the culture medium. Preferably, the culture medium of the lactobacillus fermentum strain satisfying all of the above conditions has the best ability to inhibit collagenase activity.

The incubation time takes into account the survival of the microorganism. The incubation time is about 12 hours to 72 hours, and the culture solution has excellent collagenase activity inhibiting ability while minimizing the reduction of the cell growth due to the proliferation of the lactic acid bacteria. More preferably, 48 hours, and most preferably for 24 hours.

The culture temperature is preferably in the range of 20 to 45 캜 in terms of the ability to inhibit cell growth and collagenase activity, more preferably 25 to 35 캜, and most preferably 30 캜.

The initial pH of the culture is preferably in the range of 4.0 to 8.0 from the viewpoints of the ability to inhibit the growth of cells and collagenase activity, and most preferably pH 4.5.

The above-mentioned culture medium includes any medium used for culturing the Lactobacillus strain, but it is preferable to use a MRS (deMan Rogosa Sharpe) broth medium, an APT (All Purpose with Tween) medium Or BHI (Brain Heart Infusion) medium, among which MRS broth medium is most preferable.

As the carbon source of the culture medium, various carbohydrates can be used. In view of cell growth and ability to inhibit collagenase activity, glucose (glucose, fructose, galactose, Lactose, maltose, sucrose, xylose, and arabinose may be used, and most preferably sucrose. When the carbon source is contained in the medium, the ability to inhibit cell growth and collagenase activity is excellent.

The concentration of the carbon source is preferably in the range of 0.5% to 10% (W / V) based on the total volume of the culture medium in terms of the ability to inhibit the growth of the cells and collagenase activity, and most preferably 3% to be.

As the nitrogen source of the culture medium, various organic nitrogen sources may be used, and preferably peptone, tryptone, beef extract, yeast extract, malto extract, , Urea, and most preferably beef extract. When the nitrogen source is contained in the medium, the ability to inhibit the growth of the cells and the collagenase activity is excellent.

The concentration in which the nitrogen source is contained is preferably in the range of 1.0% to 10% of the total volume of the culture medium in terms of the ability to inhibit the growth of the cells and collagenase activity, and most preferably 3% (W / V).

As a trace element source of the culture medium, various trace elements may be used. Preferably, sodium chloride (NaCl), calcium chloride (CaCl ₂ ), magnesium sulfate (MgSO ₄ ), manganese sulfate (MnO ₄ S) Potassium hydrogen (KH ₂ PO ₄ ), dipotassium hydrogen phosphate (K ₂ HPO ₄ ), zinc sulfate (ZnSO ₄ ), and most preferably sodium chloride. When the trace source is contained in the medium, the ability to inhibit the growth of the microorganism and inhibit collagenase activity is excellent.

The concentration of the trace source is preferably in the range of 0.01% to 0.5% (W / V) based on the total volume of the culture medium in view of the ability to inhibit the growth of the microorganism and collagenase activity, and most preferably 0.3% (W / V).

The term " stem cell culture solution " of the present invention is a substance containing components contained in a medium obtained by culturing stem cells, and the type of the stem cells for producing the culture solution is not limited. For example, the stem cells making up the culture may be embryonic stem cells or adult stem cells. Furthermore, adult stem cells can originate from adult stem cells of all tissues. For example, the adult stem cells may be selected from bone marrow, umbilical cord blood, blood derived, liver derived, skin derived, gastrointestinal tract derived, placenta derived, nerve derived, adrenal derived, epithelium derived and fat derived, Derived adult stem cells. In a specific example of the present invention, a culture solution was prepared using bone marrow-derived adult stem cells.

Although the stem cells are present in a very small amount in bone marrow and the like, the process of isolating and culturing them is well known in the art (for example, as disclosed in U.S. Patent No. 5,486,359), and the stem cells can be cultured according to a known method Can be obtained by proliferation of bone marrow stem cells isolated from the hematopoietic cells by their attachment characteristics without losing their differentiation ability.

The process of obtaining stem cells will be described in detail as follows. (1) Separation of stem cells from mammalian, preferably human, mesenchymal stem cell sources such as blood or bone marrow, including human or mouse (bone marrow can be extracted from tibia, femur, spinal cord or iliac bone ), (2) culturing the isolated cells in a suitable medium, and (3) removing floating cells in the culturing process and subculturing the cells attached to the culture plate to obtain finally constructed mesenchymal stem cells .

The medium for stem cell culture can be used without limitation in a basic medium known in the art. The basic medium may be prepared by artificial synthesis, or a commercially prepared medium may be used. It is preferable to use a medium containing serum (for example, fetal bovine serum, horse serum and human serum). Media available for the present invention include, for example, RPMI series, Eagles ' s MEM (Eagle ' s minimum essential medium, Eagle, H. Science 130: 432 (1959)), alpha-MEM (Stanner, CP et al. New Biol.230: 52 (1971)), Iscove's MEM (Iscove, N. et al., J. Exp. Med. 147: 923 (1978)), 199 medium (Morgan et al., Proc. 69: 1 (1950)), CMRL 1066, RPMI 1640 (Moore et al., J. Amer. Med. Assoc. 199: 519 (1967)), F12 (Ham, Proc. Natl. Acad. Dulbecco's modification of Eagle's medium, Dulbecco, R. et al., Virology 8: 396 (1963)), F10 (Ham, RG Exp. Cell Res. 29: 515 1959), a mixture of DMEM and F12 (Barnes, D. et al., Anal. Biochem. 102: 255 (1980)), Way-mouth's MB752 / 1 (Way77mouth, CJ Natl. Cancer Inst. ) And McCoy's 5A (McCoy, TA, et al., Proc. Soc. Exp. Biol. Med., 100: 115, 1959) and MCDB series (Ham, RG et al., In Vitro 14:11 ), But is not limited thereto.

The base medium preferably contains 0.1% to 20% fetal bovine serum (FBS), more preferably 2% to 5%. The medium may include other components such as antibiotics or antifungal agents (e.g., penicillin, streptomycin) and glutamine. In a specific example of the present invention, bone marrow-derived adult stem cells were cultured in DMEM-LG (low glucose DMEM with glutamine and HEPES, PAA ?, # E15-808) medium.

The term "culture medium" of the present invention means a culture solution obtained by culturing a specific microorganism or a specific stem cell in a culture medium, a concentrated culture medium, a dried product of the culture medium, a culture filtrate, a concentrated culture filtrate or a culture filtrate. A strain or a stem cell, or a culture filtrate obtained by removing a strain or a stem cell after culturing. The culture is not limited in its formulation, and may be, for example, a liquid, an emulsion, or a solid.

It is preferable that the culture medium and stem cell culture solution obtained as described above are contained in the cosmetic composition of the present invention in an amount of 0.0001 to 100% by weight. More preferably from 0.0001 to 50.0% by weight, and most preferably from 0.001 to 10% by weight, based on the whole cosmetic composition. When the content of the culture solution is 0.0001% by weight or more, the skin can be expected to be improved by inhibiting the desired collagenase activity of the present invention. When the content of the culture solution is 50.0% by weight or less, Can be effectively achieved.

The cosmetic composition according to the present invention is selected from skin antioxidation, promotion of epidermal ceramide synthesis, promotion of skin collagen synthesis, inhibition of collagenase activity, improvement of skin wrinkles, skin antioxidation, promotion of skin regeneration, alleviation of skin irritation, improvement of skin elasticity, It is possible to obtain any one skin condition improving effect.

The ' wrinkle improvement ' means maintaining or strengthening the ability to be associated with the wrinkles and elasticity of the skin. Collagen (collagen) and elastic fibers (elastin) present in the dermis in the structure of the skin play a major role in the regulation of skin elasticity, and the biosynthesis of collagen is affected by internal and external skin . Specifically, the intrinsic factor is a decrease in cellular activity due to natural aging, a decrease in collagen fiber, and an extrinsic factor such as excessive irradiation of ultraviolet rays, stress, SH) to increase the expression of collagenases such as Matrix Metalloproteinases-1 (MMP-1), an enzyme that inhibits enzymatic activity or degrades collagen and elastin, thereby increasing skin wrinkles and reducing elasticity Results.

The 'whitening' is an element related to darkening of the skin by inhibiting the ability of melanocyte, a cell for determining skin color, to synthesize melanin pigment. The stem cell culture solution of the present invention is particularly a culture solution which performs a whitening function. Preferably, the stem cell culture solution composition of the present invention has melanocyte synthesis inhibitory ability in melanocyte determination of whitening, more preferably, May be a composition comprising a bone marrow-derived stem cell culture fluid. Melanocytes are the cells located in the uvea of the middle layer of the eye and the stratum basale of the skin. Through the process called melanogenesis, the melanocytes are found in the skin, eyes, hair Is a cell that produces melanin. Generally, there are from 1000 to 2000 melanocytes per 1 mm ² of skin and occupies 5 to 10% of the bottom surface of the epidermis.

The 'antioxidant' ability refers to a function of inhibiting the oxidation of skin constituents due to highly reactive reactive oxygen and free radicals due to ultraviolet rays. Active oxygen and free radicals oxidize the constituents of the skin to produce peroxides, resulting in structurally and functionally damaging the skin to promote aging. The antioxidant function of the present invention functions to protect the skin from this .

In order to accomplish the object of the present invention, a composition containing a culture medium of stem cells can perform the function of inactivating the free radicals. Preferably, the composition comprising the stem cell cultures, It is possible to erase the superoxide radical.

The cosmetic composition according to the present invention may be in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, ≪ / RTI >

The cosmetic composition according to the present invention may be prepared into any of the formulations conventionally produced in the art, for example, a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, But are not limited to, cleansing, oil, powder foundation, emulsion foundation, wax foundation, and spray. More particularly, when the composition of the present invention is used as a cosmetic composition, a cosmetic preparation is selected from cosmetic lotion, emulsion, cream, essence, gel, pack and cleansing cream; Makeup cosmetics such as foundation, and the like.

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

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 the case of a spray, in particular, / Propane or dimethyl ether.

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component is selected from aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

In addition, the cosmetic composition of the present invention may further contain, in addition to the components described above, oil, water, a surfactant, a moisturizer, a lower alcohol, a thickener, a chelating agent, a coloring agent, an antiseptic, And can be appropriately used in combination.

For example, the number of softening times may be selected from 1 to 10 wt% of polyhydric alcohols (propylene glycol, glycerin, etc.) and 0.05 to 2 wt% of a surfactant (polyethylene oleyl ether, polyoxyethylene hydrogenated castor oil, etc.) . The nutritional lotion and nutritional cream may contain 5 to 20% by weight of oils (squalane, petrolatum, octyldodecanol, etc.) and 3 to 15% by weight of wax components (such as cetanol, stearyl alcohol and beeswax) %, And the essence may be prepared by containing 5 to 30% by weight of polyhydric alcohols such as glycerin, propylene glycol and the like in addition to the culture medium of the present invention. The massage cream is prepared by adding 30 to 70% by weight of an oil such as liquid paraffin, petrolatum, isononylisononanoate and the like in addition to the culture medium of the present invention, and the pack contains polyvinyl alcohol 5 to 20 A peel off pack or a general emulsifiable cosmetic composition containing a weight% of a microorganism according to the present invention may contain wash bases containing 5 to 30% by weight of pigment such as kaolin, talc, zinc oxide, titanium dioxide, etc., ) Pack.

In another embodiment, the present invention provides a method for producing a recombinant vector having 16S ribosomal DNA comprising the nucleotide sequence of the first sequence of SEQ ID NO: 1 and having Lactobacillus < RTI ID = 0.0 > fermentum , and culturing the stem cells. The present invention also relates to a method for producing a cosmetic composition. The preparation of the cosmetic composition may be prepared by any method conventionally used.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

[ Example ]

1. Preparation of samples

1) Preparation of Lactobacillus fermentum culture broth

Lactobacillus perfumant L1106 (KCTC 12082BP) strain was obtained and cultured according to the method described in Korean Patent Application No. 10-2012-0075523.

Specifically, lactobacillus < RTI ID = 0.0 > fermentum Miev L1106 strain was cultivated in medium containing 0.5% (w / v) sucrose, 3% (w / v) beef extract and 0.3% (w / v) sodium chloride, Lt; / RTI >

2) Preparation of stem cell culture liquid

Stem cells were isolated from human bone marrow and used for proliferation. A human bone marrow received additional consent for the isolation of human bone marrow stem cells from a donor who agreed to donate for bone marrow transplantation, and received surplus bone marrow only if it passed the donor eligibility test. All activities, including the provision of surplus bone marrow and the isolation and culture of cells therefrom, were conducted under the approval and control of the institutional review board (IRB). Catholic Cell Therapy Center Professor Jae-Deok Jung The cultured medium was thawed in a laboratory.

DMEM-LG (low glucose DMEM with glutamine and HEPES, PAA? # E15-808) was used as the basal medium for bone marrow stem cell culture. DMEM medium supplemented with 5.55 mM glucose (low glucose), 2 mM glutamine, 15 amino acids, 25 mM HEPES, and 3700 mg / L NaHCO ₃ was added with aseptically filtered fetal bovine serum (FBS) The prepared medium was used.

The 15 kinds of amino acids contained in the medium are as follows. L-arginine · HCl (arginine · hydrochloric acid) 84 mg / l, L-cystine 48 mg / L-Lysine.HCl (lysine hydrochloride) 146 mg / L, L-isoleucine (isoleucine) 105 mg / L, H-HCl.H ₂ O (histidine hydrochloric acid hydrate) L-threonine (threonine) 95 mg / l, L-Tryptophan (threonine) 30 mg / l, L-phenylalanine (phenylalanine) 66 mg / (Tryptophan) of 16 mg / l, L-Tyrosine (tyrosine) of 72 mg / l, and L-valine (valine) of 94 mg / l.

3) Preparation of sample

A sample was prepared by mixing 10 ml of the cultured Lactobacillus fermentum culture and 100 ml of the stem cell culture.

2. Experimental Method

1) Signal protein analysis

Stem cell cultures were assayed for cytokines using the RayBio human cytokine antibody array 5 map kit and quantified by ELISA kit (Quantikine ELISA) using Vascular Endothelial Growth Factor (VEGF) -8 (IL-8) were quantitatively analyzed.

In addition, the stem cell culture broth was fractionated at 100 kDa, 30 kDa, and 10 kDa using MWCO (Molecular Weight Cut-Off), which separates the stem cell culture solution by a certain range of molecular weight.

2) Measurement of collagenase inhibitory activity

0.9 ml of a buffer solution 0.1 M Tris-HCl (pH 7.0) was added to 1 mg of Azocoll (Sigma A4341) as a substrate of the collagen enzyme, homogenized, and then a solution of collagenase type enzyme (Sigma C0130) prepared at a concentration of 200 units / ml was added to make 1 ml of the total reaction solution. After the reaction was completed at 43 ° C for 1 hour, when the reaction was completed, centrifugation was carried out at 3000 rpm for 10 minutes to precipitate undegraded collagen and supernatant containing degraded collagen was taken and absorbance was measured at 550 nm. The inhibition rate was shown by comparing the absorbance of the collagenase added group and the no added group.

3) Measurement of inhibitory activity of elastase

Elastase inhibitory activity was measured by using a conventional method. 1.3 ml of 0.1 mM N-succinyl- (L-Ala) 3-p-nitroanilide / 0.12 M tris-HCl (pH 8.0) was added to 0.1 ml of the sample and 0.1 ml of 0.765 U / The reaction was carried out at 25 DEG C for a minute and then the absorbance was measured at 410 nm. The inhibition rate was shown by comparing the absorbance of the ELASase-added group with the no-added group.

4) Measurement of hyaluronidase inhibitory activity

0.05 ml of HAase (7,900 U / ml) dissolved in 0.1 M acetate buffer (pH 3.5) and 0.1 ml of the sample solution were mixed and incubated at 37 for 20 minutes. Then, 0.1 ml of 12.5 mM CaCl ₂ was added thereto, Lt; / RTI > HA (12 mg / ml) dissolved in 0.1 M acetate buffer (pH 3.5) was added as a substrate and incubated for another 40 minutes. Then, 0.1 ml of 0.4 N potassium tetraborate and 0.4 N sodium hydroxide (NaOH) Was added to the 0.1 ml reaction mixture and heated on a water bath for 3 minutes and then completely cooled. Dimethylaminoborane (DMAB) reagent (3 ml) was added as a coloring agent to the cooled reaction product, and the mixture was incubated at 37 ° C for 20 minutes. The absorbance at 585 nm was measured to calculate the inhibitory activity.

5) Confirmation of cytotoxicity (MTT analysis)

Fibroblast and Macrophage (Raw 264.7) cells were seeded at 1 × 10 ⁴ cells in a 96-well plate, and after 24 hours, the cells were replaced with fresh medium and cultured at a constant concentration (0 to 10% The cultures of lactic acid bacteria were treated for 24 hours. Subsequently, 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetra zolium bromide (0.1 mg / ml) was added to each well and incubated for 3 hours. Insoluble formazan formed was 0.04 Absorbed in N hydrochloric acid / isopropanol, and absorbance was measured at 570 nm using an ELISA reader. The cell viability was compared with the absorbance of untreated cells.

6) Cell regeneration effect of UV-damaged Fibroblast

Fibroblast cells (1 × 10 ⁵ ) were placed in a 24-well plate. After 24 hours, UV-B 3J / m ² was irradiated and the cells were cultured for 48 hours at 1% concentration. Subsequently, 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (0.1 mg / ml) was added to each well and incubated for 4 hours. Insoluble formazan formed was dissolved in 0.04 N HCl / Absorbance was measured at 570 nm through an ELISA reader and the cell viability was compared with the absorbance of the untreated cells.

7) Cell regeneration effect (wound healing)

Fibroblast cells (1 × 10 ⁵ ) were dispensed into a 24-well plate, and the stem cell cultures were treated for 48 hours at a constant concentration (0.01, 0.1%) after 24 hours. The plate surface was scratched using a tip. The degree of wound healing over time was photographed.

3. Experimental Results

1) Signal protein analysis

16 signal protein (cytokine) was confirmed (Fig. 1) as a result of analyzing the signal protein of the stem cell culture (unfractionated fraction). The results of the electrophoresis of each fraction of the stem cell culture fluid are shown in Fig.

Most of the proteins present in the stem cell culture were unable to pass through the pores of the 100 kDa MWCO filtration membrane and were found to remain in the fractions classified as over 100 kDa. Specific results for the signal proteins of the stem cell culture broth and its fractions are shown in Table 1. < tb >< TABLE >

Stem cell culture (unfractionated) 100 kDa or more 30-100 kDa 10-30 kDa Less than 10 kDa Interleukin-6 (IL-6, Interleukin-6) O O O Interleukin-8 (IL-8, Interleukin-8) O O O O Brain Derived Neurotropic Factor (BDNF) O O O O Fibroblast growth factor-6 (FGF-6, Fibroblast growth factor-6) O O O Fibroblast growth factor-9 (FGF-9, Fibroblast growth factor-9) O O O O Hepatocyte growth factor (HGF) O O O Glial cell derived neurotrophic actor (GDNF) O Leukemia inhibitory factor (LIF, Leukemia inhibitory factor) O O O O O Platelet derived growth factor-BB (PDGF-BB) O O O Vascular endothelial growth factor (VEGF) O O O O O  Angiogenin O O Eotaxin-3 < / RTI > O O O Fractalkine O GRO (Growth-related oncogene) O O O O O Insulin-like growth factor-binding protein-1 (IGFBP-1) O Insulin-like growth factor-binding protein-2 (IGFBP-2) O O O O O Insulin-like growth factor-binding protein-3 (IGFBP-3) O O O IP-10 O O O O (MCP-1, Monocyte chemotactic protein-1) O O O O Neurotrophin-3 (NT-3) O O O Oncostatin M O O O O Osteoprotegerin (OPG, OPGN, Osteoprotegerin) O O O Osteopontin (OPN, Osteopontin) O O O PARC (pulmonary and activation-regulated chemokine) O O Transforming growth factor beta2 (TGF-beta2, Transforming growth factor-beta2) O O O O TIMP-1 (Tissue inhibitor of metalloproteinase type 1) O O O O TIMP-2 (Tissue inhibitor of metalloproteinase type 2) O O O

VEGF and IL-8 were found to be 2,125 pg / mL and 370.5 pg / mL, respectively, and VEGF content was about 5.7 times higher than IL-8.

Stem cell culture fluid
(Undivided fraction) 100 kDa or more 30-100 kDa 10-30 kDa Less than 10 kDa VEGF (pg / ml) 2,125 2,419 2,214 9.4 0.0 IL-8 (pg / ml) 370.5 88.6 54.8 5.0 0.0

The total amount of protein in the culture medium was about 5 mg / mL, and it was confirmed that there was no difference in total amount of protein before and after culturing.

2) Measurement of collagenase inhibitory activity

FIG. 3 shows the results of evaluating the degree of inhibition of collagenase activity in the examples according to the present invention.

As a result, the collagenase inhibitory activity of the mixture of the stem cell culture medium and the lactic acid bacteria culture was the highest at 74.09%, and the stem cell culture medium alone treatment group (44.83%) as well as the group supplemented with ascorbic acid alone (25.50% And 57.58%, respectively, compared to the control group alone.

3) Measurement of inhibitory activity of elastase

The results of evaluation of the degree of inhibition of the elastase activity in the examples according to the present invention are shown in FIG.

As a result, the elastase inhibitory activity of the example of the present invention in which the stem cell culture solution and the lactic acid bacteria culture solution were mixed was the highest at 74.09%, and the stem cell culture medium alone treatment (70.29%) as well as the group supplemented with the ergolic acid (71.24%) and lactic acid bacteria culture treated group (20.95%), respectively.

4) Measurement of hyaluronidase inhibitory activity

The results of evaluating the hyaluronidase activity inhibition of the examples according to the present invention are shown in Fig.

As a result of the experiment, the inhibitory activity of the elastase inhibitor of the example of the present invention in which the stem cell culture solution and the lactic acid bacteria culture solution were mixed was 44.14%, and compared with the stem cell culture medium alone treatment (1.21%) and the lactic acid bacteria culture alone treatment (41.23% The degree of hyaluronidase activity inhibition was high.

5) Confirmation of cytotoxicity (MTT analysis)

The cell cytotoxicity was examined by MTT assay by treating the stem cell culture with 0, 1, 3, 5, and 10% concentrations of dibasic cells (Fibroblast), as shown in FIG. As a result, no cytotoxicity was observed up to 10% concentration.

The cultures of lactic acid bacteria were also treated with dermis cells at 0, 0.25, 0.5, 1, 3, 5, and 10% concentrations, and then cytotoxicity was confirmed. No cytotoxicity was observed up to 1% concentration, but cell death was observed at 3% or more concentration.

6) Cell regeneration effect of ultraviolet-damaged fibroblast

The results of evaluating the degree of regeneration against ultraviolet damage in the examples according to the present invention are shown in Fig.

As a result of the experiment, the survival rate was 61.23% due to about 40% of the cells killed by ultraviolet ray damage. Cell viability was improved to 69.91% in the group treated with Lactobacillus culture alone, and cell survival rate was increased to 85.09% in the group treated with stem cell culture alone. On the other hand, it was confirmed that the embodiment of the present invention in which the stem cell culture solution and the lactic acid bacteria culture solution were mixed restored all the cell damage by ultraviolet rays.

7) Cell regeneration effect (wound healing)

The results of evaluating the degree of regeneration against cell damage in the examples according to the present invention are shown in FIG. The area where the cell damage was applied is indicated by an arrow.

As a result of the experiment, the fastest wound area recovery was observed in the group treated with the lactic acid bacterium alone after 12 hours after the cell damage, but after 24 hours, the wound of the stem cell culture and the culture of the lactic acid bacterium were mixed, All of the cells were regenerated. On the other hand, in the group treated with the stem cell culture alone, only the wound area except for a part of the wound area was recovered.

When 60 hours had elapsed, the injured area remained in the distilled water-treated group, and the wound area was completely restored in the stem cell culture alone treated group and the lactic acid bacteria treated group alone, and the stem cell culture solution and the lactic acid bacteria- In the examples, it was confirmed that not only the wound healing but also the cell density were higher than the initial level.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Korea Biotechnology Research Institute KCTC12082BP 20111122

<110> Miev Co.Ltd <120> Cosmetic Composition Having Culture Fluid of Lactobacillus sp.          and Stem cells <130> DP120679 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1426 <212> DNA <213> lactobaillus fermentum <220> <221> rRNA &Lt; 222 > (1) .. (1426) <223> DNA sequnece complemenetary to 16s rRNA <400> 1 tgcagtcgaa cgcgttggcc caattgattg atggtgcttg cacctgattg attttggtcg 60 ccaacgagtg gcggacgggt gagtaacacg taggtaacct gcccagaagc gggggacaac 120 atttggaaac agatgctaat accgcataac aacgttgttc gcatgaacaa cgcttaaaag 180 atggcttctc gctatcactt ctggatggac ctgcggtgca ttagcttgtt ggtggggtaa 240 yggcctacca aggcgatgat gcatagccga gttgagagac tgatcggcca caatgggact 300 gagacacggc ccatactcct acgggaggca gcagtaggga atcttccaca atgggcgcaa 360 gt; aaagaagaac acgtatgaga gtaactgttc atacgttgac ggtatttaac cagaaagtca 480 cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgtta tccggattta 540 ttgggcgtaa agagagtgca ggcggttttc taagtctgat gtgaaagcct tcggcttaac 600 cggagaagtg catcggaaac tggataactt gagtgcagaa gagggtagtg gaactccatg 660 tgtagcggtg gaatgcgtag atatatggaa gaacaccagt ggcgaaggcg gctacctggt 720 ctgcaactga cgctgagact cgaaagcatg ggtagcgaac aggattagat accctggtag 780 tccatgccgt aaacgatgag tgctaggtgt tggagggttt ccgcccttca gtgccggagc 840 taacgcatta agcactccgc ctggggagta cgaccgcaag gttgaaactc aaaggaattg 900 acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagctacgc gaagaacctt 960 accaggtctt gacatcttgc gccaacccta gagatagggc gtttccttcg ggaacgcaat 1020 gacaggtggt gcatggtcgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 1080 cgagcgcaac ccttgttact agttgccagc attaagttgg gcactctagt gagactgccg 1140 gtgacaaacc ggaggaaggt ggggacgacg tcagatcatc atgcccctta tgacctgggc 1200 tacacacgtg ctacaatgga cggtacaacg agtcgcgaac tcgcgagggc aagcaaatct 1260 cttaaaaccg ttctcagttc ggactgcagg ctgcaactcg cctgcacgaa gtcggaatcg 1320 ctagtaatcg cggatcagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc 1380 cgtcacacca tgagartttg taacacccaa agtcggtggg gtaacc 1426

Claims (6)

A culture medium of Lactobacillus fermentum Miev L1106 (accession number: KCTC 12082BP) having a 16S ribosomal DNA containing the nucleotide sequence of the first sequence of Sequence Listing and capable of inhibiting collagenase activity, and a stem cell culture solution &Lt; / RTI &gt;
delete The method according to claim 1,
Wherein the stem cells are at least one selected from human adult stem cells and embryonic stem cells.
The method according to claim 1,
Wherein said stem cells are derived from human bone marrow.
The method according to claim 1,
The cosmetic composition is selected from the group consisting of promoting epidermal ceramide synthesis, promoting skin collagen synthesis, inhibiting collagenase activity, inhibiting elastase activity, improving skin wrinkles, skin antioxidation, promoting skin regeneration, alleviating skin irritation, Wherein the cosmetic composition has an effect of improving the skin condition.
The method according to claim 1,
The cosmetic composition may be in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, spray, Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt;

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