Skin-improving cosmetic composition comprising epidermal growth factor

Abstract

The present invention relates to a transformed Pediococcus pentosaceus strain into which a gene encoding a human epidermal growth factor (EGF) protein is introduced to express and secrete an epidermal growth factor, and a skin-improving cosmetic composition comprising same. According to the present invention, whitening, wrinkle improvement, antioxidant, and skin regeneration effects superior to those of a cosmetic composition comprising commercially available isolated / purified EGF are exhibited. In addition, the present invention facilitates large-scale production of EGF and has economical production costs, thereby providing high competitiveness as a raw material for cosmetic compositions.

Description

Cosmetic composition for skin improvement containing epidermal growth factor

[0001] The present invention relates to a cosmetic composition for skin improvement, and more specifically, to a cosmetic composition for skin improvement comprising, as an active ingredient, a culture solution of Pediococcus pentosaceus transformed to secrete Epidermal Growth Factor (EGF) protein.

[0002] As living standards improve and the trend of prioritizing appearance becomes more pronounced, the demand for clear and clean skin is increasing, and high-functional products such as whitening, anti-wrinkle, and skin regeneration are gaining popularity in cosmetic compositions.

[0003] Recently, Epidermal Growth Factor (EGF) has been receiving significant attention due to its excellent skin regeneration capabilities. Growth factors such as human epidermal growth factor and fibroblast growth factor are listed in the International Cosmetic Ingredient Dictionary (ICID) of the Cosmetic Conformities Association (CTFA) and have also been approved as cosmetic ingredients by the Ministry of Food and Drug Safety of Korea.

[0004] Epidermal Growth Factor (EGF) is present in high concentrations in saliva, urine, breast milk, tears, and blood, and is supplied from the bloodstream when a wound occurs to promote scar-free healing. In addition to this, EGF performs various other functions, playing a key role in skin regeneration by promoting the proliferation of epithelial and endothelial cells, stimulating the proliferation of fibroblasts that synthesize collagen (a component of the dermis), inducing neovascularization at damaged skin sites and the secretion of other regenerative factors, and promoting the synthesis of fibronectin.

[0005] Polypeptide growth factors present in blood and tissues are known to have very short in vivo half-lives of only a few minutes; furthermore, EGF is biologically unstable and physicochemically heterogeneous, which can lead to reduced therapeutic efficacy. Although EGF is currently produced using genetic engineering technology, it is difficult to produce sufficient quantities for industrial use, making it a considerably expensive material; moreover, there has been a critical problem in that EGF produced through genetic engineering procedures fails to exhibit sufficient activity.

[0006] [Prior Art Literature]

[0007] [Patent Literature]

[0008] (Patent Document 1) KR 1020080032477 A

[0009] (Patent Document 2) KR 1020190116693 A

[0010] The present invention aims to overcome the limitations of the aforementioned prior art, and one objective of the present invention is to provide genetically modified transgenic lactic acid bacteria that secrete EGF, which is a raw material for cosmetic compositions.

[0011] Another objective of the present invention is to provide a cosmetic composition for skin improvement containing a high amount of EGF produced by transformed lactic acid bacteria, having skin whitening, skin barrier improvement, elasticity enhancement, and wrinkle improvement effects.

[0012] Another objective of the present invention is to provide a cosmetic composition for skin improvement containing EGF as an active ingredient, which has excellent effects of improving skin whitening, skin wrinkle improvement, skin moisturization, and skin elasticity, and can be mass-produced at a low cost.

[0013] One aspect of the present invention for achieving the purpose of the present invention described above is

[0014] This invention relates to a transformed Pediococcus pentosaceus PP-EGF strain characterized by the introduction of a gene encoding human epidermal growth factor (EGF) protein to express and secrete epidermal growth factor.

[0015] Another aspect of the present invention for achieving the purpose of the present invention described above is,

[0016] The present invention relates to a cosmetic composition for skin improvement comprising a culture medium of a transformed Pediococcus pentosaceus PP-EGF strain of accession number KCCM13348P, into which a gene encoding human epidermal growth factor (EGF) protein is introduced and which secretes EGF (Epidermal Growth Factor) (hereinafter abbreviated as 'PP-EGF'), a concentrate of said culture medium, or a dried product thereof.

[0017] The concentration of EGF included in the skin-improving cosmetic composition of the present invention may be 0.5 to 10 ppm based on the total weight of the cosmetic composition.

[0018] The above cosmetic composition may have a formulation selected from the group consisting of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, spray, and mixtures thereof.

[0019] In the present invention, the skin improvement composition may be for skin moisturization, skin wrinkle improvement, skin radiance, or skin whitening.

[0020] The cosmetic composition comprising the Pediococcus pentosaceus PP-EGF (KCCM13348P) strain transformed to produce and secrete EGF according to the present invention contains a large amount of EGF and can be used to manufacture a functional cosmetic composition having skin whitening, skin moisturizing, skin wrinkle improvement, skin elasticity improvement, and antioxidant functions.

[0021] In addition, according to the present invention, the production cost of EGF, an expensive material, can be reduced and mass production can be facilitated, thereby enhancing the price competitiveness of cosmetic compositions containing expensive functional ingredients.

[0022] FIG. 1 is a schematic diagram illustrating a method for cloning an EGF-coding gene used in one embodiment of the present invention into an expression vector.

[0023] Figure 2 is a cleavage map of the expression vector (pCBT24-2-pG6Pi-EGF-pG6Pi-EGF) used for the transformation of a Pediocottus pentosaceus strain in an embodiment of the present invention.

[0024] Figure 3 is a photograph showing the selection results of pCBT24-2-pG6Pi-EGF-pG6Pi-EGF / PP(-7) transformants in an embodiment of the present invention.

[0025] Figure 4 shows the results of an ELISA test quantifying EGF in the culture supernatant of Pediococcus pentosaceus transformed to secrete Epidermal Growth Factor (EGF) protein in an embodiment of the present invention.

[0026] Figure 5 is a graph showing the results of analyzing cell viability using the fluorescence microscopy (HCS) LiveDead cell analysis method after treating fibroblast (ccd-986sk) cell lines with EGF and PP-EGF culture supernatants.

[0027] Figure 6 is a graph showing the results of analyzing cell viability using the MTT (WST-8) method after treating melanoma (SK-MEL-2) cell lines with IBMX, EGF, Kojic acid, and PP-EGF.

[0028] Figure 7 shows the results of a wound healing assay after treating a fibroblast (ccd-986sk) cell line with PP-EGF culture supernatant.

[0029] Figure 8 shows the test results comparing the antioxidant effects of EGF and PP-EGF culture supernatants using the DPPH method.

[0030] Figure 9 shows the results of analyzing changes in tyrosinase expression (whitening efficacy) using the qRT-PCR method after treating melanoma (SK-MEL-2) cell lines with EGF and PP-EGF culture supernatants.

[0031] Figure 10 shows the results of analyzing changes in elastin expression (wrinkle improvement efficacy) using the qRT-PCR technique after treating fibroblast (ccd-986sk) cell lines with EGF and PP-EGF culture supernatants.

[0032] Figure 11 shows the results of an experiment confirming Collagenase Inhibitors following treatment with EGF and PP-EGF culture supernatants.

[0033] Figure 12 shows the results of an experiment confirming Elastase inhibitors following treatment with EGF and PP-EGF culture supernatants.

[0034] The present invention will be described in detail below with reference to the attached drawings.

[0035] In describing the present invention, if it is determined that a detailed description of related prior art could unnecessarily obscure the essence of the present invention, such detailed description is omitted.

[0036] In this specification, 'and / or' includes each of the mentioned items and all combinations of one or more. In this specification, the singular form includes the plural form unless specifically stated otherwise in the text. As used in this specification, 'comprises' and / or 'comprising' do not exclude the presence or addition of one or more other components in addition to the mentioned components.

[0037] In this specification, the term “culture” includes not only live bacteria obtained from a culture medium but also any processed form of lactic acid bacteria known to those skilled in the art, such as lysed cells, dried cells, frozen cells, etc., but is not necessarily limited to these.

[0038] In this specification, the term "expression vector" refers to a recombinant vector capable of expressing a target peptide in a target host cell, and means a genetic construct comprising essential regulatory elements linked to function to express a gene insert. The expression vector includes expression regulatory elements such as a start codon, a stop codon, a promoter, and an operator.

[0039] In this specification, the term "operably linked" means a state in which a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein or RNA are functionally linked to perform a general function.

[0040] In this specification, the expression “secreted” of EGF means that EGF is transferred from the transformant to the extracellular space, including cases where the entire molecule of EGF is actually present in the medium in a completely free form, cases where all molecules of EGF are present in the cell surface layer, and cases where a portion of the EGF molecule is present in the medium and the remainder of the molecule is present in the cell surface layer.

[0041] In this specification, the term "skin improvement" comprehensively refers to the process or effect thereof of treating, alleviating, or mitigating skin damage caused by intrinsic or extrinsic factors of the skin. In the present invention, the term "skin improvement" may be interpreted to mean effects such as skin moisturization, improvement of skin wrinkles, skin whitening, improvement of skin elasticity, and antioxidant properties, which can be induced by applying a cosmetic composition containing the transformed lactic acid bacteria strain that secretes EGF according to the present invention to the skin.

[0042] One aspect of the present invention relates to a transformed Pediococcus pentosaceus PP-EGF strain characterized by having a gene encoding human epidermal growth factor (EGF) protein introduced to express and secrete epidermal growth factor.

[0043] The transformed strain of the present invention is the Pediococcus pentosaceus PP-EGF strain deposited internationally with the Korean Culture Collection of Microorganisms under accession number KCCM13348P.

[0044] The transformed Pediococcus pentosaceus strain of the present invention, which produces and secretes EGF, can express the EGF-coding gene in the Pediococcus pentosaceus strain and produce EGF by transfecting Pediococcus pentosaceus cells using an expression vector in which the EGF-coding gene is cloned.

[0045] The transformed Pediococcus pentosaceus strain of the present invention was codon-optimized using codon usage appropriate for Pediococcus pentosaceus to express the human EGF gene (NCBI_Sequence ID: 2KV4_A) in Pediococcus pentosaceus (ATUM, USA). The synthetic EGF gene (see Pediococcus pentosaceus strain below) was cloned into the expression vector pCBT24-2 (KCCM12182P), which contains an Em resistance gene, along with pG6Pi, the native promoter of Pediococcus pentosaceus. To induce secretion after protein expression, the secretion signal peptide usp45 was inserted upstream of the EGF-coding gene (Fig. 1).

[0046] Specific amino acid sequences of EGF or nucleotide sequence information of the gene encoding it can be obtained from known databases such as NCBI’s GenBank (GenBank Accession No. AAS83395.1, AAH92277.1, etc.). In the present invention, the human EGF coding gene may be the human epidermal growth factor nucleotide sequence (Sequence No. 1). The EGF may preferably be a peptide represented by the amino acid sequence of Sequence No. 2.

[0047] Sequence Number Sequence Human EGF DNA Sequence (159 bp) Sequence Number 1 AATAGTGACTCTGAATGTCCCCTGTCCCACGATGGGTACTGCCTCCATGATGGTGTGTGCATGTATATTGAAGCATTGGACAAGTATGCATGCAACTGTGTTGTTGGCTACATCGGGGAGCGATGTCAGTACCGAGACCTGAAGTGGTGGGAACTGCGC Amino acid sequence of human EGF protein (53 aa) Sequence Number 2 NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR

[0048] In the present invention, pG6Pi (Sequence No. 3), which is a self-promoter of PP in the expression vector pCBT24-2, can be used as the promoter.

[0049] In the present invention, to induce EGF protein into the secretory pathway of Pediococcus pentosaceus cells, a secretory signal peptide (S) may be provided within an expression vector. The secretory signal peptide sequence is usually located at the 5' of the DNA sequence encoding the EGF protein. In the present invention, the secretory signal peptide may include a secretory signal peptide that directs strong protein secretion, such as the USP45 secretory signal; Usp45 N4 in which the lysine at position 4 of the wild-type Usp45 secretory signal is substituted with asparagine; or the Lactobacillus brevis S-layer protein signal peptide.

[0050] In the present invention, the expression vector includes a Selective Labeling Maker (SLM) that enables the selection of strains containing EGF. The Selective Labeling Maker represents a confirmation factor, generally an antibiotic or chemical resistance gene, that can be selected based on the effect of the marker gene, namely, resistance to antibiotics, resistance to herbicides, colorimetric analysis markers, enzymes, fluorescent markers, etc. The Selective Labeling Maker may be an ampicillin resistance gene or a kanamycin resistance gene.

[0051] The arrangement order of the promoter, secretion signal peptide, EGF-coding gene, and selective marker of the present invention is not specifically limited as long as the expression of the selective marker gene is possible, but is generally arranged from upstream to downstream in the order of promoter, secretion signal peptide, EGF-coding gene, and selective marker.

[0052] In the present invention, the expression vector may include a double promoter, as shown in FIG. 2. That is, it may additionally include a second promoter, a second secretion signal peptide, and an EGF-coding gene sequence downstream of the first promoter. Such an expression vector can increase the expression and secretion of EGF protein compared to an expression vector containing a single promoter. Cloning was performed in the order of the pG6Pi promoter derived from Pediococcus pentosaceus, the secretion signal sequence peptide USP45, and the EGF-coding gene, and a two-promoter system can be applied to the expression vector pCBT24-2 having an EM resistance gene. The gene was made to repeat twice in the order of the pG6Pi promoter derived from Pediococcus pentosaceus, the secretion signal sequence peptide USP45, and the EGF-coding gene, and restriction enzyme sequences of NheI and SalI, and BamHI and NdeI were inserted before and after the genes for cloning. By applying a two-promoter system to the expression vector pCBT24-2 containing an EM resistance gene, the final pCBT24-2-pG6Pi-EGF-pG6Pi- can be cloned. After cloning the EGF gene through recombinant cloning, the Pediococcus pentosaceus transformant becomes resistant to Em and can survive in MRS medium.

[0053] The expression vector may be introduced into the desired Pediococcus pentosaceus cells by methods known in the art, for example, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), the use of a gene gun, or a DNA vector transporter. Any currently known method or any method to be developed in the future may be applied to the method of introducing the expression vector.

[0054] EGF protein can be produced by culturing the transformed Pediococcus pentosaceus strain in vitro or in vivo. The cultivation of the transformed Pediococcus pentosaceus strain is performed according to known methods.

[0055] Another aspect of the present invention relates to a cosmetic composition for improving skin comprising a culture medium of a transformed Pediococcus pentosaceus PP-EGF strain of accession number KCCM13348P, into which a gene encoding human epidermal growth factor (EGF) protein is introduced and which secretes EGF (Epidermal Growth Factor), a concentrate of said culture medium, or a dried product thereof.

[0056] The strain mentioned above is the transformed Pediococcus pentosaceus PP-EGF strain of accession number KCCM13348P.

[0057] The cosmetic composition of the present invention preferably contains a high amount of EGF, and preferably contains 0.5 to 10 ppm of EGF based on the total weight of the composition.

[0058] In the present invention, the term "transformed Pediococcus pentosaceus SL4 strain" may be replaced or included by a selection from the group consisting of: the cells of the strain; the lysate of the cells; the culture of the strain; the culture medium from which the cells have been removed from the culture of the strain; the cell extract of the strain; the extract of the culture of the strain; and the extract of the culture medium from which the cells have been removed from the culture of the strain.

[0059] The formulation of the above cosmetic composition may be selected from the group consisting of skin lotion, skin softener, skin toner, astringent, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, foot cream, neck cream, foundation, essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, hair shampoo, hair treatment, hair rinse, and body cleanser.

[0060] The transformed Pediococcus pentosaceus SL4 strain of the present invention is a safe strain that is not resistant to one or more antibiotics selected from the group consisting of ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, clindamycin, erythromycin, tetracycline, and chloramphenicol.

[0061] In the present invention, the cell lysate of the transformed Pediococcus pentosaceus SL4 strain can be obtained by centrifuging the culture medium of the strain to separate the cells from the culture supernatant, suspending them in PBS, treating them with lysozyme, and then ultrasonically treating them. At this time, when separating the cells by centrifugation, the cells may be washed with a buffer solution such as PBS and then centrifuged again, and centrifugation may be performed at 3,000 to 5,000 rpm for 5 to 60 minutes. It is preferable to treat the cells suspended in PBS with lysozyme at a concentration of 300 to 700 μg / ml at 30 to 37°C, and the ultrasonically treated conditions are preferably 50 to 90W, 20 to 60 seconds, and 20 to 40 cycles. At this time, it is advisable to heat treat the lysate to kill any bacteria that may be alive. Heat treatment can be performed at 50 to 70°C for 30 to 60 minutes, and it is preferable to heat using a water bath rather than direct heating to minimize protein denaturation. Additionally, the culture supernatant separated at this time can be adjusted to a pH of 7 to 8, filtered, and used as a composition derived from various cultures.

[0062] Each composition derived from the transformed Pediococcus pentosaceus SL4 strain in the present invention can be used without separate processing and can be purified or dried through various methods and added to cosmetic compositions.

[0063] Each composition derived from the above strain may be dried by performing a freeze-drying method and may include various excipients included when drying ordinary strains or compositions derived therefrom. The freeze-drying excipients may be selected from gluconic acid, alginic acid, sodium alginate, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, methylcellulose, carbomer, hyaluronic acid, tragacanth, karaya gum, water-soluble starch, pectin, gelatin, polyvinyl alcohol, polyvinylpyrrolidone, oligosaccharides, sugar alcohols, calcium gluconate, calcium lactate, polymethyl methacrylate, wheat protein, soybean protein, methylcellulose, aquacort, guar gum, locust bean gum, xanthan gum, gellan gum, gum arabic, trehalose, etc.

[0064] A functional cosmetic composition can be prepared by adding a suitable carrier, used in the preparation of a cosmetic composition for general skin, to the cosmetic composition of the present invention. At this time, the carrier used is not particularly limited thereto, but preferably, oils, water, surfactants, humectants, lower alcohols, thickeners, chelating agents, pigments, preservatives, fragrances, etc., may be used alone or in an appropriate combination.

[0065] The cosmetic composition of the present invention may be prepared in the form of, for example, a solution, emulsion, suspension, paste, cream, lotion, gel, powder, spray, surfactant-containing cleansing, oil, soap, liquid cleansing agent, bath additive, foundation, makeup base, essence, lotion, foam, pack, softening water, sunscreen cream, sun oil, etc., and preferably may be prepared in the form of an ointment for external skin, softening lotion, nourishing lotion, nourishing cream, massage cream, essence, pack, emulsion, or oil gel.

[0066] A carrier may be used selectively depending on the formulation of the cosmetic composition. For example, when preparing a cosmetic composition in the form of an ointment, paste, cream, or gel, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. may be used alone or in combination as a carrier component; when preparing a cosmetic composition in the form of a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, chlorofluorohydrocarbon, propane / butane, dimethyl ether, etc. may be used alone or in combination as a carrier component.

[0067] When preparing a cosmetic composition in the form of a solution or emulsion, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan, etc., may be used as carrier components, either alone or in combination.

[0068] When preparing a cosmetic composition in the form of a suspension, water, ethanol or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tracanth, etc. may be used as carrier components, either alone or in combination.

[0069] Meanwhile, the cosmetic composition of the present invention may contain auxiliary agents commonly used in the technical field to which the present invention belongs, such as hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, blockers, pigments, absorbents, dyes, etc. The amount of these various auxiliary agents is the amount commonly used in the field, for example, within the range of 0.001 to 10 weight percent with respect to the total weight of the composition.

[0070] The final form of the cosmetic composition of the present invention is not particularly limited and may be, for example, a lotion, gel, cream, etc. In addition, it is preferable to apply the cosmetic composition to skin including the scalp or hair.

[0071] The present invention will be explained in more detail below through the following examples. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited thereto.

[0072] Examples

[0073] Experimental Equipment and Materials

[0074] The culture supernatant (hereinafter abbreviated as “PP-EGF”) of the transformed Pediococcus pentosaceus PP-EGF (KCCM13348P) strain, into which a gene encoding the human epidermal growth factor (EGF) protein has been introduced to secrete EGF, was provided by the Cellbiotech Research Institute and stored frozen at -20℃ until the test. Additionally, the standard EGF used in the experiment was human epidermal growth factor (hEGF) (Sigma), and the excipient was PBS (pH 7.2; Gibco). The reagents and assay kits used in the experiment were the EGF Human ELISA Kit (invitrogen), LIVE / DEAD Viability / Cytotoxicity Kit (invitrogen), WST-8 (DOJINDO), DPPH Antioxidant Assay Kit (Colorimetric / abcam), RNeasy Mini Kit (QIAGEN), and TOPscript RT DryMIX dN6 plus (enzynomics), TB Green Premix Ex Taq II (TaKaRa), Gelatinase / Collagenase assay kit (Molecularprobes), Elastase assay kit (Molecularprobes), 1,1-Dimethylethyl hydroperoxide (Sigma-Aldrich), L-Ascorbic acid (Sigma-Aldrich), CellROX ®Deep Red Reagent (Invitrogen), 96-well cell culture plates (Corning), 96-well Micro Plate Black (SPL), LACTOBACILLI MRS Broth (BD DIFCO), BL Broth (MBcell), BL Agar (BD DIFCO), and paper discs (ADVANTEC) were used. For the equipment used in the experiment, an iMark microplate reader (BIORAD) was used for absorbance measurements, and an HCS ImageXpress micro confocal (MOLECULAR DEVICES) was used for cell viability measurements. An ECLIPSE Ts2 (Nikon) was used for the optical microscope for the cell regeneration test, a Quick Drop (MOLECULAR DEVICES) was used for RNA measurement, and a Mastercycler X50a (Eppendorf) was used for PCR. CFX96 was used for qRT-PCR. TM A Real-Time System (BIORAD) was used, and a Glomax explorer (Promega) was used for fluorescence measurement.

[0075]

[0076] Example 1: Construction of a system for the expression and secretion of lactic acid bacteria-derived EGF protein

[0077] Example 1.1: Transformation of Pediococcus pentosaceus strain

[0078] The synthetic EGF gene shown in Table 1 above was cloned into the expression vector pCBT24-2, which contains an erythromycin (hereinafter abbreviated as Em) resistance gene, along with pG6Pi, the autopromoter of Pediococcus pentosaceus. To induce secretion after protein expression, the secretion signal peptide usp45 was inserted upstream of the gene (see Fig. 1). Cloning was performed in the order of pG6Pi, usp45, and EGF in the expression vector pCBT24-2, and the cloned plasmid was named pCBT24-2-pG6Pi-EGF. To clone the EGF gene, restriction enzyme sequences were inserted upstream and downstream of the gene, and a two-promoter system was applied to further increase expression levels. The final completed plasmid was named pCBT24-2-pG6Pi-EGF-pG6Pi-EGF (see Fig. 2).

[0079] The plasmid obtained by cloning was transformed into Pediococcus pentosaceus (-7) and transformed organisms were selected using the Em resistance test method (Fig. 3). Wild-type Pediococcus pentosaceus cannot grow in MRS medium with an Em concentration of 10 μg / ml, and thus Pediococcus pentosaceus (-7) transformed organisms that grow by forming colonies with Em resistance can be selected.

[0080]

[0081] Example 1.2: Culture of Pediococcus pentosaceus transformants (PP-EGF)

[0082] Pediococcus pentosaceus transformants (PP-EGF) were inoculated into MRS culture medium and cultured at 37°C for 24 hours, followed by two subcultures using the same method. PP-EGF medium with the composition shown in Table 2 was prepared in a jar-fermentor, sterilized, and inoculated with 1% of the culture volume. The medium was cultured at 37°C and 100 rpm for 24 hours; after incubation, the culture medium was centrifuged to remove the cells, and the supernatant was obtained.

[0083] Raw material content (%) Glucose2Na2HPO41.28KH2PO40.3NaCl0.005NH4Cl0.1Yeast extract(DSM)2MgSO40.024CaCl20.00111

[0084] Culture Conditions Culture Temperature 37℃ Culture Time 24 hours Integration pH 7.0 (Uncorrected) Culture pH 6.0 Stirring Speed ​​100 rpm Inoculum 1% of culture medium

[0085] Example 1.3: Quantification of EGF in the culture supernatant of Pediococcus pentosaceus transformants

[0086] The coating solution was prepared by diluting the capture antibody in the coating buffer. 100 µl of the coating solution was dispensed per well into a 96-well plate, the plate was covered, and incubated overnight (16 hours) at 2–8°C. The contents of the wells were discarded, 300 µl of wash buffer was dispensed per well, and the plate was inverted and tapped on absorbent paper to remove excess liquid. 300 µl of blocking buffer was dispensed per well and reacted at room temperature for 1 hour. The contents of the wells were discarded, 300 µl of wash buffer was dispensed per well, and the plate was inverted and tapped on absorbent paper to remove excess liquid. The standard and sample were diluted with the blocking buffer diluent, and 100 µl of the standard and sample were dispensed into designated wells. The plate was then incubated at 500 rpm at room temperature for 1–2 hours. The contents of the wells were discarded, 300 µl of wash buffer was dispensed per well, and the plate was inverted and tapped on absorbent paper to remove excess liquid. This process was repeated 5 times. The detection antibody was diluted in blocking buffer, and 100 µl of the solution was dispensed into each well and incubated at 500 rpm at room temperature for 1–2 hours. The contents of the wells were discarded, and 300 µl of wash buffer was dispensed per well; the wells were then inverted and tapped on absorbent paper to remove excess liquid. This process was repeated 5 times.

[0087] 100 µl of TMB substrate solution was dispensed into each well, and the plate was incubated at room temperature for 30 minutes. After adding 100 µl of stop solution to each well, the absorbance was measured at 450 nm, and the results were calculated using a 4-parameter curve fit and are shown in Figure 4.

[0088] Referring to Figure 4, the results of the ELISA method for the quantification of EGF in the PP-EGF culture supernatant showed that the precision (%CV) of the EGF contained in the kit was 0.3–2.5%, the accuracy was 97.7–103.4%, and the linearity (R 2 ) was confirmed to be 1.0. In addition, the precision (%CV) of the PP-EGF culture supernatant was 0.2%, satisfying the judgment criteria of precision of 5% or less, accuracy of 95–105%, and linearity of 0.995 or higher for the standard and test substances. At this time, it was confirmed that the EGF content in the PP-EGF culture supernatant was approximately 15 μg / L. Therefore, the content of EGF in the PP-EGF culture supernatant is 0.75 ng / mL at 5%, 0.6 ng / mL at 4%, 0.3 ng / mL at 2%, 0.15 ng / mL at 1%, 0.075 ng / mL at 0.5%, 0.0375 ng / mL at 0.25%, 0.015 ng / mL at 0.1%, 0.0075 ng / mL at 0.05%, and 0.0015 ng / mL at 0.01%.

[0089]

[0090] Example 2: Cytotoxicity Test of Pediococcus pentosaceus Transformers (PP-EGF)

[0091] Example 2.1: Fibroblast (CCD-986SK) Culture and Cytotoxicity Test

[0092] The cell line used in the experiment was normal human skin fibroblast ccd-986sk (human fibroblast, CRL-1947), purchased from ATCC. This cell line was cultured in IMDM (1X) medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% penicillin 0.02 UI / mL, and streptomycin 0.02 μg / mL (Gibco) at 37°C in a 5% CO2 incubator.

[0093] Cells were seeded into a 96-well plate at a density of 2000 cells / well and cultured for 1 day. After washing once with PBS, culture medium containing the untreated group and the culture supernatant of EGF and PP-EGF at the desired concentrations was dispensed, and the cells were cultured for 3 days. Cells were stained using the Cell LiveDead analysis kit, and cell viability was calculated using 4X LiveDead cell analysis with HCS ImageXpress, and the results are shown in Figure 5.

[0094] To evaluate the efficacy of the PP-EGF growth factor, cell viability was analyzed using the LiveDead Cell Analysis (HCS) method after treating fibroblast (ccd-986sk) cell lines with EGF and PP-EGF culture supernatants. Cells were treated for 2 days with EGF (10 ng / mL) and PP-EGF culture supernatants (5%, 2%, 1%), respectively. Additionally, the EGF content in the PP-EGF culture supernatant was 0.75 ng / mL at 5%, 0.3 ng / mL at 2%, and 0.15 ng / mL at 1%.

[0095] Referring to Figure 5, the test results showed that no cytotoxicity was observed in the PP-EGF culture supernatant compared to the untreated group. In the groups treated with 2% and 1% PP-EGF culture supernatant, cell growth-promoting efficacy similar to that of the positive control (EGF) was observed. However, in the group treated with 5% PP-EGF culture supernatant, although there was no severe cytotoxicity, cell growth similar to that of the untreated group was observed, which suggests that cytotoxicity caused by the treatment with an excessive amount of culture medium actually inhibits the growth-promoting efficacy of EGF.

[0096]

[0097] Example 2.2: Melanoma cell (SK-MEL-2) culture and cell viability measurement (cytotoxicity)

[0098] The cell line used in the experiment was human skin melanoma cell SK-MEL-2 (human malignant melanoma, KCLB30068), purchased from the Korean Cell Line Bank (KCLB). This cell line was cultured in DMEM (1X) medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% penicillin 0.02 UI / mL & streptomycin 0.02 μg / mL (Gibco), in a 37°C, 5% CO2 incubator.

[0099] Cells were seeded into a 96-well plate at a density of 2000 cells / well and cultured for 3 days. After one wash with PBS, culture medium containing the untreated group and culture supernatants of EGF and PP-EGF at desired concentrations was dispensed, and the cells were cultured for 2 days. After one wash with PBS, 10% WST-8 reagent was added to the cell culture medium, and at 2 hours, absorbance was measured at an OD of 450 nm using an absorbance meter. The cell viability results were calculated and are shown in Figure 6.

[0100] Referring to Figure 6, to evaluate the efficacy of the PP-EGF growth factor, melanoma (SK-MEL-2) cells were treated with the test substance for 2 days, and cell viability was confirmed using the MTT (WST-8) method. The EGF content in the PP-EGF culture supernatant was 0.075 ng / mL at 0.5%, 0.0375 ng / mL at 0.25%, and 0.015 ng / mL at 0.1%. As a result of the test, no cytotoxicity was observed at IBMX (200 μM), EGF (10 ng / mL), Kojic acid (40 μM), or PP-EGF culture supernatant at 0.25% or less. However, cell growth efficacy was observed in the EGF-treated group, and weak cell growth efficacy was observed in the PP-EGF culture supernatant (0.25%) treated group. It is determined that the melanoma (SK-MEL-2) cell line has cytotoxicity that is more than 10 times more sensitive to PP-EGF culture supernatant than the fibroblast (ccd-986sk) cell line.

[0101]

[0102] Example 3: Wound healing assay (skin regeneration)

[0103] To evaluate the skin regeneration efficacy of EGF growth factor and PP-EGF culture supernatant, a wound healing assay was conducted using fibroblasts (CCD-986SK).

[0104] 2 x 10 CCD-986SK in a 6-well plate for cell culture 5After inoculating cells into wells and culturing for one day, the inside of the wells was neatly scratched vertically using sterile Blue tips (1000 µl), washed three times with PBS, and the scratched area was photographed using an optical microscope. After washing five times with PBS, the untreated group and culture medium containing EGF and PP-EGF culture supernatants at desired concentrations were dispensed, and the samples were cultured for one day. The skin regeneration effect was confirmed by photographing five scratched areas using an optical microscope to determine the number of cells that had entered the scratches, and calculating the average value.

[0105] Referring to Figure 7, the number of cells was checked in five images for each test group. It was confirmed that cell regeneration increased by approximately 1.3 times in the EGF (5 ng / ㎖) treated group and by approximately 1.8 times in the EGF 10 ng / ㎖ treated group compared to the untreated group. For PP-EGF culture supernatant, the values ​​were found to be approximately 1.3 times, 1.5 times, and 1.6 times for 1%, 2%, and 4%, respectively. The EGF content in the PP-EGF culture supernatant was 0.6 ng / ㎖ at 4%, 0.3 ng / ㎖ at 2%, and 0.15 ng / ㎖ at 1%. In summary, the PP-EGF culture supernatant (1-4%; EGF 0.15 - 0.6 ng / mL) showed better cell regeneration than the positive control group treated with EGF (5 ng / mL), but did not reach the cell regeneration efficacy of the positive control group treated with EGF (10 ng / mL). However, despite the EGF content in the PP-EGF culture supernatant being approximately 10 times lower than that of the EGF (5 ng / mL) single treatment group, the high cell regeneration rate relative to the EGF concentration in the PP-EGF culture supernatant is attributed to the synergistic effect of the lactic acid bacteria culture medium and EGF.

[0106]

[0107] Example 4: DPPH Antioxidant Assay

[0108] To evaluate the antioxidant efficacy of EGF and PP-EGF culture supernatants, the antioxidant efficacy was assessed by utilizing the inhibition of DPPH.

[0109] For each sample type, dilutions were performed with DPPH assay buffer to verify the EC dose-response curve. The volume was adjusted to 100 µl / well with DPPH assay buffer. 200 µl of DPPH assay buffer was added to the assay buffer control well. Trolox standard curve preparation: 0, 5, 10, 20, and 30 µl of 1 mM Trolox standard stock solution were added to each well to generate 0, 5, 10, 20, and 30 nmoles / well of Trolox standard, after which the volume was adjusted to 100 µl / well using DPPH assay buffer. 600 µM DPPH working solution was prepared by diluting 8 mM DPPH stock with DPPH assay buffer. 100 µl of 600 µM DPPH working solution was added only to the sample and standard wells, and the solutions in the wells were mixed. After incubating the plates in the dark for 10 minutes at room temperature, absorbance was measured at 517 nm. The inhibition rate (%) was calculated by subtracting the analysis buffer control readings from all standard readings and is shown in Figure 8.

[0110] Referring to Fig. 8, in this embodiment, the higher the DPPH inhibition rate, the better the antioxidant effect. Trolox (0, 5, 10, 20, 30 nmoles / well), which served as the positive control for this experiment, was treated, and DPPH was inhibited in a dose-dependent manner according to the treatment concentration. Additionally, regarding EGF, the DPPH inhibition rates were found to be very low depending on the treatment concentration, with the EGF 10 ng / ml treatment group showing a DPPH inhibition rate of 3.6% and the EGF 5 ng / ml treatment group showing a DPPH inhibition rate of 3%, respectively, so the antioxidant effect of EGF is judged to be negligible. However, the DPPH inhibition rate of the PP-EGF culture supernatant (5%) treatment group was very high at 35.5%, and the DPPH inhibition rate of the PP-EGF culture supernatant (2%) treatment group was superior to that of the positive control Trolox (10 nmoles). It was also confirmed that the DPPH inhibition rate of the PP-EGF culture supernatant (1%) treatment group was higher than that of Trolox (5 nmoles). Based on the above results, the PP-EGF culture supernatant of the present invention is considered to be an excellent synergistic partner for antioxidant efficacy that complements the low antioxidant efficacy of EGF.

[0111]

[0112] Example 5: Tyrosinase Expression qRT-PCR (Skin Whitening)

[0113] Tyrosinase is a factor involved in the synthesis of black to brown melanin, which causes skin pigmentation such as melasma and freckles, and it is known that inhibiting the expression of tyrosinase is effective for skin whitening. To evaluate the efficacy of EGF growth factor, 200 μM of 3-isobutyl-1-methylxanthine (IBMX), which induces tyrosinase expression, EGF standard (10 ng / ㎖), positive control Kojic acid (40 μM), and PP-EGF culture supernatant (0.1%, 0.05%, 0.01%) were treated to skin melanima (SK-MEL-2) cell lines, and the amount of tyrosinase expression was analyzed using qRT-PCR analysis.

[0114] 2x10 SK-MEL-2 in a 6-well plate 5 Cells were inoculated and cultured for 3 days. After a single wash with PBS, the completely untreated group and 200 μM 3-Isobutyl-1-methylxanthine (IBMX) were treated, while the untreated group and inhibitor control (IC) were treated with 40 μM Kojic Acid, EGF standard 10 ng / mL, and PP-EGF culture supernatant, followed by 2 days of culture. After cell harvesting, RNA was extracted using the RNeasy Mini Kit, and the extracted RNA concentration was verified using Nanodrop. cDNA was synthesized using TOPscript RT DryMIX dN6 plus (enzynomics). cDNA was synthesized under the following conditions: 25°C for 10 minutes, 42°C for 1 hour, 95°C for 5 minutes, and 4°C. qRT-PCR was performed using TB Green Premix Ex Taq II. At this time, qRT-PCR conditions were performed at 50°C for 5 minutes, 95°C for 15 minutes, 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds for 40 cycles. After calculating the ΔΔCT value using the confirmed CT value, the change in tyrosinase expression was analyzed, and the results are shown in Figure 9.

[0115] Primer Sequence Forward (5'→3') Reverse (5'→3') GAPDHACCACAGTCCATGCCATC (Sequence No. 3) TCCACCACCCTGTTGCTGTA (Sequence No. 4) Tyrosinase GAGGTCAGCACCCCACAAAT (Sequence No. 5) TGTTGTACTCCTCCAATCGGC (Sequence No. 6)

[0116] Referring to Figure 9, the EGF content in the PP-EGF culture supernatant was 0.015 ng / mL at 0.1%, 0.0075 ng / mL at 0.05%, and 0.0015 ng / mL at 0.01%. As a result of the test, it was confirmed that the expression of tyrosinase increased in the negative control [negative control with IBMX (200 μM)] compared to the negative control [negative control without IBMX (200 μM)], and that the expression of tyrosinase decreased in the positive controls, Kojic acid (40 μM) and EGF (10 ng / mL), respectively. In the test substance, PP-EGF culture supernatant (0.1%), the expression of tyrosinase decreased more than in the positive controls, Kojic acid (40 μM) and EGF (10 ng / mL). In addition, the PP-EGF culture supernatant showed a tendency to inhibit tyrosinase expression in a dose-dependent manner depending on the treatment concentration. In summary, since the EGF content of the PP-EGF culture supernatant (EGF 0.0015 - 0.015 ng / ml) is very low compared to the positive control EGF (10 ng / ml), the whitening efficacy of the PP-EGF culture supernatant is considered to be due to the high synergistic effect between EGF and the PP-EGF culture supernatant.

[0117]

[0118] Example 6: Elastin expression qRT-PCR (wrinkle improvement test)

[0119] Elastin is composed of elastic fibers and is involved in the flexibility, elasticity, and resilience of skin tissue. Elastin exists between collagen fibers and plays a role in binding and supporting collagen to prevent it from escaping outward. Collagen acts as a pillar that firmly supports our skin and has the efficacy to effectively improve skin elasticity and wrinkles. Therefore, in this experiment, the efficacy evaluation substance must increase the expression of elastin to be effective in improving wrinkles. In this example, the expression of elastin within fibroblasts (ccd-986sk) was analyzed using the qRT-PCR technique after treatment with the positive control EGF and PP-EGF culture supernatants compared to the untreated group.

[0120] To evaluate the wrinkle-improving efficacy of PP-EGF culture supernatant, fibroblasts (ccd-986sk) were treated with EGF (10 ng / ㎖) and PP-EGF (0.5%, 1%, 2%) for 24 hours, and changes in elastin expression were analyzed using the qRT-PCR technique.

[0121] 1 x 10⁶ CCD-986SK in a 6-well plate for cell culture 5 Cells were cultured for 1 day after inoculation into wells. A single wash with PBS was performed, followed by 1 day of culture after treatment with the untreated group and culture supernatants at desired concentrations of EGF and PP-EGF. After harvesting the cells, RNA was extracted using the RNeasy Mini Kit, and the concentration of the extracted RNA was verified using Nanodrop. cDNA was synthesized using TOPscript RT DryMIX dN6 plus (enzynomics). cDNA was synthesized under the conditions of 25°C for 10 minutes, 42°C for 1 hour, 95°C for 5 minutes, and 4°C. qRT-PCR was performed using TB Green Premix Ex Taq II. The qRT-PCR was conducted under the conditions of 50°C for 5 minutes, 95°C for 15 minutes, 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds for 40 cycles. Changes in elastin expression were analyzed after calculating ΔΔCT values ​​using the verified CT values.

[0122] Primer Name Sequence Forward (5'→3') Reverse (5'→3') GAPDHGAAATCCCATCACCATCTTCCAGG (Sequence No. 7) GAGCCCCAGCCTTCTCCATG (Sequence No. 8) Elastin AAAGCAGCAGCAAAGTTCGG (Sequence No. 9) ACTGGGAC AACTGGAATCC (Sequence No. 10)

[0123] Referring to Figure 10, the EGF content in the PP-EGF culture supernatant was 0.3 ng / mL at 2%, 0.15 ng / mL at 1%, and 0.075 ng / mL at 0.5%. As a result of the test, it was confirmed that compared to the untreated group, the elastin expression level increased by approximately 1.7 times in the EGF (10 ng / mL) treated group, while the elastin expression level increased by 1.22, 1.82, and 2.1 times in the PP-EGF (0.5%, 1%, 2%) treated groups, respectively. It was confirmed that the elastin expression level increased in a dose-dependent manner depending on the amount of PP-EGF culture supernatant applied. In particular, a higher increase in elastin expression was observed in the PP-EGF culture supernatant (1%, 2%) treated groups compared to the positive control EGF (10 ng / mL) treated group. In summary, since the EGF content of the PP-EGF culture supernatant (EGF 0.075 - 0.3 ng / ml) is very low compared to the positive control EGF (10 ng / ml), the wrinkle-improving efficacy of the PP-EGF culture supernatant is considered to be due to the high synergistic effect between EGF and the PP-EGF culture supernatant.

[0124]

[0125] Example 7: Collagenase Inhibitor Analysis (Wrinkle Improvement Test)

[0126] In a 96-well plate for fluorescence measurement, all experimental groups except the buffer control (reaction buffer) were treated with 0.2 U / ml collagenase in 100 μg / ml gelatin, and this was designated as the untreated group. As a positive control, 1,10-phenanthroline monohydrate, a general metalloproteinase inhibitor, was treated at 25, 50, and 100 μM. The experimental groups were treated with 20%, 10%, and 5% PP-EGF culture supernatant and incubated at room temperature for 2 hours; subsequently, fluorescence was measured using an emission filter at 495-505 nm and an excitation filter at 475 nm. The fluorescence intensity of each treated group was determined by subtracting the buffer control from the measured values ​​and converting the untreated group to 100%.

[0127] Collagenase breaks down collagen in the body, reducing the elasticity and moisture of skin tissue and causing wrinkle formation. An experiment to identify collagenase inhibitors was conducted; in this experiment, values ​​closer to 0% compared to the untreated group (100%) indicate a reduction in collagenase activity, which is effective for wrinkle improvement. In this cell-free experiment, 0.2 U / ml of collagenase was added to 100 μg / ml of gelatin for all experimental groups, while 1,10-phenanthroline monohydrate, a general metalloproteinase inhibitor, was added at concentrations of 25, 50, and 100 μM as a positive control. Additionally, PP-EGF culture supernatant was added to the experimental groups at concentrations of 20%, 10%, and 5%, and fluorescence was measured after incubation at room temperature for 2 hours.

[0128] As a result, the positive control, 1,10-phenanthroline monohydrate, was found to reduce collagenase activity in a dose-dependent manner, and the PP-EGF culture supernatant also reduced collagenase activity depending on the treatment amount, with collagenase activity decreasing by approximately 30% at 20% treatment. This is a similar value to that of the positive control, 1,10-phenanthroline monohydrate at 25 μM. Furthermore, no inhibition of collagenase was confirmed at EGF 10 ng / mL, and given that the PP-EGF culture supernatant reduced collagenase activity in a dose-dependent manner, it is determined that there is a substance in the PP culture medium that inhibits collagenase.

[0129]

[0130] Example 8: Elastase Inhibitor Analysis (Wrinkle Improvement Test)

[0131] In a 96-well plate for fluorescence measurement, 25 μg / ml of elastin and 0.1 U / ml of elastase were added to all experimental groups, excluding the buffer control (reaction buffer), and this was designated as the untreated group. As a positive control, the elastase inhibitor N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone was added at concentrations of 10, 5, and 2.5 μM. The experimental groups were treated with PP-EGF culture supernatant at concentrations of 20%, 10%, and 5% and incubated at room temperature for 2 hours; subsequently, fluorescence was measured using an emission filter at 495-505 nm and an excitation filter at 475 nm. The fluorescence intensity of each substance-treated group was determined by subtracting the buffer control from the measured values ​​and converting the untreated group to 100%.

[0132] Elastin is composed of elastic fibers and is involved in the flexibility, elasticity, and resilience of skin tissue. Elastin exists between collagen fibers and plays a role in binding and supporting the collagen to prevent it from escaping outward; collagen acts as a pillar that firmly supports our skin and has the efficacy to effectively improve skin elasticity and wrinkles. Therefore, in this experiment, compared to the untreated group (100%), inhibiting elastase activity indicates that the closer the fluorescence intensity value is to 0%, the better the effect on wrinkle improvement. In this cell-free experiment, all experimental groups were treated with 25 μg / ml of elastin and 0.1 μm / ml of elastase, and as a positive control, the elastase inhibitor N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone was treated at 10, 5, and 2.5 μm. In addition, PP-EGF culture supernatant was treated at concentrations of 20%, 10%, and 5% for the experimental group, and after incubation at room temperature for 1 hour, fluorescence intensity was measured. As a result, it was confirmed that the positive control, N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone, reduced elastase activity in a dose-dependent manner, and the PP-EGF culture supernatant also reduced elastase activity depending on the treatment amount; it was confirmed that elastase activity decreased by approximately 47% at 20% treatment. This value is similar to that of the positive control, N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone 2.5 μM, and elastase inhibition was confirmed to be approximately 41% at EGF 10 ng / mL. Based on the comprehensive results above, EGF inhibits elastase, and while the culture medium of Pediococcus pentosaceus PP-EGF also contains substances that inhibit elastase, it is determined that there is no synergy between the two substances and they exhibit an additive action.

[0133] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be implemented in various modified or altered forms without departing from the essential characteristics of the invention. Therefore, the true scope of protection of the present invention should be determined not by the aforementioned embodiments, but by the following claims and their equivalents.

[0134] [Consignment Number]

[0135] Name of depositing institution: Korean Culture Collection of Microorganisms

[0136] Trustee Number: KCCM13348P

[0137] Date of Trust: 20230414

[0138] [Correction pursuant to Rule 91 Dec. 27, 2024]

Claims

1. A transformed Pediococcus pentosaceus PP-EGF strain characterized by having a gene encoding human epidermal growth factor (EGF) protein introduced to express and secrete epidermal growth factor.

2. A transformed Pediococcus pentosaceus PP-EGF strain according to claim 1, characterized in that the gene is introduced using the plasmid pCBT24-2-pG6Pi-EGF-pG6Pi-EGF.

3. A cosmetic composition for improving skin comprising a culture medium of a transformed Pediococcus pentosaceus PP-EGF (KCCM13348P) strain that secretes EGF (Epidermal Growth Factor) by introducing a gene encoding human epidermal growth factor (EGF) protein, a concentrate of said culture medium, or a dried product thereof.

4. A cosmetic composition for skin improvement according to claim 3, characterized in that the strain is the transformed Pediococcus pentosaceus PP-EGF strain of deposit number KCCM13348P.

5. A cosmetic composition for skin improvement according to claim 3, characterized in that the concentration of EGF included in the cosmetic composition for skin improvement is 0.5 to 10 ppm based on the total weight of the cosmetic composition.

6. A cosmetic composition for skin improvement according to claim 3, characterized in that the cosmetic composition has a formulation selected from the group consisting of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, spray, and mixtures thereof.

7. A cosmetic composition for improving skin according to claim 3, characterized in that the skin-improving composition is for skin moisturizing, skin wrinkle improvement, skin radiance, or skin whitening.

Images