Method for preparing microorganism-derived highly functional DNA fragment mixture, and functional composition comprising DNA fragment mixture prepared thereby

Abstract

The present invention relates to: a method for preparing a highly functional DNA fragment mixture derived from microorganisms (yeast, microalgae, lactic acid bacteria, and the like); and a functional composition comprising a DNA fragment mixture prepared thereby. Specifically, the present invention relates to the preparation of a DNA fragment mixture having a uniform size within a certain range by using a specific restriction enzyme in microorganisms (yeast, microalgae, lactic acid bacteria, and the like). According to the present invention, a functional composition may be provided, which comprises the DNA fragment mixture as a main ingredient, and exhibits excellent effects in filling an interstitial scaffold, improving skin condition, regenerating tissue, healing wounds, moisturizing, promoting ECM synthesis, inhibiting cellular senescence, forming new blood vessels, alleviating inflammation, relieving pain, treating dermatitis such as atopy, inhibiting hair loss, promoting hair growth, and promoting stem cell activity.

Description

Method for preparing a mixture of high-functional DNA fragments derived from microorganisms and a functional composition comprising a mixture of DNA fragments prepared according to the same

[0001] The present invention relates to a method for preparing a high-functionality DNA fragment mixture derived from microorganisms (yeast, microalgae, lactic acid bacteria, etc.) and a functional composition comprising the DNA fragment mixture prepared according to the same.

[0002] Specifically, the present invention relates to the preparation of a DNA fragment mixture having a uniform size within a certain range using specific restriction enzymes and sonication within microorganisms (yeast, microalgae, lactic acid bacteria, etc.). Furthermore, the invention relates to a functional composition having excellent effects, such as inter-tissue scaffold filling, skin condition improvement, tissue regeneration, wound healing, moisturization, promotion of extracellular matrix (ECM) synthesis, inhibition of cellular aging, neovascularization, pain relief, anti-inflammatory effects, inhibition of hair loss, promotion of hair growth, or promotion of stem cell differentiation, using the DNA fragment mixture prepared thereby as a main component.

[0003] This application claims the benefit of priority based on Korean Patent Application No. 10-2025-0022007 filed on February 20, 2025, and all contents disclosed in the document of said Korean patent application are incorporated herein as part of this specification.

[0004] Polynucleotides included in DNA fragment mixtures are biopolymers composed of phosphate, sugar, and base; they possess viscoelasticity and exhibit tissue repair effects upon subcutaneous injection. In particular, polynucleotides form complexes with adenosine A2 receptors, which are skin regeneration signaling pathways, to promote the secretion of various growth factors. Furthermore, by inducing fibroblasts constituting skin tissue to secrete the extracellular matrix (ECM), they serve as the primary component of a novel concept of tissue repair biomaterial capable of inducing skin tissue regeneration rather than acting as mere skin fillers.

[0005] Korean registered patent number 10-2402360 discloses a method for producing polynucleotides, but the above prior art differs from the present invention in that it describes a method for producing polynucleotides derived from fish. Furthermore, the above prior art describes a method for obtaining low-molecular-weight polynucleotides of 5 kDa or less using a salting-out method (high salt) and a buffer, which differs from the present invention in that it obtains a mixture of high-functional DNA fragments having a uniform size within a certain range by using specific restriction enzymes on microorganisms (yeast, microalgae, lactic acid bacteria, etc.).

[0006] Polynucleotides, which are commonly used as core materials in cosmeceuticals and bio-cosmetics, are primarily composed of animal-derived ingredients derived from fish such as halibut, trout, and salmon. However, there is a risk of contamination with animal tissue-derived viruses in the case of mixtures of animal-derived DNA fragments, and there are difficulties in consistently supplying raw materials. Furthermore, due to the nature of aquaculture, management is cumbersome and costly, and there is a problem that pollution of the marine ecosystem can be accelerated due to feed residues and excrement from aquatic organisms discharged from aquaculture farms.

[0007] Against this backdrop, research is actively underway to produce polynucleotides from various non-fish materials, such as various plants and microalgae, and to manufacture functional compositions containing them as active ingredients.

[0008] However, research on extracting and manufacturing DNA fragment mixtures from microorganisms such as yeast and lactic acid bacteria has not been conducted, nor has research been carried out to manufacture medical device fillers, cosmetics, food, and pharmaceutical compositions or to verify their efficacy using these fragments.

[0009] In addition, Korean registered patent number 10-2584027 and Korean registered patent number 10-2252410 disclose research on manufacturing and extracting polynucleotides using microalgae, but research on manufacturing a functional composition containing these as active ingredients was not conducted. Furthermore, the size of the DNA fragment mixture manufactured and extracted in the aforementioned prior art is similar to that of salmon-derived DNA fragment mixtures used in the market, or is a DNA fragment mixture having a range of 5 kDa to 20,000 kDa, which differs from the present invention, which manufactures a DNA fragment mixture having a uniform size within a certain range.

[0010] Accordingly, the present invention aims to provide a method for producing a high-functionality DNA fragment mixture having a uniform size within a certain range by utilizing specific restriction enzymes in microorganisms (yeast, microalgae, lactic acid bacteria, etc.), and to provide a functional composition comprising the DNA fragment mixture obtained by the method of production.

[0011] The problem that the present invention aims to solve is to provide a method for preparing a mixture of high-functional DNA fragments derived from microorganisms (yeast, microalgae, lactic acid bacteria, etc.).

[0012] Another objective of the present invention is to provide a functional composition comprising a DNA fragment mixture prepared according to the method described above.

[0013] To solve the above problems, the present invention provides a composition for inter-tissue scaffold filling, skin condition improvement, tissue regeneration, wound healing, moisturization, promotion of extracellular matrix synthesis, inhibition of cell aging, neovascularization, pain relief, anti-inflammatory, inhibition of hair loss, promotion of hair growth, or promotion of stem cell differentiation, comprising a mixture of DNA fragments isolated and extracted from microorganisms (yeast, microalgae, lactic acid bacteria, etc.) and having a low molecular size of less than 1,000 bp or less than 37 kDa.

[0014] According to another embodiment of the present invention, a composition for inter-tissue scaffold filling, skin condition improvement, tissue regeneration, wound healing, moisturizing, promotion of extracellular matrix synthesis, inhibition of cellular aging, neovascularization, pain relief, anti-inflammatory, inhibition of hair loss, promotion of hair growth, or promotion of stem cell differentiation is provided, comprising a mixture of DNA fragments isolated and extracted from microorganisms (yeast, microalgae, lactic acid bacteria, etc.) and having a medium molecular size of 1,000 bp or more and less than 5,000 bp or 37 kDa or more and less than 185 kDa.

[0015] According to another embodiment of the present invention, a composition for inter-tissue scaffold filling, skin condition improvement, tissue regeneration, wound healing, moisturizing, promotion of extracellular matrix synthesis, inhibition of cell aging, neovascularization, pain relief, anti-inflammatory, inhibition of hair loss, promotion of hair growth, or promotion of stem cell differentiation is provided, comprising a mixture of DNA fragments isolated and extracted from microorganisms (yeast, microalgae, lactic acid bacteria, etc.) and having a polymerized size of 5,000 bp or more or 185 kDa or more.

[0016] According to one embodiment, the yeast used in the present invention may include the genus Saccharomyces sp., the genus Galactomyces sp., or a combination of one or more of these.

[0017] According to one embodiment, the microalgae used in the present invention may include the genus Chlorella sp., the genus Spirulina sp., or a combination of one or more of these.

[0018] According to one embodiment, the lactic acid bacteria used in the present invention may include the genus Lactobacillus sp., the genus Bifidobacterium sp., the genus Lactococcus sp., the genus Enterococcus sp., or a combination of one or more of these.

[0019] According to one embodiment, the DNA fragment in the composition of the present invention may be one or more selected from the group consisting of polydeoxyribonucleotides and polynucleotides.

[0020] According to one embodiment, the tissue may be selected from the group consisting of skin, cartilage, muscle, and ligament.

[0021] According to one embodiment, the mixture of DNA fragments having a low molecular size is

[0022] (S1) A step of adding cells of microorganisms (yeast, microalgae, lactic acid bacteria, etc.) to a lysis buffer and performing lysis and homogenization at 60 to 70°C for 1 to 3 hours;

[0023] (S2) A step of centrifuging the solution obtained in step (S1) at 9,000 to 11,000 rpm for 20 to 40 minutes to obtain a supernatant;

[0024] (S3) A step of adding 2.5 to 7.5 μL of RNase A at a concentration of 5 to 15 mg / mL to the supernatant and then placing it in an incubator at 30 to 40℃ for 10 to 30 minutes to degrade RNA and increase DNA yield;

[0025] (S4) A step of fragmenting a mixture of DNA fragments by treating the solution obtained in step (S3) with one or more restriction enzymes selected from the group consisting of Sml1, Mme1, Mly1, and Hinc2;

[0026] (S5) A step of inactivating the restriction enzyme; and

[0027] (S6) It may be manufactured by a manufacturing method that includes the step of secondary fragmentation of a mixture of DNA fragments by sonication.

[0028] According to one embodiment, the mixture of DNA fragments having a medium molecular size is

[0029] (S1) A step of adding cells of microorganisms (yeast, microalgae, lactic acid bacteria, etc.) to a lysis buffer and performing lysis and homogenization at 60 to 70°C for 1 to 3 hours;

[0030] (S2) A step of centrifuging the solution obtained in step (S1) at 9,000 to 11,000 rpm for 20 to 40 minutes to obtain a supernatant;

[0031] (S3) A step of adding 2.5 to 7.5 μL of RNase A at a concentration of 5 to 15 mg / mL to the supernatant and then placing it in an incubator at 30 to 40℃ for 10 to 30 minutes to degrade RNA and increase DNA yield;

[0032] (S4) A step of fragmenting a mixture of DNA fragments by treating the solution obtained in step (S3) with one or more restriction enzymes selected from the group consisting of Ahd1, BsrG1, Kpn1, and PshA1;

[0033] (S5) A step of inactivating the restriction enzyme; and

[0034] (S6) It may be manufactured by a manufacturing method that includes the step of secondary fragmentation of a mixture of DNA fragments by sonication.

[0035] According to one embodiment, the mixture of DNA fragments having the polymerized size is

[0036] (S1) A step of adding cells of microorganisms (yeast, microalgae, lactic acid bacteria, etc.) to a lysis buffer and performing lysis and homogenization at 60 to 70°C for 1 to 3 hours;

[0037] (S2) A step of centrifuging the solution obtained in step (S1) at 9,000 to 11,000 rpm for 20 to 40 minutes to obtain a supernatant;

[0038] (S3) A step of adding 2.5 to 7.5 μL of RNase A at a concentration of 5 to 15 mg / mL to the supernatant and then placing it in an incubator at 30 to 40℃ for 10 to 30 minutes to degrade RNA and increase DNA yield;

[0039] (S4) A step of fragmenting a mixture of DNA fragments by treating the solution obtained in step (S3) with one or more restriction enzymes selected from the group consisting of Ahd1, BsrG1, Kpn1, and PshA1; and

[0040] (S5) It may be manufactured by a manufacturing method comprising the step of inactivating the restriction enzyme.

[0041] According to one embodiment, the composition of the present invention may be a cosmetic composition.

[0042] According to one embodiment, the composition of the present invention may be a food composition.

[0043] According to one embodiment, the composition of the present invention may be a medical device filler composition.

[0044] Specific details of other embodiments according to the present invention are included in the following detailed description.

[0045] According to the composition of the present invention, a mixture of DNA fragments derived from microorganisms (yeast, microalgae, lactic acid bacteria, etc.) can be made to have a uniform size within a certain range by selective ultrasonic grinding with specific restriction enzymes. Accordingly, a functional composition with excellent effects such as inter-tissue scaffold filling, improvement of skin condition, tissue regeneration, wound healing, moisturization, promotion of extracellular matrix (ECM) synthesis, inhibition of cellular aging, neovascularization, pain relief, anti-inflammatory effects, inhibition of hair loss, promotion of hair growth, and promotion of stem cell differentiation can be provided.

[0046] FIG. 1 is a schematic diagram showing a method for preparing a mixture of high-functional DNA fragments derived from microorganisms (yeast, microalgae, lactic acid bacteria, etc.) according to the present invention.

[0047] Figure 2 is the result of confirming the size of a mixture of multi-fragmented DNA fragments derived from microorganisms (yeast, microalgae, lactic acid bacteria, etc.) by gel electrophoresis.

[0048] Figure 3 shows the results of confirming the size of a mixture of high-functional low-molecular-weight DNA fragments derived from yeast by gel electrophoresis.

[0049] Figure 4 shows the results of confirming the size of a mixture of high-performance low-molecular-weight DNA fragments derived from microalgae by gel electrophoresis.

[0050] Figure 5 shows the results of confirming the size of a mixture of high-functional low-molecular-weight DNA fragments derived from lactic acid bacteria by gel electrophoresis.

[0051] Figure 6 shows the results of confirming the size of a mixture of high-functional medium-sized DNA fragments derived from yeast by gel electrophoresis.

[0052] Figure 7 shows the results of confirming the size of a mixture of high-functional medium-sized DNA fragments derived from microalgae by gel electrophoresis.

[0053] Figure 8 shows the results of confirming the size of a mixture of high-functional medium-sized DNA fragments derived from lactic acid bacteria by gel electrophoresis.

[0054] Figure 9 shows the results of confirming the size of a mixture of high-performance polymer DNA fragments derived from yeast by gel electrophoresis.

[0055] Figure 10 shows the results of confirming the size of a mixture of high-performance polymer DNA fragments derived from microalgae by gel electrophoresis.

[0056] Figure 11 shows the results of confirming the size of a mixture of high-performance polymer DNA fragments derived from lactic acid bacteria by gel electrophoresis.

[0057] The advantages and features of the present invention, and the methods for achieving them, will become clear by referring to the embodiments described in detail below. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. The embodiments introduced herein are provided to ensure that the disclosed content is thorough and complete, and to ensure that the spirit of the present invention is sufficiently conveyed to those skilled in the art.

[0058] The term "microbial body (yeast, microalgae, lactic acid bacteria, etc.)" in the present invention refers to the entire body of a microbial body (yeast, microalgae, lactic acid bacteria, etc.). According to one embodiment, the microorganism of the present invention may include one or more of the genus Galactomyces sp., the genus Saccharomyces sp., the genus Chlorella sp., the genus Spirulina sp., the genus Lactobacillus sp., the genus Lactococcus sp., and the genus Enterococcus sp. Specifically, for example, the microorganism of the genus Galactomyces may include one or more of Galactomyces resssii and Galactomyces candidus. In addition, it may include one or more of Saccharomyces cerevisiae and Saccharomyces boulardii of the genus Saccharomyces. In addition, it may include one or more of Chlorella pyrenoidosa and Chlorella variabillis of the genus Chlorella. In addition, it may include one or more of Spirulina subsalsa and Spirulina major of the genus Spirulina. In addition, it may include Lactobacillus acidophilus of the genus Lactobacillus, Bifidobacterium animalis of the genus Bifidobacterium, Lactococcus lactis of the genus Lactococcus, and Enterococcus faecium of the genus Enterococcus.

[0059] According to one embodiment, the microorganism of the present invention may comprise yeast containing one or more of the genera Saccharomyces sp. and Galactomyces sp.; microalgae containing one or more of the genera Chlorella sp. and Spirulina sp.; or lactic acid bacteria containing one or more of the genera Lactobacillus sp., Bifidobacterium sp., Lactococcus sp. and Enterococcus sp.

[0060] The term "DNA fragment complex" in the present invention refers to one or more groups selected from polydeoxyribonucleotides and polynucleotides.

[0061] The "DNA fragment" used in the present invention may have a size of 20 bp to 300,000 bp. Specifically, for example, the molecular weight may be 1 to 10,000 kDa, for example, 1 to 37 kDa, 37 to 185 kDa, or 185 kDa to 1,000 kDa.

[0062] The term "small molecule DNA fragment mixture" in the present invention refers to a DNA fragment mixture having a size of less than 1,000 bp or 37 kDa. To prepare a high-functional small molecule DNA fragment mixture derived from microorganisms (yeast, microalgae, lactic acid bacteria, etc.), the present invention may include the steps of: treating with 5 U / μL to 15 U / μL of restriction enzyme for 5 to 7 hours under the restriction enzyme activity temperature conditions using one or more of restriction enzymes Sml1, Mme1, Mly1, and Hinc2; and sonicating the DNA fragment mixture, which was first fragmented using the restriction enzyme, for 10 to 15 minutes under conditions of 20 to 40 kHz and 30 to 50% Amp to secondarily fragment the DNA fragment mixture.

[0063] According to one embodiment, the active temperature of the restriction enzymes Mme1, Mly1, and Hinc2 is 35 to 40°C, for example, 37°C, and the active temperature of Sml1 can be 50 to 60°C, for example, 55°C.

[0064] The term "medium-sized DNA fragment mixture" in the present invention refers to a DNA fragment mixture having a size of 1,000 bp or more and less than 5,000 bp, or 37 kDa or more and less than 185 kDa. To prepare a high-functional medium-sized DNA fragment mixture derived from microorganisms (yeast, microalgae, lactic acid bacteria, etc.), the present invention may include the steps of: treating with 5 U / μL to 15 U / μL of restriction enzyme using one or more of the restriction enzymes Ahd1, BsrG1, Kpn1, and PshA1 for 5 to 7 hours under conditions of 37°C; and secondarily fragmenting the DNA fragment mixture by sonicating the DNA fragment mixture, which was first fragmented using the restriction enzyme, for 10 to 15 minutes under conditions of 20 to 40 kHz and 20 to 30% Amp.

[0065] The term "polymer DNA fragment mixture" in the present invention means that the size of the DNA fragment mixture corresponds to 5,000 bp or more or 185 kDa or more. To prepare a high-functional polymer DNA fragment mixture derived from microorganisms, the present invention may include the step of treating with 5 U / μL to 15 U / μL of restriction enzyme using one or more of the restriction enzymes Ahd1, BsrG1, Kpn1, and PshA1 for 5 to 7 hours under conditions of 37°C.

[0066] The term "multisegmented DNA fragment mixture" in the present invention means that the DNA fragment mixture is mixed in a size range of 1,000 bp to 10,000 bp.

[0067] The term "functional composition" of the present invention may be a composition having efficacy such as inter-tissue scaffold filling, tissue regeneration, wound healing, moisturization, promotion of extracellular matrix (ECM) synthesis, inhibition of cellular aging, neovascularization, pain relief, anti-inflammatory effects, inhibition of hair loss, promotion of hair growth, and promotion of stem cell differentiation, but is not limited thereto. For example, the tissue may be selected from the group consisting of skin, cartilage, muscle, and ligament.

[0068] The cosmetic composition according to the present invention can be prepared in any formulation according to conventional methods. For example, it can be prepared in a formulation selected from the group consisting of a solution, topical ointment, cream, foam, nourishing lotion, softening lotion, pack, emulsion, makeup base, foundation, essence, soap, liquid cleanser, bath additive, sun cream, sun oil, suspension, gel, lotion, powder, surfactant-containing cleansing, patch, and spray, but is not limited thereto.

[0069] The pharmaceutical composition according to the present invention can be prepared in any formulation according to conventional methods. For example, it may be used in the form of oral formulations such as capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols, as well as topical preparations, suppositories, and sterile injectable solutions, but is not limited thereto.

[0070] The food composition according to the present invention may be prepared in one formulation selected from powder, granules, pills, tablets, capsules, candies, syrups, effervescent tablets, and beverages, but is not limited thereto.

[0071] As described above, the present invention provides a functional composition comprising a mixture of DNA fragments derived from microorganisms (yeast, microalgae, lactic acid bacteria, etc.) as an active ingredient, which has excellent nucleic acid stability and excellent effects such as inter-tissue scaffold filling, improvement of skin condition, tissue regeneration, wound healing, moisturization, promotion of extracellular matrix (ECM) synthesis, inhibition of cellular aging, neovascularization, alleviation of inflammation, pain relief, anti-inflammatory, inhibition of hair loss, promotion of hair growth, and promotion of stem cell differentiation. The DNA fragment mixture prepared according to the present invention has excellent binding affinity for the A2 receptor and, through this, exhibits excellent cyclic AMP signaling activity.

[0072] According to one embodiment, the composition of the present invention may include cationic polysaccharides and cationic amino acids, etc., to enhance stability with a mixture of DNA fragments derived from isolated and purified microorganisms (yeast, microalgae, lactic acid bacteria, etc.). By including polysaccharides and cationic amino acids, etc., a composition with excellent nucleic acid stability can be provided.

[0073] According to one embodiment, the present invention may provide a medical device filler, a cosmetic, a food composition, or a pharmaceutical composition comprising the composition as described above.

[0074] The present invention will be explained in detail below through examples. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

[0075]

[0076] Comparative Example 1: Preparation of a mixture of multisegmented DNA fragments derived from yeast cells

[0077] 1) Four species of yeast, Galactomyces resssii and Galactomyces candidus belonging to the genus Galactomyces sp., and Saccharomyces cerevisiae and Saccharomyces boulardii belonging to the genus Saccharomyces sp., were cultured with shaking in a culture medium, and yeast cells were obtained using a centrifuge. At this time, YPD (Yeast extract Peptone Dextrose) medium was used as the culture medium, which was sterilized at 120°C for 20 minutes before use, and cultured under conditions of 25°C and 180 rpm. Subsequently, the cells were cultured for approximately 48 hours until the OD600 nm value reached 1.0, and then centrifuged at 4°C and 15,000 rpm to obtain the cells. Then, Galactomyces recyi, Galactomyces candidus, Saccharomyces cerevisiae, and Saccharomyces boulardii cells were mixed in a mass ratio of 1:1:1:1, and 5 mL of lysis buffer was added per 1 g of the yeast cell mixture, and a lysate was obtained through a homogenizer. At this time, the composition of the lysis buffer was 100 mM Tris-HCl (pH 8.0), 10 mM EDTA, 100 mM β-Mercaptoethanol, 1 M sorbitol, and 1% SDS per 1 L, and lysis and homogenization were performed for 2 hours under conditions of 65°C.

[0078] 2) The above lysate was centrifuged to extract a mixture of DNA fragments from the supernatant. At this time, the supernatant was obtained after centrifuging at 11,000 rpm for 20 minutes.

[0079] 3) 5 μL of RNase A at a concentration of 10 mg / mL was added to the above supernatant, and then the mixture was placed in a 37℃ incubator for 20 to 30 minutes to degrade RNA and increase DNA yield.

[0080] 4) The above supernatant was sonicated for 10 cycles (pulse; 30 / 30 sec) under 20 kHz and 25% AMP conditions to fragment the DNA fragment mixture.

[0081] 5) A precipitate was obtained from the supernatant containing the above-mentioned fragmented DNA mixture using alcohol. At this time, twice the volume of the supernatant was added as alcohol, and after precipitating overnight under 4°C conditions, the precipitate was obtained by centrifuging at approximately 7,000 rpm for 1 hour using a centrifuge. The DNA fragment mixture prepared from Comparative Example 1 contains randomly multi-fragmented DNA ranging from 1,000 to 10,000 bp. The concentration of the DNA fragment mixture obtained in this way was confirmed through DNA quantification, and its degradation was confirmed through DNA electrophoresis. For the experiment, a 1 mg / mL stock solution was prepared and used at a final concentration of 10 μg / mL, etc.

[0082]

[0083] Comparative Example 2: Preparation of a mixture of multisegmented DNA fragments derived from microalgae cells

[0084] 1) Four species of microalgae belonging to the genus Chlorella (Chlorella pyrenoidosa, Chlorella variabillis) and the genus Spirulina (Spirulina subsalsa, Spirulina major) were cultured with shaking in a culture medium, and microalgae cells were obtained using a centrifuge. At this time, BG-11 medium was used as the culture medium, sterilized at 120°C for 20 minutes, and cultured at 25°C and 150 rpm. Afterward, the cells were cultured for about 7 days until the OD600 nm value reached 1.0, and then centrifuged at 4°C and 15,000 rpm to obtain the cells. Subsequently, Chlorella pyrenoidosa:Chlorella barrierbilis:Spirulina subsalsa:Spirulina major cells were mixed in a mass ratio of 1:1:1:1, and 5 mL of lysis buffer was added per 1 g of the microalgae cell mixture, and a lysate was obtained through a homogenizer. At this time, the composition of the lysis buffer was 50 mM Tris-HCl (pH 8.0), 100 mM EDTA, 100 mM β-Mercaptoethanol, 1 M sorbitol, 1% SDS, 1% PVP (Polyvinylpyrrolidone), and 2% CTAB (Cetyltrimethylammonium bromide) per 1 L, and lysis and homogenization were performed for 2 hours under conditions of 65°C.

[0085] 2) The remaining process was manufactured in the same manner as Comparative Example 1 above.

[0086]

[0087] Comparative Example 3: Preparation of a mixture of multi-segmented DNA fragments derived from lactic acid bacteria cells

[0088] 1) Four species of lactic acid bacteria, including Lactobacillus acidophilus (belonging to the genus Lactobacillus sp.), Bifidobacterium animalis (belonging to the genus Bifidobacterium sp.), Lactococcus lactis (belonging to the genus Lactococcus sp.), and Enterococcus faecium (belonging to the genus Enterococcus sp.), were cultured with shaking on a culture medium, and lactic acid bacteria cells were obtained using a centrifuge. At this time, BHI (Brain Heart Infusion) medium was used, sterilized at 120°C for 20 minutes, and cultured under conditions of 35°C and 180 rpm. After that, the cells were cultured for about 24 hours until the OD600 nm value became 1.0, and then centrifuged at 4°C and 15,000 rpm to obtain the cells. Then, Lactobacillus acidophilus:Bifidobacterium animalis:Lactococcus lactis:Enterococcus faecium cells were mixed in a mass ratio of 1:1:1:1, 5 mL of lysis buffer was added per 1 g of the lactic acid bacteria cell mixture, and a lysate was obtained through a homogenizer. At this time, the composition of the lysis buffer was 50 mM Tris-HCl (pH 8.0), 50 mM EDTA, 20% SDS, 20 mg / mL Lysozyme, 200 μg / mL Proteinase K, and 20 mM DTT (Dithiothreitol) per 1 L, and lysis and homogenization were performed for 2 hours under conditions of 60℃.

[0089] 2) The remaining process was manufactured in the same manner as Comparative Example 1 above.

[0090]

[0091] Example 1: Preparation of Yeast-Derived DNA Fragment Mixtures of Different Sizes

[0092] 1) Four species of yeast, Galactomyces resssii and Galactomyces candidus belonging to the genus Galactomyces sp., and Saccharomyces cerevisiae and Saccharomyces boulardii belonging to the genus Saccharomyces sp., were cultured with shaking in a culture medium, and yeast cells were obtained using a centrifuge. At this time, YPD (Yeast extract Peptone Dextrose) medium was used as the culture medium, which was sterilized at 120°C for 20 minutes before use, and cultured under conditions of 25°C and 180 rpm. Subsequently, the cells were cultured for approximately 48 hours until the OD600 nm value reached 1.0, and then centrifuged at 4°C and 15,000 rpm to obtain the cells. Then, Galactomyces recyi, Galactomyces candidus, Saccharomyces cerevisiae, and Saccharomyces boulardii cells were mixed in a mass ratio of 1:1:1:1, and 5 mL of lysis buffer was added per 1 g of the yeast cell mixture, and a lysate was obtained through a homogenizer. At this time, the composition of the lysis buffer was 100 mM Tris-HCl (pH 8.0), 10 mM EDTA, 100 mM β-Mercaptoethanol, 1 M sorbitol, and 1% SDS per 1 L, and lysis and homogenization were performed for 2 hours under conditions of 65°C.

[0093] 2) The above lysate was centrifuged to extract a mixture of DNA fragments from the supernatant. At this time, the supernatant was obtained after centrifuging at 11,000 rpm for 20 minutes.

[0094] 3) 5 μL of RNase A at a concentration of 10 mg / mL was added to the above supernatant, and then the mixture was placed in a 37℃ incubator for 20 to 30 minutes to degrade RNA and increase DNA yield.

[0095] 4-1) To extract the small molecule DNA fragment mixture, the solution obtained in Step 3 was treated with a specific restriction enzyme and the DNA fragment mixture was fragmented. At this time, Mme1 was used as the restriction enzyme, and 10 U / μL of restriction enzyme was treated for 6 hours under conditions of 37°C. Afterward, the restriction enzyme was inactivated for 1 hour under conditions of 65°C. The DNA fragment mixture was secondarily fragmented by ultrasonic grinding for 10 cycles (pulse; 30 / 30 sec) under conditions of 30 kHz and 40% Amp. The specific restriction enzyme for the preparation of the yeast-derived small molecule DNA fragment mixture may include, but is not limited to, Sml1, Mme1, Mly1, and Hinc2.

[0096] 4-2) To extract the medium-sized DNA fragment mixture, the solution obtained in Step 3 was treated with a specific restriction enzyme and the DNA fragment mixture was fragmented. Ahd1 was used as the restriction enzyme, and 10 U / μL of the restriction enzyme was treated for 6 hours at 37°C. Afterward, the restriction enzyme was inactivated for 1 hour at 65°C. Subsequently, the DNA fragment mixture was secondarily fragmented by ultrasonic grinding for 10 cycles (pulse; 30 / 30 sec) at 30 kHz and 25% Amp. The specific restriction enzyme for the preparation of yeast-derived medium-sized and high-molecular-weight DNA fragment mixtures may include, but is not limited to, one or more of Ahd1, BsrG1, Kpn1, and PshA1.

[0097] 4-3) To extract the polymeric DNA fragment mixture, the solution obtained in Step 3 was treated with a specific restriction enzyme to fragment the DNA fragment mixture. Ahd1 was used as the restriction enzyme, and 10 U / μL of the enzyme was treated for 6 hours at 37°C. Afterward, the restriction enzyme was inactivated for 1 hour at 65°C.

[0098] 5) A precipitate was obtained from the supernatant containing the above-mentioned fragmented DNA mixture using alcohol. At this time, twice the volume of the supernatant was added, and after precipitating overnight under 4°C conditions, the precipitate was obtained by centrifuging at approximately 7,000 rpm for 1 hour using a centrifuge.

[0099] 6) The above purified material was dried to obtain a DNA fragment mixture. At this time, the precipitate was dried under conditions of 25–30°C for 1–3 hours to obtain a DNA fragment mixture. The concentration of the DNA fragment mixture obtained in this way was confirmed by DNA quantification, and its degradation was confirmed by DNA electrophoresis. For the experiment, a 1 mg / mL stock was prepared and used at a final concentration of 10 μg / mL, etc.

[0100]

[0101] Example 2: Preparation of a mixture of microalgae-derived DNA fragments of different sizes

[0102] 1) Four species of microalgae belonging to the genus Chlorella (Chlorella pyrenoidosa, Chlorella variabillis) and the genus Spirulina (Spirulina subsalsa, Spirulina major) were cultured with shaking in a culture medium, and microalgae cells were obtained using a centrifuge. At this time, BG-11 medium was used as the culture medium, sterilized at 120°C for 20 minutes, and cultured at 25°C and 150 rpm. Afterward, the cells were cultured for about 7 days until the OD600 nm value reached 1.0, and then centrifuged at 4°C and 15,000 rpm to obtain the cells. Subsequently, Chlorella pyrenoidosa:Chlorella barrierbilis:Spirulina subsalsa:Spirulina major cells were mixed in a mass ratio of 1:1:1:1, and 5 mL of lysis buffer was added per 1 g of the microalgae cell mixture, and a lysate was obtained through a homogenizer. At this time, the composition of the lysis buffer was 50 mM Tris-HCl (pH 8.0), 100 mM EDTA, 100 mM β-Mercaptoethanol, 1 M sorbitol, 1% SDS, 1% PVP (Polyvinylpyrrolidone), and 2% CTAB (Cetyltrimethylammonium bromide) per 1 L, and lysis and homogenization were performed for 2 hours under conditions of 65°C.

[0103] 2) The remaining process was manufactured in the same manner as in Example 1 above.

[0104]

[0105] Example 3: Preparation of a mixture of DNA fragments of different sizes derived from lactic acid bacteria

[0106] 1) Four species of lactic acid bacteria, including Lactobacillus acidophilus (belonging to the genus Lactobacillus sp.), Bifidobacterium animalis (belonging to the genus Bifidobacterium sp.), Lactococcus lactis (belonging to the genus Lactococcus sp.), and Enterococcus faecium (belonging to the genus Enterococcus sp.), were cultured with shaking on a culture medium, and lactic acid bacteria cells were obtained using a centrifuge. At this time, BHI (Brain Heart Infusion) medium was used, sterilized at 120°C for 20 minutes, and cultured under conditions of 35°C and 180 rpm. After that, the cells were cultured for about 24 hours until the OD600 nm value became 1.0, and then centrifuged at 4°C and 15,000 rpm to obtain the cells. Then, Lactobacillus acidophilus:Bifidobacterium animalis:Lactococcus lactis:Enterococcus faecium cells were mixed in a mass ratio of 1:1:1:1, 5 mL of lysis buffer was added per 1 g of the lactic acid bacteria cell mixture, and a lysate was obtained through a homogenizer. At this time, the composition of the lysis buffer was 50 mM Tris-HCl (pH 8.0), 50 mM EDTA, 20% SDS, 20 mg / mL Lysozyme, 200 μg / mL Proteinase K, and 20 mM DTT (Dithiothreitol) per 1 L, and lysis and homogenization were performed for 2 hours under conditions of 60℃.

[0107] 2) The remaining process was manufactured in the same manner as in Example 1 above.

[0108]

[0109] The low molecule DNA fragment mixture prepared in the above example was labeled A (Process 4-1), the medium molecule DNA fragment mixture was labeled B (Process 4-2), and the high molecule DNA fragment mixture was labeled C (Process 4-3).

[0110] Experimental Example 1: Quality inspection of DNA fragment mixture

[0111] 1) 1 mL of purified water was added to the DNA fragment mixture prepared from Comparative Examples 1 to 3 and Examples 1 to 3, and the purity was measured and the concentration was quantified using Nanodrop. The quantified sample was then subjected to electrophoresis on 1 μg of the sample using a 1% agarose gel to determine the range and size of the DNA fragment mixture. Pure double-stranded DNA has an OD 260 / OD 280 value of 1.8, and if contaminated with protein, the OD 260 / OD 280 value is lower than 1.8, and if contaminated with RNA, the OD 260 / OD 280 value is higher than 1.8.

[0112] 2) The results of verifying the purity of the DNA fragment mixtures prepared in Comparative Examples 1 to 3 and Examples 1 to 3 are as shown in Table 1 below. It was confirmed that the purity of all DNA fragment mixtures prepared in Comparative Examples 1 to 3 and Examples 1 to 3 was close to 1.8.

[0113] 3) The results of verifying the size of the DNA fragment mixtures prepared in Comparative Examples 1 to 3 above are as shown in Figure 2 below. As shown in Figure 2, it was confirmed that the size of the multi-segmented DNA fragment mixture prepared in Comparative Example 1 was randomly mixed in the range of 1,000 to 10,000 bp.

[0114] 4) On the other hand, it was confirmed that the low molecular weight DNA fragment mixture prepared in Example 1 was fragmented into uniform sizes within a certain range, with a size of 1,000 bp or less, the medium molecular weight DNA fragment mixture from 1,000 to 5,000 bp, and the high molecular weight DNA fragment mixture from 5,000 bp or more.

[0115] In all the tables below, the low-molecular-weight DNA fragment mixture prepared in the present invention is denoted as A, the medium-molecular-weight DNA fragment mixture as B, and the high-molecular-weight DNA fragment mixture as C.

[0116]

[0117]

[0118] Experimental Example 2: Evaluation of In Vivo Degradation Rate and Volume Change

[0119] The inventors investigated whether the mixture of DNA fragments derived from microorganisms (yeast, microalgae, lactic acid bacteria, etc.) of the present invention exhibits sustained performance within biological tissues.

[0120] 1) Experiments were conducted using 20 female SKH1 hairless mice.

[0121] 2) 100 μg / mL of the DNA fragment mixture prepared in Comparative Examples 1 to 3 and Examples 1 to 3 was intradermally injected at 0.5 ml per animal in three locations. Five animals were euthanized at 4, 8, and 16 weeks after injection, and necropsy was performed. The injection sites were excised and the size of the injection sites was measured, and the volume change was analyzed immediately after injection and after 4, 8, and 16 weeks.

[0122] 3) The experimental results are shown in Table 2 below. Among them, the polymer DNA fragment mixture treatment group prepared in Examples 1 to 3 showed the least volume change and maintained the initial injection volume for a long period. In particular, the polymer DNA fragment mixture treatment group prepared in Example 1 maintained the initial injection volume for the longest time, confirming that it had the most superior inter-tissue scaffold filling effect.

[0123]

[0124]

[0125] Experimental Example 3: Analysis of Adenosine A2 Receptor Binding Affinity

[0126] 1) Protein lysate was obtained using 293T cells overexpressing adenosine A2 receptor protein.

[0127] 2) The DNA fragment mixtures prepared in Comparative Examples 1 to 3 and Examples 1 to 3 were labeled using ThermoFisher Scientific’s Pierce Biotin 3’ End DNA labeling kit.

[0128] 3) An EMSA assay was performed using the Thermo Scientific LightShift Chemiluminescent EMSA kit. The details are as follows.

[0129] 4) A mixture of DNA fragments prepared in Comparative Examples 1 to 3 and Examples 1 to 3 was added to 200 μL of a lysate rich in A2 receptor protein lysed from cells to induce a binding reaction at a concentration of 100 μg / mL.

[0130] 5) After electrophoresis using a polyacrylamide gel, detection was performed on a membrane that had undergone transfer, crosslinking, and blocking processes. At this time, 200 μL of an HRP-conjugated solution having biotin binding affinity was added, and luminescence was induced through a substrate such as Luminol.

[0131] 6) Subsequently, the size of the emitted band was quantified using the ImageJ program (National Institutes of Health, NIH) and is shown in Table 3.

[0132] 7) As a result of the experiment, the binding affinity of the low-molecular-weight and medium-molecular-weight DNA fragment mixtures prepared in Examples 1 to 3 to the A2 receptor increased compared to the DNA fragment mixtures prepared in Comparative Examples 1 to 3, and among them, the low-molecular-weight DNA fragment mixture prepared in Example 1 showed the highest binding affinity. The following experimental example was performed using only Comparative Example 1 to compare with the DNA fragment mixture prepared in Example 1, which had the highest A2 receptor binding affinity.

[0133]

[0134]

[0135] Experimental Example 4: Analysis of Collagen and Extracellular Matrix (ECM) Gene Expression

[0136] 1) Human dermal fibroblasts (HDFs) were cultured at 70-80% in a 100 pi dish, and the medium was replaced with a mixture of 10 μg / mL of DNA fragments prepared in Comparative Example 1 and Example 1, and cultured for an additional 6 hours.

[0137] 2) Total RNA was extracted, and cDNA was synthesized using it as a template with M-MLV Reverse Transcriptase (Invitrogen).

[0138] 3) qRT-PCR was performed using primers for COL1A1, COL3A1, MMP1, and Elastase1 with the generated cDNA.

[0139] 4) As shown in Table 4, human dermal fibroblasts (HDF) showed increased collagen synthesis enzyme (COL1A1, COL3Al) mRNA expression in the group treated with the small and medium molecular weight DNA fragment mixture prepared in Example 1 compared to Comparative Examples 1 and 2, and among them, the highest COL1A1 and COL3Al mRNA expression was observed in the group treated with the small molecular weight DNA fragment mixture prepared in Example 1.

[0140] 5) Also, as shown in Table 4, collagenase (MMP1) and elastinase (Elastase1) mRNA expression decreased in the group treated with the mixture of small and medium molecular weight DNA fragments prepared in Example 1 compared to Comparative Example 1. Among them, MMP1 mRNA expression and Elastase mRNA expression decreased the most in the group treated with the mixture of small molecular weight DNA fragments prepared in Example 1.

[0141]

[0142]

[0143] Experimental Example 5: Analysis of In vitro Wound Regeneration Effect

[0144] 1) Physical scratches were made on the bottom of a 100 pi dish containing 100% culture of HaCaT, and the medium was replaced with a mixture of 10 μg / mL of DNA fragments prepared in Comparative Example 1 and Example 1. The size of the scratches was measured under a microscope after 12, 24, and 48 hours.

[0145] 2) As shown in Table 5, compared to Comparative Example 1, the HaCaT cells showed higher wound regeneration efficacy in the groups treated with the mixture of small and medium molecular weight DNA fragments prepared in Example 1, and among them, the highest wound regeneration efficacy was observed in the group treated with the mixture of small molecular weight DNA fragments prepared in Example 1.

[0146]

[0147]

[0148] Experimental Example 6: Analysis of Moisturizing-Related Gene Expression

[0149] 1) The medium was replaced with a medium containing 10 μg / mL of the DNA fragment mixture prepared in Comparative Example 1 and Example 1, and cultured for an additional 6 hours.

[0150] 2) Total RNA was extracted, and cDNA was synthesized using it as a template with M-MLV Reverse Transcriptase (Invitrogen).

[0151] 3) qRT-PCR was performed using primers for HAS1, HAS2, and HAS3 with the generated cDNA.

[0152] 4) As shown in Table 6, compared to Comparative Example 1, the mRNA expression of the HAS family (HAS1, HAS2, HAS3), which are enzymes that synthesize hyaluronic acid, increased in the group treated with the mixture of small and medium molecular weight DNA fragments prepared in Example 1.

[0153]

[0154]

[0155] Experimental Example 7: Analysis of Efficacy in Alleviating Inflammatory and Atopic-Associated Cytokines

[0156] 1) The plate containing Raw264.7 cells was replaced with a medium containing 10 μg / mL of the DNA fragment mixture prepared in Comparative Example 1 and Example 1, and cultured for an additional 6 hours.

[0157] 2) Total RNA was extracted, and cDNA was synthesized using it as a template with M-MLV Reverse Transcriptase (Invitrogen).

[0158] 3) qRT-PCR was performed using primers for IL-1β, IL-6, and IL-4 with the generated cDNA.

[0159] 4) As shown in Table 7, Raw264.7 cells showed reduced mRNA expression of inflammatory cytokines and atopic-associated cytokine genes (IL-1β, IL-6, IL-4) in the group treated with the mixture of small and medium molecular weight DNA fragments prepared in Example 1 compared to Comparative Example 1.

[0160]

[0161]

[0162] Experimental Example 8: VEGF Gene Expression Analysis

[0163] 1) Human dermal fibroblasts (HDFs) were cultured at 70-80% in a 100 pi dish, and the medium was replaced with a mixture of DNA fragments prepared in Comparative Example 1 and Example 1 at 10 μg / mL and cultured for an additional 6 hours.

[0164] 2) Total RNA was extracted, and cDNA was synthesized using it as a template with M-MLV Reverse Transcriptase (Invitrogen).

[0165] 3) qRT-PCR was performed using primers for VEGF and VEGFR with the generated cDNA.

[0166] 4) As shown in Table 8, compared to Comparative Example 1, HDF cells showed increased expression of vascular endothelial growth factor and vascular endothelial growth factor receptor (VEGF, VEGFR) mRNA in the group treated with the small and medium molecular weight DNA fragment mixture prepared in Example 1, and among them, the highest expression of vascular endothelial growth factor and vascular endothelial growth factor receptor (VEGF, VEGFR) mRNA was observed in the group treated with the small molecular weight DNA fragment mixture prepared in Example 1.

[0167]

[0168]

[0169] Experimental Example 9: Analysis of Efficacy in Alleviating Cell Aging

[0170] 1) Human epidermal keratinocytes (HEK) were cultured for 24 hours, then replaced with a medium containing 10 ppm of the DNA fragment mixture prepared in Comparative Example 1 and Example 1 and pretreated for 4 hours. Afterward, 2200 μM of H2O was added and cultured for an additional 4 hours.

[0171] 2) After removing the culture medium from the cells, they were washed twice with 1–2 mL of PBS.

[0172] 3) Add an equal amount of Fixation solution to PBS and incubate at room temperature for 5 minutes.

[0173] 4) After removing the fixation solution, the process of 2) was repeated.

[0174] 5) After adding 1~2 mL of staining solution, the plate was sealed, wrapped in aluminum foil, and incubated at 37℃ for about 10 hours.

[0175] 6) The degree of staining was checked between the control group and the negative control group treated only with H2O2. When a significant change was observed, the staining solution was removed, washed with PBS, and 70% glycerol was added.

[0176] 7) The number of stained cells was counted using a microscope.

[0177] 8) As shown in Table 9, the SA-β-gal activity increased by H2O2 decreased in the low-molecular-weight and medium-molecular-weight DNA fragment mixture treatment groups of Example 1 compared to the DNA fragment mixture treatment group of Comparative Example 1. Among them, the highest cell aging inhibitory efficacy was observed in the low-molecular-weight DNA fragment mixture treatment group prepared in Example 1.

[0178]

[0179]

[0180] Experimental Example 10: Analysis of Hair Growth-Associated Gene Expression

[0181] 1) Human dermal papilla cells (HDPCs) were cultured at 70-80% in a 100 pi dish, and the medium was replaced with a mixture of 10 μg / mL of DNA fragments prepared in Comparative Example 1 and Example 1, and cultured for an additional 6 hours.

[0182] 2) Total RNA was extracted, and cDNA was synthesized using it as a template with M-MLV Reverse Transcriptase (Invitrogen).

[0183] 3) qRT-PCR was performed using primers for FGF7, FGF10, and NOG with the generated cDNA as the template.

[0184] 4) As shown in Table 10, the mRNA levels of hair growth factors FGF7, FGF10, and NOG increased in the group treated with the low-molecular-weight and medium-molecular-weight DNA fragment mixture of Example 1 compared to the group treated with the DNA fragment mixture of Comparative Example 1 in HDP cells.

[0185]

[0186]

[0187] Experimental Example 11: Analysis of Stem Cell Potential Regulating Factor Expression

[0188] 1) Mesenchymal stem cells (MSCs) in culture were replaced with a medium containing 10 μg / mL of the DNA fragment mixture prepared in Comparative Example 1 and Example 1 and cultured for an additional 6 hours.

[0189] 2) Total RNA was extracted, and cDNA was synthesized using it as a template with M-MLV Reverse Transcriptase (Invitrogen).

[0190] 3) qRT-PCR was performed using the generated cDNA with primers for OCT4 and SOX2.

[0191] 4) As shown in Table 11, the mRNA levels of OCT4 and SOX2 in mesenchymal stem cells increased in the groups treated with the small and medium molecular weight DNA fragment mixture of Example 1 compared to the group treated with the DNA fragment mixture of Comparative Example 1. Among them, the highest mRNA expression levels of OCT4 and SOX2 were observed in the group treated with the small molecular weight DNA fragment mixture prepared in Example 1.

[0192]

[0193]

[0194] Experimental Example 12: Evaluation of Pain Level

[0195] 1) 1 mL of a mixture of DNA fragments from Comparative Example 1 and Example 1 at a time was injected into the skin of male Sprague-Dawley (SD) rats weighing 200–250 g.

[0196] 2) To determine the degree of pain immediately after injection and 24 hours later, ultrasonic sounds of 22–27 KHz generated by rats in pain, distress, and stress conditions for 10 minutes each were measured using Sonotrack (Metris).

[0197] 3) It was confirmed that the frequency of ultrasound generation was lower when the DNA fragment mixture of Example 1 was treated compared to Comparative Example 1 in the USV call measurements immediately after injection.

[0198] 4) In the USV call measurements 24 hours after injection, it was confirmed that the frequency of ultrasound generation was lower when the DNA fragment mixture of Example 1 was treated compared to Comparative Example 1. Among them, the group treated with the low molecular weight DNA fragment mixture prepared in Example 1 showed the lowest pain levels.

[0199]

[0200]

[0201] Formulation Example 1: Filler

[0202] A mixture of 5% of the DNA fragment mixture of Examples 1 to 3 of the present invention, 50% of hyaluronic acid, 0.2% of vitamin C, 1% of L-arginine, and 0.01% of chitosan was mixed, and then purified water was added to reach a maximum volume of 100% to produce a filler according to a conventional method for producing fillers.

[0203]

[0204] Formulation Example 2: Tablet

[0205] 10 mg of the DNA fragment mixture of Examples 1 to 3 of the present invention, 400 mg of lactose, 400 mg of corn starch, 2 mg of magnesium stearate, 0.1 mg of L-arginine, and 0.01 mg of chitosan were mixed, and then tablets were prepared by compressing the mixture according to a conventional method for preparing tablets.

[0206]

[0207] Formulation Example 3: Drink

[0208] 10 mg of the DNA fragment mixture of Examples 1 to 3 of the present invention, 10 g of glucose, 0.6 g of citric acid, 25 g of liquid oligosaccharide, 1 mg of L-arginine, and 0.1 mg of chitosan were mixed, and then 300 ml of purified water was added and filled into each bottle with 200 ml. After filling into the bottles, the mixture was sterilized at 130°C for 4 to 5 seconds to prepare a drink.

[0209]

[0210] Formulation Example 4: Lotion

[0211] A lotion was prepared according to a conventional method for preparing lotion by mixing 1% of the DNA fragment mixture of Examples 1 to 3 of the present invention, 15% of glycerin, 10% of butylene glycol, 1% of cetearyl alcohol, 1% of sorbitan oliveate, 3% of caprylic / capric triglyceride, 0.5% of squalane, 2% of 1,2-hexanediol, 3% of cyclopentasiloxane / cyclohexasiloxane, 5% of mineral oil, 0.01% of disodium EDTA, 0.05% of BHT, 0.5% of tocopheryl acetate, 0.2% of ethylhexyl methoxycinnamate, 0.1% of L-arginine, and 0.01% of chitosan, and then adding purified water to make up to 100% of the volume.

Claims

A composition for inter-tissue scaffold filling, skin condition improvement, tissue regeneration, wound healing, moisturizing, promotion of extracellular matrix synthesis, inhibition of cellular aging, neovascularization, pain relief, anti-inflammatory, inhibition of hair loss, promotion of hair growth, or promotion of stem cell differentiation, comprising a mixture of DNA fragments isolated and extracted from microorganisms and having a low molecular size of less than 1,000 bp, wherein the microorganisms include one or more of yeast, microalgae, and lactic acid bacteria. A composition for inter-tissue scaffold filling, skin condition improvement, tissue regeneration, wound healing, moisturizing, promotion of extracellular matrix synthesis, inhibition of cellular aging, neovascularization, pain relief, anti-inflammatory, inhibition of hair loss, promotion of hair growth, or promotion of stem cell differentiation, comprising a mixture of DNA fragments isolated and extracted from microorganisms and having a medium molecular size of 1,000 bp or more and less than 5,000 bp, wherein the microorganisms comprise one or more of yeast, microalgae, and lactic acid bacteria. A composition for inter-tissue scaffold filling, skin condition improvement, tissue regeneration, wound healing, moisturizing, promotion of extracellular matrix synthesis, inhibition of cellular aging, neovascularization, pain relief, anti-inflammatory, inhibition of hair loss, promotion of hair growth, or promotion of stem cell differentiation, comprising a mixture of DNA fragments isolated and extracted from microorganisms and having a polymerized size of 5,000 bp or more, wherein the microorganisms include one or more of yeast, microalgae, and lactic acid bacteria. A mixture of DNA fragments of microorganisms according to any one of claims 1 to 3, wherein the microorganism comprises yeast containing one or more of the genera Saccharomyces sp. and Galactomyces sp.; microalgae containing one or more of the genera Chlorella sp. and Spirulina sp.; or lactic acid bacteria containing one or more of the genera Lactobacillus sp., Bifidobacterium sp., Lactococcus sp. and Enterococcus sp. A composition according to any one of claims 1 to 3, wherein the DNA fragment is one or more selected from the group consisting of polydeoxyribonucleotides and polynucleotides. A composition according to any one of claims 1 to 3, wherein the tissue is selected from the group consisting of skin, cartilage, muscle, and ligament. In claim 1, the mixture of DNA fragments having a low molecular size is (S1) A step of adding microbial cells to a lysis buffer and performing lysis and homogenization at 60 to 70°C for 1 to 3 hours; (S2) A step of centrifuging the solution obtained in step (S1) at 9,000 to 11,000 rpm for 20 to 40 minutes to obtain a supernatant; (S3) A step of adding 2.5 to 7.5 μL of RNase A at a concentration of 5 to 15 mg / mL to the supernatant and then placing it in an incubator at 30 to 40℃ for 10 to 30 minutes to degrade RNA and increase DNA yield; (S4) A step of fragmenting a mixture of DNA fragments by treating the solution obtained in step (S3) with one or more restriction enzymes selected from the group consisting of Sml1, Mme1, Mly1, and Hinc2; (S5) A step of inactivating the restriction enzyme; and (S6) A composition prepared by a method comprising the step of secondary fragmentation of a mixture of DNA fragments by sonication. In paragraph 2, the mixture of DNA fragments having a medium molecular size is (S1) A step of adding microbial cells to a lysis buffer and performing lysis and homogenization at 60 to 70°C for 1 to 3 hours; (S2) A step of centrifuging the solution obtained in step (S1) at 9,000 to 11,000 rpm for 20 to 40 minutes to obtain a supernatant; (S3) A step of adding 2.5 to 7.5 μL of RNase A at a concentration of 5 to 15 mg / mL to the supernatant and then placing it in an incubator at 30 to 40℃ for 10 to 30 minutes to degrade RNA and increase DNA yield; (S4) A step of fragmenting a mixture of DNA fragments by treating the solution obtained in step (S3) with one or more restriction enzymes selected from the group consisting of Ahd1, BsrG1, Kpn1, and PshA1; (S5) A step of inactivating the restriction enzyme; and (S6) A composition prepared by a method comprising the step of secondary fragmentation of a mixture of DNA fragments by sonication. In paragraph 3, the above mixture of DNA fragments having a polymerized size is (S1) A step of adding microbial cells to a lysis buffer and performing lysis and homogenization at 60 to 70°C for 1 to 3 hours; (S2) A step of centrifuging the solution obtained in step (S1) at 9,000 to 11,000 rpm for 20 to 40 minutes to obtain a supernatant; (S3) A step of adding 2.5 to 7.5 μL of RNase A at a concentration of 5 to 15 mg / mL to the supernatant and then placing it in an incubator at 30 to 40℃ for 10 to 30 minutes to degrade RNA and increase DNA yield; (S4) A step of fragmenting a mixture of DNA fragments by treating the solution obtained in step (S3) with one or more restriction enzymes selected from the group consisting of Ahd1, BsrG1, Kpn1, and PshA1; and (S5) A composition prepared by a method comprising the step of inactivating the restriction enzyme. A composition according to any one of claims 7 to 9, wherein the DNA fragment mixture is prepared by a manufacturing method in which a microbial cell is cultured by shaking and then the following steps are performed (S1). A composition according to any one of claims 1 to 3, wherein the composition is a cosmetic composition. A composition according to any one of claims 1 to 3, wherein the composition is a food composition. A composition according to any one of claims 1 to 3, wherein the composition is a medical device filler composition.

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