Method for preparing microbial polynucleotide and Anti-aging microbial pdrn prepared thereby

Abstract

The present invention relates to a method for preparing a microbial polynucleotide that can be used as a functional ingredient for cosmetics and, specifically, to: a method for preparing a microbial polynucleotide, the method comprising culturing lactic acid bacteria or yeast while maintaining an appropriate temperature and pH using a mixed medium free of a meat extract, and effectively eluting and obtaining a high concentration of the microbial polynucleotide through a cell softening process and a thermal-pressure disruption process; and a microbial polynucleotide prepared by the preparation method.

Description

Method for producing microbial polynucleotides and anti-aging microbial PDRN produced by the method

[0001] The present application claims priority to Korean Patent Application No. 10-2024-0189570 filed on December 18, 2024 and Korean Patent Application No. 10-2025-0192490 filed on December 8, 2025, and the entire specification is a reference to the present application. The present invention relates to a method for producing microbial polynucleotides that can be used as functional ingredients for cosmetics, and specifically, to a method for producing microbial polynucleotides by culturing lactic acid bacteria or yeast while maintaining an appropriate temperature and pH using a mixed medium that does not contain meat extract, and effectively eluting and obtaining high concentrations of microbial polynucleotides by a cell softening process and a hot-pressure crushing process, and to microbial polynucleotides produced by said method.

[0002]

[0003] Polydeoxyribonucleotides (PDRN) are DNA fragments extracted from plants or animals and consist of low molecular weight DNA ranging from 50 to 1,500 kDa. These PDRNs can exhibit pharmacological activity without the transfer of genetic information and are known to aid in tissue repair and regeneration by promoting the production of growth factors and enhancing cell proliferation related to skin regeneration.

[0004] Currently, PDRN is used as a cosmetic ingredient, a skin injectable, and an injectable for cartilage and tissue regeneration, and is mainly extracted from the sperm of salmon or trout. However, the supply is insufficient because the amount obtainable per fish is small. Furthermore, since PDRN is extracted through chemical reactions using organic solvents such as phenol and chloroform during the extraction process, there are issues with chemical residues, and the purity of PDRN decreases due to the mixing with animal proteins, which is the cause of various side effects.

[0005] Meanwhile, various formulations using lactic acid bacteria such as Lactococcus, Bifidobacterium, and Lactobacillus are being marketed as cosmetic ingredients. However, these lysates contain cellular components of microorganisms, such as denatured proteins, and if these components are in excessive amounts, they may cause skin problems such as rashes, erythema, and atopy.

[0006] Therefore, there is a need to develop a method to produce PDRN as a functional cosmetic ingredient more efficiently and economically while compensating for the disadvantages of the lactic acid fermentation process.

[0007]

[0008] The inventors have completed the present invention by introducing a softening process using a high-molecular-weight polysaccharide as a preliminary step for the physical crushing of lactic acid bacteria or yeast, as a method for producing microbial polynucleotides having a structure and functionality similar to polydeoxyribonucleotides (PDRN). Through the softening and crushing steps, the use of chemicals is excluded from the polynucleotide elution process, thereby stabilizing and concentrating low-molecular-weight polynucleotides that are safe and have excellent skin beautifying effects, while minimizing cellular components that cause skin troubles and enabling economical production of microbial polynucleotides.

[0009]

[0010] Therefore, the objective of the present invention is to...

[0011] (a) a pre-fermentation step in which microorganisms are grown in a nutrient medium containing a carbon source, a nitrogen source, vitamins, and inorganic salts;

[0012] (b) a fermentation step in which the microorganisms proliferated in step (a) above are proliferated in a mixed medium containing a carbon source, a nitrogen source, vitamins, and inorganic salts;

[0013] (c) A cell softening step in which the microorganisms proliferated in step (b) above are separated and softened in a softening solution consisting only of high-molecular-weight polysaccharides and water;

[0014] (d) a step of hot-press crushing the softening solution containing the microorganisms from step (c) above so that the lactic acid bacteria cells are crushed and the microbial polynucleotides are eluted; and

[0015] (e) a step of removing cell wall and cytoplasmic protein components from the product obtained in step (d) above and recovering microbial polynucleotides;

[0016] The present invention provides a method for manufacturing a microbial polynucleotide containing

[0017]

[0018] Another objective of the present invention is to provide a microbial polynucleotide produced by the above-described manufacturing method.

[0019]

[0020] Another objective of the present invention is to provide a cosmetic or quasi-drug composition for skin antioxidant, skin aging inhibition, wound healing, or cell activity enhancement comprising a microbial polynucleotide prepared by the above-described manufacturing method.

[0021]

[0022] Another objective of the present invention is to provide a use of the microbial polynucleotide for preparing compositions for skin antioxidant, skin aging inhibition, wound healing, or activity enhancement.

[0023]

[0024] Another objective of the present invention is to provide a method for skin antioxidant, skin aging inhibition, wound healing, or cell activity enhancement comprising administering an effective amount of a composition containing the microbial polynucleotide as an active ingredient to an individual in need thereof.

[0025]

[0026] To achieve the above objectives, the present invention

[0027] (a) a pre-fermentation step in which microorganisms are grown in a nutrient medium containing a carbon source, a nitrogen source, vitamins, and inorganic salts;

[0028] (b) a fermentation step in which the microorganisms proliferated in step (a) above are proliferated in a mixed medium containing a carbon source, a nitrogen source, vitamins, and inorganic salts;

[0029] (c) A cell softening step in which the microorganisms proliferated in step (b) above are separated and softened in a softening solution consisting only of high-molecular-weight polysaccharides and water;

[0030] (d) a step of hot-press crushing the softening solution containing the microorganisms from step (c) above so that the lactic acid bacteria cells are crushed and the microbial polynucleotides are eluted; and

[0031] (e) a step of removing cell wall and cytoplasmic protein components from the product obtained in step (d) above and recovering the microorganism polynucleotide;

[0032] A method for producing a microbial polynucleotide comprising

[0033]

[0034] To achieve another objective of the present invention, the present invention provides a microbial polynucleotide produced by the above-described manufacturing method.

[0035]

[0036] To achieve another objective of the present invention, the present invention provides a cosmetic or quasi-drug composition for skin antioxidant, skin aging inhibition, wound healing, or cell activity enhancement comprising a microbial polynucleotide prepared by the above-described manufacturing method.

[0037]

[0038] To achieve another objective of the present invention, the present invention provides a use of the microbial polynucleotide for preparing a composition for skin antioxidant, skin aging inhibition, wound healing, or activity enhancement.

[0039]

[0040] To achieve another objective of the present invention, the present invention provides a method for skin antioxidant, skin aging inhibition, wound healing, or cell activity enhancement comprising administering an effective amount of a composition containing the microbial polynucleotide as an active ingredient to an individual in need thereof.

[0041]

[0042] Meanwhile, the term "polynucleotide" as used in this specification refers to a compound in which nucleotides are repeatedly linked through phosphate bonds and may exist in the form of DNA or RNA. Such polynucleotides may exhibit various biological activities depending on their length, molecular weight, and base composition, and are particularly useful in the fields of medicine and cosmetics due to their efficacy in tissue regeneration and skin improvement. In this specification, the term "polynucleotide" is not interpreted as a concept limited by origin, length, molecular weight, or structural characteristics, and may also be used to include or have equivalent meanings of PDRN, PDRN (polydeoxyribonucleotide), polydeoxyribonucleotide, and / or polyribonucleotide, and may include nucleotides related thereto, and may be used interchangeably with the above terms depending on the context.

[0043]

[0044] The present invention will be described in detail below.

[0045]

[0046] The first step of the method for producing lactic acid bacteria polynucleotide according to the present invention is

[0047] (a) A pre-fermentation step in which microorganisms are grown in a nutrient medium containing carbon sources, nitrogen sources, vitamins, and inorganic salts.

[0048]

[0049] Step (a) above is a pre-fermentation step in which microorganisms to elute polynucleotides are primarily proliferated. The proliferation of microorganisms in Step (a) takes place in a nutrient medium, which is a medium containing carbon sources, nitrogen sources, vitamins, and inorganic salts that are nutrients necessary for the proliferation of microorganisms; examples include MRS liquid medium, lactose liquid medium, BHI liquid medium, Todd Hewitt liquid medium, etc. In the embodiment of the present invention, Step (a) was carried out using MRS or YPD as the nutrient medium.

[0050] The above MRS liquid medium refers to a medium developed for the selective cultivation of lactic acid bacteria (LAB), and contains nitrogen sources such as beef extract, yeast extract, and peptone, as well as buffering and selective components such as glucose, sodium acetate, and ammonium citrate. The pH is low at 5.5, which inhibits the growth of competitive microorganisms and promotes the proliferation of various lactic acid bacteria such as Lactobacillus, Lacticaseibacillus, and Lactiplantibacillus.

[0051] The above YPD liquid medium is a standard medium for yeast culture composed of yeast extract, peptone, and dextrose. It refers to a highly nutritious medium designed to enable most yeast species, including Saccharomyces cerevisiae, to grow rapidly and stably, and is a medium widely used for general pre-culture and main culture of yeast.

[0052] The above-mentioned liquid lactose medium is a medium of simple composition containing lactose as the primary carbon source, primarily used for the detection of coliforms and fermentation capacity tests. It is suitable for identifying microorganisms that decompose lactose to produce acid and gas, and generally refers to a medium that supports only basic growth by including a minimal nitrogen source, such as peptone.

[0053] The above BHI liquid medium is a highly nutrient-intensive medium suitable for culturing microorganisms with high nutrient requirements, such as Streptococcus, Enterococcus, and Staphylococcus. It also supports the growth of various bacteria and fungi and is widely used as a standard medium in clinical microbiology.

[0054] The Todd Hewitt liquid medium mentioned above refers to a high-nutrient medium developed for the culture of streptococcal species such as Streptococcus and Lactococcus. It is a selectively enriched medium that is standardly used in clinical microbial culture, diagnostic tests, and studies on streptococcal toxin production.

[0055]

[0056] (a) The activity of the strain stored in a frozen state is increased due to the culture in the pre-fermentation stage, thereby facilitating the smooth progress of the subsequent (b) main fermentation stage of microorganisms. Additionally, the pre-fermentation stage (a) is performed to proliferate the strain, thereby allowing for the appropriate inoculation amount to be adjusted for the (b) main fermentation stage. For example, in the case of 1% inoculation, 0.5 mL of 1 mL frozen strain stock is inoculated (pre-fermented) into 50 mL of culture medium, and after cultivation, the pre-fermented 50 mL of culture medium is inoculated into 5 L of culture medium to perform the (b) stage of cultivation.

[0057]

[0058] Step (a) can be carried out according to a conventional culture method. Specifically, microorganisms are inoculated into a nutrient medium at room temperature sterilized by a conventional method at a volume of 0.5 to 10% (v / v) and cultured at 30 to 40°C for 10 to 30 hours. More preferably, microorganisms can be inoculated into the nutrient medium at a volume of 1 to 3% (v / v) and cultured at 34 to 38°C for 14 to 18 hours. It is preferable to perform Step (a) as a static culture without stirring.

[0059]

[0060] In step (a) of the present invention, any microorganism capable of producing polynucleotides may be used, but in the present invention, it is most preferable that the microorganism be lactic acid bacteria or yeast.

[0061] In the method for producing microbial polynucleotides according to the present invention, the lactic acid bacteria can be applied to any lactic acid bacteria that can be used in the cosmetics field, etc., as long as the polynucleotide can be eluted by cell lysis of the lactic acid bacteria. Lactic acid bacteria are also called lactic acid bacteria or lactic acid bacteria, and are a general term for Gram-positive bacteria that decompose carbohydrates into lactic acid through metabolism. The method for producing a microbial polynucleotide according to the present invention may utilize lactic acid bacteria such as Lacticaseibacillus sp., Lactobacillus sp., Limosilactobacillus sp., Lactiplantibacillus sp., Bifidobacterium sp., Streptococcus sp., Lactococcus sp., Enterococcus sp., Pediococcus sp., or Weissella sp., and preferably, Lacticaseibacillus sp., Lactobacillus sp., Limosilactobacillus sp., Lactiplantibacillus sp., Lactococcus sp., or Bifidobacterium sp. Lactic acid bacteria of the genus Streptococcus can be applied.More specifically, the above lactic acid bacteria are Lacticaseibacillus rhamnosus, Lacticaseibacillus casei, Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus bulgaricus, Limosilactobacillus reuteri, Lactiplantibacillus plantarum, Lactococcus lactis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium infantis, One or more lactic acid bacteria selected from the group consisting of Bifidobacterium bifidum and Streptococcus thermophilus may be used to obtain polynucleotides according to the method of the present invention.

[0062] In addition, the method for producing microbial polynucleotides according to the present invention may also be applied to yeast. Yeast is a unicellular eukaryotic organism with excellent energy metabolism and fermentation capabilities, and is widely used as a key model microorganism in biotechnology in general, such as industrial fermentation, protein expression, and genomic research. The method for producing microbial polynucleotides according to the present invention may be applied to yeasts of the genus Saccharomyces, such as Saccharomyces p. More specifically, one or more yeasts selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces pastorianus, and Saccharomyces boulardii may be used to obtain polynucleotides according to the method of the present invention. However, it is not limited thereto.

[0063]

[0064] Next to the pre-fermentation step of the method for producing microbial polynucleotides according to the present invention

[0065] (b) The present fermentation step is carried out in which the microorganisms proliferated in step (a) above are proliferated in a mixed medium containing a carbon source, a nitrogen source, vitamins, and inorganic salts.

[0066]

[0067] Step (b) above is a step of culturing the microorganisms primarily proliferated in the pre-fermentation step (a) in earnest in a mixed medium, and as the strain proliferates and grows, polynucleotides also accumulate within the cells. The main fermentation process of Step (b) above is carried out in a mixed medium containing a carbon source, a nitrogen source, vitamins, and inorganic salts.

[0068]

[0069] In particular, the mixed medium of step (b) above may contain glucose, yeast extract, sodium acetate anhydrous, potassium phosphate dibasic, ammonium citrate dibasic, magnesium sulfate, and manganese(II) sulfate monohydrate. It is preferable to use distilled water or filtered water from which impurities have been removed to prepare the mixed medium.

[0070] MRS medium, commonly used for culturing lactic acid bacteria, contains beef extract, making it unfriendly and uneconomical in terms of cost. Similarly, YPD, used for culturing yeast, contains expensive extracts, thus presenting limitations in both eco-friendliness and economic feasibility. To develop a medium capable of producing high concentrations of polynucleotides while replacing these MRS / YPD media, the inventors cultured lactic acid bacteria or yeast without meat extract and varied the components and composition of the medium, and compared the polynucleotide yields. As a result, they developed a mixed medium having the aforementioned components and the following composition. The mixed medium according to the present invention has components that are partially similar to MRS, a representative lactic acid bacteria medium, while minimizing side effects by excluding animal-derived ingredients (beef extract, peptone, etc.) and polysorbate 80. It also offers significant economic advantages, such as cost savings of approximately 10 times or more compared to MRS.

[0071]

[0072] In step (b) according to the present invention, the mixed medium for culturing lactic acid bacteria may preferably be a liquid medium having the following composition dissolved in distilled water: glucose 1~8% (w / v), yeast extract 1~8% (w / v), sodium acetate anhydrous 0.05~2% (w / v), potassium phosphate dibasic 0.02~1% (w / v), ammonium citrate dibasic 0.02~1% (w / v), magnesium sulfate 0.001~0.05% (w / v), manganese (II) sulfate monohydrate 0.001~0.05% (w / v), antifoam 0.01~1% (v / v).

[0073] More preferably, the mixed medium for lactic acid bacteria culture in step (b) above may be a liquid medium having the following composition dissolved in distilled water: glucose 1.5~4% (w / v), yeast extract 1.5~4% (w / v), sodium acetate anhydrous 0.3~0.7% (w / v), potassium phosphate dibasic 0.1~0.4% (w / v), ammonium citrate dibasic 0.1~0.4% (w / v), magnesium sulfate 0.005~0.02% (w / v), manganese (II) sulfate monohydrate 0.005~0.02% (w / v), antifoam 0.1~0.2% (v / v).

[0074] Most preferably, the mixed medium of the above (b) step for culturing lactic acid bacteria may be a liquid medium having the following composition dissolved in distilled water: glucose 2% (w / v), yeast extract 2% (w / v), sodium acetate anhydrous 0.5% (w / v), potassium phosphate dibasic 0.2% (w / v), ammonium citrate dibasic 0.2% (w / v), magnesium sulfate 0.01% (w / v), manganese (II) sulfate monohydrate 0.01% (w / v), antifoam 0.1% (v / v).

[0075] In step (b) according to the present invention, the mixed medium for yeast culture may preferably be a liquid medium having the following composition dissolved in distilled water: glucose 1-8% (w / v), yeast extract 1-8% (w / v), soy peptone 1-8% (w / v), sodium acetate anhydrous 0.05-2% (w / v), potassium phosphate dibasic 0.02-1% (w / v), ammonium citrate dibasic 0.02-1% (w / v), magnesium sulfate 0.001-0.05% (w / v), manganese (II) sulfate monohydrate 0.001-0.05% (w / v). Antifoam 0.01~1%(v / v).

[0076] More preferably, the mixed medium for yeast culture in step (b) above may be a liquid medium having the following composition dissolved in distilled water: glucose 1.5~4% (w / v), yeast extract 1.5~4% (w / v), sodium acetate anhydrous 0.3~0.7% (w / v), potassium phosphate dibasic 0.1~0.4% (w / v), ammonium citrate dibasic 0.1~0.4% (w / v), magnesium sulfate 0.005~0.02% (w / v), manganese (II) sulfate monohydrate 0.005~0.02% (w / v), antifoam 0.1~0.2% (v / v).

[0077]

[0078] When culturing lactic acid bacteria, step (b) above can be carried out according to a conventional lactic acid bacteria culture method. Specifically, a nutrient medium containing the lactic acid bacteria proliferated in step (a) above is inoculated into a mixed medium at room temperature sterilized by a conventional method at a ratio of 0.5 to 10% (v / v) relative to the volume of the mixed medium, and then shaken culture is performed for 10 to 30 hours under conditions of 30 to 40°C, 20 to 300 rpm, and pH 4.0 to 7.0. More preferably, a nutrient medium containing the lactic acid bacteria proliferated in step (a) above is inoculated into the mixed medium at a ratio of 1 to 3% (v / v), and then cultured for 14 to 18 hours under conditions of 34 to 38°C, 100 to 200 rpm, and pH 5.0 to 6.0.

[0079] Meanwhile, when culturing yeast, the above step (b) can be carried out according to a conventional yeast culture method. Specifically, the yeast from step (a) is inoculated into a sterilized mixed medium at room temperature using a conventional method at a concentration of 0.5 to 10% (v / v), and then cultured for 10 to 40 hours under conditions of 25 to 30°C, 20 to 300 rpm, 0.5 to 1.5 vvm, and pH 4.0 to 7.0. More preferably, the yeast from step (a) is inoculated at a concentration of 1 to 3% (v / v), and then cultured for 20 to 30 hours under conditions of 27 to 29°C, 100 to 200 rpm, 0.7 to 1.0 vvm, and pH 5.0 to 6.0.

[0080]

[0081] In the method for producing microbial polynucleotides according to the present invention, when the pre-fermentation step and the main fermentation step of lactic acid bacteria are completed,

[0082] (c) The microorganisms proliferated in step (b) above are separated and subjected to a cell softening step in which they are softened in a softening solution consisting only of high-molecular-weight polysaccharides and water.

[0083]

[0084] Step (c) above is a step to facilitate and ensure smoother hot-press crushing in the subsequent step (d) by culturing the strain that has finished proliferating in a softening solution so that the cell wall of the strain is softened, thereby allowing more strains to be crushed and high concentrations of polynucleotides to be eluted.

[0085]

[0086] Step (c) above can be performed by isolating only the proliferated microorganisms by a conventional method, washing them, and then culturing them in a softening solution. Specifically, the microbial cells can be recovered by centrifugation (8,000 rpm, 20 min), washed twice with sterile physiological saline, centrifuged again under the same conditions to recover only the microbial cells, resuspended in a softening solution, and cultured by shaking for 12 to 24 hours at 4 to 18°C ​​and 200 rpm to perform the softening step. More preferably, the microbial cells can be resuspended in a softening solution and cultured by shaking for 16 to 18 hours at 4°C and 200 rpm to perform the softening step.

[0087]

[0088] In addition, the above-mentioned softening solution can be used in a volume ranging from 0.05 to 1 times the volume of the mixed medium in the previous step (b). Most preferably, the softening solution can be used in a volume of 0.1 times the volume of the mixed medium in step (b) to carry out step (c).

[0089]

[0090] The above-mentioned polymeric polysaccharide may be included in the softening solution at a concentration of 0.1 to 4% (w / v). More preferably, it may be included at a concentration of 0.5 to 2% (w / v). The concentration of the polymeric polysaccharide included in the softening solution can be appropriately selected by a person skilled in the art within the above range to minimize denatured proteins and abnormal fermentation odors generated during microbial fermentation and the subsequent hot-press crushing process, depending on the selected polymeric polysaccharide. It is preferable to use distilled water or filtered water that does not contain impurities for preparing the softening solution. For example, the polymeric polysaccharide is dissolved in distilled water at a selected concentration and sterilized for use.

[0091]

[0092] The softening solution of step (c) above consists only of a high-molecular-weight polysaccharide and water. The high-molecular-weight polysaccharide is one that microorganisms cannot utilize in metabolic processes or is not degraded by microorganisms.

[0093] That is, during the softening step (c) above, the high-molecular-weight polysaccharides in the softening solution are not broken down into low-molecular-weight compounds by microorganisms, and perform only the function of softening the cell wall regardless of the proliferation or metabolic activity of microorganisms. The high-molecular-weight polysaccharides of the natural gum type used in the present invention have a large molecular weight and are stable against changes in pH, so the decomposition of the substance does not easily occur even if there is a change in the pH of the solution containing the polysaccharides. In addition, due to the high molecular weight of the high-molecular-weight polysaccharides, they cannot be utilized as metabolic substances by microorganisms themselves, and therefore lactic acid, a metabolic product of lactic acid bacteria, is not produced, so the pH of the softening solution in step (c) can be maintained more stably.

[0094]

[0095] The polymeric polysaccharide for preparing the above-mentioned soft nitride may be one or more selected from the group consisting of xanthan gum, gum arabic, locust bean gum, tara gum, and guar gum.

[0096] These natural gums, derived from natural sources, are widely used in the food manufacturing industry as natural thickeners, stabilizers, or gelling agents. Gum arabic is a natural gum produced by solidifying acacia sap and is a high-molecular-weight mixture of glycoproteins and polysaccharides. Xanthan gum is a natural mixture of polysaccharides produced by fermenting carbohydrates using microorganisms obtained primarily from cruciferous plants such as cabbage. Locust bean gum is obtained by crushing soybeans and lentils, or by dissolving them in hot water, filtering, and precipitating with isopropyl alcohol; its main components are polysaccharides of mannose and galactose. Tara gum is obtained from the seeds of soybeans and tara, and its main components are polysaccharides of mannose and galactose. Guar gum is obtained by crushing the endosperm of soybean and guar seeds or by extracting them with hot or warm water, and its main component is polysaccharides.

[0097]

[0098] The polymeric polysaccharide for preparing the softening solution according to the present invention may preferably be xanthan gum or gum arabic. Most preferably, it may be gum arabic.

[0099] In an embodiment of the present invention, when a softening solution was prepared with xanthan gum or gum arabic and step (c) was performed, both xanthan gum and gum arabic showed excellent polynucleotide yields, and specifically, it was observed that the effect was better in a softening solution containing gum arabic than in xanthan gum. In addition, as a result of comparing the sensory aspects, such as abnormal fermentation odor, and yield of polynucleotides produced by varying the concentration of gum arabic, it was found that when a softening solution containing 1% (w / v) gum arabic was used, high levels of polynucleotides could be obtained with less denatured protein and abnormal fermentation odor, and thus, 1% (w / v) was confirmed to be the most desirable concentration of gum arabic when using gum arabic in a softening solution.

[0100]

[0101] Additionally, glucose may be added to the softening solution of step (c) as needed. Glucose may be included in the softening solution at a concentration of 0.1 to 10% (w / v).

[0102]

[0103] In the method for producing microbial polynucleotides according to the present invention, when the lactic acid bacteria proliferation steps, such as pre-fermentation and main fermentation, and the cell softening process are completed

[0104] (d) A step is performed to hot-press crush the softened solution containing the microorganisms from step (c) above so that the lactic acid bacteria cells are crushed and the microbial polynucleotides are eluted.

[0105]

[0106] In the above (d) hot-press crushing step, the microorganisms are crushed and the internal polynucleotides are leached out. In addition, the high-molecular-weight polysaccharides contained in the softening solution are reduced to low-molecular-weight molecules and mixed with the leached polynucleotides to protect the polynucleotides and prevent loss.

[0107]

[0108] The hot-pressure crushing in step (d) above may be performed for 20 to 60 minutes under conditions of a temperature of 80 to 130°C and a pressure of 0.01 to 0.3 MPa. More preferably, it may be performed for 25 to 35 minutes at a temperature of 120 to 125°C and a pressure of 0.15 to 0.25 MPa. Accordingly, the hot-pressure crushing in step (d) above may be controlled so that the process intensity (∫P·t) using heat or pressure is in the range of 0.01 to 0.30 MPa·h. According to one embodiment of the present invention, when hot-pressure crushing was performed under the above conditions, the polynucleotide concentration was confirmed to be 1040 to 2620 μg / mL, and at this time D 90 <100 was achieved, and it was confirmed that protein and endotoxin impurities decreased sharply depending on the intensity.

[0109]

[0110] In addition, the hot-pressure crushing in step (d) above may be controlled so that the F0 value (cumulative heat treatment strength) is in the range of 3 to 8.

[0111] The above F0 value (F-zero value) is an indicator used to quantify the cumulative thermal lethality in the heat treatment process. It represents a value converted to have the same sterilization and crushing effect as treatment time at a reference temperature of 121.1°C (i.e., 250°F). In other words, it is a value calculated by integrating the temperature fluctuations during the actual hot-pressure crushing process along the time axis and converting them into an equivalent treatment time based on 121.1°C; it is a quantitative indicator that comprehensively reflects the temperature-time history of the entire process. For example, an F0 value of 3 signifies a thermal lethality equivalent to treatment at 121.1°C for 3 minutes, while an F0 value of 8 indicates a thermal effect equivalent to treatment at 121.1°C for 8 minutes. Therefore, the F0 value is an integrated process indicator that reflects all thermal effects such as processing temperature, holding time, and heating / cooling intervals. In the present invention, by controlling the hot-pressure crushing step so that F0 is in the range of 3 to 8, it is possible to prevent excessive thermal decomposition while maximizing the elution efficiency of polynucleotides and effectively suppressing the retention of impurities such as high molecular weight DNA, proteins, and endotoxins.

[0112]

[0113] In the method for producing microbial polynucleotides according to the present invention, after crushing lactic acid bacteria

[0114] (e) A step is performed to remove cell wall and cytoplasmic protein components from the product obtained in step (d) above and to recover microbial polynucleotides.

[0115]

[0116] Step (e) above is a step of obtaining polynucleotides by removing unnecessary microbial proteins and cell wall components to increase the purity of the lactic acid bacteria polynucleotides. By removing microbial cell walls and cytoplasmic proteins, unnecessary proteins that may cause microbial-derived fermentation odors, precipitate formation, and skin troubles are removed, thereby enhancing the sensory properties and safety of the raw material.

[0117]

[0118] In step (e) above, in particular, the pellet containing the cell wall can be removed through cooling and centrifugation, and the remaining pellet and cytoplasmic proteins can be removed through filtration.

[0119] To remove the cell walls and denatured protein components caused by high temperature and high pressure in step (e) above, methods known in the field of biology may be used without limitation. Preferably, after hot-pressure crushing in step (d), the cell walls and denatured proteins may be aggregated by cooling, the pellet containing the cell walls and cytoplasmic proteins may be removed by centrifugation, and the remaining denatured proteins may be further removed through filtration. The cooling may be performed in a range of 0 to 10°C where the denatured proteins may aggregate or settle, and preferably, it is cooled to 0 to 4°C. For centrifugation, any centrifugation condition and method capable of separating and removing cell walls and denatured proteins in the form of pellets from the lactic acid bacteria lysate may be used, and preferably, centrifugation is performed at a speed of 7,000 to 8,000 rpm. For the filtration process, any type of filter that filters out denatured proteins while allowing polynucleotides to pass through may be used without limitation, and preferably, a filter with a pore size of 0.2 to 1.0 μm may be used.

[0120]

[0121] In the method for producing microbial polynucleotides according to the present invention, after recovering the microbial polynucleotides, as needed

[0122] (f) A step of preparing the microbial polynucleotide recovered in step (e) into a powder formulation through freeze-drying, spray-drying, vacuum drying, or fluid bed drying may be additionally performed.

[0123]

[0124] Step (f) above is a step of preparing the microbial polynucleotide recovered in Step (e) into a powder formulation. This powdering process plays an important role in improving the storage stability of the raw material by minimizing the denaturation of the polynucleotide that may occur in the liquid state, quality degradation caused by microbial residues, and reduced stability in high temperature and high humidity environments.

[0125] According to one embodiment of the present invention, as a result of powdering the microbial polynucleotide prepared according to the present invention, D 50 and D 90 The values ​​were confirmed to be 60 bp or less and 100 bp or less, respectively, showing no change compared to the liquid state; no new DNA bands were detected even after the powdering process, nor was an increase in protein and endotoxin content observed. As a result of DNA quantification, the recovery rate remained at an excellent level compared to the liquid state immediately after manufacturing, and the moisture content was maintained at a level of 2-3%, maintaining its original characteristics even during long-term storage under room temperature conditions (25℃, relative humidity 60%) and accelerated conditions (40℃, relative humidity 75%). Thus, it was confirmed that the microbial polynucleotide according to the present invention is a safe functional ingredient that improves storage stability through the powdering process, increases applicability to various product formulations, and maintains the same quality as the liquid state even upon redissolution.

[0126] The freeze-drying described above refers to a method in which a sample is rapidly cooled to a solid state and then the ice is directly sublimated under reduced pressure. It has the advantage of obtaining high-purity powder while minimizing the degradation of heat-sensitive polynucleotides, and is advantageous for maintaining bioactivity due to minimal structural denaturation.

[0127] The above spray drying is a process in which a liquid is sprayed as fine droplets and then dried instantaneously by contacting it with drying air; it is suitable for mass production and is characterized by a very short drying time.

[0128] The vacuum drying described above refers to a process that enables drying even at relatively low temperatures by removing moisture at low pressure. This allows for the maintenance of the stability of materials sensitive to heat and / or oxidation, such as polynucleotides, and offers the advantage of a low risk of quality degradation as oxidation is suppressed during the process.

[0129] The fluidized bed drying described above is a method of drying powder or fine particles while suspended by an airflow, characterized by the ability to achieve efficient drying due to uniform contact between the air and the particles. It is advantageous for formulation development as it allows for the uniform maintenance of particle shapes and the production of powders with excellent resoluble properties.

[0130]

[0131] In addition, the present invention provides a microbial polynucleotide produced by the microbial polynucleotide production method described above.

[0132]

[0133] The inventors compared the size and activity of polynucleotides produced by the method according to the present invention, which they named microbial PDRN, with those of conventional salmon PDRN. It was found that salmon PDRN mainly has a size in the range of 200 to 800 bp and a molecular weight of approximately 130 to 520 kDa, indicating that it is a mixture of polynucleotides of relatively diverse sizes. In contrast, the polynucleotides produced according to the present invention are mostly low-molecular-weight substances with a size of less than 100 bp and a molecular weight of approximately 32.5 to 65 kDa, confirming that they are a mixture of relatively homogeneous sizes. In particular, it can be seen that the small size of the polynucleotides will allow for more effective absorption into the skin than salmon PDRN.

[0134] More specifically, as confirmed by the inventors, the microbial polynucleotide produced by the method according to the present invention is D 50 ≤60bp and D 90 It showed a distribution of ≤100 bp. Here, the above D 50 is the median (percentile 50) meaning that 50% of the total DNA fragments are less than or equal to that length, and the above D 90 The median value indicates that 90% of the total fragments are of a length less than or equal to the specified length, meaning that the polynucleotide produced by the method according to the present invention is a low-molecular-weight polynucleotide composition in which high-molecular-weight DNA fragments are almost non-existent. Furthermore, the protein content of the polynucleotide produced according to the method of the present invention was confirmed to be 200 ppm or less, and the endotoxin content was 5 EU / g or less, which means that microbial cell residues and outer membrane components were effectively removed. Since endotoxin is a lipopolysaccharide (LPS) found in the outer membrane of Gram-negative bacteria and is a substance that can induce fever and / or inflammatory responses in living organisms, low endotoxin levels are an important indicator for evaluating high-purity purification. These results demonstrate that the hot-pressure crushing and purification process according to the present invention is highly advantageous for securing high purity and high yield of low-molecular-weight polynucleotides while effectively suppressing the retention of impurities such as high-molecular-weight DNA, proteins, and endotoxins.

[0135] Accordingly, the polynucleotide according to the present invention was found to have a significant effect, exhibiting a statistically significant difference compared to salmon PDRN in terms of antioxidant capacity measured by DPPH radical scavenging ability and SOD-like activity. Furthermore, the lactic acid bacteria polynucleotide according to the present invention was found to be superior to salmon PDRN at the same concentration in terms of anti-inflammatory effect (NO production) measured by the inhibition of intracellular nitric oxide production, skin protection effect against H2O2, and skin regeneration effect measured using a skin keratinocyte cell line (HaCaT).

[0136]

[0137] The microbial polynucleotide according to the present invention demonstrated excellent efficacy in clinical skin evaluations. No skin irritation or adverse skin reactions were observed when applied to human skin. The microbial polynucleotide according to the present invention increased skin radiance with just a single application, and after 4 weeks of use, skin elasticity, density, turnover, and wrinkles all showed significant improvement compared to before use.

[0138]

[0139] Therefore, it can be seen that the microbial polynucleotide produced by the manufacturing method according to the present invention is advantageous for skin absorption due to its small and homogeneous size, and can serve as an excellent functional ingredient for anti-aging cosmetics with superior skin protection and regenerative effects. In addition, the microbial polynucleotide produced by the manufacturing method according to the present invention includes liquid formulations and / or powder formulations as described above, and in the case of a powder formulation, it can be stored more stably for a long period of time and can be applied to various formulation products.

[0140]

[0141] Accordingly, the present invention provides a cosmetic composition and a quasi-drug composition for skin antioxidant, skin aging inhibition, wound healing, or cell activity enhancement, comprising a microbial polynucleotide prepared by the above-described method for preparing microbial polynucleotides.

[0142]

[0143] In the present invention, the cosmetic composition may be prepared in any formulation that is conventionally manufactured, and may be formulated, for example, as a solution, emulsion, suspension, paste, cream, lotion, gel, powder, spray, surfactant-containing cleansing, oil, soap, liquid cleansing agent, bath additive, wax foundation, makeup base, essence, lotion, foam, pack, softening water, sunscreen cream or sun oil, but is not limited thereto.

[0144] When the formulation of the present invention is a solution or an emulsion, a solvent, a solubilizing agent, or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan.

[0145] In the case where the formulation of the present invention is a suspension, liquid diluents such as water, ethanol, or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, or tracant may be used as carrier components.

[0146] In the case where the formulation of the present invention is a paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component.

[0147] In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, it may additionally include a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether.

[0148] In the case where the formulation of the present invention is a cleansing agent containing a surfactant, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester, etc. may be used as a carrier component.

[0149] The cosmetic composition according to the present invention may further include conventional auxiliary agents and carriers, such as commonly used antioxidants, stabilizers, solubilizers, vitamins, pigments, fragrances, etc., in addition to microbial polynucleotides. For example, the cosmetic composition may further include auxiliary components such as glycerin, butylene glycol, polyoxyethylene hydrogenated castor oil, tocopheryl acetate, citric acid, panthenol, squalane, sodium citrate, and allantoin.

[0150] In the present invention, the content of the composition is not significantly limited depending on the purpose or aspect of use, and may be, for example, 0.01 to 99 weight%, preferably 0.5 to 50 weight%, and more preferably 1 to 30 weight% based on the total weight of the composition. In addition, the composition according to the present invention may further include additives such as pharmaceutically acceptable carriers, excipients, or diluents in addition to the active ingredient. The pharmaceutical composition of the present invention may contain 0.1 to 99.9 weight% of the microbial polynucleotide prepared by the method of the present invention and may contain 99.9% to 0.1 weight% of a carrier.

[0151]

[0152] In the present invention, the above-mentioned quasi-drug refers to articles used for the purpose of diagnosing, treating, improving, alleviating, managing, or preventing diseases of humans or animals, among which the effect is milder than that of pharmaceuticals. For example, according to the Pharmaceutical Affairs Act, quasi-drugs are defined as articles excluding those used for pharmaceutical purposes, and include products used for the treatment or prevention of diseases of humans or animals, and products that have a mild effect on the human body or do not act directly on it.

[0153] When the polynucleotide according to the present invention is used as an additive for quasi-drugs, the composition may be added as is or used together with other quasi-drugs or quasi-drug ingredients, and may be used appropriately according to conventional methods. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use.

[0154] The quasi-drug composition of the present invention is not limited thereto, but preferably may be one or more formulations selected from the group consisting of body cleansers, disinfectants, detergents, kitchen detergents, cleaning detergents, toothpaste, mouthwash, wet wipes, detergents, soaps, hand washes, hair cleansers, hair softeners, humidifier fillers, masks, ointments, and filter fillers. Except for differences according to the formulation, the same content as described for the cosmetic composition may be applied to the quasi-drug composition of the present invention.

[0155]

[0156] To achieve another objective of the present invention, the present invention provides a use of the microbial polynucleotide for preparing a composition for skin antioxidant, skin aging inhibition, wound healing, or activity enhancement.

[0157]

[0158] To achieve another objective of the present invention, the present invention provides a method for skin antioxidant, skin aging inhibition, wound healing, or cell activity enhancement comprising administering an effective amount of a composition containing the microbial polynucleotide as an active ingredient to an individual in need thereof.

[0159]

[0160] The "effective amount" of the present invention refers to an amount that exhibits skin antioxidant effects, inhibition of skin aging, wound healing, or enhancement of cell activity when administered to an individual. The "individual" may be an animal, preferably a mammal, particularly including humans, and may also be a cell, tissue, organ, etc. derived from an animal. The individual may be a patient requiring the said effect.

[0161]

[0162] The method for producing microbial polynucleotides according to the present invention adds a cell softening step prior to the lactic acid bacteria or yeast crushing step, thereby allowing the lactic acid bacteria or yeast to be crushed more effectively and the polynucleotides to be efficiently eluted. Additionally, the softening solution from the aforementioned step protects the eluted polynucleotides, thereby preventing loss. The microbial polynucleotides produced by the method of the present invention exhibit improved sensory properties due to reduced fermentation odor of the raw materials, are advantageous for skin absorption due to their small size, and possess excellent effects such as antioxidant and anti-inflammatory properties. Therefore, the method for producing microbial polynucleotides according to the present invention and the polynucleotides produced by said method can be usefully utilized as an economical, safe, and highly effective anti-aging functional ingredient in cosmetics.

[0163]

[0164] FIG. 1 is a process flowchart illustrating a method for producing high-concentration microbial polynucleotides according to the present invention.

[0165] Figure 2 is a graph showing the concentration (μg / mL) of lactic acid bacteria polynucleotides according to the composition of the cell softening solution.

[0166] Figure 3 is a graph showing the elution concentration (ppm) of lactic acid bacteria polynucleotides according to the concentration of gum arabic in the softened solution.

[0167] Figure 4 is a graph showing the concentration (ppm) of lactic acid bacteria polynucleotides prepared under different culture conditions.

[0168] Figures 5a and 5b show agarose gel photographs comparing the sizes of lactic acid bacteria polynucleotide (LACTO) and salmon PDRN (salmon 1, salmon 2) according to the present invention.

[0169] Figures 6a and 6b show agarose gel photographs comparing the sizes of lactic acid bacteria polynucleotides (L. rhamnosus, L. plantarum, B. longum), yeast polynucleotides (S. cerevisiae), and salmon-PDRN (salmon) according to the present invention.

[0170] Figure 7 shows the GPC chromatogram, cumulative distribution, and molecular weight distribution curve of a lactic acid bacteria polynucleotide according to the present invention.

[0171] Figure 8 is a graph of experimental results comparing the DPPH free radical scavenging activity of lactic acid bacteria polynucleotide (LACTO) and salmon PDRN (PDRN) according to the present invention. * indicates p<0.05.

[0172] Figure 9 is a graph of experimental results comparing the SOD-like activity of lactic acid bacteria polynucleotide (LACTO) and salmon PDRN (PDRN) according to the present invention. *** indicates p<0.001.

[0173] Figure 10 is a graph showing experimental results confirming the anti-inflammatory effect of lactic acid bacteria polynucleotide (LACTO) and salmon PDRN (PDRN) according to the present invention as the degree of inhibition of nitric oxide production.

[0174] Figure 11 is a graph of experimental results measuring the cytotoxicity of lactic acid bacteria polynucleotide (LACTO) and salmon PDRN (PDRN) according to the present invention as cell viability.

[0175] Figure 12 is a graph of experimental results measuring the degree of skin protection against cell stimulation by H2O2 of lactic acid bacteria polynucleotide (LACTO) and salmon PDRN (PDRN) according to the present invention as cell viability.

[0176] Figures 13a and 13b show a graph (Figure 13a) showing the would area indicating the degree of cell wound healing in the HaCaT scratch assay and a photograph (Figure 13b) showing the change in the would area.

[0177] Figure 14 is a graph of the results of a clinical trial on skin radiance. GU is a parameter of skin radiance, and * indicates p<0.05.

[0178] Figures 15a and 15b are a graph of the clinical trial results for skin density (Figure 15a) and a photograph of the ultrasound measurement results (Figure 15b). Skin density is expressed in %, and * indicates p<0.05.

[0179] Figure 16 is a graph of the results of a clinical trial on skin elasticity. R2 is the parameter of skin elasticity, and * indicates p<0.05.

[0180] Figures 17a and 17b are the results graph of a skin turnover clinical trial (Figure 17a) and a UV image (Figure 17b). The degree of skin turnover is indicated by intensity, and # indicates p<0.05.

[0181] Figures 18a and 18b show the results of a clinical trial on skin wrinkles and photographs of the changed wrinkles. Skin wrinkles are indicated by maximum depth (mm), and # indicates p<0.05.

[0182] Figures 19a to 19c show the survey evaluation items of the skin clinical trial and the survey results of the subjects.

[0183] FIG. 20 shows an agarose gel photograph comparing the sizes of the liquid formulation (Liquid), freeze-dried powder redissolved solution (FD-powder), and spray-dried powder redissolved solution (SD-powder) of the lactic acid bacteria polynucleotide according to the present invention.

[0184]

[0185] The present invention will be described in detail below.

[0186] However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0187]

[0188] Example 1

[0189] Preparation of lactic acid bacteria polynucleotides using glucose + xanthan gum softening solution

[0190] Lactic acid bacteria (Lacticaseibacillus rhamnosus) isolated from domestic soybeans were inoculated into MRS liquid medium at a concentration of 2% (v / v) and pre-cultured at 37°C for 16–18 hours, then inoculated into MRS liquid medium at a concentration of 2% (v / v) and pre-cultured at 37°C for 7–9 hours. After inoculating the secondary pre-culture medium at a concentration of 2% (v / v) into a mixed medium composed of yeast extract, sodium acetate anhydrous, potassium phosphate dibasic, ammonium citrate dibasic, magnesium sulfate, and manganese (II) sulfate monohydrate, the main culture was carried out for 16 to 18 hours at 37℃, 100 rpm, and pH 5.6, followed by centrifugation (8,000 rpm, 20 min) to recover only the cells, which were then washed twice with 0.85% (w / v) saline solution and centrifuged (8,000 rpm, 20 min) to recover only the cells. The recovered bacterial cells were suspended in a sterile softening solution (glucose + xanthan gum aqueous solution) and softened at 4°C and 200 rpm for 16–18 hours. Afterward, the cells were lysed under hot-pressure crushing conditions for 30 minutes, and after cooling at 2–4°C, the lactic acid bacteria lysate was centrifuged (8,000 rpm, 20 min) to recover only the supernatant. After being left at room temperature for 16–24 hours, it was finally filtered through a 0.20 μm filter.

[0191]

[0192] Comparative Example 1

[0193] Polynucleotide production at different softening reaction process temperatures

[0194] Cells were cultured and recovered in the same manner as in Example 1, washed twice with 0.85% saline solution, and centrifuged (8,000 rpm, 20 min) to recover only the cells. The recovered cells were suspended in a sterile softening solution (glucose + xanthan gum aqueous solution) and softened at 10°C, 200 rpm, for 16 to 18 hours. Afterward, the cells were crushed under hot-pressure crushing conditions for 30 minutes, and after cooling at 2 to 4°C, the lactic acid bacteria lysate was centrifuged (8,000 rpm, 20 min) to recover only the supernatant.

[0195]

[0196] Comparative Example 2

[0197] Polynucleotide production at different softening reaction process temperatures

[0198] Cells were cultured and recovered in the same manner as in Example 1, washed twice with 0.85% saline solution, and centrifuged (8,000 rpm, 20 min) to recover only the cells. The recovered cells were suspended in a sterile softening solution (glucose + xanthan gum aqueous solution) and softened at 25°C, 200 rpm, for 16–18 hours. Afterward, the cells were crushed under hot-pressure crushing conditions for 30 minutes, and after cooling at 2–4°C, the lactic acid bacteria lysate was centrifuged (8,000 rpm, 20 min) to recover only the supernatant.

[0199]

[0200] Comparative Example 3

[0201] Polynucleotide production at different softening reaction process temperatures

[0202] Cells were cultured and recovered in the same manner as in Example 1, washed twice with 0.85% saline solution, and centrifuged (8,000 rpm, 20 min) to recover only the cells. The recovered cells were suspended in a sterile softening solution (glucose + xanthan gum aqueous solution) and softened at 37°C, 200 rpm, for 16–18 hours. Afterward, the cells were crushed under hot-pressure crushing conditions for 30 minutes, and after cooling at 2–4°C, the lactic acid bacteria lysate was centrifuged (8,000 rpm, 20 min) to recover only the supernatant.

[0203]

[0204] The polynucleotide concentrations according to the softening reaction process temperature were analyzed using the methods of Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3, respectively, and were confirmed to be 1,530 μg / mL, 1,425 μg / mL, 1,250 μg / mL, and 1,103 μg / mL, respectively. Although a decrease in concentration due to contamination occurring during the room temperature manufacturing process was predicted, preservative treatment was excluded due to its impact on the quality of the final product.

[0205]

[0206] Example 2

[0207] Preparation of lactic acid bacteria polynucleotides using a softened solution of glucose and gum arabic

[0208] Lactic acid bacteria (Lacticaseibacillus rhamnosus) were inoculated into MRS liquid medium at a concentration of 2% (v / v) and pre-cultured at 37°C for 16–18 hours. Then, the bacteria were inoculated into MRS liquid medium at a concentration of 2% (v / v) and pre-cultured at 37°C for 7–9 hours. After inoculating the secondary pre-culture medium at a concentration of 2% (v / v) into a mixed medium composed of glucose, yeast extract, sodium acetate anhydrous, potassium phosphate dibasic, ammonium citrate dibasic, magnesium sulfate, manganese (II) sulfate monohydrate, and antifoam, the main culture was carried out for 16 to 18 hours at 37℃, 100 rpm, and pH 5.6. The culture was then centrifuged (8,000 rpm, 20 min) to recover only the cells, washed twice with 0.85% (w / v) saline solution, and centrifuged again (8,000 rpm, 20 min) to recover only the cells. The recovered bacterial cells were suspended in a sterile softening solution (glucose + arabic gum aqueous solution) and softened at 4°C and 200 rpm for 16–18 hours. Afterward, the cells were lysed at 121°C and 0.12 MPa for 30 minutes, and after cooling at 2–4°C, the lactic acid bacteria lysate was centrifuged (8,000 rpm, 20 min) to recover only the supernatant. After being left at room temperature for 16–24 hours, it was finally filtered through a 0.20 μm filter.

[0209]

[0210] Example 3

[0211] Preparation of lactic acid bacteria polynucleotides using xanthan gum softening solution

[0212] Lactic acid bacteria (Lacticaseibacillus rhamnosus) were inoculated into MRS liquid medium at a concentration of 2% (v / v) and pre-cultured at 37°C for 16–18 hours. Then, after inoculating into MRS liquid medium at a concentration of 2% (v / v), pre-cultured at 37°C for 7–9 hours. After inoculating the secondary pre-culture medium at a concentration of 2% (v / v) into a mixed medium composed of glucose, yeast extract, sodium acetate anhydrous, potassium phosphate dibasic, ammonium citrate dibasic, magnesium sulfate, manganese (II) sulfate monohydrate, and antifoam, the main culture was carried out for 16 to 18 hours at 37℃, 100 rpm, and pH 5.6, followed by centrifugation (8,000 rpm, 20 min) to recover only the cells, which were then washed twice with 0.85% (w / v) saline solution and centrifuged (8,000 rpm, 20 min) to recover only the cells. The recovered bacterial cells were suspended in a sterile softening solution (xanthan gum aqueous solution, 1 / 10th the volume of the culture medium) and softened at 4°C and 200 rpm for 16–18 hours. Afterward, the cells were lysed at 121°C and 0.12 MPa for 30 minutes, and after cooling at 2–4°C, the lactic acid bacteria lysate was centrifuged (8,000 rpm, 20 min) to recover only the supernatant. After being left at room temperature for 16–24 hours, it was finally filtered through a 0.20 μm filter.

[0213]

[0214] Example 4

[0215] Preparation of lactic acid bacteria polynucleotides using gum arabic softening solution

[0216] Lactic acid bacteria (Lacticaseibacillus rhamnosus) were inoculated into MRS liquid medium at a concentration of 2% (v / v) and pre-cultured at 37°C for 16–18 hours, then pre-cultured at 37°C for 7–9 hours. After inoculating the secondary pre-culture medium at a concentration of 2% (v / v) into a mixed medium composed of glucose, yeast extract, sodium acetate anhydrous, potassium phosphate dibasic, ammonium citrate dibasic, magnesium sulfate, manganese (II) sulfate monohydrate, and antifoam, the main culture was carried out for 16 to 18 hours at 37℃, 100 rpm, and pH 5.7, followed by centrifugation (8,000 rpm, 20 min) to recover only the cells, which were then washed twice with 0.85% (w / v) saline solution and centrifuged (8,000 rpm, 20 min) to recover only the cells. The recovered bacterial cells were suspended in a sterile softening solution (arabic gum aqueous solution) and softened at 4°C at 200 rpm for 16–18 hours. Afterward, the cells were lysed at 121°C at 0.12 MPa for 30 minutes, and after cooling at 2–4°C, the lactic acid bacteria lysate was centrifuged (8,000 rpm, 20 min) to recover only the supernatant. After being left at room temperature for 16–24 hours, it was finally filtered through a 0.20 μm filter.

[0217]

[0218] Example 5

[0219] Yeast polynucleotide production

[0220] After inoculating yeast into YPD liquid medium at 5% (v / v) and performing a first pre-culture at 30°C for 16–18 hours, the yeast was inoculated into YPD liquid medium at 5% (v / v) and performed a second pre-culture at 30°C for 7–9 hours. After inoculating the secondary pre-culture medium at 5% (v / v) into a mixed medium composed of glucose, yeast extract, soy peptone, sodium acetate anhydrous, potassium phosphate dibasic, ammonium citrate dibasic, magnesium sulfate, manganese (II) sulfate monohydrate, and antifoam, the main culture was carried out for 16 to 18 hours at 30℃, 100 rpm, and pH 5.7, followed by centrifugation (8,000 rpm, 20 min) to recover only the cells, which were then washed twice with 0.85% (w / v) saline solution and centrifuged (8,000 rpm, 20 min) to recover only the cells. The recovered bacterial cells were suspended in a sterile softening solution (Arabic gum aqueous solution) and softened at 4°C, 200 rpm, for 16–18 hours. Afterward, the cells were lysed at 121°C, 0.12 MPa, for 30 minutes. After cooling to 2–4°C, the lactic acid bacteria lysate was centrifuged (8,000 rpm, 20 min) to recover only the supernatant. After being left at room temperature for 16–24 hours, it was finally filtered through a 0.20 μm filter.

[0221]

[0222] Comparative Example 4

[0223] Preparation of lactic acid bacteria polynucleotides excluding the softening process

[0224] Lactic acid bacteria (Lacticaseibacillus rhamnosus) were inoculated into MRS liquid medium at a concentration of 2% (v / v) and pre-cultured at 37°C for 16–18 hours. Then, after inoculating into MRS liquid medium at a concentration of 2% (v / v), pre-cultured at 37°C for 7–9 hours. After inoculating the secondary pre-culture medium at a concentration of 2% (v / v) into a mixed medium composed of glucose, yeast extract, sodium acetate anhydrous, potassium phosphate dibasic, ammonium citrate dibasic, magnesium sulfate, manganese (II) sulfate monohydrate, and antifoam, the main culture was carried out at 37°C for 16 to 18 hours, then centrifuged (8,000 rpm, 20 min) to recover only the cells, washed twice with 0.85% (w / v) saline solution, and centrifuged (8,000 rpm, 20 min) to recover only the cells. The recovered bacterial cells were suspended in sterile distilled water and lysed for 30 minutes at 121°C and 0.12 MPa. After cooling to 2–4°C, the lactic acid bacteria lysate was centrifuged (8,000 rpm, 20 min) to recover only the supernatant. After being left at room temperature for 16–24 hours, it was finally filtered through a 0.20 μm filter. Figure 2 is a graph of polynucleotide concentration according to the type of softening solution.

[0225]

[0226] Experimental Example 1

[0227] Selection of optimal soft solution for lactic acid bacteria polynucleotide production

[0228] To compare and evaluate the efficacy of the softening process optimized for the elution of lactic acid bacteria polynucleotides, the yield was compared after eluting lactic acid bacteria polynucleotides by either eluting polynucleotides without softening (no softening) or by performing the softening process using aqueous solutions of glucose + gum arabic, glucose + xanthan gum, gum arabic alone, or xanthan gum alone as softening solutions. For the analysis of polynucleotide concentration, the solution was diluted to obtain absorbance values ​​in the range of 0.1 to 1.0 at a single wavelength of 260 nm using a spectrophotometer, placed in a quartz cell to determine the absorbance value at 260 nm, and the polynucleotide concentration was analyzed according to the following formula.

[0229] DNA Concentration = 260 nm Absorbance × Dilution Ratio × 50 (DNA Measurement Constant) μg / mL

[0230]

[0231] In the case of the softening solution mixed with glucose, it was expected that lactic acid bacteria would utilize glucose in metabolism to proliferate more and elute a higher concentration of polynucleotides, but unexpectedly, the softening did not proceed well. As confirmed in Figure 2, in the case of the softening solution of xanthan gum alone, a higher concentration of polynucleotides was elute than in the softening solution mixed with glucose, and the highest concentration of lactic acid bacteria polynucleotides was elute in the single softening solution using only gum arabic.

[0232]

[0233] Based on the above experimental results, in order to select the optimal concentration of gum arabic softening solution, a softening process was performed using gum arabic softening solutions at concentrations of 0.5% (w / v), 1.0% (w / v), 1.5% (w / v), and 2.0% (w / v), and lactic acid bacteria polynucleotides were eluted. For the analysis of polynucleotide concentration, the solution was diluted to obtain an absorbance value in the range of 0.1 to 1.0 at a single wavelength of 260 nm using a spectrophotometer, placed in a quartz cell to determine the absorbance value at 260 nm, and the polynucleotide concentration was analyzed according to the following formula.

[0234] DNA Concentration = 260 nm Absorbance × Dilution Ratio × 50 (DNA Measurement Constant) μg / mL

[0235]

[0236] Sensory quality was evaluated using color changes, the formation of denatured proteins, and abnormal odors occurring under conditions of polynucleotide elution as sensory elements. During the process of eluting lactic acid bacteria polynucleotides, heat and pressure under certain conditions are applied, causing denaturation of structural proteins constituting the cell wall and proteins. Consequently, the color of the finished product darkens, becomes cloudy rather than transparent, and abnormal fermentation odors occur. Therefore, after the filtration process, the softened liquid was selected by leaving it at room temperature for one week and observing it, and the liquid was selected for which no denatured protein or abnormal fermentation odor occurred. Through sensory evaluation, if the color was dark, cloudy in a colloidal form due to denatured protein precipitates, and a strong abnormal fermentation odor occurred, it was marked as "+++"; if there was color, denatured protein precipitates settled at the bottom of the container, and an abnormal fermentation odor occurred, it was marked as "++"; if the color was light, there were denatured protein precipitates settled at the bottom, and an abnormal fermentation odor occurred, it was marked as "+"; and if there was no color and no denatured protein precipitates or abnormal fermentation odor occurred, it was marked as "-".

[0237] Sensory Evaluation of Lactic Acid Bacteria Polynucleotides According to Softening Solution Composition and Gum Arabica Concentration Cell Softening Color Denaturation Protein Production Abnormal Fermentation Odor lucose + Xanthan gum++++ lucose + Gum arabic++++ anthan gum+-+ um arabic+-+ um arabic 0.5%+++++ um arabic 1.0%+-+ um arabic 1.5%+-+ um arabic 2.0%+++

[0238]

[0239] As confirmed in Figure 2, the highest concentration of polynucleotides was eluted from a single gum arabic nitriding solution, and there were no abnormalities in the sensory evaluation in Table 1. In addition, as confirmed in Figure 3, the concentration of polynucleotides increased as the gum arabic concentration decreased; however, as a result of the sensory evaluation listed in Table 1, it was confirmed that denatured proteins and abnormal fermentation odors were produced at a gum arabic concentration of 0.5% (w / v), so the nitriding solution was finally selected at a gum arabic concentration of 1% (w / v).

[0240]

[0241] Experimental Example 2

[0242] Selection of optimal culture conditions for lactic acid bacteria polynucleotide production

[0243] MRS nutrient medium contains beef extract, making it unfriendly and not economical in terms of cost. To select optimal culture conditions capable of producing high concentrations of lactic acid bacteria polynucleotides while replacing this MRS nutrient medium, culture was conducted while adjusting the medium composition and pH. The culture conditions were carried out as shown in Table 2, and the softening solution was prepared with a gum arabic concentration of 1% (w / v) based on the results of Experimental Example 1. Subsequently, culture was carried out according to Table 2, and the experiment was conducted using the same process as in Example 2.

[0244]

[0245] Lactic Acid Bacteria Culture Conditions Main Culture ABCDEF Medium Conditions Glucose (g / L) 20 20 20 20 20 Yeast extract 20 20 20 20 20 Sodium acetate anhydrous (g / L) 10 10 10 10 5 5 Potassium Phosphate dibasic (g / L) 22 22 2 Ammonium Citrate dibasic (g / L) 0 0 0 0 2 Magnesium sulfate (g / L) 0 10 10 10 10 10 1 Manganese (II) sulfate monohydrate (g / L) 0 10 10 10 10 10 10 1 Antifoam (ml / L) 11 11 1 Culture Conditions pH -5.3 5.5 5.7 5.6 5.6 Stirring Speed ​​(rpm) 0100 100 100 100 100 Temperature (°C) 37.0 37.0 37.0 37.0 37.0 37.0

[0246]

[0247] As confirmed in Figure 4, the highest concentration of lactic acid bacteria polynucleotides was eluted under F culture conditions, which was even higher than the concentration of polynucleotides eluted from lactic acid bacteria grown in MRS nutrient medium (compared to "Gum arabic 1.0%" in Figure 3). Therefore, it can be seen that high concentrations of lactic acid bacteria polynucleotides can be produced from lactic acid bacteria using a lower-cost medium without using MRS nutrient medium with added meat extract.

[0248]

[0249] Experimental Example 3

[0250] Manufacture of various lactic acid bacteria and yeast polynucleotides

[0251] To examine the preparation of polynucleotides by applying the present invention to various types of lactic acid bacteria and yeast, the lactic acid bacteria included Lacticaseibacillus rhamnosus, Lacticaseibacillus casei, Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus bulgaricus, Limosilactobacillus reuteri, Lactiplantibacillus plantarum, Lactococcus lactis, Bifidobacterium lactis, Bifidobacterium longum, and Bifidobacterium Polynucleotides were prepared and their concentrations analyzed using lactic acid bacteria Bifidobacterium infantis, Bifidobacterium bifidum, and Streptococcus thermophilus, and yeasts Saccharomyces cerevisiae, Saccharomyces pastorianus, and Saccharomyces boulardii. The results are shown in Table 3 below.

[0252] Bacterial species Polynucleotide concentration (μg / mL)Lactobacillus rhamnosus6,605 ± 40Lactobacillus bulgaricus4,670 ± 15Limosilactobacillus reuteri3,362 ± 43.80Lactiplantibacillus plantarum5,982 ± 27.53Lactococcus lactis5,295 ± 32.78Bifidobacterium lactis4,725 ± 35Bifidobacterium longum4,018 ± 36Bifidobacterium infantis3,117 ± 52.66Bifidobacterium bifidum3,756 ± 22.91Streptococcus thermophilus2,107 ± 36.11Saccharomyces cerevisiae3,682 ± 19Saccharomyces pastorianus3,175 ± 35.60Saccharomyces boulardii3,315 ± 30

[0253] As can be seen in [Table 3] above, it is possible to produce high concentrations of polynucleotides when producing lactic acid bacteria polynucleotides and yeast polynucleotides using the method according to the present invention.

[0254]

[0255] Experimental Example 4

[0256] Differences in raw material quality due to differences in hot-pressure crushing process intensity

[0257] In the method of Experimental Example 1, lactic acid bacteria (Lacticaseibacillus rhamnosus) were inoculated into MRS liquid medium at a concentration of 2% (v / v), and after primary pre-culture at 37°C for 16–18 hours, they were inoculated into MRS liquid medium at a concentration of 2% (v / v) and secondary pre-cultured at 37°C for 7–9 hours. After inoculating the secondary pre-culture medium at a concentration of 2% (v / v) into a mixed medium composed of glucose, yeast extract, sodium acetate anhydrous, potassium phosphate dibasic, ammonium citrate dibasic, magnesium sulfate, manganese (II) sulfate monohydrate, and antifoam, the main culture was carried out for 16 to 18 hours at 37℃, 100 rpm, and pH 5.6, followed by centrifugation (8,000 rpm, 20 min) to recover only the cells, which were then washed twice with 0.85% (w / v) saline solution and centrifuged (8,000 rpm, 20 min) to recover only the cells. The recovered bacterial cells were suspended in a sterile softening solution (glucose + xanthan gum aqueous solution) and softened at 4°C, 200 rpm, for 16–18 hours. The correlation between polynucleotide concentration (μg / mL), protein, and endotoxin according to the hot-pressure crushing process intensity was then examined. Protein was tested using the BCA method, and endotoxin was tested using the LAL test with Nexgen PTS-150 (Charles River Laboratories). The results are listed in [Table 4].

[0258] Conditions (Temperature / Pressure / Time) ∫ Pㆍt (MPaㆍh) Polynucleotide Concentration (μg / mL) Protein (ppm) Endotoxin (EU / g) 80℃ / 0.01MPa / 1h 0.01 10 40 36 0 4.01 00℃ / 0.10MPa / 1h 0.10 12 30 19 5 2.7 12 1℃ / 0.12MPa / 0.5h 0.06 15 30 18 0 1.1 130℃ / 0.30MPa / 1h 0.30 26 20 14 0 0.9

[0259] As confirmed in Table 4, polynucleotide concentrations were observed to range from 1040 to 2620 μg / mL in the range of ∫P·t of 0.01 to 0.30 (MPa·h), and at this time, D 90 <100 was achieved, and protein and endotoxin impurities decreased sharply according to intensity.

[0260]

[0261] Experimental Example 5

[0262] Analysis of lactic acid bacteria polynucleotide molecular weight

[0263] To compare the molecular weights of eluted lactic acid bacteria polynucleotides and salmon polynucleotides, DNA fragment sizes were analyzed using agarose gel electrophoresis. A 1.5% agarose gel was used, and electrophoresis was performed for 25 minutes using a 100 bp marker and Dyne Loading STAR as the staining reagent. The molecular weight of the polynucleotides was approximately 650 Da per fragment, and the molecular weight was calculated according to the following formula.

[0264] Nucleotide molecular weight = Fragment size × 650 Da

[0265]

[0266] As can be seen in FIGS. 5a and 5b, salmon PDRN mainly has a size of 200 to 800 bp and a molecular weight of about 130 to 520 kDa, whereas the lactic acid bacteria polynucleotide of the present invention mainly has a size of less than 100 bp and a molecular weight of about 32.5 to 65 kDa.

[0267] Subsequently, to compare the molecular weights of lacto-PDRN (Lacticase-Bacillus rhamnosus, Lactiplantibacillus rhamnosus), bifida-PDRN (Bifidobacterium longum), and yeast-PDRN (Saccharomyces cerevisiae), electrophoresis was performed for 30 minutes using a 2% agarose gel and a 50 bp DNA marker.

[0268] As a result, as confirmed in Figures 6a and 6b, the DNA fragment sizes of both lacto-PDRN and yeast-PDRN are 100 bp or less (D 90 ≤100 bp), and most were 50 bp to 60 bp or less (D 50 It was confirmed that it has a size of ≤60bp and a molecular weight of 65kDa or less, and that it has a smaller molecular weight than salmon PDRN. This means that the lactic acid bacteria polynucleotide prepared by the present invention is advantageous for skin absorption compared to salmon PDRN.

[0269]

[0270] Experimental Example 6

[0271] Analysis of molecular weight of eluted lactic acid bacteria polynucleotides

[0272] Gel permeation chromatography (GPC) was performed to analyze the molecular weight of eluted lactic acid bacteria polynucleotides. 0.1M NaNO3 was used as the mobile phase, and after completely dissolving the sample in the mobile phase and filtering it through a 0.45μm nylon filter, molecular weight analysis was conducted using an EcoSEC HLC-8420 GPC as the analyzer, an RI-detector as the detector, and PEG / PEO as the standard.

[0273] As a result, as confirmed in Fig. 7, the peak showing the largest molecular weight of the sample showed an average molecular weight of 58.5 kDa, followed by a size of 4.7 kDa. Upon reviewing the agarose gel electrophoresis images, it was analyzed that the lactic acid bacteria polynucleotide according to the present invention has a size between approximately 10 bp and 100 bp.

[0274]

[0275] Experimental Example 7

[0276] Evaluation of the Antioxidant Effects of Lactic Acid Bacteria Polynucleotides

[0277] To evaluate the antioxidant effects of lactic acid bacteria polynucleotides, DPPH radacal scavenging activity and SOD activity experiments were conducted.

[0278] DPPH radical scavenging activity was prepared by diluting the sample (PDRN, LACTO) and ascorbic acid to different concentrations, adding 12.5 μl of the sample to 50 μl of ethanol, adding 62.5 μl of 0.1 ml of DPPH (2,2-diphenyl-1-picrylhydrazyl), reacting for 30 minutes at 4°C in a dark state without light, measuring the absorbance at 520 nm using a spectrophotometer to determine the concentration of DPPH radicals, and calculating according to the following formula.

[0279]

[0280]

[0281]

[0282] SOD activity was tested using the Superoxide Dismutase (SOD) Colorimetric Activity kit (Thermo Fisher Scientific, USA) according to the manufacturer's test method.

[0283]

[0284]

[0285] As shown in Figures 8 and 9, it was confirmed that the antioxidant activity of lactic acid bacteria polynucleotide is superior to that of salmon PDRN.

[0286]

[0287] Experimental Example 8

[0288] Evaluation of lactic acid bacteria polynucleotide anti-inflammatory

[0289] To evaluate the anti-inflammatory effects of lactic acid bacteria polynucleotides, the ability to inhibit nitric oxide (NO) production was measured. Raw264.7 cells were placed in a 96-well plate at a rate of 1 x 10⁻⁶ 4 After aliquoting, the samples were incubated for 24 hours, and each sample was treated and incubated for 24 hours. Distilled water or LPS was used as a control, and NO content was analyzed using the Promega Griess Reagent system (Promega Corporation, Medison, USA) according to the manufacturer's instructions. After the reaction, the nitrite concentration was determined by measuring the absorbance at a wavelength of 540 nm using a spectrophotometer.

[0290] As shown in Figure 10, NO activity decreased in a concentration-dependent manner for both salmon PDRN and lactic acid bacteria polynucleotide, and it was confirmed that the anti-inflammatory ability of lactic acid bacteria polynucleotide was superior to that of salmon PDRN at the same concentration.

[0291]

[0292] Experimental Example 9

[0293] Evaluation of lactic acid bacteria polynucleotide cytotoxicity

[0294] An MTT assay was performed to evaluate the cytotoxicity of lactic acid bacteria polynucleotides. HaCaT cells were placed in a 96-well plate at a ratio of 1 x 10⁶ per 1 ml. 4 Cells were dispensed to form and cultured for 24 hours, then the medium was removed. After removing the medium, culture medium containing each sample was dispensed and cultured. Then, 10 μL of MTT (5 mg / mL) solution dissolved in PBS was dispensed, the cells were shielded from light, and the mixture was allowed to react for 4 hours to confirm the formation of formazan. After removing the MTT solution, DMSO was added and stirred for 30 minutes, after which the absorbance was measured at a wavelength of 540 nm using a spectrophotometer.

[0295] As can be seen in Figure 11, the lactic acid bacteria polynucleotide showed a tendency for cell viability to decrease as the concentration increased, but the cell viability was over 90%, so it was determined that there was no toxicity.

[0296]

[0297] Experimental Example 10

[0298] Evaluation of lactic acid bacteria polynucleotide cell protection

[0299] To evaluate the skin cell protective ability of lactic acid bacteria polynucleotides, skin protection evaluation was conducted using H2O2 as a cell stimulator in the same manner as in Experimental Example 9.

[0300] As shown in Figure 12, both salmon PDRN and lactic acid bacteria polynucleotide had a concentration-dependent cytoprotective effect against H2O2, and it was confirmed that the efficacy of lactic acid bacteria polynucleotide was superior to that of salmon PDRN.

[0301]

[0302] Experimental Example 11

[0303] Evaluation of the skin regeneration efficacy of lactic acid bacteria polynucleotides

[0304] To confirm the skin regeneration effect of lactic acid bacteria polynucleotides, HaCaT cells were placed in a 24-well plate at a ratio of 1 x 10 5 After dispensing cells into a well and culturing for 24 hours until the well was full, the bottom of the well was cut longitudinally with a 1 mL pipette tip to induce cell wounding. Subsequently, the samples and control (PBS) were applied, and the appearance at 0 and 18 hours was observed. TNF-α was used as the cell stimulant, and the wound area was measured using the Image J program.

[0305] As shown in Figures 13a and 13b, it was confirmed that lactic acid bacteria polynucleotide has superior skin regeneration functionality compared to salmon PDRN.

[0306]

[0307] Experimental Example 12

[0308] Microbial polynucleotide human application test

[0309] To evaluate the efficacy of the lactic acid bacteria polynucleotide (LACTO) prepared according to the present invention as a functional cosmetic ingredient, clinical subjects were selected, the product was applied directly to the skin, and the effects were observed from various angles.

[0310] After applying the test substance to the backs of selected subjects for 24 hours, the degree of skin irritation was evaluated according to the PCPC Guideline (Personal Care Products Council) judgment criteria at 30 minutes and 24 hours after patch removal. The research staff explained the purpose and methods of the study, expected efficacy, and adverse skin reactions to at least 30 subjects who met the selection criteria and did not fall under the exclusion criteria.

[0311] In addition, gloss (radiance) was evaluated by measuring the facial area before using the 'LACTO-PDRN' (50ppm) product and after one use. Skin gloss was evaluated as GU, a parameter of skin surface gloss (gloss), by measuring reflectance based on the principle of light reflection using the Skin-Glossymeter GL 200 (Delfin technologies Ltd, Finland).

[0312] Skin density, elasticity, and wrinkles were evaluated by measuring the facial area before product use and after 4 weeks of use. Skin density (%) was measured using an ultrasound probe from the Derma-Lab® Series SkinLab Combo (Cortex Technology, Denmark). Acoustic pulses were projected onto the skin using an ultrasound imaging device to measure the responsiveness; the effect was verified by displaying the signal intensity, with low density appearing in dark colors and high density in bright colors. Skin elasticity was assessed using a Cutometer® MPA580 (C+K, Germany), which continuously suctioned the skin three times for 2 seconds each at a constant sound pressure of 450 mbar, and the results were presented as graphs and numerical values.

[0313] The skin regeneration effect was verified by the following method. The skin was stained with Dansyl chloride, and UV images captured using Ghost in the Mirror (PSIplus, Korea) were analyzed based on the intensity values ​​of the measurement area using the Image-pro® 10 (media Cybermetics, USA) program. When the skin is stained using Dansyl chloride and then photographed, the skin surface brightens, causing the intensity value to increase; conversely, as skin turnover occurs in the stained area due to 'LACTO-PDRN', the skin surface darkens, causing the intensity value to decrease. Skin turnover was evaluated by measuring the forearm area before staining, 24 hours after staining, and after 4 weeks of product use.

[0314]

[0315]

[0316]

[0317] In addition, adverse skin reactions were evaluated through surveys by the study participants and observations and interviews by the research staff. Statistical significance was verified using the SPSS Package Program (IBM, USA), and the statistical significance level was set at a p-value of less than 0.05.

[0318]

[0319] 1. Gloss (Radiance) Evaluation

[0320] As shown in Figure 14, the analysis results confirmed that skin radiance improved, with the parameter value (GU) for radiance increasing from 8.34 before product use to 11.23 after one use, showing a significant increase of 34.65% (p<0.05).

[0321]

[0322] 2. Skin Density Assessment

[0323] As shown in Figures 15a and 15b, the analysis results confirmed that skin density improved, with the parameter value (%) for skin density increasing significantly from 17.14 to 18.85 at the time point after 4 weeks of product use, showing a change rate of 9.98% (p<0.05).

[0324]

[0325] 3. Flexibility Evaluation

[0326] As shown in Figure 16, the analysis results indicated that skin elasticity improved, with the parameter value (R2) for elasticity increasing significantly from 73.77 to 76.01 after 4 weeks of product use, a change rate of 3.04% (p<0.05).

[0327]

[0328] 4. Skin Turnover Assessment

[0329] As shown in Figures 17a and 17b, the analysis results confirmed that skin turnover and regeneration were improved, with the parameter value (Intensity) for skin turnover decreasing significantly from 183.62 to 89.02 (a 51.52% change rate) compared to 24 hours after dyeing (p<0.05).

[0330]

[0331] 5. Skin Wrinkle Evaluation

[0332] As shown in Figures 18a and 18b, the analysis results indicated that skin wrinkles improved, with the parameter value for skin wrinkles (Maximum depth (mm)) decreasing significantly from 0.10 to 0.09 at the time point after 4 weeks of product use, a change rate of 10.00% (p<0.05).

[0333]

[0334] 6. Survey evaluation by study participants

[0335] As a result of the survey evaluation regarding the efficacy and usability of the product, approximately 95% to 100% of the study subjects responded positively to all items at the point after 4 weeks of product use. They responded positively to the effects and usability of the product. The survey content and results are as described in Figures 19a to 19c.

[0336]

[0337] 7. Evaluation of adverse skin reactions

[0338] No adverse skin reactions were observed in any of the study subjects during the study period. It was confirmed that there were almost no side effects, such as skin irritation or redness, which are prone to accompanying skin functional ingredients, while using the lactic acid bacteria polynucleotide according to the present invention for 4 weeks.

[0339]

[0340] Based on the above clinical results, the inventors' 'LACTO-PDRN' was evaluated as a product with five anti-aging functions that help with radiance (glow) after one use and, after 4 weeks of use, radiance (glow), skin density, elasticity, skin turnover, and skin wrinkles.

[0341]

[0342] Experimental Example 13

[0343] Comparison of physicochemical properties of microbial polynucleotides before and after powdering

[0344] We intended to verify whether the characteristics of low molecular weight polynucleotides and high purity characteristics are maintained even when the microbial polynucleotides prepared according to the present invention are powdered.

[0345] First, the liquid lactic acid bacteria polynucleotide prepared in Example 2 above was frozen at -40°C or lower using a freeze dryer and then dried under vacuum to produce a powdered polynucleotide. Meanwhile, the spray-dried powder was prepared using a spray dryer under conditions of an inlet temperature of 160°C and an outlet temperature of 80°C. The powdered polynucleotide was redissolved in sterile distilled water to achieve the same concentration.

[0346] Immediately after preparation, liquid microbial polynucleotide (Liquid), freeze-dried powder (FD-powder) redissolved solution and spray-dried powder (SD-powder) redissolved solution were compared, and DNA fragment size, concentration recovery rate, protein content, and endotoxin content were analyzed using the same method as in Experimental Examples 4 and 5 above.

[0347] As a result, as confirmed in Fig. 20, D 50 and D 90 The value is 60bp or less for Liquid, FD-powder, and SD-power respectively (D 50 ) and 100bp or less (D 90It was found that no new DNA bands were generated even after the powdering process. In addition, DNA quantification results showed that the concentration recovery rate of the freeze-dried powder remained in the range of approximately 91-97% compared to the liquid phase immediately after preparation, and the spray-dried powder remained at a level of approximately 80-90%.

[0348] Protein content (BCA assay) was less than 200 ppm in all groups, and endotoxin (LAL test) was confirmed to be less than 5 EU / g, so no increase in protein or endotoxin due to the powdering process was observed. In addition, the moisture content of freeze-dried and spray-dried powders was measured to be low at 2.2% and 3.8%, respectively, confirming that the powder formulation is advantageous for long-term preservation.

[0349] Thus, it was confirmed that the microbial polynucleotide according to the present invention stably maintains low molecular weight structural characteristics and high purity characteristics even after pulverization.

[0350]

[0351] Experimental Example 14

[0352] Accelerated and long-term stability test of powdered microbial polynucleotides

[0353] We intended to evaluate the stability of the polynucleotide formulation according to the present invention in high temperature and high humidity environments.

[0354] First, the liquid microbial polynucleotide prepared in Example 2 above and the freeze-dried powder prepared in Experimental Example 13 above were each stored under the following conditions.

[0355] - Room temperature conditions: 25℃, relative humidity 60%

[0356] - Acceleration conditions: 40℃, relative humidity 75%

[0357] - Duration: Room temperature 0, 1, 3, 6 months / Accelerated 0, 1, 2, 3 months

[0358]

[0359] At each end of storage, the liquid group was analyzed as is, and the powder group was analyzed after being redissolved in sterile water.

[0360] DNA size analysis results showed that D in all periods of the liquid and powder groups (PE vinyl primary packaging and aluminum secondary packaging) 50 ≤60bp, D 90 The value was maintained at ≤100 bp, and no new DNA bands were observed.

[0361] In addition, when comparing the concentration retention rates of the liquid and powder groups, after 3 months under 40℃ conditions, the liquid group maintained 93% of the initial concentration and the powder group maintained 98%. Regarding changes in color and fermentation odor, there were some changes in the liquid group, but no significant changes in the powder group.

[0362] There were no changes in endotoxin and microbial contamination across all powder groups, and no increase in endotoxin was observed even with high-temperature storage.

[0363] Accordingly, it was confirmed that the powdered microbial polynucleotide according to the present invention maintains the structural stability of DNA even under high temperature and high humidity conditions, and that storage stability is significantly improved compared to the liquid form.

[0364]

[0365] Experimental Example 15

[0366] Evaluation of the redissolvability and formulation applicability of powdered microbial polynucleotides

[0367] In order to confirm whether the powdered polynucleotide of the present invention prepared according to Experimental Example 13 above has properties favorable for cosmetic formulation, the following experiment was performed.

[0368] The lyophilized powder formulation of polynucleotide was added to sterile distilled water at 25°C and stirred at 500 rpm to measure the time until complete dissolution. As a result, it was completely dissolved within 1-3 minutes, and no aggregation or precipitation was observed.

[0369] The redissolved powdered microbial polynucleotide showed no difference from the liquid state immediately after preparation in terms of transparency (600 nm absorbance) and pH measurements, and when added to basic essence and cream formulations, no separation, discoloration, off-odor, viscosity, or pH change was observed even after storage for 1-3 months under conditions of 40°C and 75% relative humidity.

[0370] Accordingly, it was confirmed that the powdered microbial polynucleotide according to the present invention has excellent redissolvability and is a safe functional ingredient applicable to various formulations.

[0371]

[0372] As described above, the method for producing microbial polynucleotides according to the present invention adds a cell softening step prior to the lactic acid bacteria or yeast crushing step, thereby allowing the lactic acid bacteria or yeast to be crushed more effectively and the polynucleotides to be efficiently eluted. Furthermore, the softening solution in the above step has the effect of protecting the eluted polynucleotides to prevent loss. The microbial polynucleotides produced by the method of the present invention have improved sensory properties due to reduced fermentation odor of the raw materials, are advantageous for skin absorption due to their small size, and exhibit excellent effects such as antioxidant and anti-inflammatory properties. Therefore, the method for producing microbial polynucleotides according to the present invention and the polynucleotides produced by said method can be usefully utilized as an economical, safe, and highly effective anti-aging functional ingredient in cosmetics.

Claims

1. (a) A pre-fermentation step in which microorganisms are grown in a nutrient medium containing a carbon source, a nitrogen source, vitamins, and inorganic salts; (b) a fermentation step in which the microorganisms proliferated in step (a) above are proliferated in a mixed medium containing a carbon source, a nitrogen source, vitamins, and inorganic salts; (c) A cell softening step in which the microorganisms proliferated in step (b) above are separated and softened in a softening solution consisting only of high-molecular-weight polysaccharides and water; (d) a step of hot-pressing the softening solution containing the microorganisms from step (c) above to crush the cells and elute the microbial polynucleotides; and (e) a step of removing cell wall and cytoplasmic protein components from the product obtained in step (d) above and recovering microbial polynucleotides; A method for producing a microbial polynucleotide comprising 2. A method for producing a microbial polynucleotide according to claim 1, characterized in that the microorganism is a lactic acid bacterium or yeast.

3. In paragraph 2, the lactic acid bacteria are Lacticaseibacillus rhamnosus, Lacticaseibacillus casei, Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus bulgaricus, Limosilactobacillus reuteri, Lactiplantibacillus plantarum, Lactococcus lactis, Bifidobacterium lactis, Bifidobacterium longum, and Bifidobacterium inpantis A method for producing a microbial polynucleotide, characterized in that it is one or more selected from the group consisting of infantis), Bifidobacterium bifidum, and Streptococcus thermophilus, and the yeast is one or more selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces pastorianus, and Saccharomyces boulardii.

4. A method for producing a microbial polynucleotide according to claim 1, wherein the mixed medium of step (b) comprises glucose, yeast extract, sodium acetate anhydrous, potassium phosphate dibasic, ammonium citrate dibasic, magnesium sulfate, and manganese(II) sulfate monohydrate.

5. A method for producing a microbial polynucleotide according to claim 1, characterized in that the polymeric polysaccharide of step (c) is included in the softening solution at a concentration of 0.1 to 4% (w / v).

6. A method for producing a microbial polynucleotide according to claim 1, characterized in that the polymeric polysaccharide in step (c) is not metabolized or degraded by lactic acid bacteria.

7. A method for producing a microbial polynucleotide according to claim 6, characterized in that the polymeric polysaccharide is one or more selected from the group consisting of xanthan gum, gum arabic, locust bean gum, tara gum, and guar gum.

8. A method for producing a microbial polynucleotide according to claim 1, wherein step (c) is performed at 4 to 18°C ​​for 12 to 24 hours.

9. A method for producing a microbial polynucleotide according to claim 1, characterized in that the volume of the softening solution in step (c) is 0.05 to 1 times the volume of the mixed medium in step (b).

10. A method for producing a microbial polynucleotide according to claim 1, wherein the softening solution of step (c) further comprises glucose.

11. A method for producing a microbial polynucleotide according to claim 1, characterized in that the hot-pressure crushing in step (d) is performed under conditions of a temperature of 80 to 130°C and a pressure of 0.01 to 0.3 MPa.

12. A method for producing microbial polynucleotides according to claim 1, wherein the hot-pressure crushing in step (d) is controlled such that the process strength (∫P·t) using heat or pressure is in the range of 0.01 to 0.30 MPa·h.

13. A method for producing a microbial polynucleotide according to claim 1, wherein the hot-pressure crushing in step (d) is controlled such that the F0 value (cumulative heat treatment intensity) is in the range of 3 to 8.

14. A method for producing a microbial polynucleotide according to claim 1, characterized in that, in step (e), the pellet containing the cell wall is removed by cooling and centrifugation, and the remaining pellet and cytoplasmic protein are removed by filtration.

15. In the method for producing microbial polynucleotides according to paragraph 1, (f) a step of preparing the microbial polynucleotide recovered in step (e) into a powder formulation by freeze-drying, spray-drying, vacuum drying, or fluid bed drying; A method for producing microbial polynucleotides characterized by further including 16. Microbial polynucleotide produced by a method according to any one of claims 1 to 15.

17. In paragraph 16, the above-mentioned microbial polynucleotide is D 50 ≤60bp and D 90 A microbial polynucleotide characterized by having a distribution of ≤100 bp, a protein content of 200 ppm or less, and an endotoxin content of 5 EU / g or less.

18. A cosmetic composition for skin antioxidant, skin aging inhibitory, wound healing, or cell activity enhancement comprising a microbial polynucleotide according to claim 16.

19. A quasi-drug composition for skin antioxidant, skin aging inhibitory, wound healing, or cell activity enhancement comprising a microbial polynucleotide according to Paragraph 16.

20. Use of microbial polynucleotides according to claim 16 for preparing compositions for skin antioxidant, skin aging inhibition, wound healing, or cell activity enhancement.

21. A method for skin antioxidant, skin aging inhibition, wound healing, or cell activity enhancement comprising administering an effective amount of a composition containing a microbial polynucleotide according to claim 16 as an active ingredient to an individual in need thereof.

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