阅读说明：本技术 源自于熔岩海水的矿物质与源自于皮肤微生物的核苷酸的结合物的制造方法以及利用所述结合物的功能性皮肤微生物化妆品组合物 (Method for producing conjugate of mineral derived from lava seawater and nucleotide derived from skin microorganism, and functional skin microorganism cosmetic composition using the same ) 是由 李昌琓 梁舒真 金头星 金景珉 金艺香 车昭润 崔志辉 李昇勋 于 2020-03-31 设计创作，主要内容包括：本发明涉及一种源自于利用熔岩海水培养的皮肤微生物的矿物质-核苷酸结合物的制造方法,所述矿物质-核苷酸结合物没有皮肤毒性且具有皮肤细胞再生、皮肤屏障强化、皮肤舒缓效果以及皮肤常居菌群维持功能,因此可以作为含有所述结合物的功能性化妆品组合物或皮肤外用剂等形态提供。(The present invention relates to a method for producing a mineral-nucleotide conjugate derived from skin microorganisms cultured in lava seawater, which is free from skin toxicity and has skin cell regeneration, skin barrier enhancement, skin soothing effects, and a skin resident flora maintaining function, and thus can be provided in the form of a functional cosmetic composition or an external skin preparation containing the conjugate.)

1. A method for producing a mineral-nucleotide conjugate derived from a skin microorganism cultured in lava seawater, comprising:

(first step) a step of obtaining a first culture solution by culturing skin microorganisms using a culture medium using lava seawater as culture water;

(second step) a step of obtaining a second culture solution by culturing the cells in lava seawater mineral concentrate after recovering the cells from the culture solution; and the number of the first and second groups,

(third step) a step of recovering a supernatant containing mineral-nucleotide by subjecting the second culture solution to hot-pressure extraction and cold purification.

2. The method for producing a mineral-nucleotide conjugate derived from skin microorganisms cultured using lava seawater according to claim 1, characterized in that:

the medium in the first step is a medium containing glucose, yeast extract, soy peptone, and casein as components, and sterilized after dissolving the components in water containing 30-70% (v/v) lava seawater.

3. The method for producing a mineral-nucleotide conjugate derived from skin microorganisms cultured using lava seawater according to claim 1, characterized in that:

the skin microorganism in the first step is one or more strains selected from the group consisting of Lactobacillus (Lactobacillus sp.), Bifidobacterium (sp.), Streptococcus (Streptococcus sp.), Lactococcus (Lactobacillus sp.), Enterococcus (Enterococcus sp.), Pediococcus (Pediococcus sp.), Weissella (Weissella sp.), Saccharomyces sp.), Bacillus (Bacillus sp.), Staphylococcus (Staphylococus sp.), and Propionibacterium (Propionibacterium sp.).

4. The method for producing a mineral-nucleotide conjugate derived from skin microorganisms cultured using lava seawater according to claim 1, characterized in that:

the concentrated water in the second step is mineral concentrated water containing calcium of 220mg or more, magnesium of 1,130mg or more, and selenium of 0.013mg or more per 1L, and is obtained by concentrating lava seawater under reduced pressure or by osmotic concentration.

5. The method for producing a mineral-nucleotide conjugate derived from skin microorganisms cultured using lava seawater according to claim 1, characterized in that:

the hot-pressing extraction in the third step is carried out at 120-130 ℃ and 0.12-0.30 MPa for 15-200 minutes.

6. The method for producing a mineral-nucleotide conjugate derived from skin microorganisms cultured using lava seawater according to claim 1, characterized in that:

the cooling purification in the third step is performed by performing centrifugation at 3,000 to 8,000rpm for 10 to 30 minutes while maintaining 3 to 5 ℃ and then collecting the supernatant containing the mineral-nucleotide using a nanomembrane filtration process using a nanomembrane having a pore size (pore size) of 200nm or less.

7. The method for producing a mineral-nucleotide conjugate derived from skin microorganisms cultured using lava seawater according to claim 1, characterized in that:

the concentration of the nucleotide in the supernatant in the third step is in the range of

8. A mineral-nucleotide conjugate, characterized by:

manufactured by the method of claim 1.

9. A cosmetic composition for skin barrier enhancement characterized by:

comprising the mineral-nucleotide conjugate of claim 8.

10. Cosmetic composition for skin barrier strengthening according to claim 9, characterized in that:

the composition enhances the expression of 1 or more genes or proteins selected from the group consisting of Serine Palmitoyltransferase (SPT), Filaggrin (Filaggrin), and Claudin-1.

11. Cosmetic composition for skin barrier strengthening according to claim 9, characterized in that:

the composition also has a repair effect on Ultraviolet (UV) damaged skin cells.

12. The cosmetic composition for skin barrier enhancement according to claim 11, characterized in that:

the composition has the effect of enhancing the expression of Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha).

13. Cosmetic composition for skin barrier strengthening according to claim 9, characterized in that:

the composition has the function of maintaining or increasing the total bacterial count of skin-resident bacteria, i.e., Staphylococcus epidermidis (Staphylococcus epidermidis).

14. Cosmetic composition for skin barrier strengthening according to claim 9, characterized in that:

the composition has the function of maintaining or increasing the total bacterial count of skin-resident bacteria, namely Propionibacterium acnes.

Technical Field

The present invention relates to a method for preparing a mineral-nucleotide conjugate (conjugate) having excellent thermal stability by a hot press extraction method, wherein beneficial skin microorganisms (dermabitics) are cultured at a high concentration using lava seawater rich in natural minerals as culture water in order to simultaneously solve intrinsic factors (endocrinsic factors) and extrinsic factors (externalics) causing skin aging (skin aging), and then the cells are recovered and re-cultured in lava seawater mineral concentrate water to generate a large amount of mineral-nucleotides in the cells. In addition, the present invention provides a method for selectively removing cell wall components (e.g., cell walls, lipopolysaccharides, lipoproteins, etc.) of bacterial cells inducing inflammatory reactions of skin cells by membrane filtration after hot-pressing extraction by cooling centrifugation, thereby purifying mineral-nucleotides, and a mineral-nucleotide conjugate derived from skin microorganisms cultured in lava seawater, which is produced by the above-mentioned method, has thermal stability, and has an expression of functional proteins involved in skin barrier enhancement and a regulatory function of a skin microorganism group, which is a third skin layer.

Background

The skin is composed of a triple multi-layer structure of an epidermal layer, a dermal layer, and a horny layer, wherein the epidermal layer is positioned just below the horny layer of the skin and functions as a permeation barrier, innate immunity, protection against ultraviolet rays, wound repair, synthesis of vitamin D, and the dermal layer is a layer that supplies nutrients to the epidermis and regenerates the skin and functions to suppress invasion of pathogens, repair wounds, maintain the skin structure and elasticity, and the like. The outermost stratum corneum, which is located in the skin structure, is composed of very thin layers with a thickness of about 0.5 mm. The stratum corneum, which is composed of 25 to 30 apoptotic skin cells, can protect human skin, and the differentiation of keratinocytes (keratinocytes) is composed of four steps, i.e., division of basal cells, synthesis in spinous cells, autolysis in granular cells, and reconstitution in keratinocytes, to form a skin barrier. In the process of forming the skin barrier as described above, cell regeneration (regeneration) occurs about four weeks based on the young skin. The skin can protect the human body from physical and chemical stimuli from the outside, but when it is continuously exposed to ultraviolet rays, which is a representative external factor of skin aging, it causes damage to deoxyribonucleic acid (DNA) of skin cells and generation of Reactive Oxygen Species (ROS), thereby inhibiting and promoting decomposition of collagen synthesis of the skin, and finally causing skin aging and generation of wrinkles. Intrinsic factors of skin aging depend on genetic factors inherited from parents, which have been determined to be the cause of natural aging, but the rate and state of skin aging will also vary according to changes in the skin microbiome (skin microbiome) called the third skin layer.

Further, it has been reported that the skin state and type can be confirmed statistically to be different depending on the region, race and age. Kompaore et al (Skin Pharmacol., 1993; 6: 200-. Marrakchi et al (Contact demattis, 2007; 57:28-34) reported that comparing the moisture content, sebum content, etc. of young people who are 24-34 years old and old people who are 66-83 years old shows that the moisture content of forehead and cheek of young people is higher than that of old people, and the sebum content does not show a large difference between young people and old people.

Different country skin feature library construction career reports indicate that korean women have skin types in the order of composite > dry > neutral > oily, while men have skin types in the order of oily > composite > neutral > dry.

The skin microbiome (Skinmicrobiome), which is the subject of recent attention, refers to the ecosystem of the resident flora distributed in the skin, and it has been reported that microorganisms existing in the skin, such as Staphylococcus (Staphylococcus), Propionibacterium (Propionibacterium), and the like, mainly maintain a balanced and healthy skin state through interaction with skin cells. The resident flora constituting the skin microbiome varies depending on age groups, women and men, race, living area, and the like, and it has been reported that particularly dominant species of skin in different age groups can maintain skin health.

In recent industrial fields, cosmetics using microorganisms as core source materials have been developed, and attempts have been made to improve the skin barrier by directly reacting with immune receptors inside the skin by making microorganisms into lysate form (lysate) and applying the microorganisms to cosmetics to avoid problems of skin permeation due to the size of microorganisms, apoptosis due to preservatives, and competitive inhibition. Among commercially available raw materials using microorganisms, lactic acid bacteria such as those belonging to the genus Bifidobacterium (Bifidobacterium) or the genus Lactobacillus (Lactobacillus) are sold as raw materials suitable for skin, and among them, a yeast fermentation concentrate of schizosaccharomyces, etc. are exemplified as a representative example, and a cell lysate of lactic acid bacteria is used as a main component. However, the cell wall contains peptidoglycan (peptidoglycan), lipopolysaccharide (lipopolysaccharide), lipoprotein (lipoprotein) and the like, which are known components that cause an immune reaction with the skin among cell constituents, and when the above components are excessive, skin problems such as rash, atopic dermatitis, erythema and the like may be caused. In addition, since the thermal stability of microbial nucleotides (bacterial nucleotides), which are active ingredients in cytoplasm, is 70 to 105 ℃ based on the melting temperature (Tm) (temperature at which the double helix structure of deoxyribonucleic acid (DNA) is unwound), when a temperature of 121 ℃ or higher, which is a temperature at which cell walls can be completely broken, is applied, the active ingredients are modified and thus cannot contribute to improvement of skin barrier. Furthermore, another problem when applying microbial raw materials for skin protection is that when applying cell lysates to skin, because the skin resident flora is not a growth factor (growth factors), this leads to a balanced destruction of the healthy skin microbiome if the skin harmful bacteria use it as a growth nutrient source. Since cell lysates of various microorganisms, which have been registered as raw materials for international cosmetics, contain a large amount of components of a regulator unit, which may have an influence on the human skin microbiome, the imbalance of the skin microbiome cannot be solved.

The lava seawater is a long-time aged water in which seawater is naturally filtered by a basalt layer and a sand muddy layer and simultaneously infiltrates into the deep stratum, is a unique water resource only in the Jizhou island, is actively used as feeding water for flatfish farms because of the characteristics of low temperature, cleanliness and the like of the lava seawater since the 1980 s, and is also used as water for saunas and the like in health, beauty and the like from 5 to 6 years ago and is attracting much attention. The lava seawater may be desalted lava seawater from which salts are removed or may be used as it is in an originally collected state, depending on the purpose of use.

Compared with common seawater, deep water or Sanduo water, lava seawater not only contains more minerals such as sodium, magnesium, calcium and potassium, but also contains more common beneficial mineral components (such as iron, manganese, zinc, molybdenum, selenium, etc.). In particular, vanadium contained in lava seawater, which has effects of stabilizing insulin secretion or improving diabetes, hyperlipidemia, etc., germanium having effects of promoting blood circulation, enhancing immunity, and preventing cancer, selenium having synergistic effects of inhibiting oxidation of fat, maintaining heart and liver, radical scavenging ability, and improving anticancer, infertility, aging, and cholesterol values, is a unique feature of lava seawater, which has not been reported in deep seawater. Furthermore, the minerals are in an ionized state, and the ionized minerals can be more easily digested and absorbed by human body or other animals. Further, lava seawater is a clean groundwater resource in which no harmful components such as arsenic, mercury, and cadmium are detected or only a trace amount of lead is detected, and thus is a clean raw material without any hindrance when applied to industrialization. In particular, minerals such as magnesium and calcium contained in the lava seawater are essential elements for the proliferation of skin microorganisms and are also essential nutrients recommended to be taken by the human body.

The eluted deoxyribonucleic acid (DNA) has a Temperature-based modification value (Tm) in a decomposition factor, which is an intermediate value in a Temperature change for converting Double-stranded deoxyribonucleic acid (Double strand DNA) into single-stranded deoxyribonucleic acid (single strand DNA). The currently known melting temperature (Tm) value of deoxyribonucleic acid (DNA) eluted after cell lysis is 70 to 105 ℃, and nucleotides, which are composed of purine and pyrimidine and are decomposed under the temperature condition, can be stabilized by internal hydrogen bonding. However, the phosphate groups in the constituent elements of the decomposed nucleotides exhibit strong negative charges and thus have repulsive forces with respect to each other. In order to extract nucleotides having the above-described characteristics, the nucleotides need to be maintained in a stable state even under a hot-pressing condition of 121 ℃ which is a temperature higher than a melting temperature (Tm) value, i.e., 70 to 105 ℃, in order to cleave cell walls and cytoplasm.

For this reason, the present inventors confirmed that mineral-nucleotides isolated from skin microorganisms cultured using lava seawater can significantly improve skin barrier strengthening and skin damage inhibition functions compared to nucleotides derived from skin microorganisms cultured using existing general culture media, and completed the present invention.

Prior art documents

Patent document

(patent document 1) Korean registered patent No. 10-1347694 (title of the invention: method for producing a fermentation product of desalted lava seawater, fermentation product obtained by the method, and cosmetic composition using the fermentation product, applicant: national institute of science and technology, registration date: 2013, 12/27/12/2013)

(patent document 2) Korean registered patent No. 10-1693574 (name of the invention: composition for moisturizing skin or improving wrinkles, containing killed bacteria of intermittent sterilized lactic acid bacteria as an active ingredient, applicant: Korean medical researcher, registration date: 2017, 01 month 02 day)

Non-patent document

(non-patent document 1) Zakostelska Z, Kverka M, Klimenova K, Rossmann P, Mrazek J, et al (2011) salt of biological Lactobacillus casei DN-114001 amino acids vitamins by Strength helium the Gut Barrier Function and Changing the Gut microorganism PLoS ONE 6(11)

Disclosure of Invention

The purpose of the present invention is to provide a technique whereby lava seawater rich in natural minerals can be used as water for culturing skin microorganisms without separate pretreatment, and thereby natural minerals derived from lava seawater can be accumulated in the cytoplasm of the skin microorganisms and mineral-nucleotide conjugates can be induced to be produced.

The mineral-nucleotide conjugate is manufactured through a hot-pressing extraction process of skin microorganisms, so that it is excellent in thermal stability, and thus it is possible to achieve more excellent skin barrier strengthening function and protection effect of skin cells of ultraviolet rays, and improve skin conditions by regulating skin microbiome.

The present invention relates to a method for producing a mineral-nucleotide conjugate derived from skin microorganisms cultured using lava seawater, comprising:

(first step) a step of obtaining a first culture solution by culturing skin microorganisms using a culture medium using lava seawater as culture water;

(second step) a step of obtaining a second culture solution by culturing the cells in lava seawater mineral concentrate after recovering the cells from the culture solution; and the number of the first and second groups,

(third step) a step of recovering a supernatant containing mineral-nucleotide by subjecting the second culture solution to hot-pressure extraction and cold purification.

The invention is characterized in that: the medium in the first step is a medium containing glucose, yeast extract, soy peptone, and casein as components, and sterilized after dissolving the components in water containing 30-70% (v/v) lava seawater. Preferably, the medium in the first step may be a medium for propagation culture prepared by dissolving each component in water containing 30 to 70% (v/v) lava seawater in a state of containing 1 to 5% (w/v) of glucose, 0.5 to 5% (w/v) of yeast extract, 0.5 to 5% (w/v) of soytone, and 0.5 to 3% (w/v) of casein in a total volume of 1l and sterilizing the solution.

In the first step, a method of maintaining the pH of the culture medium at 6.0 to 7.5 by dropping a mixed solution of 10 to 50% (w/v) glucose and 10 to 50% (w/v) sodium hydroxide is used. The mixed solution may be a product obtained by mixing a glucose solution and a sodium hydroxide solution at a volume ratio of 1:0.5 to 1: 2.

The skin microorganism to be used in the culture of the first step may be one or more strains selected from the group consisting of Lactobacillus (Lactobacillus sp.), Bifidobacterium (Bifidobacterium sp.), Streptococcus (Streptococcus sp.), Lactococcus (Lactococcus sp.), Enterococcus (Enterococcus sp.), Pediococcus (Pediococcus sp.), Weissella (Weissella sp.), Saccharomyces sp.), Bacillus (Bacillus sp.), Staphylococcus (Staphylococcus sp.), and Propionibacterium (Propionibacterium sp.).

The invention is characterized in that: the lava seawater mineral concentrate water in the second step is mineral water obtained by concentrating lava seawater under reduced pressure or by osmotic concentration in a state where 1L of the lava seawater contains at least 220mg of calcium, at least 1,130mg of magnesium, and at least 0.013mg of selenium.

The hot-pressing extraction in the third step can be performed at 120-130 ℃ and 0.13-0.30 MPa for 15-200 minutes.

The cooling purification in the third step may be performed by performing centrifugation at 3,000 to 8,000rpm for 10 to 30 minutes while maintaining 3 to 5 ℃, and then the mineral-nucleotide conjugate may be collected by a filtration process using a nanomembrane having a pore size (pore size) of 200nm or less. The pore size of the nano-film is preferably 20 to 200 nm.

The concentration of the nucleotide in the supernatant in the third step may be in the range of 200 to 2,000. mu.g/ml.

Preferably, the mineral-nucleotide in the supernatant in the third step contains 50-200 mg/L of calcium, 300-800 mg/L of magnesium and 0.001-0.01 mg/L of selenium.

The present invention provides a mineral-nucleotide conjugate derived from skin microorganisms cultured using lava seawater, manufactured by the method, and provides a skin barrier-strengthening cosmetic composition comprising the conjugate.

The invention is characterized in that: the conjugate or the cosmetic composition containing the conjugate strengthens the skin barrier by enhancing the expression of Serine Palmitoyltransferase (SPT), Filaggrin (Filaggrin), and tight junction-related protein-1 (Claudin-1).

The invention is characterized in that: the conjugate or the cosmetic composition containing the same also has a repair effect on Ultraviolet (UV) -damaged skin cells. The invention is characterized in that: the cosmetic composition achieves the repairing effect of skin cells by enhancing the expression of Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha).

The conjugate or the cosmetic composition containing the conjugate may also have the effect of regulating the flora of skin microbiome. In particular, it has an effect of increasing the number of bacteria or maintaining the stability of the skin-resident flora, i.e., Staphylococcus epidermidis (Staphylococcus epidermidis) and propionibacterium acnes (propionibacterium acnes), which are the skin microbiome.

The present invention relates to a method for producing a mineral-nucleotide conjugate derived from skin microorganisms cultured in lava seawater, and a cosmetic composition having excellent skin barrier enhancing function, skin microbiome regulating function, and thermal stability, which is produced by including the conjugate.

Therefore, the lava seawater without desalting is used as the first culture water for skin microorganism culture, thereby inducing the high concentration proliferation of skin microorganisms by natural minerals, and then the skin microorganism flora is recovered and subjected to the second culture using the lava seawater mineral concentrated water. With this, minerals can be accumulated in the cytoplasm of skin microorganisms, the amounts of nucleotides and constructs, which are cell-activating substances such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), ribosomes, endoplasmic reticulum, and the like, present in the cytoplasm can be increased, and the conjugates of mineral ions and nucleotides in lava seawater can be obtained and collected by hot-pressing extraction, thereby collecting mineral-nucleotide conjugates having excellent thermal stability at high concentrations. Next, the mineral-nucleotide conjugate derived from skin microorganisms cultured in lava seawater, which has been purified by efficiently removing cell walls, cytoplasm, proteins, and protein salt components of skin microorganisms having an influence on the toxicity of skin and microbiome and the generation of fermentation odor of raw materials, through a filtration process using a nanomembrane filter having a pore size of 200nm or less (nanometer), is used as a skin external preparation or the like which is a cosmetic composition or other pharmaceutical product that has no skin toxicity and can realize skin cell regeneration, skin barrier enhancement, skin soothing effect, and maintenance of skin resident flora.

Drawings

FIG. 1 is a schematic engineering drawing showing a method for producing a mineral-nucleotide conjugate derived from skin microorganisms cultured in lava seawater to which the present invention is applied.

FIG. 2 is a graph showing the results of comparison of the correlation between the number of viable skin microorganisms in a culture solution cultured using the culture media of different lava seawater concentrations of example 1 and comparative example 1 and nucleotides extracted from the skin microorganisms.

Fig. 3 is a graph comparing the results of nucleotide concentrations in supernatants collected at different hot press extraction times of skin microorganisms obtained after performing a differentiated second culture using general tap water or lava seawater mineral concentrate water in a first culture solution cultured using lava seawater 30 (v/v)% as culture water.

FIG. 4 is a graph of the results of confirming that the mineral-nucleotide conjugate derived from the skin organism cultured with lava seawater was not cytotoxic up to a treatment concentration of 5 (v/v)% by thiazole blue colorimetry (MTT assay).

Fig. 5 is a result of gene expression confirmation of the repair effect of mineral-nucleotide conjugates derived from skin organisms cultured in lava seawater by activation of mitochondrial function (mitogenic function) of fibroblasts (fibroblastic cells) irradiated with medium wave Ultraviolet (UVB) measured by JC-1 staining (staining).

Fig. 6 is a graph confirming the results of increasing the expression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha) decreased upon skin aging in fibroblasts (fibroblastictell) treated with medium wave Ultraviolet (UVB) of mineral-nucleotide conjugates derived from skin microorganisms cultured with lava seawater.

FIG. 7 is a diagram showing that a mineral-nucleotide conjugate derived from a skin microorganism cultured using lava seawater can be expressed by a ceramide (ceramide) synthesis-associated enzyme Serine Palmityl Transferase (SPT) gene (a); silk polymerization protein (filaggrin) gene expression (b) and protein expression (c); and Tight Junction associated protein-1 (claudin-1) gene expression to Tight Junction protein (TJ protein) (d); the skin barrier-strengthening function achieved by the promoting function of (a).

Fig. 8 is an evaluation result of confirming the effect of a mineral-nucleotide conjugate derived from skin microorganisms cultured using lava seawater on the proliferation of skin resident flora.

FIG. 9 is a photograph of a mass-produced sample using nucleotides in a conventional bacterial lysate (preparation example 2) and a mineral-nucleotide conjugate derived from a skin microorganism cultured in lava seawater.

FIGS. 10a and 10b show the results of Ultraviolet (UV) value measurement of the purity of the nucleotide in the cell lysate (preparation example 2) and the mineral-nucleotide conjugate derived from skin microorganisms cultured in lava seawater according to the prior art.

Fig. 11 is a result of confirming skin barrier-strengthening efficacy by comparing gene expression of silk polymeric protein (filaggrin) derived from a mineral-nucleotide conjugate of skin microorganisms cultured using lava seawater, which is manufactured using various strains applicable as skin microorganisms.

Detailed Description

The present invention relates to a method for producing a mineral-nucleotide conjugate derived from skin microorganisms cultured using lava seawater.

Preferably, the manufacturing method includes:

(a) production step of high-concentration skin microorganisms by using lava seawater

1x10 was obtained by preparing a culture medium prepared by using water containing 30 to 70% (v/v) lava seawater as culture water and inoculating cells¹⁰A first culture step of a viable bacteria culture medium having a concentration of CFU/ml or more;

(b) step of formation of mineral-nucleotide conjugates in the body of skin microorganisms

A second culturing step of, after recovering skin microbial cells from the culture solution, suspending the recovered cells in a limited medium (containing 220mg/L calcium, 1,130mg/L magnesium, and 0.013mg/L or more selenium) containing only lava seawater mineral concentrate and culturing the cells to form mineral-nucleotide conjugates in the cells;

(c) recovery step of mineral-nucleotide conjugates purified by hot-pressing extraction, cooling

The mineral-nucleotide conjugate derived from skin microorganisms cultured in high-purity lava seawater is produced by lysing cells with a secondary culture solution under a hot-press extraction condition of 0.12MPa or more to elute mineral-nucleotide, cooling and precipitating cell walls of the lysed cells, proteins and glycoprotein components in cytoplasm at 3 to 5 ℃, centrifuging the precipitate, and filtering the precipitate with a nanomembrane.

The medium in the step (a) may be any medium that can be used for culturing a skin microorganism strain, and more preferably may be a culture medium prepared by dissolving glucose, yeast extract, soybean peptone, and casein as components in water containing 30 to 70% (v/v) lava seawater and sterilizing the dissolved components.

More preferably, the medium in the first step may be a medium for propagation culture prepared by dissolving each component in water containing 30 to 70% (v/v) lava seawater in a state of containing 1 to 5% (w/v) of glucose, 0.5 to 5% (w/v) of yeast extract, 0.5 to 5% (w/v) of soytone, and 0.5 to 3% (w/v) of casein in a total volume of 1l and sterilizing the solution. The sterilization of the medium is preferably performed at 121 to 123 ℃ for 30 minutes. The skin microorganism suitable for use in the culture of the first step may be one or more strains selected from the group consisting of Lactobacillus (Lactobacillus sp.), Bifidobacterium (Bifidobacterium sp.), Streptococcus (Streptococcus sp.), Lactococcus (Lactobacillus sp.), Enterococcus (Enterococcus sp.), Pediococcus (Pediococcus sp.), weissel (weissel sp.), Saccharomyces (Saccharomyces sp.), Bacillus (Bacillus sp.), Staphylococcus (Staphylococcus sp.), Bacillus sp.), and Propionibacterium (Propionibacterium sp.), preferably Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus bulgaricus (Lactobacillus sp.), Lactobacillus casei (Lactobacillus), Lactobacillus rhamnosus (Lactobacillus sp.), Lactobacillus plantarum), Lactobacillus rhamnosus (Lactobacillus sp.), Lactobacillus plantarum), Lactobacillus acidophilus (Lactobacillus sp.), Lactobacillus rhamnosus (Lactobacillus sp.), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus acidophilus (Lactobacillus sp.), Lactobacillus acidophilus (Lactobacillus lactis), Lactobacillus acidophilus (Lactobacillus sp.), Lactobacillus lactis), Lactobacillus sp Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus reuteri (Lactobacillus reuteri), Bifidobacterium bifidum (Bifidobacterium bifidum), Bifidobacterium breve (Bifidobacterium breve), Bifidobacterium infantis (Bifidobacterium infantis), Bifidobacterium lactis (Bifidobacterium lactis), Bifidobacterium longum (Bifidobacterium longum), Streptococcus faecalis (Streptococcus faecalis), Streptococcus faecium (Streptococcus faecalis), Lactococcus lactis (Lactobacillus lactis), Enterococcus faecalis (Enterococcus faecalis), Enterococcus faecalis (Streptococcus faecalis), Pediococcus acidilici), Pediococcus pentococcus pentosaceus (Pediococcus pentosaceus), Leuconostoc (Leuconostoc granulosum), Leuconostoc (Leuconostoc luteum), Leuconostoc (Leuconostoc, and its, Leuconostoc, and its, and, Leuconostoc mesenteroides (Leuconostoc parameterioides), Weissella comedonii (Weissella cibaria), Weissella confusa (Weissella convulsa), Weissella koreana (Weissella koreensis), Weissella tumefaciens (Weissella soli), Weissella viridis (Weissella virescens), Staphylococcus aureus (Staphylococcus aureus), Staphylococcus epidermidis (Staphylococcus epidermidis), Propionibacterium acnes (Propionibacterium acnes), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Saccharomyces carlsbergensis (Saccharomyces boulardii), Bacillus subtilis (Bacillus subtilis), Bacillus coagulans (Bacillus licheniformis), Bacillus licheniformis (Bacillus licheniformis), Bacillus subtilis (Bacillus licheniformis), and the like. The culture conditions in the first step are preferably 70-150 rpm and 18-24 hours at 30-40 ℃.

The recovery of the bacteria in step (b) may be performed by centrifugation or ultrafiltration (ultrafiltration), preferably centrifugation at 5,000-8,000 rpm for 10-30 minutes. The recovered cells were finally suspended in lava seawater mineral concentrate (limiting medium) after being washed twice with sterilized distilled water (washing). Next, the bacterial cells suspended in the limiting medium are subjected to a second culture at 30 to 45 ℃ for 15 to 20 hours, thereby forming mineral-nucleotides. More preferably, the culture is carried out at 37 ℃ for 16 hours after the inoculation of the cells. Since the limiting medium means a medium which does not contain sufficient nutrients required for proliferation but whose composition is limited, in the examples of the present invention, the limiting medium is used after sterilizing lava seawater mineral concentrate water in which lava seawater is concentrated by a reduced pressure or osmotic concentration method.

The limiting medium may further contain any functional liquid or powder biological material that can be used in the field of cosmetics, high molecular functional biological materials, low molecular functional biological materials, and the like.

The invention is characterized in that: in order to recover the formed mineral-nucleotide, the hot-pressing extraction conditions in the step (c) are 15-200 minutes time for cracking the skin microbial cells, 120-130 ℃ and 0.12-0.30 MPa pressure. The method comprises the steps of thermally modifying protein components in cell walls and cytoplasm outside a lava seawater mineral-nucleotide conjugate (conjugate) under the conditions, cooling and precipitating the thermally modified components at 3-5 ℃, centrifuging at 3,000-8,000 rpm for 10-30 minutes to recover the mineral-nucleotide, and removing residual components by using a nanomembrane to produce a mineral-nucleotide conjugate derived from skin microorganisms cultured in lava seawater. Through the process as described above, proteins derived from bacterial cell walls, cytoplasm and microorganisms, which may induce rash, erythema and atopic dermatitis when applied to the skin, can be removed, thereby reducing side effects on the skin and skin resident bacteria. In the centrifugal separation, conditions for removing spherulites contained in cell walls and cytoplasm that are disrupted by the hot-press extraction conditions may be, for example, 3,000 to 8,000rpm, and the nanomembrane may be filtered through a membrane filter of 200 nanometers (nanomoter) or less, thereby providing a production method for purifying only mineral-nucleotides derived from skin microorganisms.

In the present invention, it is preferable to provide a skin-improving skin microbial cosmetic composition having skin barrier strengthening, skin vitality improving and skin wound healing properties.

The present invention can provide a mineral-nucleotide conjugate derived from skin microorganisms cultured using lava seawater, which is produced by the production method of the present invention as described above.

The cosmetic may contain any of the ingredients generally used. For example, it may contain common auxiliary components such as emulsifiers, thickeners, emulsions, surfactants, lubricants, alcohols, water-soluble polymers, gelling agents, stabilizers, vitamins, inorganic salts, emulsifiers, and perfumes. The amount of the above-mentioned components may be selected within a range not to impair the intrinsic effects of the cosmetic, depending on the form and the intended use. For example, the amount of the component added may be 0.1 to 100% by weight, preferably 0.1 to 30% by weight, based on the total weight of the composition, but is not limited thereto.

The type of cosmetic is not particularly limited, and examples thereof include skin care cosmetics such as lotions, milky lotions, gels, creams, essences, masks, ampoules, lotions, detergents, soaps, body products, soaps, and so forth; makeup cosmetics such as lipsticks, foundations, and the like; hair care cosmetics, etc., but the formulation thereof is not particularly limited.

Hereinafter, preferred embodiments of the present invention will be described in detail in order to facilitate easier understanding of the present invention. However, the comparative examples and examples described herein are only for illustrating the present invention, and the scope of the present invention should not be construed as being limited thereby. But rather as a representative basis for teaching one skilled in the art to more fully convey the concept of the invention to others skilled in the art and to thereby enable the present invention to be more fully and completely described herein.

< production example 1: production of skin microorganism culture solution in minimal Medium containing general tap Water >

In the present invention, as a minimal medium (general tap water medium) for culturing a skin microorganism strain, each component was dissolved in water (distilled water) in a state of containing 3% (w/v) of glucose, 2% (w/v) of yeast extract, 2% (w/v) of soybean peptone, and 1% (w/v) of casein per 1l, and sterilized at 121 to 123 ℃ for 30 minutes.

A skin microorganism species culture solution was inoculated into a sterilized minimal medium of a fermenter using a sterilized ordinary tap water culture medium as described above, and cultured at 100rpm and 37 ℃ for 20 hours, wherein the culture was performed while maintaining pH at 5.0 to 7.5 by dropping an aqueous solution (hereinafter, referred to as a glucose-sodium hydroxide solution) in which 40% (w/v) of glucose and 40% (w/v) of sodium hydroxide were mixed at a volume ratio of 1:1 at regular time intervals (Fed-batch culture was performed). Skin microbial species culture solution: the skin microorganism species culture solution in the following is a product obtained by culturing a lactic acid bacterium, i.e., a lactobacillus plantarum strain, in lactobacillus MRS Broth (BD) at 37 ℃ for 24 hours.

< production example 2: production of nucleotide derived from skin microorganism >

The skin microorganism culture solution obtained in production example 1 was centrifuged (6,000rpm, 20 minutes) to recover only the bacterial cells, and then washed (washed) with sterilized tap water (distilled water), and the procedure was repeated once.

Next, the cells were suspended in sterilized tap water, and a second culture was performed at 37 ℃ for 16 hours. Next, skin microorganism cells were lysed by performing hot-pressing extraction at a temperature of 121 ℃ and a pressure of 0.15MPa for 120 minutes, and after cooling at 4 ℃ (freezing), nucleotides derived from the skin microorganisms were recovered by centrifugation (6,000rpm, 20 minutes) from the skin microorganism lysate.

< production example 3: production of purified product of nucleotide derived from skin microorganism >

Only the bacterial cells were recovered by centrifugation (6,000rpm, 20 minutes) of the skin microorganism culture solution obtained in production example 1, and then washed (washed) with sterilized tap water (distilled water), and the procedure was repeated once.

Next, the cells were suspended in sterilized tap water, and a second culture was performed at 37 ℃ for 16 hours. Next, skin microorganism cells were lysed by performing hot-pressing extraction at a temperature of 121 ℃ and a pressure of 0.15MPa for 120 minutes, and only the final supernatant was recovered by centrifugation of a skin microorganism lysate after cooling at 4 ℃ (freezing). The recovered supernatant was finally filtered using a nanomembrane filter of 200nm or less (nanomiter), thereby recovering a nucleotide purified product derived from skin microorganisms.

< example 1: production of skin microorganism culture solutions having different lava seawater concentrations >

A lava seawater mixed solution containing 10 to 90% (v/v) of lava seawater or lava seawater itself (100%) was prepared as culture water, and the culture water was used in place of water (in a state of 0% lava seawater) in the minimal medium production conditions used in production example 1. The culture medium was sterilized in the same manner as in production example 1 using culture water containing lava seawater at different concentrations as described above.

Next, a culture solution of a skin microorganism species was inoculated into a sterilized lava seawater culture medium in the same manner as in production example 1, and cultured for 20 hours while maintaining pH at 5.0 to 7.5 by dropping a glucose-sodium hydroxide dissolution solution at regular time intervals under conditions of 100rpm and 37 ℃ (Fed-batch culture) was performed).

< example 2: production of mineral-nucleotide conjugates derived from skin microorganisms cultured using lava seawater >

The skin microorganism culture solution cultured in the lava seawater in example 1 was centrifuged (6,000rpm, 20 minutes) to recover only the bacterial cells, and then washed (washed) with sterilized tap water (distilled water), and the procedure was repeated once. Then, the cells were suspended in mineral-concentrated water (calcium 220mg, magnesium 1,130mg, and selenium 0.013mg or more) prepared by concentrating lava seawater to 1/10 volumes or more, and then subjected to a second culture at 37 ℃ for 16 hours. Next, skin microbial cells were lysed by performing a hot-pressing extraction at a temperature of 121 ℃ and a pressure of 0.15MPa for 120 minutes, and after cooling at 4 ℃ (freezing), only the final supernatant was recovered by centrifugation (20 minutes at 6,000 rpm). The recovered supernatant was finally filtered using a nanomembrane filter of 200nm or less (nanomiter) to recover the mineral-nucleotide derived from the skin microorganism.

< test example 1: identification of viable count of skin microorganisms and nucleotide content at different lava seawater concentrations >

The culture solutions of production example 1 and example 1 were diluted with physiological saline, and then the diluted solutions were dispensed into 1ml petri dishes (petridish) and 20ml of sterilized lactobacillus MRS Agar (lactobacillus MRS Agar, BD, USA) was mixed and solidified. The number of viable skin microorganisms was confirmed by counting colonies (colony) cultured in a static culture vessel at 37 ℃ for 48 hours, and the results are shown in FIG. 2 (hereinafter, referred to as "viable skin microorganism count measurement method").

Meanwhile, the culture solutions of production example 1 and example 1 were centrifuged (6,000rpm, 20 minutes), only the bacterial cells were collected, and then washed with sterilized tap water (distilled water) (washing), and the above-described process was repeated once. Next, the cells were suspended in sterilized tap water, and a second culture was performed at 37 ℃ for 16 hours. Next, the nucleotide was confirmed by lysing skin microbial cells by performing hot-pressing extraction at a temperature of 121 ℃ and a pressure of 0.15MPa for 120 minutes, and after cooling at 4 ℃ (freezing), centrifuging the skin microbial lysate (6,000rpm, 20 minutes), and then finally filtering the residual components using a nano-membrane filter of 200 nanometers (nanomter) or less.

The Ultraviolet (UV) wavelength absorbances (230nm, 260nm, 280nm) of nucleotides produced from skin microorganism culture solutions with different lava seawater concentrations were confirmed, and the nucleotide concentrations are shown in FIG. 2 (hereinafter, referred to as a nucleotide assay method derived from skin microorganisms).

In fig. 2, the lava seawater concentration of 0% means the number of viable skin microorganisms cultured in the culture solution obtained in production example 1 or nucleotides derived from skin microorganisms.

As can be seen from the analysis of the results in fig. 2, when the number of viable skin microorganisms cultured in the medium to which 30 to 70 (v/v)% of lava seawater was applied and the content of nucleotides derived from skin microorganisms in example 1 and production example 1 were confirmed, the number of viable skin microorganisms cultured in the medium to which 80 (v/v)% or more of lava seawater was applied and the nucleotides derived from the skin microorganisms were increased as compared with the cells cultured in production example 1 to which none of lava seawater was applied, and the number of viable skin microorganisms cultured in the medium to which 80 (v/v)% of lava seawater was applied and the nucleotides derived from the skin microorganisms showed similar results to those of the skin microorganisms cultured in production example 1.

It was found that the cultured skin microorganisms could not be increased unconditionally by increasing the addition amount of lava seawater, and that the culturability of skin microorganisms was hindered when the salt concentration was increased to 80 (v/v)% or more by the lava seawater. From this, it can be seen that an appropriate mineral concentration in the lava seawater is an important factor for the culture of skin microorganisms and the formation of nucleotides.

In addition, in the above results, as conditions which can achieve the optimum effects of increasing the number of viable bacteria of skin microorganisms and increasing the concentration of nucleotide formation, it is preferable to use 30 to 70 (v/v)% of lava seawater as the culture water for the medium for the first culture, and it is particularly preferable to use 30 (v/v)% of lava seawater.

< test example 2: identification of nucleotide and mineral contents derived from skin microorganisms with different water for second fermentation and extraction time under hot pressing >

In this experiment, after skin microorganisms were recovered by centrifugal separation from a skin microorganism culture solution cultured using lava seawater as culture water, the amounts of nucleotides recovered according to the next second culture conditions were compared.

For this reason, only the bacterial cells were recovered by centrifuging (6,000rpm, 20 minutes) the skin microorganism culture solution cultured with lava seawater at a concentration of 30 (v/v)% obtained in example 1, and then washed (washing) with sterilized tap water (distilled water), and the process was repeatedly performed once. Then, the cells were suspended in tap water and lava seawater mineral concentrate (Ca 220mg/L, Mg 1,130mg/L, and Se 0.013mg/L or more), respectively, and then cultured for a second time at 37 ℃ for 16 hours. Next, hot-press extraction was performed at a temperature of 121 ℃ and a pressure of 0.15Mpa, wherein hot-press extraction time was divided into 30 minutes and 120 minutes for confirming the optimal hot-press extraction conditions.

After lysis of the skin microbial cells by performing a hot-pressing extraction, the lysate was cooled at 4 ℃ (freezing) and only the final supernatant was recovered by centrifugation (6,000rpm, 20 minutes). The recovered supernatant is finally filtered using a nanomembrane filter of 200nm or less (nanomiter) to recover nucleotides or mineral-nucleotides derived from skin microorganisms. The Ultraviolet (UV) wavelength absorbance was confirmed by Nanodrop 2000(Thermo, USA) for the nucleotide content under different conditions, and the concentration of the nucleotide content derived from skin microorganisms under different conditions of water for the second fermentation and hot-pressing extraction is shown in fig. 3.

From the results in fig. 3, it can be confirmed that, when the content of nucleotides derived from skin microorganisms by the second cultivation using lava seawater mineral concentrate water is confirmed, the content of nucleotides is increased by about 1.5 times as compared with the case of the second cultivation using tap water, which indicates that a large amount of mineral-nucleotides can be generated by allowing the minerals to be absorbed into the inside of cells by the second fermentation using the lava seawater mineral concentrate water-limited medium. The results will be described again by table 1 below.

Further, it was found that the mineral-nucleotide formed as described above was extracted in the maximum amount at the autoclaving time of 120 minutes, which indicates that the mineral ions inside and outside the skin microorganism cells could be increased in reactivity by heating at the second cultivation with lava seawater mineral concentrate and autoclaving, thereby densely cracking cytoplasm and cell walls and binding to mineral-nucleotide inside and outside the cells, and thereby exhibiting thermal stability. That is, lava seawater mineral concentrate is an important element for culturing skin microorganisms and cell lysis, and the hot press extraction time is also an important element.

Next, in order to confirm the concentration of minerals that migrate from lava seawater and bind to nucleotides, the samples manufactured by the method were analyzed for their mineral content. Inorganic (calcium, magnesium, selenium) standard reagents in the range of about 0.1 to 1g were titrated and diluted (nitric acid) and then used as a standard solution. The sample produced by the method is diluted with nitric acid. The standard solution and the sample were decomposed for one hour by microwave (microwave) and then cooled, followed by filling with 50mL of triple distilled water (fill-up) and analysis with ICPOES (Perkin Electron precision optical Emission spectrometer, Optima 5300DV) together with the standard solution, and the mineral contents thereof were as shown in Table 1.

[ TABLE 1 ]

As shown in table 1, it was confirmed that the amount of minerals bound to nucleotides in skin microorganisms was significantly increased when the second culture was performed using lava seawater mineral concentrate water. In addition, when the second cultivation is performed according to the method of the present invention by repeated experiments, a supernatant containing mineral-nucleotide containing 50 to 200mg/L of calcium, 300 to 800mg/L of magnesium, and 0.001 to 0.01mg/L of selenium can be obtained.

< test example 3: confirmation of skin cytotoxicity of mineral-nucleotide conjugates derived from skin microorganisms cultured using lava seawater >

In this test example, it was attempted to demonstrate that the cytotoxicity of mineral-nucleotide conjugates derived from skin microorganisms cultured in lava seawater can be improved by effectively removing cell walls, cytoplasm, proteins, and protein salt components of skin microorganisms that affect the toxicity of skin and microbiome and the fermentation odor generation of raw materials by using a nanomembrane filter of 200 nanometers or less (nanometer).

For this purpose, a mineral-nucleotide derived from skin microorganisms obtained by performing a first culture using 30 (v/v)% lava seawater and performing a second culture using lava seawater mineral concentrate water was used as a representative sample of example 2 and referred to as a mineral-nucleotide conjugate derived from skin microorganisms cultured using lava seawater or a mineral-nucleotide conjugate derived from skin microorganisms cultured in lava seawater.

The nucleotide obtained in production example 2 is referred to as a nucleotide derived from a skin microorganism cultured in general tap water or a nucleotide derived from a skin microorganism cultured in general tap water.

In addition, since the conjugate was prepared by treating with 2.5 (v/v)% of 1,2-hexanediol (1,2-hexanediol) as a preservative in order to impart preservability to the conjugate, the conjugate was applied to a Control group (Control) by treating with 1,2-hexanediol (1,2-hexanediol) in sterilized tap water in order to be used as an empty test group in a skin cytotoxicity test, and the conjugate was applied to 1,2-hexanediol (1,2-hexanediol) at the same concentration in production example 3.

Thiazole blue colorimetry (MTT assay) is a commonly used assay for cell proliferation or toxicity by measuring the number of surviving cells, and employs the principle that succinate dehydrogenase (or mitogenic dehydrogenase) in mitochondria of cells in a surviving state can reduce a water-soluble yellow salt, namely thiazole blue (MTT, 3- [4, 5-dimethylthiozole-2-yl ] -2,5-diphenyltetrazolium bromide), to a water-insoluble blue formazan (formazan) derivative.

For this purpose, fibroblasts (Human fibroplasts) and keratinocytes (Human keratinocytes) were diluted to 1.5X 10 in 24-well plates (well plates)⁵Cell/well (cells/well) density, followed by the use of Dulbecco's Modified Eagle Medium (DMEM) containing 10% (v/v) bovine serum at 37 ℃ with 5% CO₂The culture was carried out in a medium for one day. After the culture, all the medium was removed and replaced with a Dartbuck Modified Eagle's Medium (DMEM) containing no bovine serum, and then the culture was performed for one day after adding test samples at different concentrations, respectively.

Next, the medium was removed and treated with a 0.25 mg/ml thiazole blue (MTT) solution, and a reaction was performed at 37 ℃ for 4 hours. Next, thiazole blue formazan (MTT formazan) was dissolved by adding dimethyl sulfoxide (DMSO) to the cells from which the thiazole blue (MTT) solution was removed, and then the solution was measured by absorbance at 570 nm.

As a result thereof is shown in fig. 4a and 4b, it can be found that cytotoxicity was not observed until the concentration of the mineral-nucleotide conjugate production liquid (supernatant) derived from skin microorganisms cultured using lava seawater reached 5 (v/v)%, whereas cytotoxicity was exhibited from the 3% (v/v) treated group starting with nucleotides cultured using general tap water without filtration through a nanomembrane filter of 200nm (nanometer) or less. The sample concentration% from fig. 4 to fig. 8 indicates (v/v)%.

That is, it was confirmed that since the sample of production example 2 contains cell walls, cytoplasm, proteins, and protein salt components, which are cellular structures inducing skin inflammation, and is not purified, when the sample was treated with 3% (v/v) or more in the basic skin toxicity evaluation, apoptosis occurred in both skin fibroblasts and skin keratinocytes.

In contrast, it was confirmed that in the group of treatment of mineral-nucleotide conjugates derived from skin microorganisms cultured in lava seawater of example 2, since the inflammation-inducing factors were removed by the nanomembrane filter of 200nm or less (nanometer), cytotoxicity was not exhibited even when each skin cell was treated at a concentration of 5% (v/v), and skin safety (skin safety) could be confirmed. From the results described above, it was confirmed that the viable cell count of skin microorganisms produced by culturing the mineral-nucleotide complex derived from skin microorganisms cultured in lava seawater was 2.0x10 even when cultured in 30% (v/v) lava seawater¹⁰The high concentration state of CFU/ml does not exhibit cytotoxicity, so that more usage can be achieved and thereby maximization of skin health efficacy is achieved.

In addition, in order to compare samples before and after the nano-membrane filtration process, skin fibroblasts were treated and cytotoxicity thereof was confirmed by using production examples 2, 3, and 2, and the results are shown in table 2 below. For comparison, a sample was produced without filtration using a nanomembrane of 200nm or less in the production method of example 2 and used as a control.

From the results, it was confirmed that in order to produce mineral-nucleotides derived from skin microorganisms cultured in lava seawater according to the method of the present invention, samples having almost no cytotoxicity could be produced only by performing the nanomembrane filtration process.

[ TABLE 2 ]

< test example 4: confirmation of the Effect of cellular Activity of mineral-nucleotide conjugates derived from skin microorganisms cultured with lava seawater >

To verify the change in cell membrane potential of mitochondria, fibroblasts (human fibroplasts) were aliquoted into 35mm plates (plates) and stabilized for 24 hours, and then replaced with serum-free medium and treated with samples (example 2 and production example 3).

Next, the nucleotide obtained in production example 3 is referred to as a purified nucleotide derived from a skin microorganism cultured with general tap water or a purified nucleotide derived from a skin microorganism cultured in general tap water.

After one hour had elapsed, the cells were washed with a salt buffer solution (HBSS), and then a plate (plate) was put with the same amount of the salt buffer solution as the cell culture medium, and then irradiated with medium-wave ultraviolet light (UVB) until the total irradiation amount reached 50mJ/cm 2. After irradiation with medium-wave ultraviolet light (UVB), the medium was replaced with Serum-free medium (Serum-free medium), and then the sample (example 2 and production example 3) was treated with 5% CO at 37 ℃₂The culture was carried out in an incubator (incubator) for 24 hours. After removal of the medium, it was treated with 1. mu.g/ml JC-1 solution (solution) and treated at 37 ℃ with 5% CO₂The reaction was carried out in an incubator (incubator) for 2 hours in the absence of light, and then the medium was removed and observed with a fluorescence microscope.

The ratio of red to green (green) observed in the state of healthy mitochondria (mitochondria) is 1:1 by staining with JC-1 solution (solution) as described above, while the ratio of red to green (green) shows a state in which the green (green) value is not changed but the red (red) value is significantly reduced, for example, 0.5:1 in the state of stress (stress) that may induce mitochondrial dysfunction (mitochondrion) or the state of cell damage or apoptosis.

Accordingly, it can be confirmed from fig. 5 that the treated group of mineral-nucleotide conjugates derived from skin microorganisms cultured using lava seawater can repair cells in which damage to mitochondria increases due to irradiation of medium wave Ultraviolet (UVB) rays, thereby allowing mitochondria to maintain a healthy state at a level comparable to that of the control group not subjected to medium wave Ultraviolet (UVB) rays. However, the values of the nucleotide purification products derived from skin microorganisms cultured with ordinary tap water could not be increased to the control level, indicating that the state of mitochondrial repair was slow.

< test example 5: cytothesis effect induced by Mitochondrial biogenesis (mitochondarial biogenesis) of mineral-nucleotide conjugates derived from skin microorganisms cultured using lava seawater >

After fibroblast (human fibroblast) cells were separated into 35mm plates (plates) and stabilized for 24 hours, they were replaced with serum-free medium and treated with the samples. After one hour had elapsed, the mixture was washed with a saline buffer solution (HBSS), and then irradiated with medium-wave ultraviolet rays (UVB) at 50mJ/cm after the addition of the saline buffer solution². The medium was replaced with Serum-free medium (Serum-free medium), and the sample (example 2 and production example 3) was treated with 5% CO at 37 ℃₂The culture was carried out in an incubator (incubator) for 24 hours. The cells after the culture were collected by using 1ml of lysine reagent (Qiagen), and then ribonucleic acid (RNA) was extracted by using chloroform (chloroform), 2-propanol (2-propanol) and ethanol (ethanol). After the extracted ribonucleic acid (RNA) was put into purified water treated with diethyl pyrocarbonate (DEPC), quantification was performed using a quintflorometer (Invitrogen, USA) and a quint RNA BR Assay kit (Invitrogen, USA), followed by synthesis of complementary deoxyribonucleic acid (cDNA) and execution of real-time polymerase chain reaction (real-time PCR). Synthesis of complementary deoxyribonucleic acid (cDNA) was performed using a high capacity RNA-to-cDNA kit (Applied Biosystems, USA), and the test was performed according to the method of the kit (kit). Real-Time polymerase chain reaction (Real-Time PCR) was performed using kit (qpcrbar SyGreen Blu Mic Lo-ROX, pcrbiasystems, London, UK), and the test was performed according to the method of kit (kit), and then the amplification product was quantitatively analyzed after amplifying the gene using applied Biosystems 7500FAST Real-Time PCR System. Primers (primers) used in Polymerase Chain Reaction (PCR) were synthesized from cosmogenetech (korea) and used as shown in table 3.

[ TABLE 3 ]

As a result shown in fig. 6, in order to repair damaged cells in cells treated with medium wave Ultraviolet (UVB), the expression of peroxisome proliferator-activated receptor gamma coactivator-1 α (PGC-1 α) decreased upon skin aging was increased by 16% at the same concentration of 5 (v/v)% as compared to a nucleotide purified product derived from skin microorganisms cultured with general tap water, whereby it could be confirmed that the mineral-nucleotide conjugates derived from skin microorganisms cultured with lava seawater exhibited the effects of repairing damaged cells and skin vitality.

< test example 6: skin barrier-strengthening effects of mineral-nucleotide conjugates derived from skin microorganisms cultured using lava seawater >

Keratinocytes (human keratinocytes) were seeded into cell culture dishes (cell culture dish) and cultured for 24 hours. Then, the medium was changed to serum-free (serum-free) episile (gibco) medium and cultured for 3 days after the treatment with the specimen (example 2 and production example 3). Using 1ml of QIAzol^TMCells after culture were collected by lysine reagent (Qiagen), and ribonucleic acid (RNA) was extracted using chloroform (chloroform), 2-propanol (2-propanol) and ethanol (ethanol). After the extracted ribonucleic acid (RNA) was put into purified water treated with diethyl pyrocarbonate (DEPC), quantification was performed using a Qubit flometer (Invitrogen, USA) and a Qubit RNA BR Assay kit (Invitrogen, USA), followed by synthesis of complementary deoxyribonucleic acid (cDNA) and execution of real-time polymerase chain reaction (real-time PCR). Synthesis of complementary deoxyribonucleic acid (cDNA) was performed using a high capacity RNA-to-cDNA kit (Applied Biosystems, USA), and the test was performed according to the method of the kit (kit). The Real-time polymerase chain reaction (Real-time PCR) is carried outThe assay was performed with kit (qpcrbar SyGreen blue Mic Lo-ROX, pcrbiasystems, London, UK) and the assay was performed as per the kit (kit) method, followed by quantitative analysis of the amplified product after amplification of the gene using applied Biosystems 7500FAST Real-Time PCR System. Primers (primers) used in Polymerase Chain Reaction (PCR) were synthesized from cosmogenetech (korea) and used as shown in table 4.

[ TABLE 4 ]

As shown in fig. 7a, the results of confirming the effect of the ceramide (ceramide) synthesis-related enzyme, Serine Palmitoyltransferase (SPT), which functions as a cement (lipid) in the skin barrier (brick and mortar) function, of the mineral-nucleotide conjugate derived from skin microorganisms cultured in lava seawater, showed that the skin barrier-strengthening effect was 53% higher than that of the nucleotide-purified product derived from skin microorganisms cultured in normal water when treated with 5 (v/v)% of the mineral-nucleotide conjugate derived from skin microorganisms cultured in lava seawater.

As shown in the results in fig. 7b, as a result of confirming the efficacy of Filaggrin (FLG), which is one of terminal differentiation markers, constituting a skin barrier while being one of Natural moisturizing Factor (Natural molecular moisturizing Factor) transcriptomes, the expression amount of Filaggrin (FLG) was about 3.5 times higher at a treatment concentration of 5 (v/v)% as compared to a nucleotide purification derived from a skin microorganism cultured with general tap water.

In addition, silk fibroin target (filaggrin target) immunofluorescent cell staining was performed based on the results as described above. Immunofluorescent cell staining was carried out by the following method.

Human normal keratinocytes (human normal keratinocytes) were seeded into cell culture dishes (cell culture dish) and cultured for one day, followed by treatment with samples (example 2 and manufacturing example 3) at different concentrations and culturing for three days after replacement with serum free (serum free) episile (gibco) medium. For cell fixation, cells were treated with 4% (w/v) formaldehyde solution (formaldehyde solution), then treated with 0.1% (v/v) Triton X-100 solution (solution), and cultured for 10 minutes. Next, it was changed to a 1% bovine serum albumin solution (BSA solution) and treated and reacted with a silk fibroin antibody (Invitrogen) of 2 μ g/ml after one hour had elapsed. Next, Alexa was used488conjugate antibody (Invitrogen) was processed and reacted, and then photographed after charging 1. mu.g/ml of 4', 6-diamidino-2-phenylindole (DAPI, sigma).

From the results, it was confirmed that, as in the above-mentioned results, the most distinct differentiation pattern was observed in cell morphology (cell morphology) by visual evaluation and protein fluorescent staining at a treatment concentration of 5% (v/v) for the mineral-nucleotide conjugate derived from skin microorganisms cultured in lava seawater, and the results were quantified as shown in fig. 7 c.

It was confirmed thereby that the mineral-nucleotide conjugate derived from skin microorganisms cultured using lava seawater exhibited more excellent effects on silk Fibroin (FLG) and Serine Palmitoyltransferase (SPT) at a concentration of 5 (v/v)% than the nucleotide extract derived from skin microorganisms cultured using general tap water, which indicates that it exhibited more excellent effects in terms of moisture barrier (moisture barrier) that can bring a feeling of wetness to sensitive skin and strengthen the barrier.

Furthermore, it can be confirmed from the results in fig. 7d that the mineral-nucleotide conjugate derived from skin microorganisms cultured using lava seawater can express the tight junction-associated protein (claudin-1) gene of the tight junction protein (TJ) that regulates the passage of water (water) and ions (ion) and participates in the skin barrier strengthening function.

From the test results, it was confirmed that the most significant synergistic effect was exhibited in 5 (v/v)% of the treated group, as compared with the nucleotide purification derived from skin microorganisms cultured with general tap water as a control group, for the expression of the claudin-1 gene under the corresponding conditions (24 hours of treatment). That is, lava seawater mineral-nucleotide skin microorganisms can induce 45% increase in expression of the claudin-1 gene of the claudin, compared to general tap water, thereby enhancing the skin barrier function.

< test example 7: skin microbiome regulating function derived from mineral-nucleotide conjugates of skin microorganisms cultured using lava seawater >

In order to compare the stability (hemeostasis) of mineral-nucleotide conjugates derived from skin microorganisms cultured with lava seawater in relation to the equilibrium regulation of the skin microbiome, the proliferation impact associated with skin-resident bacteria, Staphylococcus epidermidis (Staphylococcus epidermidis) and propionibacterium acnes (propionibacterium acnes), was evaluated.

Specifically, 1% (v/v) of each glycerol stock of skin resident bacteria was inoculated into Trypticase Soy Broth (TSB), followed by shaking culture at 37 ℃ for 18 to 20 hours, and the culture solution was diluted with physiological saline to adjust the O.D.600nm to 0.02 to 0.04 (about 1X 10)⁷CFU/ml), 3 (v/v)% of each of the samples of example 2 and production example 3 was added to physiological saline, and then 100. mu.l of each dilution of dermal microbial inoculum (final count of cells: x 10) was inoculated into 9.9ml of test dilution diluted at a given concentration⁵CFU/ml) and 10-fold multi-stage dilution of viable cell numbers at day 0, day 3, and day 6 after inoculation, followed by inoculation of 1ml to trypticase soy broth (trypticase soy), respectivelyagar, TSA) and pour-plates (pour-plates) were made and incubated at 37 ℃.

As can be confirmed from fig. 8, with the passage of time, the skin-resident bacteria, Staphylococcus epidermidis (Staphylococcus epidermidis) and propionibacterium acnes (Cutibacterium acnes), proliferate and maintain their stability by the mineral-nucleotide combinations derived from skin microorganisms cultured with lava seawater, but it is difficult to adjust the stability of the flora by the nucleotide purification products derived from skin microorganisms cultured with general tap water.

< test example 8: mass production of mineral-nucleotide conjugates derived from skin microorganisms cultured using lava seawater >

The mass production of each sample was performed on a 100L scale (scale) by the production process of the mineral-nucleotide conjugate derived from skin microorganisms cultured using lava seawater of example 2 and the production process of production example 2, respectively, and the results are shown in fig. 9. The purity of the product was measured by nucleotide analysis of skin microorganisms after mass production, and the results are shown in fig. 10.

As shown in fig. 9, since various cell wall components, lipoproteins, glycoproteins, and the like are contained in the supernatant (microbial lysate) containing the nucleotides of production example 2, which is a conventional technique, caramelization reaction (caramelization) occurs during the hot-pressing extraction, resulting in a change in color to dark brown and an abnormal fermentation odor. In contrast, when the method for producing a mineral-nucleotide conjugate derived from skin microorganisms cultured using lava seawater of the present invention is applied (lava seawater culture, microbial lysis, centrifugal separation, and membrane separation processes are performed), a stable raw material that is transparent and free from abnormal fermentation odor is produced.

Regarding the purity (purity) of mineral-nucleotide conjugate raw materials derived from skin microorganisms cultured with lava seawater in large quantities, pure (pure) can be considered as long as the Absorbance ratio (Absorbance ratio)260/280 reaches 2.0 to 2.2 and the Absorbance ratio (Absorbance ratio)260/230 reaches 1.8 to 2.0.

As a result of measurement by the nucleotide analysis method for skin microorganisms, as shown in fig. 10 and table 5, the concentration of nucleotides was high by concentration, and the concentration measured at each wavelength of Ultraviolet (UV) was high, and the purity thereof was confirmed by the ratio (ratio). In particular, by removing impurities such as proteins mixed in the production process, the uniformity of the graph can be maintained.

[ TABLE 5 ]

< test example 9: comparison of mineral-nucleotide conjugate efficacy derived from skin microorganisms cultured using lava seawater, produced using a variety of different skin microorganism strains >

In order to confirm the efficacy of the mineral-nucleotide conjugate derived from skin microorganisms cultured in lava seawater using a plurality of different strains of skin microorganisms, the mineral-nucleotide conjugate production process derived from skin microorganisms cultured in lava seawater of example 2 was applied to skin microorganisms described in table 6 below.

[ TABLE 6 ]

Strain numbering	Strain name
		1	Lactobacillus plantarum (Lactobacillus plantarum)
2	Bifidobacterium longum (Bifidobacterium longum)
		3	Streptococcus thermophilus (Streptococcus thermophilus)
4	Lactococcus lactis (Lactococcus lactis)
		5	Enterococcus faecalis (Enterococcus faecalis)
6	Pediococcus pentosaceus (Pediococcus pentosaceus)
		7	Weissella (Weissella cibaria)
8	Saccharomyces cerevisiae (Saccharomyces cerevisiae)
		9	Bacillus subtilis (Bacillus subtilis)
10	Staphylococcus epidermidis (Staphylococcus epidermidis)
		11	Propionibacterium acnes (Propionibacterium acnes)

Next, as a result of confirming the expression of the silk fibroin (Filagrin) gene in the method of test example 6, as shown in FIG. 11, it was confirmed that the gene was present in the presence of 1.2mM calcium chloride (CaCl) as a positive control group₂) Compare it withThe expression of Filaggrin (Filaggrin) gene on the body is increased, and the method for producing a mineral-nucleotide conjugate derived from skin microorganisms cultured in lava seawater according to the present invention is a specialized production method that can achieve effects such as skin barrier enhancement from a variety of different skin microorganisms.

< cosmetic preparation example 1 preparation of skin softening toning lotion >

A skin softening lotion (cream, 100g) comprising the supernatant containing the mineral-nucleotide conjugate of example 2 was produced by a general method according to the composition shown in table 7 below.

[ TABLE 7 ]

Raw materials	Content (g)
		Supernatant containing the mineral-nucleotide conjugate of example 2	3.0
Glycerol	3.0
		Butanediol	2.0
Propylene glycol	2.0
		Polyoxyethylene (60) hydrogenated castor oil	1.0
Ethanol	10.0
		Triethanolamine	0.1
Preservative	Micro-scale
		Pigment	Micro-scale
Perfume	Micro-scale
		Purified water	Balance of

< cosmetic preparation example 2 preparation of nutrient cosmetic lotion >

After the supernatant containing the mineral-nucleotide conjugate of example 2 was extracted with 70 (v/v)% ethanol aqueous solution to obtain a concentrated solution from which the solvent was removed, and then a nutritional lotion (skin lotion, 100g) containing the concentrated solution according to the composition shown in fig. 8 was manufactured by a general method.

[ TABLE 8 ]

Raw materials	Content (g)
		Supernatant containing the mineral-nucleotide conjugate of example 2	1.0
Sitosterol	1.7
		Polyglycerol-2 oleate	1.5
Ceteareth	1.2
		Cholesterol	1.5
Dicetyl phosphate ester	0.4
		Concentrated glycerin	5.0
Sunflower seed oil	10.0
		Carboxyvinyl polymer	0.2
Xanthan gum	0.3
		Preservative	Micro-scale
Perfume	Micro-scale
		Purified water	Balance of

< cosmetic preparation example 3 preparation of nutrient cream >

A nutrient cream (100g) comprising a supernatant containing the mineral-nucleotide conjugate of example 2 was manufactured by a general method according to the composition shown in table 9 below.

[ TABLE 9 ]

Raw materials	Content (g)
		Supernatant containing the mineral-nucleotide conjugate of example 2	5.0
Sitosterol	4.0
		Polyglycerol-2 oleate	3.0
Ceramide	0.7
		Ceteareth-4	2.0
Cholesterol	3.0
		Dicetyl phosphate ester	0.4
Concentrated glycerin	5.0
		Sunflower seed oil	22.0
Carboxyvinyl polymer	0.5
		Triethanolamine	0.5
Preservative	Micro-scale
		Perfume	Micro-scale
Purified water	Balance of

As described above, the present invention has been described in detail with reference to the manufacturing examples, the embodiments, and the test examples, but the description is for illustrative purposes only, and it will be understood by those having ordinary skill in the art to which the present invention pertains that the present invention can be implemented by various modifications and equivalent other embodiments. Therefore, the true technical scope of the present invention should be defined by the technical matters of the claims of the whole body.