KR102133689B1 - Manufacturing method of conjugate between lava seawater-originated minerals and dermabiotics-derived nucleotide, and functional dermabiotics cosmetic composition using the mineral-nucleotide - Google Patents

Manufacturing method of conjugate between lava seawater-originated minerals and dermabiotics-derived nucleotide, and functional dermabiotics cosmetic composition using the mineral-nucleotide Download PDF

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KR102133689B1
KR102133689B1 KR1020200022439A KR20200022439A KR102133689B1 KR 102133689 B1 KR102133689 B1 KR 102133689B1 KR 1020200022439 A KR1020200022439 A KR 1020200022439A KR 20200022439 A KR20200022439 A KR 20200022439A KR 102133689 B1 KR102133689 B1 KR 102133689B1
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mineral
nucleotide
dermabiotics
lava seawater
cultured
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KR1020200022439A
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Korean (ko)
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이창완
양서진
김두성
김경민
김예향
차소윤
최지휘
이승훈
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에스케이바이오랜드 주식회사
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K8/00Cosmetics or similar toilet preparations
    • A61K8/18Cosmetics or similar toilet preparations characterised by the composition
    • A61K8/96Cosmetics or similar toilet preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99Cosmetics or similar toilet preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K8/00Cosmetics or similar toilet preparations
    • A61K8/18Cosmetics or similar toilet preparations characterised by the composition
    • A61K8/30Cosmetics or similar toilet preparations characterised by the composition containing organic compounds
    • A61K8/60Sugars; Derivatives thereof
    • A61K8/606Nucleosides; Nucleotides; Nucleic acids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K8/00Cosmetics or similar toilet preparations
    • A61K8/18Cosmetics or similar toilet preparations characterised by the composition
    • A61K8/96Cosmetics or similar toilet preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/965Cosmetics or similar toilet preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of inanimate origin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILET PREPARATIONS
    • A61Q19/00Preparations for care of the skin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K2800/00Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/57Compounds covalently linked to a(n inert) carrier molecule, e.g. conjugates, pro-fragrances

Abstract

The present invention relates to a method for producing a dermabiotics-derived mineral-nucleotide complex cultured in lava seawater, wherein the mineral-nucleotide complex has no skin toxicity and regenerates skin cells, strengthens skin barriers, soothes skin, maintains a superficial flora. Since it has a function, it can be provided as a functional cosmetic composition containing it or as an external preparation for skin.

Description

Manufacturing method of lava seawater-derived minerals and dermabiotics-derived nucleotides and functional dermabiotics cosmetic composition using the mineral-nucleotides and functional dermabiotics cosmetic composition using the mineral-nucleotide }

The present invention is useful for solving both intrinsic factors and extrinsic factors of skin aging at the same time, useful skin microorganisms (dermabiotics, dermabiotics) are used as culture water for lava sea water rich in natural mineral content. After cultivation at high concentrations, the cells are recovered, and lava seawater mineral concentrated water is cultivated to generate a large amount of mineral-nucleotides in the cells, and heat-pressure extraction method is used to prepare a mineral-nucleotide conjugate having excellent thermal stability. It is about. In the present invention, after thermal pressure extraction, a method of purifying mineral-nucleotides by selectively removing cell wall components (cell walls, glycolipids, lipid proteins, etc.) that cause inflammatory reactions of skin cells through cooling centrifugation through membrane filtration methods Provided, Dermabiotics-derived mineral-nucleotide conjugate cultured in lava seawater thus prepared is thermally stable and suggests that it has the function of regulating the skin micro-biome, the third skin layer, and the expression of functional proteins related to skin barrier strengthening. do.

The skin is composed of three layers of epidermal layer, dermal layer, and stratum corneum. The epidermal layer is directly under the stratum corneum and is responsible for the function of permeation barrier, innate immunity, protection against ultraviolet rays, wound healing, and synthesis of vitamin D. The dermal layer is the epidermis. It is a layer responsible for supplying nutrients to the skin and regenerating the skin. The outermost stratum corneum in the skin structure is formed of a very thin layer about 0.5 mm thick. The stratum corneum composed of 25 to 30 dead skin cells serves to protect our skin and the differentiation of keratinocytes is the division of basal cells-synthesis in polar cells-autolysis in granule cells-in keratinocytes It consists of 4 stages of the rebuilding process of and forms a skin barrier. In the process of forming this skin barrier, cell regeneration occurs for about 4 weeks based on young skin. Skin protects the human body from physical and chemical stimuli from the outside, but when exposed to ultraviolet rays, a representative external factor of skin aging, it damages the DNA of skin cells and releases reactive oxygen species (ROS). To inhibit the collagen synthesis of the skin and accelerate the decomposition, thereby aging the skin to create wrinkles. Although it is accepted as the theory of natural aging that the internal factors of skin aging depend on genetic factors inherited from parents, the rate and condition of skin aging due to changes in skin microbiome called the third skin layer Is different.

In addition, there have been statistically confirmed reports of differences in skin condition and type according to region, race, and age. Kompaore et al. (Skin Pharmacol., 1993;6: 200-207) reported percutaneous moisture loss in Asians, Caucasians and African Americans, and reported that Asians and African Americans were higher than Caucasians. Skin Pharmacol.Physiol.,200;22:190-199) measured sebum levels by age group in Chinese men and women, and women's foreheads had lower sebum levels than men's foreheads, and women tended to decrease significantly after their 30s. However, males reported similar tendencies until their 50s, and then gradually decreased. Marrakchi et al. (Contact dermatitis, 2007; 57:28-34) compared the moisture content and sebum content of young people aged 24 to 34 with those aged 66 to 83. As a result, the moisture content of young people was higher than that of older people on the forehead and cheeks. The amount of sebum was reported to have no significant difference between the young and old.

According to the national skin characteristics bank construction business report, in the case of Koreans, female skin types were in the order of complexity>dry>neutral>oily, and male skin types were in the order of oily>complex>neutral>dry.

Skin microbiome, which has been spotlighted as a topic of recent research, refers to the ecosystem of the flora that is distributed in the skin, and is mainly present in the skin of the genus Staphylococcus , Propionibacterium , etc. It is reported that microorganisms maintain a healthy skin condition by maintaining a balance through interaction with skin cells. The skin flora that constitutes the skin microbiome varies depending on the age group, woman and man, race, and region of residence, and it is reported that skin dominant species by age group maintain skin health.

Recently, with the development of cosmetics using microorganisms as a core material in the industry, the microorganisms are manufactured in lysate and used in cosmetics to avoid skin penetration problems caused by microbial size, problems caused by preservatives, and competitive inhibition problems. It was intended to improve skin barriers by directly reacting with immunoreceptors. Of using a commercially available microbial material Bifidobacterium (Bifidobacterium) in or Lactobacillus bacteria (Lactobacillus) There are commercially available as the material used for the genus of lactic acid bacteria to the skin of which, bipyridinium the lactic acid bacteria cell lysate Examples include fermentation concentrate representative It has as a main component. However, among the cell components, peptidoglycans (peptidoglycan), glycolipids (lipopolysaccharide), and lipoproteins, which are known to cause an immune reaction with the skin, are present in the cell wall. , May cause skin problems such as erythema. In addition, since the thermal stability of bacterial nucleotides, which are active ingredients in the cytoplasm, is 70-105°C based on the Tm value (the temperature at which the DNA double helix is released), it is an effective ingredient to apply at least 121°C, the temperature that can completely break the cell wall. It becomes denatured and does not help improve the skin barrier. In addition, one of the problems in the application of the microbial material for skin protection is that it is not a selective growth factor for the skin bacterial flora when applying cell debris to the skin, so if harmful skin is used as a growth nutrient, healthy skin micro The balance of the biome is broken. Various microbial cell debris currently listed as ingredients for international cosmetics do not solve the imbalance problem of skin microbiome because they contain a large amount of the components of the regulator unit that can affect human skin microbiome.

Lava seawater is water that has been aged for a long time as the seawater is naturally filtered into basalt and sandy layers and matured for a long time.It is a unique water resource possessed only by Jeju Island. In Jeju, lava seawater has been developed from the characteristics of low temperature and cleanliness of lava seawater since 1980s. Has been actively used as a breeding water for halibut farming, and has been in the spotlight since it was used as water for saunas in terms of health and beauty since 5-6 years ago. Depending on the purpose of use, lava seawater may be used as desalted lava seawater from which salt has been removed or as it is collected.

Lava seawater contains more essential minerals (sodium, manganese, zinc, molybdenum, selenium, etc.) as well as essential minerals such as sodium, magnesium, calcium, and potassium than ordinary seawater, deep water, and samdasoo water. Among them, vanadium, which is known to stabilize insulin secretion or improve diabetes, hyperlipidemia, etc., promote blood circulation, enhance immunity, germanium with anticancer activity, inhibit oxidation of fat, synergistic effect to maintain heart and liver, radical scavenging The inclusion of selenium, which has the effect of improving ability, anti-cancer, infertility, aging and cholesterol levels, is characteristic of lava seawater that has never been reported in deep ocean water. In addition, these minerals are in an ionized state, and the ionized minerals can be easily digested and absorbed by the human body or other animals. In addition, lava seawater is a clean groundwater resource in which E. coli, nitrate nitrogen, phosphate phosphorus, phenols, etc. are not detected, and harmful elements such as arsenic, mercury, and cadmium are not detected, or trace amounts of lead are detected, which causes obstacles to industrial application. There is no clean raw material. In particular, minerals such as magnesium and calcium contained in lava seawater are essential elements for the proliferation of Dermabiotics, and it is recommended for human consumption as an essential nutrient.

The eluted DNA has a melting temperature (Tm) among the decomposition elements, which refers to the intermediate value in the temperature change from double strand DNA to single strand DNA. After cell crushing, the eluted DNA is decomposed into nucleotides at this temperature condition with known Tm values of 70 to 105°C, and these are composed of purine and pyrimidine to stabilize between nucleotides by internal hydrogen bonding. However, the decomposed nucleotides have a strong negative charge among the components, so they have repulsion. In order to extract nucleotides having these characteristics, in order to crush cell walls and cytoplasm, the nucleotides must be maintained in a stable state even at a thermal pressure of 121° C. higher than the Tm value of 70 to 105° C.

Accordingly, the present inventors confirmed that mineral-nucleotides isolated from Derma Biotics cultured in lava seawater have significantly better skin barrier strengthening and skin damage suppression function than Derma Biotics-derived nucleotides cultured in conventional medium. Was completed.

Republic of Korea Registered Patent No. 10-1347694 (Invention name: Method for manufacturing fermentation product of desalted lava seawater and cosmetic composition using the fermentation product obtained by the method and the fermentation product, Applicant: Jeju Techno Park, Foundation Date: December 2013 Month 27) Republic of Korea Registered Patent No. 10-1693574 (Invention name: Composition for improving skin moisturizing or wrinkles, which contains a tindalized lactic acid bacterium as an active ingredient, Applicant: Korea Institute of Oriental Medicine, Registration Date: January 02, 2017)

Zakostelska Z, Kverka M, Klimesova K, Rossmann P, Mrazek J, et al. (2011) Lysate of Probiotic Lactobacillus casei DN-114 001 Ameliorates Colitis by Strengthening the Gut Barrier Function and Changing the Gut Microenvironment. PLoS ONE 6(11)

An object of the present invention is to use natural nutrient-rich lava seawater as a culture water to proliferate Dermabiotics without additional pre-treatment to induce the generation of mineral-nucleotide complexes as natural minerals derived from lava seawater accumulate in the cytoplasm of Dermabiotics. It is about providing technology.

The mineral-nucleotide complex is manufactured through the thermal pressure extraction process of Derma Biotics, and thus has excellent thermal stability. Through this, it has a skin barrier strengthening function and a protective effect of skin cells due to ultraviolet rays, and by controlling the skin microbiome. Improve skin condition.

The present invention (first step) culturing Derma Biotics in a medium using lava seawater as culture water to obtain a primary culture solution;

(Second step) After recovering the cells from the culture medium, culturing in lava seawater mineral concentrate to obtain a secondary culture medium; And

(3rd step) recovering the supernatant containing the mineral-nucleotide by extracting and cooling and purifying the secondary culture solution by thermal pressure;

It relates to a method for producing a mineral-nucleotide conjugate derived from Derma Biotics cultured in lava seawater containing a.

The medium of the first step is a medium containing glucose, yeast extract, soypeptone, and casein, and dissolving the component in water containing 30 to 70% (v/v) of lava seawater and sterilizing the medium. It is characterized by. Preferably, the medium in the first step is 1 to 5% (w/v) glucose, 0.5 to 5% (w/v) yeast extract, 0.5 to 5% soypeptone (w/v), casein Each component may be a medium for propagation culture prepared by dissolving and sterilizing each component in water containing 30 to 70% (v/v) of lava seawater so as to be 0.5 to 3% (w/v).

When incubating the first step, 10 to 50% (w/v) of glucose and 10 to 50% (w/v) of sodium hydroxide mixed solution are added dropwise to maintain a pH of 6.0 to 7.5 during culture. Can be used. The mixed solution may be a mixture of glucose solution and sodium hydroxide solution in a volume ratio of 1:0.5 to 1:2, respectively.

Dermabiotics applied to the first step culture is Lactobacillus sp., Bifidobacterium sp., Streptococcus sp., Lactococcus sp., Enterococcus sp., Pediococcus sp., Weissella sp., Saccharomyces sp., Bacillus sp., Staphylococcus It may be one or more strains from the group consisting of the genus ( Staphylococcus sp.) and the genus Propionibacterium sp.

The lava seawater mineral concentrate in the second step is characterized in that it is mineral water obtained by concentrating the lava seawater under reduced pressure or osmosis to include more than 220mg of calcium per 1L, more than 1,130mg of magnesium, and 0.013mg of selenium.

The thermal pressure extraction of the third step may be performed at 120 to 130° C. and 0.12 to 0.30 MPa for 15 to 200 minutes.

Cooling and purification of the third step can be performed by centrifugation at 3,000 to 8,000 rpm for 10 to 30 minutes while being maintained at 3 to 5°C, and thereafter, nano having a pore size of 200 nm or less. Mineral-nucleotide conjugates may be collected using a membrane filtration process. The pore size of the nano-membrane may be preferably 20 to 200 nm.

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

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

The present invention provides a dermabiotics-derived mineral-nucleotide conjugate cultured in lava seawater prepared by the above method, and provides a cosmetic composition for strengthening the skin barrier comprising the conjugate.

The conjugate or a cosmetic composition containing the same is characterized by enhancing the expression of SPT (Serine palmitolytransferase), Filaggrin, and Claudin-1 to strengthen the skin barrier.

The combination or the cosmetic composition containing the same is characterized in that it has a more effective efficacy of skin cells due to UV damage. The cosmetic composition is characterized by enhancing the expression of PGC-1α (Peroxisome proliferator-activated receptor gamma coactivator 1α), which has the effect of restoring skin cells.

The conjugate or cosmetic composition containing the same may further have an effect of controlling the microflora of the skin microbiome. In particular, as the skin microbiome, there is an effect of increasing the number of bacteria of the skin flora, Staphylococcus epidermidis and Cutibacterium acnes , or maintaining homeostasis.

The present invention relates to a method for preparing a dermabiotics-derived mineral-nucleotide conjugate cultured in lava seawater and a cosmetic composition having excellent skin barrier strengthening function, skin microbiome control function and thermal stability prepared by containing the conjugate.

To this end, demineralized lava seawater is applied as the primary culture water for cultivation of Dermabiotics to induce high-density proliferation of Dermabiotics through intrinsic natural minerals, and then recover Dermabiotics cells to use lava seawater mineral concentrate. Second culture. By doing so, it is possible to obtain mineral accumulation in the dermabiotics cytoplasm and increase the amount of substances of nucleotides and constructs, such as DNA, RNA, ribosomes, and vesicles, which are the cell-activating substances present in the cytoplasm, and obtain a combination of mineral ions and nucleotides in lava seawater, By collecting through thermal pressure extraction, mineral-nucleotide conjugates having excellent thermal stability can be collected at a high concentration. The cell wall, cytoplasm, protein and protein salt components of Derma Biotics affect the toxicity of skin and microbiome and the production of fermentation odor of raw materials through a filtration process using a nano-membrane filter having a pore size of 200 nanometers or less. By effectively removing the Dermabiotics-derived mineral-nucleotide complex cultured in purified lava seawater, it has no skin toxicity and is capable of skin cell regeneration, skin barrier strengthening, skin soothing effect, and skin cosmetic composition maintenance. It can be provided as an external skin preparation for pharmaceuticals.

1 is a process diagram showing a method for producing a mineral-nucleotide conjugate derived from Derma Biotics cultured in lava seawater according to the present invention.
FIG. 2 is a graph comparing the correlation between the number of Dermabiotics viable cells and nucleotides extracted from the Dermabiotics in the culture medium cultured in the lava seawater concentration medium of Example 1 and Comparative Example 1.
FIG. 3 shows the thermal pressure extraction time of Derma Biotics obtained after differentiating the secondary culture after the primary culture incubated in lava seawater 30 (v/v)% as cultured water with normal constant or lava seawater mineral concentrate. It is a graph comparing the concentration of nucleotides in the supernatant obtained according to.
Figure 4 is a graph confirming that Derma Biotics-derived mineral-nucleotide conjugates cultured in lava seawater through MTT assay have no cytotoxicity up to a treatment concentration of 5 (v/v)%.
Figure 5 is a result confirming the cell's recovery effect through gene expression by activating the mitochondrial function of the fibroblast cell irradiated with UVB, dermabiotics-derived mineral-nucleotide conjugate cultured in lava seawater through JC-1 staining measurement.
6 is a graph confirming the result of increasing the expression of PGC-1α, which is reduced by derma biotics-derived mineral-nucleotide conjugates cultured in lava seawater, in UVB-treated fibroblast cells.
7 is a dermabiotics-derived mineral-nucleotide conjugate cultured in lava seawater, a ceramide synthesis-related enzyme Serine palmitolytransferase (SPT) gene expression (a); filaggrin gene expression (b) and protein expression (c); And Tight Junction protein (TJ protein) for claudin-1 gene expression (d); is a result of confirming that the skin barrier strengthening function through the promoting function.
8 is an evaluation result confirming the proliferation of dermatophytes by derma biotics-derived mineral-nucleotide conjugates cultured in lava seawater.
Figure 9 is a photograph of a mass production sample of nucleotides (Preparation Example 2) and derma biotics-derived mineral-nucleotide conjugates cultured in lava seawater using a prior art technique.
10A and 10B are results obtained by measuring the purity of nucleotides (Preparation Example 2) and derma biotics-derived mineral-nucleotide conjugates cultured in lava seawater using a conventional technique as UV values.
11 is a result of confirming the effect of strengthening the skin barrier through a comparison of filaggrin gene expression of dermabiotics-derived mineral-nucleotide conjugates cultured in lava seawater prepared with various strains applicable to dermabiotics.

The present invention relates to a method for preparing a mineral-nucleotide conjugate derived from Dermabiotics cultured in lava seawater.

Even more preferably, the manufacturing method,

(a) Production stage of high concentration derma biotics using lava seawater

A primary culture step of preparing a culture medium prepared by using water containing 30-70% (v/v) of lava seawater as the culture water, and inoculating the cells to obtain a viable culture solution of 1x10 10 CFU/ml or more;

(b) Formation of mineral-nucleotide complex in Dermabiotics cells

After recovering the Dermabiotics cells from the culture solution, the recovered cells are suspended and cultured in a limited medium (containing calcium 220 mg/L, magnesium 1,130 mg/L, selenium 0.013 mg/L or more) containing only lava seawater mineral concentrate. A secondary culture step of forming a lava seawater mineral-nucleotide conjugate in a cell;

(c) Step of recovery of mineral-nucleotide complex through heat pressure extraction and cooling purification

Cells are crushed in a secondary culture solution with a heat pressure extraction condition of 0.12 MPa or more to elute mineral-nucleotides, and the cell wall of the crushed cells, the protein and glycoprotein components in the cytoplasm are cooled and precipitated at 3~5°C, and centrifuged and filtered through a nanomembrane It comprises a step of preparing a mineral-nucleotide conjugate derived from Derma Biotics cultured in high-purity lava seawater.

The medium of step (a) is preferably, may be any medium capable of culturing the Dermabiotics strain, more preferably, as a component, glucose, yeast extract, soypeptone, casein is included, and the composition The component may be a culture medium prepared by dissolving and sterilizing lava seawater in water containing 30 to 70% (v/v).

Even more preferably, the medium of the first step is based on a total volume of 1 ℓ, glucose 1-5% (w/v), yeast extract 0.5-5% (w/v), soypeptone 0.5-5% (w/v) ), casein 0.5 to 3% (w / v) so that each component may be a medium for growth culture prepared by dissolving and sterilizing lava seawater in water containing 30 to 70% (v/v). The medium sterilization is preferably performed at 121 ~ 123 ℃ for 30 minutes. Dermabiotics applied to the first step culture is Lactobacillus sp., Bifidobacterium sp., Streptococcus sp., Lactococcus sp., Enterococcus sp., Pediococcus sp., Weissella sp., Saccharomyces sp., Bacillus sp., Staphylococcus One or more fungi from the group consisting of the genus Staphylococcus sp. and Propionibacterium sp., preferably Lactobacillus acidophilus , Lactobacillus bulgaricus , lacto Bacillus Kasei (Lactobacillus casei), Lactobacillus buffer momentum (Lactobacillus fermentum), Lactobacillus is Serena (Lactobacillus gasseri), Lactobacillus helveticus (Lactobacillus helveticus), Lactobacillus ramno suspension (Lactobacillus rhamnosus), Lactobacillus zone soniyi (Lactobacillus johnsonii ), Lactobacillus plantarum , Lactobacillus reuteri , Bifidobacterium bifidum , Bifidobacterium breve , Bifidobacterium ins. infantis ), Bifidobacterium lactis , Bifidobacterium longum , Streptococcus pecalis ( S) treptococcus faecalis , Streptococcus faecium , Lactococcus lactis ssp. lactis ), Enterococcus faecalis , Enterococcus faecium , Pediococcus acidolacticii , Pediococcus pentosaceus , Leuconostock carnosum ( Leuconostoc carnosum ), Leuconostoc citreum , Leuconostoc gasicomitatum , Leuconostoc gellidum , Leuconostoc inhae , Leuconostoc inhae , Kimchi ( Leuconostoc inhae ) Leuconostoc kimchii), current Kono Stock lactis (Leuconostoc lactis), current Kono Stock mesen teroyi death (Leuconostoc mesenteroides subsp. mesenteroides), current Kono Stock Farah mesen teroyi death (Leuconostoc paramesenteroides), by cibaria (Weissella cibaria), by Weissella confusa , Weissella koreensis , Weissella soli , Weissella viridescens , Staphylococcus aureus , and Staphylococcus aureus dummy disc (Staphylococcus epidermidis), propynyl sludge tumefaciens arc Ness (Propionibacterium acnes), saccharose in my process celebrity bicyclic on (Saccharomyces cerevisiae), saccharose in my process Carlsbad Bergen sheath (Saccharomyces carlsbergensis), saccharose in my process carambola Woody (Saccharomyces boulardii ), Bacillus subtilis , Bacillus coagulans ( Bacillus coagulans ), Bacillus licheniformis ( Bacillus licheniformis ), etc. One or more may be selected from the group consisting of. The culture conditions of the first step is preferably 70 to 150 rpm, 30 to 40 ℃ conditions for 18 to 24 hours.

In the step (b), centrifugation, ultrafiltration, etc. may be used to recover the cells, but it is preferable to perform centrifugation at 5,000 to 8,000 rpm for 10 to 30 minutes. The recovered cells are washed twice with sterile distilled water and suspended in final lava seawater mineral concentrate (limited medium). Thereafter, the cells suspended in the restricted medium are cultured for 2 to 15 hours at 30 to 45° C. to form mineral-nucleotides. More preferably, the cells are cultured at 37° C. for 16 hours after inoculation. The term “restricted medium” refers to a medium that does not contain enough nutrients required for proliferation and has limited components. In the examples of the present invention, As a limiting medium, lava seawater mineral concentrated water in which lava seawater was concentrated under reduced pressure or osmotic concentration was used as a limiting medium.

Thereafter, any functional liquid or powdered biomaterials that can be used in the cosmetic field, high molecular weight and low molecular weight functional biomaterials can be used in the restricted medium.

In step (c), the thermo-pressure extraction condition is characterized in that the time of 15 to 200 minutes, the temperature of 120 to 130° C., and the pressure of 0.12 to 0.30 MPa are crushed to derma biotics cells to recover the formed mineral-nucleotide. do. Under the above conditions, the cell wall other than the lava seawater mineral-nucleotide conjugate is thermally denatured to protein components in the cytoplasm, and the thermally denatured components are cooled and precipitated at 3 to 5°C, and centrifuged at 3,000 to 8,000 rpm for 10 to 30 minutes. -By recovering the nucleotides and removing the remaining components using a nano-membrane, a mineral-nucleotide conjugate derived from Dermabiotics cultured in lava seawater is prepared. Through this process, when used on the skin, skin rashes, erythema, and atopic skin cells that can cause atopy are removed, and proteins derived from the cytoplasm and microorganisms can be reduced and side effects on the skin and skin flora can be reduced. The centrifugation can be used, for example, 3,000 to 8,000 rpm as a condition that can remove pellets contained in the cell wall and cytoplasm, which are crushed through thermo-pressure extraction conditions, and the nano-membrane is filtered with a membrane filter of 200 nanometers or less. By providing a manufacturing method for purifying only the dermabiotics-derived mineral-nucleotide.

In the present invention, it is preferred to provide a dermabiotics cosmetic composition for skin improvement, which is characterized by having skin barrier strengthening, skin vitality, and skin wound healing.

The present invention can provide a mineral-nucleotide conjugate derived from Dermabiotics cultured in lava seawater prepared by the method of the present invention.

The cosmetic composition may include all of the commonly used ingredients. For example, it may include general auxiliary ingredients such as emulsifiers, thickeners, emulsions, surfactants, lubricants, alcohols, water-soluble polymers, gelling agents, stabilizers, vitamins, inorganic salts, emulsifiers, and fragrances. The above ingredients can be selected depending on the formulation or purpose of use, within the range that does not impair the inherent effects of cosmetics. The added amount of the components may be, for example, 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.

In addition, the type of the cosmetic is not particularly limited, for example, lotion, latex, gel, cream, essence, pack, ampoule, lotion, cleaning agent, soap, body products, soap, oil and other skin care cosmetics, lipstick, Makeup cosmetics such as foundations, cosmetics for hair, and the like, and the formulation is not particularly limited.

Hereinafter, preferred embodiments of the present invention will be described in detail so that the present invention may be more easily understood. However, the comparative examples and examples described herein are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by them. Rather, it is provided to sufficiently convey the spirit of the present invention to those skilled in the art so that the contents introduced herein are thorough and complete.

<Production Example 1: Preparation of Dermabiotics culture medium in a basic medium containing general constants>

In the present invention, 3% (w/v) of glucose per 1 liter, 2% of yeast extract (w/v), and 2% of soypeptone (w/v) as a basic medium (general constant medium) for culturing Dermabiotics strains, Each component was dissolved in water (distilled water) so that casein was 1% (w/v) and sterilized at 121 to 123°C for 30 minutes.

The Dermabiotics seed culture solution was inoculated into the sterilized basic medium of the fermenter in a sterilized general constant medium and incubated for 20 hours at 100 rpm and 37° C., but 40% (w/v) glucose and 40% (w/v) An aqueous solution in which sodium hydroxide was mixed at a volume ratio of 1:1 (hereinafter, referred to as a glucose-sodium hydroxide solution) was added dropwise at regular intervals while maintaining the pH at 5.0 to 7.5 and incubating (performed fed-batch culture). * Dermabiotics seed culture solution: Lactobacillus plantarum strain, a lactic acid bacteria, was cultured in Lactobacilli MRS Broth (BD) for 24 hours at 37°C, hereinafter referred to as Dermabiotics seed culture solution.

<Production Example 2: Preparation of nucleotides derived from Derma Biotics>

The Dermabiotics culture solution obtained in Preparation Example 1 was centrifuged (6,000 rpm, 20 minutes), and only the cells were recovered, washed with sterile constant (distilled water) and repeated once.

Thereafter, the cells were suspended in sterilized constant water and cultured for 2 hours at 37°C for 16 hours. Then, the dermabiotic cells were crushed by thermal pressure extraction at a temperature of 121°C and a pressure of 0.15 MPa for 120 minutes, and after cooling to 4°C, the dermabiotic crushing solution was centrifuged (6,000 rpm, 20 minutes). Dermabiotics-derived nucleotides were recovered.

<Production Example 3: Preparation of purified product of nucleotides derived from Derma Biotics>

The Dermabiotics culture solution obtained in Preparation Example 1 was centrifuged (6,000 rpm, 20 minutes), and only the cells were recovered, washed with sterile constant (distilled water) and repeated once.

Thereafter, the cells were suspended in sterilized constant water and cultured for 2 hours at 37°C for 16 hours. After thermal pressure extraction for 120 minutes at a temperature of 121°C and a pressure of 0.15 MPa, the Dermabiotics cells were crushed, and after cooling to 4°C, the Dermabiotics crushed solution was centrifuged (6,000 rpm, 20 minutes) to the final supernatant. Bays were recovered. The recovered supernatant was finally filtered through a nano-membrane filter of 200 nanometers or less to collect a nucleotide purified product derived from Dermabiotics.

<Example 1: Preparation of culture medium of Derma Biotics by lava seawater concentration>

A lava seawater mixture containing 10 to 90% (v/v) of lava seawater or lava seawater itself (100%) was prepared as culture water, and water was used in the basic medium preparation conditions using the culture water in Preparation Example 1 (lava seawater 0 % State) instead. In this way, the culture medium containing lava seawater for each concentration was sterilized as in Preparation Example 1.

Next, the Dermabiotics seed culture solution was inoculated into the sterile lava seawater medium as in Preparation Example 1, and the pH was dropped at intervals of 5.0 to 7.5 using glucose-sodium hydroxide solution for 20 hours at 100 rpm and 37°C. While maintaining the pH at 5.0 ~ 7.5 and cultured (Fed-batch culture performed)

<Example 2: Preparation of mineral-nucleotide conjugate derived from Derma Biotics cultured in lava seawater>

The Dermabiotics culture medium cultured in the lava seawater of Example 1 was centrifuged (6,000 rpm, 20 minutes), and only the cells were recovered, washed with sterile constant (distilled water) and repeated once. After that, the cells were suspended in mineral concentrated water (calcium 220 mg, magnesium 1,130 mg, selenium 0.013 mg or more) prepared by concentrating the lava seawater to 1/10 or more by volume, followed by secondary culture at 37°C for 16 hours. Then, the dermabiotic cells were crushed by thermal pressure extraction at a temperature of 121°C and a pressure of 0.15 MPa for 120 minutes, and after cooling to 4°C, the dermabiotic crushing solution was centrifuged (6,000 rpm, 20 minutes). Only the final supernatant was recovered. The recovered supernatant was finally filtered with a nano-membrane filter of 200 nanometers or less to collect mineral-nucleotides derived from Dermabiotics.

<Experimental Example 1: Confirmation of the number of living cells and nucleotides of Derma Biotics by concentration of lava seawater>

The culture solution of Preparation Example 1 and Example 1 was taken, diluted with physiological saline, 1 ml of the diluted solution was dispensed in petridish, and 20 ml of sterilized Lactobacilli MRS Agar (BD, USA) was mixed and hardened. The number of colonies cultured for 48 hours in a stationary incubator at 37°C was counted to confirm the number of Dermabiotics viable cells, and this is shown in FIG. 2 (hereinafter, referred to as a method for measuring the number of Dermabiotics viable cells).

In addition, at the same time, the culture solution of Preparation Example 1 and Example 1 was centrifuged (6,000 rpm, 20 minutes), and only the cells were recovered, washed with sterile constant (distilled water) and repeated once. Thereafter, the cells were suspended in sterilized constant water and cultured for 2 hours at 37°C for 16 hours. After thermal pressure extraction at a temperature of 121°C and a pressure of 0.15 MPa for 120 minutes, the Dermabiotics cells were crushed and cooled to 4°C, followed by centrifugation of the Dermabiotics crushing solution (6,000 rpm, 20 minutes). The nucleotide was confirmed by final filtration of the residual component with a nano-membrane filter of 200 nanometers or less.

The nucleotide concentration is shown in FIG. 2 by checking the UV wavelength absorbance (230 nm, 260 nm, 280 nm) of the nucleotides prepared by the Dermabiotics culture solution for each lava seawater concentration (hereinafter referred to as a nucleotide measurement method derived from Dermabiotics).

In Figure 2, the lava seawater concentration of 0% means the number of Dermabiotics viable cells or Dermabiotics-derived nucleotides cultured using the culture solution obtained in Preparation Example 1.

Analyzing the results of FIG. 2, when confirming the number of Dermabiotics viable cells and nucleotides derived from Dermabiotics cultured in lava seawater according to concentrations of Example 1 and Preparation Example 1, 30 to 70 (v/ v)% Dermabiotics viable cells cultured in a medium to which lava seawater was applied, the nucleotides derived from the Dermabiotics can be seen to increase compared to cells cultured through Preparation Example 1 in which no lava seawater was applied, and 80(v /v)% Dermabiotics viable cells cultured in a medium to which lava seawater was applied, and the nucleotides derived from Dermabiotics were found to be prepared similarly to Dermabiotics cultured through Preparation Example 1.

This can be seen that the cultivation of Dermabiotics does not increase unconditionally by increasing the amount of lava seawater added, and when the salt concentration is increased by 80 (v/v)% or higher by lava seawater, the Dermabiotics culture is inhibited. Is confirmed. Accordingly, it can be seen that the proper mineral concentration in the lava seawater is an important factor in culturing Dermabiotics and forming nucleotides.

In addition, among the above results, it is preferable to use 30-70 (v/v)% of lava seawater as the culture medium for primary culture as the most optimal condition for increasing the number of Dermabiotics viable cells and increasing nucleotide formation. , Of these, it can be confirmed that the most optimal condition is to use 30(v/v)%.

<Experimental Example 2: Determination of nucleotides and minerals derived from Dermabiotics according to the second fermentation water and the time of hot pressure extraction>

In this experiment, the amount of nucleotides recovered according to the subsequent secondary culture conditions was compared after collecting Derma Biotics through centrifugation from Derma Biotics culture medium cultured using lava seawater as culture water.

To this end, the lava seawater obtained in Example 1 was centrifuged (6,000 rpm, 20 minutes) and cultured with Derma Biotics culture at a concentration of 30 (v/v)%, and only the cells were recovered and washed with sterile constant (distilled water). And this was repeated once. After that, the cells were suspended in normal constant water and lava seawater mineral concentrate (calcium 220 mg/L, magnesium 1,130 mg/L, selenium 0.013 mg/L or more), respectively, and then incubated at 37°C for 16 hours. Thereafter, hot-pressure extraction was performed at a temperature of 121° C. and a pressure of 0.15 MPa, but the hot-pressure extraction time was divided into 30 minutes and 120 minutes in order to confirm optimal heat pressure extraction conditions.

After the Dermabiotics cells were crushed by thermal pressure extraction, only the final supernatant was recovered by centrifugation (6,000 rpm, 20 minutes) after cooling the crushing solution to 4°C. The recovered supernatant was finally filtered with a nano-membrane filter of 200 nanometers or less to collect nucleotides or mineral-nucleotides derived from Dermabiotics. The nucleotide content of each condition was confirmed by using Nanodrop 2000 (Thermo, USA) to confirm the UV wavelength absorbance, and the concentration of nucleotide content derived from Dermabiotics according to the secondary fermentation water and the time of heat extraction was shown in FIG. 3.

According to the results of FIG. 3, when the nucleotide content derived from Derma Biotics secondary cultured with lava seawater mineral concentrate was confirmed, it was confirmed that the nucleotide content was increased by about 1.5 times compared to the constant secondary culture, which is the concentration of lava seawater mineral. By performing secondary fermentation through a limited number of medium, it is proved that minerals are absorbed into Derma Biotics cells and that mineral-nucleotides are formed in large quantities. This result will be explained once again through Table 1 below.

In addition, it can be seen that the largest amount of the mineral-nucleotide thus formed is extracted when the heat pressure treatment time is 120 minutes. When the lava seawater mineral concentrate is secondaryly cultured and the pressure is extracted, mineral ions inside and outside the Dermabiotics cells are reactive by heating. This increase can be said to be a result that proves that the cytoplasm and the cell wall are densely crushed inside and outside the cell, and combined with mineral-nucleotides to exhibit thermal stability. That is, lava seawater is an important factor in culturing Dermabiotics and crushing cells, and mineral concentration water is also an important factor in the time of extracting heat.

Next, the mineral content of the sample prepared by the above method was analyzed in order to confirm the concentration of minerals that are introduced from lava seawater and bound to nucleotides. Standard reagents of mineral (calcium, magnesium, selenium) in the range of about 0.1 to 1 g were titrated and diluted (nitric acid), and taken as standard solutions. The sample prepared by the above method was diluted with nitric acid. After dissolving and cooling the standard solution and sample for 1 hour in a microwave, 50 mL fill-up with tertiary distilled water is analyzed with ICPOES (PerkinElmer precisely optical emission spectrometer, Optima 5300DV) with the standard solution to show the mineral content in Table 1. Did.

Heat treatment time Second culture method Mineral content (mg/L) 30min General constant
Second culture
calcium 10
magnesium 120 Selenium 0 Lava seawater minerals
Second culture of concentrated water
calcium 72
magnesium 221 Selenium 0.002 120min General constant
Second culture
calcium 22
magnesium 232 Selenium 0 Lava seawater minerals
Second culture of concentrated water
calcium 132
magnesium 532 Selenium 0.006

As shown in Table 1, it can be seen that the amount of the mineral binding to the nucleotide in Dermobiotics is significantly increased during the secondary culture of lava seawater mineral concentrate. In addition, repeated experiments were performed to obtain supernatant containing mineral-nucleotides of calcium 50-200 mg/L, magnesium 300-800 mg/L, and selenium 0.001-0.01 mg/L during the second culture according to the method of the present invention.

<Experimental Example 3: Confirmation of dermal cytotoxicity of derma biotics-derived mineral-nucleotide complex cultured in lava seawater>

In this experimental example, dermabiotics cell wall, cytoplasm, protein and protein salt components, which affect the toxicity of skin and microbiome and fermentation of raw materials, are effectively removed with a nano-membrane filter of 200 nanometers or less. The aim was to prove that the cytotoxicity of the dermabiotics-derived mineral-nucleotide complex cultured in lava seawater was improved .

To this end, Dermabiotics-derived mineral-nucleotide obtained by primary cultivation of lava seawater 30 (v/v)% and secondary cultivation with lava seawater mineral concentrate is used as a representative sample of Example 2, and said,'Lava seawater culture Dermabio It is called'Tix derived mineral-nucleotide conjugate' or'Dermabiotics derived mineral-nucleotide conjugate cultured in lava seawater'.

In addition, the nucleotide obtained in Preparation Example 2 is referred to as'nucleotide derived from Dermabiotics in general constant culture' or'nucleotide derived from Dermabiotics cultured in normal constant'.

On the other hand, in order to impart preservation in the manufacture of the conjugate, preservatives of 2.5 (v/v)% of 1,2-hexanediol were prescribed and prepared, respectively. 1,2-hexanediol was prescribed as a control and applied as a control, and 1,2-hexanediol of the same concentration was applied to Preparation Example 3.

MTT assay is an experimental method that is frequently used for cell proliferation or toxicity by measuring the number of living cells. Living cells are water-soluble and yellow salt MTT(3-[4,5-dimethlythiazole-2-yl]-2,5 in mitochondria. The principle is that diphenyltetrazolium bromide is reduced to a water-insoluble blue formazan derivative by succinic dehydrogenase or mitochondrial dehydrogenase.

To this end, DMEM (Dulbecco's Modified Eagle) containing 10% (v/v) bovine serum by diluting fibroblasts (human fibroblasts) and keratinocytes (Human keratinocytes) at a density of 1.5×10 5 cells/well in a 24 well plate Medium, Welgene, Korea) cultured at 37° C. in a 5% CO 2 incubator for 1 day. After incubation, all of the medium was removed, replaced with DMEM medium without bovine serum, and each test sample was added for each concentration, followed by incubation for 1 day.

Thereafter, the medium was removed, and a 0.25 mg/ml MTT solution was treated to react at 37° C. for 4 hours. Thereafter, dimethyl sulfoxide (DMSO) was added to the cells from which the MTT solution was removed to dissolve MTT formazan and measured by absorbance at 570 nm.

The results for this are shown in FIGS. 4A and 4B, but cytotoxicity was not confirmed up to a concentration of 5 (v/v)% of the Derma Biotics-derived mineral-nucleotide conjugate preparation solution (supernatant) cultured in lava seawater, but 200 nanometers. Dermabiotics-derived nucleotides cultured in normal constants that were not filtered with nanometer filters below a nanometer showed cytotoxicity from the 3% (v/v) treatment group. ※ All sample concentrations in FIGS. 4 to 8 mean (v/v)%.

That is, since the sample of Preparation Example 2 does not contain cell walls, cytoplasm, protein and protein salt components, which are cell structures that cause inflammation of the skin, 3% (in both fibroblasts and keratinocytes in skin basic toxicity evaluation) v/v) It can be seen that apoptosis occurs during sample processing.

On the other hand, in the treatment group of dermabiotics-derived mineral-nucleotide conjugate cultured in lava seawater of Example 2, the inflammation-causing factors were removed through a nano-membrane filter of 200 nanometers or less to 5% (v/v). It was confirmed that skin safety was not observed even when treated with each cell of the skin. As a result of this, Dermabiotics-derived mineral-nucleotide conjugates cultured in lava seawater have improved cytotoxicity despite being produced at a high concentration of 2.0x10 10 CFU/ml of Dermabiotics viable cells through 30% (v/v) lava seawater culture. As it is manufactured in a non-representative form, it is possible to expect maximization of skin health efficacy through implementation of more usage.

Meanwhile, in order to compare samples before and after the nano-membrane filtration process, Preparation Example 2, Preparation Example 3, and Example 2 were treated with skin fibroblasts to confirm cytotoxicity and are shown in Table 2 below. For comparison, a sample without performing a nano-membrane filter of 200 nanometers or less in the manufacturing method of Example 2 was prepared and used as a comparison group.

As a result, it can be confirmed that the preparation of a sample with little cytotoxicity is possible only by performing a nano-membrane filtration process for the production of mineral-nucleotides derived from lava seawater culture Dermabiotics according to the method of the present invention.

sample Sample concentration
(%, v/v)
Cell viability average (%)
Control 3.0% 100.00 Nucleotide derived from Dermabiotics
-Before nano-membrane filtration
(Production Example 2)
3.0% 77.24
Nucleotide purified from Dermabiotics in general constant culture-after nano-membrane filtration
(Production Example 3)
3.0% 94.32
From lava seawater culture Derma Biotics
Mineral-nucleotide-before nano-membrane filtration
3.0% 78.30
From lava seawater culture Derma Biotics
Mineral-nucleotide complex-after nano-membrane filtration
(Example 2)
3.0% 101.40

<Experimental Example 4: Confirmation of cell activity effect of mineral-nucleotide conjugate derived from Derma Biotics cultured in lava seawater>

To verify the mitochondrial cell membrane potential change, fibroblasts (human fibroblasts) were dispensed into 35 mm plates, stabilized for 24 hours, replaced with serum-free medium, and treated with samples (Example 2 and Preparation Example 3).

The nucleotides obtained in Preparation Example 3 are referred to as'general constant culture Dermabiotics-derived nucleotide purification' or'general constant cultured Dermabiotics-derived nucleotide purification'.

After 1 hour, after washing with a salt buffer solution (HBSS) buffer, the salt buffer solution was added to the plate as much as the cell culture solution, and then irradiated until the total irradiation amount reached 50 mJ/cm 2 of UVB. After UVB irradiation, the medium was replaced with a serum-free medium, and then treated with a sample (Example 2 and Production Example 3) at 37°C and incubated for 24 hours in a 5% CO 2 incubator. After removing the medium, 1 μg/ml of JC-1 solution was treated and reacted for 2 hours without light in a 37° C., 5% CO 2 incubator to remove the medium and observed under a fluorescence microscope.

Through staining with JC-1 solution, the ratio of red:green is confirmed to be 1:1 when healthy mitochondria is present, but red when stress is applied to induce mitochondrial dysfunction or cell damage or death is induced. There is no change in the green value, such as 0.5:1 :green ratio, and the red value is significantly reduced.

As shown in FIG. 5, the dermabiotics-derived mineral-nucleotide conjugate culture group cultured in lava seawater restores cells with increased damage to mitochondria due to UVB irradiation, and healthy mitochondria as a control level without UVB treatment It is confirmed to remain in the state. However, the nucleotide purification derived from Dermabiotics cultured in a general constant does not increase its value to the control level, indicating that the recovery state of mitochondria is slow.

<Experimental Example 5: Cell recovery effect through induction of mitochondrial biogenesis of dermabiotics-derived mineral-nucleotide complex cultured in lava seawater>

After fibroblasts (human fibroblast) were dispensed on a 35 mm plate and stabilized for 24 hours, the cells were replaced with serum-free medium and samples were treated. After 1 hour, after washing with a salt buffer solution (HBSS) buffer, a salt buffer solution (HBSS) was added, and then UVB 50 mJ/cm 2 was irradiated. After replacing the medium with a serum-free medium, the samples (Example 2 and Preparation Example 3) were treated and incubated for 24 hours in a 37°C, 5% CO 2 incubator. After incubation, cells are collected with 1 ml of lysis reagent (Qiagen), and RNA is extracted using chloroform, 2-propanol, and ethanol. After adding the purified water treated with DEPC to the extracted RNA, quantitation was performed using a Qubit florometer (Invitrogen, USA) and a Qubit RNA BR Assay kit (Invitrogen, USA), and then cDNA was synthesized to perform real-time PCR. For cDNA synthesis, a high capacity RNA-to-cDNA kit (Applied Biosystems, USA) was used, and experiments were performed by the method of the kit. For real-time PCR, a kit (qPCRBIO SyGreen Blu Mic Lo-ROX, PCRBIOSYSTEMS, London, UK) was used, and experiments were performed by the method of the kit.After amplification of the gene using applied Biosystems 7500 FAST Real-Time PCR System The amplification product was quantitatively analyzed. The primers used for PCR are shown in Table 3, and were synthesized and used by Cosmogenetech (KOREA).

Mitochondrial biosynthesis
Primer for gene identification
Forward Reverse
PGC-1α 5’-CTTTGCCCAGATCTTCCTGAAC-3’ 5’-CACTGCACCACTTGAGTCCAC-3’ GAPDH 5’-CAATGACCCCTTCATTGACC-3’ 5’-AAATGAGCCCC