KR102238733B1 - Novel Paenisporosarcina quisquiliarum 17mud 1-1541 strain and its use - Google Patents

Abstract

The present invention relates to a novel Paenisporosarcina quisquiliarum 17mud 1-1541 strain and its use, having skin whitening and wound healing effects isolated from mud, and the use of the Paenisporosarcina Quisquiliarum Riarum ( Paenisporosarcina quisquiliarum ) 17mud 1-1541 strain or a culture thereof has excellent whitening and wound healing effects and can be usefully used as an active ingredient in pharmaceutical compositions, cosmetic compositions, and health functional foods for skin improvement.

Description

Novel Paenisporosarcina quisquiliarum 17mud 1-1541 strain and its use {Novel Paenisporosarcina quisquiliarum 17mud 1-1541 strain and its use}

The present invention relates to a novel Paenisporosarcina quisquiliarum 17mud 1-1541 strain and its use, having skin whitening and wound healing effects isolated from mud, and the use of the Paenisporosarcina Quisquiliarum Riarum ( Paenisporosarcina quisquiliarum ) 17mud 1-1541 strain or a culture thereof has excellent whitening and wound healing effects and can be usefully used as an active ingredient in pharmaceutical compositions, cosmetic compositions, and health functional foods for skin improvement.

Recently, the tendency to improve the quality of life has become clear, and interest in appearance is increasing. Accordingly, the general public's interest in plastic surgery, beauty, and makeup is rapidly increasing. In particular, interest in whitening cosmetics and wrinkle-improving cosmetics continues to increase explosively due to the hot air and preference for white faces.

It is everyone's constant desire to have such white and fine skin. It is genetically determined according to the concentration and distribution of melanin in human skin, but it is also affected by environmental or physiological conditions such as solar ultraviolet rays, fatigue, and stress. Melanin is made through a non-enzymatic oxidation reaction after an enzyme called tyrosinase acts on tyrosine, a kind of amino acid, and is converted into dopa and dopaquinone. As such, the pathway by which melanin is produced is known, but the mechanism that induces melanin synthesis, which is the previous step in which tyrosinase acts, is still not elucidated in detail.

Meanwhile, as a commonly known whitening component, substances that inhibit tyrosinase enzyme activity such as Kojic acid and arbutin, hydroquinone, vitamin-C (L-Ascorbic acid) and derivatives thereof, and various There are plant extracts. These are known to be capable of realizing skin whitening or improving skin hyperpigmentation such as spots and freckles by inhibiting the synthesis of melanin pigments. However, when applied to the skin, there is a problem in that the amount of use is limited due to safety problems such as irritation and redness, or the effect is insignificant, so that a practical effect cannot be expected.

In addition, the skin tissue consists of the epidermis, the dermis, and the subcutaneous (hypodermis). The part that determines the nature of the skin is the epidermis, and the epidermis is directly and frequently damaged from the outside, so its recovery is very important. In general, when a wound occurs in the body, a creature has the property of self-defense and self-healing to some extent. In particular, if the skin is damaged by trauma such as a wound, skin damage below a certain level can be recovered with self-healing ability, but skin damage above a certain level is caused by other diseases due to other factors such as bacterial infection. It can also spread. In order to prevent this, a wound healing composition is used, and the wound healing composition has excellent biocompatibility so that it has less rejection at the wound site, must be able to adhere to the wound site, and can prevent bacterial infection at the wound site. It should be, and it should not be reactive even if it is mixed with other medicines. Drugs for skin regeneration or wound treatment have been developed so far, but the development of new ingredients that are safer to the body than existing treatments and, above all, have superior skin regeneration than substances known to be effective in skin regeneration or wound treatment. This is in desperate need.

Therefore, there is still a need to develop a component that is safe for the living body, has a stable active ingredient, and, above all, has superior whitening, skin barrier improvement, and wound healing ability than conventional materials.

Korean Patent Registration No. 1869275 (Registered on June 14, 2018) Korean Patent Publication No. 2018-0108209 (published on Oct. 4, 2018)

The present invention was conceived to solve the above problems, and the present inventors have identified a novel Paenisporosarcina quisquiliarum (Paenisporosarcina quisquiliarum) 17mud 1-1541 strain from mud very excellent whitening and wound healing or recovery It was discovered that the effect was exhibited and the present invention was completed.

Accordingly, it is an object of the present invention to provide a wave Ennis captive Sar key extractor Squeal Ria room (Paenisporosarcina quisquiliarum) 17mud 1-1541 strain deposited as accession number KCTC13860BP.

It is another object of the present invention provides the wave Ennis captive Sar key extractor Squeal Ria room (Paenisporosarcina quisquiliarum) 17mud 1-1541 strain, or pharmaceutical composition for lightening skin pigmentation disorders or the prevention or the treatment thereof, including the culture as an active ingredient There is it.

Another object of the present invention to provide such a wave Ennis captive Sar key extractor Squeal Ria room (Paenisporosarcina quisquiliarum) 17mud 1-1541 skin-lightening or pigmentation prevention or cosmetic composition for the improvement comprising the strain or culture thereof as an active ingredient There is it.

Of the invention A further object is the wave Ennis captive Sar key extractor Squeal Ria room (Paenisporosarcina quisquiliarum) 17mud 1-1541 strain, or health food for skin whitening or discoloration prevention or improvement containing the culture thereof as an active ingredient It is in providing.

A further object of the present invention to provide a pharmaceutical composition for wound healing containing the wave Ennis captive Sar key extractor Squeal Ria room (Paenisporosarcina quisquiliarum) 17mud 1-1541 strain, or a culture thereof as an active ingredient.

A further object of the present invention to provide a cosmetic composition for wound healing containing the wave Ennis captive Sar key extractor Squeal Ria room (Paenisporosarcina quisquiliarum) 17mud 1-1541 strain, or a culture thereof as an active ingredient.

A further object of the present invention to provide a dietary supplement for wound healing, including the wave Ennis captive Sar key extractor Squeal Ria room (Paenisporosarcina quisquiliarum) 17mud 1-1541 strain, or a culture thereof as an active ingredient.

In order to solve the above problems, the present invention provides a Paenisporosarcina quisquiliarum 17mud 1-1541 strain having excellent skin whitening and wound healing effects deposited under the accession number KCTC13860BP.

In addition, the present invention provides a pharmaceutical composition for preventing or treating skin whitening or pigmentation diseases comprising the strain or a culture thereof as an active ingredient.

In addition, the present invention provides a cosmetic composition for preventing or improving skin whitening or pigmentation diseases comprising the strain or a culture thereof as an active ingredient.

In addition, the present invention provides a health functional food for preventing or improving skin whitening or pigmentation diseases comprising the strain or a culture thereof as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for wound healing comprising the strain or a culture thereof as an active ingredient.

In addition, the present invention provides a cosmetic composition for wound healing comprising the strain or a culture thereof as an active ingredient.

In addition, the present invention provides a health functional food for wound healing comprising the strain or a culture thereof as an active ingredient.

Paenisporosarcina Quisquiliarum 17mud 1-1541 strain according to the present invention inhibits the activity of tyrosinase and alpha-glucosidase, and has excellent melanin synthesis inhibitory effect. It has an excellent effect without irritation.

In addition, since the strain has a wound closing effect by inhibiting the action of heme oxygenase-1 (HO-1), the wound healing or healing effect is excellent.

Therefore, the strain and its culture according to the present invention are non-toxic and thus have no side effects, so they can be usefully used as active ingredients of pharmaceutical compositions, cosmetic compositions, and health functional foods for skin improvement.

1 is a final selection in the present invention Paenisporo Sarkina Quisquilia room 17mud 1-1541 It shows the 16s rRNA gene sequence of the strain.
FIG. 2 is a phylogenetic tree of the Paenis porosarcina genus Gongsi strain and the Paenis porosarcina quisquiliarum 17mud 1-1541 strain.
Figure 3 is a result of comparing the skin whitening improvement effect of the Paenisporosarcina Quisquiliarum 17mud 1-1541 strain and arbutin according to the present invention, Figure 3 (a) confirms the pigmentation effect in melanocytes Results, Figure 3(b) is a result of confirming the effect of reducing tyrosinase activity in melanocytes, and Figure 3(c) is a result of confirming the effect of inhibiting tyrosinase activity.
4 is a result of confirming the whitening effect according to the experimental method required by the KFDA, FIG. 4 (a) is the result of confirming the effect of inhibiting tyrosinase activity, and FIG. 4 (b) is the result of confirming the effect of inhibiting alpha-glucosidase activity. It is the result.
FIG. 5 is a result of confirming the wound healing effect on keratinocytes of the Paenisporosarcina Quisquilia room 17mud 1-1541 strain according to the present invention, and FIG. 5(a) shows the wound closure ratio (wound closure, %). 5(b) is a comparison of the effect of the 17mud 1-1541 strain and the zinc protoporphyrin (Znpp), and the result of confirming the inhibitory effect of heme oxidase (HO-1) activity of the 17mud 1-1541 strain. to be.
6 is a result of a cytotoxicity test of the 17mud 1-1541 strain of Paenisporosarcina Quisquilia room according to the present invention.
7 is a result of confirming the antioxidant effect of the Paenisporosarcina Quisquilia room 17mud 1-1541 strain according to the present invention, FIG. 7 (a) is a result of confirming the DPPH radical scavenging ability, and FIG. 7 (b) is This is the result of confirming the hydrogen peroxide radical scavenging ability.

Accordingly, the present inventors discovered and identified a strain of Paenisporosarcina Quisquiliarum 17mud 1-1541 belonging to a new Paenisporosarcina sp. isolated from mud, and investigated the mycological properties of the strain. It has been identified, and its use as a pharmaceutical composition, cosmetic composition, and health functional food was suggested by confirming the effect of skin whitening and wound healing as a new use thereof.

According to one aspect of the present invention, the present invention is a novel strain deposited with accession number KCTC13860BP, specifically Paenisporosarcina quisquiliarum excellent in skin whitening and wound healing effect (Paenisporosarcina quisquiliarum) 17mud 1-1541 strain to provide.

Preferably, the strain may be isolated from mud, but is not limited thereto.

The strain has the 16S rRNA nucleotide sequence of SEQ ID NO: 1, the G+C base content of DNA is 46.0 mol%, and is characterized by resistance to gamma rays of 3 KGy radiation dose.

Preferably, the strain may have a skin whitening effect by reducing the amount of melanin through inhibition of tyrosinase activity or α-glucosidase activity. At this time, the inhibition of alpha-glucosidase inhibits the glycosylation process in the activation step of tyrosinase, thereby modifying the structure of tyrosinase to inactivate tyrosinase into melanosomes. By making it move, it makes it impossible to produce melanin. Therefore, it can be used to inhibit or prevent melanin from being deposited on the skin by inhibiting the production of melanin. Specifically, it may exhibit effects on spots, freckles, spots, sun pigmentation, pigmentation after drug use, pigmentation after inflammation, and gestational pigmentation.

In addition, the strain has a wound-closing effect in keratinocytes, and is predicted to inhibit the action of heme oxygenase-1 (HO-1), and in particular, a HO-1 specific inhibitor (HO-1 specific inhibitor), zinc protoporphyrin (Znpp; Zinc protoporphyrin) showed excellent wound closing effect. Here, the zinc protoporphyrin is a protoporphyrin-based compound present in a small amount in the body, and the protoporphyrin is coordinated with zinc ions. Zinc protoporphyrin is known to inhibit the action or activity of heme oxidase (HO-1), an antioxidant enzyme that protects cells from oxidative damage caused by reactive oxygen species present in high concentrations in cancer cells and tissues.

On the other hand, the strain was confirmed to have no DPPH radical scavenging ability and hydrogen peroxide radical scavenging ability, and is a strain that does not have an antioxidant effect.

According to another aspect of the invention, it quinolyl wave Ennis Sar captive keys according to the invention Syrian Squeal room (Paenisporosarcina quisquiliarum) preventing or treating 17mud 1-1541 strain, and skin lightening or pigmentation diseases, including cultures thereof as an active ingredient To provide pharmaceutical compositions for use. In addition, the present invention wave Ennis captive Sar key extractor Squeal Ria room (Paenisporosarcina quisquiliarum) 17mud 1-1541 provides a strain and a pharmaceutical composition for wound healing comprising a culture thereof as an active ingredient according to the.

In the present invention, "skin whitening" generally refers to any action that inhibits or prevents skin deposition of melanin by inhibiting the production of melanin.

In the present invention, "pigmentation" is medically or cosmetically, as long as it falls within the category of pigmentation, a specific disease name is not particularly limited, but preferably, spots, freckles, spots, sunlight pigmentation, pigmentation after drug use, and after inflammation Pigmentation and gestational pigmentation.

In the present invention, "wound" refers to a physical rupture of continuity or completeness of tissue structure, and "wound healing" refers to restoration of tissue integrity, and may include partial or complete restoration of tissue integrity, and treatment ( treat). The healing (cure or heal) may be a wound healing in a shorter time compared to natural healing. The healing may include improvement and/or alleviation of the wound. Improving the wound means reducing the severity of the wound. In addition, the healing may include all of the healing of wounds and/or wound-related diseases. The healing may mean healing and/or regeneration of damaged tissue caused from a wound. The healing may include the meaning of skin regeneration. In addition, the healing may be to maintain the original composition of the damaged tissue. In addition, the healing may be to promote the treatment and/or regeneration of the damaged tissue while minimizing complications and/or scars of diseases related to the wound. Thus, wound healing refers to the promotion, improvement, progression, acceleration, or progression of one or more steps or processes involved in the wound healing process.

Further, the wound can be any internal wound or external wound, in particular a skin wound, in which the external structural integrity of the skin is maintained, for example a bruise or internal ulceration, and thus the tissue can be internal or external body tissue. . In one example, the tissue is skin (eg, human skin), and the wound may be a skin wound such as a dermal or epidermal wound.

In the present invention, "treatment" refers to, or includes alleviation, inhibition or prevention of one or more symptoms thereof, a disease, disorder or condition, and the "active ingredient" refers to a disease, disorder or condition, or one or more symptoms thereof. It can mean any amount of the composition used in the practice of the invention provided herein sufficient to alleviate, inhibit or prevent progression.

The pharmaceutical composition comprises 0.001% to 80% by weight of Paenisporosarcina quisquiliarum 17mud 1-1541 based on the total weight of the composition. Strains. Further, the wave Ennis captive Sar key extractor Squeal Ria room (Paenisporosarcina quisquiliarum) dose of 17mud 1-1541 strain is 0.01mg to about 10,000mg, 0.1mg to 1000mg, 1mg to 100mg, 0.01mg to 1000mg, 0.01mg to 100mg, 0.01 It may be from mg to 10 mg, or from 0.01 mg to 1 mg. Also, Paenisporosarcina quisquiliarum 17mud 1-1541 The dosage of the strain may be 1.0×10 ⁵ to 1.0×10 ⁸ cells/kg (body weight). However, the dosage may be variously prescribed depending on factors such as the formulation method, the mode of administration, the patient's age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate, and response sensitivity. In consideration of these factors, the dosage can be appropriately adjusted. The number of times of administration may be one time or two or more times within the range of clinically acceptable side effects, and the administration site may be administered to one or two or more sites. For non-human animals, the same dose per kg as human or, for example, the volume ratio (e.g., average value) of the target animal and human organs (heart, etc.) One amount can be administered. Possible routes of administration include oral, sublingual, parenteral (e.g., subcutaneous, intramuscular, intraarterial, intraperitoneal, intrathecal, or intravenous), rectal, topical (including transdermal), inhalation, and injection, or implantable devices. Or the insertion of material.

The pharmaceutical composition may contain a pharmaceutically acceptable carrier and/or additive. For example, sterile water, physiological saline, conventional buffers (phosphoric acid, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (ascorbic acid, etc.), surfactants, suspending agents, isotonic agents, or preservatives, etc. can do. For topical administration, organic substances such as biopolymers, inorganic substances such as hydroxyapatite, specifically collagen matrix, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers, and chemical derivatives thereof, etc. may be included. I can.

When the pharmaceutical composition is formulated in a dosage form suitable for injection, Paenisporosarcina quisquiliarum 17mud 1-1541 strain is dissolved or dispersed in a pharmaceutically acceptable carrier, or dissolved or dispersed. It may be frozen in the state of a solution.

The pharmaceutical composition is a suspension agent, a solubilizer, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, a painless agent, a buffering agent, a reducing agent, if necessary depending on the administration method or formulation. Antioxidants and the like may be appropriately included. In addition, the pharmaceutical composition according to an embodiment is formulated using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the technical field to which the present invention pertains. It may be prepared in a dosage form or may be prepared by placing it in a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of powder, granules, tablets, or capsules.

In addition, the pharmaceutical composition may be applied to an external skin preparation for application to the skin. The external preparation for skin may be any one selected from the group consisting of a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal delivery patch, a drug-containing bandage, and a lotion.

The external preparations for the skin include metal sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, bellapamil, licorice extract, glavidine, and caline. Hot water extract of fruit, various herbal medicines, drugs such as tocopherol acetate, glytilithic acid, tranexamic acid and derivatives or salts thereof, vitamin C, ascorbic acid magnesium phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, Sugars such as trehalose can also be appropriately blended.

According to another aspect of the invention there is provided wave Ennis captive Sar key extractor Squeal Ria room (Paenisporosarcina quisquiliarum) 17mud 1-1541 strain, or skin-lightening or pigmentation prevention or cosmetic composition for the improvement comprising a culture thereof as an active ingredient The composition is provided. In addition, the present invention wave Ennis captive Sar key extractor Squeal Ria room (Paenisporosarcina quisquiliarum) 17mud 1-1541 provides a cosmetic composition for wound healing comprising the strain or culture thereof as an active ingredient.

In the present invention, the "cosmetic composition" refers to an article used in the human body to clean and beautify the human body to add attractiveness, change appearance, or maintain or promote the health of skin and hair.

The cosmetic composition may be provided in any formulation suitable for topical application. For example, a solution, an emulsion obtained by dispersing an oil phase in an aqueous phase, an emulsion obtained by dispersing an aqueous phase in an oil phase, a suspension, a solid, a gel, a powder, a paste, a mask pack or sheet, a foam or an aerosol composition Can be. Compositions of such formulations can be prepared according to conventional methods in the art.

The cosmetic composition may further include a moisturizing agent, an emollient agent, an ultraviolet absorber, a preservative, a bactericide, an antioxidant, a pH adjuster, an organic and inorganic pigment, a fragrance, a cooling agent or a limiting agent. The blending amount of additional ingredients such as the moisturizing agent can be easily selected by a person skilled in the art within a range that does not impair the object and effect of the present invention, and the blending amount is 0.01 to 5% by weight, specifically 0.01 to 3, based on the total weight of the composition. It may be weight percent.

The cosmetic composition is from 0.001% to 80% by weight based on the total weight of the composition, for example, from 0.01% to 60% by weight, from 0.01% to 40% by weight, from 0.01% to 30% by weight, from 0.01% to 20% by weight Wt%, 0.01 wt% to 10 wt%, 0.01 wt% to 5 wt%, 0.05 wt% to 60 wt%, 0.05 wt% to 40 wt%, 0.05 wt% to 30 wt%, 0.05 wt% to 20 wt% , 0.05% to 10% by weight, 0.05% to 5% by weight, 0.1% to 60% by weight, 0.1% to 40% by weight, 0.1% to 30% by weight, 0.1% to 20% by weight, 0.1 Wt% to 10% by weight, or 0.1% to 5% by weight of Paenisporosarcina quisquiliarum (Paenisporosarcina quisquiliarum) 17mud 1-1541 strains may be included.

According to another aspect of the present invention, the present invention comprises Paenisporosarcina quisquiliarum (Paenisporosarcina quisquiliarum) 17mud 1-1541 strain or a culture thereof as an active ingredient for preventing or improving skin whitening or pigmentation disorders Provide functional food. The present invention also provides a wound healing dietary supplement that includes the par Ennis prisoners Sar key thistle Liao Squeal Room (Paenisporosarcina quisquiliarum) 17mud 1-1541 strain as an active ingredient.

In the present invention, "health functional food" is the same term as food for special health use (FoSHU), and has a medical and medical effect processed so that a bioregulatory function is effectively displayed in addition to nutritional supply. Means high food.

In the present invention, "culture" refers to a product obtained by culturing a strain in a known liquid medium or solid medium, and is a concept including a strain.

The health functional food may be formulated in a formulation of a conventional health functional food known in the art. The health functional foods may be prepared in general formulations such as powders, granules, tablets, pills, capsules, suspensions, emulsions, syrups, needles, liquids, extracts, etc., meat, sausages, bread, chocolate, candies, It may be prepared in the form of any health food such as snacks, confectionery, pizza, ramen, other noodles, gums, jelly, dairy products including ice cream, various soups, beverages, teas, drinks, alcoholic beverages, and vitamin complexes. For the formulation of the health food, a food pharmaceutically acceptable carrier or additive may be used, and any carrier or additive known to be usable in the art may be used to prepare a formulation to be prepared. As the additive, various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, used in carbonated beverages It may contain a carbonation agent and the like.

The dietary the far Ennis captive Sar key extractor Squeal Ria room (Paenisporosarcina quisquiliarum) content of 17mud 1-1541 cells in foods may be determined as appropriate depending on the purpose of use (prevention or amelioration). In general, it may be contained in an amount of 0.01 to 15% by weight of the total food weight, and as an example, when a beverage is prepared, it may be contained in an amount of 0.02 to 10 g based on 100 mL.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely describe the present invention to those of ordinary skill in the art.

<Example 1> Isolation and identification of strains

1. Sample collection and separation of strains

Mud samples were collected from the coastal area of Boryeong, Chungcheongnam-do. The collected mud samples were subjected to a pretreatment process of irradiating gamma rays at a dose of 3 kGy using a ^{60 Co gamma irradiator (point source AECL, IR-79) before strain isolation.} A 50 mL saline (0.85% NaCl) solution was added to the gamma-ray-treated soil, sufficiently settled, stirred sufficiently, and diluted with a 10-fold volume of saline.

The diluted aliquot of 100 μL was spread on R2A Agar (Difco. Co. USA) medium, and the process of forming a colony by standing culture at 25° C. for a week was repeated twice. The strain was isolated from the three colonies thus formed, stored in 20% (w/v) glycerol, and frozen at -80°C for use.

2. Identification of strains

The isolated genomic DNA of the strain was isolated according to the manufacturer's protocol of a commercially available DNA extraction kit (Solgent), and the 16S rRNA gene was PCR amplified using 9F/1492R using the genomic DNA as a template (Weisburg et al. , 1991). The amplified PCR product was purified, and the purified DNA was sequenced using universal bacterial primer sets 9F, 518F, 785F and 800R, and the 16S rRNA gene sequence was determined using the SeqMan software (DNASTAR) program. 1 shows the 16S rRNA gene nucleotide sequence of this strain.

The analyzed sequence was identified by the global alignment analysis method using the EzTaxon program to identify the closest strain isolated to date, and a phylogenetic tree was created by collecting the adjacent sequences. When calculating the phylogenetic tree, the minimum-evolution and maximum-likelihood algorithms were used, if necessary, based on the neighbor-joining provided by the MEGA6 program. Figure 2 shows a phylogenetic tree made by analysis of the rRNA gene sequence of the 17mud 1-1541 strain of Paenisporosarcina Quisquilia room.

As a result, the strain of the present invention was analyzed as an unrecorded strain belonging to the genus Paenisporosarcina sp. Accordingly, the present inventors have identified the strain as a domestically grown Paenisporosarcina Quisquiliarum strain, and Paenisporosarcina Quisquiliarum 17mud 1-1541 As the initial excavation was named, it was deposited with the depository institution KCTC (Korea Research Institute of Bioscience and Biotechnology Biological Resources Center) on June 17, 2019 (accession number KCTC13860BP).

3. Physicochemical analysis of the isolated strain

For the mycological properties and physicochemical analysis of the isolated strains, first, the Gram staining reaction was performed by a traditional Gram staining reaction according to the method of Doetsch (1981). In addition, the morphological characteristics of the strain were observed at 1,000 times magnification using an optical microscope (Olympus BX51). In addition, the motility of the strain was 25 ℃, after culturing the strain for 3 days on the R2A agar medium, TEM (Transmision Electron Microscope) was used.

The 17mud 1-1541 strain is aerobic, gram-positive, and motile, grows stably under a temperature condition of 25°C, and is characterized by resistance to gamma rays at a dose of 3 kGy. In addition, it showed an oval strain shape, formed opaque white colonies, and the G+C base content of genomic DNA was found to be 46.0 (mol%).

The 17mud 1-1541 strain of the present invention, which was identified as above and analyzed for physicochemical properties, was confirmed to have skin whitening and wound healing effects through the following examples.

<Example 2> Whitening effect of 17mud 1-1541 strain

In order to confirm the whitening effect on the cells of the 17mud 1-1541 strain according to the present invention, the effect of reducing melanin, inhibiting tyrosinase activity, and inhibiting alpha-glycosidase activity were confirmed.

1. Whitening effect

1) Confirmation of pigmentation and melanin reduction effect

In order to investigate the pigmentation and melanin reduction effect of the 17mud 1-1541 strain according to the present invention, B16F10 cells, which are melanoma cells, were treated with 100 nM of α-melanocyte stimulating hormone (α-MSH) for 48 hours. Stimulated. At this time, the concentration range of 17mud 1-1541 strain was used as 10 μg/ml, 50 μg/ml, and 100 μg/ml without cytotoxicity. B16F10 cells were washed with PBS (phosphated buffer saline), and the cells were recovered using a scraper. The recovered cells were counted using a hematocytometer, centrifuged at 1,000 rpm for 3 minutes, and then the supernatant was removed to obtain a pellet. The obtained pellet was photographed and shown in Fig. 3(a), and it was confirmed that the 17mud 1-1541 strain according to the present invention had a pigmentation effect together with arbutin.

In the cell pellet thus obtained, 200 μl of 1N sodium hydroxide solution was added and dissolved in a 60° C. thermostat to obtain intracellular melanin. With this solution, the absorbance was measured at 490 nm with a microplate reader, and the amount of melanin per a certain number of cells was calculated and shown in Fig. 3(b). Here, as a control, arbutin, which is known to inhibit melanin production, was used.

As a result of FIG. 3(b), it was found that the 17mud 1-1541 strain according to the present invention has superior melanin production inhibitory effect than arbutin, and it can be seen that the whitening effect is superior to the 17mud 1-1541 strain arbutin.

2) Confirmation of tyrosinase activity

In order to investigate the tyrosinase activity of the 17mud 1-1541 strain according to the present invention, B16F10 cells, which are melanoma cells, were treated with 100 nM of α-melanocyte stimulating hormone (α-MSH) for 48 hours. Stimulated. At this time, the concentration range of 17mud 1-1541 strain was used as 10 μg/ml, 50 μg/ml, and 100 μg/ml without cytotoxicity. B16F10 cells were washed with PBS (phosphated buffer saline), and the cells were recovered using a scraper. The recovered cells were centrifuged at 1,000 rpm for 3 minutes, and then the supernatant was removed to obtain a pellet. The cell pellet was pulverized using a lysis buffer, and then centrifuged at 15,000 rpm for 20 minutes to collect the supernatant. After quantifying this solution by the BCA protein assay, 40 μg of this solution and 10 mM L-DOPA were added. The plate to which all the reaction solutions were added was subjected to a dark reaction at 37° C. for 1 hour, and then absorbance was measured at 475 nm, and the tyrosinase activity was obtained and shown in FIG. 3(c). Here, arbutin was used as a control. As a result of FIG. 3(c), it was confirmed that the 17mud 1-1541 strain according to the present invention reduced tyrosinase activity.

2. Confirmation of tyrosinase activity inhibition effect

In accordance with the guidelines provided by the Ministry of Food and Drug Safety, the inhibition of tyrosinase activity was confirmed by the in vitro tyrosinase activity inhibition test method. At this time, the concentration range of 17mud 1-1541 strain was used as 10 μg/ml, 50 μg/ml, and 100 μg/ml without cytotoxicity. 100 μL of 175 mM sodium phosphate buffer (pH 6.5), 20 μL of sample solution, 40 μL of mushroom tyrosinase solution (110 U/mL), and 40 μL of 1.5 mM L-tyrosine were added in order. As shown in Figure 4 (a), it was confirmed that the 17mud 1-1541 strain according to the present invention inhibits tyrosinase activity.

3. Confirmation of the effect of inhibiting alpha-glycosidase activity

In order to investigate the inhibitory effect of the alpha-glycosidase activity of the 17mud 1-1541 strain according to the present invention, an experiment using 10 μg/ml, 50 μg/ml, and 100 μg/ml concentrations of the 17mud 1-1541 strain without cytotoxicity Was implemented. 10 μL of the sample solution diluted by concentration and 90 uL of 1 U/mL α-glucosdiase were added to the test tube and reacted at 37° C. for 15 minutes, and then 100 uL of 1 mM p-NPG was further added and reacted for 5 minutes. The absorbance was measured at. As shown in FIG. 4(b), it was confirmed that the 17mud 1-1541 strain inhibited alpha-glycosidase activity.

<Example 3> Wound healing effect of 17mud 1-1541 strain

In order to confirm the wound healing effect on the cells of the 17mud 1-1541 strain according to the present invention, a wound recovery ability test was performed on HaCaT cells, which are skin keratinocytes.

1. Confirmation of wound closing effect on keratinocytes

After physically wounding HaCaT cells, which are skin keratinocytes, with a pipette tip, 17mud 1-1541 strain 10 μg/ml, 17mud 1-1541 strain 50 μgml, 17mud 1-1541 strain 100 μg/ml, Znpp 20 μg/ml, Znpp 20 μg/ml and 17mud 1-1541 strain 100 μg/ml were added and the degree of closure of the wound was calculated every 12 hours for 24 hours. After measuring the length of the wound for 0, 12, and 24 hours, the degree of wound closure was calculated as a percentage compared to the 0 hour for each group.

As a result of Figure 5 (a), it was confirmed that the 17mud 1-1541 strain according to the present invention has a wound closing effect.

2. Confirmation of heme oxygenase-1 (HO-1) activity involvement

After physically wounding HaCaT cells, keratinocytes, with a plastic pipette tip, 17mud 1-1541 strain 10 μg/ml, 17mud 1-1541 strain 50 μg/ml, 17mud 1-1541 strain 100 μg/ml, Znpp 20 μg /ml, Znpp 20 μg/ml and 17mud 1-1541 strain 100 μg/ml were added and the degree of closure of the wound was calculated every 12 hours for 24 hours. After measuring the length of the wound for 0, 12, and 24 hours, the degree of wound closure was calculated as a percentage compared to the 0 hour for each group.

As a result of FIG. 5(b), it was confirmed that the wound closing effect was more excellent when the 17mud 1-1541 strain was treated as compared to Znpp. Through this, it can be predicted that the 17mud 1-1541 strain according to the present invention may be involved in the inhibition of HO-1 activity like Znpp.

<Example 4> Cytotoxicity measurement (MTT assay)

In order to confirm the irritation to the cells of the 17mud 1-1541 strain according to the present invention, a cytotoxicity test was performed on B16F10 cells, which are melanocytes, and HaCaT cells, which are skin keratinocytes.

1. Toxicity test using melanocyte B16F10 cells

B16F10 cells (2x10⁴ cells/well), which are melanogenic cells, were dispensed into a 96-well plate, and then cultured for 24 hours or 48 hours in an environment of _{37°C and 5% CO 2.} Then, the cultured cells were treated with 10 μg/ml, 50 μg/ml, and 100 μg/ml of 17mud 1-1541 strain, respectively, for 24 hours or 48 hours. After incubation for 24 hours or 48 hours, the supernatant was removed, and the MTT (formazan) solution was treated and incubated in an environment of _{37° C. and 5% CO 2 for 4 hours.} Absorbance was measured at 540 nm using an ELISA Reader (BioTek). Cell viability was calculated using Equation 1 below in comparison with the treated group (untreated group) not treated with the sample.

[Equation 1]

Cell viability (%) = (absorbance of the sample treated group / absorbance of the untreated group) X 100

As a result of FIGS. 6(a) and 6(b), 17mud 1-1541 strains according to the present invention showed little overall cytotoxicity. These results prove that the 17mud 1-1541 strain according to the present invention can be used in various ways as therapeutic agents, cosmetics, health functional foods, etc. without side effects.

2. Toxicity test using keratinocyte HaCaT cells

Cytotoxicity experiments were performed in the same manner, except that skin keratinocytes HaCaT cells were used instead of melanocytes B16F10 cells.

As a result of FIG. 6(c), it was confirmed that the 17mud 1-1541 strain according to the present invention had a high cell viability of 85% or more, and was quite stable to cells.

<Example 5> Free radical scavenging effect

_{DPPH and hydrogen peroxide (H 2} O ₂ ) radical scavenging ability was confirmed to determine whether the 17mud 1-1541 strain according to the present invention is capable of preventing skin aging due to active oxygen having free radicals.

1. Measurement of antioxidant activity using DPPH

To confirm the DPPH radical scavenging ability of the 17mud 1-1541 strain according to the present invention, specifically 100 μL of the 17mud 1-1541 strain diluted by concentration in a test tube and 100 μl of 99% ethyl alcohol, 0.4 mM DPPH (2,2-diphenyl After adding 100 μl of -1-picrylhydrazyl) solution, it was allowed to stand in a dark room at room temperature for 30 minutes, and the absorbance was measured at 517 nm. In addition, DPPH radical scavenging ability was calculated according to Equation 2 below, and ascorbic acid was used as a positive control.

[Equation 2]

Radical scavenging ability (%) = (absorbance without sample-absorbance with sample)/absorbance without sample x 100

As a result of FIG. 7(a), it was confirmed that the 17mud 1-1541 strain according to the present invention had no effect on radical scavenging ability when compared with L-ascorbic acid, which is widely known as a natural antioxidant.

2. Measurement of antioxidant activity using hydrogen peroxide

_{Hydrogen peroxide (H 2} O ₂ ) radical scavenging activity was confirmed in the same manner as the DPPH radical scavenging activity. 17mud 1-1541 diluted by concentration in a test tube 20 μl of the strain, 100 μl of phosphate buffer, and 20 μl of 1.0 MH ₂ O ₂ were added, and then reacted at 37° C. for 5 minutes. After adding 30 μl of 1.25 mM ABTS and 30 μl of 1 U/mL peroxidase, the reaction was further performed at 37° C. for 10 minutes, and the absorbance was measured at 405 nm.

As a result of FIG. 7(b), it was confirmed that the 17mud 1-1541 strain according to the present invention had no effect on _{hydrogen peroxide (H 2} O _{2) radical scavenging ability.} Therefore, it was confirmed that the 17mud 1-1541 strain according to the present invention had no antioxidant effect.

The novel Paenisporosarcina quisquiliarum (Paenisporosarcina quisquiliarum) 17mud 1-1541 strain according to the present invention inhibits melanin production and tyrosinase and alpha-glycosidase activity, thereby inhibiting pigmentation, thereby exhibiting excellent whitening effect, and It has excellent stability against the skin and has no side effects on the skin, and is very useful in improving spots and freckles, skin whitening, and further healing wounds, and thus can be widely used as a composition for improving skin.

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it will be apparent that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Korea Research Institute of Bioscience and Biotechnology KCTC13860BP 20190617

<110> Seoul Women's University Industry-University Cooperation Foundation <120> Novel Paenisporosarcina quisquiliarum 17mud 1-1541T strain and its use <130> ADP-2019-0110 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1482 <212> DNA <213> Artificial Sequence <220> <223> 16S rRNA of Paenisporosarcina quisquiliarum 17mud 1-1541T <400> 1 tagacttcac cccaatcatc tgtcccacct tcggcggctg gctccaaaaa ggttacctca 60 ccgacttcgg gtgttacaaa ctctcgtggt gtgacgggcg gtgtgtacaa ggcccgggaa 120 cgtattcacc gcggcatgct gatccgcgat tactagcgat tccggcttca tgtaggcgag 180 ttgcagccta caatccgaac tgagaacgat tttatgggat tggctccccc tcgcgggttt 240 gcagcccttt gtatcgtcca ttgtagcacg tgtgtagccc aggtcataag gggcatgatg 300 atttgacgtc atccccacct tcctccggtt tgtcaccggc agtcacctta gagtgcccaa 360 ctaaatgctg gcaactaaga tcaagggttg cgctcgttgc gggacttaac ccaacatctc 420 acgacacgag ctgacgacaa ccatgcacca cctgtcacca ctgtccccga agggaaaagc 480 atatctctat gccggtcagt gggatgtcaa gacctggtaa ggttcttcgc gttgcttcga 540 attaaaccac atgctccacc gcttgtgcgg gcccccgtca attcctttga gtttcagcct 600 tgcggccgta ctccccaggc ggagtgctta atgcgttagc tgcagcacta aggggcggaa 660 accccctaac acttagcact catcgtttac ggcgtggact accagggtat ctaatcctgt 720 ttgctcccca cgctttcgcg cctcagcgtc agttacagac cagaaagccg ccttcgccac 780 tggtgttcct ccacatctct acgcatttca ccgctacacg tggaattccg ctttcctctt 840 ctgtactcaa gtcccccagt ttccaatgac cctccacggt tgagccgtgg gctttcacat 900 cagacttaaa ggaccgcctg cgcgcgcttt acgcccaata attccggaca acgcttgcca 960 cctacgtatt accgcggctg ctggcacgta gttagccgtg gctttctaat aaggtaccgt 1020 caaggtacga gcagttactc tcgtacgtgt tcttccctta caacagagtt ttacgatccg 1080 aaaaccttct tcactcacgc ggcgttgctc catcagactt tcgtccattg tggaagattc 1140 cctactgctg cctcccgtag gagtctgggc cgtgtctcag tcccagtgtg gccgatcacc 1200 ctctcaggtc ggctacgcat cgtcgccttg gtaggccatt accctaccaa ctagctaatg 1260 cgccgcgggc ccatctcgta gtgacagccg aagccgcctt ttaacatctc gccatgaagc 1320 aaaatggatt attcggtatt agccccggtt tcccggagtt atcccaatct acaaggtagg 1380 ttgcccacgt gttactcacc cgtccgccgc taaaatcaga agaagcaagc ttcttcatca 1440 ttccgctcga cttgcatgta ttaggcacgc cgccagcgtt cg 1482

Claims (16)

Paenisporosarcina Quisquiliarum (Paenisporosarcina quisquiliarum) 17mud 1-1541 strain, deposited with accession number KCTC13860BP.
The method of claim 1,
The strain is a strain, characterized in that isolated from mud.
The method of claim 1,
The strain is a strain, characterized in that having the 16S rRNA nucleotide sequence of SEQ ID NO: 1.
delete The method of claim 1,
The strain is characterized in that it has a skin whitening and wound healing effect.
The method of claim 1,
The strain is a strain characterized in that the inhibition of tyrosinase (tyrosinase) activity or alpha-glucosidase (α-glucosidase) activity.
The method of claim 1,
The strain, characterized in that it has a wound-closing effect in skin keratinocytes.
The method of claim 1,
The strain is a strain characterized in that inhibiting the activity of heme oxygenase (Heme oxygenase-1; HO-1).
A pharmaceutical composition for the prevention or treatment of skin whitening or pigmentation disorders, comprising the strain according to any one of claims 1 to 3, and 5 to 8 or a culture thereof as an active ingredient .
The method of claim 9,
The pharmaceutical composition is used as a composition of any one external preparation for skin selected from the group consisting of creams, gels, ointments, skin emulsifiers, skin suspensions, transdermal patches, drug-containing bandages, and lotions. A pharmaceutical composition for the prevention or treatment of deposition disorders.
A cosmetic composition for preventing or improving skin whitening or pigmentation disorders, characterized in that it comprises the strain according to any one of claims 1 to 3, and 5 to 8 or a culture thereof as an active ingredient.
A health functional food for preventing or improving skin whitening or pigmentation diseases, characterized in that it comprises the strain according to any one of claims 1 to 3, and 5 to 8 or a culture thereof as an active ingredient .
A pharmaceutical composition for wound healing comprising the strain according to any one of claims 1 to 3, and 5 to 8 or a culture thereof as an active ingredient.
The method of claim 13,
The pharmaceutical composition is used as a composition of any one external preparation for skin selected from the group consisting of creams, gels, ointments, skin emulsifiers, skin suspensions, transdermal patches, drug-containing bandages, and lotions. Composition.
A cosmetic composition for wound improvement comprising the strain according to any one of claims 1 to 3, and 5 to 8 or a culture thereof as an active ingredient.
A health functional food for wound improvement comprising the strain according to any one of claims 1 to 3, and 5 to 8 or a culture thereof as an active ingredient.

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