KR101879852B1 - Novel Streptomyces sp. strain and use thereof - Google Patents

Abstract

According to an embodiment of the present invention, provided is a novel specified Streptomyces sp. strain isolated from soil and a use thereof. According to an embodiment of the present invention, various whitening effect materials, produced by the specified Streptomyces sp. strain, prevent the generation of melanin or inhibit activity of tyrosinase, and have a more profound effect than arbutin, a commercially available and effective whitening material. Therefore, according to an embodiment of the present invention, the specified Streptomyces sp. strain, a culture medium of the specified Streptomyces sp. strain, or a culture medium extract of the specified Streptomyces sp. strain can be used as an active ingredient of a whitening composition.

Description

A novel strain of Streptomyces sp. And its use {Novel Streptomyces sp. strain and use thereof}

The present invention relates to a novel Streptomyces sp. Strain, and its use, and more particularly, to a strain of Streptomyces sp. Isolated from soil and having different mycological properties from conventional strains and a whitening composition using the same.

Melanin, which determines the color of a person's skin, is made in skin cells called melanocytes and then transferred to epidermal cells called keratinocytes. Here, melanin forms a hat-like structure around the nucleus, which protects genes from ultraviolet rays and eliminates free radicals, thereby protecting the intracellular proteins. In vivo, there is no enzymes that degrade melanin, but when keratinocytes fall off the epidermis, they are removed from the skin along with melanin. However, when melanin is produced more than necessary, it induces hypercholesterolemia such as spots, freckles, and dots, resulting in poor cosmetic results. In addition, as the number of people enjoying outdoor activities increased due to the increase in the leisure population, the demand for preventing melanin pigmentation caused by ultraviolet rays was further increased. Therefore, it has become necessary to develop a whitening agent that prevents excessive melanin production, and many efforts have been made.

The development of whitening agents has consisted primarily of finding substances that reduce the amount of melanin by inhibiting the activity of tyrosinase, an essential and essential enzyme essential for melanin biosynthesis. There are kojic acid, arbutin, glutathione, vitamin A and vitamin C, but consumers do not have a satisfactory whitening effect and the usage is limited due to side effects. Therefore, it is urgent to develop a whitening agent that is more effective than the whitening agent so far and safer.

Regarding the whitening agent derived from natural products, Korean Patent Registration No. 10-1072641 discloses a skin whitening composition containing Streptomyces subrutilus strain or a culture extract of the strain as an active ingredient . Korean Patent Registration No. 10-1167606 discloses a cosmetic composition for skin whitening comprising a fermentation broth obtained by fermenting Yedoaceae with a microorganism selected from the group consisting of yeast, lactic acid bacteria, Bacillus, and a mixture thereof. . Korean Patent Registration No. 10-1207562 discloses a cosmetic composition for skin whitening comprising an extract of mist tree as an active ingredient.

The present invention has been made in view of the background of the prior art, and it is an object of the present invention to provide a novel strain of Streptomyces sp. That produces a variety of whitening-efficacious substances having different mycological properties from those of conventional strains. It is also an object of the present invention to provide a novel strain of Streptomyces sp.

The inventors of the present invention have screened various actinomycetes capable of producing a whitening-efficacious substance from the soil, and found that a certain Streptomyces sp. Strain produces a whitening-effect substance having more efficacy than arbutin, which is a commercial whitening effect substance And completed the present invention.

In order to solve the above object, an example of the present invention provides Streptomyces longisporus MBB1001 strain (accession number: KACC 92136P).

In order to solve the above object, an example of the present invention provides a culture medium of Streptomyces longisporus MBB1001 strain (accession number: KACC 92136P).

In order to solve the above object, an example of the present invention provides a culture extract of Streptomyces longisporus MBB1001 strain (accession number: KACC 92136P).

In order to solve the above object, another aspect of the invention is Streptomyces long Kish porous (Streptomyces longisporus) MBB1001 strain (accession number: KACC 92136P), whitening, including its culture, their debris or its extract as an active ingredient Lt; / RTI >

In order to solve the above object, an example of the present invention is a culture of Streptomyces longisporus MBB1001 strain (accession no. KACC 92136P) or a strain of Streptomyces longisporus MBB1001 : KACC 92136P) as an active ingredient.

The various whitening effect substances produced in a specific Streptomyces sp. Strain according to an embodiment of the present invention are more effective than albutin (Arbutin), which inhibits the production of melanin or inhibits the activity of tyrosinase and is a commercial whitening agent. Therefore, a specific Streptomyces sp. Strain, a culture solution of a Streptomyces sp. Strain or a culture solution extract of a Streptomyces sp. Strain according to an example of the present invention can be used as an effective ingredient of a whitening composition.

Figure 1 is a phylogenetic tree of MBB1001 strain.
Figure 2 shows the morphological characteristics of MBB1001 and reference strains.
3 is a schematic diagram showing a step of isolating, culturing and producing an extract from MBB1001 strain.
4 is a graph showing the cytotoxicity measurement results of the samples SM-I-1 and SM-I-2.
Fig. 5 is a photograph showing the results of measurement of the ability of the sample SM-I to inhibit melanin formation, Fig. 6 is a photograph showing the results of measurement of the ability of the samples SM-I-1 and SM- FIG. 8 is a graph showing the results of measuring the inhibition of tyrosinase activity of samples SM-I-1 and SM-I-2.
9 is a photograph showing the results of measuring the ability of the samples SM-I-1-1-1 to SM-I-1-1-10 (ten fractions obtained through the second fraction in the example of the present invention) to inhibit melanin formation .
Fig. 10 shows the results of measurement of the ability of the samples SM-I-1-1-4-1 to SM-I-1-1-4-8 (eight fractions obtained from the third fraction in the example of the present invention) FIG.
Fig. 11 is a graph showing the distribution of melanin in samples SM-I-1-1-4-8-1 to SM-I-1-1-4-8-9 (nine fractions obtained from the fourth fraction in the example of the present invention) This is a photograph showing the results of measurement of inhibition of production.

Hereinafter, terms used in the present invention will be described.

The term "culture product " used in the present invention means a product obtained by culturing a microorganism in a known liquid medium or solid medium, and includes a microorganism.

The term "culture liquid " used in the present invention means a product obtained by culturing a microorganism in a known liquid medium or solid medium, and does not include microorganisms.

The term "composition" used in the present invention means a material in which two or more components are uniformly mixed, and includes not only an article but also an intermediate material for producing an article.

As used herein, the term "pharmaceutically acceptable" means not significantly stimulating an organism and not interfering with the biological activity and properties of the administered active substance.

As used herein, the term "prophylactic " means any act that inhibits the symptoms of a particular disease or delays its progress by administration of the composition of the present invention.

The term "treatment" as used herein refers to any action that improves or alleviates the symptoms of a particular disease upon administration of the composition of the present invention.

As used herein, the term "improvement" means any action that at least diminishes or alleviates the parameters associated with the condition being treated, for example, the severity of the condition.

The term "administering" as used herein is meant to provide any desired composition of the invention to an individual by any suitable method. The term " individual " means any animal such as a human, a monkey, a dog, a goat, a pig, or a mouse having a disease in which symptoms of a specific disease can be improved by administering the composition of the present invention.

The term " pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit or risk rate applicable to medical treatment, including the type of disease, severity, activity of the drug, The time of administration, the route and rate of excretion of the drug, the duration of the treatment, factors including drugs used simultaneously and other factors well known in the medical arts.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention relates to a novel strain of Streptomyces sp. Which is industrially very useful.

Streptomyces longisporus MBB1001 strain (accession number: KACC 92136P) is a novel strain of Streptomyces sp . Which is isolated from soil and produces various whitening effect substances. Streptomyces longisporus MBB1001 strain (Accession No .: KACC 92136P) is preferably 16S rDNA and comprises the nucleotide sequence shown in SEQ ID NO: 1. The Streptomyces longisporus strain MBB1001 (accession number: KACC 92136P) is preferably a carbon source and is selected from the group consisting of glucose, mannitol, galactose, inositol and maltose, (maltose) is used. The strain Streptomyces longisporus MBB1001 (accession number: KACC 92136P) is preferably resistant to starch and casein but is resistant to tyrosine and xanthine, The resolution is not shown.

One aspect of the present invention relates to various uses of novel strains of Streptomyces sp.

An example of the present invention provides a culture of Streptomyces longisporus strain MBB1001 (accession number: KACC 92136P) as a novel strain of Streptomyces sp . The culture liquid may have various effects such as whitening, and may be used as a material for medicines, foods, cosmetics and the like. In the present invention, the culture liquid is a liquid product obtained by culturing a predetermined strain in a culture medium and then removing the cells through centrifugation or the like. The culture medium may be selected from known liquid culture media or solid culture media used for actinomycetes culture, For example, GYSM medium, Humic acid agar medium, or Bennett's agar medium.

In addition, an example of the present invention provides a culture extract of Streptomyces longisporus MBB1001 strain (accession number: KACC 92136P) as a novel strain of Streptomyces sp . The culture medium extract may have various effects as well as whitening depending on the extraction solvent or extraction method, and may be used as a material for medicines, foods, cosmetics and the like. The culture broth extract may be prepared by a conventional extraction method known in the art, for example, a solvent extraction method. The extraction solvent used in the solvent extraction may be water, a lower alcohol having 1 to 4 carbon atoms (for example, methanol, ethanol, propanol and butanol), or a mixture thereof, a lower alcohol such as propylene glycol, The solvent may be selected from the group consisting of glycols, glycerin, acetone, diethyl ether, ethyl acetate, butyl acetate, dichloromethane, chloroform, hexane and mixtures thereof, and water, alcohols, hydrated alcohols, diethyl ether, , Butyl acetate, chloroform or hexane. When a functional alcohol is used as the extraction solvent, the alcohol content is preferably 50 to 90%, more preferably 60 to 85%. On the other hand, it is obvious to those skilled in the art that a culture medium extract exhibiting substantially the same effect can be obtained by using not only the above-mentioned extraction solvent but also another extraction solvent. In addition, the above-mentioned culture medium extract can be obtained not only by the above-described extract by the extraction solvent, but also by other conventional extraction methods. For example, extraction with supercritical fluid extraction using carbon dioxide, extraction with extraction using ultrasound, separation using an ultrafiltration membrane with a constant molecular weight cut-off value, or various chromatography (size, charge, Such as separation by centrifugation, centrifugation, centrifugation, centrifugation, centrifugation, centrifugation, centrifugation, centrifugation, centrifugation, hydrophobicity, or affinity separation.

One example of the present invention is a strain of Streptomyces longisporus MBB1001 (Accession No .: KACC 92136P), a culture thereof, a crushed product thereof or an extract thereof as an active ingredient for use as a novel strain of Streptomyces sp . ≪ / RTI > Streptomyces longisporus MBB1001 strain (accession number: KACC 92136P) has a whitening effect by inhibiting the production of melanin or inhibiting the activity of tyrosinase when cultured. In the present invention, the culture is a product obtained by culturing a predetermined strain in a culture medium. The culture medium may be selected from known liquid culture media or solid culture media used for actinomycetes culture. Examples of the culture media include GYSM medium, Humic acid agar medium, or Bennett's agar medium.

Further, still another aspect of the invention is a novel Streptomyces for purposes of my process in the strain Streptomyces long Kish porous (Streptomyces longisporus) MBB1001 strain (accession number: KACC 92136P) culture or Streptomyces long Kish porous (Streptomyces longisporus) MBB1001 of (Accession number: KACC 92136P) as an active ingredient. The culture medium extract used as an active ingredient of the whitening composition has a whitening effect by inhibiting the production of melanin or inhibiting the activity of tyrosinase. The culture broth extract used as an active ingredient of the whitening composition is preferably an alkyl acetate soluble fraction of the culture broth. The alkyl acetate may have an alkyl group having 1 to 5 carbon atoms, and may be selected from methyl acetate, ethyl acetate, propyl acetate or butyl acetate. The culture medium extract used as an active ingredient of the whitening composition is more preferably a secondary fraction obtained by primary fractionating the culture with alkyl acetate and secondary fractionating the primary fraction with methanol or hydrated methanol. The methanol concentration of the hydrous methanol to obtain the second fraction may be selected from 80 to 95% (w / w).

In the present invention, the whitening composition can be used for preventing, ameliorating or treating a symptom or a disease in which the skin or the like turns black. In addition, the whitening composition according to the present invention may be formulated into a pharmaceutical composition, a cosmetic composition, a personal care product, and the like depending on the intended use or aspect, and the content of the active ingredient in the composition may also be a specific form, And may be adjusted in various ranges depending on the intended use or aspect.

The content of the active ingredient in the pharmaceutical composition according to the present invention is not particularly limited and is, for example, 0.01 to 99% by weight, preferably 0.5 to 50% by weight, more preferably 1 to 30% by weight, Lt; / RTI > In addition, the pharmaceutical composition according to the present invention may further contain, in addition to the active ingredient, an additive such as a pharmaceutically acceptable carrier, excipient or diluent. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical composition of the present invention can be formulated into a formulation for oral administration or parenteral administration by a conventional method, and can be formulated into a pharmaceutical composition such as a filler, an extender, a binder, a wetting agent, a disintegrant, Diluents or excipients. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions and syrups. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used. Further, it can be suitably formulated according to each disease or ingredient, using appropriate methods in the art or by the method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA. The pharmaceutical composition of the present invention may be administered orally or parenterally to a mammal including a human according to a desired method. Examples of the parenteral administration method include external dermal application, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravenous injection, Intravenous injection or intra-thoracic injection. The dosage of the pharmaceutical composition of the present invention is not limited as long as it is a pharmacologically effective amount and is not limited as long as it depends on the body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, Varies. The typical daily dose of the pharmaceutical composition of the present invention is not particularly limited, and is, for example, in the range of 1 to 3000 占 퐂 / kg, preferably 10 to 2000 占 퐂 / kg on the basis of the active ingredient, 20 to 1000 占 퐂 / kg. In addition, the pharmaceutical composition of the present invention can be administered once a day or divided into several times. The pharmaceutical composition according to the present invention is preferably prepared in the form of a skin external preparation such as a cream, a gel, a patch, a spray, a research agent, a warning agent, a lotion, a liniment, a pasta agent or a cataplasma agent Do.

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used in the form of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. Specifically, the cosmetic composition of the present invention can be prepared in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder. When the formulation of the cosmetic composition of the present invention is a paste, a cream or a gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide Can be used. When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid esters of sorbitan. When the formulation of the cosmetic composition of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester , Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used. When the formulation of the cosmetic composition of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. Especially, in the case of a spray, Propellants such as carbon, propane / butane or dimethyl ether. When the formulation of the cosmetic composition of the present invention is a surfactant-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, Fatty acid amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester. The cosmetic composition according to the present invention can be used as a skin tonic, skin conditioner, skin essence, skin lotion, skin nutrition lotion, skin cream, skin nutrition cream, skin moisturizing cream, skin massage cream, skin pack, , A soap, a body cleanser, and the like.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to clearly illustrate the technical features of the present invention and do not limit the scope of protection of the present invention.

1. Isolation and screening of new strains

1 g of the soil collected in Korea is pretreated at 50 ° C for 30 minutes and then suspended in 10 ml of physiological saline. The suspension is suspended in a humic acid agar medium (composition: Humic acid 1 g, Na ₂ HPO ₄ 0.5 g, KCl , 0.5 g of MgSO ₄ , 0.01 g of FeSO ₄ , 0.02 g of CaCO ₃ , 1 mg of B-vitamin, 18 g of agar, 1 L of distilled water, pH 7.0) and incubated at 28 ° C for about 7 days to 14 days. Then, candidate strains forming a single colony from the culture were separated, cultured on the medium, and then the strain was purified. Then, the final strain was selected based on the whitening efficacy of the purely isolated strains, and the strain name was designated as "MBB1001 ".

2. MBB1001  Identification of the strain

(1) Analysis of 16S rDNA sequence

The 16S rDNA sequence was firstly analyzed for the identification of MBB1001 strain. Specifically, MBB1001 strain was cultured for 7 days on Bennett's agar medium (composition: glucose 10 g, Bacto peptone 2 g, Yeast extract 1 g, Beef extract 1 g, agar 15 g, distilled water 1 L), and MBB1001 strain colonies were dissolved in a liquid Bennett's medium . After the cells were cultured for 24 hours, genomic DNA was extracted using Promega Wizard Genomic DNA purification kit. Then, PCR was performed using universal primer 27F (5'-AGA GTT TGA TCM TGG CTC AG-3 ') and 1492r (5'-TAC GGY TAC CTT GTT ACG ACT T-3' 16S rDNA gene was amplified. The nucleotide sequence analysis of the obtained PCR product was carried out with the request of Biopharmaceuticals Co., As a result, the 16S rDNA sequence of the MBB1001 strain was confirmed to have the nucleotide sequence of SEQ ID NO: 1. Thereafter, the 16S rDNA sequence of the MBB1001 strain was identified by the BLAST search of Genebank (http://www.ncbi.nlm.nih.gov/). As a result, strains having the same 6S rDNA sequence were not found. Streptomyces long And was found to have 97.7% homology with the strain Streptomyce logisporus . Then, the 16S rDNA sequence of the strain related to MBB1001 strain was downloaded from NCBI and the phylogenetic tree of MBB1001 strain was completed using MEGA 6 software (Tamura et al. 2013). Figure 1 is a phylogenetic tree of MBB1001 strain.

(2) Morphological characterization

In order to compare morphological characteristics of MBB1001 and reference strains, each strain was cultured in International Streptomyces Project (ISP) medium [Reference: EB Shirling and D. Gottlieb, 1964, Methods for characterization of Streptomyces species. The cells were cultured at 28 ° C for 7 to 14 days, and then growth of cells, formation of mycelia, pigment production and sporulation were observed. Figure 2 shows the morphological characteristics of MBB1001 and reference strains. As shown in FIG. 2, the strain MBB1001 showed good growth in the BN medium, ISP2 medium, ISP3 medium, ISP5 medium, ISP6 medium and ISP7 medium, but not in the ISP4 medium. In addition, it was confirmed that MBB1001 strain produced a small amount of melanin pigment in ISP6 medium and ISP7 medium. The MBB1001 strain showed better growth in the BN medium, ISP2 medium and ISP5 medium as compared with the Streptomyce logisporus strain KCCM40531, which is a near reference medium, and the melanin pigment production in the ISP6 medium and ISP7 medium was much less Showed a big difference.

(3) Carbon source availability analysis

In order to analyze the carbon source availability of MBB1001 and reference strains, 1% of various carbon sources were added to the minimum medium and the final medium [Reference: EB Shirling and D. Gottlieb, 1964, Methods for characterization of Streptomyces species. The strain was inoculated with each strain and incubated at 28 ° C for 7 to 14 days, and the growth state was observed. In order to evaluate the availability of carbon sources, a medium supplemented with glucose as a carbon source was used as a positive control, and a medium supplemented with water in place of a carbon source was used as a negative control. The results are shown in Table 1 below. As shown in Table 1, the MBB1001 strain showed the same availability as the reference strain Streptomyce logisporus KCCM40531 in the remaining carbon sources except for cellulose.

Carbon source Strain classification MBB1001 S. longisporus KCCM40519 S. flavidovirens KCCM40519 S. subtilis KCCM40337 S. lavendulae KCCM32799 water - - - - - glucose + + + + + mannitol + + - ± - fructose ± + - + - cellulose ± - - - - 가카성 + + - - - inositol + + + - - maltose + + + + +

In the above Table 1, "+" indicates the same or similar state as the positive control, "-" indicates that the growth state is the same as or close to the negative control, and "±" indicates the state between the positive control and the negative control Lt; / RTI >

(4) Enzymatic Characterization

 The enzymatic characteristics of MBB1001 and reference strains were analyzed by the following methods, and the results of enzymatic characterization of MBB1001 strain and reference strain are shown in Table 2 below.

- xanthine or tyrosine resolving ability: Agar plate was prepared by adding 0.5% xanthine or tyrosine to Bennett's agar medium, and the strain was inoculated and cultured at 28 ° C for 14 days. When xanthine or tyrosine crystals are decomposed and become transparent around the colony of the strain, it is regarded as having resolution.

Casein degradation: Casein agar was prepared with Difco skim milk 5% and agar 2%, and the strain was inoculated and cultured at 28 ° C for 14 days. When a transparent ring is formed around the colony of the strain, it is considered to have casein decomposition ability.

- Starch resolution: Strain was inoculated in a starch resolving medium (Soluble starch 10 g, Difco beef extract 3 g, agar 12 g, distilled water 1 L) and cultured at 28 ° C for 14 days. When a transparent ring is formed around the colony of the strain, it is regarded as having resolution.

temperament Strain classification MBB1001 S. longisporus KCCM40519 S. flavidovirens KCCM40519 S. subtilis KCCM40337 S. lavendulae KCCM32799 xanthine - - + - - tyrosine - + + + + casein + + + + + starch + + + + +

As shown in Table 2, the MBB1001 strain showed casein and starch resolution but no tyrosine and xanthine resolution. On the other hand, reference strain S. longisporus KCCM40531 showed casein, starch, and tyrosine resolution but no xanthine resolution.

(5) Mycological nomenclature and consignment information of MBB1001 strain

As described above, the MBB1001 strain was highly similar to Streptomyces longisporus, which is a actinomycetes. The inventors of the present invention finally named the strain MBB1001 as Streptomyces longisporus MBB1001. The inventors of the present invention have also found that a strain Streptomyces longisporus MBB1001 was cultivated on July 21, 2016 in the Korean Agricultural Culture Collection (KACC, Streptomyces in the name 370) sp. MJMWHT, and granted the accession number KACC 92136P. In addition, the inventors of the present invention have filed the microorganism name of accession number KACC 92136P on Apr. 27, 2017 as Streptomyces sp. The application was made to change from MJMWHT to Streptomyces longisporus MBB1001.

3. MBB1001  Isolation of the strain, culture and preparation of the extract from the culture

3 is a schematic diagram showing a step of isolating, culturing and producing an extract from MBB1001 strain.

Preparation Example 1: Isolation and culture of MBB1001 strain and preparation of culture liquid

As shown in Figure 3, Streptomyces sp. The MBB1001 strain was cultivated in Bennett's agar medium for 7 days and then agar block was cut into 1 cm x 1 cm and 50 ml of GYMS medium (constituent: 4 g of glucose, 4 g of yeast extract, 10 g of malt extract, 10 g of Soytone, In an amount of 1% and cultured at 28 DEG C for 24 hours under shaking at 180 rpm (seed culture). Thereafter, the cultured seeds were inoculated in 500 ml of GYMS medium in an amount of 1%, and shake cultured at 180 rpm for 7 days (main culture) at 28 ° C. Thereafter, the culture was recovered and centrifuged under the condition of 7000 rmm to separate the supernatant of the solid phase and the liquid phase (SM-I; corresponding to the culture solution).

Production Example 2: Preparation of Extract of MBB1001 Culture Medium through the First Fraction

Water and ethyl acetate were added to the supernatant (SM-I) obtained in Preparation Example 1 at a weight ratio of 1: 3, followed by mixing and solvent fractionation. Thereafter, the solvent fraction layer was recovered and concentrated under reduced pressure using a rotary vacuum evaporator (ETELA, Japan), followed by lyophilization to obtain a culture solution extract SM-I-1 corresponding to an ethyl acetate fraction layer and a water fraction layer And the corresponding culture medium extract SM-I-2 was obtained.

Production Example 3: Preparation of Extract of MBB1001 Culture Medium by Secondary Fraction

90% methanol and? -Hexane were added to the culture solution extract SM-I-1 corresponding to the ethyl acetate fraction obtained in Preparation Example 2 at a weight ratio of 1: 3 and mixed to prepare a solvent fraction Respectively. The recovered 90% methanol fraction layer (SM-I-1-1) was dissolved in methanol and subjected to MPLC (medium pressure liquid chromatography) using silica gel 40 (SIL-25,, YMC). Specifically, 10 fractions (SM-I-1-1-1 to SM-I-1-1-10) were eluted with mobile phase solvent under the condition of gradient of methanol and chloroform (0:10 to 7: 3) ≪ / RTI > The concentration gradient conditions of the mobile phase solvent used in the second fraction are shown in Table 3 below.

Time (min) Mobile phase solvent Fraction No. chloroform(%) Methanol (%) 0 100 0 SM-I-1-1-1 to SM-I-1-1-8 60 30 70 70 30 70 SM-I-1-1-9 Washing 0 100 SM-I-1-1-10

Production Example 4: Preparation of Extract of MBB1001 Culture Medium by Third Fraction

The culture broth extract SM-I-1-1-4 obtained in Production Example 3 was further fractionated using a YMC-Pack ODS-A column for prep-HPLC, and 8 fractions (SM-I-1-1-4-1 To SM-I-1-1-4-8). Specific conditions of the third fractionation process are as follows.

* Sample SM-I-1-1-4 Input amount: 200 μl

* Eluent: 55% methanol with 2mM ammonium acetate added

* Dissolution rate: 10 ml / min

* Detector: UV detector (254 nm)

Production Example 5: Preparation of Extract of MBB1001 Culture Medium by Fraction 4

The culture broth extract SM-I-1-1-4-8 obtained in Production Example 4 was further fractionated by using a YMC-Pack ODS-A column for prep-HPLC and 9 fractions (SM-1-1-4 -8-1 to SM-I-1-1-4-8-9). The specific conditions of the fourth fractionation process are as follows.

* Sample: 54 mg of SM-I-1-1-4-8 fraction was dissolved in methanol to make 200 μl,

* Eluent: 80% methanol with 2mM ammonium acetate added

* Dissolution rate: 10 ml / min

* Detector: UV detector (254 nm)

4. MBB1001  Identification of skin whitening efficacy of the culture extract of the strain

(1) Cell culture

B16F10 cells, melanocytes, were cultured in MEM alpha modification medium containing 10% FBS and 1% pen / strep in an incubator at 37 ° C and 5% CO ₂

(2) Cytotoxicity assay (MTT assay)

A B16F10 cells cultured in 96-well plate was inoculated so that 2.0 × 10 ³ / 200㎕ per well. At 24 hours after inoculation, the MBB1001 culture extract samples were treated for 48 hours. The positive control group was also treated with arbutin. Subsequently, 20 μl of the MTT reagent dissolved in PBS at a concentration of 5 mg / ml was dispensed into each well. Thereafter, the cells were reacted for 3 hours in an incubator, the medium was removed, 200 μl of DMSO was dispensed, and the absorbance was measured at 570 nm.

(3) Measurement of inhibition of melanin formation by melanocyte

B16F10 cells were plated at a density of 3.0 × 10 ⁵ cells / well in a 6-well plate and pre-incubated for 24 h at 37 ° C and 5% CO ₂ . And a sample of the culture extract of MBB1001 strain was treated for 48 hours by concentration. The positive control group was also treated with arbutin. Then, the medium was removed, washed with PBS, treated with 0.05% trypsin-EDTA, and the cell pellet recovered. The recovered pellet was transferred to a 1.5 ml eppendorf tube and centrifuged at 13000 rpm for 15 minutes to remove the supernatant. 50 ㎕ of 1 N NaOH solution was added to the pellet from which the supernatant was removed, and the melanin in the cells was dissolved by treatment at 60 캜 for 2 hours. The dissolved melanin was dispensed into a 96-well plate and absorbance was measured at 405 nm.

(4) Inhibition of tyrosinase activity by using melanocyte

B16F10 cells were plated at a density of 3.0 × 10 ⁵ cells / well in a 6-well plate and pre-incubated for 24 h at 37 ° C and 5% CO ₂ . And a sample of the culture extract of MBB1001 strain was treated for 48 hours by concentration. The positive control group was also treated with arbutin. Subsequently, the medium was removed and washed with PBS. 100 μl of RIPA buffer 100 × complex was added to the prepared ICE box, and the mixture was reacted for 30 minutes. Then, the scraper was scraped off and the cell attached to the plate was dropped. The supernatant was centrifuged at 13000 rpm for 15 minutes, and the supernatant was dispensed into a 96-well plate. N, N, Dimethyl formamide, 3,4-Dihydroxy-L-phenylalanine and 3-methyl-2-benzothiazolinone hydrazone were dissolved in sodium phosphate buffer (pH 7.1) After incubation with the supernatant for 30 min., The absorbance was measured at 505 nm.

(5) Experimental results

4 is a graph showing the cytotoxicity measurement results of the samples SM-I-1 and SM-I-2. Both samples SM-I-1 and SM-I-2 showed little cytotoxicity at concentrations of 10 ^-4 ㎍ / ㎖ to 1 ㎍ / ㎖.

Fig. 5 is a photograph showing the results of measurement of the ability of the sample SM-I to inhibit melanin formation, Fig. 6 is a photograph showing the results of measurement of the ability of the samples SM-I-1 and SM- FIG. 8 is a graph showing the results of measuring the inhibition of tyrosinase activity of samples SM-I-1 and SM-I-2.

9 is a photograph showing the results of measuring the ability of the samples SM-I-1-1-1 to SM-I-1-1-10 (ten fractions obtained through the second fraction in the example of the present invention) to inhibit melanin formation .

Fig. 10 shows the results of measurement of the ability of the samples SM-I-1-1-4-1 to SM-I-1-1-4-8 (eight fractions obtained from the third fraction in the example of the present invention) FIG.

Fig. 11 is a graph showing the distribution of melanin in samples SM-I-1-1-4-8-1 to SM-I-1-1-4-8-9 (nine fractions obtained from the fourth fraction in the example of the present invention) This is a photograph showing the results of measurement of inhibition of production.

In Figs. 4 to 11, the unit concentration of the sample treatment is / / ml. As shown in FIGS. 4 to 11, the culture extract of MBB1001 strain showed almost no cytotoxicity and showed better whitening effect than arbutin, a commercial whitening agent.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Therefore, the scope of the present invention should be construed as including all embodiments falling within the scope of the appended claims.

Korea Agricultural Microbiology Resource Center KACC92136P 20160721

<110> MANBANGBIO Co., Ltd. <120> Novel Streptomyces sp. strain and use thereof <130> DP-17-305 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 1391 <212> DNA <213> Unknown <220> <223> 16S rDNA of Streptomyces longisporus MBB1001 strain <400> 1 tgcagtcgaa cgatgaagcc tttcggggtg gattagtggc gaacgggtga gtaacacgtg 60 ggcaatctgc ccttcactct gggacaagcc ctggaaacgg ggtctaatac cggatatgag 120 ccggggaggc atctccctgg ttggaaagct ccggcggtga aggatgagcc cgcggcctat 180 cagcttgttg gtgaggtagt ggctcaccaa ggcgacgacg ggtagccggc ctgagagggc 240 gaccggccac actgggactg agacacggcc cagactccta cgggaggcag cagtggggaa 300 tattgcacaa tgggcggaag cctgatgcag cgacgccgcg tgagggatga cggccttcgg 360 gttgtaaacc tctttcagca gggaagaagc ggaagtgacg gtacctgcag aagaagcgcc 420 ggctaactac gtgccagcag ccgcggtaat acgtagggcg cgagcgttgt ccggaattat 480 tgggcgtaaa gagctcgtag gcggcttgtc acgtcgggtg tgaaagaccg gggcttaacc 540 ccggttctgc attcgatacg ggctggctag agtgtggtag gggagatcgg aattcctggt 600 gtagcggtga aatgcgcaga tatcaggagg aacaccggtg gcgaaggcgg atctctgggc 660 cattactgac gctgaggagc gaaagcgtgg ggagcgaaca ggattagata ccctggtagt 720 ccacgccgta aacggtggga actaggtgtt ggcgacattc cacgtcgtcg gtgccgcagc 780 taacgcatta agttccccgc ctggggagta cggccgcaag gctaaaactc aaaggaattg 840 acgggggccc gcacaagcgg cggagcatgt ggcttaattc gacgcaacgc gaagaacctt 900 accaaggctt gacatacacc ggaaacggcc ggagacggtc gcccccttgt ggtcggtgta 960 caggtggtgc atggctgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 1020 agcgcaaccc ttgtcccgtg ttgccagcaa ctcttcggag gttggggact cacgggagac 1080 cgccggggtc aactcggagg aaggtgggga cgacgtcaag tcatcatgcc ccttatgtct 1140 tgggctgcac acgtgctaca atggccggta caatgagctg cgatgccgtg aggtggagcg 1200 aatctcaaaa agccggtctc agttcggatt ggggtctgca actcgacccc atgaagtcgg 1260 agtcgctagt aatcgcagat cagcattgct gcggtgaata cgttcccggg ccttgtacac 1320 accgcccgtc acgtcacgaa agtcggtaac acccgaagcc ggtggcccaa cccccttgcg 1380 gggaggagct t 1391

Claims (8)

Streptomyces longisporus MBB1001 strain (accession number: KACC 92136P).
The Streptomyces longisporus MBB1001 strain (accession number: KACC 92136P) according to claim 1, which is characterized by inhibiting the production of melanin or inhibiting the activity of tyrosinase.
Streptomyces long Kish porous (Streptomyces longisporus) MBB1001 of the strain that obtained by removing the cells from the culture, Streptomyces long Kish porous (Streptomyces longisporus) MBB1001 strain: culture medium of (accession No. KACC 92136P).
A culture medium is obtained by removing the microbial cells from a culture of Streptomyces longisporus strain MBB1001 to obtain a culture medium. In this culture medium, the content of methyl acetate, ethyl acetate, propyl acetate, butyl acetate, methanol and methanol is 55 to 90% A culture extract of Streptomyces longisporus MBB1001 strain (accession number: KACC 92136P), which is obtained by fractionating components soluble in methanol or a mixed solvent of methanol and chloroform.
A whitening composition comprising Streptomyces longisporus strain MBB1001 (Accession No: KACC 92136P), a culture thereof, a lysate thereof or an extract thereof as an active ingredient.
Containing a culture extract of: (KACC 92136P accession No.) as an active ingredient: Streptomyces long Kish porous (Streptomyces longisporus) MBB1001 strain culture or Streptomyces long Kish porous (Streptomyces longisporus) MBB1001 strain (accession No. KACC 92136P) / RTI &gt;
7. The whitening composition according to claim 6, wherein the culture solution extract is an alkyl acetate-soluble fraction of the culture solution, and the alkyl group of the alkyl acetate has 1 to 5 carbon atoms.
[Claim 7] The method according to claim 6, wherein the culture medium extract is a secondary fraction obtained by firstly fractionating a culture solution with an alkyl acetate and then fractionating a first fraction with methanol or dihydrate methanol, wherein the alkyl group of the alkyl acetate has 1 to 5 carbon atoms , And the methanol concentration of the hydrous methanol is 80 to 95% (w / w).

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