KR20190024835A - Novel tenacibaculum sp. and composition comprising thereof - Google Patents

Abstract

The present invention relates to a novel Tenacibaculum sp. microorganism and a composition comprising the same, and more specifically, to a novel Tenacibaculum sp. microorganism exhibiting antioxidant, anti-inflammatory and skin whitening effects, and a composition comprising the same. A novel Tenacibaculum sp. Apep Microorganism Jeju 170240 (Accession No: KCTC18595P) strain or a culture medium thereof, provided by the present invention, exhibits excellent anti-inflammatory, antioxidant and skin whitening effects, and can be used in manufacturing medicines, functional foods and cosmetics.

Description

NOVEL TENACIBACULUM SP. AND COMPOSITION COMPRISING THEREOF}

The present invention relates to a novel microorganism in the tenacibraculum and a composition containing the microorganism and more particularly to a novel microorganism in the tenaci cabbum showing antioxidative, anti-inflammatory and skin whitening effects and a composition containing the same.

Fermentation refers to the process by which microorganisms possess enzymes that change raw materials and produce beneficial components. During the fermentation process, active ingredients are extracted and the content of functional materials increases.

And fermentation technology means the technology to produce desired products from precursors by using the enzymatic functions of microorganisms with semantically overlapping terms such as bioconversion, biosynthesis, biocatalyst, etc. Therefore, The fermentation technology used in the enzyme stage is one of the main process steps of the present biotechnology industry with the main purpose of producing useful materials. The second generation fermentation using merely traditional fermentation method was the development of the practical use technology of the efficent substance in the first generation fermented ramen and the bioreactor fermentation which laid the foundation of the functional material industry through the advanced fermentation production technology capable of mass- Technology has become a third-generation fermentation technology.

The Korean people use fermented foods such as kimchi, soybean paste and so on, so the concept of fermentation is very familiar and cosmetics using fermented raw materials are used without any objection. It is also known to the general public that when a new material is developed by using fermentation technology, many new ingredients exhibiting completely new effects or harmful ingredients are transformed into safe ingredients. Therefore, the preference is high for fermented materials, However, many fermentation materials currently applied to pharmaceuticals, functional foods or cosmetics are known to use natural fermentation or a fermentation method close to natural fermentation.

Natural fermentation refers to the natural production of ingredients that are useful to the skin by using natural forces such as water, wind, earth, and seasons without leaving unprocessed various raw materials intact. Although it takes a long time, However, natural fermentation or fermentation in a natural fermentation method is difficult to obtain a fermentation product of a certain quality per batch, and it is also difficult to control the quality of the fermentation product.

On the other hand, active oxygen introduced from the outside of the living body or generated in the living body promotes aging of the living body or causes cancer. Therefore, studies on antioxidants that inhibit oxidation by active oxygen are under way, and phenolic compounds, flavonoids, tocopherol, vitamin C, selenium and the like are known. However, antioxidants present in nature can not be expected to have practically sufficient effects when applied to skin. On the other hand, antioxidant activity is excellent and low-priced synthetic antioxidants have a problem that their use is limited due to safety concerns such as human side effects.

In addition, inflammation is caused by oxidative stress such as ultraviolet rays, active oxygen, and free radicals, which activates inflammatory factors and causes aging of various diseases and skin. To date, substances used as anti-inflammatory drugs include non-steroidal drugs such as flufenamicacid, ibuprofen, benzydamine, indomethacin, and steroids such as prednisolone, dexamethasone ). There are other allantoin, azene, hydrocortisone and the like. However, all of them have a problem of safety on the skin, there is a limitation in the use amount, or the effect is insignificant, so that the inflammation relieving effect can not be expected substantially.

As a whitening ingredient, there are substances inhibiting tyrosinase enzyme activity such as kojic acid and arbutin, hydroquinone, vitamin C (L-Ascorbic acid), derivatives thereof and various plant extracts . By inhibiting the synthesis of melanin pigment, they are known to realize skin whitening or to improve skin hypercholesterolemia such as spots and freckles. However, when applied to skin, there is a problem that the use amount is limited due to safety problems such as irritation and redness, or the effect is insignificant, so that a practical effect can not be expected.

Accordingly, there is a demand for a method for stably producing a microorganism-derived fermentation substance that is safe in vivo, has excellent anti-inflammatory and antioxidative effects, and exhibits an excellent effect on skin beauty.

Accordingly, the present inventors have made a careful effort to develop a microorganism that is safe for living organisms, has excellent anti-inflammatory and antioxidative effects, and can produce a fermented product showing an excellent effect on skin beauty. As a result, The present inventors have found that the fermented product of the new Tenacibaculum sp. Microorganism isolated from the local seawater exhibits excellent antiinflammation, antioxidant and skin whitening effect.

Accordingly, an object of the present invention is to provide a strain of Tenacibaculum sp. Apep Microorganism Jeju 170240 (accession number: KCTC18595P).

Another object of the present invention is to provide a novel Tenacibaculum sp. Strain having 16S rRNA having the nucleic acid sequence shown in SEQ ID NO: 1.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating an inflammatory disease comprising as an active ingredient a cell, a cell lysate, a culture supernatant or a mixture thereof obtained by culturing the above-mentioned strain.

Another object of the present invention is to provide a composition for antioxidation comprising a cell, a cell lysate, a culture supernatant or a mixture thereof obtained by culturing a strain as an active ingredient.

Another object of the present invention is to provide a composition characterized in that the antioxidant composition is a pharmaceutical composition, a functional food composition or a cosmetic composition.

Another object of the present invention is to provide a cosmetic composition comprising as an active ingredient a cell, a cell lysate, a culture supernatant or a mixture thereof obtained by culturing a strain.

Another object of the present invention is to provide a cosmetic composition which is a cosmetic composition for whitening.

Another object of the present invention is to provide a cosmetic composition which is characterized in that the composition is an anti-inflammatory cosmetic composition.

To achieve the above object, the present invention provides a strain of Tenacibaculum sp. Apep Microorganism Jeju 170240 (accession number: KCTC18595P).

In order to achieve another object of the present invention, the present invention provides a novel Tenacibaculum sp. Strain having 16S rRNA having a nucleic acid sequence represented by SEQ ID NO: 1.

In order to accomplish still another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease comprising as an active ingredient a cell, a cell lysate, a culture supernatant or a mixture thereof obtained by culturing the above strain.

In order to accomplish still another object of the present invention, there is provided an antioxidative composition comprising as an active ingredient, a cell, a cell lysate, a culture supernatant or a mixture thereof obtained by culturing a strain.

In order to accomplish still another object of the present invention, the present invention provides a composition, wherein the antioxidant composition is a pharmaceutical composition, a functional food composition or a cosmetic composition.

In order to accomplish still another object of the present invention, there is provided a cosmetic composition comprising as an active ingredient, a cell, a cell lysate, a culture supernatant or a mixture thereof obtained by culturing a strain.

Another object of the present invention is to provide a cosmetic composition which is a whitening cosmetic composition.

In order to achieve another object of the present invention, the present invention provides a cosmetic composition, wherein the composition is an anti-inflammatory cosmetic composition.

Hereinafter, the present invention will be described in more detail.

The present invention provides a new Tenacibaculum sp. Apep Microorganism Jeju 170240 (Accession No .: KCTC18595P) strain. In the present invention, the strain may be a new Tenacibaculum sp. Strain having 16S rRNA having the nucleic acid sequence shown in SEQ ID NO: 1.

The inventors of the present invention have been developing microorganisms that are safe for living organisms, have excellent anti-inflammatory and antioxidative effects, and can produce fermented water exhibiting excellent effects on skin beauty. While developing microorganisms, ( Tenacibaculum sp.) Microorganisms were found in the seawater of Yongmori shore (the area where fresh water and sea water meet), Yongmuri shore (area where geological activity is active) and Taedong coastal area (general coastal area) Tenacibaculum sp.) Apep Microorganism Jeju 170240 strain was deposited on August 17, 2017 at the Korea Research Institute of Bioscience and Biotechnology, and was given accession number KCTC18595P .

The novel strain of the tenascibacillum is excellent in protease activity (proteolytic activity), agarolytic activity, cellulase activity, amylase activity (carbohydrate degrading ability), etc., and various substances and enzymes produced by the microorganism during fermentation are various. It is more effective in decomposition of proteins and carbohydrates possessed by natural products. Therefore, when used in cosmetics, skin irritation is significantly lower than when natural products alone are used, and thus it is confirmed to be a suitable strain for producing cosmetic fermented products .

According to one embodiment of the present invention, the cultured product obtained by culturing the strain of the new Tenacibaculum sp. Strain was found to have excellent anti-inflammatory, antioxidative and skin whitening effects.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, comprising as an active ingredient, a cell, a cell lysate, a culture supernatant or a mixture thereof obtained by culturing the above-mentioned strain.

In the present invention, the microorganism may be a culture medium in which a new Tenacibaculum sp. Strain according to the present invention is cultured in a suitable microbial culture, or a pure microbial cell in which the microbial culture medium is separated from the culture medium. Further, the above-mentioned cell disruption solution may mean a culture solution containing the cell degradation product obtained by a method such as ultrasonication of the culture solution of the strain or treatment of the culture solution with a lysozyme. In the present invention, the cells, the cell lysate, the culture supernatant, or a mixture thereof may include both the concentrate and the dried product thereof.

In the present invention, the inflammatory diseases are selected from asthma, eczema, psoriasis, allergies, allergic dermatitis, rheumatoid arthritis, psoriatic arthritis, contact dermatitis, atopic dermatitis, acne, atopic rhinitis, , Chronic sinusitis, seborrheic dermatitis, gastritis, gout, gouty arthritis, ulcer, chronic bronchitis, lung inflammation, Crohn's disease, ulcerative colitis, ankylosing spondylitis, sepsis, vasculitis, bursitis, rheumatism Inflammatory bowel disease, epidermodysplasia verruciformis, convulsive encephalitis (e. G., Chronic obstructive pulmonary disease), multiple sclerosis, multiple sclerosis, neurodegeneration, neutrophilic alveolitis, Alzheimer's disease, arteriosclerosis, obesity, inflammatory bowel disease Herpes simplex encephalitis), chronic mucocutaneous candidiasis, Trypanosomiasis, Scotland may be selected from the group consisting of inflammatory diseases, infections and musamagwi, may preferably be inflammatory skin disease, eczema, psoriasis, contact dermatitis, atopic dermatitis, acne, seborrheic dermatitis or allergic dermatitis.

The term " prophylactic, " as used herein, refers to any action that inhibits or delays the progression of an inflammatory disease upon administration of a composition of the present invention.

As used herein, the term " treatment " refers to any action by which administration of the composition of the present invention improves or alleviates the symptoms of an inflammatory disease.

The composition of the present invention may further comprise a pharmaceutically acceptable additive. Examples of the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, Starch glycolate, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, calcium stearate, magnesium stearate, White sugar, dextrose, sorbitol and talc may be used. The pharmaceutically acceptable additives according to the present invention are preferably included in the composition in an amount of 0.1 to 90 parts by weight, but are not limited thereto.

In addition, the composition of the present invention can be administered in various formulations of oral and parenteral administration at the time of actual clinical administration. In the case of formulation, a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, . ≪ / RTI >

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 or gelatin And the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions and syrups, and various excipients such as wetting agents, sweetening agents, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used .

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

On the other hand, injecting agents may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives and the like.

In addition, the therapeutic compositions of the present invention may be formulated with any physiologically acceptable carrier, excipient or stabilizer (Remington: The Science and Practice of Pharmacy, l9th Edition, Alfonso, R., ed., Mack Publishing Co. : 1995)). Acceptable carriers, excipients or stabilizers are nontoxic to the recipient at the dosages and concentrations employed and include buffer solutions such as phosphoric acid, citric acid and other organic acids; Antioxidants including ascorbic acid; Low molecular weight (less than about 10 residues) polypeptides; Proteins, such as serum albumin, gelatin or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, arginine or lysine; Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose or dextrin; Chelating agents such as EDTA; Sugar alcohols such as mannitol or sorbitol; Salt-forming counterions such as sodium; And / or non-ionic surfactants such as tween, pluronics or polyethylene glycol (PEG).

The dosage of the pharmaceutical composition of the present invention to the human body may vary depending on the patient's age, body weight, sex, dosage form, health condition and disease severity, and is generally 0.01 to 100 mg / kg / day, 0.1 to 20 mg / kg / day, and more preferably 5 to 10 mg / kg / day. It may also be administered at a fixed interval according to the judgment of a doctor or pharmacist.

The present invention also provides a composition for antioxidation comprising, as an active ingredient, a cell, a cell lysate, a culture supernatant or a mixture thereof obtained by culturing the above-mentioned strain.

In the present invention, 'antioxidation' refers to the oxidation of cells with highly reactive free radicals or reactive oxygen species (ROS) according to oxidative stress caused by intracellular metabolism or ultraviolet rays And includes removal of free radicals or reactive oxygen species, thereby reducing damage to the cells.

In the present invention, the antioxidant composition may be a pharmaceutical composition, a functional food composition or a cosmetic composition, preferably a functional food composition or a cosmetic composition, and most preferably a cosmetic composition.

When the functional food composition of the antioxidant composition is added to food materials such as beverage, tea, spice, gum, and confectionery, or encapsulated, powdered, or suspended, the food contains certain health effects However, unlike general medicine, there is an advantage that there is no side effect that can occur when a food is used as a raw material for a long period of taking the medicine.

Since the functional food composition of the present invention thus obtained can be taken on a daily basis, a high antioxidative effect can be expected and is very useful.

Generally, the antioxidant composition of the present invention is added in an amount of not more than 70 parts by weight, preferably not more than 50 parts by weight, based on the raw material, when the food or beverage is produced. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range .

In the present invention, there is no particular limitation on the kind of the functional food. Non-limiting examples of these include dairy products including meats, sausages, breads, chocolates, candies, snacks, confectionery, pizza, ramen noodles, other noodles, gums, ice cream, various soups, beverages, tea, And a combination thereof, all of which contain functional foods in a conventional sense.

The functional food composition of the present invention may be prepared as a functional beverage by adding various flavors or natural carbohydrates as additional components such as ordinary beverages. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the composition of the present invention.

In addition to the above, the functional food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the functional food composition of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

When the composition according to the present invention is a cosmetic composition, it may be applied to a gel type, a skin type, a cream type, an ointment type, and the like, but is not limited thereto. The above composition can be suitably prepared by known methods, adding appropriate conventional softening agents, emulsifying agents, thickening agents or other materials known in the art depending on the type thereof.

The gel-type composition may be prepared by adding a softening agent such as trimethylolpropane, polyethylene glycol or glycerin, a solvent such as propylene glycol, ethanol, isocetyl alcohol, or the like.

The skin-type composition may contain at least one of fatty alcohols such as stearyl alcohol, myristyl alcohol, behenyl alcohol, arachidyl alcohol, isostearyl alcohol and isocetyl alcohol, butylene glycol, 2-sodium, xanthan gum, dimethicone, polyethylene glycol-60 hydrogeneate castor oil, polysorbate 60 and purified water.

The cream-type composition may be formulated by dissolving or dispersing the above-mentioned cream type composition in an aqueous medium such as alcohols such as stearyl alcohol, myristyl alcohol, behenyl alcohol, arachidyl alcohol, isostearyl alcohol, isocetyl alcohol and the like, lecithin, phosphatidylcholine, phosphatidylethanolamine, Emulsifiers such as glyceryl stearate, sorbitan palmitate, and sorbitan stearate; natural fats or oils such as avocado oil, apricot oil, barba water oil, glass fat oil and camellia oil; Propylene glycol and the like, purified water, and the like.

The ointment type composition may be prepared by adding a softening agent, an emulsifying agent, and a wax such as microcrystalline wax, paraffin, ceresin, wax, spermaceti, vaseline or the like.

The cosmetic composition of the present invention may further contain at least one ingredient showing the same or similar functions in addition to the effective ingredient. Non-limiting examples include, but are not limited to, one or more selected from the group consisting of vitamin C, retinoic acid, TGF, protein from animal placenta, betulinic acid and chlorella extract. The cosmetic composition of the present invention may further contain an excipient including a fluorescent substance, a fungicide, a hygroscopic substance, a moisturizing agent, a fragrance, a fragrance carrier, a protein, a solubilizer, a sugar derivative, a sunscreen, a vitamin, .

The present invention also provides a cosmetic composition comprising, as an active ingredient, a cell, a cell lysate, a culture supernatant or a mixture thereof obtained by culturing the above-mentioned strain. Preferably, the cosmetic composition may be a whitening cosmetic composition or an anti-inflammatory cosmetic composition.

A detailed description of the cosmetic composition is as described above.

The novel Tenacibaculum sp. Apep Microorganism Jeju 170240 (Accession No .: KCTC18595P) strain or its culture provided by the present invention exhibits excellent anti-inflammatory, antioxidant and skin whitening effects and is useful for the production of pharmaceuticals, functional foods and cosmetics Can be very useful.

Fig. 1 is a gene sequence of 16s rRNA of a strain of the new Tenaciabetes genome identified in Example 1 of the present invention.
Fig. 2 is a phylogenetic tree prepared by sequencing the 16S rRNA gene of the strain of the genus Tenaciabculum identified in Example 1 of the present invention.
Fig. 3 is a graph showing the results obtained in Example 3 of the present invention in the case of a new tenacibulum strain, tenascibacillum lutimaris (KCTC 123020), tenascibacillum isoturia (KCTC 12569), and tenascibaculum soluria Protease activity was confirmed and photographed with DSLR (D3000, Nicon, Japan).
Fig. 4 is a photograph showing the agar-resolving ability of the strain of the new tenascibaculum in Example 4 of the present invention and photographing it with DSLR (D3000, Nicon, Japan).
FIG. 5 is a graph showing the results obtained by comparing the results of Example 5 of the present invention with that of the strain of the new tenacibaculum, tenascibacillum lutrimaris (KCTC 123020), tenacibaculum isoturia (KCTC 12569) Cellulase activity was confirmed and photographed with DSLR (D3000, Nicon, Japan).
Fig. 6 is a graph showing the results obtained by comparing the results obtained in Example 6 of the present invention with that of the strain of the new tenacibaculum, tenascibacillum lutimaris (KCTC 123020), tenascibacillum isoturia (KCTC 12569) and tenascibaculum soluria The amylase activity was confirmed and photographed with DSLR (D3000, Nicon, Japan).
FIG. 7 is a graph comparing the anti-inflammatory effects of the fermented culture broth of the new strain prepared in Example 7 and the M2A medium.
8 is a graph comparing the antioxidant efficacy of the fermented culture broth of the new strain prepared in Example 7 and the M2A medium (before fermentation).
FIG. 9 is a graph comparing the inhibitory activity of tyrosinase of the fermented culture broth of the new strain prepared in Example 7 and M2A medium (before fermentation).
FIG. 10 is a photograph of a fermented product in a cream formulation taken in Example 11 of the present invention and then taken with DSLR (D3000, Nicon, Japan).

Hereinafter, the present invention will be described in detail with reference to preferred embodiments. Prior to this, terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms, and the inventor should appropriately interpret the concepts of the terms appropriately The present invention should be construed in accordance with the meaning and concept consistent with the technical idea of the present invention. Accordingly, it is to be understood that the constituent features of the embodiments described herein are merely the most preferred embodiments of the present invention, and are not intended to represent all of the inventive concepts of the present invention, so that various equivalents, And the like.

< Example  1>

Isolation and identification of new strains

In order to discover new strains usable as functional cosmetic compositions in the present invention, strains were isolated from seawater in various clean areas of Jeju Island. Specifically, sea water was plated on marine agar 2216 (MA 2216, Difco, USA) and R2A agar plates (R2A agar, Difco, USA) and cultured at 25 ° C for 48 hours, The strain was firstly screened. Then, the cultured strains were each plated on a medium of various conditions and cultured at 25 DEG C for 48 hours, and the strains were secondly selected on the plate. The finally selected strains were stored at -70 &lt; 0 &gt; C in a 15 wt% glycerol medium.

16S rRNA was isolated from a strain isolated from purified seawater according to the above Example and the nucleic acid sequence thereof was determined (FIG. 1). To compare the homology between the 16S rRNA and the known sequence, NCBI BLAST NCBI (www.ncbi.nlm.nih.gov/BLAST/) was used. As a result of the analysis, it was judged that the isolated strain had 95.0% homology with the Tenacibaculum sp strain as a gram negative bacterium (Fig. 1). Based on these results, the isolated isolate was designated as Apep Microorganism Jeju 170240, which was deposited on August 17, 2017 in the Korean Collection for Type Culture (KCTC) , And assigned accession number KCTC18595P. As a result of comparison with the relatives, it was confirmed that the strain was a new strain (Fig. 2).

< Example  2>

Tenaciabakulum  Culture of the genus

M2A plate (medium containing 1: 1 mixture of marine medium and R2A medium + 1.5% agar), R2A plate and marine plate were cultivated at 22 ℃ ~ 30 ℃ for 3 days, As a result, the highest growth rate was observed at 25 ° C and M2A plate, and the culture medium of Tenacibaculum was cultured at 25 ° C using M2A medium. Through the present cultivation, it was confirmed that the strain of the new Tenaciabculum can grow normally.

< Example  3>

Evaluation of Protease Activity (Protein Resolution)

After culturing the new strain in a marine medium, 1 ml of the culture broth was taken, and the number of viable cells was measured at 600 nm using a spectrophotometer. All of the strains were used for the measurement of the enzyme activity. The above strains were compared with the above-mentioned novel strains. Tenaciabaculumum lutimaris (KCTC 123020), Tenacibaculumum isoteri (KCTC 12569) and Apent Microorganism Jeju (KCTC 12569), a strain isolated by the applicant, 170219, A &amp; Peptide isolate) was used.

A solid medium supplemented with Skim Milk was used to measure the protease activity of each strain. The solid medium contained 0.5 g of peptone (Difco, USA), 0.25 g of yeast extract (Difco, USA), skim milk (Difco, USA) (Difco, USA), 15 grams of agar (Difco, USA) and 1 liter of distilled water. The protease activity of each strain was measured using the characteristics of treating the above-mentioned solid medium with the above-mentioned each strain at the same concentration, culturing at 25 ° C for 5 days, and forming a zona around the colonies.

As a result, the novel strain of the present invention has the protease activity of tenascibacillum lutimaris, b shows the protease activity of tenaciabaculum isotypes, c shows the protease activity of tenasiba (Proteolytic activity) of the new strain was higher than that of the other strains, as shown in the right photograph, the protease activity of the new strain. And as a result, it was confirmed that the novel strain according to the present invention is suitable for various kinds of fermentation.

< Example  4>

Agar Resolution Evaluation

The identified strain was cultured in a marine agar solid medium at 25 DEG C for 2 days. The solid medium was used as a minimal nutrient medium, R2A (Difco, USA) medium, for agar resolving ability evaluation. The modified R2A medium contained 0.5 g of proteose peptone No. 3, 0.5 g of casamino acids, 0.5 g of yeast extract, 0.5 g of glucose, 0.5 g of soluble starch, 0.3 g of sodium pyruvate, 0.3 g of K ₂ HPO ₄ , 0.05 g of MgSO ₄ .7H ₂ O, 30 g of NaCl, 10 g of agarose and 1 L of distilled water.

In order to evaluate the agar resolving ability of the identified strains, the cultured new strains were incubated in an agar-resolving ability medium at 25 占 폚 for 3 days to observe the resolving ability of the agar.

As a result, it was confirmed that the agar was decomposed in the vicinity of the cells, and that the agar-resolving ability was observed in the agar medium, and thus the newly identified strain secreted an agarase enzyme outside the cell (FIG.

< Example  5>

Evaluation of cellulase activity (cellulase resolution)

In order to evaluate the cellulase activity of the novel strain, the new strain and the known strains for comparison with the strain, Tenascibaculum lutrimaris (KCTC 123020), Tenacibaculum isoturia (KCTC 12569) and a strain isolated by the applicant (Apep Microorganism Jeju 170219, A &amp; Peptide isolate) was used in liquid culture on Marine broth medium.

The cellulase activity of each strain was measured using solid medium supplemented with carboxymethyl cellulose (CMC). The solid medium is carboxymethyl cellulose (CMC, Difco, USA) 5g, yeast extract (Difco, _{USA) 0.5g, K 2 HPO 4 ·} 3H 2 O 0.25g, MgSO 4 · 7H 2 O 0.2g, NaCl 30g, MnSO _4. 7H ₂ O, 2.5 g of (NH ₄ ) ₂ SO ₄ , 15 g of agar (Difco, USA) and 1 L of distilled water. The culture broth of each strain was measured at 600 nm using a spectrophotometer, and then the same concentration was used. The thus-prepared solid medium was treated with the same concentration of the strain, incubated at 25 ° C for 5 days, stained with 0.1% congo red for 30 minutes, discolored with 1M NaCl for 5 minutes, To measure cellulase activity.

As a result, the novel strain of the present invention showed the cellulase activity of tenascibaculum lumilaris, b shows the cellulase activity of tenascibacillum isoturia, c shows tenascibaculumum (Cellulase activity of the strain), d shows the cellulase activity of the new strain, and the photograph on the right shows the cellulase activity of the new strain. It was confirmed that the cellulase activity (cellulase resolution ability) Thus, it was confirmed that the novel strains were suitable for various fermentations.

< Example  6>

Evaluation of Amylase Activity (Carbohydrate Resolution)

In order to evaluate the amylase activity of the new strain, the new strain and tenasibaculumurutumaris (KCTC 123020), Tenaciabaculumum isoturia (KCTC 12569) and strains isolated by the applicant, which are known strains for comparison with the strains (Apep Microorganism Jeju 170219, A &amp; Peptide isolate) was used in liquid culture on Marine broth medium.

 The amylase activity of each strain was evaluated using solid medium supplemented with 0.5 wt% solubilized starch, 3 wt% NaCl and 1.5 wt% agar. The culture broth of each strain was measured at 600 nm using a spectrophotometer, and then the same concentration was used. Each strain was treated at the same concentration in the prepared solid medium and cultured at 25 ° C for 5 days. Amylase activity was measured according to the degree of zona pellucida using Gram's Iodione solution.

As a result, the novel strain of the present invention showed the amylase activity of tenascibacillum lutimaris, b shows the amylase activity of tenaciabaculum isotyri, c showed tenasibacillum (Amylase activity of the strain), d shows the amylase activity of the new strain, and the photograph on the right shows the amylase activity of the new strain), the amylase activity (carbohydrate degrading ability) Respectively.

< Example  7>

Fermentation production

The M2A medium in which the marine culture and the R2A medium were mixed in a one-to-one manner was inoculated with the new strain identified above and cultured at 25 DEG C for 48 hours, and then used as a fermentation strain for fermentation. For the culture of liquid medium, the identified new strain cultured in M2A solid medium in M2A liquid medium was inoculated and cultured at 25 DEG C for one week. The cells were centrifuged to separate the culture medium of the strain and the cells. The separated culture supernatant was filtered with a filter having a pore size of 0.2 mu m to prepare a new strain culture supernatant. After the new strain was pulverized by using ultrasonic waves, the pulverized new strain was centrifuged to separate the pellet and the lysate, and the pulverized liquid alone was collected and filtered with a pore size filter of 0.2 탆 Then, the culture supernatant and the disruption solution were mixed to prepare a fermentation product of the culture broth of a new strain.

< Example  8>

Evaluation of anti-inflammatory efficacy

The anti-inflammatory efficacy was evaluated using the fermented culture broth of the new strain prepared in Example 7 above. The macrophage cell line used for the anti-inflammatory efficacy evaluation was Raw 264.7. The medium was DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. After the Raw 264.7 was cultured at 37 ° C for 24 hours, 99% (v / v)% ethanol was added to the fermentation product to dilute the fermentation product to a concentration of 10% by volume, 1% by volume and 0.1% ) Were added to the Raw 264.7 culture medium, respectively, and cultured at 37 DEG C for 24 hours. After incubation, the cell culture supernatant was mixed with the Griess reagent at a ratio of 1: 1. After 10 minutes of reaction, the absorbance was measured at 540 nm to evaluate the anti-inflammatory effect. For comparison, 99 (v / v)% ethanol was added to the M2A medium The medium concentration was adjusted to 10% by volume, 1% by volume and 0.1% by volume, and the absorbance of M2A medium was measured at 540 nm to evaluate the anti-inflammatory effect. The results are shown in FIG.

 As a result, it was confirmed that the anti-inflammatory efficacy of the fermented product was excellent in all the concentration ranges when compared with the M2A medium (FIG. 7).

< Example  9>

Antioxidant efficacy assessment

The antioxidant efficacy was evaluated using the fermented culture broth of the new strain prepared in Example 7 above. Antioxidant efficacy was evaluated by DPPH method. DPPH (1,1-diphenyl-2picrylhydazyl) contains an intramolecular radical. When the radical is cleaved, the deep purple color is discolored. The DPPH method is a method of measuring the degree of decolorization by the hydrogen donating ability of an antioxidant . The decolorization process can be explained by the reduction process. The larger the reducing power, the greater the antioxidant capacity. The DPPH used for the measurement of hydrogen donating ability is a safe free radical and exhibits the maximum absorption value at around 517 nm due to its non-covalent electrons. First, the DPPH was dissolved in ethanol to prepare 0.2 mM DPPH. 99 (v / v)% ethanol was added to the fermentation product, and 800 희 of a dilution diluted to respective concentrations, namely 10 vol%, 1 vol% and 0.1 vol%, and 200 쨉 l of a 0.2 mM DPPH solution dissolved in ethanol were added After incubation at room temperature for 30 minutes, the absorbance was measured at 517 nm and the antioxidant efficacy was evaluated. For comparison, 99 (v / v)% ethanol was added to the M2A medium to adjust the medium concentration to 10 volume%, 1 volume% %, The absorbance of the M2A medium was measured at 517 nm, and the antioxidant efficacy was evaluated. The results are shown in FIG.

 As a result, it was confirmed that the antioxidant efficacy of the above fermented product was excellent in all the concentration ranges when compared with the M2A medium (before fermentation) (FIG. 8).

< Example  10>

Whitening efficacy  evaluation

The whitening efficacy evaluation was carried out using the fermented culture broth of the new strain prepared in Example 7 above. The whitening efficacy test was verified using experiments measuring tyrosinase inhibitory activity. The experiment was carried out by adding 220 [mu] l of 0.1 M phosphate buffer (pH 6.5) and 99 (v / v)% ethanol to the fermentation broth and diluting them to respective concentrations, namely 10 vol%, 1 vol% and 0.1 vol% Add 20 μl of a sample dilution, mushroom tyrosinase (1500 U / ml to 2000 U / ml) or 20 μl of human tyrosinase solution in that order. To this solution, 40 μl of 1.5 mM tyrosine was added and reacted at 37 ° C. for 10 to 15 minutes. Absorbance was measured at 490 nm using an ELISA reader. For comparison, 99 (v / v) The medium concentration was adjusted to 10% by volume, 1% by volume and 0.1% by volume, and the absorbance of the M2A medium was measured at 490 nm to evaluate the whitening efficacy. The results are shown in FIG.

As a result, it was confirmed that the tyrosinase inhibitory activity of the fermented product was very excellent when compared with the M2A medium (before fermentation) (FIG. 9).

< Example  11>

cream In the formulation Fermentation product  stability

10% by weight of the fermented culture broth of the new strain prepared in Example 7 was added to the cream formulation, and the cream formulation was stored at 4 캜 and 50 캜, respectively, and were stored at 0 캜, 1 nd, 2 nd, 3 nd, Color, odor, phase change and content. As a result, all of the cream formulations stored at 4 캜 and 50 캜 maintained the stability for 4 weeks without discoloration, breakage, and odor (Fig. 10).

As described above, the strain Tenacibaculum sp. Apep Microorganism Jeju 170240 (Accession No .: KCTC18595P) according to the present invention has protease activity (proteolytic activity), agarase activity, cellulase activity and amylase activity (carbohydrate degradation activity) And the fermented product fermented using the strain Tenacibaculum sp. Apep Microorganism Jeju 170240 (Accession No .: KCTC18595P) in the above-mentioned new Tenacibaculum sp. Were found to have anti-inflammatory, antioxidative and whitening effect Was superior to the M2A medium (before fermentation).

The present invention has been described in detail with reference to the production examples, experimental examples and drawings. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the appended claims.

Accordingly, the scope of the present invention is defined by the appended claims rather than the foregoing detailed description, and all changes or modifications derived from the meaning, range, and equivalence of the claims are included in the scope of the present invention Should be interpreted.

Korea Research Institute of Bioscience and Biotechnology KCTC18595P 20170817

<110> A and PEP INC. <120> NOVEL TENACIBACULUM SP. AND COMPOSITION COMPRISING THEREOF <130> NP17-08078 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 1433 <212> DNA <213> Artificial Sequence <220> <223> 16s rDNA of Tenacibaculum sp. Apep Microorganism Jeju 170240 <400> 1 ggggaaggcg gcagctacac atgcagtcga ggggtaacag agtagcttgc tattgctgac 60 gaccggcgaa cgggtgcgta acgcgtatag aatctgcctt gtacaggagg atagccttta 120 gaaatgaaga ttaatactcc ataatgtaga gttgtggcat cacaatttta ttaaagattt 180 attggtacaa gatgactatg cgtcctatta gctagatggt aaggtaacgg cttaccatgg 240 caacgatagg tagggggtct gagaggatta tcccccacac tggtactgag acacggacca 300 gactcctacg ggaggcagca gtgaggaata ttggtcaatg gaggcaactc tgaaccagcc 360 atgccgcgtg caggaagact gccctatggg ttgtaaactg cttttataca ggaagaaaca 420 gatctacgtg taggtccttg acggtactgt aagaataagc accggctaac tccgtgccag 480 cagccgcggt aatacggagg gtgcaagcgt tatccggaat cattgggttt aaagggtccg 540 caggcggtca attaagtcag aggtgaaatc ccatagctta actatggaac tgcctttgat 600 actggttgac ttgagttata cggaagtaga tagaataagt agtgtagcgg tgaaatgcat 660 agatattact tagaataccg attgcgaagg cagtctacta cgtatatact gacgctcatg 720 gacgaaagcg tggggagcga acaggattag ataccctggt agtccacgcc gtaaacgatg 780 gacactagtt gttggattat ttcagtgact aagcgaaagt aataagtgtc ccacctgggg 840 agtacgatcg caagattgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc 900 aagtggttta attcgatgat acgcgaggaa ccttaccagg gcttaaatgt agtatgacag 960 gtttagagat agacttttct tcggacatat tacaaggtgc tgcatggttg tcgtcagctc 1020 gtgccgtgag gtgtcaggtt aagtcctata acgagcgcaa cccctatcgt tagttgctag 1080 caggtaaagc tgaggactct agcgagactg ccggtgcaaa ccgtgaggaa ggtggggatg 1140 ggtacacacc atgagcagcc actatgcgaa taggagcgaa tctataaacc atatcacagt tcggatcgga 1260 gtctgcaact cgactccgtg aagctggaat cgctagtaat cggatatcag ccatgatccg 1320 gtgaatacgt tcccgggcct tgtacacacc gcccgtcaag ccatggaagt tgggggtgcc 1380 tgtagagtct cgtacaccga gacgagctga actaggtaga ccgttaactt ccg 1433

Claims (8)

Tenacibaculum sp.) Apep Microorganism Jeju 170240 (Accession No .: KCTC18595P) strain. The method according to claim 1,
The strain is a new Tenacibaculum sp. Strain having 16S rRNA having the nucleic acid sequence shown in SEQ ID NO: 1. A pharmaceutical composition for preventing or treating an inflammatory disease comprising as an active ingredient a cell, a cell lysate, a culture supernatant, or a mixture thereof obtained by culturing the strain of any one of claims 1 or 2. A composition for antioxidation, comprising as an active ingredient, a cell, a cell lysate, a culture supernatant or a mixture thereof obtained by culturing the strain of claim 1 or 2. 5. The method of claim 4,
Wherein the antioxidant composition is a pharmaceutical composition, a functional food composition or a cosmetic composition. A cosmetic composition comprising, as an active ingredient, a cell, a cell lysate, a culture supernatant, or a mixture thereof obtained by culturing the strain of claim 1 or 2. The method according to claim 6,
Wherein the composition is a whitening cosmetic composition. The method according to claim 6,
Wherein the composition is an anti-inflammatory cosmetic composition.

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