KR20160099163A - Novel Chryseobacterium sp. P1-3 producing protease and method using the same - Google Patents

Abstract

The present invention relates to: a novel Chryseobacterium sp. p1-3 (KCTC 12594BP) strain producing protease; a culture medium thereof; and compositions for decomposing organic waste, for a detergent, and for removing dead skin cells comprising the strain and the culture medium thereof. According to the present invention, the Chryseobacterium sp. p1-3 strain or the culture medium thereof produce protease and have excellent activities of keratinase among the protease, and thus have excellent proteolytic ability to decompose non-biodegradable keratin, thereby being effectively used for various industrial uses, such as processing of forage, fabric, natural leather, etc., organic waste treatment, washing, dead skin cell removal, etc.

Description

A novel strain of the genus Chrysobacterium belonging to the genus P1-3 which produces a proteolytic enzyme and its use {Novel Chryseobacterium sp. P1-3 producing protease and method using the same}

The present invention relates to a novel strain of genus Chrysobacterium p1-3 which produces a protease, a culture solution of the strain, and a composition for decomposing organic wastes, detergents and exfoliating compositions containing the same as an effective ingredient.

In Korea, about 150 million chickens are produced annually (about 8.5 billion in the US), and chicken feathers, which are between 5 and 7 percent of the weight of a chicken, (About 1 billion kilograms in the United States), and the chicken worms are used for livestock and cattle feed because of their high protein content. Keratin, the main constituent of chicken wool, is a fibrous structural protein that constitutes skin, nails, hair, wool, horseshoe, horns, and feathers that protect the organism. Peptide bonds that form β-structures and numerous hydrogen Bonded by a disulfide bond, and is very stable in terms of physical and chemical properties. Because of its structural stability, it is a by-product, which is not easily decomposed by chemical treatments such as acid or alkali, or by proteolytic enzymes such as pepsin, trypsin and chymotrypsin in animal digestive organs, and insoluble in water. For this reason, only a small part of the discharged chicken wool is used as a filling material for insulation and cushion, and most of the waste is discharged as waste and incinerated or buried for disposal. However, when incinerating, many soot is generated, And it is one of the main factors of environmental pollution because it has many problems due to the degradation.

Protease has various functions such as digestion, absorption and defense in vivo, and is divided into serine, cysteine, aspartic acid and metallo protease depending on the structure of the active site. In addition to the treatment of human diseases such as thrombus dissolution, proteolytic enzymes are used as a component of detergent for clothes, as a component of contact lens cleaner, as well as for modification of milk proteins, silk degumming of silk fibers, , Deodorization, dehairing, recovery of silver from x-ray film, and regeneration of ultrafiltration membrane for protein solution concentration. In addition, proteolytic enzymes can be used for a variety of purposes, such as degrading feathers from the poultry industry.

The enzyme that degrades the keratin protein in the protease is called a keratinolytic protease or keratinase. The enzyme has been distinguished from other proteolytic enzymes in that it decomposes a substrate with a highly stable structure.

So far, researches have been carried out to decompose feathers of poultry including chicken worms effectively and industrialize them.

Accordingly, the present inventors have identified a novel strain P1-3 of the genus Chrysobacterium during the isolation of a strain having excellent keratin protein degradability, and found that the strain and its culture have excellent keratinase activity, And thus the present invention has been completed.

It is an object of the present invention to provide a strain of Chrysosobacterium sp. P1-3 (KCTC 12594BP) producing a protease.

It is also an object of the present invention to provide a culture broth of the strain.

It is also an object of the present invention to provide a method for culturing the strain.

It is also an object of the present invention to provide a composition for decomposing organic wastes containing the strain and a culture solution thereof as an effective ingredient.

It is another object of the present invention to provide a method for decomposing organic wastes using the strain and a culture solution thereof.

It is also an object of the present invention to provide a detergent composition containing the above strain and a culture solution thereof as an active ingredient.

It is also an object of the present invention to provide a cosmetic composition for removing skin exfoliation comprising the above-mentioned strain and a culture solution thereof as an active ingredient.

In order to solve the above problems, the present invention provides a strain of Chrysosobacterium sp. P1-3 (KCTC 12594BP) producing a protease.

In addition, the present invention provides a culture solution of the strain.

The present invention also provides a method of culturing the strain.

The present invention also provides a composition for decomposing organic wastes comprising the strain and a culture solution thereof as an effective ingredient.

In addition, the present invention provides a method for decomposing organic wastes using the strain and the culture solution thereof.

The present invention also provides a detergent composition comprising the strain and a culture solution thereof as an active ingredient.

In addition, the present invention provides a cosmetic composition for removing skin exfoliating comprising the above strain and a culture solution thereof as an active ingredient.

The strain of the genus Chrysobacterium sp. P1-3 (KCTC 12594BP) or a culture thereof produces protease, and the keratinase activity is excellent, and the resolving ability of the keratin protein is excellent. Therefore, the strain or its culture may be useful for various industrial applications such as processing feeds, fabrics, natural leather, treating organic wastes, cleaning and exfoliation.

Brief Description of the Drawings Fig. 1 is a phylogenetic diagram showing the 16s rRNA base sequence (1,400 bp) of the strain P1-3 of the genus Chrysobacterium in comparison with a known strain.
FIG. 2 is a view showing a culture of the strain P1-3 of the genus Chrysobacterium in a McCony medium. FIG.
FIG. 3 is a graph showing the results of using API Kit 20 to analyze the biochemical characteristics of the strain P1-3 of the genus Chrysobacterium.
FIG. 4 is a graph showing the fatty acids content of the strain P1-3 of the genus Chrysobacterium.
FIG. 5 is a graph showing the degree of growth of the strain P1-3 of the genus Chrysosobacterium according to the culture temperature.
FIG. 6 is a diagram showing the growth of the strain P1-3 of the genus Chrysobacterium according to the change in pH. FIG.
FIG. 7 is a diagram showing the results of foliar decomposition of the strain P1-3 of the genus Chrysobacterium according to the lapse of 1, 4, and 7 days of culture.
FIG. 8 is a graph showing the keratinase digestion rate of the strain P1-3 of the genus Chrysobacterium according to the elapsed time of 1 to 7 days.

The present invention provides a strain of Chrysosobacterium sp. P1-3 (KCTC 12594BP) producing a protease.

The protease is preferably a keratinase.

In one embodiment of the present invention, soil samples from five wastewater treatment sites of a chicken farming area were collected to isolate strains having excellent degradation keratin protein degradability. Neighbor-joining methods by comparing the isolated strains with a 16S rRNA gene sequence comparison with a Chrysobacterium type strain; As a result of comparing with Bioedit and Mega5 program, it was confirmed that the strain having excellent resolving ability of the degradable keratin protein was a novel strain showing 97% homology with the previously reported Chrysophyte bacterium strain. Therefore, the strain is named "Chrysosobacterium sp. P1-3" On May 16, 2014, the strain P1-3 was deposited at the Microbiological Resource Center of the Korea Biotechnology Research Institute, which is the depository of the Korean patented strain, and received the deposit number KCTC 12594BP. The nucleotide sequence of the 16S rRNA of the strain is shown in SEQ ID NO: 1.

Through the analysis of the morphological characteristics of the strain, it was confirmed that the bacterium was a gram-negative bacterium. Through the analysis of sugar fermentation characteristics, it was confirmed that the strain used D-glucose and L-maltose sugar as an energy source As a result of analysis of fatty acid characteristics, it was confirmed that the strain contained 13 unsaturated fatty acids and 2 saturated fatty acids.

According to still another embodiment of the present invention, the strain P3-3 belonging to the genus Chrysobacterium was added to the medium supplemented with the dry hair, and then the hair follicle degradation ability was calculated. As a result, it was found that about 65% The weight reduction rate is shown, and Chrysobacteria sp. The strain P1-3 was found to be excellent in the degradation of hair follicles.

In addition, the present invention provides a culture solution of the strain.

The term "cultivation" in the present invention means cultivation of microorganisms under moderately artificially controlled environmental conditions.

The P3-3 strain of the genus Chrysobacterium can be grown in a conventional medium, and can be cultured in an inorganic salt medium supplemented with, for example, wheat powder. The culture medium may contain nutrients required for culturing, that is, a microorganism to be cultured in order to cultivate a specific microorganism, and may be a mixture in which a substance for a special purpose is further added and mixed. The medium is also referred to as an incubator or a culture medium, and is a concept including both natural medium, synthetic medium and selective medium. The strain P1-3 of the genus Bacterium can be cultured according to a conventional culture method.

The medium used for cultivation should meet the requirements of a specific strain in a suitable manner while controlling temperature, pH, etc. in a conventional medium containing a suitable carbon source, nitrogen source, amino acid, vitamin, and the like. The carbon sources that can be used include glucose and xylose mixed sugar as main carbon sources, and sugar and carbohydrates such as sucrose, lactose, fructose, maltose, starch and cellulose, soybean oil, sunflower oil, castor oil, Oils and fats such as oils and the like, fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture. Nitrogen sources that may be used include inorganic sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine and glutamine, and organic substances such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or their decomposition products, defatted soybean cake or decomposition products thereof . These nitrogen sources may be used alone or in combination. The medium may include potassium phosphate, potassium phosphate and the corresponding sodium-containing salts as a source. Potassium which may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used. Finally, in addition to these materials, essential growth materials such as amino acids and vitamins can be used.

In addition, suitable precursors may be used in the culture medium. The above-mentioned raw materials can be added to the culture in the culture process in a batch manner, in an oil-feeding manner or in a continuous manner by an appropriate method, but it is not particularly limited thereto. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid can be used in a suitable manner to adjust the pH of the culture.

In addition,

a) culturing said strain of the genus Chrysobacterium P1-3; And

b) inoculating the seed culture into a culture medium containing 0.1 to 5% of organic nitrogen source and 0.1 to 5% of carbon source, followed by culturing at a temperature of 28 to 32 ° C and a pH of 5.5 to pH 8.5 (KCTC 12594BP) strain of the genus Chrysobacterium.

In the step b), the incubation temperature is preferably 28 to 32 ° C, more preferably 29 to 31.

In the step b), the pH is preferably from pH 5.5 to pH 8.5, more preferably from pH 6 to pH 8.

Since the organic nitrogen source and the carbon source in the step b) include the carbon source and the organic nitrogen source of the culture medium used in the culturing described above, it is preferable that the organism nitrogen source and the carbon source used in the cultivation of the above-mentioned strain of the genus Chrysobacterium P1-3 (KCTC 12594BP) The overlapping of the nitrogen source and the carbon source is omitted in order to avoid the excessive complexity of the present specification.

Also, the present invention provides a composition for decomposing organic wastes comprising a strain of the genus Pseudomonas aeruginosa P1-3 (KCTC 12594BP) or a culture solution of the strain as an active ingredient.

The term "organic waste " in the present invention means wastes mainly composed of organic matter, among general and industrial wastes.

Also, the present invention provides a method for decomposing organic wastes comprising the step of hydrolyzing a poorly decomposable keratin using a strain of the genus Pseudomonas aeruginosa P1-3 (KCTC 12594BP) or a culture thereof.

The refractory keratin is a poultry feather.

The poultry is at least one member selected from the group consisting of a chicken, a duck, a goose, a turkey, a geese and a pigeon, but preferably a chicken.

The present invention also provides a composition for a detergent comprising a strain of the genus Pseudomonas aeruginosa P1-3 (KCTC 12594BP) and a culture thereof as an active ingredient.

The detergent composition according to the present invention is preferably in a liquid state, but it may be prepared in a solid or paste form.

The composition for a detergent of the present invention may contain at least one of an anionic surfactant, a cationic surfactant, a nonionic surfactant or a positive surfactant as an essential component in a detergent composition. The structure of the anionic surfactant in the surfactant is generally composed of two moieties having a C ₁₂ -C ₁₈ lipophilic linear carbon chain and a polar group ionized at the chain end. Here, the long chain of linear carbon is a long tail in adsorption micelles (admicelle), is nonpolar and adsorbs the contaminating organic material, and the ionized polar group attached to the end of the chain is attached to the nucleus of adsorption micelles It acts as a hair which is adsorbed and arranged uniformly.

In the liquid state, the surfactant can generally be, for example, an anionic surfactant. Examples of anionic surfactants include, but are not limited to, carboxylates, carboxylates such as ethoxycarboxylates, ester carboxylates, alcohol sulfates, alcohol ester sulfates, ethoxysulfates, alpha-olefin sulfates, polyethoxyl alcohol sulfates , Sulfonates such as alkylbenzenesulfonates and alkyl sulfonates, sodium salts such as sodium laurylsulfate, ammonium laurylsulfate, ethoxylated alcohol ethers, alkylpolyglucosides, fatty acid amides, sodium cocoylisothiovate, di Sodium laureth sulfinate, acyl-N-methyl laurate, acyl glucomate and the like.

Cleaning adjuncts may include other components, including amphoteric surfactants such as amine oxides or betaines, as is well known to those skilled in the art. Nonionic surfactants are not ionized in aqueous solution but polarity is dispersed in several functional groups. Usually, oxyethylene (-CH ₂ CH ₂ O-) or oxypropylene (-CH ₂ -CH (CH ₃ ) O-) Repeated several times and shows the structure of the hydrophilic head and the nonpolar tail. And the solubility of nonionic surfactant increases with the number of EO (ethylene oxide, ethylene oxide, or oxyethylene) groups. EO unit useful for adsorption adsorption micelles is 8 ~ 30. Examples include, but are not limited to, alcohol ethoxylates (AE) such as ethoxylated fatty alcohols, monoalkyl polyethylene glycol ethers, polyoxyethylene alcohols, alkyl polyoxyethylene glycols, alkyl alcohol polyethers, alkylpolyglycosides ), Alkylphenol ethoxylates (APE), polyoxyethylene fatty acid esters, polyoxyethylene glycol esters, fatty acid ethoxylates such as fatty acid polyethers, ethoxylated amines, alkylpolyoxyethyleneamines, polyoxyethylene alkylamines, alkyl Fatty amines such as amine polyethers, ethoxylated alkanolamides such as amide ethoxylates, polyethoxylated monoalkanolamides, monoalkanolamidoethoxylates, fatty amide polyglycol ethers, fatty acid amide polyethers, And the like.

In addition to the above components, an acidity modifier, a suitable fragrance and a pigment may be selectively added at a known compositional ratio to produce a conventional kitchen liquid detergent composition. For example, the pH can be adjusted by using at least one of citric acid, malic acid, succinic acid, ascorbic acid, polyoxyethylene, lauryl epoxylate and the like.

The present invention also provides a cosmetic composition for skin exfoliating comprising a strain of Chrysobacterium sp. P1-3 (KCTC 12594BP) and a culture thereof as an active ingredient.

The term "horny" in the present invention means that microorganisms are grown under moderately artificially controlled environmental conditions. The keratin is a type of fibrous structural protein and is a major structural material constituting epithelial cells of skin, hair and nails. The size is very diverse, charged and insoluble, and the primary function is to improve the ability of cells to withstand trauma. In addition, it is responsible for functions such as cell signaling and cell proliferation regulation. Typically, it exists in the epidermis of the skin, in particular, the skeleton of keratinocytes, and is present in the hair follicle, hair follicle, and hair follicle. It is also present in the cornea of the eye, the upper basal layer of the mucosa of the mouth, epithelial cells of the intestine, and the like.

The cosmetic composition of the present invention may further contain conventional auxiliary agents and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments, fragrances and the like which are conventionally used in cosmetic compositions, in addition to the above-mentioned effective ingredients. For example, the cosmetic composition may further contain an auxiliary component such as glycerin, butylene glycol, polyoxyethylene hardened castor oil, tocopheryl acetate, citric acid, panthenol, squalane, sodium citrate, allantoin and the like .

Since the cosmetic composition of the present invention is basically applied to the skin, it can be prepared into any formulation conventionally produced by referring to the cosmetic composition of the related art. For example, they may be formulated as solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations and sprays, But is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritive cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a mask pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, the carrier component may include an animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, have.

When the formulation of the present invention is a powder or a spray, the carrier component may include lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder and the like. Particularly in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane, dimethyl ether, and the like.

When the formulation of the present invention is a solution or an emulsion, the carrier component may include a solvent, a solubilizing agent, and an emulsifying agent. Specific 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, fatty acid esters of sorbitan, and the like.

When the formulation of the present invention is a suspension, a liquid diluent such as water, ethanol, propylene glycol or the like as a carrier component; Suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester; Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, trachant, and the like.

When the formulation of the present invention is an interfacial active agent-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 sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, ethoxylated glycerol fatty acid esters, and the like.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be obvious to you.

Example  1. Isolation and identification of novel strains with excellent degradation keratin protein degradation

In order to isolate and isolate a novel strain having excellent degradation keratin protein degradability, the following experiment was conducted.

More specifically, soil samples were collected from five soil samples in a chicken farming area, and microorganisms isolated from soil samples were treated with inorganic salt medium (ammonium chloride (NH ₄ Cl) 0.05%, sodium chloride (NaCl) 0.05% (1.0%) was added to 0.03% potassium phosphate (K ₂ HPO ₄ ), 0.03% potassium phosphate monobasic (KH ₂ PO ₄ ), 0.01% magnesium chloride (MgCl ₂揃 6H ₂ O), and 0.01% yeast extract. (w / v) at 30 DEG C for 2 days And the microorganism having the ability to hydrolyze the hair was isolated. For identification of isolated microorganisms, genomic DNA was isolated and identified by 16s rRNA PCR and sequencing.

Specifically, for the analysis of the 16S rRNA nucleotide sequence of the isolated strain, the cells were recovered from the minimal culture medium of the strain and then extracted with the Bioneer genomic DNA extraction kit to extract genomic DNA from the recovered cells. 16S rRNA was amplified by polymerase chain reaction (PCR) using the separated genomic DNA as a template. The universal primer used was 27F (5'-AGAGTTTGATCCTGGCTCAG-3 ', SEQ ID NO: 2) 1492R (5'-GGTTACCTTGTTACGACTT-3 ', SEQ ID NO: 3), respectively. The amplified PCR product was purified using PCR Purification system (Solgent, Daejon, Korea). Sequencing to analyze the whole nucleotide sequence of the purified PCR was performed by Solgent, Korea, and the results of the sequencing were compared using BLASTN of NCBI. Sequence homology and phylogenetic tree analysis are based on neighbor-joining methods by comparing sequences of 16S rRNA genes with Chryseobacterium type strains; Bioedit and Mega5 program. The results are shown in Fig.

As shown in FIG. 1, the strain having excellent resolving ability of the degradable keratin protein showed 97% homology with the previously reported genus of Chrysosobacterium. However, it was confirmed that it was a novel strain belonging to the above species because it was not completely identical. Therefore, the strain was named as Chrysosobacterium genus P1-3, and the strain P1-3 of the genus Chrysosobacterium was deposited on May 16, 2014 at the Microbiological Resource Center of the Korea Biotechnology Research Institute And received the deposit number KCTC 12594BP. The sequence of the 16S rRNA of the strain is shown in SEQ ID NO: 1.

Example  2. Cryptobacterium  genus P1 Morphological, sugar fermentation and fatty acid characterization of -3 strains

In order to analyze the morphological, sugar fermentation and fatty acid characteristics of the strain P1-3 of the genus Chrysobacterium isolated and identified in Example 1, the following experiment was conducted.

More specifically, in order to understand the morphological characteristics of the genus Pseudomonas aeruginosa P1-3, Nutrient borth, 30 占 폚, 48 hours (initial pH 7.5) , Streaked on a MacConkey agar, and colonies formed were subjected to Gram staining and observed under an optical microscope (see FIG. 2).

In order to analyze the sugar fermentation characteristics of the genus Pseudomonas aeruginosa P1-3, the API 20 NE kit (Bio Merioux) was used according to the supplier's method and cultured in a 30 ° C incubator for 48 hours . As shown in FIG. 3, the fermentation characteristics of the saccharides were verified and compared using color change and turbidity depending on the growth of the strain (see Table 1).

In order to analyze the fatty acid content and content of the strain P1-3 of the genus Chrysobacterium, a Sherlock MIDI system was used according to the manufacturer's manual. 1 ml of reagent 1 was added to the tube, and 40 strains (loops) of the strains cultured on NA medium were vortexed. The strain was reacted at 100 ° C for 5 minutes, then vortexed again and reacted at 100 ° C for 25 minutes. After cooling the reaction solution, 2 ml of Reagent 2 was added to the reaction mixture, followed by reaction in a constant temperature water bath at 80 ° C for 10 minutes. After the reaction solution was cooled again, 1.25 ml of Reagent 3 was added, and the mixture was stirred for 10 minutes using an orbital shaker. After stirring, the layers were allowed to completely separate. The layer-separated supernatant was transferred to a new tube using a Pasteur pipette, and 3 ml of Reagent 4 was added and stirred. 5 ml of Reagent 5 was added and stirred again. The supernatant was loaded into a vial for GC using a Pasteur pipette. The fatty acid was measured by HP 6890 Gas Chromatograph and H ₂ was flowed into the carrier gas at an ultrafiltration rate of 0.4 ml / min using an ultra-2 capillary column The initial temperature of the column was increased from 170 ° C to 260 ° C at a rate of 5 ° C per minute. The FID was used as a detector, and the fatty acid content and content thereof were analyzed using MIS Sherlock software (see Table 2 and FIG. 4).

The above results are shown in Figs. 2 to 4 and Table 1 and Table 2 below.

Active ingredient result Active ingredient result Potassium nitrate - D-mannose - L-tryptophan + D-mannitol - D-glucose (fermentation) + N-acetyl-glucosamine - L-arginine - D-maltose + Element + Potassium gluconate - Esculin ferric citrate + Capric acid - Gelatin (from cattle) + Adipic acid - 4-nitrophenyl-bD-galactopyranoside - Malian - D-glucose (fairy tale) + Sodium citrate - L-arabinose - Phenylacetic acid + -

Phospholipid  fatty acid compounds Relative abundance of total 포스포이드  fatty acid (%) 10: 0 4.82 11: 0 2OH 1.04 13: 0 ISO 0.60 13: 1 AT 12-13 0.30 14: 0 ISO 0.69 14: 0 1.11 15: 0 ISO 19.78 15: 0 0.36 16: 0 ISO 1.07 16: 0 14.42 15: 0 ISO 3 OH 3.02 ISO 17: 1 w9c 2.94 16: 0 ISO 3 OH 1.19 16: 0 3OH 1.07 18: 0 7.26 17: 0 ISO 3 OH 11.97

As shown in Fig. 2, colonies of P1-3 of the genus Chrysobacterium showed pink or pale red colonies on the MacConkey medium. Gram staining revealed that P1-3 of the genus Pseudomonas aeruginosa Negative bacillus.

Further, as shown in Table 1 and Fig. 3, it was confirmed that the strain P1-3 of the genus Bacterium used D-glucose and L-maltose sugar as an energy source

Further, as shown in Table 2 and Fig. 4, it was confirmed that the strain P1-3 of the genus Bacterium contained 13 kinds of unsaturated fatty acids and 2 kinds of saturated fatty acids.

Example  3. Depending on the culture temperature and pH Cryptobacterium  genus P1 -3 Confirmation of growth of strain

In order to confirm the growth of P1-3 strain of the genus Chrysobacteria according to the change of culture temperature and pH, the following experiment was carried out.

More specifically, the growth rate was observed at 28, 30, 32 and 37 캜 for 48 hours using Nutrient broth, which is a nutrient medium, in order to confirm the optimal growth temperature of strain P1-3 of the genus Chrysobacterium (Fig. 5) . In order to measure the optimum growth pH, the growth was observed at pH 4, 6, 8 and 10 for 48 hours (FIG. 6). The results are shown in Fig. 5 and Fig.

As shown in FIG. 5, it was confirmed that the strain P1-3 of the genus Bacterium could be cultured for 48 hours at 30 ° C.

Also, as shown in FIG. 6, the strain P1-3 of the genus Chrysobacterium was not well grown when the pH was weak (pH 4), but was found to exhibit the maximum activity at pH 6.

Example  4. Cryptobacterium State of Stay P1 -3 Determination of chicken feather resolution

In order to measure the chicken feather resolution of the strain P1-3 of the genus Chrysobacterium, the following experiment was carried out.

More specifically, chicken hams were weighed in an inorganic salt medium by drying in a drying oven at 70 캜 for 24 hours, and the P1-3 strain of Chrysophyte bacterium strain was inoculated and shaking incubator at 37 ° C and 200 rpm for 4 days and 7 days. The filter paper (Whatman filter paper No. 2, 185 mm Φ) was dried in a 70 ° C dry oven for 24 hours and then the dry weight was determined in order to reduce the error as much as possible before filtration of the decomposed chicken wool. The decomposed chicken wool was filtered using filter paper and washed five times with distilled water. The chicken wool, which was filtered on filter paper, was dried in a dry oven at 70 ℃ for 24 hours. The results are shown in Fig.

As shown in FIG. 7, it was confirmed that the late drying weight of the chicken wool showed a weight reduction rate of about 65%, and the strain P1-3 of the genus Chryseobacterium was found to be excellent in the resolution of chicken wool .

Example  5. Cryptobacterium  genus P1 -3 strain Keratinase ( Keratinase ) Active measurement

In order to measure the keratinase activity of the strain P1-3 of the genus Chrysobacterium, the following experiment was carried out.

More specifically, the strain P1-3 of the genus Chryseobacterium was cultured for 7 days in an inorganic salt medium supplemented with 1.0% (w / v) of whey powder, and samples were collected at intervals of 1 to 7 days. The culture broth of the cultured strain of the genus Chrysobacterium sp. P1-3 was recovered by centrifugation at 13,000 rpm and 20 minutes at 4 ° C in a centrifuge (Vision Scientific Co., Top-rim, Yu-sung, Daejeon, Korea) The supernatant was used as crude enzyme solution (Kim Se-jong et al., 2010). The activity of the crude enzyme solution was measured by the method of Anson (1938) and the Folin-Ciocalteu method (Ledoux and Lamy, 1986; Margesin and Schinner, 1991). The substrate solution was used by dissolving 50 mM Tris-HCl buffer to a pH of 7.0 to make the substrate casein 0.6%. 2.5 ml of the substrate solution and 0.5 ml of the crude enzyme solution were mixed and reacted at 37 ° C for 20 minutes. Then, 2.5 ml of 50% (w / v) trichloroacetic acid (TCA) was added to stop the reaction. The supernatant was centrifuged at 13,000 rpm for 20 minutes at 4 ° C to obtain a supernatant. To the supernatant was added 1 ml of 0.55M Na ₂ CO ₃ solution (2.5 ml) and a 5-fold dilution of Folin reagent (SIGMA, USA) 0.5 ml of the solution was reacted at room temperature for 30 minutes and absorbance was measured at 660 nm with a UV-spectrophotometer. Enzyme activity (Unit) was determined by the amount of enzyme liberating 1 μmol of tyrosine from the substrate for 1 minute under the above conditions using the tyrosine standard chart. The results are shown in Fig.

As shown in Fig. 8, it was confirmed that the strain of P1-3 belonging to the genus Chrysobacterium exhibited a maximum keratinase activity at 9.8 unit / ml on the 2nd day of culture. In addition, it was confirmed that the activity tended to decrease overall after keratinase activity.

According to the above results, the strain P1-3 of the genus Chrysobacterium according to the present invention or the culture thereof has excellent decomposing ability of the degradable keratin protein and can be used for processing feeds, fabrics and natural leather, treating organic wastes, It can be used for a variety of industrial applications.

Hereinafter, formulation examples of the cosmetic composition of the present invention will be described, but the present invention is not to be construed as limiting the present invention .

Formulation example  One. Cosmetics  Preparation of composition

1-1. Flexible longevity manufacturing

0.1% by weight of the strain P1-3 of the genus Chrysobacterium or a culture thereof, 5.2% by weight of 1,3-butylene glycol, 1.5% by weight of oleyl alcohol, 3.2% by weight of ethanol, 3.2% by weight of polysorbate 20, -9, 2.0% by weight of carboxyl vinyl polymer, 3.5% by weight of glycerin, a small amount of fragrance, a small amount of preservative, and a remaining amount of purified water were mixed to prepare a softening water by a conventional method.

1-2. Milk lotion  Produce

0.1% by weight of the strain P1-3 of the genus of Chrysosi bacterium or a culture thereof, 5.1% by weight of glycerin, 4.2% by weight of propylene glycol, 3.0% by weight of tocopheryl acetate, 4.6% by weight of liquid paraffin, 1.0% by weight of triethanolamine, , 2.5% by weight of macadamia nut oil, 1.6% by weight of polysorbate 60, 1.6% by weight of sorbitan sesquioleate, 0.6% by weight of propylparaben, 1.5% by weight of carboxyl vinyl polymer, Milk lotion was prepared by mixing purified water with a conventional method.

1-3. Nutrition cream manufacturing

0.5% by weight of glycerin, 3.5% by weight of petroleum jelly, 2.1% by weight of triethanolamine, 5.3% by weight of liquid paraffin, 3.0% by weight of squalane, 2.6% by weight of beeswax, 5.4% by weight of peryl acetate, 3.2% by weight of polysorbate 60, 1.0% by weight of carboxyl vinyl polymer, 3.1% by weight of sorbitan sesquioleate, a small amount of fragrance, a small amount of preservative, Cream.

1-4. Massage cream manufacturing

0.5% by weight of glycerin, 3.5% by weight of petroleum jelly, 3.5% by weight of triethanolamine, 24.0% by weight of liquid paraffin, 3.0% by weight of squalane, 2.1% by weight of beeswax, 0.1% by weight of peryl acetate, 2.4% by weight of polysorbate 60, 1.0% by weight of carboxyl vinyl polymer, 2.3% by weight of sorbitan sesquioleate, a small amount of fragrance, a small amount of preservative and a remaining amount of purified water were mixed, Cream.

1-5. For cleaning Body Cleanser  Produce

1 g of the culture of P1-3 of the genus Chrysobacterium or a culture thereof, 18 g of an anionic surfactant, 5 g of a nonionic surfactant, 7 g of glycerin, 3 g of sodium chloride, 1.5 g of natural olive liquid soap, 1 g of a fragrance and 100 g of water were mixed, To prepare a cleansing body cleanser.

Korea Biotechnology Research Institute KCTC12594BP 20140516

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Novel Chryseobacterium sp. P1-3 producing protease and method          using the same <130> 1-215 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 1399 <212> RNA <213> Chryseobacterium sp. P1-3 16S rRNA <400> 1 cggggagcta ccatgcagcc gagcggtaga ttgctttcgg gcaattgaga gcggcgtacg 60 ggtgcggaac acgtgtgcaa cctgccttta tctgggggat agcctttcga aaggaagatt 120 aataccccat aatataagag acggcatcgt tttttattga aaactccggt ggatagagat 180 gggcacgcgc aagattagat agttggtgag gtaacggctc accaagtcga cgatctttag 240 ggggcctgag agggtgatcc cccacactgg tactgagaca cggaccagac tcctacggga 300 ggcagcagtg aggaatattg gacaatgggt gaaagcctga tccagccatc ccgcgtgaag 360 gacgacggcc ctatgggttg taaacttctt ttgtataggg ataaaccttt ccacgtgtgg 420 gt; acggagggtg caagcgttat ccggatttat tgggtttaaa gggtccgtag gctgatttgt 540 aagtcagtgg tgaaatctca cagcttaact gtgaaactgc cattgatact gcaagtcttg 600 agtgttgttg aagtagctgg aataagtagt gtagcggtga aatgcataga tattacttag 660 aacaccaatt gcgaaggcag gttactaagc aacaactgac gctgatggac gaaagcgtgg 720 ggagcgaaca ggattagata ccctggtagt ccacgccgta aacgatgcta actcgttttt 780 gggatgtaag tttcagagac taagcgaaag tgataagtta gccacctggg gagtacgaac 840 gcaagtttga aactcaaagg aattgacggg ggcccgcaca agcggtggat tatgtggttt 900 aattcgatga tacgcgagga accttaccaa ggcttaaatg ggaaatgaca ggcttagaaa 960 taggcttttc ttcggacatt tttcaaggtg ctgcatggtt gtcgtcagct cgtgccgtga 1020 ggtgttaggt taagtcctgc aacgagcgca acccctgtca ctagttgcca tcattaagtt 1080 ggggactcta gtgagactgc ctacgcaagt agagaggaag gtggggatga cgtcaaatca 1140 tcacggccct tacgccttgg gccacacacg taatacaatg gccggtacag agggcagcta 1200 cacagcgatg tgatgcaaat ctcgaaagcc ggtctcagtt cggattggag tctgcaactc 1260 gactctatga agctggaatc gctagtaatc gcgcatcagc catggcgcgg tgaatacgtt 1320 cccgggcctt gtacacaccg cccgtcaagc catggaagtc tggggtacct gaagtcggtg 1380 accgtaaaag gagctgcct 1399 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27F primer <400> 2 agagtttgat cctggctcag 20 <210> 3 <211> 19 <212> DNA <213> 1492R primer <400> 3 ggttaccttg ttacgactt 19

Claims (10)

( Chryseobacterium sp. P1-3) (KCTC 12594BP) strain producing protease. The method according to claim 1,
Wherein the protease is a keratinase, a strain of the genus Chrysobacterium P1-3 (KCTC 12594BP). A culture of the strain of the genus Chrysobacterium P1-3 (KCTC 12594BP) of claim 1. a) culturing the strain of claim 1; And
b) inoculating the seed culture into a culture medium containing 0.1 to 5% of organic nitrogen source and 0.1 to 5% of carbon source, and culturing at a culturing temperature of 28 to 32 ° C under a condition of pH 5.5 to pH 8.5 (KCTC 12594BP), which comprises the step of culturing a strain of the genus Chrysobacterium P1-3 (KCTC 12594BP). A composition for decomposing organic wastes comprising the strain of the genus Chrysobacterium P1-3 (KCTC 12594BP) or the culture of the strain of claim 3 as an active ingredient. A method for decomposing organic waste, comprising the steps of: (a) culturing the deficient keratin using the strain of the genus Chrysobacterium P1-3 (KCTC 12594BP) or the culture of the strain of claim 3; The method according to claim 6,
Characterized in that the refractory keratin is a poultry feather. 8. The method of claim 7,
Wherein the poultry is at least one selected from the group consisting of chicken, duck, goose, turkey, geese and pigeon. A composition for a detergent comprising the strain of the genus Chrysobacterium P1-3 (KCTC 12594BP) or the culture of the strain of claim 3 as an active ingredient. A cosmetic composition for skin exfoliating comprising the strain of Chrysobacterium sp. P1-3 (KCTC 12594BP) of claim 1 or the culture medium of claim 3 as an active ingredient.

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