PSEUDOMONAS PROTEGENS JR52 STRAIN HAVING ANTIMICROBIAL ACTIVITY AND USES THEREOF

In the embodiment hereinafter embodiments through one or more corresponding business are provided as follows. However, in the embodiment of the present invention to account for one or more of these embodiments illustratively and not the limited to these range in the embodiment.

In the embodiment 1: On derivatives and identification

1 - 1: Strain of isolating and culturing

Antimicrobial activity for separating microorganisms in the soil collecting a sample of fine powder was also ch'unch'on gangwon gangwon university one. 10 G sample is a soil sample after 30 ml total volume 50 ml conical tube so packed PBS (NaCl 8. 5%) Was added to a 30 minutes agitating (shaking). After PBS sample using continuous dilution (1/10³ , 1/10⁴ ) And, Nutrient agar (peptone 5, beef extract 1. 5, Yeast extract 1. 5, And agar 15 g/L NaCl 5; hereinafter NA medium by transcribing) soil sample dilution was a continuous plate 100 micro l human herpesvirus. Human herpesvirus in a sample plate 30 °C 24 hours (colony) have grown by culturing a variety separating, new medium was isolated colony passaging (subculture).

1 - 2: Identification of strains (identification)

16S rDNA sequence (sequence number 1) to mark said in the embodiment 1 - 1 strains isolated agents result in that it exhibits a homology of 99% Pseudomonas pro [...] analysis request checks, separating strain Pseudomonas pro reel [keyn (hereinafter, by transcribing JR52) identifying JR52 finally to him. 2 December 2016 JR52 identifying Pseudomonas strains by using biomass center reel [keyn Korean Institute bio-technology KCTC18518P strain strain profile for impurities.

In the embodiment 2: Strain isolated JR52 identifying antimicrobial activity

2 - 1: JR52 billion number identifying microbial growth of a strain effect

To make sure that the antimicrobial activity of a strain which is separated in said in the embodiment 1 JR52 flat medium diffusion method (agar well diffusion) by water.

Recombinant Candida albicans (CandidaAlbicans), Bacillus subtilis (Bacillus subtilis), Outer [...] (Staphylococcus Aureus), The wetting ability Aspergillus age (Aspergillus niger), Pseudomonas base labor and management (PseudomonasAeruginosa) Is using the low of the passenger domains (Escherichia coli) 48 Hours LB him as a medium. Then, OD₆₀₀ 1 Corrected to a microorganism culture NA plate and each said 6 species a 100 micro l herpesvirus, LB medium (Luria Bertani; tryptone 10, and NaCl 10 g/L yeast extract 5) 100 micro l JR52 strains are cultured in a culture of each drip-gate. Plate 18 to 24 hours culturing, the resulting transparent band diameter were measured.

As a result, and also to table 1 1 6 species such as shown in the growth of the microorganisms in strains of JR52 verify billion number cream, in particular B. Subtilis, S. Aureusand C. AlbicansFor been excellent antimicrobial activity can be ascertained.

The disclosure to non-patent document 1 Lactobacillus Plantarum FL9 FL9 strains compared to the strain S. Aureus, E. Coli and P. AeruginosaRespect to the 18 mm, 14 mm and 18 mm and therefore is transparent block or an active microbial growth can be know JR52 strain billion number significantly.

2 - 2: Minimum growth billion (minimum inhibitory concentration; MIC) identifying number concentration JR52 strain

3 Strains in optimum medium JR52 LB 30 °C during culturing, 40 ml culture on screen centrifuge (7,500 g, 10 minutes, 4 °C) culture supernatants after a obtained. The culture supernatants obtained ethyl acetate (99. 5% By weight) in 40 ml by adding 25 °C extracted from the 30 minutes. Said extract (rotary evaporator) and concentrated using rotary evaporator 40 °C reducing pressure in the culture supernatants extract obtained. Culture supernatants obtained using extract B. Subtilis and S. AureusMinimum inhibitory concentration for a long time.

NB optimum medium Mcfaland haze 0. 5 A by a user B. Subtilis Or S. Aureus A 96 well plate (well plate) dispensing a culture 100 micro l respectively, JR52 strain culture supernatant extract 6. Concentration of 67 mg/Ml beginning at 2 times after dilution of the grooves 100 micro l his four magnetic segments. After 24 hours in culture it starved 30 °C [ley person (resazurin; Sigma non-Aldrich co. , American) each well by dispensing a solution 30 micro l, 15 minutes in 30 °C minimum inhibitory concentration are observed to color change after further culturing were measured.

As a result, strains such as JR52 2 also appears in B. Subtilis and S. Aureus6 Against. Verify activity at concentrations of 67 mg/Ml were.

The disclosure to non-patent document 2 Staphylococcus Sp. 1E strain is compared, MIC is 1E 6 strains. 75 JR52 strain can be know to excellent antimicrobial activity.

In the embodiment 3: It is sour with gun [e(Siderophore) Confirmation

3 - 1: JR52 strain It is sour with gun [e Production method for checking

2 Strains in culture during 30 °C JR52 LB optimum medium after centrifuging (7,500 g, 10 minutes, 4 °C) a few supernatant obtained.

Next CAS medium (60 mg CAS (Chrome Azurol S), 1 mm FeCl3, 10 mm HCl 10 ml, 72. 9 Mg HDTAB, 16 g agar, 30. 2 G pipes and pH 6. 8) Plate and small number, using 10 mm diameter cork see (cork borer) went well. Adding 100 micro l JR52 well culture supernatants strain, plate 30 °C 48 hours of culturing cancer conditions well to production therefrom whether it is sour with gun [e whether or not a yellow ring around.

As a result, such as adding a yellow ring around 3 JR52 strain also appears in the larger well confirmed it is sour with gun [e strains to produce JR52 his car.

3 - 2: JR52 strain It is sour with gun [e Identifying production

JR52 OD after 2 to 3 strains during cell culture₆₀₀ 1 A new medium 10% concentration is corrected is provided which reduces, after retrieving the 24 hour culture of 15 ml centrifuge (3,400 g, 30 minutes) to a few supernatant obtained. 1 M hydrochloric acid (HCL) culture supernatants obtained using a pH of pH 2. 9 The output buffer, comparable in 30 minutes by adding 300 rpm of culture supernatants as well as ethyl acetate extraction shaker (Recipro shaker RS provided 1, Jeio Tech) extracted from the times using 1. Centrifuging the extracted solution (3400 g, 15 minutes, 4 °C) to 5 ml ethyl acetate layer and an upper layer Hathway reaction solution (0. 1 M FeCl₃ 1 Ml, 0. 1 M potassium ferricyanide 1 ml, 0. 1 L distilled water) with respect to a 5 ml 30 minutes reaction. For improving substances while using the OD₇₀₀ In absorbance when the measuring, quantifying the tooth using a standard curve is dihydroxy benzo (dihydroxy benzoic acid) was.

As a result, such as about 0 2 4 also appears in culture JR52 strain difference. It is sour with gun [e produce the average concentration of 13 mm it is sour with gun [e production were confirmed.

The disclosure to non-patent document 3 PseudomonasFluorescensPFMDU 3, strain PFMDU 8 and PFMDU 9 is compared, it is sour with gun [e since it is sour with gun [e JR52 said cells 12 to 13 umol/Ml concentration of strains producing PFMDU 3 from producing strains, strains possess a significantly higher than PFMDU 8 and PFMDU 9 can be boosted.

3 - 3: JR52 producing strains It is sour with gun [e Analysis

It is sour with gun word meaning JR52 using tetra nicotineamid inspection (Tetrazolium test) would strain the kind for him. Specifically, a small amount of sodium hydroxide (NaOH) solution drop 2N Tetrazolium chloride 1 - 2 added 1 ml culture supernatants strains when the JR52 after processing, it is confirmed whether instantaneous red color developing reaction are observed to vary.

As a result, 5 such as also appears in red color of solution 1 ml culture supernatants of a strain JR52 vary in making sure that the instantaneous processing queue. The result of experiment it buys may [thu JR52 (Hydroxamates) strains of hydroxy-based it is sour with gun [e to produce his car.

In the embodiment 4: JR52 time dependent strain (Time-a killing assay) identifying antimicrobial activity

3 Strains in optimum medium JR52 LB 30 °C during culturing, a 40 ml culture after retrieving the centrifugal separator (7,500 g, 10 minutes, 4 °C) a few supernatant obtained. Culture supernatants obtained after centrifuging using a hydrochloric acid 6N pH2 to regulate (10000 g, 20 minutes, 4 °C) separating the pellet (pellet), adding 15 ml methanol separated the pelletizing of the chains suspensions. Using pH7 1N NaOH methanol suspension is then regulated so as to extract a hydrophobic filter (hydrophobic filter; 0. 22 Micro m pore) with respect to the conduit. Using rotary evaporator (rotary evaporator) reducing pressure in the filtered extract 40 °C and concentrated culture supernatants extract JR52 strain obtained.

B. Subtilis 100 Micro l culture (10⁵ CFU/Ml) dispensing a micro tube, 2 hours 30 minutes by adding 100 micro l said JR52 strain culture supernatants extract recovery interval 100 micro l culture was. Retrieving the inoculating the culture medium after a few NB B. SubtilisOf determined the CFU.

As a result, also 6 such as shown in the B. Subtilis Culture supernatants performance JR52 strain when required, B. Subtilis The individual water excellent antimicrobial activity before arcing occurs verify JR52 strain been can be ascertained.

The number into the tank by the present invention to the preferred embodiment the transformed for flaws. The present invention is in the field of the present invention is provided essentially from deviating from a person with skill in the art of the present invention is embodied in the form of modified inputted properties may be understand it will rain. The definitive aspect as well as the descriptive disclosure in the embodiment are contemplated aspect should. The aforementioned range of the present invention description and claim rather than as shown, and the present invention is in a range equal to all differences may be carried on an will be interpreted.