KR20020051803A - Bacillus Amyloliquefaciens CHO104 with Antagonic Property and Antimicrobial Compound Produced by This Strain - Google Patents

Abstract

The present invention has an antimicrobial active material isolated from a culture medium of microorganisms identified as Bacillus amyloliquefaciens CHO104 by separating microorganisms having antagonism against mold and bacteria from soil and having an antagonism It relates to a microbial agent. More specifically, an antimicrobial activity named amyloliquetone was discovered from a culture of Bacillus amyloliquifaciens CHO104 (Accession Number: KCTC 18060P) having antagonism.

Centrifuge the culture medium of Bacillus amyloliquifaciens CHO104 to obtain the culture filtrate from which the cells were removed. Finally, the antimicrobial active material was isolated through the steps of, and the structure of the antimicrobial active material was determined by MS and NMR analysis.

Amyloquiquitone and Bacillus amylopiquifaciens CHO104 microbial agent of the present invention will provide a wide range of applications, such as natural antibacterial agent with antimicrobial effect, such as pharmaceuticals, pesticides, foods and the like.

Description

Antagonist microorganism Bacillus amyloliquefaciens crude 104 and antibacterial substance produced by this strain {Bacillus Amyloliquefaciens CHO104 with Antagonic Property and Antimicrobial Compound Produced by This Strain}

The present invention relates to an antimicrobial active substance amyloliquetone (amyloliquetone) isolated from a culture medium of microorganisms isolated and identified with Bacillus amyloquifaciens CHO104 as soil-derived antagonist microorganisms, and a method for separating the same. More specifically, this microorganism (accession number: KCTC 18060P) selected and identified as Bacillus amyloliquifaciens CHO104, which was selected and identified as an excellent antagonistic strain that inhibits the growth of microorganisms such as fungi and bacteria, has an excellent antagonistic effect. The antimicrobial active substance named amyloquiquitone was isolated from the culture medium of microorganisms.

Bacillus amyloliquifaciens CHO104 isolated from soil showed an antagonistic effect of inhibiting the growth of fungi and bacteria, and experimentally confirmed that the bacteria produce antimicrobial actives that inhibit the growth of microorganisms. Since the antimicrobial activity causative agent is unknown, the present inventors have isolated the active substance which is an antagonist contained in the culture obtained by culturing this strain, and succeeded in the structure determination. There is no report on amyloliquitone, which is an antimicrobial active substance produced by Bacillus amyloliquifaciens of the present invention.

The present invention isolates and selects antagonistic microorganisms that strongly inhibit the growth of fungi, bacteria, etc. in the soil, and identifies this strain as Bacillus amyloliquifaciens CHO104. By separating and structuring the natural antimicrobial active substance named quiton, the antimicrobial active substance, which is an antagonist of this strain, was found, and also the separation method was found.

Amyloquiquitone and Bacillus amyloliquifaciens CHO104 microbial agent of the present invention will provide a wide range of applications, such as pharmaceuticals, pesticides, foods, etc. as an antimicrobial agent having an antimicrobial effect.

The present invention is isolated from the soil antagonistic bacteria CHO104 excellent in inhibiting the growth of pathogenic fungi and bacteria in the soil, and identified as Bacillus amyloliquefaciens (Nov. 18, 2000 ) Commissioned to the Institute of Engineering (Bacterial name: Bacillus amyloliquefaciens CHO104, Accession No .: KCTC 18060P).

The culture solution obtained by culturing the microorganism was centrifuged to obtain the culture filtrate from which the cells were removed. Then, the following steps were performed: solvent fractionation, silica gel adsorption column chromatography, Sephadex LH-20 column chromatography, octadecyl column chromatography, and HPLC. Finally, the antimicrobial active material was isolated and the structure of the antimicrobial active body was determined by MS and NMR analysis.

Hereinafter, the present invention will be specifically described based on the following examples.

Example 1 Isolation of Antagonistic Microorganisms from Nature

In order to select microorganisms having antagonism to the pathogenic microorganisms of plants, pathogens were isolated from plants infected with the phytopathogens shown in Table 1.

Antagonist microbial selection for various pathogens was mixed well with 100 mL of tris-HCl buffer solution (pH 7.5) collected from all over the country and incubated in nutrient broth (Nutrient Broth) and incubated for 30 minutes at 25 ℃. A portion of the culture solution was taken and diluted in physiological saline (0.85% NaCl) stepwise and applied to a proper concentration on nutrient agar (Nutrient Agar) and incubated for 3 days at 25 ℃ to isolate the bacterial flora. In order to antagonize the pathogenic pathogens of plants, inoculate each plant pathogenic fungus in the center of potato dextrose agar, incubate at 25 ° C for 1 day, and then inoculate the isolated antibacterial bacteria at four sites. Strains that formed inhibition of growth (Inhibition Zone) after daily replacement culture were selected as antagonistic bacteria. At this time, the degree of antagonism was determined by the following formula by measuring the diameter of mold growth ring in case of replacement culture and in case of only mold culture. As a result, antagonistic bacteria CHO104 excellent in antagonism were selected and their antagonistic power is shown in Table 2.

Zone of Inhibition = (NT-T) / NT × 100

NT: colony diameter of no treatment (mm)

TN: colony diameter of treatment (mm)

Example 2 Inhibition of Microbial Growth of Selected Antagonist Bacteria CHO104

Various plant pathogenic fungi were inoculated on potato glucose agar medium, incubated for 7 days, and 1 mL of sterile Tween 80 solution (0.1%, v / v) and 5 mL of sterile distilled water were added to wash the spores and filtered through a filter paper. . After three times washing the resulting semi spores in sterile distilled water so that it contains 10 ⁷ to 10 ⁸ spores per mL, it was then suspended in Tween 80 and distilled water solution. Antagonist bacteria were inoculated with 1 mL of antagonistic bacteria in 50 mL of SDBP medium (3% soy sauce, 5% glucose, 0.1% beef extract, 0.1% proteose peptone) and incubated at 30 ° C. for 3 days.

1) Inhibition of harmful fungi by spraying antagonistic broth

Inoculate each plant pathogenic fungus with potato glucose agar medium and incubate for 1 day. Inoculate each of the fungus with potato glucose agar medium and incubate for 1 day. Then, pour 1 mL of the culture solution containing the antagonist CHO104 onto the surface of the growing fungus. Incubation for 7 days was compared with the control group to check the antagonism. As a result of the experiment, as shown in Table 3, all pathogenic fungi showed more than 95% inhibition rate, in particular, rot rot (rot), anthrax, spot deciduous (spot deciduous), ovule, stem blight (bronchial), blue Fungi, wilting disease, diarrhea, gray mold, root rot, purple-wing disease, and white-wing disease were all suppressed.

2) Inhibitory Effect of Plant Pathogenic Fungus on Media Containing Antagonistic Bacteria CHO104 Filtrate

A medium containing antagonistic bacteria CHO104 culture filtrate was prepared. That is, 39 g of potato glucose medium was dissolved in 250 mL of water and sterilized. After cooling, 750 mL of CHO104 culture filtrate was added thereto to prepare a medium. Inoculation of plant pathogenic fungi and incubation at 30 ° C. for 7 days was carried out to investigate the degree of growth inhibition of the fungus and expressed as the degree of antagonism (Zone of Inhibition). Phytopathogenic fungi were cultured in solid medium containing antagonistic bacterial culture filtrates; rotted rot, anthracnose, spotted deciduous disease, spot deciduous disease, ovule, stem blight, rot, rot, root rot The fungus of wing-pattern disease (marrow disease) and white-wing disease (white-moon disease) was completely inhibited as shown in Table 4.

3) Inhibitory Effect of Plant Pathogenic Fungi on Medium Containing Heat Treatment Culture Solution of Antagonist Bacteria CHO104

A medium containing antagonistic bacteria heat-treated culture was prepared. That is, after the culture supernatant obtained by centrifugation after culturing for 15 minutes at 121 ℃ dissolved in powder 39 g of potato glucose medium corresponding to the total volume of the culture medium in a heat treatment culture medium was prepared after sterilization. Inoculation of plant pathogenic fungi and incubation at 30 ° C. for 7 days was carried out to investigate the degree of growth inhibition of the fungus and expressed as the degree of antagonism (Zone of Inhibition). As a result of culturing plant pathogenic fungi in solid medium containing antagonistic bacterial heat-treatment broth, rot rot (rot), anthrax, ovulation, stem blight (warring disease), wilting disease, gray mold, root rot, purple wing pattern disease ( Fungal growth, such as advisory disease) and white-wing disease (white-moon disease) were completely suppressed as shown in Table 5.

Example 3 Identification of Antagonistic Microorganism CHO104

In order to identify CHO104, which was selected as an antagonistic strain, the morphological properties, culture characteristics, and physiological biochemical properties of microorganisms were examined. Biolog MicroLog 34.01A was identified as Bacillus amyloliquefaciens as a result of 99% likelihood (PROB) and similarity (SIM) 0.74. According to these results and the classification criteria described in Bergey's manual of systematic bacteriology and microbiological methods, the CHO104 strain was identified as Bacillus amyloliquefaciens . This antagonist ( Bacillus amyloliquefaciens CHO104) was entrusted to the Institute of Biotechnology (Accession No .: KCTC 18060P).

Example 4 Culture of Antagonist Bacteria Bacillus amyloliquefaciens CHO104 (Accession Number: KCTC 18060P)

1) Growth according to culture temperature

The growth of Bacillus amyloliquefaciens CHO104 was investigated in the temperature range of 25 ~ 45 ℃. Experimental results showed the best growth at 33 ℃.

2) Effect of pH on Growth

As a result of culturing the antagonists while changing the pH of the medium, the most suitable pH range was determined to be about 7.

3) Bulk Cultivation

Bacillus amyloliquefaciens CHO104 subcultured in a Nutrient Agar medium for 2 to 3 times as a parent strain, and 1 platinum strain was added to 5 mL of SDBP (Soy Dextrose Beef ex Proteos Peptone) medium ( Soy sauce 3%, glucose 5%, beef extract 0.1%, proteose peptone 0.1%) was incubated for 33 hours at 33 ℃. After incubation in SDBP medium, 1% of the strains were collected from the cultured strains, transferred to a new SDBP medium, and cultured for 24 hours to be used for identification of antagonists.

Example 5 Isolation of Natural Antibacterial Active Material

The culture filtrate of Bacillus amyloliquifaciens CHO104 was centrifuged (5,000 rpm, 15 minutes), and the culture filtrate (7 L) from which the cells were removed was adjusted to pH 3 with 1 N HCl, and then partitioned with ethyl acetate. Neutral fraction (EtOAc-soluble acidic and neutral fraction) and aqueous fraction were obtained. The ethyl acetate soluble acidic and neutral fractions were partitioned with 2% NaHCO ₃ adjusted to pH 8.0 to obtain an ethyl acetate soluble neutral fraction and an aqueous phase fraction. The antimicrobial activity of the obtained fractions was analyzed by Staphylococcus aureus and mold by the paper disk method (Zaika, LL, J. Food safety , 9, p. 97-118, 1988). Botrytis cinerea) showed strong antimicrobial activity in the ethyl acetate soluble neutral fraction, and the antimicrobial activity assay was then performed by Staphylococcus aureus to purify the active material.

Elution fractions of ethyl acetate soluble neutral fraction (421 mg) were gradually eluted by increasing the concentration of methanol solution in the ethyl acetate solution using silica gel adsorption column chromatography. v / v) fraction, yielding 127 mg of active fraction. This active fraction was fractionated by Sephadex LH-20 column chromatography (methanol-chloroform 4: 1, v / v) and assayed for activity.Ve/Vt (elution volume / column bed volume) was found at 0.64-0.78. Activity was shown in the eluted fractions. This active fraction (120 mg) was eluted with octadecsilane column chromatography (methanol-water) to assay for activity. As a result, a fraction eluted with a solvent of methanol-water 80:20 (v / v) (14.6 mg) was extracted. ) Showed activity. This active fraction (14.6 mg) was separated into single peaks at a retention time of 19 minutes by HPLC (Senshu pak, 8 × 250 mm, 70% methanol, 1.5 mL / min) equipped with a reversed phase column, and was formed into a white powder of 3.36. mg was successfully isolated. As a result of assaying the activity against Streptococcus aureus by placing 10 μg of the isolated active substance on a 6 mm paper disc, the size of the growth inhibitory ring was 12 mm, and benzoic acid (200 μg) used as a control was 10 mm. In addition, the minimum inhibitory concentration (MIC) of the isolated active body was 5 μg or less.

Example 6 Structure Determination of Amyloquiquitone by Instrumental Analysis

The mass spectrometry measured by mass spectrometer (MS, Jeol JNM AX 505 WA, 30 eV) to determine the structure of amyloriquitone was severe molecular cleavage at 70 eV employed in conventional EI-MS. Although not possible, the molecular ion [M +] was measured in m / z ( rel. Int. ) At 402 (1.3), and the fragment ions were 384 (15.4), 366 (18.6), 273 ( 15.6), 255 (16.0), 227 (9.0), 172 (20.7), 133 (35.1), 107 (54.8), 81 (82.0), 67 (base peak), and high resolution mass spectrometry (HR-MS). ) Was m / z 402.2401, and the measured molecular formula was C ₂₄ H ₃₄ O ₅ . In addition, the results of analysis and mass spectrometry such as ¹ H-NMR, ¹³ C-NMR, and HMBC (spectral presentation) measured by a nuclear magnetic resonance analyzer (NMR, Varian Unity Inova 500, 500 MHz, CD ₃ OD) Based on the results, the structure of amyloriquitone was determined as follows.

Example 7 Antibacterial Activity Assay of Amyloquiquitone

The antimicrobial activity of amyloriquitone isolated from the culture solution of Bacillus amyloliquifaciens CHO104 by the method described in Example 5 was determined by the paper disk method (Zaika, LL, J. Food safety , 9, p. 97-118, 1988). Was carried out. That is, 0.1 mL of the preculture solution of Staphylococcus aureus KCTC1928 was mixed with 20 mL of sterile medium adjusted to 45 ° C. by the pour plate method, and then solidified in a Petri dish. , 6 mm paper disk containing the antimicrobial active material was put up and diffused with 0.85% saline, and then cultured at 30 ° C. for 16 hours to measure the effect of growth inhibition ring size (mm). Nutrient medium (Difco) was used as the medium. In addition, the antifungal activity test against the fungus was assayed by forming an inhibition zone on potato glucose agar plates (4 mm thick) coated with spores of Botrytis cinerea . In other words, using a sterilized cork borer (diameter 9 mm), after making a round agar hole and put amyloriquitone incubated at 28 ℃ for 4 days, the activity was measured by the size of the growth inhibition. Table 6 shows the results measured using benzoic acid as a control in Staphylococcus aureus and Botrytis sinerea.

That is, amyloriquitone showed strong antimicrobial activity against fungi and bacteria.

The present invention has been selected as antagonistic microorganisms in soil, identified as Bacillus amyloliquifaciens Bacillus amyloliquifaciens CHO104 (Accession Number: KCTC 18060P) and structured amyloid separated from the culture medium of this microorganism as an antimicrobial active material Loriquiton and the culture medium containing this microorganism are natural antimicrobials and can be widely used in medicines, pesticides, and foods.

Claims (8)

An antagonistic microorganism Bacillus amyloliquefaciens CHO 104 (Accession No .: KCTC 18060P) isolated from soil and a microbial agent consisting of this microbial culture. The pathogen of the plant which can be applied to the microbial preparation is a rot, anthrax, spotted deciduous disease, spot deciduous disease, stem blight, rhythm blight, wilted fruit disease, wilt disease Microorganisms characterized by plant pathogens, including pathogenic fungi, such as (disease disease), gray mold disease, root rot disease, purple-winged disease, and white-winged disease. The method of claim 1, wherein the strain is incubated at 30 to 35 ℃ in Soy Dextrose Beef ex Proteos Peptone medium (soy 3%, glucose 5%, beef extract 0.1%, proteose peptone 0.1%) Microbial agent characterized in that. Amyloliquetone, an antimicrobial active material obtained from a culture of Bacillus amyloliquifaciens CHO104 (Accession No .: KCTC 18060P). Culture step of Bacillus amyloliquifaciens CHO104, separation of the culture filtrate, and the aqueous phase fraction and the ethyl acetate soluble acidic and neutral fraction (EtOAc-soluble acidic / neutral fraction) distributed by adjusting the culture medium to pH 3 (1 N HCl) Separation step and the step of diluting the above ethyl acetate soluble acidic and neutral fractions with a weak alkaline 2-5% NaHCO ₃ (pH 8.0) solution and separating the aqueous fractions and the ethyl acetate soluble neutral fractions, and Elution fractions of the ethyl acetate neutral fractions were gradually eluted by increasing the concentration of methanol in the ethyl acetate by silica gel adsorption column chromatography to obtain the active fractions, and the active fractions were separated by Sephadex LH-20 column chromatography. A gel filtration step for separating and separating the active fraction from the active fraction by octadecyl column chromatography; The method of antibacterial material disclosed is Bacillus amyl quinone Lowry Pacific Enschede CHO104 characterized in that it comprises the step of isolating the water-based solvent by the production - with the group of the purified active fractions of the reverse phase HPLC of methanol. A method of using Bacillus amyloliquifaciens CHO104 culture drug containing amyloliquitone, the active substance of claim 4, as a microbial agent for medicines, pesticides, and foods. A method of using the amyloriquitone, the antimicrobial active substance of claim 4, as a leading substance of various active substances or medicines, pesticides, foods, etc. A method of attaching or substituting other functional groups to the structure of amyloriquitone according to claim 4 for use.