WO2020161760A1

WO2020161760A1 - Thermosporothrix hazakensis-derived antimicrobial active material

Info

Publication number: WO2020161760A1
Application number: PCT/JP2019/003813
Authority: WO
Inventors: 英晃 ▲高▼野; 実人佐藤; 昭一天野; 初美 ▲高▼野; 賢志上田; 一夫降旗; 庄平作田; 明横田; 修平矢部
Original assignee: 株式会社県南衛生工業
Priority date: 2019-02-04
Filing date: 2019-02-04
Publication date: 2020-08-13
Also published as: JPWO2020161760A1

Abstract

The present invention provides: a novel compound having antimicrobial activity; and a method for producing same. Provided is a compound represented by formula (I), or a salt or an ester thereof.

Description

Antibacterial active substance derived from Thermosporothrix hazakensis

The present invention relates to a novel compound having antibacterial activity and a method for producing the same.

Thermosporothrix hazakensis (Thermosporothrix hazakensis, hereinafter also referred to as "T. hazakensis") is a bacterium belonging to the order of the phytochlorophyllous phylum Cteddonobacteria and is an aerobic gram-positive bacterium. The present inventors have isolated the ^T. hazakensis SK20-1 ^T strain (= NBRC 105916T= JCM 16142T= ATCC BAA-1881T) and shown that it has the ability to degrade cellulose, xylan, and chitin ( Non-Patent Document 1). So far, antibacterial active substances derived from T. hazakensis have been reported (Non-patent documents 2 and 3), but further antibacterial active substances have been demanded.

The present invention aims to provide a novel compound having antibacterial activity and a method for producing the same.

As a result of repeated intensive studies to solve the above problems, the present inventors found a novel antibacterial active substance from ^T. hazakensis SK20-1 ^T strain (= NBRC 105916T= JCM 16142T= ATCC BAA-1881T), The present invention has been completed.

The present invention includes the following inventions.
Formula (I):

Or a salt or ester thereof.
(2) culturing Thermosporothrix hazakensis in a medium and collecting the compound of formula (I), or a salt or ester thereof from the culture, or a compound of formula (I) Method for producing salt or ester.
(3) The method according to (2) above, wherein Thermosporothrix hazakensis is Thermosporothrix hazakensis SK20-1 ^T strain.
(4) An antibacterial agent containing the compound of formula (I) described in (1) above, or a salt or ester thereof as an active ingredient.

According to the present invention, it is possible to provide a novel antibacterial active substance derived from ^T. hazakensis SK20-1 ^T strain (= NBRC 105916T= JCM 16142T= ATCC BAA-1881T), and a method for producing the same.

FIG. 1-1 is a chart showing ¹ H-NMR spectrum of HK-1. FIG. 1-2 is a chart showing ¹³ C-NMR spectrum of HK-1. FIG. 1-3 is a diagram showing a COZY spectrum of HK-1. FIG. 1-4 is a diagram showing an HSQC spectrum of HK-1. FIG. 1-5 is a diagram showing an HMBC spectrum of HK-1. FIG. 1-6 is a diagram showing a ROESY spectrum of HK-1. FIG. 1-7 is a diagram showing the result of NMR spectrum analysis of HK-1. FIG. 1-8 is a diagram showing a ¹³ C-NMR spectrum in which isotope shift of HK-1 due to deuterium substitution is observed. FIG. 2 is a diagram showing the monomer structure of HK-1. FIG. 3 is a diagram showing a planar structure of HK-1. FIG. 4-1 is a diagram showing the results of highly heat-treated HK-1 antibacterial activity measurement after pH fluctuation. FIG. 4-2 is a diagram showing the results of measuring the antibacterial activity against B. subtilis with changes in pH, storage temperature, and the passage of time. FIG. 4-3 is a diagram showing the results of antibacterial activity measurement against G. stearothermophilus with pH changes and storage temperature and the passage of time.

The present invention provides formula (I):

And a salt or ester thereof (hereinafter, also referred to as a compound of the present invention). The compounds of the present invention have high antibacterial activity. The compounds of the invention have a high antibacterial activity, especially against Gram-positive bacteria.

The compound of formula (I) has the following structure:

Has a dimeric structure. The dimer is due to peroxide formation via oxygen at the C-8 position. When the compound of formula (I) has an asymmetric carbon, all enantiomers and mixtures thereof are included, and when stereoisomers are present, all stereoisomers and mixtures thereof are included. .. The compounds of formula (I) may form hydrates, solvates or crystalline polymorphs. The glycosidic bond at the C-10 position may be either an α bond or a β bond.

The salt of the compound of formula (I) is not particularly limited and various salts can be used. Examples thereof include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium and magnesium, ammonium salts, organic amine salts such as triethanolamine salts and triethylamine salts. Furthermore, these salts may form hydrates, solvates or crystalline polymorphs.

The ester of the compound of the formula (I) is not particularly limited, and examples thereof include esters with fatty acids such as acetic acid and aromatic carboxylic acids such as benzoic acid. Fatty acids include saturated fatty acids and unsaturated fatty acids, short chain for the chain length, including medium and long chains, specifically acetic acid, propionic acid, butyric acid, caproic acid, caprylic acid, lauric acid, myristylic acid, Examples include, but are not limited to, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, decenoic acid, oleic acid, erucic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid and the like. Further, the aromatic fatty acid includes those having a substituent such as benzoic acid, ortho, meta and/or para position of benzoic acid, such as amino group and hydroxyl group, and also includes polycyclic aromatic carboxylic acid such as naphthoic acid. .. The compound of formula (I) may form an ester at 1 to 12 positions among the hydroxyl groups in formula (I). These esters can be obtained by a conventional method, for example, by reacting a corresponding carboxylic acid with a compound of formula (I) under an acidic catalyst.

The present invention comprises culturing Thermosporothrix hazakensis in a medium and collecting the compound of formula (I), or a salt or ester thereof from the culture, or a compound of formula (I), or It also relates to a method for producing the salt or ester (hereinafter, also referred to as the production method of the present invention). According to the production method of the present invention, the compound of the present invention can be obtained in a high yield.

For culturing T. hazakensis in the production method of the present invention, a normal microbial culture method is used. As the medium, either a synthetic medium or a natural medium can be used as long as it appropriately contains an assimilable carbon source, a nitrogen source, an inorganic substance and necessary growth/production promoting substances. As the carbon source, glucose, starch, dextrin, mannose, fructose, maltose, lactose, molasses, etc. may be used alone or in combination. Furthermore, if necessary, hydrocarbons, alcohols, organic acids, amino acids (tryptophan, etc.) and the like are also used. As the nitrogen source, ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, urea, peptone, meat extract, yeast extract, dry yeast, corn steep liquor, soybean powder, cottonseed meal, casamino acid, etc. may be used alone or in combination. .. In addition, inorganic salts such as sodium chloride, sodium chloride, potassium chloride, magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous sulfate, calcium chloride, manganese sulfate, and zinc sulfate may be added as necessary. Add. Further, trace components that promote the growth of the microorganism used and the production of the compound of the present invention can be added appropriately, and those skilled in the art can select appropriate components.

Culturing T. hazakensis in such a nutrient medium can be performed according to the method usually used in the production of antibiotics by general microorganisms. Usually, it is suitable to culture under aerobic conditions, and usually, it can be performed with stirring and/or aeration. As the culture method, any of static culture, shaking culture, and liquid culture accompanied by aeration and stirring can be used, and shaking culture is suitable.

The culture temperature that can be used is not particularly limited and can be appropriately selected as long as the growth of T. hazakensis is not substantially inhibited and the antibiotic can be produced. Particularly preferable is a culture temperature in the range of 35 to 50°C. The medium can be cultured at a pH of 3 to 11, preferably 6 to 10. Culturing can usually be continued until the antibiotic is fully accumulated. The culturing time varies depending on the composition of the medium, the culturing temperature, the working temperature, the used producing strain, etc., but usually the desired antibiotic is produced and accumulated in the culture solution and the microbial cells by culturing for 2 to 8 days.

The amount of new antibiotics accumulated in the culture can be quantified by the paper disk method used for usual antibiotic activity tests using Geobacillus stearothermophilus 111499 strain and/or Bacillus subtilis NBRC 3134 ^T strain as an assay bacterium. it can.

For novel antibiotics accumulated in the culture and cells, collect them from the culture. After culturing, if necessary, after separating the bacterial cell and the supernatant by a separation method known per se such as filtration or centrifugation, the supernatant is subjected to solvent extraction using an organic solvent, particularly acetone or the like, adsorption or ion exchange ability. The novel antibiotics can be isolated and purified from the culture supernatant and collected by using the chromatography utilizing the above, the gel filtration, the chromatography utilizing the liquid-liquid partition, alone or in combination. As a carrier for chromatography having adsorption or ion exchange ability, activated carbon, silica gel, porous polystyrene/divinylbenzene resin or various ion exchange resins can be used. The cells are extracted with 50% acetone and then isolated and purified by using various chromatographies alone or in combination as in the case of the supernatant. Thus, a new antibiotic with the above mentioned properties is obtained.

As a result of investigating the antibacterial activity of the compound of formula (I), Geobacillus stearothermophilus 111499 strain, Bacillus subtilis NBRC 3134 ^T strain, Streptomyces griseus NBRC 15744 ^T strain, Escherchia coli NBRC 102203 ^T strain, Thermosporothrix hazakensis SK20-1 ^T /NBRC It was found that they have particularly excellent antibacterial activity against 105916 ^T strain, Thermosporothrix narukonensis F4 ^T strain, Streptomyces sp. AGRN-7 strain, Streptomyces sp. AGRN-8 strain, and Streptomyces sp. AGRN-9 strain.

The present invention also relates to an antibacterial agent containing the compound of formula (I), or a salt or ester thereof as an active ingredient (hereinafter, also referred to as an antibacterial agent of the present invention). The antibacterial agent of the present invention is prepared by mixing the compound of formula (I), or its salt or ester as an active ingredient, with a conventional liquid or solid carrier such as ethanol, water, starch and the like. You may use it. The effective concentration is not particularly limited as long as it can exert antibacterial properties, and can be, for example, 1 to 1000 μg/ml.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

[Example 1] Purification of antibacterial active substance "HK-1" produced by Thermosporothrix hazakensis SK20-1 ^T <Used strain>
Thermosporothrix hazakensis SK20-1 ^T /NBRC 105916 ^T
<Use medium>

<Experimental method>
1. Cultivation of T. hazakensis 1.1 Inoculation of bacteria, 250 ml of YS medium was prepared in a 300 ml Erlenmeyer flask, and sterilized (121°C, 20 min) in an autoclave. After cooling to 50°C or lower, 25 ml was dispensed into a petri dish (10 plates were prepared). A piece of ice from a glycerol stock of T. hazakensis stored at -80°C was radially inoculated on one YS medium with a toothpick. Static culture was carried out at 50°C for 4 days.

1.2 Seed culture 100 ml of LB(+M) medium was prepared in a 500 ml baffled Erlenmeyer flask and sterilized by autoclave (121°C, 20 min). After cooling to 50°C or lower, 1 cm ^{2 of} toothpick was used to extract T. hazakensis from the plate inoculated with "1.1 Inoculation" and inoculated. It was cultured in a shaking incubator at 45° C. and 135 rpm for 24 hours.

1.3 Culture by jar fermenter DIAION HP-20 (Nippon Rensui Co., Ltd.) is used for two 4 L mini jars (ABLE Co., Ltd.) and one 5 L mini jar (Maruhishi Bio Engine Co., Ltd.), 10 g and 15 g, respectively. added. Make 7 L of R ₂ A(+L) medium in 10 L plastic beaker, add 2 L each to 4 L mini jar, add 3 L to 5 L mini jar, and sterilize by autoclave (121 °C, 15 min) .. After sterilization, the medium was left to stand for 1 day to cool. Seed-cultured T. hazakensis was inoculated at 0.1% of the medium amount in each mini jar. After connecting to the control device, half the amount of the medium was introduced, and shaking culture was carried out at 45° C. at a rotation speed of 160 rpm for a 4 L mini jar and 135 rpm for a 5 L mini jar for 4 days.

2. Extraction Experiment 2.1 Extraction The total volume of 28 L of the culture solution containing HP-20 in each mini jar was combined and sieved through a sieve of DIAMETER 315/OPENING 200 μm and DIAMETER 200/OPENING 300 μm to recover bacterial cells and HP-20. An equal amount was dispensed into four 500 ml centrifuge tubes, and the same amount of 100% acetone as the sample was added. After vigorous stirring, it was shaken in a reciprocal shaking incubator (135 rpm) for 30 minutes. The centrifuge tube was centrifuged with a cooling centrifuge (4°C, 6,000 rpm, 10 min). The supernatant obtained from the centrifugation was suction-filtered with a Kiriyama funnel, the filtrate was transferred to Nascoruben (for evaporator), and then distilled by an evaporator to remove acetone in the sample. A total of 2.8 L of concentrated solution was stored in a 4°C room.

2.2 Liquid-liquid distribution
(1) Acetone aqueous solution The extracted sample was concentrated to 800 ml with an evaporator to completely remove acetone.
(2) The obtained sample was adjusted to pH 5.7 with 6 N hydrochloric acid.
(3) The whole amount was put into a 3,000 ml separating funnel, and 400 ml of ethyl acetate was added.
(4) The separating funnel was vigorously stirred for 15 to 30 seconds, the mouth was directed upward, the stopper was opened, and the gas was discharged.
(5) After repeating the operation of (4) 5 or 6 times, the solution was allowed to stand until it was separated.
(6) The lower layer and the upper layer were divided into 2,000 ml Erlenmeyer flasks, and the upper layer was used as an ethyl acetate extract.
(7) The lower layer was repeated twice in the same procedure as (3) to (6) above.
(8) All the ethyl acetate extracts were combined, placed in a 3,000 ml separating funnel, and 500 ml of 5% aqueous sodium hydrogen carbonate solution was added.
(9) After performing the above operations (4) to (5), the lower layer was used as an extract and 500 ml was collected.
(10) The upper layer was repeated again in the same procedure as (8) to (9) above.
(11) The obtained lower layer (1,000 ml) was adjusted to pH 5.7 with 6 N hydrochloric acid.
(12) The whole amount of the lower layer was placed in a 3,000 ml separating funnel, and an equal amount of ethyl acetate was added.
(13) After performing the above operations (4) to (6), the upper layer was used as an extract and 500 ml was collected.
(14) The lower layer was repeated twice in the same procedure as (3) to (6) above.
(15) All of the ethyl acetate extracts were combined, an appropriate amount of anhydrous sodium sulfate was added (to the extent that water disappeared to become a solid), and the mixture was allowed to stand at room temperature for 15 minutes.
(16) After filtering with dew paper, it was dried using an evaporator.
(17) The yield was measured using an electronic balance.

3. Silica gel column chromatography 50 g of C-200 silica gel (Wakogel(R) C-200, Fujifilm Wako Pure Chemical Industries, Ltd.) was suspended in 100 ml of ethyl acetate and poured into the column. The column was tapped from the outside and the silica gel suspension was added dropwise. The dried ethyl acetate extract was dissolved with 6 ml of ethyl acetate and loaded onto the column. Ethyl acetate was added from the upper part, and when the color (yellow color) of the target substance reached the lower part, the eluate was collected in 15 ml portions in a test tube. When the fractions no longer showed coloration, the solvent was changed to ethyl acetate:methanol=1:1 and fractions were collected until the eluate had no color.

4. Thin layer chromatography Every other fraction of the ethyl acetate eluate collected was spotted on a TLC plate (Silica gel 60 F254, MERCK KGaK) using a capillary. After drying with a drier, development was performed using a developing solution (ethyl acetate:acetic acid=4 ml:0.5 ml). After the developing solution reached about 1 cm above the TLC plate, it was taken out from the developing layer and dried with a drier. Spots were detected on the TLC plate under UV254 nm and UV325 nm irradiation. The fraction with few contaminated spots was concentrated using an evaporator and then dried in a desiccator. The yield was measured using an electronic balance.

<Results>
HP-20 added to the culture solution and a 50% acetone extract of the bacterial cells were concentrated and then extracted with ethyl acetate to obtain 1.0 g of an extract. The obtained extract was subjected to silica gel column chromatography and eluted with ethyl acetate. The eluate was fractionated, and as a result of TLC analysis of each fraction, a single spot (hereinafter referred to as HK-1) was confirmed in the 18th to 25th fractions. 240 mg of HK-1 was obtained from the 18th to 25th fractions.

[Example 2] Structural analysis of HK-1 [1. Physical and chemical properties of HK-1]
<Experimental method>
1. MS
A small amount of HK-1 was dissolved in methanol. It was subjected to ESI(-)/TOF-MS spectrum analysis. The analytical equipment is as follows.
Agilent 1110 series (pump)
The Accu TOF JMC-T100LC(MS)
2. HRMS
We asked the National Institute of Pharmaceutical Sciences for measurement. HRMS is (MH) ^- was to analyze the profile of the ion. The analytical equipment is as follows.
Agilent 1200 series (pump)
Agilent 6530 Q-TOF (MS)
3. UV
A small amount of HK-1 was dissolved in methanol and the absorption maximum was measured with a JASCO V-630 Spectrophotometer.
4. pH stability 20 mg of HK-1 was weighed in a 50 ml Falcon tube with an electronic balance, 20 ml of methanol was added, and the mixture was lightly stirred (1000 γ). Dispense into two 15 ml falcon tubes, 3.3 ml each. 20 μl of 1 N sodium hydroxide was added to one of them and stirred gently. The pH was confirmed with pH test paper. 10 μl of each sample was subjected to HPLC. The HPLC analysis conditions are as follows.
Column: CAPCELL PAK C ₁₈ 5 μm 4.6 mm lD × 150 mm
Mobile phase A: 0.1% trifluoroacetic acid aqueous solution Mobile phase B: acetonitrile Column temperature bath temperature: 40°C
Flow rate: 1 ml/min
UV: 254 nm
Elution method: Concentration gradient 0 to 18 minutes B concentration 50% to 100%
18.1 minutes to 23 minutes B concentration 100%
23.1 to 25 minutes B concentration 50%

<Results/Discussion>
HK-1 is a light yellow powder, and its methanol solution also showed similar color. In the UV spectrum of HK-1 measured with an ultraviolet-visible spectrophotometer, an absorption maximum was observed at 340 nm, and the molar extinction coefficient was 30630. The ESI / TOF / MS spectra measured in negative mode, in the vicinity of m / z 1645 (MH) ^- ion believed ions were observed, it was suggested that contain halogen atoms from the signal pattern. Furthermore, as a result of analyzing the profile of ions around m/z 1645 by HRMS, it was found that HK-1 is a compound of the molecular formula C ₇₄ H ₅₈ Cl ₈ O ₂₆ having 8 chlorine atoms. It was found that the solution of HK-1 adjusted to pH 9 and adjusted to pH 9 was stable because no decomposition product peak was observed in HPLC.

[2. HK-1 NMR spectrum]
<Experimental method>
80 dimethyl sulfo mg of HK-1 to 0.75 ml dimethylsulfoxide -d _6, 99.9% D (for NMR, Kanto Chemical Co., Ltd.) was dissolved in. Quartz cotton was packed in a Pasteur pipette, the dissolved HK-1 solution was passed through, and the NMR tube was filled. The cap was closed, and the film was sealed with parafilm and protected from light (NMR instrument, Varian Inova 500).

<Results/Discussion>
¹ H-NMR, ¹³ C-NMR, COSY, HSQC, HMBC and ROESY spectra of HK-1 are shown in FIGS. 1-1 to 1-6. Analysis of these NMR spectra revealed that HK-1 had the partial structure shown in FIG. 1-7. This partial structure includes all 37 carbon signals observed in the ¹³ C-NMR spectrum, suggesting that HK-1 has a structure in which this partial structure is a dimer.

[3. Hydrolysis reaction with hydrochloric acid]
<Experimental method>
1. Hydrolysis 1 mg of HK-1 was placed in a 20 ml eggplant flask containing a stir bar. While adding 1 ml of acetonitrile and slowly stirring, 1 ml of 1 M hydrochloric acid was slowly added drop by drop. The mixed solution was heated under reflux for 30 minutes on an oil bath, returned to room temperature, and then freeze-dried.

2. HRMS
The lyophilized hydrochloric acid hydrolyzate was requested to be measured by the National Institute of Health Sciences. HRMS is (MH) ^- the analysis of the profile in the vicinity of the ion. The analytical equipment is as follows.
Agilent 1200 series (pump)
Agilent 6530 Q-TOF (MS)

<Results/Discussion>
HK-1 was hydrolyzed by heating under reflux in 50% acetonitrile containing hydrochloric acid at a concentration of 0.5 M, and then the hydrolyzate was directly analyzed by negative mode Q-TOF/MS. In the obtained mass spectrum, an ion having a maximum intensity of m/z 1413 was observed, and HRMS revealed that the molecular formula of the hydrolyzate was C ₆₄ H ₄₂ Cl ₈ O ₂₀ . The molecular formula of this hydrolyzate was consistent with that of the aglycone that results when the two 5-deoxyfuranosides of HK-1 are removed by hydrolysis. Therefore, it was shown that a hydroxyl group is bonded to the 3-position carbon of 5-deoxyfuranoside of HK-1.

[4. ¹³ C-NMR spectrum analysis]
From the analysis so far, it was considered that HK-1 has a structure in which a hydroxyl group is bonded to C-3″ of the partial structure in FIG. 1-7 to form a dimer. Addition of these will satisfy the molecular formula of the monomer structure part of HK-1.In Fig. 1-7, carbons to which a chlorine atom can be bonded are C-2, C-4, C-8, C-. 2'and C-5', but since oxygen is bonded to C-8, a chlorine atom can be bonded to C-2, C-4, C-2' and C-5'. It became clear.

[5. Confirmation of hydroxyl bonding position]
The bonding positions of the six hydroxyl groups present in HK-1 differ from those of the hydroxyl group-bonded carbons in the ¹³ C-NMR spectrum due to the isotope shifts in D ₂ O and H ₂ O. Was confirmed using a method for determining the carbon bonded to the. When measured using a double-tube NMR tube containing D ₂ O and H ₂ O solutions, respectively, the ¹³ C-NMR spectrum shown in Fig. 1-8 was obtained, and C-3' and C-6' with a hydroxyl group bonded were obtained. , C-8', C-10', C-2" and C-3", two signals were observed, and the binding positions of 6 hydroxyl groups were confirmed.

[6. Structure of HK-1]
From the above, it was revealed that HK-1 has a dimer structure shown in FIG. 3 which forms a peroxide between oxygen at the C-8 position of the monomer structure shown in FIG. Table 2 shows the attribution of each signal in the ¹ H-NMR and ¹³ C-NMR spectra. Natural products with diphenyl peroxide structure are rare and known only in bungein A isolated as a plant component (Hui Yang, Ai-Jun Hou, Shuang-Xi Mei, Han-Dong Sun and Chun-Tao Che, 2002 , Constituents of Clerodendrum Bungei, Journal of Asian Natural Products Research, 4 165-169). 5-Deoxyfuranoside 5 carbon chemical shift (99.78(C-1”), 72.75(C-2”), 70.21(C-3”), 77.15(C-4”) and 15.90(C-5 ))), the glycosidic bond at the C-10 position is likely to be 5-deoxyl-β-lyxofuranose (Joseph R, Snyder and Anthony S Serianni, 1987, Synthesis and NMR-spectral analysis of unenriched and [1- ¹³ C]-enriched 5-deoxypentoses and 5-O-methylpentoses, Carbohydrate Research, 163(1987) 169-188). (5-deoxylyxose) has never been reported as a natural product so far, and is the first in HK-1.

[Example 3] HK-1 activity measurement test [1. Antibacterial spectrum test]
The antibacterial activity of HK-1 was assayed for the following cells using a paper disc.

<Used strain>
Geobacillus stearothermophilus 111499
Bacillus subtilis NBRC 3134 ^T
Streptomyces griseus NBRC 15744 ^T
Escherchia coli NBRC 102203 ^T
Thermosporothrix hazakensis SK20-1 ^T /NBRC 105916 ^T
Thermosporothrix narukonensis F4 ^T
Streptomyces sp. AGRN-7
Streptomyces sp. AGRN-8
Streptomyces sp. AGRN-9
<Use medium>

<Experimental method>
1. G. stearothermophilus antibacterial assay One piece of ice was inoculated with a toothpick from a G. stearothermophilus glycerol stock stored at -80°C on LB agar medium (see Example 1). The cells were cultured at 60°C for 18 hours. After culturing, 1/2 platinum loop was taken and suspended in 500 μl of sterilized water. The suspended bacterial solution was diluted 10 times with sterile water. 100 μl of the diluted solution was added dropwise to LB agar medium, and spread on a spreader until dry. One paper disk was placed on an exposed paper, 50 μl of HK-1 solution adjusted to 1000 γ with methanol was dropped, dried and then placed on an agar medium coated with bacterial cells. After culturing at 60° C. for 18 hours, the diameter of the inhibition circle was measured.

2. B. subtilis, E. coli antibacterial assay Assay was performed in the same manner as in 1 above except that the culture temperature was changed to 37°C.

3. S. griseus antibacterial assay One piece of ice from S. griseus glycerol stock stored at -80°C on YMPD agar was inoculated on one side with a toothpick. It was cultured at 28°C for 5 days. After culturing, a small amount of spores were scraped off with a cotton swab and the whole surface was inoculated in YMPD medium. One paper disk was placed on an exposed paper, 50 μl of HK-1 solution adjusted to 1000 γ with methanol was dropped, dried and then placed on an agar medium coated with bacterial cells. After culturing at 28°C for 4 days, the diameter of the inhibition circle was measured.

4. T. hazakensis, T. narukonensis antibacterial assay YS medium (see Example 1) was assayed in the same manner as 3 above except that the culture was changed to 50°C for 2 days.

5. Streptomyces sp. AGRN-7 antibacterial assay Assay was carried out in the same manner as 3 above except that the culture was changed to ISP2 medium at 45°C for 2 days.

6. Streptomyces sp. AGRN-8, 9 antibacterial assay Assay was carried out in the same manner as 3 above except that the culture was changed to BM medium at 45°C for 2 days.

<Results/Discussion>
The diameter of the inhibition circle in each Gram-positive bacterium is B. subtilis 32.0 mm, G. stearo-thermophilus 34.0 mm, S. griseus 38.0 mm, and the HK-1 producing bacterium T. hazakensis is 10.0 mm and its related strains T. narukonensis also had the same 10.0 mm. Further, Streptomyces sp. AGRN-7 (Streptomyces thermodiastaticus 99% similar), which is a thermophilic actinomycete isolated from the compost of Agora Landscaping Co., Ltd., is 22.0 mm, Streptomyces sp. AGRN-8 (Similar to Streptomyces mexicanus 99%) is 16.0 mm. mm, Streptomyces sp. AGRN-9 (similar to Streptomyces leeuweenhoeeckii 99%) formed an inhibition circle of 19.0 mm. In addition, E. coli, a gram-negative bacterium, formed a 14.0 mm stop circle.

From the size of the inhibition circle, it was shown that HK-1 has high antibacterial activity against Gram-positive bacteria. In addition, it has been found that it exhibits antibacterial activity against various thermophilic actinomycetes. It is known that the group of actinomycetes that produce antibiotics is highly resistant to the drug, but the results show that HK-1 is an effective antibiotic against the actinomycetes. It was Moreover, since it also exhibits antibacterial activity at a high concentration against T. hazakensis, which is a producing bacterium, it is possible that the producing bacterium may control the production amount of HK-1 to be below the lethal concentration. ..

[2. Measurement of minimum growth inhibitory concentration]
The minimum inhibitory concentration (MIC) is one of the antibiotic susceptibility tests and means the minimum concentration of an antimicrobial substance that inhibits visible growth of microorganisms. In the field of research, MIC measurement is also used to compare the effects of new antibiotics and conventional substances and to verify the effects on several strains, and it is an important criterion for evaluating antibiotics. The MIC of the antibacterial active substance "HK-1" obtained this time was measured by a dilution method using a liquid medium.

<Used strain>
Geobacillus stearothermophilus 111499 MERCK
Bacillus subtilis NBRC 3134 ^T
<Use medium>

<Experimental method>
1. Preparation of G. stearothermophilus bacterium solution One piece of LB agar medium was inoculated with a toothpick on a piece of ice from a glycerol stock of G. stearothermophilus stored at -80°C. The cells were cultured at 60°C for 18 hours. 1 cm ² of the medium was cut out with a toothpick, placed in a long test tube containing 10 ml of liquid LB medium, and cultured by shaking at 60° C., 300 rpm for 18 hours. The culture solution was diluted to about McF standard solution No. 1 with sterile water.

2. Preparation of B. subtilis bacterial solution Preparation assay was performed in the same manner as in 1 above except that only the temperature was changed to 37°C.

3. Preparation of HK-1 supplemented medium 2 mg HK-1 was weighed into a 2 ml Eppendorf tube using an electronic balance. To an Eppendorf tube, 2 ml of methanol was added and dissolved to obtain a 10 kγ HK-1 solution. It was passed through a sterile 0.22 μm filter. Using 100 μl of HK-1 solution, 50 μl of 2-fold dilution series (10 kγ to 19 γ) was prepared using methanol. Each 1% was added to a test tube containing 5 ml of LB liquid medium. The culture solutions prepared in 1 and 2 above were added to the test tube in an amount of 1% each. The culture was performed at each temperature (G. stearothermophilus, 60°C, B. subtilis, 37°C), 135 rpm, 18 h. After culturing, visual observation and OD ₆₀₀ were measured using a test tube obtained by adding 1% methanol and sterilized water to a 5 ml LB liquid medium as a control.

<Results/Discussion>
Both strains inhibited the growth of both strains to a concentration of 0.78 μg/ml. From the OD ₆₀₀ measurement results, no significant difference was observed in test tubes with turbidity of 0.78 μg/ml or less. The high-concentration HK-1-supplemented medium exhibited a slight yellow color compared to the normal LB medium. The color did not change after the culture.

From the above results, it was shown that HK-1 has a relatively strong antibacterial effect on Bacillus subtilis B. subtilis and food spoilage-causing bacterium G. stearother-mophilus.

[3. Antibacterial activity test when pH is changed]
The stability of a compound is regarded as one of the important information for evaluating a substance. This is because if the compound itself is stable even if the external environment changes, it can be applied in various situations and the information can lead to the acquisition of biological knowledge. Therefore, the stability of the activity was investigated by measuring the antibacterial activity by G. stearothermophilus and B. subtilis using the solution prepared in the above "1. Antibacterial spectrum test".

<Used strain>
Geobacillus stearothermophilus 111499
Bacillus subtilis NBRC 3134 ^T
<Use medium>

<Experimental method>
1. Preparation and Assay of Antibacterial Activity Assay Medium 1.1 Bacillus subtilis Antimicrobial Activity Assay Medium (10 plates)
<Preparation of Bacillus subtilis spore suspension>
(1) 250 ml of LB medium was prepared in a 300 ml Erlenmeyer flask and sterilized by autoclave (121°C, 20 min).
(2) After cooling to 50°C or lower, 25 ml was dispensed into a petri dish (10 plates were prepared).
(3) 30 μl of B. subtilis spore solution was inoculated on 10 sheets of LB agar medium (see Example 1) with a cotton swab.
(4) Incubation was carried out at 37°C for 1 week.
(5) 3 ml of sterilized water was added to one piece of sterilized absorbent cotton, and the spores for one plate were scraped off.
(6) About 3 ml of the bacterial solution was sucked from the absorbent cotton with a 1 ml syringe and transferred to a sterilized 50 ml Falcon tube.
(7) The above (5) and (6) were performed 10 times in total.
(8) A 50 ml Falcon tube containing about 30 ml of the bacterial solution was heated for 30 minutes in a bath at 60°C.
(9) Centrifugation (6,000 rpm, 10 min, 4° C.) with a cooling centrifuge and discarding the supernatant.
(10) To the precipitate, 30 ml of sterilized water was added and stirred.
(11) The above (9) and (10) were performed twice in total.
(12) After centrifuging with a cooling centrifuge (6,000 rpm, 10 min, 4° C.) and discarding the supernatant, a small amount of sterilized water to dissolve the precipitate was added.
(13) The mixture was heated in a bath at 60°C for 30 minutes.
(14) Stored at 4°C and used as a suspension of B. subtilis spores.

<Preparation of Bacillus subtilis antibacterial activity assay medium>
(1) 100 ml of NB medium was prepared in a 200 ml Erlenmeyer flask and sterilized by autoclave (121°C, 20 min).
(2) After cooling to 50° C. or lower, 100 μl of B. subtilis spore suspension was added.
(3) After stirring lightly, 10 ml each was dispensed into a petri dish.
(4) After coagulation, it was stored at 4°C.

During assay, incubated at 37°C for 18 hours.

1.2 Geobacillus stearothermophilus medium for antibacterial activity assay (10 plates)
(1) 10 ml of LB medium (see Example 1) (autoclaved) was dispensed into two long test tubes.
(2) Each long test tube was inoculated with 50 μl of a commercially available G. stearothermophilus spore suspension.
(3) Each long test tube was cultured by shaking at 60° C. and 300 rpm for 24 hours.
(4) 100 ml of AM2 medium was prepared in a 200 ml Erlenmeyer flask and sterilized by autoclave (121°C, 20 min).
(5) After cooling the above medium to 50° C. or lower, 20 ml of G. stearothermophilus culture solution was added.
(6) After gently stirring, 10 ml of each was dispensed into a petri dish.
(7) After coagulation, it was stored at 4°C.

During assay, incubated at 50°C for 18 hours.

1.3 Method for measuring activity of medium for assaying antibacterial activity: Paper disc assay A paper disc in which 50 μl of the sample was dipped was placed on each bacterial weight layer medium (B. subtilis, G. stearothermophilus). The medium was incubated. The medium was taken out of the incubator, and the diameter of the inhibition circle was measured with a caliper (kanon hardened stainless 150 mm).

<Results/Discussion>
In B. subtilis, the diameter of the inhibition circle of the unadjusted pH solution was 18.5 mm, while that of the solution adjusted to pH 9 was 20.2 mm. In G. stearothermophilis, the diameter of the inhibition circle of a pH-unadjusted solution was 21.9 mm, whereas that of a solution adjusted to pH 9 was 22.6 mm. The solution adjusted to be basic had a slightly larger inhibition circle than the control.

[4. Stability test during temperature fluctuation]
Typical tests for measuring the stability of a compound include a pH stability test, a heat resistance test, and a storage test. This information is also important in evaluating the compound as described above. Using the sample of HK-1 prepared in the above "3. Antibacterial activity test when pH was changed", G. stearothermophilus and B. subtilis were used as an index to examine whether the compound is stable or not.

<Used strain>
Geobacillus stearothermophilus 111499
Bacillus subtilis NBRC 3134 ^T

<Experimental method>
1. Heat resistance test A
300 μl of the 1000 γHK-1 solution (pH 9 and unadjusted) prepared in “3. Antibacterial activity test when pH was changed” was dispensed into a 1 ml assist tube. The mouth of the tube was sealed with Parafilm, and the mixture was heated in a bath at 98°C for 5 minutes. The assay was carried out using the antibacterial activity assay medium prepared in "3. Antibacterial activity test when pH was changed".

2. Heat resistance test B
The two samples prepared in the above “3. Antibacterial activity test when pH was changed” were dispensed into 1-ml assist tubes in 1-ml aliquots. The mouths of a total of four tubes were sealed with parafilm, and each tube was stored in a freezer set at -28°C and an incubator set at 28°C and 50°C, protected from light with aluminum foil. Each sample was assayed using the antibacterial activity assay medium prepared in "3. Antibacterial activity test when pH was changed" 1, 3, 6, 12, 24 days after the start of storage.

<Results/Discussion>
Regarding the heat resistance test A, HK-1 was adjusted to pH 9 and heated at high temperature. As a result, the two samples did not lose their antibacterial action, although there were some differences in the numerical values (Fig. 4-1).

Regarding the above-mentioned heat resistance test B, the antibacterial activity of the two samples was not lost even after 24 days, indicating that they are compounds resistant to high temperature. Even with unadjusted HK-1, no significant difference due to storage temperature was observed (Figs. 4-2 and 4-3).

The compound of the present invention is useful as an antibacterial agent.

All publications cited in this specification are incorporated by reference in this specification as they are.

Claims

Formula (I):

Or a salt or ester thereof.
A compound of formula (I), or a salt or ester thereof, comprising culturing Thermosporothrix hazakensis in a medium and collecting the compound of formula (I), or a salt or ester thereof from the culture. Manufacturing method.
The method according to claim 2, wherein the Thermosporothrix hazakensis is Thermosporothrix hazakensis SK20-1 T strain.
An antibacterial agent containing the compound of formula (I) according to claim 1 or a salt or ester thereof as an active ingredient.