JPS6265684A - Antibiotic substance and production and use thereof - Google Patents

Abstract

NEW MATERIAL:A white powdery substance produced from Hypocrea schweinitzii and having the following properties. Melting point, 272-275 deg.C (decomposition); solubility, soluble in methyl alcohol and ethyl alcohol and insoluble in water; color reaction, negative to ninhydrin reaction and Beilstein reaction and positive to Liebermann reaction; Rf value of thin-layer chromatography (silica gel), 0.53-0.67 (chloroform:methanol:ammonia=35:15:1); elemental analysis, C 54.79%, H 7.39%, N 16.7%, free from halogen and sulfur; molecular weight, 2,540 by equilibrium vapor pressure method. USE:Fungicide and herbicide for the protection of plant. PREPARATION:For example, Hypocrea schweinitzii is aerobically cultured in a medium, the body of fungus is separated and homogenized with methanol and the homogenized mixture is filtered to separate the fungal cell. A crude extract produced by removing methanol from the above filtrate is treated with chloroform and the insoluble fraction is fractionated in a silica gel column with a mixture of ethyl acetate and methanol. The obtained active fraction is concentrated to obtain the objective antibiotic substance.

Description

[Detailed Description of the Invention] [Field of Industrial Application] This invention relates to an antibiotic extracted from fungi produced during the culture production of mushroom fungi, a method for producing the same, and its use in the field of protecting and growing plants. It's about how to do it.

[Conventional technology]

Shiitake mushrooms are generally cultivated using oval trees.

In other words, seed pieces or sawdust seeded with shiitake fungi are inoculated onto the crossbars of shiitake mushrooms, and the cultivation of fungal cells and the propagation of mycelia depend on the oval wood to supply nutrients, which in turn promotes the formation of fruiting bodies. The method is to collect the fruiting bodies that have developed.

By the crosspiece. In Shiitake cultivation.

Bacteria that are harmful to Shiitake mushrooms often occur.

These harmful bacteria attack oval trees and cause a significant drop in the yield of shiitake mushrooms, but the harmful bacteria that cause this are:
Many Hypoflaea genus bacteria were detected.

It has been recognized that this is the main cause of damage.

A typical example of these harmful bacteria is the black button mushroom (
Hypocrea schweinitzii) r
Hypocrea nigricans
gricans) + hypoflare and Muroiana (H
ypocrea muroiana).

Such harmful bacteria generally consist mainly of 2-(4-thiazolyl)benzimidazole, for example.

It is known that damage can be suppressed by spraying pest control agents.

[Problem that the invention seeks to solve]

The inventor of this invention focused on the above-mentioned growth-inhibiting effect of Hypophleia genus bacteria on Shiitake mushrooms, took into consideration the active use of this Hypophleia genus bacteria, and investigated whether this could be used in the field of plant protection. The antibiotic compounds contained in this Hypophleiae bacterium have a bactericidal effect against one bacterium.

They discovered that it has an inhibitory effect on the growth of plant radicles, leading to the completion of this invention.

[Means for solving problems]

That is, the present invention relates to Hyp.

crea schweinitzii).

(a) Melting point: 272-275°C (decomposed).

(b) Soluble in methyl alcohol, ethyl alcohol, chloroform, benzene, carbon tetrachloride.

It is sparingly soluble in n-hexane and insoluble in water.

(c) Ninhydrin reaction, Beilstein reaction =
It was negative for 6- and positive for Liberman reaction.

(d) Rf on thin layer chromatograph (silica gel)
(aLl:, 0.53-0.67 (Rio o Hol L: J-J-ru alcohol: Ammonia-35
:5:1).

(e) Elemental analysis values are carbon = 54.79%, hydrogen = 7.39%, nitrogen - 16.71%, and does not contain halogen or sulfur.

(f) The molecular weight measured by vapor pressure equilibrium method is about 2.5
It's 40.

(g) It has an infrared absorption spectrum and a nuclear magnetic resonance spectrum substantially as shown in FIGS. 1 and 2.

A first invention is an antibiotic characterized by this, a second invention is a method for producing this antibiotic, and a third invention is a method for using such an antibiotic.

The antibiotic of the present invention is obtained from Hypocrea 5chiveinitzii, which belongs to the above-mentioned Hybocrea genus.
itzii is a harmful fungus that occurs on the wood of shiitake mushrooms, and is also known as Kurobotantake.It is extremely difficult to obtain because it grows as a harmful fungus on the crossbars of shiitake mushrooms. There is no gender.

In order to obtain the antibiotic of this invention from this black button mushroom, first, the black button mushroom is
uraud) liquid medium (polypeptone 1.0% by weight,
PH6.0 medium containing 2.0% by weight of glucose),
After 10 days of aerobic culture at a temperature of 25°C, the bacterial cells are separated, homogenized with methanol, and further filtered to remove methanol to obtain a crude extract.

The crude extract obtained was then treated with chloroform and the residue was purified using silica gel.

Fractionate with ethyl acetate/methyl alcohol mixture, concentrate the active fraction, further fractionate with ethyl acetate/ethyl alcohol using an alumina column, concentrate the active fraction, and concentrate the active fraction. It can be obtained by dissolving the concentrate in methyl alcohol, adding the same amount of water, filtering the precipitate, and drying it.

The product thus obtained is a white powder and can be characterized by the data described below.

(a) Melting point: 272-275°C (decomposition).

(b) Solubility: Soluble in methyl alcohol, ethyl alcohol, chloroform, benzene.

Slightly soluble in carbon tetrachloride and n-hexane.

Insoluble in water.

(c) Properties: Negative for the ninhydrin reaction used to color amino acids using a ninhydrin reagent, negative for the Beilstein reaction used for the qualitative determination of halogens, Liberman reaction used for the color reaction of steroids It shows positive for.

(d) Measured value Thin layer chromatography using Nijiri gel as a thin film = ratio of movement of each substance to the length from the starting point (chloroform: methanol: ammonia = 35:15:1) to the final development 9 part The Rf value indicating this is 0.53 to 0°67.

(e) Elemental analysis: Carbon = 54.79% of the total,
Hydrogen = 7.39%, nitrogen - 16.71%.

Contains no halogen or sulfur.

(f) Molecular weight: Molecular weight measured by vapor pressure equilibrium method.

It is approximately 2.540.

(G) Infrared absorption spectrum, nuclear magnetic resonance spectrum
: Figure 1 (infrared absorption spectrum).

As shown in Figure 2 (nuclear magnetic resonance spectrum), the maximum absorption of the infrared absorption spectrum is 3,500 (strong) and 3,300 (strong).
Strong) + 3,050 (weak), 2,970 to 2.92
0 (medium) + 2,860 (weak), 1,660~1
, 650 (strong) + 1,540 ~ L530 (weak),
1,460.1,450゜1.380.1,360.1
, 290.1, 215.1, 190 (weak), 700~
500 (broad slightly) cm-'.

(h) Certain Durham-positive bacteria, Durham-negative bacteria and
〇- As described below, it has the effect of killing basidiomycetes with an excellent minimum inhibitory concentration.3 It also inhibits the growth of plant radicles, especially dicotyledonous plant radicles. It has a blocking effect.

In addition, at this concentration that inhibits the radicle of dicotyledonous plants, it does not exhibit any growth inhibiting effect on rice, so it is useful as a herbicide for rice plants.

The use as fungicides and herbicides for plants can be carried out in the usual electrical formulations, eg solutions, emulsions, suspensions, powders, pastes, granules, etc.

Such fungicides and herbicides can be effectively used using a diluent as a liquid, a carrier as a solid, or a bulking agent.

It can also be used in the form of spraying, spraying, coating, etc.

Regarding the antibiotic of this invention, the measurement results of the ultraviolet absorption spectrum did not show a clear spectrum diagram, and circle I was insoluble in water, so accurate measurement results could not be obtained, but it is neutral. It is estimated to be.

[For production]

As mentioned above, according to the research of one of the inventors, the antibiotic of this invention is effective against Gram-positive bacteria. basidiomycetes and plant radicles,
In particular, since it has the effect of inhibiting the growth of dicotyledonous plants, it is effective as a fungicide and as a herbicide for exterminating dicotyledonous plants as harmful plants.

That is, first, the MIC value (minimum inhibitory concentration) for various bacteria was measured using a white antibiotic powder obtained by the culture and isolation method.

In this case, Staphylococcus aureus (St
aphyllococcus aureus FDA 2
09P) + Bacillus subtilis
s ATCC6633), and as a Durham-negative bacterium, Escherichia coli (NIH
J JC-2), Pseudomonas aeruginosa
eruginosa IFO13578), and as a basidiomycete, shiitake mushroom (Lentjnus edod
es), Nameko (Pholiota nameko) +
Oyster mushroom (Pleulotus ostreatus)
) was used.

Additionally, for bacterial strain culture, sensitive broth medium (
Eiken), and regarding basidiomycete culture.

Potato Dextrose Agar(Dirc
Mueller-Hinton medium (Eiken) and Potato Dextrose Aga
This was carried out by the agar plate dilution method using r (Dirco) medium.

These results were as shown in Tables 1 and 2.

Table 1 Table 2 Next, regarding the plant growth inhibiting effect of the antibiotic of the present invention, Seeds of Mitsukuna were inoculated onto filter paper containing various concentrations of the antibiotic of the present invention and 5IIll of sterilized water. When cultured aerobically at a temperature of 25°C, the length of the radicle at the time of germination and growth (mm)
, and elongation rate (%) were measured.

The results are shown in Table 3.

〔Example〕

Hereinafter, this invention will be explained in more detail with reference to Examples.

Example: Hypocrea schweinit, which was separated from Shiitake Matsumoto in Hirai-cho, Kiryu City, Gunma Prefecture.
zii) was aerobically cultured in Sabouraud medium (polypeptone 1.0%, low glucose 2.0%, pH 6.0, 21#) at a temperature of 25°C for 10 days, and the bacterial cells were collected. Approximately 300 g of the wet bacterial cells obtained were homogenized with 21 methyl alcohol, and after filtering the bacterial cells, the crude extract was concentrated to remove methyl alcohol to obtain approximately 50 g of a crude extract.

The obtained crude extract was dissolved in chloroform at 100 IllI! 20.89 g of residue was obtained.

The residue was transferred to a silica gel column (200 g, 31 mmφ
After fractionation using acetic acid ethyl ester/methyl alcohol (the concentration of methyl alcohol was increased by 10% for every 200 ml), the active fraction was concentrated, and further.

Alumina column (150g, 22mmφX 400n
+ m), add 1 ml of ethyl acetate/ethyl alcohol (100 II) to 1 ml of ethyl alcohol.
After fractionation (increasing by 0%), the active fraction was concentrated, the resulting extract was dissolved in methyl alcohol, the same amount of water was added, the precipitate was collected by filtration, and it was dried. 3.56 g of the antibiotic of this invention in the form of a white powder was obtained.

The obtained antibiotic has the above-mentioned physicochemical properties, and mainly uses Corynebacterium sp., which occurs in plants.

), Bacillus sp. +
Clostridium spp.
p, ) + Streptomyces spp.
It has excellent bactericidal activity against gram-endowing bacteria such as S. yces sp, ), and has also been shown to have an effective bactericidal action against gram-endowing bacteria associated with the human body, such as Staphylococcus aureus, Mycobacterium diphtheriae, and Mycobacterium tuberculosis. It is something.

It has also been confirmed that the antibiotic obtained by the above-mentioned isolation method has a herbicidal effect on dicotyledonous plants, in particular, through tests on the radicle of Mitsuzuka cabbage.

〔Effect of the invention〕

The antibiotic of this invention is extracted from harmful bacteria that occur during the cultivation of edible mushrooms such as shiitake mushrooms, and can be obtained at low cost and with high yield through relatively simple operations. It has effective uses as a disinfectant for plant protection and as a herbicide.

[Brief explanation of drawings]

FIG. 1 shows an infrared absorption spectrum of the antibiotic obtained by this invention, and FIG. 2 shows a nuclear magnetic resonance spectrum. 171 Procedural Juichi Official Book (Method) December 1985 Item 9 1. Indication of the case 1985 Patent Application No. 191145 2. Name of the invention Anti-reactive substance and its manufacturing method and its method of use 3. Amendment Patent applicant: 1-2-23 Nishikugata-cho, Kiryu-shi, Gunma Prefecture Mori Sangyo Co., Ltd. Company representative: Yoshimi Mori, male 4, agent ■105 5-12 Shinbashi, Minato-ku, Tokyo No. 1 No. 6, Subject of amendment Drawings (all drawings) 7. Contents of amendment As per the engraving and attached sheet of the drawings originally attached to the application (no change in content)

Claims (3)

[Claims] (1) Hypocrea schwe
(b) soluble in methyl alcohol, ethyl alcohol, chloroform, benzene, carbon tetrachloride, n- Slightly soluble in hexane and insoluble in water; (c) negative for ninhydrin reaction and Beilstein reaction; positive for Liberman reaction; (d) Rf value on thin layer chromatograph (silica gel) is 0.53 to 0.67 (chloroform: methyl alcohol: ammonia = 35:15:1), and (e) elemental analysis values are carbon = 54.79%, hydrogen = 7.
39%, nitrogen = 16.71%, does not contain halogen or sulfur, and (f) has a molecular weight of approximately 2,540 as measured by vapor pressure equilibrium method.
(g) having an infrared absorption spectrum and a nuclear magnetic resonance spectrum substantially as shown in FIGS. 1 and 2. (2) Hypocrea schwe
Initzii) was cultured under aerobic conditions, and the resulting culture revealed that (a) it has a melting point of 272-275°C (decomposition), and (b) it is soluble in methyl alcohol and ethyl alcohol, chloroform, benzene, and tetrachloride. Slightly soluble in carbon, n-hexane, and insoluble in water; (c) Negative for ninhydrin reaction and Beilstein reaction, positive for Liberman reaction; (d) Thin layer chromatography (silica gel) The above Rf value is 0.53 to 0.67 (chloroform: methyl alcohol: ammonia = 35:5:1), and (e) elemental analysis values are carbon = 54.79%, hydrogen = 7.
39%, nitrogen = 16.71%, does not contain halogen or sulfur, and (f) has a molecular weight of approximately 2,540 as measured by vapor pressure equilibrium method.
and (g) isolating a white powdery substance having an infrared absorption spectrum and a nuclear magnetic resonance spectrum substantially as shown in FIGS. 1 and 2. Manufacturing method. (3) Hypocrea schwe
(b) soluble in methyl alcohol, ethyl alcohol, chloroform, benzene, carbon tetrachloride, n- Slightly soluble in hexane and insoluble in water; (c) negative for ninhydrin reaction and Beilstein reaction; positive for Liberman reaction; (d) Rf value on thin layer chromatograph (silica gel) is 0.53 to 0.67 (chloroform: methyl alcohol: ammonia = 35:5:1), and (e) elemental analysis values are carbon = 54.79%, hydrogen = 7.
39%, nitrogen = 16.71%, does not contain halogen or sulfur, and (f) has a molecular weight of approximately 2,540 as measured by vapor pressure equilibrium method.
and (g) has an infrared absorption spectrum and a nuclear magnetic resonance spectrum substantially as shown in Figures 1 and 2, and is used as a plant fungicide or herbicide with an antibiotic as an active ingredient. A method of using an antibiotic characterized by: