JPS6231905B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a novel macrolide antibiotic-producing bacterium that exhibits extremely excellent antibacterial properties against Staphylococcus and Mycoplasma. Means for Solving the Problems The present invention provides a new macrolide antibiotic-producing actinomycete, Micromonospora griseorvida.
A11725 strain (Micromonospora griseorubida
A11725) is cultured in a medium, and the culture is used to develop new macrolide antibiotics (hereinafter referred to as A11725 and A11725).
A11725) is extracted and isolated from a new macrolide antibiotic producing bacterium. The novel antibiotics A11725 and A11725 of the present invention are basic nitrogen-containing compounds having the following physicochemical properties, respectively.

[Table] Antibacterial spectrum (indicating minimum inhibitory concentration) of the antibiotic of the present invention (free base) by agar dilution method
is as shown in the table below.

[Table] From the above properties, A11725 and
A11725 is a 16-membered ring macrolide substance containing one sugar each of desosamine and 6-deoxy-2,3-di-O-methyl-hexose. (where R ₁ represents a hydrogen atom or a hydroxyl group) and does not have an aldehyde group in the molecule _, A11725 has a hydrogen atom in its R ₁ , and A11725 has a hydroxyl group in its R Among the substances shown, based on these facts, this substance is judged to be a new substance as there is no known substance among antibiotics belonging to the basic macrolide group. The substance of the present invention is usually a pharmaceutically acceptable mineral acid,
It can be used in the form of salts with organic acids, etc., such as tartrates, citrates, succinates, etc. The antibiotics of the invention can be taken orally as tablets and powders or can be applied by intravenous injection. The amount used is approximately 400 to 2000 mg per day for an adult, and it is effectively used against respiratory infections caused by Gram-positive bacteria such as Staphylococcus. Furthermore, the antibiotic of the present invention can be used as an antibiotic for feed addition or for animal treatment. The novel macrolide antibiotic-producing actinomycetes of the present invention are actinomycetes isolated from soil in a potato field in Unazuki-cho, Shimoshinkawa-gun, Toyama Prefecture, and belong to the genus Micromonospora. - Referred to as Micromonospora griseorubida A11725. (Feikoken Article No. 705) The mycological properties are described below. [] Morphological characteristics The basal hyphae are long, wavy, and simply branched, with a diameter of 0.6 to 0.8μ, and no hyphal divisions are observed. Spores are formed individually at the tips of short sporophores that arise from basal hyphae, and are spherical or ovoid in size, 1.0 to 1.5μ in size, and their surfaces have thorn-like protrusions, which at first glance does not appear. This is Mr. Compeito. Also, on an agar medium, depending on the medium composition, undeveloped aerial mycelia may be formed, or a black spore layer may be formed on the colony surface. [] Growth status on various media The results of culturing at 30°C for 20 days on various media are shown in Table 2. Color indications are based on the Color Harmony Manual, 4th edition 1958 (Container Corporation of
It follows the color classification by America).

【table】

【table】

【table】 [] Physiological properties Physiological properties are shown below. (1) Assimilation of carbon sources

【table】

[Table] (Pridham Codolive agar medium contains D-glucose and the growth of this bacteria was poor, so 0.5% yeast extract and 1.5% agar medium was used as the basal medium.) (2) Growth temperature range : 15-45℃ (3) Liquefaction of gelatin: glucose, peptone,
Positive on gelatin medium (4) Hydrolysis of starch: Positive on starch/inorganic salt agar medium (5) Skimmed milk: Positive on both peptonization and coagulation (6) Production of melanin-like pigment: Tyrosine agar and peptone yeast/iron agar Negative in culture medium (7) Salt tolerant [Inter.J.System.Bacteriol.21, 240
-247 (1971) method was followed. ]

[Table] (8) Decomposition of cellulose: Negative (9) Production of nitrous acid [Inter.J.System.
The organic medium of Bacteriol. 21, 240-247 (1971) was used. ]: Negative As mentioned above, the strain attaches one spore to the tip of the sporophyte produced from the branched basal hyphae,
Since it does not form true aerial hyphae and is a mesophilic bacterium, it is a strain of Micromonospora griseorubida, which belongs to the genus Micromonospora. The present invention will be explained below with reference to examples, but it is not limited thereto. Example [A] 100 ml of a medium (PH7.0) containing 1% dextrin, 1% glucose, 0.5% casein hydrolyzate, 0.5% yeast extract, and 0.1% calcium carbonate was dispensed into a 500 ml Erlenmeyer flask, 120℃,
Heat sterilized for 20 minutes. One platinum loop from a slant culture of Micromonospora griseorbida A11725 strain was inoculated into 10 bottles of this medium, and incubated at 30°C.
Shaking culture was performed for 120 hours. This was then mixed with 30% of heat-sterilized medium containing 20% of the same composition as above.
The cells were transplanted into a jar fermenter and cultured with aeration at 30°C for 72 hours under the conditions of sterile air at 300 rpm/min. Then heat-sterilized medium containing 5% dextrin, 0.5% glucose, 3% defatted soy flour, and 0.2% calcium carbonate (PH 7.2)
Transfer the above culture to a 250 volume tank containing 200
Transplant 10, 30℃, 120 hours, 250r.pm/min
The culture was carried out with aeration under sterile air conditions of 100 °C to obtain a culture of 190 °C. [B] A liquid 160 was obtained by filtering the culture 190 to remove bacterial cells and other solid substances.
This liquid was extracted with the same amount of ethyl acetate to obtain 160 ml of an ethyl acetate solution containing the target product. This was concentrated under reduced pressure to a concentration of 50% and then mixed with 20% of an aqueous solution of hydrochloric acid having a pH of 2.5 to transfer and dissolve the target product into the aqueous layer. Furthermore, the pH of the hydrochloric acid aqueous solution is adjusted using concentrated ammonia.
8.5, extracted with 20 chloroform, concentrated and dried the chloroform layer to obtain 8.5 g of crude product.
I got it. [C] 8.5 g of the above crude product was dissolved in 50 ml of chloroform and adsorbed onto a silica gel column (3 cm x 55 cm) that had been filled with chloroform in advance. It was then developed with a solvent consisting of chloroform-methanol-28% ammonia (20:1:0.1) and
It was fractionated by ml. Antibacterial activity and chloroform-methanol-7 using Bacillus subtilis for each fraction
The target product was confirmed by thin layer chromatography using % aqueous ammonia (40:12:20: lower layer) as a developing solvent, and fractions containing it were collected. The 78th fraction contains only the substance identified as A11725- from the 61st fraction, and this fraction is concentrated and dried.
A11725-1.2g was obtained. The 160th fraction contains only the substance identified as A11725 from the 126th fraction,
This fraction was concentrated and dried to obtain 1.7 g of A1172. Effects of the Invention The novel macrolide antibiotic-producing bacterium of the present invention can be effectively used against respiratory infections caused by Gram-positive bacteria, and can also be used as a novel antibiotic A11725 and an antibiotic used as feed additives and animal treatment antibiotics. Produce.

[Brief explanation of the drawing]

Figures 1 and 2 are infrared absorption spectrum diagrams of the novel antibiotic A11725 of the present invention, and Figures 3 and 4 are diagrams of the novel antibiotic A11725 and Figure 4, respectively.
The ultraviolet absorption spectrum diagrams of A11725 and A11725, Figures 5 and 6, respectively, are of this antibiotic.
A11725 and nuclear magnetic resonance spectrum diagrams of A11725.

Claims (1)

[Claims] 1. Macrolide antibiotic A11725 or
Micromonospora that has the ability to produce A11725.
Griseorrhida (Micromonospora・
griseorubida), which is a novel macrolide antibiotic-producing bacterium A with the following mycological properties. No division is observed, and spores are formed individually at the tips of short sporophores arising from basal hyphae. They are spherical or ovoid in size, 1.0 to 1.5μ in size, and have spiny projections on their surfaces. B. Growth status on various media Cultured on various media at 30°C for 20 days and observed.
Color display is based on Color Harmony Manual 4th edition
According to the color classification according to 1958, (a) Sucrose/nitrate agar medium Growth: Good Base hyphae color: Cedar (6le) or brick red (6ng) Spore layer: None Irrigated aerial hyphae: Poor;・Pink (5ca), Dusty Peach (5ec) Soluble pigment: Dusty Coral (6gc)
Or not (6ie) (b) Glucose-asparagine agar medium Growth: Slight to poor Color of basal hyphae: Nude tan (4gc) Spore layer: None Immature aerial hyphae: None Soluble pigment: None (c) Glycerol-asparagine Agar medium Growth: Slight Base hyphae color: Nude tan (4gc) or bisque (4ec) Spore layer: None Immature aerial hyphae: None Soluble pigment: None (d) Starch/inorganic salt agar medium Growth: Moderate to Good Color of basal hyphae: Brick red (5ng) Spore layer: Moderate; Rump black (p) Undeveloped aerial hyphae: None Soluble pigment: None (e) Tyrosine agar medium Growth: Slight Color of basal hyphae: Bisque (3ec) or Beige (3gc) Spore layer: few; lamp black (p) Undeveloped aerial hyphae: none Soluble pigment: none (f) Oatmeal agar medium Growth: good to moderate Base hyphae color: brick black Red (5ng) or Copper Brown (5pi) Spore layer: Good; Rump Black (p) Irregular Aerial Hyphae: None Soluble Pigment: Produces Copper Tan (5ie) around the colony (g) Yeast/Malt Agar Medium Growth: Good Substrate mycelium color: Light rose brown (7lg) or rose brown (7ni) Spore layer: Very little; lamp black (p) Irregular aerial mycelium: None Soluble pigment: Cedar (6le) slightly (h) Glucose yeast extract agar medium Growth: Moderate Base hyphae color: Cocoa brown (5lg) or dark red/wet (6lg) Spore layer: Poor or few; lamp black (p) Not yet Growth aerial hyphae: few; shell pink (5ba) Soluble pigment: none (i) Glucose/nitrate agar medium Growth: moderate to poor Baseline hyphae color: cedar (61/2le) or brick red (61/2le) ) Spore layer: None Undeveloped hyphae: None Soluble pigment: None (j) Nutrient agar medium Growth: Slight Base hyphae color: Colorless to ride tan (3gc) Spore layer: Lamp black (p) Irrigated aerial hyphae : None Soluble pigment: None (k) Emerson agar medium Growth: Good, wrinkled Base hyphae color: Cedar (6le to 61/2le) Spore layer: None Irrigated aerial hyphae: None Soluble pigment: None (l) Bennet Agar medium Growth: Moderate to good Base hyphae color: Light rose brown (7lg) or rose brown (7ni) Spore layer: Moderate; lamp black (p) Irregular aerial hyphae: None Soluble pigments: Produces light rose brown (7lg) or rose brown (7ni) around the colony (m) High-key Tresner agar medium Growth: Good Substrate mycelium color: Cocoa brown (5lg) Spore layer: None Undeveloped Aerial mycelium: Poor; Bisque (4ec) Soluble pigment: Seeders (6lg) are produced around the colony (n) Starch/NZ amine/yeast extract agar medium (ATCC No. 172) Growth: Good Color of basal mycelium: Dark Wine (8pi) or Maub Wine (8ni) Spore layer: Good; Rump Black (p) Undeveloped aerial mycelium: None Soluble pigment: Old Wine (8ng) (o) Glucose/NZ amine agar (1% glucose , 3% NZ amine type A, 1.5% agar) Growth: Moderate to poor Base hyphae color: Cedar (6le) or rust tan (5le) Spore layer: None Irrigated aerial hyphae: None Soluble pigments: None (p) Glucose-peptone agar medium Growth: Moderate Base hyphae color: Rose brown (7ni) Spore layer: Moderate to poor, lamp black (p) Irregular aerial hyphae: None Soluble pigment: Old wine (7ng) (q) Ginger piece medium Growth: No or slight growth (r) Ginger piece + CaCO ₃ medium Growth: Good, wrinkled Substrate mycelia color: Dark rose brown (7pn) Sporolayer: Medium; Lamp black (p) Undeveloped aerial mycelium: None Soluble pigment: Dark rose brown (7pn) Slightly occurring (s) Peptone yeast iron agar medium Growth: Very little and poor Color of base mycelia: Light tan (3gc) Spore layer: few; lamp black (p) Undeveloped aerial hyphae: None Soluble pigment: None C Physiological properties Physiological properties are as shown below (1) Assimilation of carbon sources [Table ] [Table] (2) Growth temperature range: 15-45℃, (3) Liquefaction of gelatin: glucose, peptone,
Positive on gelatin medium, (4) Hydrolysis of starch: Positive on starch/inorganic salt agar medium, (5) Skimmed milk: Positive for both peptonization and coagulation, (6) Production of melanin-like pigment: Tyrosine agar and peptone yeast.・Negative on iron agar medium, (7) Salt tolerance [Table] (8) Decomposition of cellulose: Negative, (9) Production of nitrous acid: Negative, 2. The producing bacteria is specified in the Mycology of Microtech Institute No. 705 2. The bacterium according to item 1 above, which has the following characteristics.