JPS60130394A - Novel antibiotic sf-2324 substance, preparation, and use thereof - Google Patents

Abstract

PURPOSE:To obtain a novel antibiotic SF-2324 showing strong coccidium action, by cultivating a specific strain belonging to the genus Actinomadura. CONSTITUTION:Actinomadura.sp. SF-2324(FERM-P 7346) is cultivated under aerobic conditions especially by submerged culture. Preparation of SF-2324 substance reaches the maximum accumulation usually in 3-10 days by shaking culture or tank culture. The minimum growth inhibition concentration of SF-2324 substance to various microorganism measured by agar dilution method is shown as follows. Staphylococcus aureus FDA209JC-1; 1.56, Staphylococcus epidermidis ATCC14990; 1.56, Bacillus anthracis No.119; 0.78, Salmonella typhi O-901-W; >100, Pseudomonas aeruginosa MB-3829; >100, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibiotic SF'-2324 substance, its production method and uses. We discovered that a substance with a growth-inhibiting effect was produced and accumulated in culture, and succeeded in collecting the effective substance.

Furthermore, the present inventors isolated and purified this effective substance, and as a result of examining its properties, discovered that it is a new antibiotic different from known substances.
Four substances were named.

Therefore, the first invention is a novel antibiotic SF-23.
The 5F-2324 substance of the present invention has the following physical and chemical properties.Color and properties: Colorless acicular crystals.

(2) Elemental analysis value: Free acid C60, 17%, H8,87.

NQ% Sodium salt C59,80%, H8,76%, NO%
(3) Molecular weight, potassium salt 105581M8 [
Lium salt 103981M8 (4) Melting point: Free acid 128~+30'C Potassium salt 155~l 57'C (5) Specific rotation: [α]25+15.7° (c=l
, Mel/-/L, )1G) Ultraviolet absorption is
No characteristic absorption is shown above 210 mμ.

(7) Infrared absorption spectrum: The absorption spectrum of the free acid in KBr tablets is shown in FIG. KB of potassium salt
The absorption spectrum by the r tablet is shown in Figure 2. O +81 Nuclear magnetic resonance spectrum: The hydrogen nuclear magnetic resonance spectrum of the potassium salt is shown in Figure 3. The carbon nuclear magnetic resonance spectrum of potassium salt is shown in Figure 4.0 (9) Rf value of silica gel thin layer a madogram: R
f=0.28 (ethyl acetate) Rf=0.23 (ethyl acetate:chloroform-=S
: + ) R, = o, ag (7 tons of ethylnia acetate =
5:I) [1 Color reaction: sulfuric acid and Lemieux reagent K11a
Characteristics: Negative to ninhydrin reagent〇αυ i M Characteristic: Easily soluble in hexane, chloroform, ethyl acetate, acetone and methanol 0 Slightly soluble in water 〇Minimal growth inhibition against various microorganisms measured by agar dilution method of 5F-2324 substance It was also found that this substance had a strong anti-coccidiosis effect. The toxicity of substance 81'i'-2324 is LD5° (mouse, subcutaneous injection) value of 6.2η/ky. Compared to that of 1, it is considered to be classified as a so-called polyether ionophore antibiotic. However, among the polyether ionophore antibiotics, the substance with a molecular weight of 1000 is Ni-41A (f+zi 962). , Ni-41B (molecular weight + o
5F-2324 substance is a new antibiotic.

The novel antibiotic 5F-2324 substance of the present invention is produced by culturing an antibiotic 5F-2324 substance-producing bacterium belonging to the genus Actinomadeula and collecting the 5F-2324 substance from the culture. The method for producing the 5F-2324 substance constitutes the second invention. As an example of the producing bacteria of the novel antibiotic 5F-2324 substance of the present invention, newly separated 5
There is F-2324 strain.

The mycological properties of the water droplets are as follows.

(2) Morphological properties The basal hyphae are well elongated and branched, and their diameter is approximately 0.5 μm.No division of the basal hyphae is usually observed in either agar medium or liquid medium. Generally, the attachment of aerial mycelium is poor, but white aerial mycelium is attached on starch agar, and spore layer formation is also observed. In addition, the formation of aerial mycelia and spores is 28
37°C is better than 37°C. The branches of the aerial hyphae are simple branches, and no axle branches are observed. When observed with an electron microscope, spore chains are seen on the aerial mycelium, and the shape is loop-like to short spiral. Chains of spores often have 3 to IQ pieces @Spores are oval to oval, 0.6 to 1, OXO
, 8 to 1.2 μm in size, and its surface is smooth (slightly wrinkled) 〇2. Growth status on various media Table 2 shows the growth status of strain 8F-2324 on various media. @Observation was carried out after 14-21 days of culture.

Although the culture temperature was 28°C, conditions at 37°C were also described for some of the culture media.

8. Physiological properties +11 Growth temperature range: 15 in yeast malt agar
It grows in a temperature range of ~45°C, with an optimum temperature of 28-40°C.

(2) Liquefaction of gelatin: Positive (cultured at 20°C for 121 days) (3) Hydrolysis of starch: Negative (28°C, 137°
C914 day culture) (4) Nitrate reduction: Positive (28°C137°C1
14 days of culture) (5) Skim milk is transformed and coagulated (280C
, 37”C.

14-day culture)・(6) Salt tolerance: Grows in the presence of 5% salt, but does not grow in the presence of 7% salt.

(7) Formation of melanin-like pigments: Negative with triditone yeast extract broth, peptone yeast extract, iron agar, and tyrosine agar.

4. Utilization of carbon source (use of Prydham-Gottries celestial medium) - Rule (2) Sugars whose use is questionable: D-7 Lacdose,
i-inositol, raffinose (3) Unused sugars: L-arabinose, D-xylose, L-rhamnose, sucrose 5, cell wall composition 8 Meso-diaminopimelic acid was observed in the whole cell hydrolyzate of strain F-2324 Based on the above characteristics, strain 8F-2324 is most similar to the genus Acttnomadura among actinomycetes. Therefore, we S F
-2324 strain Actinomadeura sp. 5F-2
324 (AcLtnoma, dura sp, 5F-2
This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology since November 18, 1981, and its deposit number is Kikoken Bacteria Deposit No. 7346 (F'l1M). P-7346
).

The 8F-2324 strain is one that can be mutated [2] by artificial mutagenesis methods using ultraviolet rays, X-rays, radiation, chemicals, etc. in many other strains of actinomycetes, and any mutant strain is Any strain of the genus Acunano 1 dura that has the ability to produce the 8F-2324 substance can be used in the method of the present invention.

In the method of the present invention, the nutrient source for culturing the P. spp. in a medium containing nutrients that can be used by ordinary microorganisms is:
Known materials conventionally used for culturing Actinobacteria can be used. For example, glucose, starch syrup, glycerol, sugar syrup/vegetable oil, animal oil, etc. can be used as the carbon source.

Nitrogen sources include soybean flour, wheat germ, meat extract, cornstarch, yeast extract, cornstap liquor, cottonseed gas, etc.
Fishmeal, ammonium sulfate, sodium nitrate, urea, etc. may be used. Other calcium carbonate as required.

In addition to adding inorganic salts such as sodium chloride, magnesium sulfate, cobalt chloride, and phosphates, it helps the growth of bacteria and
As the O 2 culture method, in which organic and inorganic substances that promote and promote the production of the F-2324 substance can be appropriately added, a culture method under aerobic conditions, particularly a deep culture method, is most suitable. The appropriate temperature for culturing is 15-45
°C, but in most cases a range of 28 to 37 °C is desirable.

The production of SF'-2324 substance differs depending on the culture medium and culture conditions, but in both shaking culture and tank culture, the accumulation usually reaches its maximum within 3 to 1° days.The 5F-2324 substance produced in this way is Since it has physical and chemical properties, it is possible to extract and purify it from the culture solution according to its properties, but it can be particularly efficiently extracted and purified by the following method. In other words, it can be extracted and purified efficiently by the following method. Add an organic solvent that is immiscible with water and extract with stirring. Alternatively, a solution that does not mix with water, such as ethyl acetate, is added to the P solution obtained by separating the solid matter from the culture, and the mixture is stirred and extracted. On the other hand, the solid substance is stirred with a solvent such as acetone that warms freely with water, the active ingredient is extracted from the solid substance, the organic solvent is distilled off, and the active ingredient is extracted with an organic solvent such as ethyl acetate. .

The oily substance obtained by distilling off the solvent of the extract is extracted by adding a solvent such as n-hexane, and the residue is filtered. 6. Dissolve the isolated 8F-2324 substance in an organic solvent such as ethyl acetate, and isolate the 5F-2324 substance using an appropriate combination of carriers such as silica gel, alumina, and gel filtration agents. and stir. After washing the organic solvent with pure water, the organic solvent is concentrated under reduced pressure, and the resulting residue is crystallized using a solvent system such as methanol-water, ethanol-water, acetone-water, etc. to obtain 8F-2324 substance. Obtain crystals. In addition, SF-2324 substance can be obtained as various salts, for example, sodium salt, potassium salt, and ammonium salt. In assaying O8t'-2324 substance, Bacillus stearoLhe -rmoph
A biological assay method using S. illus ) as the test bacteria is used.

In this assay method, 5F-2324 substance is 62.5μ? /
In the concentration range of ml-500 μf/-, there is a direct relationship between the logarithm and the inhibition circle diameter, and I 7,
9 mm-20,8vm blocking (faint zone
e).

Furthermore, the present invention provides, as a third invention, an anti-coccidiosis agent containing the antibiotic 5r-2324 substance as an active ingredient.

When the 8F-2324 substance of the present invention is used as an anticoccidial agent, it can be administered orally directly as it is, or it can be administered by adding it to feed. Examples of possible feeds include compound feed, barley flour, flour,
Bare flour, corn flour.

Soybean flour, soybean meal, rapeseed meal, rice bran, defatted rice bran, horse flour, Kansho powder, other starches, tofu meal, yeast,
Examples include fishmeal, fermentation residues, etc.6 Also, commonly used feed additives, such as various vitamins,
Minerals, preservatives.

Enzyme preparations, proteins, carbohydrates, amino acids, antipyretics,
It may be used in combination with sedatives, anti-inflammatory agents, bactericidal agents, etc.

Next, an actual example of the method for producing the 5F-2324 substance according to the present invention will be shown, but it goes without saying that many modifications or modification methods not exemplified here may be used.Example 1 Actinomadeura sp.
strain (Kikoken Bacteria No. 7346) was used. Starch 1.0%, glucose 1.01, hefton 0.5 as seed medium
%, meat extract 0.2%, yeast extract I), soybean flour 0.21%, calcium carbonate 0.2%.
Also, the production medium was 2.5% glucose and 1.5% corn steep liquor.

A medium consisting of 1.0% Sungrain (manufactured by Suntory), Pharmamedia (Traders Oil Mill Co., Ltd.), 0.51% cobal chloride, 0.001% zinc sulfate, and 0.0005% zinc sulfate was adjusted to pH 7.0. After the adjustment, one inoculum grown on the O yeast malt slant agar medium using a medium supplemented with 0.3% calcium carbonate was scraped off and placed in a 100-capacity Erlenmeyer flask containing the seed medium 20-20. 1
The cells were inoculated into 280C cells and cultured with shaking for 16 days. Furthermore, this was inoculated at a rate of 10% into two 500-volume Erlenmeyer flasks containing 80 m of seed medium.

A 500-capacity Erlenmeyer flask 3 containing 80 ml of a production medium of 0.5 mm was cultured with shaking at 28°C for 13 days and used as a seed culture.
Five bottles were inoculated with 6% each of the above seed cultures and cultured with shaking at 288C for 6 days. The bacterial cells were extracted with 70% thiacetone water to obtain 3.5 tons of bacterial cell extract. Furthermore, the bacterial cell extract was concentrated under reduced pressure to remove acetone, and the aqueous layer was added to 1.5 L.
1.56 ml of ethyl acetate was added and shaken, and the active ingredients were extracted into the ethyl acetate layer.

This ethyl acetate layer was concentrated under reduced pressure, and a blackish brown oily substance 94 was obtained.
50-hexane was added to 942.7 ~ of the O oily substance obtained from Cape 2.7, extracted with stirring for 30 minutes, and the residue was filtered. This operation was repeated twice and the obtained hexane layer was concentrated under reduced pressure to obtain 650 cm of an oily substance.
was dissolved in 5 ml of chloroform, applied to a 65TntO column of silica gel (Fuko-gel C-20O, manufactured by Wako Pure Chemical Industries, Ltd.), and added to a mixed solution of chloroform-ethyl acetate (IQ: l).
After washing with 260ml of a mixture of chloroform and ethyl acetate (4:I), it was developed with 500ml of a mixture of chloroform and ethyl acetate (1:10).The active substance was eluted. Among the active fractions, a thin layer of silica gel (Kieselgel 5714, manufactured by Merck & Co., Ltd.) using a mixture of acetone and ethyl acetate (+:S) as a developing solvent gave a single spot using a chromatography method, and both fractions were combined and concentrated under reduced pressure. As a result, S-2324' rostrum with t' +14.7η was obtained as a white powder.

The remaining active fraction was concentrated to dryness to obtain a crude powder of 361.9 cm, which was dissolved in a small amount of methanol, applied to a 100 d Sephadex L +-1-20 column filled with methanol, and developed with methanol. The active fraction was concentrated to dryness, and 33
9.31 ng of coarse powder was obtained. This coarse powder 339.3
q was dissolved in chloroform-filled silica gel (Wakogel C-200, manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.), and the tower was washed with 670 d of a mixed solution of chloroform-acetone (+0:+). Afterwards, the mixture was eluted with a mixture of chloroform-acetone (5:l). Among the eluted active fractions, the silica gel thin layer chromatography described above revealed a single Sze-Soto-J? , 1 rhyme 1
- I'lA, 2 scoops of 5F-2324 substance were obtained as white θ powder@Example 2 446 mg of the crude powder obtained by culturing and making silk in the same manner as in Example 1 was mixed with a small amount of acetic acid. Dissolve in ethyl.

Thin layer of silica gel for branch weirs (Kieselgel 5744.20X
2Qcm, Merck & Co., Inc. a) 5 modified was charged linearly, and ethyl acetate and chloroform were mixed at a ratio of 5:10/ζ development solution.

After the development path r, both sides of the 4 layers of silica gel were cut into a width of 1 crn, and after spraying 10% sulfuric acid water, heating was performed to detect the -C spot. The silica gel was scraped off according to the detected spots, and the scraped silica gel was extracted using 100 ml of ethyl acetate.The ethyl acetate layer was concentrated to dryness to obtain a +90% white powder. Add this white powder +00Lη to 50rn1. of ethyl acetate and 50 g of O,IN
Around that time, the enemy poured out stirring oil. Furthermore, the ethyl acetate layer was washed with 150 g of pure water and concentrated to dryness to obtain 79 l of white powder. This white powder was dissolved in acetone water, the acetone was distilled off using a vacuum knife, and then cooled. 5F-232
The excellent activity of the four substances against coccidiosis in poultry will be explained by the test, but the present invention is of course not limited thereto.

Trial example Test method: Six groups of 8-day-old male white Leghoe chicks of 7 species were used, with 5 chicks per group. 5F-23 for 1st to 4th groups
0.002 and 0.0 of each pure powder of 24 substances
Feed with feed uniformly added at a ratio of 0.03%, 0.004chi, and 0.005φ (-).The 5th and 6th groups were fed feed without chemical additives.The day after the feeding of each &11 old was started. In addition, the first to fifth groups include Eimeri adenella.
a tenellera) +7) 5XIO' of mature oocysts were orally infected per bird.Infection 8
Days later, the weights of the chicks in all groups were measured and necropsied to determine the strength of the cecal lesions. Judgment is based on the basics of Tsunoda et al. -~+
+++ and converted it into a political situation of 0 to 4.Furthermore, the number of oocysts in 1f of cecal stool was measured and billed according to Table 3. In addition, the relative weight gain rate of each group was calculated as the weight gain rate 'r+00 of the non-infected control group by calculating the weight gain rate for each group. Based on the above results, the anticoccidial index (ACI) was calculated using the following formula ◇ ACI = (relative gain rate + survival rate) - (lesion value + oocyst value) Table 3 Oocyst value classification 0 to 0. 1 0 0.1~1.0 1 2.0~5.0 10 6.0~ + 0.0 20 ≧11.0 40 Test results: 4th ACI of 5F-2324 substance
Shown in the table. S←'-2324 substance is 0.0 in chick feed
When 03 or more was added uniformly and administered, it significantly inhibited coccopanodiasis and showed no toxic effects.

[Brief explanation of the drawing]

1 and 2 are infrared absorption spectra of the free acid and potassium salt of SF'-2324 substance by KBr tablet, respectively, and FIG. 3 is a hydrogen nuclear magnetic resonance spectrum of potassium salt of 8F-2324 substance, Figure 4 is 5F-2
324 is a carbon nuclear magnetic resonance spectrum of the potassium salt of substance 324. Procedural amendment (spontaneous) December 28, 1980 Mr. Commissioner of the Patent Office1, Indication of the case Patent Application No. 237917 of 19822, Title of the invention Novel antibiotic 5F-2324 substance, its manufacturing method and uses3, Relationship with the case of the person making the amendment Patent application fire pillar Address: Bussan Building Annex, 2-4-16, Kyotachibana, Chuo-ku, Chuo-ku Telephone: (591) 0261Lccwl rC# Baju ITI Nae M5, subject of amendment Ming Wholesale] Cao's +6 Details of Ming +1: Contents of Nl Shukumei Nozu Aya 6 Sode correction tl) Specification 1- Actinobacteria in line 8 of the 12th Teikyuu is corrected as ``actinobacteria.'' (2) Same, I-(faint
zone) Delete J (3) rEimeria te on the 15th line of the 20th
gimeria tenell
a Correct as J.

Claims (1)

[Claims] 1. Novel antibiotic 8F'-232 having the following properties:
4 substances and their salts: +t+ g and properties: colorless needle crystals (2) original cumulative analysis values: free acid C60, 17 width, H8, 131%. 80% Sodium salt C5940%, H8,76%. N0% (3) Molecular weight: Potassium salt 1055'sIMs
Sodium salt 1039 SIMS 141 Melting point: Free tifG @ 12g ~ 13o°
C potassium salt 155~+57'c (5) Specific optical rotation -[α)%7+-15;7°(c
=1. Methanol) (6) Ultraviolet absorption is a cutter:
No characteristic absorption is shown above 210-'μ. KB of potassium salt
The absorption spectrum of the r-tablet is shown in Figure 2.0 (8) Nuclear magnetic resonance spectrum: The hydrogen nuclear magnetic resonance spectrum of potassium salt is shown in Figure 3. The carbon nuclear magnetic resonance spectrum of potassium salt is shown in Figure 4. 0 (91 Color reaction: positive to WI acid and Lemieux reagent, negative to ninhydrin reagent. 01 Solubility: Hemusan, chloroform, ethyl acetate, Easily soluble in acetone and methanol. Slightly soluble in water. 2. Antibiotic 5F-232 belonging to Acunano 1 Deyura.
8F-2324 was obtained from the culture by culturing 4 substance-producing bacteria.
SF-23'24 characterized by collecting a substance
Method of manufacturing a substance. B. Anticoccidial agent containing antibiotic 5F-2324 substance as an active ingredient.