JPS5831327B2 - Veterinary pharmaceutical composition - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to antibiotic A-2315, and more particularly to a non-human veterinary pharmaceutical composition containing the novel neutral nitrogen-containing antibiotic A-2315.

The antibiotic A-2315 of the present invention is a novel microorganism belonging to the genus Actinoplanes, particularly Actinoplanes philippinensis (Actinoplanes philippinensis).
NRRL 5462 under deep aerobic conditions until the active substance is obtained in substantial quantities.

The produced antibiotic can be isolated as an amorphous solid by filtering the culture solution, preferably extracting it with an organic solvent, and purifying it by ram chromatography over activated alumina or silica gel.

The antibiotic A-2315 of the present invention is useful as an antibacterial agent, and exhibits remarkable antibacterial activity particularly against Durham-positive Streptococcus bacteria.

Furthermore, antibiotic A-2315 has the effect of suppressing microorganisms that induce dental palpation and periodontal ligament disease.

Furthermore, antibiotic A-2315 has the effect of promoting growth when added to poultry feed, and is additionally useful as a fungicide for plants.

Antibiotic A-2315 is a white, amorphous solid with a molecular weight of 503.2638 according to mass spectrometry and the following elemental analysis: C161,51
%; H17, 51%; N, 3.69%: 0.21.53
%.

Based on the above molecular weight and elemental analysis results, A-2315
The empirical formula: C26H37N307 is given for.

Electrometric titration of A-2315 in 66% water-dimethylformamide reveals no measurable groups.

The specific optical rotation of A2315 at 27° C. in methanol solvent (concentration of 0.375% by weight of A-2315 per volume of methanol) is −132°.

The infrared absorption spectrum of Ogero A-2315 in chloroform has the following maximum absorption (unit wavelength micron, W
( stands for “weak”, m stands for “medium”, S stands for “strong”):
2.81 (w), 2.99 (w~m), 3.38 (
m), 5.82 (m), 6.02 (s), 6.20 (w
), 6.30 (m), 6.40 (w), 6.66 (r
rr-s), 6.82 (w), 6.94 (w~m),
7.05 (w), 7.30 (w-m), 7.71
(w), s, s s (w-m), 9.11 (w
-m), 10.41 (m), 10.89 (w-
m), 11.14 (W ~ m) μO The ultraviolet absorption spectrum of antibiotic A-2315 in 95% ethanol shows maximum absorption at 214 mμ (ε40.200), and its absorption value (E1%) is 799. .

1crI'L Antibiotic A-2315 is soluble in methanol, ethanol, higher alcohols, and most other organic solvents, but is slightly soluble in water.

Sarcina lutea was used as the detected microorganism in paper chromatography using the following solvent system, and antibiotic A-2315 was used.
The results of measuring the Rf value are shown below: Rf value Solvent System 0.19yl ethyl ethyl ketone:benzene:water (
1:5:1), (upper layer).

0.91 water saturated butanol 0.91 Water saturated butanol and 2% p-) Ensulfonic acid 0.56 water saturated methyl isobutyl ketone 0.60 water saturated methyl isobutyl ketone and 2% p-toluenesulfonic acid 0.83 Water: methanol: acetone (12:3: 1) to pH with ammonium hydroxide pH adjusted to 10.5 and then with phosphoric acid Adjusted to 7.5.

E*0.75 Methyl isobutyl ketone 1%, hydroxide Ammonium 0.5% - water.

0.72 Potassium hydrogen phosphate 17.4 gul 30 ml/water 100017110 Ethano 0.65 Sodium chloride 7%-Methyl ethyl ketone 2.5%-Water 0.84 Propartur: Water (1:9) 0.90
Butanol:ethanol:water (13,5:15:150
) Amino acid analysis of antibiotic A-2315 releases alanine in the device.

Antibiotic A-2315 contains two hydroxyl groups, which can be acetylated with acetic anhydride in hahiridine to form its ester derivative using conventional methods.

This ester derivative is also useful as an antibiotic.

Based on the above physical data and research to elucidate the structure, the following hypothetical structure can be proposed for A-2315.

Although this structure cannot be determined with certainty, it can be understood as simply expressed as a hypothesis based on facts.

Antibiotic A-2315 has the effect of inhibiting the growth of certain microorganisms.

The concentration of antibiotic A-2315 that inhibits microbial growth can be determined by various test methods.

Test 1 The test method and test results are shown below.

(1) Circular plate screening test method An agar plate with test microorganisms attached is used.

A 6-diameter circular plate (volume 0.02 rrLl) is saturated with fog'2 dilution of antibiotic solution.

The content of the circular plate is 1150 of the concentration of the solution used, i.e. 1
500μ? /ml solution of antibiotic 30μ2
A solid plate content of is obtained.

Using a disk plate like the one above, antibiotic A-
Table 1 shows the results of the microbial inhibition test for 2315.

Prior to testing the therapeutic efficacy of experimentally infected mice, tests were conducted to confirm the effectiveness of antibiotic A-2315 in diluted cultures against pathogenic Gram-positive microorganisms.

vinegar.

The results are shown in Table 2. Antibiotic A-23 was administered to mice experimentally infected with bacteria.
As a result of subcutaneous administration of A-15 and in vivo testing, A-23
15 was found to have antimicrobial activity.

ED5o value when two doses of antibiotic A-2315 are administered to infected mice (effective dose that allows 50% of test animals to survive) (Journal by Warren Wick et al.
Op Bacteriology, Vol. 81, pp. 233-235 (1)
961)) is shown below.

Staphylococcus arale 90■ / 9 us 3055 Streptococcus pyogae 831nfj / 9 nes Shiphlococcus pneumoniae > 1 '66 m9
/ky Engineering (Park I) Antibiotic A-2315 was administered orally or subcutaneously to mice, and its acute toxicity was tested.
As a result of administering 2315 into the shoe cavity and testing its acute toxicity, its LD.

The value was 9 to 400 dogs.

Another utility of antibiotic A-2315 is to treat microorganisms that are pathogenic to poultry: Mycoplasma gallisepticum (M
ycoplasma gallisepticum
) has antibacterial activity.

Of particular importance is the activity of antibiotic A-2315 against the above-mentioned bacteria that are resistant to tylosin.

In this regard, the minimum inhibitory concentrations determined by in vitro testing of diluted cultures are shown in Table 2.

Treatment of Mycoplasma gallisepticum infection in chicks: Chicks infected with Mycoplasma gallisepticum (MG) were treated with 5 Iv/dose of A-2315 dissolved in ethanol.
- Treated by injection at a rate of 150 m9/9 feathers.

The chicks were killed after 16 days and A-23
15 was found to be effective.

Furthermore, when the antibiotic A-2315 is administered to chickens infected with Mycoplasma gallisepticum, there is a slight increase in body weight and fewer cases of air sac damage among the surviving chickens.

When we prepared an antibiotic preparation by mixing it with polyethylene glycol 200 and injected it subcutaneously into the necks of chickens, all of the chickens that died at the beginning showed slight bleeding in the neck area.
No signs of toxicity were observed.

The results are shown in Table ■. As is clear from Table 2, the present invention provides an antibacterial agent that is effective against symptoms caused by Mycoplasma in poultry.

Poultry are protected against Mycoplasma when the antibiotic A2315 is administered parenterally or orally at a dose of about 50 to 500 μ/9 (animal body weight).

When used for prophylactic or therapeutic purposes against respiratory diseases, it is convenient to incorporate an appropriate concentration of antibiotic A-2315 into normal animal feed.

Another advantage of the present invention is that antibiotic A-2315 has the effect of inhibiting the growth of microorganisms that promote the progression of periodontal ligament disease.

According to the diluted culture solution test, A-2315 is 1-2μ
Streptococcus mutans (5treptococcus muta)
ns), and suppresses odontal fibrous bacilli at concentrations of 0.1 μf/rul or less.

Antibiotic A-2315 solution is effective in inhibiting tooth-tactile microorganisms, as exemplified by the following test: A nutrient solution containing 5% sucrose was placed in a test tube and inoculated with tooth-touching microorganisms. Cultivate this.

A thin glass rod was inserted into the test tube so as to reach below the surface of the nutrient solution, and cultured overnight at 37°C to form a layer of artificial spots (mainly composed of cells and dextran).

) is formed on the surface of the glass rod. The glass rods are then inserted into solutions containing various concentrations of A-2315 and left in contact with the antibiotic solution for 5.10 and 15 minutes.

The glass rod was then rinsed in sterile deionized water.
Grow overnight at 37°C in unseeded medium containing sucrose and bromothymol blue.

The presence or absence of microorganism growth is detected using the change in color of bromothymol blue from green to yellow due to the acid produced when microorganisms grow in a medium containing sucrose through this operation.

No growth detected indicates that the antibiotic has killed the microorganism.

According to the above test method, a glass rod with a speckled layer formed using Streptococcus sp. was exposed to a 0.1% solution of antibiotic A-2315 for about 5 minutes. -2315 was found to be effective against the above-mentioned tooth-tactile bacteria.

Since this antibiotic A-2315 has activity against odontobacterial microorganisms and microorganisms associated with periodontal ligament, it can be used as a cavity prophylactic agent such as a toothpaste, powder or gel, or in the oral cavity at concentrations that can suppress these symptoms. It is suitable to be added to detergents, etc.

In general, formulations containing about 1-15% A-2315 are effective for inhibiting dental microorganisms.

Another useful property of antibiotic A-2315 is its ability to improve animal fattening purposes.

When A-2315 was added to the feed of fattened chickens at a concentration of 45.4P/), the average weight gain 10 days after addition was 1591, while the average weight gain without addition was 1431. Met.

The feed conversion ratio (feed amount/weight gain) of chickens fed antibiotic A-2315 was 1.44, while that of chickens not fed antibiotics was 1.53.

The weight gain promoting properties of antibiotic A-2315 make it particularly useful for fattening purposes.

When the antibiotic is used as a growth promoter, it is convenient to incorporate it into normal animal feed in appropriate concentrations.

Antibiotic A-2315 has typical effects as a growth promoter when administered to animals at concentrations of about 20-50 f/ton of feed.

Another advantage of the antibiotic A-2315 of the present invention is its usefulness as a plant fungicide.

Antibiotic A-2315 at a concentration of 400 ppm inhibits bean rust (Uromyces phaseoii va.
It protects legume plants inoculated with Colletotrichum lagenarium (Colletotrichum lagenarium).
1- Inoculation of Etotric Colletotrichu )) can protect the Japanese cucumber plant.

In all cases, only slight toxicity to plants was observed.

To protect plants, effective germicidal doses of antibiotic A-2315 can be administered in a variety of ways, for example as liquid sprays or powders, using suitable solvents, carriers, emulsifiers, wetting agents or surfactants as desired. can be used.

It is necessary to consider individual conditions, but approximately 200 to 500
A-2315 is typically useful as an antimicrobial agent when administered to plants in ppm amounts.

As is clear from the above characteristics, antibiotic A-2315
is useful because it inhibits the growth of pathogenic microorganisms.

Therefore, in addition to the above uses, solutions containing about 2% antibiotics can be used to sterilize glassware, dental and surgical equipment, instruments, and maintain sterility in hospitals, food processing machines, etc. Effective for disinfecting surfaces such as walls, floors, and tables wherever needed.

The novel antibiotic of the present invention can be produced by culturing the A-2315 ecological strain of a microorganism belonging to the genus Actinoplanenes in a suitable medium under aerobic deep culture conditions until the medium substantially contains antibiotic activity. I can do it.

Antibiotics accumulated in the culture medium can be recovered by applying various separation and purification methods understood and used in the art.

The novel microorganism of the present invention, Actinoplanes philippinensis NRRL5462, was isolated from a soil sample collected from a grain field in Belville, Kansas.

A small amount of soil, no more than a spoonful, was placed in a sterile helium dish and soaked with sterile water.

As soon as the mud subsides, add boiled millet (Paspa).
lum) 2.3 blades of grass were placed in a dish so that they were just floating on the surface or only partially submerged.

After 4 to 7 days, the leaves were examined under strong direct light using a binocular dissecting microscope (approximately 100x).

Actinoplanes were identified by the white, shimmering sporangia that formed in the air above the water's surface.

Small pieces of sporozoated leaves were then cut out and transferred to 3% flat agar.

One sporangium was excised and a pure culture was induced.

This pure stock culture was transferred to various culture media using conventional methods to examine growth characteristics and sporangium formation.

Actinoplanes is also known as Li quid.
It can also be isolated using the pollen of A. ambar.

This pollen floats on or just below the surface, which is more convenient than boiled leaves, and actinoplanes appear on the pollen surface in 3 to 4 days.

The actenoplanetized pollen is easy to pick up under a binocular dissecting microscope, transfer to flat agar, and cut out 5 of 1 VC spores to prepare a pure culture.

This separation method has been described by Couch K. (Trans.

of N-Y, Acad-of Sci., 16
315-318 (1954)).

The microorganism useful for the production of antibiotic A-2315 is taxonomically known as Actinoplanes philippinensis.
It has the characteristics of a new strain named ``pinensis Couch''.

The genus Actinoplanes is a genus of the Actinoplaneceae family of the order Actinobacteria.

The Actinoplanet family was first described by Couch (Journal of Elisha Mitchell Scientific Society Vol. 65, 31).
pp. 5-318 (1949), vol. 66, 87-92
(1950), Transactions of the New York Academy of Sciences, Vol. 16, 315.
~318 pages (1954), Journal of Elisher Mitchell Scientific Society Vol. 71, pp. 148-155 and 269 (1955), Versey's Manual of Determinative Bacteriology 7th Edition pp. 825-829 (1957
), Journal of Elisher Mitchell Scientific Society Vol. 79, 53-70 (1
963)).

The methods adopted for the mycological study of the A-2315 live and friable strain of the genus Actinoplanes are similar to those recommended by The International Streptomyces Project as well as supplementary test methods commonly used in taxonomy. It is something.

(Shirling (E, B, Shirling) and Gottlieb (D.

"Methods for Characterization of Streptomyces spacies" International Press
Bacteriology, Vol. 16, pp. 313-340 (1966)
).

Next, the mycological characteristics of Actinomyces philippinensis NRRL 5462 used in the present invention will be described.

Morphological characteristics: Development of mycelia on synthetic and semi-synthetic agar media are initially short, slightly branched, with septa, the diameter of which is 0.2-1.
It is 2 μm.

After about 2 weeks, the colonies consist of well-discernible clumps extending vertically from the mycelial substrate that extends deeper into the agar medium, or the vertical mycelia are indistinct and shorter;
It may consist of slightly mucoid masses.

In colonies with well-distinguished vertical mycelial masses, the mycelia form dense, slightly elongated crusts.

The mycelia of colonies with shorter hyphae form distinct dome-shaped growths.

Aerial mycelium does not grow on any medium.

Many spore grooves are formed on the dome-shaped colony surface.

Spore grooves Abundantly form spore grooves on semi-synthetic Amiohensen agar.

This appears about 2-4 weeks after inoculation at 17-19°C.

One spore furrow is formed on the very short sporangium stalk (terminal part of the hyphae on the agar surface).

The spore sulcus may be spherical, slightly spherical, or slightly fan-shaped, and is relatively small, 4 to 10 μm in diameter.

The sporangium contains 20 to 40 nearly spherical sporangia, which are arranged in one or more ill-defined coils in the sporangium.

Sporangiophores are approximately 1.0 μm in diameter when mature.

Spore furrow dehiscence is thought to occur by complete or partial destruction of individual spore furrows.

After 10-15 minutes in water, it begins to swell into a typical spherical shape and releases spores by collapse of the capsule/wall.

Usually, the spores are not immediately motile, but within about 5 minutes they become active due to the lower terminal hairs.

Appearance on medium: Zapetsk solution - agar Growth is good.

Colonies with a diameter of approximately 0.75 Cr/L are formed from one inoculation point in 4 weeks.

Aerial hyphae are not observed.

Colonies are generally flat with a slightly raised center.

The color of the colony is a mild orange.

Spore grooves are rarely observed. Peptone-zapeck solution
Good agar growth.

Colonies approximately 1.0 degrees in diameter are formed from one inoculation point within 4 weeks.

Aerial hyphae are not observed.

The colony is slightly raised and has some spiral or irregular bumps.

No spore grooves are observed.

Amio Hensen-Agar Growth is quite good.

Colonies with a diameter of about 0.5 CrrL are formed from one inoculation point in 4 weeks.

Aerial hyphae are not observed.

Colonies are circular, cross-shaped, or star-shaped with a depression or crevice in the center.

Colony color is dark gray to grayish white.

A large number of spore grooves are formed on colonies cultured at 17 to 19°C, and a small number are formed on colonies cultured at a relatively high temperature.

The culture characteristics on various media are shown in Table 7 (abbreviation ICP stands for International Streptomyces Project Media (Scharring & Gottlieb)).

Cell membrane chemistry in NRRL 5462
EnsisSzaniszlo and Goode
r) very similar to that of 1967.

After hydrolyzing cell membranes with 1 ml of 6N hydrochloric acid at 100°C for 18 hours, automatic amino acid and amino-sugar analysis of the resulting residue revealed that glucosamine, muramic acid, glycine, alanine and 2.6 - It was found that a large amount of diaminopimelic acid * * (DAP) was present.

2,6-diamitu-3-hydroxy-pimelic acid (HD
The absence of AP) is a notable feature.

Actinophrenes philippinensis NRRL54
Table 2 shows the results of comparing the amino acids in the cell membranes of Actinoplanenes philippinensis stuamisturo and bladder.

After hydrolyzing the cell membrane with 2 ml of 2N sulfuric acid for 2 hours at 100°C, paper chromatography of the resulting residue revealed detectable amounts of glucose, galactose, mannose, arabinose, xylose, and rhamnose in the cell membrane. These saccharides are also found in Actinoplanes philippinensis stamisturo and bladder.

Physiological properties: (1) Catalase production *1 + (2)
Liquefaction of gelatin + (3) Generation of melanin-like pigment *2 ISP 應1 + ISP A6 + ISP A, 7 + improved ISP
167 (4) NaCl resistance (%) *31 (5) Starch hydrolysis *4 + (6)
Hydrolysis of skim milk + (7) Peptonization of skim milk (8) Growth temperature range *5 10-37°C *1: Blazevic's method (B 1azevic's method
ic-D-J.

and G-M, Ederer, l 975,'
Pr1nciples □f Biochemical
Tests in Diagnostic Micro
Biology? John Wi lay and
Sons・Inc-1NewYork,pl
H2O2 was added to the surface of a fresh agar slant according to 36).
was added and the release of oxygen was observed.

*2: Melanin-like pigment production (chromodynecity) is produced using ISP Al (tryptone-yeast extract broth medium), 15PA6 (peptone-yeast extract iron agar medium), 15PA7 (tyrosine agar medium), and improved ISP&7 (tyrosine removed). culture medium) was investigated.

*3: NaCl resistance was measured by adding NaCl to the ISp A2 medium until the desired concentration was reached.

*4: Starch hydrolysis was measured by detecting the presence of starch with iodine on an ISP A, 4 (inorganic salts-starch agar medium) plate.

*5: Growth temperature range was determined by incubating 15PA2 plates at a predetermined temperature for 14 days.

The microorganism producing A-2315 was classified as a new strain called Actinoplanes philippinensis cochi based on the above taxonomic description.

This bacterium differs from previous publications on this species in that it does not produce spore grooves on glucose-asparagine agar and has slightly different colors on the three types of media, but these differences are due to the strain It indicates variation between species, not differences between species.

Strains of Actinoplanenes microorganisms useful for producing antibiotic A-2315 are available from the Department of Agriculture, Agricultural Research Service, Northern Marketing and Nutrition Research, Peoria, Illinois 61604, United States of America. Actinoplanes philippinensis NRR in division
It has been deposited as L5462 and is generally separable.

In addition, this strain has been deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology, 5-8-1 Inage Higashi, Chiba City, Japan, under consignment number 2181.

As mentioned above, Actinoplanenes philippinensis NRRL5462 can produce antibiotic A-2315 by growing it in a culture medium.

Although various media can be used as the medium, it is preferable to use a specific medium from the viewpoints of economic efficiency in production, amount of antibiotic produced, ease of antibiotic isolation, and the like.

For example, glucose is preferred as the carbohydrate source and soybean flour is preferred as the nitrogen source.

Nutrient inorganic salts that should be added to the culture medium include sodium, potassium,
It may be a conventional salt capable of generating ions such as ammonium, calcium, phosphoric acid, hydrochloric acid, sulfuric acid, acetic acid, carbonic acid, etc.

In addition, advantageous results can be obtained by including sources of growth factors such as desteller's solubles, yeast extracts.

Trace amounts of essential elements required for the growth and development of other common microorganisms must also be included in the culture medium for the Actinoplanes microorganisms used in the present invention.

Such trace elements are usually provided as impurities with the addition of other medium components.

The initial pH of the medium can vary over a wide range.

However, prior to inoculation of microorganisms, the pH of the culture medium should be adjusted to 6.5-6.5.
It is preferred to adjust to a range of 7.3, this range depending on the medium used.

The final pH value is determined, at least in part, by the initial pH of the medium, the buffer present in the medium, the time of incubation of the microorganism.

To produce antibiotic A-2315 in substantial yields, preferably large tanks are used and fermentation is carried out under deep aerobic conditions.

When obtaining small quantities of antibiotics, fermentation is carried out in shake flasks.

When large tanks are inoculated with microorganisms in spore form, there is usually a time lag between inoculation and antibiotic production, so in such cases it is preferable to use a growth inoculum.

This growth inoculum is prepared by inoculating a small volume of medium with spore forms of the microorganism or small pieces of mycelium to obtain a fresh and growthally active culture.

This growing inoculum is then transferred to a larger tank.

The medium for growing this growth inoculum may be the same as that used for larger-scale fermentation, but
Other media may also be used.

Microorganisms that produce A-2315 can be grown at about 20-40°C.

Optimal production of A2315 can be achieved at about 30°C.

Sterile air is blown through the medium as is commonly done in submerged aerobic culture methods.

In order to efficiently grow microorganisms and obtain antibiotic A-2315, the volume of air introduced into the A-2315 production tank is preferably about 0.1 volume per volume of medium.

Optimal growth is achieved when the air volume is approximately 0.2 to 0.8 volumes per volume of medium.

During the fermentation process, antibiotic production can be monitored by taking samples of the fermentation broth and testing for antibiotic activity against microorganisms known to be sensitive to the antibiotics of the invention.

One example of a microorganism used in this test is Sarcina lutena.

This biological test method can be conveniently carried out by the agar plate over paper pattern test method.

In large tank or shake flask fermentations, typically 2-
Maximum production of the antibiotic of the invention is given within 6 days.

Maximum antibiotic activity is usually given within 20-96 hours.

Extraction and adsorption methods allow the separation and recovery of antibiotic A-2315 from other substances that may be present in the culture medium.

Extraction methods are preferred to initially recover A-2315 after filtration.

A suitable solvent for separating antibiotics from filtered basic fermentation broth is chloroform, although other commonly used solvents can also be used with satisfactory results.

A-2315 is then conveniently purified by adsorption and elution methods using adsorbents such as activated carbon, polyamide resin, silica gel, Sephadex LH-20, and Amberlite XAD polymeric adsorbents to further purify A-2315.
15 can be separated.

Next, the present invention will be explained in more detail with reference to Reference Examples and Examples.

However, it should not be understood that the technical scope of the method of the present invention is limited to the scope described in the Examples.

Reference Example I A, Production of A-2315 by shake flask fermentation: Composition: 60.1' of o'meal, dried after pre-boiling treatment; 2.52 of completely crushed dry yeast for brewing;
Potassium hydrogen phosphate 1.0f; Czapek
) mineral raw materials *5. Oml; agar 2540g; deionized water 1100.0rn10 (* Composition of Zapetsk mineral raw materials: ferrous sulfate heptahydrate (
(in 2 ml of concentrated hydrochloric acid solution) 2.0P; potassium chloride 100.
0P: Magnesium sulfate heptahydrate ioo, oy; Add deionized water to adjust the total volume to 1000 rfLl.

) Prepare Actinoplanes philippinensis NRRL5462 cultures on agar slants having the above composition.

That is, the medium having the above composition was adjusted to pH approximately 6 with sodium hydroxide solution.
．． Adjust to 2-7.3.

In autoclave, 120℃ (pressure 15-20 bond)
Sterilize by treating for 30 minutes.

This treatment produces a medium with a pH of 6 or 7. A slant medium is prepared from this medium according to a conventional method.

The obtained slant medium was inoculated with Actinoplanes philippinensis NRRL5462, and incubated at 30°C for 10 to 40 min.
Incubate for days.

Since the culture is not intended to allow it to sporulate on the medium, it is necessary to soften the mycelium with a flat, sharp inoculation needle to increase the number of potential growth centers.

The broth is added to the culture and this is carefully mixed in a sterile frame to form a mycelial suspension.

This suspension is placed in six freeze-drying tubes to prepare freeze-dried strains.

Growth medium 507721 having the following composition is inoculated with 1 freeze-dried pellet above: 10.0 g of Nigel course; 30.0 g of potato starch; Ot; Soy flour 20.0? : Absorbent cottonseed powder 20.0? : Calcium carbonate 2.0f; tap water 1100rIl 10 Inoculated growth medium was placed in a 250rnl flask on a rotary shaker at 250ml.
Incubate for 72 hours at 30°C with rotation at rpm.

Growth medium 200 in the second step having the same composition as above
ml is inoculated with 10 ml of the growth medium cultured in the previous step.

The medium from the second stage step is cultured in a 10100O flask at 30° C. for 24 hours while rotating at 25 Orpm on a rotary shaker.

B. Preparation of A-2315 by tank fermentation Nigel course 1.0%: potato starch 4.0%; molasses 0.5%; soluble meat peptone 4.0%; enzyme-hydrolyzed casein 0.
4%: Soy flour 1.0%: Magnesium sulfate 03%; Calcium carbonate 0.2%; Antifoaming agent (Gura Corning) 0.
02%; Add tap water to make the total volume 25,000ml.

Sterile medium for antibiotic production 25000T having the above composition
Ll is inoculated with 200 ml of the growth medium prepared in the second step described in A above and cultured.

That is, first, the culture medium is sterilized in an autoclave at 15-20 pounds of pressure and 120°C for 30 minutes, and then the culture medium is adjusted to pH
Adjust to 6.4.

The inoculated production medium was placed in a 40,000 m1 fermentation tank for 3
Ferment at 0°C for 4 days.

During this time, the fermentation medium is aerated with sterile air at a rate of 1/2 volume per minute relative to the fermentation medium volume and stirred with a conventional stirrer (400 rpm).

C. Separation of A-2315 A filter aid is added to the total fermentation liquor obtained from the 25000 ml tank culture according to the procedure B above, and the mixture is filtered.

The filtrate is adjusted to pH 8.5 with 2N sodium hydroxide.

The resulting solution is extracted with 2/3 volume of chloroform.

The chloroform extract is concentrated under reduced pressure and 20 volumes of petroleum ether (Skelisorp F) are slowly added with stirring.

The precipitate is collected by filtration and dried under reduced pressure to obtain crude antibiotic 3131.

This crude antibiotic was dissolved in 100 ml of ethyl acetate, and a basic alumina (manufactured by M Welm, Eschwege, Germany) column (diameter 2, length 100 CrrL) was prepared.
Pass it through.

The garum was washed with 300 TIll of ethyl acetate and eluted with ethyl acetate:ethanol (19:1) to remove active substance A-23.
A fraction containing 15 is obtained.

These fractions are collected and evaporated to dryness under reduced pressure.

The residue is dissolved in a small amount of chloroform and the solution is added to 20 volumes of petroleum ether (Skelisorp F) to precipitate the active substance.

The precipitate is filtered and dried under reduced pressure to obtain 1,631 ml of antibiotic A2315.

Sartina lutena was used as the test microorganism by the filter paper-circular agar-plate test method and as a microorganism for thin layer chromatography and bioautography on silica gel using ethyl acetate:ethanol (9:1) as the developing system. The elution of the active substance is monitored by a test method using Sartina lutena, or an iodine vapor treatment or sulfuric acid spray method is carried out to detect the active substance.

Reference Example 2 Purification of Antibiotic A-2315 using Silica Gel: The active substance is precipitated with petroleum ether and the crude product 51 of Antibiotic A-2315 is isolated according to the procedure of Example IC above.

This product is dissolved in approximately 10 rnl of ethyl acetate and sufficient methanol to obtain a clear solution.

Add this solution to silica gel (manufactured by Merck) (filled with ethyl acetate).
Column containing 3005' (diameter 5crrL, length 90c
Add to m).

The column is then washed with 25000 mA' of ethyl acetate to remove impurities.

Ether acetate:ethanol (95:5) was used as the eluent, and A
-2315 active ingredient is eluted from the column.

These active fractions were combined and concentrated to dryness under reduced pressure to form a white powder. Obtain A-2315 of Of.

Elute using the above solvent system and further release as a pale yellow powder.
, 8g A-2315 is obtained.

Example 1 Control of porcine dysentery with A-2315: Swine dysentery is a diarrheal disease of pigs that is limited to the colon and is caused by Treponema hyodysenteriae.
yodysenteriae).

The disease is found worldwide and causes economic losses due to death and stunted growth.

Swine dysentery can be easily caused by infecting pigs by orally administering a saline suspension of colonic scrapings and contents prepared from pigs suffering from swine dysentery.

Thirty-two weaned pigs were placed 4 in each pen so that the average weight of the pigs in each pen was similar to that in the other pens.

Pigs in two pens were fed a diet containing A-2315 at a rate of 100 g/l.

Similarly, pigs in two other pens were fed a diet containing 50 S'/l and pigs in two more pens were fed a diet containing 25 P/l of A-2315.

Pigs in the remaining two pens were fed the same diet without A-2315.

After 2.3 days, all pigs were given colonic material orally.

The pigs were observed daily to see if they were alive or dead, and the surviving pigs were killed after 6 weeks.

All dead pigs were examined for colonic lesions indicative of swine dysentery.

The results are shown in Table 2 below.

As is clear from Table 2, A-2315 is effective in controlling swine dysentery at a dose of 25 P/l.

Using another lot of A-2315, 50.25 and 1
When the same test as above was conducted except that the drug was administered at a rate of 0 f/l, results were obtained that all doses were extremely effective.

Claims (1)

[Claims] 1. An antibacterial agent composition for animals other than humans, containing antibiotic A-2315 as an essential component.