JPS5948813B2 - Animal pathogen control agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides an antibiotic A-28086 complex consisting of Factor A, Factor B and Factor D, or any combination thereof, as an essential component. This invention relates to pathogenic microorganism control agents.

Antibiotic A-28086 complex and its constituent factors are:
Anti-bacterial, anti-filamentous, anti-viral, antibiotic pleuropneumococcus (anti-P
PLO), is useful as an anticoccicidal, insecticide, and acaricide, and is effective for increasing feed utilization efficiency in ruminants.

The present invention relates to Streptomysis aureophaciens (
Streptomyces aureofaciens)
NRRL5758 or Streptomyces aureofaciens ( Streptomycesaureo
Faciens faciens) NRRL8092 was cultured in a culture medium containing carbon sources, nitrogen sources and inorganic salts available to the strain and subjected to agitated aerated fermentation conditions until a significant amount of antibiotic activity was developed in the medium by the microorganisms. Antibiotic A-28086 complex comprising factor A) factor B and factor D obtained by culturing under the following conditions: The purpose is to provide an agent for controlling pathogenic microorganisms.

Antibiotic A-28086 Factor A is a white crystalline compound (
acetone (crystallized from water), lower alcohols,
Soluble in dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone and benzene, slightly soluble in hexane, insoluble in water, melts at about 98-100°C, re-solidifies and further Approximately 195
This antibiotic has the following physical and chemical properties ((a) to (n1)). (a) Molecular weight 764 (measured by spectrometry), (b) Elemental analysis value (approximate value), C, 66.69%: H, 9.85
(F6; 0.23, 10%0 (c) Empirical formula: C43H7
2On (measured by mass spectrometry), (d) (
(4M:-547 (C=0.2, methanol), (e)
In the infrared absorption spectrum (measured in chloroform), the following distinct absorption band peaks appear: 02.85, 3.
34, 5.83, 6.82, 7.22, 7.53 (weak)
, 7.78 (weak), 8.75 (strong), 8.95 (strong),
9.15, 9.50 (strong), 9.55 (strong), 9.60
, 9.85, 10.15, 10.45, 10.70 (weak) microns.

(f) In the ultraviolet absorption spectrum (ethanol solution), 2
Only shows terminal absorption below 00 mμ〇(g) Features of nuclear magnetic resonance spectrum (measured in deuterated chloroform): δ6.01, 4.21, 4.113.993.8
93.803.673.653.573.552.83
2.762.742.682.662.582.5
62.302.222.172.102.051.96
1.901.851.701.621.601.471
．． 391.311.251.180.950.930.
900.880.850.770.750.730.6
80.66P Ceramic (11) Has a titratable group with a PKa value of 7.9 in 80% dimethylformamide water (1) Characteristic X-line with the following lattice spacing (unit: Angstrom combs) Powder diffraction pattern (Cu++ irradiation, 1
．． 0(j
) Bacillus subtilis
1-1s) ATCC6633 strain was used as the detection organism, and benzene monoethyl acetate (3:
In the thin layer chromatography method tried with the system 2), R of 0.24 was obtained.
f value〇(k) Rf value as shown in the table below in the paper chromatography system using Bacillus subtilis ATCC6633 strain as the detection organism.

(1) The number of acid groups capable of forming salts and ester derivatives is 1
There are two.

- There is at least one hydroxyl group that can be esterified〇 In this specification, the term (antibiotic A-28086) Factor A refers to the above-mentioned Factor A and its C2, unless otherwise specified from the surrounding text. ~C6 acyl ester derivatives as well as physiologically acceptable salts thereof.

Antibiotic A-28086 factor B is a white crystalline compound (
It is soluble in lower alcohols, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone and benzene, but only slightly soluble in hexane and insoluble in water.
This substance has the following physical and chemical properties (a) to (k): (a) a melting point of approximately 150 to 153°C; (b)
Molecular weight 762 (measured with a high-resolution mass spectrometer), (c)
Empirical formula C43H7OOll (measured by high-resolution mass spectrometry), (d) In the infrared absorption spectrum (in chloroform) the following distinct absorption band peaks appear: 2
．． 82, 3.30, 5.77, 5.85, 6.80, 7
．． 20, 7.50 (weak), 7.72 (weak), 7.80 (
Weak), 8.57 (Strong), 8.68, 8.90 (Strong) 9.
10, 9.50, 9.83 (strong), 9.90, 10.1
0, 10.17 (strong), 10.43 (weak), 10.80
(weak), 11.20 (weak), 11.35 (weak), 11.
73 (weak 6), 12.03 (weak) microns 〇 (e) In the ultraviolet absorption spectrum (ethanol solution), the maximum absorption is at 220 mμ (E1% = 137.5:1 crIL゜ε = 10
．． 477).

(f) Nuclear magnetic resonance spectrum (in deuterated chloroform) shows the following features 〇δ7. ,20,7.09,
6.26, 6.15, 4.19, 4.12, 4.05,
3.95, 3.89, 3.78, 3.62, 3.59,
3.52, 3.48, 2.81, 2.73, 2.63,
2.54, 2.52, 1.99, 1.91, 1.84,
1.71, 1.67, 1.64, 1.55, 1.43,
1.33, 1.18, 1.11, 0.96, 0.94,
0.90, 0.87, 0.84, 0.77, 0.74,
0.68pIn(g) Bacillus subtilis ATCC6
633 as the detection microorganism, the Rf value of thin layer chromatography on silica gel performed in a system of benzene monoacetate (3:2) is 0.42.〇(h) Detection of Bacillus subtilis ATCC 6633 The Rf values in a paper chromatography system using microorganisms are as follows: 〇(1) One acid group capable of forming salts and ester derivatives is present 〇(j) Two ketone residues, (k ) At least one hydroxyl residue is present. In this specification, the term (antibiotic A-28086) Factor B refers to the above-mentioned Factor B and its physiologically acceptable o Antibiotic A-28086 Factor D is a white crystalline compound (crystallized from acetone monowater).

It is soluble in methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone and benzene, but only slightly soluble in hexane and insoluble in water. This substance is 96
It melts at ~98°C and has the following properties (a) ~ - (a) Molecular weight 778 (measured by high-resolution mass spectrometry) (b) Elemental analysis value (approximate value), C,6
7.59%; H, 9.38%; 0.22.77%, (c
) Empirical formula C44H74Oll (measured by high-resolution mass spectrometry), (d) (6) M:-56O (C=0.
1. Methanono (c), (e) Infrared absorption spectrum (
(measured in chloroform), the following absorption band peaks are observed: 2.89, 3.39, 3.43, 3.50, 5.
88, 6.90, 7.27, 7.60, 7.84, 9.
00,9.26,9.62,10.31,10.58,
11.10, 11.49 microns.

(f) No ultraviolet absorption is observed in 95% aqueous ethanol (g) Nuclear magnetic resonance spectrum in chloroform has the following characteristics, δ6.00, 4.20, 4.1
0, 4.00, 3.98, 3.92, 3.86, 3.8
3, 3.79, 3.67, 3.64, 3.57, 3.5
4, 2.88, 2.81, 2.71, 2.62, 2.5
8, 2.48, 2.43, 2.37, 2.29, 2.2
1, 2.15, 2.10, 2.04, 1.97, 1.8
9, 1.83, 1.76, 1.68, 1.61, 1.5
8, 1.55, 1.47, 1.39, 1.30, 1.2
5, 1.18, 0.95, 0.90, 0.88, 0.8
4,0.74,0.68 pF0(h) 80% dimethylformamide in water with one titratable group with a PKa value of 8.67.

(1) A characteristic X-line powder diffraction pattern having the following lattice spacing (unit: angstroms). (Cu
++ irradiation, 1.5405λ, nickel filter).
(j) Using Bacillus subtilis ATCC6633 as the detection organism, in the silica gel thin layer chromatography system shown below, the following Rf value〇Rf value Solvent system 0.26 Benzene monoacetate (3 :2)
0.66 ethyl acetate-diethylamine (95:5) (k
) The following Rf value determined by the paper chromatography system shown below using Bacillus subtilis ATCC6633 as the detection organism.

(1) The number of acid groups capable of forming salts and ester derivatives is 1
In this specification, the term (antibiotic A-28086) Factor D refers to the above Factor D and its C2 to C6, unless there is a contradiction from the surrounding text. Although many antibacterial agents are known, there continues to be a need for new and progressive antibiotics.

One problem with current antibiotic therapy is the fact that antibiotics differ in their efficacy against pathogenic organisms. Another problem is the emergence of strains of organisms that are resistant to standard antibiotics. An additional problem is the fact that patients individually suffer from certain antibiotics, often experiencing dangerous side effects due to hypersensitivity or toxicity. Because of these problems with current therapies, there continues to be a need for new antibiotics. In addition to the need for new antibiotics useful in treating human diseases, advances in antibiotics are also needed in the field of treating livestock diseases. 0 required
One important aspect is that advanced antibiotics are also needed to promote growth in poultry and livestock.
Growth promotion is achieved by reducing disease and increasing feed utilization efficiency. One of the diseases that is important in livestock disease treatment, especially in the livestock breeding industry, is the protozoal disease coccidiosis (COc).
It is well known that coccidiosis is caused by infection with one or more species of Eimeria or IsOspOra. (See also Lund and Hua 1 in 6 Daisies of Poultry 1, 5th edition, edited by Schwarte).

In view of the huge economic losses caused by coccidiosis and the disadvantages of existing anti-coccidial drugs, the search for even better anti-coccidial drugs continues.
Enteritis is another disease that causes significant economic losses to livestock producers. Enteritis occurs in young chickens, pigs, cows, and sheep, and is mainly caused by anaerobic bacteria, especially Chromatridium perfringens (Colstridium perfringens).
s) and viruses.

Intestinal toxicosis in ruminant animals, such as hyperphagia in sheep, is a symptom caused by infection with Clostridium perfringens.Next, a new challenge in veterinary medicine is to promote the growth of ruminant animals such as cattle. There is particular interest in achieving growth promotion by increasing the efficiency of zero feed utilization, which is considered an economic goal for humans.

The mechanism by which the main nutrients (carbohydrates) in the feed of feathered animals are utilized is well known. Microorganisms in the rumen of the animals break down carbohydrates into monosaccharides, and these monosaccharides are Pyruvate is metabolized by microbiological action and converted into volatile fatty acids (VFA) such as acetate, butyrate, or propionate.
). For a more detailed discussion, please refer to Leng (Philippson et al.: Physiology 1.
Of Digestion and Metapolism in
The Luminant (pages 408-410, published by Newcastle upon Tyne Oriel Publishing House, UK (1970))
). The relative effectiveness of the use of VFA is discussed in Feedstaffs, June 19, 1971, p. 19, by Matsukarav, and by J. An. Sci. 33, p. 282 (1971), and 1 Digestive Physiology and Nutrition of Luminance, vol. 2, p. 622-625 (197
1), acetate and butyrate are also used, as discussed by Church et al., but the use of propionate has even more important significance, and if the utilization efficiency of propionate decreases significantly, animals will lose ketone. Therefore, it stimulates the animal to produce a high proportion of propionic acid from carbohydrates, which increases the efficiency of carbohydrate utilization in the animal and also reduces the effects of ketosis.

Antibiotic A-28086 Factors A, B and D are new members of the polyether antibiotic family; Examples of members of this group include monensin (U.S. Pat. No. 3,500)
No. 1,568), dianemycin (Hamino KR.L.
; Whaen, M. M. ; Pitutenga, G. E. ; Chiambarin J. : and Gorman M. : Jiya)) Nar of Antibiotics Vol. 22, p. 161 (1
969)] Nigericin [L. K. Mari Pinkerton and J. Steinlau. W. Chiambarin, Biohimica Biofijica Research Communication, 33
Vol. 29 (1968)], and salinomycin [Japanese Patent Publication No. 47-25391, dated October 20, 1972, application number 19620/1971; Derwent NO
．． 7696OTl U.S. Pat. No. 3,857,948, and H. Kinashi, N., Otake, H. Yonehara, S. Satoichi and Y. Site 1, Tetrahedron Letters, 49
Volume 4955-4958 (1973)].

Fermentology and the 6-antibiotic complex (AntibiOticcOplex) as used herein
The term ゛ does not refer to a complex in chemistry;
Refers to a mixture of individual antibiotic factors produced in conjunction with each other.The fact that the ratio of individual factors produced in an antibiotic complex changes depending on the fermentation conditions is a key factor in the fermentative production of antibiotics. Those who are familiar with the matter will be able to approve 〇A-2
The 0A-28086 antibiotic complex is produced by culturing the novel strain Streptomysis aureofaciens NRRL5758 under agitated aerated fermentation conditions until the development of moderate antibiotic activity. , also produced by culturing another new strain of Streptomysis aureophaciens, NRRL8O92. Even when the A-28086 antibiotic complex is extracted from the fermentation process and mycelium with a polar organic solvent, the extracted antibiotic mixture is separated by concentrating the solvent, and the excess is added to the concentrate. Addition of petroleum ether to precipitate impurities, filtration and evaporation of the filtrate yields the A-28086 antibiotic mixture. The antibiotic mixture is further purified by column chromatography and individually purified. It is separated into factors.

A-28086 compounds inhibit the growth of organisms that cause pathogens to animals and plants. One aspect of this inhibitory activity is that A-2
In addition to the anti-coccidio effect of the 8086 compounds, the A-28086 compounds are anti-bacterial, anti-viral, anti-PPLO, insecticidal, and acaricidal substances. , and has the effect of increasing the feed utilization efficiency of ruminant animals〇The infrared absorption spectrum taken in chloroform is shown as follows〇A-28
The 086 factors are structurally related to each other.

During fermentation, at least four antibiotic factors are produced together, the factors obtained as a mixture are separated from each other, and factors A, B and D are separated from each other as described below. A mixture of 0A-28086 factors isolated as compounds is soluble in most organic solvents but insoluble in water.
The physical and spectral properties of B and D are described. Antibiotic A-28086 Factor A crystallizes from acetone monohydrate. A-28086 Factor A melts at about 98-100°C, resolidifies and melts again at about 195-200°C. As a result of elemental analysis of factor A, the following average percentage composition is obtained: C, 66.69%; H, 9.85%
;0,23.10% o Factor A has the empirical formula C43H72O
ll is proposed. The apparent molecular weight, determined by mass spectrometry, is 764. The infrared absorption vector of factor A in chloroform is shown in the attached figure (Figure 1).
It was shown to.

The following absorption band peaks are observed at 02.85,3
．． 34, 5.83, 6.82, 7.22, 7.53 (weak), 7.78 (weak), 8.75 (strong), 8.95 (strong)
, 9.15, 9.50 (strong), 9.55 (strong), 9.6
0,9.85,10.15,10.45,10.70(
Weak) Micron. The ultraviolet absorption vector of factor A in ethanol has only terminal absorption below 220 mμ. A-2 measured in deuterated chloroform
8086 田显Δ01罃彪硼此191PEJkノ1Ar力小pus驤h{水゜δ6.01,4.21,4.11,3.
99, 3.89, 3.80, 3.673.653.57
3.552.832.762.742.682.662
．． 582.562.302.222.172.102.
051.961.901.85 1.701.621.
601.471.391.311, 251.180.9
50.930.900.880.850.770.75
0.730.680.66p) Antibiotic A-28086 Factor A crystallized from acetone monowater exhibits an X-line powder diffraction pattern with the following characteristics: 0(Cu++ irradiation, 1.
5405λ, Nickel filter, d is lattice spacing (unit: angstroms).

The specific rotation of antibiotic A-28086 factor A is at a temperature of 25
-54 ratio (C=0.2, methanol) when measured at °C
This specific optical rotation is an average value based on several measurements.

Factor A in 80 (fl) dimethylformamide water
An electrotitration method showed the presence of titratable groups with a PKa value of 7.9.

Antibiotic A-28086 Factor A is soluble in various organic solvents (methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone, and benzene), but is poorly soluble in nonpolar organic solvents such as hexane. soluble and insoluble in water 〇 Antibiotic A-28086 Factor A has one acid group capable of forming salts and ester derivatives, and at least one hydroxyl group capable of esterification. Based on zero or more listed physical properties present, antibiotic A-280
86 The structure of Factor A can be proposed. However, the structure determination is merely an estimate, and the structure proposed here is merely an expression based on a hypothesis based on one experiment. 0A-2 should be understood
The predicted structure of 8086 Factor A is shown in Formula 1. Antibiotic A-28086 Factor B is a white crystalline compound (from acetone monohydrate) with a melting point of about 150-153°C.

Factor B, as determined by high-resolution mass spectrometry
has an apparent molecular weight of 762, and C43H7OOll
The infrared spectrum of factor B in chloroform is shown in Figure 2 of the appendix.
The following absorption band peaks are observed: 2.82, 3.
30, 5.77, 5.85, 6.80, 7.20, 7.
50 (weak), 7.72 (weak), 7.80 (weak), 8.5
7 (strong), 8.68, 8.90 (strong), 9.10, 9.
50, 9.83 (strong), 9.90, 10.10, 10.
17 (strong), 10.43 (weak), 10.80 (weak), 1
1.20 (weak), 11.35 (weak), 11.73 (weak)
, 12.03 (weak) microns.

7. The nuclear magnetic resonance spectrum of A-28086 factor B (in deuterated chloroform) shows the following characteristics: Oδ7.2
0, 7.09, 6.26, 6.15, 4.19, 4.1
24.053.953.893.783.623.59
3.523.482, 812.732.632.542
．． 521.991.911.841.711.671.
641.551.431.331.181.110.9
60.940.900.870.840.770.74
0.68PF0 Antibiotic A-28086 Factor B is soluble in various organic solvents such as methanol ethanol dimethylformamide dimethyl sulfoxide, ethyl acetate, chloroform, acetone and benzene, but not in non-polar organic solvents such as hexane. A-280 is extremely slightly soluble in solvents and insoluble in water.
86 Although the chemical structure of the antibiotic factor B has not been clarified, the physicochemical data obtained so far indicate that factor B
has only one carboxylic acid residue, two ketone residues and one
Antibiotic A-28086 factor D shows the presence of more than 3 hydroxyl residues.
A-28086 factor D is a minor factor when produced by S. aureofaciens NRRL5758; however, when produced by S. aureofaciens NRRL8O92, factor D is Reaching 10%〇Antibiotic A-2808
6 Factor D is a white crystalline substance (from water-acetone) with a melting point of approximately 96-98°C. 0 Its apparent molecular weight is 778 as determined by high-resolution mass spectrometry. ○ A-28086 Factor D The molecular weight of the peak in the mass vector of the sodium salt of was observed to be 800.5050 (C44H73OllNa=800.5050 is the calculated value).

In the mass spectrum of the free acid of A-28086 factor D, 7
78 has a small peak at 760.5117 (C44H72
m/E7 in the mass spectrum of the 0 free acid, where a large peak was observed at
6O is the result of losing water from the molecular ion 〇 Therefore, the molecular ion composition of the free acid of factor D is C number 4H740110 A-28086 The empirical formula of factor D is C44H74
The results of the elemental analysis of the 0 factor D for which Oll is presented show the following percentage composition: C, 67.59%: H, 9.38%
The theoretical percentage composition as 0,22.77%0C44H74011 is C, 67.87%; H, 9.5l%;
It is 0.22.77%.

The infrared spectrum of A-28086 factor D (Figure 8) contains the following observed maximum absorption: 0:2.89
, 3.39, 3.43, 3.50, 5.88, 6.90
,7.27,7.60,7.84,9.00,9.26
,9.62,10.31,10.58,11.10,1
1.49 microns.

A-28086 Factor D in 95% aqueous ethanol shows no absorption band in the ultraviolet region. The nuclear magnetic resonance spectrum of A-28086 Factor D in deuterated chloroform showed the following characteristics: δ6.00 ,4.20,4.10,4.
00,3.98,3.923.863.833.793
．． 673.643.573.542.882.812.
712.622.582.482.432.372.2
92.212.152.102.041.971.89
1.831.761.681.611.581.551
．． 471.391.301.251.180.95,0
．． 900.880.840.740.68 Antibiotic A-28086 Factor D crystallized from acetone monohydrate has the following characteristic X-line powder diffraction 'f turn (Cu++ irradiation, 1.5405λ, Nickel filter, d = lattice spacing (angstroms).

The specific optical rotation of antibiotic A-28086 factor D is -56 anti (C=0.1, methanol) when measured at a temperature of 25°C.

A-28 in 80% aqueous dimethylformamide
As a result of electrotitration of 086 factor D, the presence of a titratable group with a PKa value of 8.67 was observed.

Antibiotic A-28086 Factor D is soluble in various organic solvents such as methanol, ethanol, dimethylformamide, dimethyls)S sulfoxide, ethyl acetate, chloroform, acetone as well as benzene.
86 Factor D is soluble in very small amounts in non-polar organic solvents such as hexane and insoluble in water o Antibiotic A-28086 Factor D is an acid that can form salts and ester derivatives. The structure of the antibiotic A-28086 factor D has been proposed based on the physical properties listed above. However, the structure has not been determined. As it has only been estimated, it should be understood that the structure presented here merely represents a hypothesis based on one experiment.The estimated structure of 0A-28086 Factor D is shown in Eq. .

[In the formula, R1 is CH3 and R2 is C2H5, or R1 is C2H5 and R2 is CH3.

] Basurus subtilis ATCC663 as a detected organism
Antibiotic A-28086 factors A, B and DORf in filter paper chromatography of various systems when using 3
The values are listed in Table 1. In Table 2, Bacillus subtilis ATCC 6633 was used again as the detection organism, and silica gel (a flat plate pre-coated with a layer thickness of 0.25 m, F
-254,E. Antibiotic A-28086 factors A, B and D (7) Rf values in two types of thin layer chromatography systems performed on Talc Inc. product) A-28086
Another substance tentatively named -1 is antibiotic A-28086.
It is produced as a by-product along with the complex.

This A-28086-1 has no microbiological activity but is structurally related to A-28086 antibiotics.
-28086-1 is a white crystalline compound (crystallized from acetone/water) and has a melting point of about 160-162°C. A-2 prepared by synthesis with A-28086-I
Comparative studies of NMR spectra and other properties of the methyl ester of 8086 Factor A provided evidence that A-28086-1 is a closely related compound such as the methyl ester or stereoisomer of A-28086 Factor A. . A-280
86-1 is mixed with the active A-28086 antibiotic factors and precipitates first, but it is easily separated from the others by silica gel chromatography and detected using ethyl acetate as the elution solvent. Vanillin spray reagent (3% vanillin in methanol with concentrated H2SO for 100 d of this solution)
4O. 5! ! When sprayed with vanillin and heated, A-28086-I gives blue spots, whereas A-28086-I gives blue spots; -28086 Antibiotics give a bright pink spot, which quickly turns dark brownish blue. Antibiotic A-2
8086 Factors A, B, and D1 and the acyl ester derivatives of Factors A and D described herein are capable of forming salts. Physiologically acceptable salts include pharmacologically acceptable salts. , meaning a salt whose overall toxicity to mixed-breed animals is not increased compared to the non-salt state.

Representative and suitable alkali metal and alkaline earth metal salts of A-28086 factors A and B include sodium;
0 or A, including potassium, lithium, cesium, rubidium, barium, calcium, and magnesium salts
-28086 Suitable amine salts of Factors A and B include ammonium salts and quaternary C1-C4-alkylammonium salts and hydroxy-C2-C4-alkylammonium salts.Specific examples of amine salts include: , A-28086 factors A and B are ammonium hydroxide, methylamine, secondary butylamine, isopropylamine, diethylamine, diisopropylamine, ethanolamine, triethylamine, 3-amino-1-
There are salts that are generated by reacting with propanol etc.〇A-28
The alkali metal and alkaline earth metal cationic salts of Factors A, B and D1 and the acyl ester derivatives of Factors A and B are prepared by conventional methods for the preparation of cationic salts, e.g. Dissolving the antibiotic factor or ester derivative in acid form in a suitable solvent such as warmed methanol or ethanol;
To this solution can be added a solution of the exact calculated amount of the desired inorganic base in methanol and water. The salt thus formed can be isolated by conventional methods such as filtration or evaporation of the solvent.

Salts formed by reaction with organic amines can be prepared in a similar manner.

For example, a gaseous or liquid amine can be added to a solution of the antibiotic agent in a suitable solution such as acetone, and the solvent and excess amine can be removed by evaporation. When treating livestock with antibiotics, the form of the antibiotic is not very important.

In most cases, depending on the internal state of the animal, the drug changes to a state other than the state in which it is administered.Therefore, administering the drug in the form of salt is meaningless as a treatment method itself. 0
However, for reasons of economy, convenience, as well as toxicity, the salt form may be chosen. 0A-28086 Factor A forms acyl ester derivatives.

Esterification occurs at one location of the hydroxyl group of A-28086 Factor A when treated with any C2-C6 monoanhydride or acid chloride. For example, a typical method for preparing such esters is There is a method in which A-28086 factor A is reacted with the anhydride of the partner acid at room temperature. These ester derivatives are also effective as antibiotics and are useful as agents to increase feed utilization efficiency.
Describing the properties of ester derivatives 0A-28086 Factor A acetyl ester derivatives have a melting point of about 100-10
It is a white crystalline substance (from acetone and water) at 3°C.

The empirical formula of the A-28086 Factor A acetyl ester derivative is C4,H74Ol2, and the molecular weight is approximately 807, but both are based on the empirical formula proposed for A-28086 Factor A. Elemental analysis values of acetyl ester derivatives are shown. Calculated value, C45H74Cl2, C, 66.97%: H, 9.24%; 0,23.
79%, actual value, C, 67.67(f); H, 8.7l
%; 0.23.13% 0A-28086 The infrared absorption spectrum of the acetyl ester derivative of Factor A taken in chloroform is shown in FIG.

The following maximum absorption bands are observed at 02.85, 3.36,
3.38 (strong), 5.80, 6.83, 7.25, 7.
52 (strong), 7.60 (weak), 7.80 (strong), 8.4
5 (strong), 8.80 (strong), 8.95 (strong), 9.10
(strong), 9.20, 9.63, 9.80 (strong), 10.
12 (weak), 10.25 (weak), 10.50 microns.
In the ultraviolet absorption spectrum of A-28086 factor A acetyl ester in ethanol, only the terminal absorption was observed.
Electrotitration of 28086 Factor A acetyl ester derivative showed the presence of titratable groups with a PKa value of 8.5. 0A-28086 Factor A propionyl ester derivative is a white crystalline compound with a melting point of about 96-98° Based on the empirical formula proposed for A-28086 Factor A, the propionate derivative of Factor 0A-28086, which is a compound (from acetone monohydrate), has an empirical formula of C46H7
6Ol2 with a molecular weight of approximately 821 The following percentage composition values were obtained from the elemental analysis values of a 0 factor A propionic acid ester derivative, C46H76Ol2
as, C, 67.29Ol); H, 9.33%; 0,
23.38Cff), actual value, C, 66. O6%; H,
9. l7%; 0,23.41%0A-28086 factor A
Figure 4 shows the infrared absorption vector of the propionic acid ester derivative in chloroform. , 5.75 (strong), 5.82, 6.
81, 7.22, 7.30 (strong), 7.50 (weak), 7
．． 60 (weak), 7.80, 8.43, 8.75 (strong),
8.90, 9.05, 9.15 (strong), 9.50 (strong)
, 9.63, 9.83 (weak), 10.05 (strong), 10
．． 13, 10.20 (strong), 10.45, 10.68 microns. Only terminal absorption was observed in the ultraviolet absorption spectrum of the propionic acid ester derivative of A-28086 Factor A in ethanol.

Antibiotics A-28086 Factor A butyrate ester derivative is a white crystalline (from acetone monohydrate) compound with a melting point of about 96-98°C. As inferred from the proposed empirical formula, the empirical formula is C47H7
8Ol2, the molecular weight is approximately 835, and C, 67.6O
Figure 5 shows the infrared absorption spectrum in chloroform of the butyrate ester derivative of A-28086 factor A, which has an approximate composition of %; H, 9.4 lO/); 0.22.99 (I). .

02.89, 3.40, 3 where the following maximum absorption was observed
．． 45, 3.51, 5.85, 5.92 (strong), 5.9
7 (strong), 6.90, 7.30, 7.84 (weak), 8.
55, 8.85 (weak), 9.01 (strong), 9.26, 9
．． 76, 9.95, 10.31, 10.64 microns.

Antibiotic A-28086 factor A citric acid ester derivative is a white crystalline compound (from acetone monohydrate) with a melting point of about 173-175°C. A-28086 factor A citric acid ester derivative is The experimental formula inferred from the experimental formula of A-28086 factor A is C48H8OOl2
, molecular weight approximately 849 and C, 67.89%; H, 9.5OO
I) has an approximate elemental composition of 0.22.61%.

Figure 6 shows the infrared absorption spectrum of the tight acid ester derivative of A-28086 Factor A in chloroform.
40, 3.45, 3.51, 5.87, 5.92 (strong)
, 5.99 (strong), 6.91, 7.30, 7.69 (weak), 7.87 (weak), 8.16, 8.58, 8.85 (
Weak), 9.26, 9.76, 10.00 (weak), 10.
31, 10.64 microns.

Antibiotic A-28086 Factor A caproic acid ester derivative is a white crystalline compound (from acetone monohydrate) with a melting point of about 163-167°C. A-28086 Factor A caproic acid ester derivative is A- Empirical formula C49H estimated from the experimental formula presented as 28086 factor A
82Ol2, molecular weight approximately 863 and C,68. l8%;
Figure 7 shows the infrared absorption spectrum in chloroform of the caproic acid ester derivative of 0A-28086 factor A, which has an approximate elemental composition of H, 9.58%; 0.22.24%. was observed 02
．． 90, 3.40, 3.45, 3.51, 5.87, 5
．． 92 (strong), 5.97 (strong), 6.90, 7.30,
7.66 (weak), 7.84 (weak), 8.16, 8.58
, 8.85 (weak), 9.05 (strong), 9.17, 9.7
2, 9.95, 10.29, 10.62 micron〇A-
It was found that when 28086 factor A was acylated, the following changes occurred in the 1H nuclear magnetic resonance spectrum.
The carbinyl resonance occurs at about 4PP and about 5.3PF
1 (the exact position varies slightly for different acyl derivatives) to the lower region.

As a result, the vinyl proton signal is also transferred. This shift is characteristic of the changes appearing in the substructure shown below (in this figure R represents C1-C5-alkyl). Release (homonuclea dekatsuprinkri experiment results and
13C from acetic acid and propionic acid ester derivatives
This is in complete agreement with the nuclear magnetic resonance experimental results.

A-28086 Factor A(7) C2-C6-acyl ester derivatives are soluble in various organic solvents such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone, and benzene. However, it is only slightly soluble in nonpolar organic solvents such as hexane, and insoluble in water. A
All of the C2-C6-acyl ester derivatives of -28086 have one acid group that can form salts and ester derivatives.

The novel antibiotic of the present invention is a novel antibiotic for Streptomysis
It is produced by culturing a productive strain of A. aureophaciens A-28086 in a suitable culture medium under aerobic agitation conditions until substantial antibiotic activity is produced.

The produced antibiotics are recovered by various known separation and purification methods. One of the new microorganisms useful for the production of antibiotic A-28086 was isolated from soil samples collected on Mount Ararat in Turkey.

This organism has been described by Shearing (E.B.) and Gottlib (D.) as a cooperative description of the type culture of streptomysis, and as an additional species description of streptomysis. First and Second Studies 0 International゜Biuretin Ob゜Systematic Bacteriology Volume 1, 18, 279-39
2 (1968), Streptomysis aureophaciens datugai (Streptomysis aureophaciens)
tiOmycesauresfaciensDugga
Classified as one strain of r)
→ t engineering small kasuri na n convex style touch resistance Z damaged international゜streptomysis゜project
E. B. ) and Gottlieb (D), Methods of Streptomysis Species International Biology Volume 16, Volume 31.
3-340 (1966)).

The color names were selected according to the ISCC {BS method.
K. L. ) and J.D.B.'s 6 The ISC
C-NBS Method of Designing Color and Dictionary of Color Names 1U-SJePLOfCOlTlOleree.
CirC. 553, l955, WashingtOn.
D. C. ]. The numbers and symbols in parentheses refer to the Tresna 1 and Butkus color series [Tresna 1 (H.D.
) and Butkus (S.J.), 6 Systems of Color Wheels for Streptomysis.
Taxonomy JA Pride Microbiology 1.11
Vol. 335-338 (1966)]. I drew a line under the color naming chart. Color plots of Mertz and Bowl are in parentheses [Mertz (4) and Paul (M.R.
), 6 Dictionary, Oba Color, 1, MacGrowniHill Book Company, Inc., New York, NY]0 Cultures were grown for 14 days at 30°C unless otherwise noted. .

Characteristic Description of A-28086 Production Strain NRRL5758 Morphology Spore-forming aerial hyphae consist of hook-shaped, ring-shaped, and spiral-shaped ones.

A typical flexorectal morphology is also observed. The spores are short and cylindrical, with 10 to 50 spores connected in a chain. The spores were 1.3μXl. It has a size of 75μ (1.
The surface structure of the spores is smooth when observed using an electron microscope in the range of 3μ to 1.95×1.3μ. Culture characteristics of NRRL5758 on various media/ISP
β2 (Yeast Exhaust - Malt Process Scratch) Medium: Strong growth, medium yellow on the underside [11K3]. Moderate to good aerial mycelial development, sporulation, white Wl3ba and dark gray (Gy)
31h, no soluble dye.

・ISPβ3 (Automino (Ha) medium: Good growth, back side is grayish yellow [11B2], sufficient formation of aerial mycelia, dark gray (Gy) 31h, some brown soluble pigment.

・ISPβ4 (inorganic salts monostarch agar) medium: vigorous growth,
The underside is pale yellowish brown [12E5], aerial mycelia and spores are white with a purple 13ba IJ. Gy) Light gray d, no soluble dye.

- ISPβ5 (glycerin-asparagine agar medium: good growth, back side is yellow-green with a bluish tinge [10B0 good aerial mycelium development, spores (Gy) yellow-gray 2dc, to bright gray-reddish-brown 5fe, no soluble pigment.

・Tomato paste oatmeal agar medium: vigorous growth,
The underside is pale yellowish brown [13H7L aerial mycelia and spore production moderate to good w white a to (Gy) medium is gray g: very small amount of brown soluble pigment.

・Glycerin-glycine agar medium: vigorous growth, dark gray-yellow underside [1216], good aerial mycelium and spore formation (Y)
Pale yellow 2db, no soluble pigment.

●Glucose-Ahasparagine agar medium: Strong growth, back side grayish-green-yellow [12E2], aerial bacteria [? [
)" Bowl? Bowl gray 2dC, very small amount of brown/ordinary agar medium: Good growth, back side is gray-yellow [12B2L Aerial mycelia and spores are present, but color is not specified due to poor production, no soluble pigment.

●Bennet's agar medium: Strong growth, back side gray-yellow [12K3L aerial hyphae and poor spore production (Gy), yellow-gray 2dc, no soluble pigment.

●Calcium malate agar medium: Good growth, back side grayish brown [15C8], no aerial mycelium or spore formation, brown soluble pigment, transparent inoculation position.

・Tsabetsu agar medium: No color specified due to poor growth and poor growth.

- Emerson's agar medium: vigorous growth, gray-yellow underside [11J5], no aerial mycelium or spores, no soluble pigment.

●Tyrosine agar: Strong growth, pale olive brown on the underside [14C4]. Aerial mycelia and many spores, CW) b (center) to (Gy) pale brown-gray 3fe (periphery), slight brown soluble pigment.

・Trifton yeast agar medium: Poor growth, no color specified.

Next, the physiological properties of NRRL5758 bacteria were investigated according to the standard recipe.

The observed properties and known characteristics are as follows. Furthermore, the results of examining the carbon source utilization ability of the NRRL5758 bacterium are as shown below, and the symbols used to indicate the degree of growth of the bacterium indicate that another new A-28086 producing microorganism is After mutation treatment, it was derived from Streptomysis aureofaciens MRRL5758 by a series of natural selection methods.

This microorganism, identified as NRRL8O92, is also Streptomysis aureophaciens (St.
ReptOmycesaureOfaciens) was classified as a strain of Dagaichi. This classification is based on previously described Streptomyces taxonomy and studies of the NRRL8O92 bacterium using similar culture conditions. The characteristics of the NRRL8O92 bacteria observed in these studies are summarized in the following items. A-28086 production strain NR
Characterized form of RL8O92 On ISPβ7 medium (tyrosine agar), the main culture often forms hook-shaped aerial hyphae (HOOk), but mainly short erect sporophores (SpOrephOre).

Spore chains have no more than 10 spores per series, usually 4 to 7 spores per series.

Short, upright spore chains were observed in the following media: ISP
β3, Zurbeck's solution agar and ISPβ5. In addition, numerous condensed hyphae were observed on Emerson's agar. Electron microscopy was performed on tyrosine agar (ISPβ7) and glucose-asparagine agar. Spores are smooth, 1.2 long
It is distributed in a size range of μ to 2.0 μ and a width of about 1.0 μ. The average spore size is 1.6μ x 51.0μ.
Culture characteristics of NRRL8O92 in various media: ・I
SPβ2 (yeast exhaust - malt factory scratch) medium: sufficient growth,
The underside is pale yellowish brown [12H8], sufficient aerial hyphae, poor spore formation, aerial hyphae pale gray [11A0, no soluble pigment.

・SPβ3 (Automino (ha) medium: Sparse growth, transparent underside, no aerial mycelium.

No soluble dyes.・ISPβ4 (inorganic salt-starch agar) medium: Medium growth, gray-yellow underside [11B2]. Aerial hyphae are poor and spore-forming, and the aerial hyphae are bluish-yellow gray [10A1
). No soluble dyes.

●ISPβ5 (glycerin-asparagine agar medium: medium growth, back side bluish yellow [10F2], sufficient aerial hyphae, poor spore formation, aerial hyphae white [10A1L no soluble pigment).

●Tomato paste-oatmeal agar medium: medium growth, green-yellow with gray underside, sufficient aerial mycelium, medium spore formation, pale blue-gray [53A2L no soluble pigment.

・Glycerin-glycine agar medium: Strong growth, back side gray-yellow [11EA]. Medium aerial hyphae and white [10A1]. No sporulation, no soluble pigment.

・Glucose-asparagine agar medium: Medium growth, pale yellow underside [10F2]. Aerial mycelial spore formation is moderate, white [10A1]. No soluble dyes.

・Ordinary agar medium: Sparse growth (back side with sparse is bluish yellow [10
B2). No aerial mycelia, no soluble pigments.

・Bennet's agar medium: sufficient growth, medium yellow-pink on the back side [
11A7). Very poor aerial hyphae, no sporulation, no soluble pigments.

●Calcium malate agar medium: Extremely poor growth, transparent, no aerial mycelium, no soluble pigment.

・Zavetzke liquid agar medium: Very poor growth, transparent back surface, no aerial mycelium, no soluble pigment.

●Emerson's agar medium: Medium growth, gray-yellow underside [1115]. Aerial mycelium scattered, no sporulation, no soluble pigment.

・Tyrosine agar medium: medium growth, light yellowish brown on the back [12H6], medium aerial mycelium,
The surrounding area is light blue-gray [53A2]. Near white center, moderate sporulation, no soluble pigment.

・Trifton Yeast Agar Medium: Very poor growth, transparent,
No aerial mycelia, no soluble pigments.

The NRRL8O92 bacterium was also tested for certain physiological properties according to standard formulations.

The observed properties and known characteristics are as follows. Growth temperature (ISP medium β2 yeast, malt, scratched slope): 2
Grows vigorously at 5℃, sufficient (fair) aerial mycelium production, light brown on the underside, no soluble pigment, grows vigorously at 30℃, sufficient aerial mycelium, light brown on the reverse, no soluble pigment, grows vigorously at 37℃, Aerial mycelium is abundant, the back side is brown, soluble pigment brown, 40℃,
Strong growth, sparse aerial mycelium, reddish brown underside, deep reddish brown soluble pigment, 45°C, good growth, no aerial mycelium, reddish brown underside, medium reddish brown pigment.

Next, the carbon source utilization ability of the NRRL8O92 bacteria was examined, and the results are as shown below, and the symbols used indicate the degree of growth.

Some properties of the A-28086 productive Streptomysis aureofaciens strain differ from those of the organism described by Shearing and Gottlieb.

These differences are summarized in Table 3. NRRL575
Differences in specificity between the 8 bacteria and the NRRL8O92 bacteria are summarized in Table 4.

A-28086 Cultures of Streptomysis aureofaciens useful in the production of antibiotics have been deposited with the Northern Marketing and Nutritional Research Division, Agricultural Research Service, United States Department of Agriculture, Peoria, Illinois 61604, USA. TheNOr-ThernMBrkettingand
NutritiOnResearchDivisiOn
．． U. S. Dept. OfAgrjcuIture・A
griculturalService, PeOria
2lllnOis, 6l6O4), from which NRRL5758 and NRR
It is available under accession number L8O92.

Also, Streptomysis aureofaciens NR
RL5758 and Streptomysis aureofaciens NRRL8O92 are located at 5-8-1 Inage Higashi, Chiba City.
FERM-P7f63O98 and FERM-
It has been deposited as P-Taki No. 3099.

Streptomysis aureofaciens NRRL5
758 or Stoptomysis aureofaciens NRRL8O92 can be grown on any of a number of culture media.

However, the economics of production, that is, the pros and cons of yield,
In addition, there are culture media that are suitable in terms of the difficulty of product separation. The preferred carbohydrate source for large-scale fermentation is
For example, tapioca dextrin and sucrose are used, but other substances such as glucose, corn starch, fructose, mannose, maltose, and lactose can also be used. Other advantageous carbon sources include corn oil, peanut oil, soybean oil, and fish oil.

The preferred nitrogen source is enzymatically degraded casein, but
Peptone soy flour, cottonseed flour, amino acids such as glutamic acid, etc. 4 can also be used. Add to culture medium. The inorganic nutrient salts include sodium 1, madanesium 1,
Calcium-1, Ammonium-1, Hydrochloric acid-, Carbonic acid-monosulfuric acid-
There are conventional soluble salts that form ions, such as nitrate monoion. The microorganisms necessary for the growth and multiplication of the microorganisms should also be included in the medium, and such trace elements are usually present as contaminants of other components in the medium in amounts sufficient to meet the growth requirements of the microorganisms. .

If foaming is a problem in large-scale fermentation cultures, add a small amount of an antifoam agent such as polypropylene glycol (
For example, it may be necessary to add 0.2 d/t).

Others, although not absolutely necessary, A-28086
It is noted that the antibiotic production of productive Streptomyces aureofaciens strains is enhanced by the addition of small amounts of oil, such as soybean oil.

Second, to produce substantially significant amounts of A-28086 antibiotics, aerated fermentation in tanks is preferred, although small amounts of A-28086 antibiotics may be obtained by shake flask culture. Probably.

Because of the time lag (1 ag) in antibiotic production associated with inoculating microorganisms in the form of spores into large tanks, it is preferable to inoculate with viable seed bacteria. A viable inoculum is a fresh, actively growing culture of a microorganism that is prepared by inoculating a small volume of culture medium with sporeforms or pieces of mycelia. This viable seed culture is then transplanted into larger tanks. The medium used to grow the viable seed culture may be the same as that used in the large tank, but other media may also be used. Next, the present A-28086-producing microorganism can grow at a temperature of about 20-40°C, but A-28086-producing microorganisms can grow at a temperature of about 27-30°C.
The most favorable production of 8086 appears to occur.

In aerobic agitation cultures, sterile air is conventionally blown into the culture medium.

In order to sufficiently propagate microorganisms, it is generally said that the amount of air used in tank production is preferably at least 0.1 volume of the culture solution per minute. This A-28086
In order to effectively produce antibiotics, it is particularly preferable that the amount of air used for tank production is at least 0.25 volume of the culture process in the tank. The production of the antibiotic is inhibited and does not decrease due to the high degree of dissolved oxygen. Antibiotic production is carried out during the fermentation period by using the culture process or cultured moss solid extract as a sample and conducting an antibiotic activity test against microorganisms known to be sensitive to the target antibiotic. can be tracked.

One assay organism useful in testing the antibiotics of the present invention is Bacillus subtilis ATCC 6633. This biological assay is performed on a paper disk (Paper disk) on an agar plate.
This is preferably carried out using a perdisc (filter paper flat disk). The initial pH of the medium without culture inoculation is
It varies depending on the medium used. Generally, its pH is 6.
It is sufficient that the pH is in the range of 0 to 7.5, and the pH at the final stage of fermentation (harvesting of the product) is slightly higher and is usually in the range of 6.5 to 8.0. Generally, antibiotic activity is detected from the second day of fermentation, usually about 6 to 10 days.
The greatest results will be seen during the day.

Following production under aerated agitated fermentation conditions, the A-28086 antibiotic can be recovered from the fermentation liquor by methods known in the fermentation art.

Antibiotic activity produced during fermentation of A-28086-producing microorganisms is found in both the mycelial mass and the culture filtrate. Therefore, maximum recovery of A-28086 antibiotics is achieved by a combination of methods including filtration, extraction, and adsorption chromatography. The preferred solvent for separating A-28086 antibiotics from the entire fermentation process or its filtrate is ethyl acetate, but other commonly used solvents are also sufficient. A-
One particularly advantageous method for separating 28086 factors A, B and D is to lower the pH of the entire fermentation process to about 3.0. This PH3. In O, A-28086 factors A, B and D can be conveniently filtered out from the mycelial mass.
Another advantageous method of separating the A-28086 factors is to add a bicarbonate, such as sodium bicarbonate, to the entire fermentation process at a rate of approximately 1 gram per liter of process. Thereby, the A-28086 factors are conveniently separated from the mycelial mass in salt form. Methanol is the preferred solvent for separating the antibiotics from the mycelial mass, but other lower alcohols and ketones may also be used. Next, the azeotropic distillation method is also A-28
It is advantageously used for the recovery of 086 antibiotics. In this method, an organic solvent is added to the fermentation process which forms a suitable azeotrope with water. When this solvent-pros mixture is azeotropically distilled to remove at least half of the water from the pros
A water-solvent mixture is obtained in which A-28086 antibiotics are dissolved in an organic solvent. Insoluble by-products are separated by suitable methods such as filtration or centrifugation and A-280
86 antibiotics are recovered from organic solvents by known methods such as evaporation of the solvent, precipitation by addition of a non-solvent, or extraction methods. Organic solvents which form suitable azeotropes with water for carrying out such recovery methods include, for example, butyl alcohol, amyl alcohol, hexyl alcohol, benzyl alcohol, butyl acetate, amyl acetate, 1,2-
Examples include dichloroethane, 3-pentanone, 2-hexanone, benzene, cyclohexanone, toluene, and other xylenes.

Recovery by azeotropic distillation is particularly advantageous in large-scale fermentative production processes.

The water and solvent retained as an azeotropic distillate can be separated by known techniques and released for further use. Also, the water thus removed is free of contaminants and does not require any waste treatment operations. Moreover, the solvent separated in this way can be reused in the same process. next,
Purification of the A-28086 antibiotic is progressed by adding extraction and adsorption operations.

Silica gel, carbon, Florisil (trademark Magnesium Silicate, manufactured by Floridine Inc., P.O. Box No. 989, Tallahassee, Fla., USA), and others are advantageously used. Alternatively, since the culture solid contains various components of the culture solution and mycelia, it is possible to remove only the water content without extraction or separation.
-28086 Used as an antibiotic source.

That is, after the antibiotic activity of A-28086 is achieved, the cultured product can be lyophilized and directly mixed into the feed premix. As another point of view, after the A-28086 activity is generated in the culture medium, the bacterial cells are separated and dried, resulting in a product that can be used directly as a feed premix.

When separating bacterial cells for such uses, adding lime carbonate (approximately 10 9 /t) facilitates filtration and provides an improved dry product. Under the conditions that have been used for many years, the NRRL5758 and NRR. The Streptomysis aureofaciens strain designated L8O92 produces antibiotic factor A-28086 as a product.

The ratio of the production factors varies depending on the fermentation conditions at the time, but in general, factor A accounts for more than 99% of the total antibiotic activity recovered from the NRRL5758 strain, and NRRL8O9
The two strains accounted for more than 90% of the total recovered antibiotic activity. A-28086 factor B accounts for most of the remaining antibiotic activity from strain NRRL5758, and factor D is a minor factor. On the contrary, A-28086 factor D is NRRL
Approximately 8-10% of the total antibiotic activity recovered from strain 8O92 occurs, with factor B being the minor factor. Antibiotic A-2
8086 Factors A, B, and D are separated from each other as individual compounds using column chromatography, thin layer chromatography, or other known methods.

For example, factors A, B and D are separated using column chromatography on silica gel by eluting the column with various solvent mixtures, such as benzene monoethyl acetate. When a benzene monoethyl acetate solvent mixture is used on a silica gel column, factor B is eluted first, and factor A (5-D) is eluted later. It is an easy method to detect.A-28086 compounds have the effect of inhibiting the growth of bacteria and filamentous fungi that are pathogenic to animals and plants.

Table 5 below compares the microbial activities of factors A and B of A-28086. The test method is the usual disk diffusion method (
Disc-Diff-UsiOnmethOd), [6
This method involves soaking 11 gt of blotting paper in a solution of 1 tbsp of product/1 d of solution and placing this blotting paper on an agar plate inoculated with the test organism.

The next important thing is that A-28086 compounds inhibit the growth of anaerobic bacteria.

The minimum inhibitory concentration (MIC) at which A-28086 Factor A inhibits various anaerobic bacteria was determined by a standard agar dilution method, and the results are summarized in Table 6.

The end point was determined after 24 hours of culture. A-2
The 8086 factor AO)C2-C, ester derivatives also have antibacterial and antifungal activity. Also,
From a certain point of view, these derivatives were also seen to have enhanced activity against Gram-positive bacteria compared to factor A. Therefore, the relative Gram-positive bacterial activity of some of these derivatives was compared with that of Factor A. Compounds as a control were R. Kuzel and F. W. A semi-automated system (Elanco's AutoTurb-type microbial testing system) was used to test It was tested by the degree measurement test method. The following test parameters were used in this A-28086 antibiotic assay. The bacterium Staphylococcus aureus (H-heatre) NRRLB-314 for assay was placed in a nutrient medium (P
The cells cultured at 37° C. for 4 hours in H7) were used.

Test sample and standard were mixed with methanol and water (10:90)
dissolved in. Standard product A-28086 factor A 2,3,
The samples were run on an AutoTurb color cell (AutoTurbcafOusel) at concentrations of 4, and 5mc γ1.

The analyte compounds were diluted to contain approximately 3-4 md of active substance per ml before application to the color cell. The relative activity of the test compound determined in comparison with the activity of the standard product is shown below. C person 1'
``Another useful aspect of the antimicrobial activity of the A-28086 compounds is mycoplasma or mycoplasma (MycO
plasma) is the activity against.

The species of Mycoplasma, also known as Buro Loproimonia (PPLO), is pathogenic to humans and various animals. Agents effective against this PPLO organism are particularly desired by livestock producers. The minimum inhibitory concentrations (MICs) of antibiotic A-28086 against various Mycoplasma species determined by the in vitro Pross dilution study are summarized in Table 7 below. Even a solution containing a trace amount of antibiotic A-28086, about 10mc9 per 1a, is effective as an external disinfectant for protecting animals from Myconolazma species.

Second, A-28086 compounds are also antiviral agents.

A-28086 Factor A is a type of polio virus (Typ.
ellpOIiOvirus), cowpox virus (Va
cciniavirus Herpes
virus) and Semliki Forest virus (S
emlikiFOrestvirus), which were described by Siminoff [Applied Microbiology 19(1), pp. 66-72].
1961)] In vitro plaque inhibition test similar to the test method (
InvitrOplaquesu-PpressiOn
Confirmation was obtained through tests). A-280
Factor 86 A also exhibits activity against infectious gastroenteritis virus, Newkatus virus, and infectious bovine nasal bronchitis virus, which were also confirmed in similar tissue culture studies. Therefore, in some respects, A-
Any of the 28086 compounds can be administered orally, topically, or by injection to clear mammals for the purpose of controlling viruses.

The effective dosage for the prevention or treatment of virus disease varies from approximately 1 to 5 mf per 1k9 body weight of a clear mammal, depending on the virus to be used and whether the purpose of the drug is prophylactic or therapeutic. is normal. In addition to the above, A-2
Solutions containing the 8086 compound, preferably in conjunction with surfactants, can also be used to disinfect in vitro habitats in which viruses such as poliomyelitis or vesicae are present.

In such a case, about 1 to 1500 mcf1/WLl (7)
A liquid preparation containing A-28086 compound is effective in suppressing viruses. The acute toxicity of the antibiotic A-28086 factor A was determined by intraperitoneal injection administration in mice.
D5O is 7.15TI9/Kg.

Second, the A-28086 compounds are also insecticides and acaricides.

A-28086 compounds are effective against insects such as Mexican beetles, milkweed bugs, and house flies, and are effective against insects such as the Mexican beetle, milkweed bugs, and house flies, as well as TwO-SpOtted spider mites.
It is also effective against mites such as e), and only a small amount of about 500 pieces is sufficient. Furthermore, the A-28086 compound is also effective against MO squitelarvae, and the amount used may be as small as 1 PI]l. This A-280
One important property of the 86 compound is its anticoccidial activity.

For example, as a result of breeding experiments, when a small amount of 0.003% of any compound of this A-28086 was added to the feed of chicks (chicken chickens), weight gain was improved;
? This small amount prevents the death of chicks attacked by coccidiosis agents and reduces the number of losses. Tables 8 to 10 show the results of factor A efficacy tests in groups of chicks fed various Eimeria pathogens. For the prevention or treatment of coccidiosis in poultry, A-2808
Preferably, one of the six compounds is orally administered daily to the bird in an amount that is non-toxic and effective against coccidia protozoa.

There are many ways to feed A-28086 compound to birds;
The simplest method is to give it together with a physiologically acceptable carrier, and particularly preferred is to give it together with the bird's inoculated feed. Additionally, various factors must be considered in determining the appropriate amount of the A-28086 compound;
The rate of administration is usually 0.003 to 0 of drug-free feed.
．． It will be in the range of 0.04%, preferably in the range of 0.005-0.02%. Another important property of the A-28086 compounds is their ability to improve animal feed utilization efficiency.

For example, A-28086 compounds improve feed utilization efficiency in ruminants with developed ruminant function.

As previously discussed, the efficiency of carbohydrate utilization in ruminants is such that they stimulate the distributed biota within the animal's rumen to produce propionate rather than acetate or butyrate. It can be increased by manipulating the

Feed utilization efficiency is detected by observing the production and concentration of propionate compounds in the rumen using the following method. First, rumen secretions are produced in the Fist.
Obtained from male calves in which the ula) have been surgically expanded and placed in the lumen.

The calf is raised on a grain-rich diet. Samples of rumen fluid were made using four layers of cheesecloth.
eclOth) and collect the filtrate. Particulate matter remaining in the cheesecloth is suspended in a physiological buffer sufficient to restore the original rumen fluid volume and leached out again. According to the description by Chien et al. in Journal of Research and Science, Vol. 38, pp. 1225-1230 (1955), the buffer solution used in this case has the following composition. The two filtrates are mixed and allowed to stand, allowing suspended solids to float to the surface.

The clear layer is separated, diluted with the same buffer (1:1) and then adjusted between...6.8 and ZO. Add the diluted rumen fluid (10ml) to the feed 4077? , 1 wt of soy protein supplement, and the test compound into the 25d flask. Four replicate flasks are used per treatment. In addition, two sets of four flasks are used as controls, and a control at 0 hours and a control at 16 hours of culture are used. All test flasks are incubated at 38°C for 16 hours. When the incubation is complete, measure the... of each flask and add 25% metaphosphoric acid (2d) to each flask. After each sample is allowed to settle, the supernatant is analyzed by gas chromatography to measure propionate, acetate, and butyrate compounds. A compound that shows an increase in propionate production more than the control is significant as an active compound. Test compound results are compared stochastically to control results.

Table 11 shows the ratio of the concentration of volatile fatty acids (FA) in the treated flasks to the concentration in the control flasks. Carbohydrate utilization efficiency is determined by the presence of a fibril in the rumen (rumen) so that samples of the contents of the rumen can be extracted.
In-vivo studies conducted in animals equipped with
ivO) will be further elucidated by testing.

The test results shown in Table 12 each have a weight of approximately 10
00tbS. This test was carried out using mature cattle that had been fitted with a living tube. Two layer cows were fed a normal diet and four cows in each treatment group were fed the same diet plus A-28086 Factor A.

10% metaphosphoric acid (100 d) was added to each 100 d of rumen fluid taken out, the sample was allowed to stand, and then the transparent liquid was analyzed by gas chromatography to measure the propionic acid concentration. The results in Table 12 are the average percent increase in propionic acid concentration in the rumen averaged over five times over the 14 day treatment period. The control at this time was approximately 20 mole percent propionic acid. Furthermore, in the case of sheep, A-2
Evidence that 8086 Factor A enhances feed utilization efficiency was demonstrated in an InvivO study.

These tests were carried out over a period of 5 or 6 days, and the results are summarized in Table 13. A-28086 Factor-D compounds are antiviral agents.

A-28086 factor D is associated with Maryland virus type B, polio virus type, COE virus, and foveal virus (H
erpesvir IlS) and Semiriki Forest Virus, as described by Siminoff [Applied Microbiology 1, Vol. 9(1), 66-7].
2 (1961)] was confirmed by an in vitro Braak inhibition test similar to that of [Page 2 (1961)]. A-28086 Factor D is also effective against infectious gastroenteritis virus, Newkatus virus, and infectious bovine nasal bronchitis virus, as confirmed by similar tissue culture studies. The fact that A-28086-Factor-D compounds have inhibitory activity against both viruses and anaerobic bacteria makes them useful for the treatment of enteritis in chickens, pigs, cows, and sheep. If it is useful for preventive purposes, it is recommended.

The A-28086-Factor-D compounds are also effective in treating intestinal toxicity in ruminants. Next, one of the important properties of the A-28086 factor-D compounds according to the present invention is anti-coccidiosis activity.

As a result of the InvitrO experiment, A-28086 factor D
has been shown to have anticoccidial activity against Eimeria tenella. Its usage is described below [for details, see Experimental Parasitology 34
L. Vol. 189-196 (1973). R. McDougald and R. B. See Galloway's report]. Cultures of host saturation are grown in Leighton tubes (LeightOntIlb).
e), prepared by a simple method using plastic dishes or other suitable culture vessels.

Growth for 2-3 days in any suitable medium (e.g., Eagle's Minimum Requirement Medium, Erle's or Hang's lactalbumin hydrolyzate in balanced saline, Medium 199) usually produces a suitable monolayer. This facilitates infection treatment and drug treatment. Primary cultures of chick spleen cells were used in these studies. Eimeria Tenera (who has the power to live)
The oocysts are collected from the stool of chicks infected with Eimeriatenella and cleaned and sterilized prior to use. The oocysts are suspended and allowed to germinate by aerating the suspension or by gently infiltrating it with a shuttlecock or other suitable device while maintaining the suspension at room temperature. During the germination process, potassium dichromate, sulfuric acid or other chemicals can be used to prevent bacterial and mold growth. The infective sporozoites are completely ejected from the ascus by mechanically breaking the outer wall of the oocyst and treating with trypsin and bile salts to activate the five sporozoites that have emerged from the ascus. .

The cell culture is infected by introducing viable seeds into the cell culture in the container.

The test compound can then be introduced thereto.
Test compounds are preferably introduced using 10% dimethylformamide in saline. Suppression at the end of the study is for the asexual stage. Infection 9
After 6 hours, the culture is fixed, stained, and its termination time determined by microscopic observation. Depending on whether second-generation schizonts (SchizO-Nt) are present and whether toxicity to single cell layers is evident, the results can be classified as active (A), inactive (N), or cytotoxic ( C)
Record as Toshino. A-28 proven by the above test
The anti-coccidial activity of 086 Factor D is summarized in Table 14.

Another feature of the A-28086 Factor D compound is that it can enhance rumen function in ruminants and increase feed utilization efficiency.

The mechanisms of utilization of major nutrients (carbohydrates) in anti-animal feeds are well known. Microorganisms in the animal's rumen break down carbohydrates into monosaccharides, which are then converted into pyruvate compounds. Pyruvate compounds are metabolized by microorganisms to form volatile fatty acids (FAs) known as acetate, bunarate, or propionate (more specifically, in Pouliprin et al., Physiology of Digestion and Metabolism). In the Luminant (published by Auriel Press, Newcastle upon Turn, UK (1970), pp. 408-410). Regarding the relative efficiency of FA usage,
McGlove (Food Stuffs (June 19, 1971)
(J. An. Sci. Vol. 33, p. 282 (1971)), Esklund et al. ) There are reports on pages 622 and 625.

Acetate and butyrate compounds are also utilized, but propionate compounds are more efficient. In addition,
Animals can suffer from ketosis when propionate compounds are very low. Therefore, useful compounds stimulate the production of higher amounts of propionate from carbohydrates in the animal body, increasing the efficiency of carbohydrate utilization and reducing the prevalence of ketosis. By observing the effect of a useful compound on the formation and concentration of propionate compounds in the rumen in the same manner as for Factor A, the activity of the useful compound can be determined. The results for the test compound are compared statistically with the results for the control compound. Table 15 shows the ratio of volatile fatty acid concentrations in flasks treated with A-28086 Factor D to those in control flasks. The compounds of the present invention have a typical effect in increasing the propionate compound, thus making this compound about 0.
Feed utilization efficiency can be increased when administered orally to rebellious animals at a rate of 0.05 to 5.0 per day per day.

Best results are obtained by administering the compounds of the invention at a rate of about 0.1-2.5 7 kg/day. A preferred method of administering the compound of the present invention is by mixing it with animal feed, but it can also be administered by other methods, such as tablets,
It can be administered in the form of trench tablets, large pills, and capsules. These various dosage forms can be formulated by methods well known in veterinary pharmacology. The individual dosage unit forms should contain an amount of the compound of the invention that is directly related to the daily dosage of the animal to be treated. Household feed and its 30 per ton A-28086
The compounds according to the present invention can be utilized by feeding a feed composition comprising 9 to fattening stock.

As is known in the art, cattle feed is different from other feeds such as poultry feed. Cattle feed includes forage such as cottonseed, corn silage, etc. In addition, cattle feeds contain higher percentages (often 15-25%) of non-protein nitrogen sources. Usually, this non-protein nitrogen source is urea. As mentioned above, the A-2808 of the present invention
6 compounds are antiviral agents and are also effective against anaerobes, especially Clostridium perfringens (ClOstrid).
It has activity against i-11mperfringens). Therefore, it is useful for treating and preventing enteritis in chickens, pigs, cattle, sheep, etc. The A-28086 compounds are also useful for treating enteric toxicosis in separated animals. Next, specific embodiments of the present invention will be described with reference to Examples.

Example 1 (A) Streptomysis aureofaciens (S
．． Shake flask fermentation of A-28086 using NRRL 5758: Streptomysis aureofaciens N.
A culture of RRL5758 was created.

Add distilled water to make 1 t.

That is, an agar slant medium having the above composition was inoculated with Streptomysis aureofaciens NRRL5758, and the inoculated medium was cultured at 30°C for 6 to 10 days.

The slant culture on which the bacteria had grown was protected with bovine serum and rubbed with a sterilized platinum loop to scatter the spores. The bovine serum suspension of spores and mycelial pieces thus obtained was freeze-dried to produce six pellets. One freeze-dried pellet obtained was inoculated into 50 ml of a growth medium having the following composition.

Add tap water to make 1 t.

The inoculated growth medium contained in a 250 mJ Erlenmeyer flask was incubated at 30°C for 24 hours with orbital shaking at 250 rpm on a 2 inch diameter arc motion shaker.

(B) Streptomysis-rheofaciens NRR
Tank fermentation of A-28086 using L5758: To make a large inoculum, 10 ml of the growth culture cultivated as described above is inoculated into 400 ml of the next stage growth medium having the same composition as the growth medium described above. This second inoculation medium was placed in a 2 t flask and incubated for 24 hours at 250 rpm on a 2 inch diameter circular motion shaker.
Cultured.

1 t of this second growth culture solution was used to inoculate 100 t of a sterilized production medium having the following composition. Make up to 1 t with deionized water.

This medium had a... of 6.7 after autoclaving at 120° C. for 30 minutes at 15-20 pounds of pressure.

The production medium prepared in this manner and inoculated with bacteria was fermented in a 165 t fermentation tank at a temperature of 29°C for 10 days.

During this time, sterile air was aerated through the fermentation liquid at a rate of 0.4 volume of the culture liquid per minute. The culture solution was stirred at a rate of 250 rpm using an ordinary stirrer. Example 2 Example 1 except that a flask culture medium with the following composition was used.
A-28086 antibiotic was produced according to the method of .

Add tap water to make 1 t.

Example 3 Streptomysis aureofaciens NRRL5
Separation of A-28086 antibiotic complex produced by A-28086: Fermentation liquid 132 obtained by the method described in Example 1
Filter all of it with a filter aid (Hyflo Supercell, a type of diatomaceous earth, manufactured by Zion Zuman Building Manufacturing Company),
97 tons of filtrate was obtained.

Extraction of this filtrate with approximately the same amount of ethyl acetate was attempted. The ethyl acetate extract was separated from the aqueous layer and concentrated to a volume of about 500ml. Excess petroleum ether (SkellySOlveF: about 10 t) was added to this concentrated ethyl acetate extract, and the resulting waste material was precipitated and separated. The concentrated solution after separation was evaporated under reduced pressure to obtain A-28086 antibiotic complex 6.9f1.
The 8086 antibiotic complex was obtained by extracting the filtered bacterial cells twice with about half the amount of methanol (62t and 59t).

After combining these two methanol extracts, methanol was concentrated and removed under reduced pressure. Approximately 10 tons of aqueous layer remained after this concentration. The aqueous layer was adjusted to pH approximately 7.5 with dilute sodium hydroxide solution. The solution thus obtained was extracted twice (9t and 10t) with approximately the same amount of ethyl acetate. The ethyl acetate extracts were combined and concentrated to a volume of about 400 ml. This concentrated ethyl acetate extract was added to a large excess of petroleum ether to remove unnecessary substances, using the method described above for concentrated extracts of filtered culture fluids. The weight of the A-28086 antibiotic complex obtained from the bacterial cells in the filtrate was 20.69.

Example 4 Isolation of individual factors A and B in A-28086: A-
28086 antibiotic conjugate (prepared as described in Example 3) 2359 was dissolved in approximately 80 ml of benzene.

Silica gel column chromatography was applied to this benzene solution (9 x 130 (177!, 8t,.Math
es0nqrade62 silica gel). The column was eluted with a mixture of benzene monoethyl acetate in various proportions. The elution behavior was followed by thin layer chromatography. Using benzene monoacetate (90:10) as the solvent system, factor B
was first eluted and isolated as one of the individual factors.
Factor B (4377V) was crystallized from acetone monohydrate.
When elution was continued with a benzene monoethyl acetate mixture while gradually increasing the proportion of Mpl5O~153°C0 ethyl acetate, Factor A was eluted.

The various fractions containing factor A are combined and concentrated under reduced pressure, the residue left behind is dissolved in about 150w11 of acetone,
Approximately 150w11 of water was added to this acetone solution. The pH of the solution thus obtained was adjusted to 3 by adding 1N hydrochloric acid.
The acidified mixture is stirred for about 1 hour, during which time a precipitate forms. Filter this precipitate and add about 150ml of acetone.
and recrystallized by adding about 60 TILI of water. The resulting crystals were dried under vacuum overnight to obtain a factor A of approximately 6.69.
Partial evaporation of the acetone from the crystal filtrate gave a secondary crop of Factor A of approximately 1.29. Example 5
Preparation of acetyl ester derivative of A-28086 factor A: Dissolve 7.49 of the antibiotic A-28086 factor A in 150 d of pyridine, and add 501 d of acetic anhydride to this solution. Ll was added.

The solution was thoroughly mixed and then left at air temperature overnight.
200 Tn1 of water was added and thoroughly mixed, and the mixture was left at room temperature for 4 hours. A white solid precipitated and was filtered off, washed with water and air dried. The solid thus obtained was dissolved in 100 ml of acetone, and the acetone solution was then evaporated to dryness under vacuum (this operation was repeated three times). The residue thus obtained was crystallized from 1007n1 acetone, 50TfL11 water, and A-28086 factor A
An acetyl ester derivative of 6.149 was obtained. melting point 10
0-103°C0 Example 5 According to the method of Example 5, propionic anhydride was reacted with Factor A in the presence of pyridine to produce antibiotic A-28086.
A propionyl ester derivative of Factor A was obtained.

Melting point: 96-98°C0 Following the method of Example 5, Factor A was reacted with n-butyric anhydride in the presence of pyridine to yield the antibiotic A-28086 Factor AO) n-butyryl ester derivative.

Melting point: 79-81°C. Antibiotic A-28086 Factor A(7) n-caproyl ester derivative was prepared by reacting Factor A with caproic anhydride in the presence of pyridine according to the method of Example 5.

Melting point 163-167°C. Following the method of Example 5, factor A was reacted with valeric anhydride in the presence of pyridine to produce antibiotic A-28086 factor AOn-valeryl ester derivative.

Melting point 173-175°C0 Example 10 Preparation of sodium salt of A-28086 factor A: 1 Antibiotic A-28086 factor A5OOη in acetone 50wL1
dissolved in.

50 ml of water was added to this solution, and 5N sodium hydroxide solution was further added to bring the ... of the liquid to 10.5-11. The solution thus obtained was stirred for 1 hour and then extracted with ethyl acetate. The ethyl acetate extract was evaporated to dryness under vacuum. The residue was precipitated from a solution of acetone and water and A-280
378 mf of the sodium salt of 86 factor A was obtained. melting point 12
0 to 123 NaC0 Examples 11 to 15 Using the method of Example 10, Antibiotic A! The barium salt of 28086 Factor A, 369Ri, was prepared.

Melting point: 188-190°C. Using the method of Example 10, 363W of A-28086 Factor A was prepared from Antibiotic A-28086 Factor A (500η) and 5N gallium hydroxide solution.
The potassium salt of

Melting point: 165-167°C0 Using the method of Example 10, 540% of A-28086 factor A cesium salt was obtained from antibiotic A-28086 factor A5OO'Mg and 1N cesium hydroxide.

Melting point 190-21'CO Sodium salt of antibiotic A-28086 factor B was prepared from antibiotic A-28086 factor B and 5N sodium hydroxide by the method of Example 10.

Example 16 Streptomysis aureofaciens (S.aI
A using NRRL8O92
Shake flask fermentation of -28086: Streptomasis aureofaciens N on an agar slant medium with the following composition:
A culture of RRL8O92 was prepared.

Add deionization to 1 t.

That is, this slant medium was inoculated with Streptomysis aureophaciens NRRL8O92, and the inoculated medium was
The cells were cultured at 0°C for about 7 days.

A suspension of spores and hyphae was prepared from the slant culture by rubbing it with a sterilized platinum loop while protecting the slant culture with sterile bovine serum. The resulting suspension was freeze-dried to prepare six pellets. One of the freeze-dried pellets thus produced was used to inoculate 50TILI growth medium with the following composition.

Add tap water to make 1 ton. pH to 6 with dilute HaOH
．． Adjusted to 5.

The inoculated growth medium in a 250 ml Erlenmeyer flask was incubated at 30-C for 48 hours at a speed of 250 rpm on a 2-inch arc motion orbital shaker.

Next, the medium in which the above-mentioned bacterial cells had grown (0.5TfL 111%) was inoculated into fermentation medium 507711 having the following composition.

Add tap water to make 1t PH (unadjusted) 6.6 *StaleyDextrin #11 (A.E-Sta
leyCO (manufactured by Dekatool, Illinois, USA).

※※A[TlberEHC(.A[T]BerLabO
ratOries (manufactured in Jiuniyu, Wisconsin, USA).

※※※NZA[111neA(SheffieldCh
chemicalCO. (Made in Norwich, New York, USA).

Example 17 Tank fermentation of A-28086 using Streptomysis aureofaciens NRRL8O92: - The method described at the beginning of Example 16 as a shake flask fermentation method of A-28086 was also applied in the case of tank fermentation.

Growth medium 10 in which bacteria were cultured to make a large amount of inoculated bacteria
ml was used to inoculate 400 ml of the next stage growth medium with the same composition as the first growth medium. This second stage culture solution in a 2 ton Erlenmeyer flask was placed on a shaker rotating in a 2 inch arc at 250 rpm.
The cells were cultured at 30°C for 24 hours.

800 ml of a secondary growth medium in which this bacterium was cultured was used to inoculate 100 t of a sterile fermentation medium having the following composition. Add tap water to make 1 ton.

*StaleyDextrin #11 (A.E. Staley
leyCO. (manufactured in Decatur, Illinois, USA).

※※AnlberEHC(AOlberLabOrat
Manufactured by Ories (located in Jiuniyu, Wisconsin, USA)
.

※※※NZAmineA(SheffieldChem
icalCO. (Made in Norwich, New York, USA).

After pressure sterilization at 121° C. for 30 minutes at a pressure of 15-20 Bonds, the ... value of this medium was 6.8±0.1.

The production medium inoculated with bacteria was fermented at 28±1° C. for 10 to 12 days in a 165 ton fermentation tank.

Sterile air was also bubbled through the fermentation medium at a rate of 0.4 volume of culture solution per minute. In addition, this culture solution is 300rpn1 (
7) Stir with a regular stirrer in proportion. Example 18 A-2808 according to the method of Example 17 except using a shake flask or tank production medium with the following composition:
6 antibiotics were produced.

Tap water was added to make 1 ton.

*StaleyDextrin7l6ll (A.E.S.
(manufactured by taleyCO (located in Decatur, Illinois, USA)).

※※AlberEHC(A[NberLabOratO
ries (manufactured in Jiuniyu, Wisconsin, USA).
※※※NZAnllneA(SheffieldChe
micalCO. (based in Norwich, New York, USA)
manufactured by).

As described in Example 17, the ... of the medium after being sterilized by the pressure method was 6.4.

Example 19 Streptomysis aureofaciens NRRL8
Separation of the A-28086 antibiotic complex produced by O92: Total fermentation liquor 6 obtained by the method described in Example 17
The pH was adjusted to 3 by adding dilute HCl to 0 liter.

The solution thus obtained was filtered using a filter aid (Hv-FlOSllp).
A type of er-Cell diatomaceous earth, JOhns-Manvl
It was filtered using lleprOdllClSCOrp.). The separated mycelium mass was extracted with 30 tons of methanol, and 1.56 Kf0) NaHCO was added to the extract with stirring. After separating this extract, the mycelial mass was further extracted again with 30 tons of methanol. Both methanol extracts were combined and concentrated in vacuo to remove methanol. Approximately 7 tons of residual aqueous solution was adjusted to pH 7.5 with dilute HCl. The solution thus obtained was extracted twice with 7 tons of ethyl acetate each time. The ethyl acetate extracts were combined and concentrated in vacuo to give an oily residue. This oily residue was washed with 1500 ml of acetone.
m1, and add 1500Tf11 of water to this acetone solution.
was added. The resulting solution was adjusted to pH 3 with dilute HCl and stirred for 1 hour. The resulting precipitate was filtered, then dissolved in 1500 d of acetone, and 4 ml of water was added to this solution.
00ml was added. The solution thus formed was left for 16 hours to precipitate crystals. The produced crystals were dried in a vacuum under vacuum to obtain a crude crystalline product 749 containing A-28086 factors A and D and other crystalline impurities. This crude crystalline product 409
Tn1 was dissolved in benzene.

Next, silica gel column chromatography was applied to this benzene solution (9-×120 column; Grac
using e-DavidsOn grade 62 silica gel). The column was sequentially eluted with 40 tons of each of the following solvents. 7)
Fractions of 1 ton of methanol were collected from these.

In order to identify the eluted compounds in each portion, tests were conducted using a Bacillus subtilis growth inhibition test method and a thin layer chromatography method. A-280861 was eluted with benzene:ethyl acetate (4:1). A-28086 factor B
was eluted with benzene:ethyl acetate (7:3). A-
28086 factors A and D are benzene:ethyl acetate (7:
3 and 1:1) (parts from 119 to 156). The parts were combined and evaporated to dryness under vacuum. The residue thus obtained was mixed with 50% acetone.
Dissolved in 0ml. Add 500 ml of water to this acetone solution, and adjust the pH of this solution to 3 with dilute HCl.
Stir for hours. The formed precipitate was crystallized from 1 liter of Namita and further from 500 TfL of acetone and 180 ml of water. The crystals thus formed were filtered and dried under vacuum.A-28
20.19 of a mixture of factors A and D of 0.086 was obtained.

Example 20 Separation and purification of single substances of factors A and D: The crystalline mixture 18, 89 of factors A and D of A-28086 obtained in Example 18 was dissolved in 50 ml of benzene.

Silica gel column chromatography (71XlOO-a column: E. Merck grade 60 silica gel, finer than 230 mesh (ASTM)) was applied to this benzene solution.
was applied. The column was eluted sequentially with the following solvents at a flow rate of 90 ml per hour. 1) 12 tons of benzene 2) 12 tons of benzene:ethyl acetate (9:1) mixture 3)
12 t of benzene: ethyl acetate (4:1) mixture 4) 12 t of benzene: ethyl acetate (7:3) mixture 5) 10 t of methanol Merck Darmstadt cellulose, on an aluminum support plate).

The solvent system used was as follows. Water: Methanol Liaton (12:3:1). First, dilute this solution with NH4OH.
HlO. 5 and then adjust the pH to 7.5 with HCl. Collect 1 to 2 tons until activity is detected, and then collect 200 tons.
Tf11 each was collected.

The portions containing only A-28086 factor D were combined and vacuum distilled to obtain a residue. This residue was dissolved in acetone and water (1:1).
) was crystallized from The crystals were separated and dried under vacuum to obtain crystalline A-28086 factor Dl4O77V. Slightly A-
If a portion containing A-28086 factor D mixed with 28086 factor A is treated in the same manner, an even smaller amount of A-28
Crystalline A containing 086 factor A-28086 factor D is 1
501T9 was obtained.

A portion containing only A-28086 Inko A was treated in the same manner, and a crystalline A-28086 factor of 4.7f was obtained! I got it. Example 21 Example 16 except that a slant medium with the following composition was used.
A-28086 antibiotics were produced according to the method of .

Make up to 1 t with deionized water.

Adjust the pH to 7.0 with KOH.

In this case, the slope planted with the bacteria was incubated at 28°C for about 7 days.

Example 22 A-280 was produced according to the method of Example 17, except that both the flask medium and the production medium had the following composition.
86 antibiotic substances were produced.

*StaleyDextin #11 (A.E.Stal
eyCO. (manufactured in Decatur, Illinois, USA). ※※
AnlberEHC(.AnlberLabOratO
Manufactured by ri-Es (located in Jiuniyu, Wisconsin, USA)
. Example 23 The method of Example 21 was followed except that a medium with the following composition was used as the intermediate third stage growth medium, -
28086 series antibiotics were produced.

A. Manufacture of baby bird feed improved with A-28086 for the purpose of controlling coccidiosis: A balanced, high-performance feed suitable for raising baby birds so that they gain weight quickly, with the following formulation: Create with.

do.

There are no restrictions on the amount of water administered. Chicks fed this diet are protected against coccidiosis infection and have weight gain comparable to coccidiosis-free chicks fed a similar diet without the drug. Example 25A-28086 Improved Beef Cattle Feed Production: A balanced high performance beef cattle feed is prepared as follows.

*Content per pound: 2,000,000 international units of vitamin Al227, 200 international units of vitamin D, and 385.79 soybean feed supplemented with 1% oil.

※※When making corn whiskey, a soluble material containing 20,00 international units of d-alpha tocopheryl acetate is added to each pound of grain to dry the grain.

The mixed feed is compressed into small pieces.

Assuming an average daily intake of 15 lbs of feed per animal, this feed provides about 300 lbs of feed per animal.
This means that the A-28086 factor A of Wa is supplied.
Example 26 Preparation of an acetyl ester derivative of A-28086 Factor D: The antibiotic A-28086 Factor D is dissolved in pyridine.

Add the calculated amount of acetic anhydride to this solution. Mix this solution thoroughly and leave it at room temperature overnight. Then water is added in excess and the mixture is left at room temperature for several hours. The precipitate formed is filtered off, washed with water and dried. The solid thus obtained was dissolved in acetone and evaporated to dryness under vacuum to form A-2808.
An acetyl ester derivative of 6 factor D was obtained. Examples 27-30 A-28 in the presence of pyridine according to the method of Example 26
Propionyl, ester derivatives of antibiotic A-28086 Factor D were prepared by reacting 086 Factor D with propionic anhydride.

A-28 in the presence of pyridine according to the method of Example 26
By reacting 086 factor D with n-monobutyric anhydride,
An n-monobutyric acid ester derivative of antibiotic A-28086 factor D was prepared. According to the method of Example 26, A-28086 Factor D was reacted with caproic anhydride in the presence of pyridine to form the antibiotic A-28086 Factor DOn-caproyl.
Ester derivatives were produced.

A-2 in the presence of pyridine according to the method of Example 26
8086 factor D was reacted with valeric anhydride to form antibiotic A-
28086 Factor D(7) n-valeryl ester derivative was prepared.

Example 31 Preparation of Sodium Salt of A-28086 Factor D: - Dissolve the antibiotic A-28086 Factor D in acetone.

To this solution is added an equal volume of water and enough 5N sodium hydroxide to bring the pH of the solution to about 11. The resulting solution is stirred for about 1 hour and then extracted with ethyl acetate.
Evaporation of the ethyl acetate under vacuum yields the sodium salt of A-28086 Factor D. Examples 32-34 A-28086 Factor D and 5N using the method of Example 31
The potassium salt of antibiotic A-28086 factor D was prepared from potassium hydroxide.

A barium salt of antibiotic A-28086 Factor D was prepared from A-28086 Factor D and saturated barium hydroxide using the method of Example 31.

A-28086 factors D and 1N using the method of Example 31
Cesium salt of antibiotic A28O86 factor D is produced from cesium hydroxide.

Example 35 Production of chick feed containing factor A-28086 for the purpose of suppressing coccidiosis: A balanced, high-performance feed suitable for raising chicks so that they gain weight quickly is as follows: It is prepared using a combination recipe.

These materials are mixed according to standard feed formulation techniques.

There are no restrictions on the amount of water administered. Chicks fed this diet are protected against coccidiosis infection and their weight gain is comparable to chicks fed a similar diet without the drug, which has never been affected by coccidiosis. Example 36 Preparation of Beef Cattle Feed Containing A-28086 Factor D: A balanced high grain beef cattle feed is produced as follows.

*Content per pound: 2,000,000 international units of vitamin Al227, 2OO of international units of vitamin D,
and soybean feed with 1% oil 385.79.

※※When producing corn whiskey, a soluble substance containing 20,000 international units of d-alpha tocopheryl acetate is added to each pound of grain to dry the grain.

The mixed feed is compressed into small pieces.

Assuming an average daily intake of 15 pounds of feed per animal, this feed has approximately 3001 f9 of A per animal.
-28086 factor D is supplied.

[Brief explanation of drawings]

All drawings are drawings showing infrared absorption spectra of antibiotic A-28086 of the present invention, and FIG.
-28086 factor A, Figure 2 shows antibiotic A-28086
Factor B, Figure 3 is an acetyl ester derivative of antibiotic A-28086 factor A, Figure 4 is antibiotic A-28086
Figure 5 shows a propionyl ester derivative of antibiotic A-28086 factor A, Figure 6 shows a valeryl ester derivative of antibiotic A-28086 factor A, and Figure 7 shows antibiotic A-28086 factor A. Caproyl ester derivative, Figure 8 shows antibiotic A-2808
Each of the six factors D.

Claims (1)

[Claims] 1 Antibiotic A- consisting of factor A, factor B and factor D
28086 complex, the factor A, the factor B, the factor D, or any combination of these factors as essential components, an agent for controlling pathogenic microorganisms for animals other than humans.