JPS5942320A - Animal growth promotor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention contains as an essential component the antibiotic A-28086 complex consisting of Factor Δ, Factor B, and Factor B, or any individual factor thereof or any combination of such factors. This invention relates to a growth promoting agent for animals and a feed utilization efficiency improving agent.

Antibiotic A-28086 complex and its constituent factors are:
Anti-bacterial, anti-filamentous, anti-viral, anti-Bovine pleuropneumonia (anti-P
PLO) is useful as an anticoccidial, insecticidal, and acaricide, and is effective for increasing feed utilization efficiency in reflex animals.

The present invention is directed to three treptomyces aureofaciens plants.
ns) N RRl-5758 or S1 ~ Streptomyces aureofacens
s aureofaciens) NRRL 80
92 is cultivated in a culture medium containing carbon sources, nitrogen sources and inorganic salts available to the strain and cultivated under agitated aerated fermentation conditions until the development of valuable parent antibiotic activity in the medium by the microorganisms. Particularly J: Antibiotic A-28086 containing constituent factor A1 factor B and factor
The object of the present invention is to provide an agent for promoting animal growth and improving feed utilization efficiency, which contains a complex, its individual factors, or any combination of the factors as an essential ingredient.

Antibiotic A-28086 Convict A is a white crystalline compound (
acetone (crystallized from water), lower alcohols,
It is soluble in dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone and benzene, but slightly soluble in hexadiene and insoluble in water. Melts at about 98-100°C, re-solidifies, and further melts at about 195°C
Remelt at ~200°C. This antibiotic has the following chemical properties ((a) to (m)).

(a) Molecular weight 76/l (measured by Subek 1-L measurement method), <1>) Elemental analysis value (approximate value), C, 66, 69%
;G1. 9. 85%: 0.
23. 10%.

(C) Empirical formula: Caq I-(7, On (measured by quality spectrum analysis), (d) [α) o Knee 57! I° (C=0.2.methanol), (e) Infrared absorption spectra 1 to 1 (measured in chloroborm), the following distinct absorption band peaks appear. 2.8
5.3.34.5.83.6. f12.7.22.7.
53 (weak>, 7.78 (weak), 8°75 strong), 8.9
5 (strong), 9.15.9.50 (strong), 9.55 (strong), 9.60.9.85.10.15.1 ('), 45
．． 10.70 (weak) microns.

([) Ultraviolet absorption spectra 1 to 1 (ethanol solution 'fj,
) only shows terminal absorption below 200 s.

(CI>Characteristics of nuclear magnetic resonance spectra 1 to 1 (measured in chloroborum hydride): δ6.01.4..21.4,
．． 11.3.99.3.89.3.80.3゜67.3
．． 65.3.57.3.55.2.83.2.76.2
．． 74.2.68.2.66.2゜58.2.56.2
．． 30.2.22.2.17.2.10, 2.05.1
．． 96.1.90,1゜85.1.70.1.62.1
．． 60,1. /1.7.1.39.1.31.1.25
．． 1.18.0゜95.0.93.0.90.0.88
．． 0.85.0.77.0.75.0.73.0.68
．． 0°66 ppm.

(11) p in 80% dimethylbormamide water
It has a titratable group with a Ka value of 7.9.

(i) Characteristic X-ray powder diffraction pattern (C1+
For Jusho, 1.5405λ Shoken, Nickel filter).

(1 relative strength 12.00 100 10.10 50 9.25 90 8.00 /10 7.50 15 6.92 90 6.40 40 5.98 05 5.68 15 5.20 /10 /1.98 40 4.62 /l.

4.21 20 3, /18 10 (j) Bacillus subtilis (13aQi l 1u
ssubtilis) AT CC6633 strain was used as the detection organism, and benzene-
An Rf value of 0.24 was obtained by thin layer chromatography using an ethyl acetate (3:2) system.

(1) Rf values as shown in the table below in the paper chromatography system shown in the table below using Bacillus subtilis ATC06633 strain as the detection organism.

Rf value Solvent system 0, 11 (methyl isobutyl quinone (MI
water saturated with MIBK) 0,4.1 (2% p- in water saturated with MIBK)
Added toluenesulfonic acid and 1% piperidine) 0,! M (water: methanol: acetate (12:
3:1) to pH 10.5 with NH40H, and after IC, 13P
Lower the pH to 7.5 with Oa. ) 0, 48. (1% MIBK, 0.5% NHaOH in water) 0,15 (17.4(]K2HP in 1 liter of water)
Oa and 30 ethanol added) 0, 24 (benzene saturated with water) 0, 24
(Water only) 6-0.75 (Water: MIB: Ethyl acetate ("18:
1:1)) (1) There is one acid group capable of forming salts and ester derivatives.

(m) At least one hydroxyl group capable of esterification is present.

In the Suimei wI circle, the term (antibiotics △-28'086) factor Δ refers to the above-mentioned factor Δ and its C2 □ C6 acyl ester derivatives and their physiologically acceptable It shall include salts that are

Antibiotic Δ-28086 factor B is a white crystalline compound (
(crystallized from water) and is soluble in lower alcohols, dimethylformamide, dimethylsulfo:1:side, ethyl acetate, chloroform, arene, and benzene; It is only slightly soluble in 1 J-ton and insoluble in water. And heavy substances are as follows <a)
It has +1 physical and chemical characteristics of ~(1).

(a) 1 sub point of about 150-153°C, (b) molecular weight 7
62 (measured with a high-resolution quality analyzer), (c) Empirical formula C, H-, , Ot+ (measured with high-resolution mass spectrometry), (d) Infrared absorption spectrum (in chloroform) The following distinct absorption band peak appears at: 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, 10.10, 10.1
7 (strong), 10°43 (weak), 10.80 (weak), 1
1.20 (weak), 11.35 (weak), 11.73 (weak>
, 12°03 (weak) microns.

<a> In the ultraviolet absorption spectra 1 to 1 (ethanol solution), the maximum absorption is at 220 ge (E, i-137,5: ε = 10
．． /1.77).

([) Nuclear magnetic resonance spectrum (in deuterated chloroform) shows the following features:

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°4B, 2.8
1, 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.
1 B, 1. IT, 0. 96.0°94
．． 0. 90.0. 87.0. 8/I, 0. 7
7.0, 74.0. 68 ppn+ (a) Thin layer deposition on silica gel using TCC6633 as the detection microorganism to Bacillus subtilis in a benzene-ethyl acetate (3:2) system [171~Graphei's Rr value is 0.42.

(h) Rf values in Paper Chroma 1 to Graph System conducted using Bacillus subtilis ATCC 6633 as the detected microorganism are as follows.

Rr value Solvent system 0.16 (2% p~1~ in water saturated with MTBK
Added luenesulfonic acid and 1% piperidine. ) 0.46 (Water; methanol:acetone (19-2:3:1) brought to pl-1 of 10,5 with NH40H and then reduced to of-17,5 with H3POa.) 0.36 (in water 1% MIBK, 0.5% NH40
1-(added) 0.51 (benzene saturated with water) 0.11
(Water only) 0.61 (Water: M r f3 K : Ethyl acetate (98:1:1)).

(1) 1 acid group capable of forming salts and ester derivatives
There are two.

(,i) 1 to 2 K residues, (k) At least one hydroxyl residue is present.

In Suimei 1, (antibiotic △-28086) factor B
Unless otherwise contradicted by the surrounding text, the term shall include the above-mentioned Factor B and its biologically acceptable salts.

Antibiotic Δ-28086 is a white crystalline compound (crystallized from acetone-water).It contains methanol, ethanol, dimethylformamide, dimyl sulfur oxide, ethyl acetate, chloroform, and alcohol. It is soluble in hexane and benzene, but only slightly soluble in hexane and insoluble in water.
It melts at 6 to 98°C and has the following properties (a) to (river).

(a) Molecule 1i778 (measured by high-resolution mass spectrometry) (b) Elemental analysis IFO (approximate fil'T), C,67
, 59%; H, 9,38%; 0.22.77%, (c)
Empirical formula 〇 44+-174On (measured by high-resolution mass spectrum method), (d)(α)o: -56° (C=0.1, methanol), (0) Infrared absorption spectrum I~ (measured in chloroborum) shows the following absorption band peaks: 2.89.3.
39.3.43.3.50,5゜88.6.90.7.
27.7.60, 7.84.9.00.9.26.9.
62.10.31.10.58.11.10111. ．．
49 microns.

(f) No ultraviolet absorption is observed with 95% aqueous ethanol.

(g) Nuclear magnetic resonance spectrum in chloroform has the following characteristics, δ6.00.4.20.4°1o, 4. o
o, 3.98.3.92.3.86.3.83.3.7
9.3.67.3.64.3゜57.3.54.2.8
8.2.81.2.71.2.62.2.58.2.4
8.2,43.2゜37.2.29.2.21.2.1
5.2.10゜2.04.1,97.1.89.1.8
3.1゜76.1.68.1.61.1.58.1.5
5.1.47.1.39.1.30,1.25.1゜1
8.0.95.0.90.0.88.0.84.0.7
4.0.681)I)m.

(h) 8.6 in 80% dimethylformamide water
One titratable group with a pKa value of 7 is identified.

(i) A characteristic X-ray powder diffraction pattern with the following (6-element spacing (unit: angstroms). (C
U+10 irradiation, 1.5405λ, nickel filter)
.

(Relative strength 12, /lo 100 10.20 70 8.85 90 7.80 30 6.80 10 6.30 100 5.70 20 5.35 20 5, 10 20 4.90 10 4.65 20 4 , .45 40 4.20 30 3.30 10 3.15 10 2.99 05 2.77 05 2.28 05 13- (j) Using Bacillus subtilis ATCC6633 as the detection organism, J: Eel Silica shown below・The following Rf values in gel thin layer chromatography system.

-Color LL (0,26 benzene-ethyl acetate (3:2) 0.66
Ethyl acetate-diethylamine (95:5) (k) The following Rf value determined by the paper chroma 1-graph system shown below using Bacillus subtilis AT CC6633 as the detection organism.

Rf value? WIs series 0.10 Methyl isobutyl ketone (MI B
K) Saturated water 0.26 Water saturated with MIBK with addition of 2% p-toluenesulfonic acid and 1% piperidine.

0.36 water: methanol niacetone (12:3:1
) was adjusted to l1l-(10°5) with NHaOH, and then
3 Lower DH to 7.5 with PO4.

14- 0.29 1% MTBK, Q, 5% NH/101-
2 HPo, 4.17.4 (
1 and 30 liters of ethanol (0.26 liters of saturated water), benzene (0.09), water alone (0.64), water: MIBK: ethyl acetate (98:1:1)
) I! ,) one acid group capable of forming salts and ester derivatives m) at least one water M group capable of T1 stellation In the present invention, (Antibiotics A-28086) the term factor n is , unless otherwise indicated by the preceding text, is intended to include the agents of article F and their C2''□C6 acyl esters, as well as their physiologically acceptable salts.

Although many antibacterial agents are known today, there is still a continuing need for new and progressive antibiotics. One problem with current antibiotic therapy is the fact that antibiotics differ in their effectiveness against pathogenic organisms. Another problem is the emergence of biological strains that are resistant to standard antibiotics. An additional problem is the fact that individual patients often experience dangerous side effects due to hypersensitivity, toxic trL, etc. from specific antibiotics. 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, there is also a need for advanced antibiotics in the field of livestock disease treatment. One important aspect is that advanced antibiotics are also needed to promote growth in poultry and livestock.

For example, promotion of growth can be achieved by reducing disease.
and is achieved by increasing feed utilization efficiency.

One of the economically important diseases for livestock disease treatment experts, and especially for the poultry breeding industry, is the protozoal disease coccidiosis (coccidiosis).
coccidiosis) is well known. Coccidiosis is caused by E imeri.
a) or due to infection by one or more species of Isospora (summary in “° Daisies or Boultry”, edited by Biester and Schwarte, 5th edition, published by Iowa State University, Ameth, Iowa, USA) (See Lund OJ: Bifur in the middle).

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.

Another disease that causes significant economic losses to livestock producers is heat haze. Enteritis occurs in incisions, pigs, cattle and sheep and is primarily caused by anaerobic bacteria, especially Clostridium perfringens.
s) and viruses. Intestinal toxicosis in resistant animals, such as hyperphagia in sheep, is a symptom caused by infection with Clostridium bellfringens.

Next, promoting the growth of rebel animals such as cows is another economic goal in veterinary medicine. There is particular interest in achieving growth promotion by increasing feed utilization efficiency.

The mechanism by which the main nutrients (carbohydrates) in ruminant feed are utilized is well known. Microorganisms within the animal's rumen break down carbohydrates into monosaccharides, which in turn convert these monosaccharides into pyruvate-related compounds. Pyruvate is metabolized by microbiological action to produce volatile fatty acids (VFA) such as acetic acid, acetic acid, or propionic acid.
). For a more detailed discussion, see Renge (Philippson et al.: Physiology).
Or Digestion and Metabolism in
The Luminand (pages 408-410, published by Newcastle upon Tyne Oriel Publishers, UK (1970))
).

The relative effectiveness of using VFA was determined by Feedstaffs 1971.
by Ruff Matsuri in the June 19 issue, p. 19; by Esgaland et al. in J. He, SOi, vol. 33, p. 282 (1971); and in Digestive Physiology and Nutrition or Luminants' 2, pp. 6218-2-625 (1971), J:C is discussed by Church et al. If the utilization efficiency is significantly reduced, the animal will suffer from ketosis.Therefore, the animal will be stimulated to absorb propionic acid at a high rate from the vJ water crude product, and this will cause the animal's isthmic water to increase. This is to avoid increasing the chemical utilization efficiency and also to reduce the effects of 1-1 syndrome.

Antibiotics Factors Δ-28085, B and D are prominent members of the polyester antibiotic group 71.

Examples of members of this JAY include monensin (U.S. Pat. No. 3,501,568), guianemycin [Hamill, R.L.; Whaen, M.M.

; Bittenger, G, E', : Chimbalin, J, ;
and Gorman, M.; Digital Antibiotics, Vol. 22, 161 ■ Title (1≦) 69)] Nigericin (L., Steinlauf, Marie Binka)
1 ~ N O J: Bi J, W, Chamberlin, Biohimi Power・Vi゛A Physics・S→J---Chi・]]Munikeshi]n, 3
Vol. 3, p. 29, 1968)), and Zarinomycin [Japanese Patent Publication No. 47-25391, dated October 20, 1972, application number 19620/1971; l) erwent
No. 7 (3960T, U.S. Patent No. 3,857.9
No. 48, and l-1, = 1 = Nashi, N, Otake, 1''
, Yonehara, S., Su1~-, and Y.

Zayt, Te 1 ~ Rahedron Letters, Volume 49, 495
5-4958 (1973)).

``Antibiotic compl
The term "ex)" does not refer to a button body in chemical terms, but refers to a mixture of individual antibiotic factors that are produced together.Individual factors that are produced in an antibiotic complex Those familiar with the fermentative production of antibiotics will agree that the ratio of

Δ-28086 antibiotic conjugate is a novel bacterial strain Streptomysis aureofaciens NRRL 5758.
is produced by culturing under agitated 1'1'-aerated fermentation conditions until a moderate amount of antibiotic activity occurs. /l-
The 28086 antibiotic conjugate also contains
The current strain of Mysis Δ-reofacininus, NRRL8092, is produced annually. S1- Leptomysis aureofaciens N
The Δ28086 antibiotic conjugate, whether produced by RRL5758 or S1-Leptomysis A-refaciens N RR+-8092, is extracted from the fermentation broth as well as the mycelium with polar organic solvents.

Extracted antibiotics? 1 mixture, concentrate the solvent I7 and divide 1i
i11, add excess petroleum ether to the concentrate to precipitate impurities, and 1Iiii! After filtration and evaporation of the filtrate of A, the A-28086 antibiotic mixture is obtained.

The antibiotic conjugate was further purified by column chromatography and separated into individual factors.

A-28086 compounds inhibit the growth of organisms that cause pathogens to plants and animals. One aspect of this soil activity is △-2
This is the anti-ccidial effect of 8086 compounds. In addition, A-28086 compounds 21- are antibacterial-1 anti-viral-1 anti-Bovine pleuropneumoniae (anti-P
PLO), Insecticide-1, is an acaricidal substance and has the effect of increasing the feed utilization efficiency of insecticides.

The infrared absorption spectrum taken in chloroform is shown below.

Figure 1 Antibiotic Δ-28086 Prisoner A Figure 2
〃 Factor B Figure 3
Acetate ester derivative of factor Δ Figure 4 Propionate ester derivative of antibiotic A-28086 factor A Figure 5 Acetate ester derivative of antibiotic A-28086 factor A Figure 6 Tightness of antibiotic Δ-2808'6 factor Δ Acid T ster derivatives Fig. 7 Antibiotic A-28086 factor A caproic acid ester derivatives Fig. 8 Antibiotic A, -28086 convict DA -,
The 28,086 factors are structurally related to each other. During the fermentation period, at least four kinds of antibiotic τ'1 factors h
It will be done.

The factors are separated into phases n, and the factors Δ, B and D are 4y individual compounds as described below. Ill do it.
Kongo 1171 (,1) is soluble in most solvents, but is insoluble in water.

Δ-28086 PrisonerΔ in the item 1 space below, [3 Onipi D
The physical and spectral properties of

Antibiotic A-28086 is crystallized from water on Day 1-1. △-28086 factor 8 is about 98-100℃
It melts at about 195-200°C, re-solidifies and melts again at about 195-200°C. As a result of elemental analysis of the factor Δ, the following 5v uniform percentage composition was obtained: C166, 69%; l-1,
9,85%; 0,23,10%.

Experiment 2〇a:+H7EOn is proposed for v1 child△.

The numerator B of IC1 or tJ is determined by the buying analysis method.
1 is 764.

The infrared absorption spectrum of factor Δ in chloroform is shown in the attached figure (Figure 1). The following absorption band peaks are observed. 2,85.3.34.5.83,G,8
2.7.22.7.53 (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.

Ultraviolet absorption spectrum at d3 in ethanol with factor △1-
The terminal absorption is below 22071//.

The nuclear magnetic resonance spectrum for the Δ-28086 factor measured in small chloroborum hydride has the following features: 66.01 , i 21.4.1 1.3.99.3
゜89.3.80.3, 67.3.65.3.57.3
．． 55.2.83.2.76.2.74.2゜68.2
．． 66.2.58.2.56.2.30.2, 22.2
．． 17.2.10.2.05.1゜96.1.90.1
．． 85.1. 70,1.62.1.60,1. /7
．． 1.39.1.31.1゜25.1.18.0.95
．． 0.93.0.90.0.88.0.85.0,77
．． 0.75.0゜73.0.68.0.66+)I)I
I10 Acetone - Antibiotic crystallized from water △-280
86 Convict A exhibits an X-ray powder diffraction pattern with the following characteristics. (CLI 100 irradiation, 1.5405λ, nickel filter, (1 is lattice spacing (unit: angstrom)).

d vs. 2m 12.00 100 10.10 50 9.25 90 8.00 4.0 7.50 15 6.92 90 6.40 40 5.98 05 5.68 15 5.20 40°4.98 40 4. 62 40 25- /1. ．． 21 20 3.718 10 The specific optical rotation of antibiotic A-28086 factor A is
-5'l' (C=0.2, methanol) when measured at °C, this specific rotation is an average value based on several measurements.

Electrotitration of Factor A in 80% dimethylformamide water showed the presence of a titratable group with a pKa fa 7,9.

Antibiotic A, -28086 Convict A can be prepared using various organic solvents (
It is soluble in methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone, and benzene), but sparingly soluble in nonpolar organic solvents such as hexazide, and insoluble in water.

In the antibiotic Δ-28086 Factor A, there is one acid group capable of forming salts and ester derivatives, and at least one gram of water that can be esterified. Based on the physical properties listed above, antibiotic Δ-28
The structure of the 086 factor Δ has been proposed. However, it should be understood that the structure determination is merely inferred and that the structure presented herein is expressed solely on the basis of one experimental hypothesis. The Jfg definite structure of A-28086 factor A is shown in formula (2).

Antibiotic Δ-28086 Factor B is a white crystalline compound (from acetate-water) with a melting point of about 150-153°C.

Factor B has an apparent 1762 molecules M, as determined by high-resolution quality m1 analysis, with C43)-17a
An empirical formula is presented.

The infrared spectrum of factor B in chloroform is shown in Figure 2 of the appendix, and 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°57 (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), 11.20 (weak), 11.3
5 (weak), 11.73 (weak), 12.03 (weak) microns.

The ultraviolet absorption spectrum of factor B in ethanol is 22
The maximum absorption (E = 137.5: ε1 = 10.477) is shown at 0.

The nuclear magnetic resonance spectrum of 29-Δ-28086 factor B (in deuterated chloroform) shows the following features:

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
．． 0.96.0.94.0.90S0.87.0.84
．． 0°77.0.74.0.68ppm.

Antibiotic A-28086 factor B is, for example, methanol,
It is soluble in various organic solvents such as ethanol, dimethylformamide, dimedyl sulfoxide, ethyl acetate, chloroform, acetone and benzene, but has very little solubility in non-polar organic solvents such as hexane, and Insoluble in water.

Although the chemical structure of A-28086 antibiotic factor B has not been clarified, the physicochemical data that have been obtained so far indicate that factor B contains only one carboxylic acid residue (1).
It shows that there are two key residues and one to seven hydroxyl residues.

Antibiotic A-28086 is a microfactor when produced by Leptomysis aureofaciens NRRL 5758. However, Sl~Rep] ~ Mysis O-Off 1 Science NRR+ 809
2, the A-28086 factor accounts for 10% of the recovered antibiotic activity.

Antibiotic Δ-28086 is a white crystalline substance (from water-acetone) with a melting point of about 96-98°C. Its apparent molecular weight is 778 as determined by high resolution mass spectrometry.

The molecular weight of the peak in the mass spectrum of the A-28086 factor Rinosu[~Rium salt was observed to be 800.5050 (CuH730u Na = 800).

5050 is the calculated value). In the buying spectrum of the free acid of A-28086, there is a small peak at 778, 760,
5117 (Cu H7, Otl= 760, 512
A large peak was observed at 5 (Wi calculated value).

'm/e760 in the mass spectrum of FF1Wn acid
is the result of the loss of water from the molecular ions. Therefore, the play of the factor f! The molecular ion composition of Ji acid is Caa F (740
It is n.

A-28086 Prisoner's experimental formula Teha, c4AH74
0I+ is presented. The results of elemental analysis show the following percentage composition: C, 67,59%: H, 9,38%
;0.22.77%.

The theoretical percentage composition as Ct, A8740+1 is C
167.87%: H, 9.51%: 0.22.77%.

The infrared spectrum of A-28086 (Figure 8) contains the following observed maximum absorption. :2.89
．． 3.39.3.43.3.5015.88.6.90
．． '7.27.7.60,7゜84.9.00.9.2
6.9.62.1o, 31.10.58.11.10,
11.49 microns.

In 95% aqueous ethanol, A-28086 particles show no absorption band in the ultraviolet region.

The nuclear magnetic resonance spectrum of /l-28086 in deuterated chloroborum showed the following properties: 66.0
01, 4.20, 4.10.4.00, 3°98.3.
92.3.86.3.83.3.79.3.67.3.
64.3.57.3, 54.2°88.2.81.2.
71.2.62.2.58.2.48.2.43.2.
37.2.29.2゜21.2.15.2.10.2.
04.1.97.1.89.1.83.1.76.1.
68.1゜61.1.58.1.55.1.47.1.
39.1.30.1.25.1.18.0.95.0゜90.0.88.0.8'l, 0.74.0.68pm
0 Antibiotic A-28086 crystals crystallized from acetone-water have the following characteristic X-ray powder diffraction pattern (Cl4→irradiation, 1.5405λ, nickel filter, d = lattice spacing ( Onges 1-Rome))
.

33- (MLMUJL 12.40 100 10, 20 70 8.85 90 7.80 30 6.80 10 6, 30 100 5, 70 20 5.35 20 5, 10 20 4.90 10 4.65 20 4, 45 40 4, 20 30 3.30 10 3.15 10 2, 99 C) 52, 77
05 2, 28 05 When measured at a temperature of 25°C, the specific optical rotation of antibiotic A-28034-86 is -56° (C=0.1, methanol).

Δ-2 in 80% water-containing dimebyl amide
As a result of electrometric titration using 8086 factors, the IIKa value was 8. ('
Titration 1 with +7! Recognized the existence of the group I.

It is soluble in various organic solvents such as methanol, ethanol, dimethylformamide, dimethylsulfoxide, ethyl acetate, chloroform, acetone and benzene. . Δ-280
86 The reason is that Hekylene's: Eel 1 Pole 1) Only a very small amount of G1 dissolves in organic solvents and is insoluble in water.

Antibiotic Δ-28086 Factor D (1) has one acid group capable of forming salts and ester derivatives, and has at least one water g 34t that can be esterified.

Based on the physical f(1) listed above, antibiotic A
A structure with -28086 factors is proposed.

However, unlike I, the structure of A is determined by A.
Therefore, it should be understood that the structure presented here merely represents a hypothesis based on one experiment. The phase-determined structure of Δ-28086 is shown in equation (2).

Basurus subtilis ΔTC06633 as the detected organism
Antibiotic Δ-28086 factor Δ in divine system filter paper chromatography when using.

The Rr values of B and D are listed in Table 1.

0.11 0.09 0.10 Methyl isobutyl ketone (MIBK) 6.・Seen 0,41 0,16 0.26 M T B K
Add 2% D-toluenesulfonic acid and 1% peridine to water saturated with 0.54 0.46 0.36 water:methanol:acetone (12:3:1).

IIHll with NHaOH After what happened H3POa Adjust to pl-17.5.

0,/18 0,3f3 0.29 1%M in water
IBK, O.

5% NH4,0H addition 0.15 0.33 0.25 17 to 1j2 water
．． 4gK 21-l P 04 and 30 cod 0.75 0.61 0.04 Water: M I
B K: Ethyl acetate In Table 2 again, Bacillus subtilis ΔTCC663
3 was used as the detection organism, and silica gel (layer thickness 0.2
The Rr values of antibiotics Δ-28086 factors A, F3 and D in two thin layer chromatographic systems performed on 5 mm pre-coated plates, F-254, E, Merck & Co. product) are listed.

6.24 0.42 0.26 Benzene-ethyl acetate (3:2> 0.54 0.34 0.(i6 Ethyl acetate-
Another substance tentatively named diethylamine (95:5) A-28086-I is a by-product with the antibiotic-28086 complex.

This Δ-28086-1 has no microbiological activity but is structurally related to A-28086 antibiotic 5R.

△-28086-I is a white crystalline compound (acetate
(crystallized from water) and FJ of approximately 160-162℃! Also point out. As a result of a comparative study of the NMR spectra and other properties of A-28086-■ and the synthetically prepared methyl ester of Δ-28086 factor Δ, A-28086
Evidence was obtained that -, I is a methyl ester of A-28086 factor A or a stereoisomer J: a closely related compound.

A-28086-I is the first to precipitate mixed with the active A-28086 antibiotic agents, but is easily separated from the others by silica gel chromatography.

Ethyl acetate was used as the elution solvent, and vanillin @ fog test was used for detection (3% vanillin in methanol solution, wood'a100.
The rough Rf value of the silica gel thin layer 071 using a thin layer of silica gel (added with f13H2sO40,5yβ) is 0.53 using a graph method. When sprayed with vanillin and heated, Δ~28086-I gives blue spots, whereas A-28086 antibiotics give bright pink spots, which quickly turn dark brownish blue. Changes to

To antibiotics - 28086 factor A, Bi)3. and Dl and the acyl ester derivatives of factors A and D described herein are salt-forming.

Physiologically acceptable salts (well, pharmacologically acceptable salts, i.e. salts whose overall toxicity to warm-blooded animals is not increased compared to the non-salt state) .A.
Representative and suitable alkali metal and alkaline earth metal salts of -28(')86 prisoners Δ and jζ and B include thorium, potassium, lithium, cesium, rubidium, barium, calcium, and magnesium salts. etc. Suitable amine salts of Δ-28086 factors A and B include ammonium 'n and quaternary C1-C71-
Alkylammonium salts and hydroxy-〇2-4l
- C4-alkylammonium salts, etc. Specific examples of amine salts include ammonium hydroxide, methylamine, secondary butylamine, isopropylamine, diethylamine, diisopropylamine, ethanolamine, triethylamine, 3-
There are salts produced by reacting with amino-1-propatol and the like.

The alkali metal and alkaline earth metal cationic salts of Factors Δ, B and J: and Dl and the acyl ester derivatives of Factors A and B are prepared by conventional methods for preparing cationic salts. for example,
Dissolve the antibiotic factor or ester derivative in the free acid state in a suitable solvent such as humidified methanol or ethanol, and add to this solution a solution containing exactly the calculated heat of the desired inorganic base in methanol water. Just add it. The salts 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 removed by evaporation.

J: Sea urchin, well known for its pharmacological techniques for treating livestock diseases.
When treating livestock with antibiotics, the form of the antibiotic is not very important.

In most cases, the internal state of the animal causes the drug to change to an open state other than the ingested state.

Therefore, the use of 811 in its salt form is meaningless as a therapeutic method per se. However, the salt form is sometimes chosen for reasons of economy, convenience, and green bamboo.

A, -28086 Concentrator A forms an acyl ester derivative. Esterification occurs at one location in the hydroxyl group of A, -28086 Factor A when treated with any C2-C6 acid anhydride or acid chloride.

For example, a typical method for preparing such esters is to react Δ-28086 factor 8 with the anhydride of the other acid at room temperature. These ester derivatives are also effective as antibiotics and useful as agents to increase feed utilization efficiency.

The following sections describe the properties of these A-28086 Factor A acyl ester derivatives.

The acedel ester derivative of A-28086 factor Δ is a white crystalline (from acetate-water) material with a melting point of about 100-103°C. The empirical formula of the Δ-28086 Factor A acetyl ester derivative is C458740t2, and the molecular weight is about 807, both of which are based on the empirical formula proposed for A-28086 Factor A. Figure 2 shows elemental analysis values of acetyl ester derivatives of Factor A.

Calculated value, C46) '1740, C,66,97
%: H, 9, 24%: 0, 23.79%, actual value, C,
67,67%: H, 8,71%: 0,23°13%.

The infrared absorption spectrum of the acetyl ester derivative of Factor A of A-28086 taken in chloroform is shown in FIG. The following maximum absorption band is observed. 2.85.3
．． 36.3.38 (strong), 5.80.6.83.7.
25.7.52 (strong), 7.60 (weak>, 7.80 (strong), 8.715 (strong), 8.80 (strong), 8.95 (
Strong), 9.10 (Strong), 9.20, 9.63.9.8
0 (strong), 10.12 (weak), 10.25 (weak), 1
0.50 micron.

Terminal absorption was observed in the ultraviolet absorption spectrum of Δ-28086 Convict A acetyl ester in ethanol.

A-28 in 80% hydrated dimethylbormamide
Electrotitration of factor 086 Δacetylnisder M1 showed the presence of a titratable group with a pKa value of 8.5.

The propionyl ester derivative of A-28086 Factor A is a white crystalline (acetate-water) compound with a melting point of about 96-98°C. Based on the empirical formula proposed for A-28086 factor 8, the propionate derivative of factor A-,28086 has the empirical formula (/s H
7c Ot2 and has a molecular weight of approximately 821. The following percentage composition values were obtained from the results of elemental analysis of the propionic acid ester derivative 45 of Factor A.

Calculated value, Cs H7A O12t L/T, C, 6'7
．． 29%: H, 9.33%; 0.23.38%, actual value.

C, 66,06%: l-1,9,17%: 0.23°4
1%.

The infrared absorption spectrum of the propionic acid ester derivative of factor Δ-28086 in chloroform is shown in FIG. The following maximum absorption was observed. 2.85.3
．． 33.3.38 (strong), 3.45 (strong), 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, 9.05.9.1
5 (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 propionate ester derivative of A-28086 Factor A in ethanol.

Butyric acid ester derivatives to the 46-antibiotic factor A-28086 are white crystalline (from acetone-water) compounds with a melting point of about 96-98°C. The butyrate ester derivative of Δ-28086 factor A is inferred from the empirical formula proposed for Δ-28086 factor 8.
H, 2012F, molecule (fl is approximately 835, C,
It has an approximate composition of 67.60%; 1.9°41%: 0.22.99%.

The infrared absorption spectrum of the butyric acid ester derivative of A-28086 factor Δ in chloroform is shown in FIG. The following maximum absorption was observed.

2.89.3.40.3.45.3.51.5゜85.
5.92 (strong), 5.97 (strong), 6.90.7.30
．． 7.84 (weak), 8.55.8°85 (weak), 9.0
1 (strong), 9.26.9.76.9, 95.10.3
1.10.64 microns.

The citric acid ester derivative of the antibiotic MA-28086 Factor A is a white crystalline compound (from acetone-water) with a melting point of about 173-175°C.

The tight acid ester derivative to factor Δ-28086 is
The experimental formula inferred from the experimental formula of A-28086 factor A is:
C411HmO12, molecule m'llJ 849 T:
C.

67.89%;) -1,9,50%: 0.22.61%
It has an approximate elemental composition of

The infrared absorption spectrum of the tight acid ester derivative of Δ-28086 factor A in chloroform is shown in FIG. The following maximum absorption was observed. 2.90.3.
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.

The caproic acid ester derivative of the antibiotic Δ-28086 Factor A is a white crystalline compound (from acetone-water) with a melting point of about 163-167°C.

A-28086 factor A caproic acid ester derivative is
Empirical formula ○aH202 estimated from the experimental formula presented as A-28086 factor A, molecular weight approximately 863 and C,6
It has an approximate elemental composition of 8,18%:)-1,9,58%;0.22.24%.

The infrared absorption spectrum of the caproic acid ester derivative of A-28086 factor A in chloroform is shown in FIG. The following maximum absorption was observed. 2.90.3.
40, 3.45.3.51.5.87.5.92 (strong)
, 5.97 (strong), 6°9017.30, 7.66 (
Weak), 7.84 (weak), 8.16, 8, 58
, 8.85<weak), 9.05 (strong), 9
．． 17.9.72.9.95.10゜29.10.62
micron.

It was found that when A-28086 factor Δ was acylated, the following changes occurred in the 1F-1 nuclear magnetic resonance spectrum.3 The carbinyl resonance occurred at about 4 ppm, and the carbinyl resonance occurred at about 5.3 ppm (exactly The position changes slightly with different acyl derivatives). As a result, the vinyl proton signal is also transferred. This shift is characteristic of the changes appearing in the substructures shown below.

49- (in this figure, λ or R represents C1-C5-alkyl). This partial structure was derived from the IH-homonuclear decoupling experiment results and acetic acid and propionic acid ester derivatives. This is completely consistent with the 13c-nuclear magnetic resonance experimental results.

The C2-C6-acyl ester derivatives of A-28086 Factor A 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 hexadiene, and is insoluble in water.

All C2-C6-acyl ester derivatives of A-28086 have one M group capable of forming salts and ester derivatives.

The novel antibiotic according to the present invention is a strain of A-28086 produced by A. faciens that produces substantial antibiotic activity:
So, let's stir the air! It is produced by cultivating it in a culture medium of 1 to 1 liters and 18 to 10 liters. The antibiotics produced are recovered by various known purification methods.

One of the new microorganisms useful for the production of Δ-28086 antibiotics 71 was isolated from soil samples collected on the mountain to Arara 1 in 1-1-2). This creature is called Shearing (E
,B,) and bok1~rib(D,)j: tsuteko Δ
Perabub description or type culture or streptomysis,■,Additional species description from farth 1~ and second studies''internasi 31 pull biraretin or systema Teitsuku Bacteriology, Vol. 18, pp. 279-392 (19'68)
Mysis aureofaciens datsuger (3treptomyces) to strip 1 as described in
auresfaciens l)uggar)-
classified as a bacterial strain. This bundle method has some additional IIE! International Streptomysis Project [Scharling (E, B,) and Gottlieb (D), Methods for Characterizing or Systematic Bacteriology of Streptomysis Species] , Volume 16, 313-340 (
This method is based on the 111 methods listed in 1966). Color names were selected according to the 15CC-NBS method [
Kelly (K, I-,) and Shutt (D, B,)'
The l5CC-NBS Method or Designing Color and a Dictionary or
Color Names” U, S, Dept, of
Convercc.

C1rc, 553.1955. WaShington
,D,C.

].

Numbers and symbols in parentheses are Tresner and Backusian (
Referring to the t22 series [1 to Lesnar (+-1, D.

) and Backus (S, J.), “System Ab Color Wheels for Streptomysis.”
Taxonomy °′ Applied Microbiology 1
1, 335-338 (1966)). I drew a line under the color naming table. Melf and bowl color blocks are in parentheses [Melf (△) and bowl (M,
R,), ``Dictionary ΔB Color'' Matsukugrownihill Book Company, Inc., Niko]-ri, N, Y,).

Cultures were grown at 30°C for 140 days unless otherwise noted.

1L sex distribution of △-28086 producing bacteria I□R15758) 4
10 Morphology Spore-forming aerial hyphae are hook-shaped, ring-shaped, and spiral-shaped. A typical flexorectal morphology is also observed. The spores have a single mouth and are cylindrical in shape, with 10 to 50 spores connected in a chain. The spores are 1.3μ53-Xl, 75μ in size (1.3μ~1.95X1
．． 3μ range). When observed using an electron microscope,
The surface structure of the spore is smooth.

On various media (Culture characteristics of NRRL5758 - ISP C2 (yeast extract - malt extract) medium: vigorous growth, medium yellow on the underside (3 on 11), moderate to good occurrence of aerial mycelia, Sporulation, white (W) 13 ha and dark gray (GV) 3 ih, no soluble pigment.

・ISP C3 (oatmeal) medium: Good growth, back side is gray-yellow (11B2), sufficient formation of aerial mycelia, dark gray (Gy) 3ih, some brown soluble pigment.

- ISP C4 (salt-free starch agar) medium: vigorous growth, back side pale yellowish brown (12E5), aerial mycelia and spores (W) purplish white (13ba or Gy) light gray d, no soluble pigment.

・ISP C5 (glycerin-asparagine agar) medium: 54-Good growth, back side bluish yellow-green (10B1) Aerial mycelium (i-good ti'r, spores (GV) yellow-gray 2 (1c,
to light brown (', 5fe, no soluble dye.

・i・71～・Pace 1～A-Tomil agar medium 2 Strong growth, back side light F37 brown (131-17), aerial mycelia A3 and spore production medium to good I'(W>white a or GV ) The medium is gray 0: microscopic brown soluble pigment of 15.

・Glycerin-glycine agar medium: Strong growth, dark gray yellow (12+6) on the underside, good formation of aerial mycelia and spores (Y), bluish (yellow 2 (Ib)).

No soluble dyes.

・Glucose-Ahasparagine agar medium: Strong growth, normal yellow on the back (12E2), aerial mycelium OJ
: A lot of spores GV) Seedling Gray 2 dc, ultrafine ff3 brown soluble pigment.

・Normal agar medium: Good growth, back side is gray-yellow (12B2), aerial mycelia and spores are present, but color is not specified due to poor production, soluble pigment 1
．． “K L/.

- Bennett's agar medium: vigorous growth, gray-yellow underside (12 to 3), poor aerial mycelia and spore production (GV) 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.

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

・Emmerthun's agar medium: Strong growth, back side yellow (11J5), no aerial mycelium or spores, no soluble pigment.

・Dyrosine agar: Strong growth, pale olive brown on the back (14C4), many aerial mycelium and spores, <W)b (center) or GV)
Pale brown-gray 3 fe (periphery), slight brown soluble pigment.

・j~Lipton-F'4) Agar medium: Poor growth, no color specified.

Next, the physiological 4!1 quality of NRR15758 was examined according to the standard prescription. The characteristics of the observed 141 cases are as follows.

Observed f1 Characteristics Effect on milk Converts milk into peplon, producing a white growth ring, the transparent part is yellowish brown, I]1-15.7 nitrate) 7 element positive meat juice geladin 14 [30% liquefied melanin pigment productivity in 1: Dirosine obstructed slope weak positive fl: (pigment production after 4 days) tryptone-yeast negative 1 no 1 Extract growth temperature (ISP 26-30℃ Good growth T: 3 medium β2 'M mother extract, vigorous growth at 0-37°C, malt extract slope) Pre-formation: Only 57°C mycelia grow, pigment production with red dissolution, carbon source utilization ability conducted on N RRL 5758 bacteria The results are as shown below, and the symbols used to indicate the degree of bacterial growth are: + Good growth, positive carbon utilization (+) Poor to fair growth (-) Weak growth, no carbon utilization ~ No growth, no carbon utilization, carbon source availability 11--arabinose + D-xylose + D-gluta1--su + sucrose -D-
Mannilla 1- - 1-Inosit + Rhamnose + Raffinose - -C71 Trol 158- (No carbohydrates added) Another novel Δ-2B (186 Bovine microorganisms were subjected to a series of chemical transformation treatments, Stoshib 1 ~ Mysis A-Leofaciens N RRL by the method of natural selection
It is derived from 5758.

The organism identified as NRR+-8092 is also S. streptomyces aureofaciens.
cns) was classified as a strain of Dagar. This classification was performed using the previously described Streptomyces taxonomy as well as NRRL80921i1! using similar culture conditions. Based on research by i. NRRL8 observed in these studies
The characteristics of 092 Kan are summarized in the following items.

A-28086-producing fungus Zhu N RR+-8092 (TSP β7 medium (Dirosin agar)) The cultured fungus often forms hook hyphae, but mainly short erect spore stalks. (5porepore) is formed.

Spore chains have no more than 10 spores per series, usually 4 to 7 spores per series. Short, upright spore chains were observed on the following media: ISP β3. A large number of condensed hyphae were observed on Czapek's solution agar and ISP β5° and Emmerthun's agar.

Electron microscopy was performed on dirosine agar (ISP β7) and glucose-asparagine agar. The spores are smooth and distributed in a size range of 1.2μ to 2.0μ in length and about 1.0μ in width. The average spore size is 1.6μ x 1.
0μ.

Cultivation characteristics of NRR18092 in various media: - TSP β2 (Ml mother extract - malt extract) medium - sufficient growth, light yellowish brown on the underside (12+-18L aerial hyphae sufficient, sporulation poor, aerial hyphae pale gray [11A1 ], no soluble dye.

・ISP β3 (oatmeal) medium: Sparse growth, transparent back surface, no aerial mycelia. No soluble dyes.

・ISP β4 (inorganic salt-starch agar) medium: Medium growth, back side yellow (11B2), lack of aerial mycelium, spore formation, aerial mycelium is bluish yellow-gray (10A1), no soluble pigment.

- ISP β5 (glycerin-asparagine agar) medium: Medium growth, back side bluish yellow (10F2), sufficient aerial hyphae, poor sporulation, aerial hyphae white (10△1), no soluble pigment.

- Tomato base oatmeal agar medium: medium growth, grayish green-yellow underside, sufficient aerial mycelium, medium sporulation, pale blue-gray (53A2), no soluble pigment.

- Glycerin-glycine agar medium: Strong growth, back side grayish-yellow (11EA), medium aerial mycelium and white (10A1), no spore formation, no soluble pigment.

- Glucose-asparagine agar medium: medium growth, pale yellow on the back (10F2), medium aerial mycelia spore formation, white [10△1], no soluble pigment.

61- ・Ordinary agar medium: Sparse growth, bluish yellow on the m side (
'1OB2), no aerial mycelium, no soluble pigment.

・Bennett's agar medium: Good growth, medium yellow-pink color (11A7) on the back side, very poor aerial mycelium, no sporulation, no soluble pigment.

・Calcium malate agar medium: Very poor growth, transparent, no aerial mycelium, no soluble pigment.

・Zupek liquid agar medium: Very poor growth, transparent back side, no aerial mycelia, no soluble pigment.

・Emmerthun's agar medium: Moderate growth, back side yellow (1115), scattered aerial mycelium, no sporulation, no soluble pigment.

- Dirosin agar medium: medium growth, light yellowish brown on the back (12H6), medium aerial mycelium,
The periphery is light blue-gray C53A2), the center is almost white,
Sporulation is moderate, no soluble pigment.

・Tryptone-M mother agar medium: 62- Extremely poor growth, transparent, tertiary threads, soluble M
f! No dye.

NRR+ 8092 bacteria were also tested for certain physiological properties according to standard formulations. The observed properties and known characteristics are as follows.

Observed sex 1 person - Characteristics Effect on milk Peptonization (90%), producing a bluish-yellow growth ring, transparent part tit pale yellow, pl-14,6 Nitrate reduction Positive Meat juice gelatin 14 days Productivity of 50% liquefied melanin pigment: Dirosine agar slant Extremely weak positive tryptone - negative Yeast extract juice Carrot piece Full growth, pale yellow, no aerial mycelium Potato piece Strong growth, grayish white, no aerial mycelium. The plug hasn't changed either.

Growth temperature (ISP medium β2 yeast extract malt extract slope)
= Strong growth at 25℃, fairly good aerial mycelium production, light brown on the w side, no soluble pigment, vigorous growth at 30℃, sufficient aerial mycelium, light brown on the back side, no soluble pigment, growth at 37℃ Vigorous, sufficient aerial mycelium, brown underside, soluble pigment brown, 40
At 45℃, vigorous growth, sparse aerial mycelium, reddish brown on the underside, with brownish-brown soluble pigment; at 45°C, good growth, no aerial mycelium, reddish brown on the underside, medium reddish brown pigment.

Next, the results of examining the carbon source utilization ability of NRRL8092 Kan are as shown below, and the symbols used indicate the degree of growth.

+ Good growth, positive availability (+) Poor growth or -1 minute (fair) (-)
Weak growth, estimated to be unusable - No growth, unusable carbon source Usable capacity: 10 L-arabinose + D-xylose + D-fulc 1--sucrose - Sucrose - D-Mannitol - i-Inosit + Rhamnose + Raffinose - -C Control - (No carbohydrates added) Some properties of the A-28086 productive Streptomysis aureofaciens strain are similar to those described by Schillering and Gott-Leeb. is different from the nature of These differences are summarized in Table 3.

Table 4 summarizes the differences in specificity between the 65-66-NRRl-5758 bacteria, the NRR+ bacteria, and the -8092 bacteria.

Growth on several types of media Color on several types of media
Yellow Cream to yellow spore formation 1-71~・Pace 1~. Often hooked, with ohmy mill and erect sporophyte inorganic 1-starch medium.

``Two-screw embryo (form 1, gamma-stalk later (1 liter malic acid), fully grown and brightly colored; growth sparse, peripheral calcila; surrounding transparent; not transparent.

Live on medium - No Iff JX2 - Moderate sporulation Almost 111
Starch medium for forming starch Aerial mycelia are purple-white None, aerial mycelia grow on top 41 to gray Pale yellow method (Δ Benet 1 to
The reverse side is pale yellow C. The reverse side is pink Kuni Iku on agar Δ-28086 Cultures of Streptomysis aureofaciens useful for the production of antibiotics have been deposited at the U.S.A., Peoria, Illinois, 61601 U.S.A. Department of Agriculture's Agricultural Research Service, Northern Market Trading and Nutrition Research Division
na and n ujr! [! On Resea
rch D 1vi-sion, (J, S, D
ept, of Δgriculture, A g
ri-cultural 5service, Peo
ria, Ilinois, 6160/I), from which NRRL5758
and NRRL8092, deposit number M'r.

In addition, S1 Heliptomysis aureofaciens N
RRL 5758 and Streptomysis aureofaciens NRRL 8092 are FERM-P No. 3098 and J: and F, respectively, to the Institute of Microbial Technology, Agency of Industrial Science and Technology, address 5-8-1 Inage Higashi, Chiba City.
It has been deposited as ERM-P No. 3099.

[S1~Rip] Hemysis O-Off 1 Science NRR
I 5758 or Streptomysis aureofaciens N RRL 8092 can be grown on many media.
'l can be used. However, there is a culture medium that is suitable in terms of productivity, ie, the quality of storage, and the ease of product separation. Preferred carbohydrate sources for large-scale fermentation are, for example, Tavimeca's dex-1-phosphorus sucrose, but others such as glutinous sugar, corn starch, soybean sugar, mannose, maltose, lactose, etc. can also be used.

Other useful carbon sources include corn oil, peanut oil, soybean oil, and fish oil. Qγ The most desirable nitrogen source is the power decomposed by enzymes! Although it is a pair of soybean flour, cottonseed flour,
Amino acids such as glutamic acid and the like may also be used.

The inorganic nutrient salts added to the culture section 1 include conventional ions that produce ions such as sodium, magnesium, calcium, ammonium, sulfuric acid, nitric acid, and sulfuric acid. There are soluble salts.

69- The micronutrients necessary for the growth and multiplication of microorganisms should also be included in the culture medium, and such microelements are usually sufficient to meet the growth requirements of the microorganisms as contaminants of the microorganisms in the culture medium. It's mixed in.

If foaming is a problem in large-scale fermentation cultures, consider adding an antifoam agent such as polypropylene glycol (
For example, it may be necessary to add 0.2ff6/l.

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

Second, for substantially meaningful production of Δ-28086 antibiotics, aeration-stirred fermentation in tanks is preferred, although small amounts of Δ-28086 antibiotics may be obtained by shake flask culture. Dew. Because of the time lag in antibiotic production (Iao) associated with inoculating microorganisms in the form of spores into large tanks, it is preferable to inoculate with viable seed bacteria; . A (-seed with resultant force and l;
1. , a newly developed microorganism, is an actively growing 111 nutrient, which is prepared by inoculating a small 81 culture medium with spore forms or pieces of mycelia. This viable seed culture is then transplanted into a larger tank. The medium used to propagate the viable seed culture is the same as that used in the large tanks; however, other media may also be used.

Next, the microorganisms producing wood 28086 can grow at humidity of about 20-40℃, but in the humidity of about 27-30℃,
The most preferred production of Δ-28086 occurs in J:U.

Aerobic) Sterilization] In F1 culture, sterile air is blown into the culture medium in 10 cases. In order to grow the microorganisms sufficiently, the amount of air used in tank production is usually 11 times the culture solution per minute.
It is said that it is preferable that the amount is 0.1 volume or more. In order to effectively produce this Δ-28086 antibiotic, it is particularly preferable that the amount of air used in tank production is equal to or greater than 0.225 volumes of culture broth in the tank. The production of the antibiotic is suppressed and does not deteriorate due to the high level of dissolved oxygen.

Antibiotic production is carried out using cultured moss solid extracts during the fermentation period. Can be tracked by substance activity testing.

One assay organism useful for testing the antibiotics of the present invention is Bacillus subtilis ΔTCC6633. This bioassay is performed using paper disks (pa
This is preferably carried out using a per-disc (filter paper flat disk).

The initial pH of the medium for culture-free inoculation port 1 varies depending on the medium used. Generally, its OH is 6°0.7.
It is sufficient that the DH is in the range of 5, and the DH at the final stage of fermentation (harvesting stage of the product) is slightly higher and is in the range of 6.5 to 8.0.

Generally, antibiotic activity becomes detectable from the second day of fermentation, with maximum results usually occurring within about 6 to 10 days.

The A-28086 antibiotic described above can be recovered from the fermentation broth following production in an aerated, agitated fermentation row '1' by methods known to fermentation engineers. Antibiotic activity produced during fermentation of the A28086 bovine microorganism is found in both the mycelial mass and the culture filtrate. Therefore, by combining methods including filtration, extraction, and adsorption chroma-graph methods, maximum recovery of A-28086 antibiotic class 71 is achieved. The preferred solvent for extracting Δ-28086 from the entire fermentation broth or its filtrate is ethyl acetate, but other commonly used solvents are also sufficient.

A-2B One particularly advantageous method for separating O86 factors A, B, and D is to lower the pH of the entire fermentation broth to about 3.
It is to lower it to 01i7. This pH3.

01- can be separated by filtration from the mycelial mass of Δ-28086 convicts Δ, B and D. Another advantageous method for separating the A-28086 factors is to add small carbonates to the whole fermentation broth at a rate of approximately 1 gram per liter of broth, e.g. It is to be. Thereby, the Δ-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 lower alcohols and ketones are also used.

Next, the co-existence distillation method is also advantageous when used to recover Δ-28086 antibiotics. In this method, an organic solvent is added to the fermentation broth that forms a suitable azeotrope with water. This solvent-broth mixture is azeotropically distilled to remove at least half of the water from the broth, resulting in a water-solvent mixture in which the A-28086 antibiotics are dissolved in the organic solvent. Insoluble by-products are separated by suitable methods such as filtration or centrifugation, and A-28086 antibiotics are separated from organic solvents by known methods such as evaporation of the solvent, precipitation by addition of a non-solvent, or extraction. It will be collected.

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, ethyl acetate, itl amyl, 1,
Examples include 2-dichloroethane, 3-penquinone, 2-hexylone, benzene, cyclohylionone, toluene, and other xylenes.

In large-scale fermentation production processes, recovery by distillation of boiling heat is particularly advantageous. The water removed in this way does not contain any contaminants and does not require any waste liquid treatment.Also, the solvent separated in this way can be used in the same way.
11 available for culm.

Next, the Δ-28086 antibiotic is purified by adding extraction and adsorption operations. Silica gel, carbon, Florisil (trademark for magnesium silicate) [1 Product of Lysine Co., Ltd. (P.O. Box No. 989, Tallahassee, Florida, USA)
) Others are used for right-handedness.

Alternatively, since the culture solid contains the 811 component of the culture solution and mycelia, it can be used as a source of A-28086 antibiotics without extraction or separation, preferably by removing only the water content. That is, once the antibiotic activity of Δ-28086 is achieved, the culture broth can be lyophilized and directly mixed into the feed premix.

As another point of view, after the Δ-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 isolating bacterial cells for such uses, adding lime carbonate (approximately 10a/β) facilitates filtration and provides an improved dry product.

Under the conditions that have been used for many years, the above-mentioned N R
The S1-Lyptomysis ornoA faciens strains designated RL 5758 and N RR+-8092 produce antibiotic factor A-28086 as a major product. The ratio of production factors varies depending on the fermentation conditions at the time, but in general, factor A is N RRL 575
Accounting for over 99% of the total antibiotic activity recovered from 8 strains, N
It accounts for more than 90% of the total antibiotic activity recovered from strain RR18092.

Δ-28086 convict B accounts for most of the remaining antibiotic activity from strain NRRl-5758, with factor D tj being a minor factor. On the other hand, the C1Δ-28086 factor is N
Total recovered antibiotic activity from RRL8092 strain: Approximately 8 to 1 of ↑1
0%, and this ■, umami factor B, is a trace factor.

Antibiotics A-28086 factors Δ, B and D are separated from each other as individual compounds using column chromatography, thin layer chroma 1 Hegelaf method, a method known in the art. For example, using column chromatography on silica gel, the factor Δ
, B and D are separated. When a benzene-ethyl acetate solvent mixture is used in a silica gel column, factor B is eluted first, and factor D is eluted later. As described above, thin layer chromatography is a convenient method for detecting the elution process.

A-28086 compounds are 77- It has the effect of inhibiting the growth of certain bacteria and fungi.

Table 5 below compares the microbial activities of Factors A and 8 of A-28086.

The test method is the usual disk diffusion method (disc-diH-
(method) (a method in which a 6 mm blotting paper is immersed in a solution of 1 ma of product/1 πβ solution and the blotting paper is placed on an agar plate inoculated with the test organism).

78- Table 5-79- The next important thing is that A-28086 compounds are anaerobic+!
1. It is to clarify the growth of bacteria.

Table 6 summarizes the results of determining the minimum inertia (MIC) at which 1-28086 Factor A kills various anaerobic bacteria using a standard agar dilution method. Terminal fish was determined after 24 hours of culture.

-80- Table 81 - A-28086 The C2-C6 factor A derivatives also have antibacterial and antifungal activity. Furthermore, from a certain point of view, these derivatives were seen to have enhanced activity against Durham-positive genes compared to factor A. Therefore, the relative Gram-positive bacterial activity of some of these Mp9 bodies was compared with that of factor A. Control compounds were N, R, Kuzel and “
,W.

Kavanara, Journal of Pharmaceutical Sciences, Vol. 60 (5), pp. 764-767 (197
The samples were tested by the turbidimetric test method on a semi-automated system (Elanco Autotarp type microbial test system) as described in 1998). The following test parameters were used in this A-28086 antibiotic assay.

Staphylococcus aureus (H-P1 to R) NRRLB-31'4 for assay was added to a nutrient medium (pl-
The cells cultured in (7) at 37°C for 4 hours were used. Test sample, standard, and product A were mixed with methanol and water (10:90).
dissolved in. The Δ-28086 factor Δ of the mark t1L product is set to 2.
3,4. OJ: 5 mcg/J-1-Turp 0/Colorzel (△u t o t ur b c
arousel). Sample compounds are diluted to 1
The color bill should contain about 3-4 mcg of active substance per πβ. 4! The relative activity of the test compound determined in comparison with Active Nano 1 of A111-crystal is shown below.

Compounds relative to each other, i
i! ! ff: 1111;-A-28086 Δ (standard product) 1 Δ-28086-A acetate ester derivative 2.5Δ-28086
-A propionic acid]-stell derivative 7 △-28086-Δ・'1- butyrohahi [scar derivative 8 A-28086-Δ-n - propionic acid di[stell derivative 1/IA-28086
-he-n- Caproic Acid Ester Derivative 20 Another useful aspect of the antimicrobial activity f1 of A-28086 compounds is Mycoplasma or Mycoplasma (He4
ycoplasma). Mycoplasma species, also known as Broilobromonia-like (PPL○), are pathogenic to humans and various animals.

A mulberry agent that is effective against PPL○ organisms is particularly desired by livestock producers. Anti-cattle products against various mycoplasmas measured by in vitro broth dilution study method 1
The minimum inhibition temperature (MIC) of fA-28086 is summarized in Table 7 below.

Mycoplasma gallisepodicum 12.5 Mycoplasma hyorhinis 12.5 Mycoplasma synobiae 6.25 My] Plasma hyobobunicomonia 6.25 Mycoplasma hyacinobiiani U 6.25 Part 17I
A trace amount of antibiotic A-280 of about 10mcg per β
A solution containing 86 is also effective as a topical disinfectant to remove animals from Mycoplasma species.

Second, the Δ-28086 compounds are also antiviral agents. △-28086 factor 8 is a type of poliomyelitis virus (tVl) em poliovirus), vaccinia virus (vaccinia virus), herpes virus (herpes virus) and Semlitov A rest virus (3emliki Fore)
st viruses) and are active against two types of bacteria, and these are used in an in vitro plaque inhibition test (in vitro v:tr
o pla(llIe 5tlpp-ression
LOsts).

85-△-28086 Convict Δ is also infectious gastritis virus,
It also shows activity against Newcastle disease virus and infectious bovine genus I/porous bronchitis virus, which was also confirmed by similar tissue tube tests.

Thus, in one aspect, any of the A-28086 compounds can be administered orally, topically, or by injection to mammals for the purpose of controlling viruses. .

The effective dosage for the prevention or treatment of virus disease is approximately 1 to 5 mg per 11 kg of body weight of a mammalian animal, depending on the target virus and whether the purpose of the drug is prophylactic or therapeutic.
It is normal for it to fluctuate and not be constant.

In addition to the above, liquid preparations containing the Δ-28086 compound include:
It can also be used, preferably in conjunction with a surfactant, to remove in vitro habitats where viruses such as those of polio or herpes rash are present. In such cases, approximately 1 to 1500
A liquid preparation containing mcg/xx of the Δ-28086 compound is effective in suppressing viruses.

To antibiotics - 28086 prisoner Δ's sudden 14th voice 111 is given by 1Ω by intraperitoneal sex in mice, and its lD'a+ is 7
, 15 +ng/kgT”.

Second, the A-28086 compounds are also insecticides and acaricides. The 8-28086 compounds are effective against insects such as Mexican bean beer, milk creed bag, and houseplants, and are effective against insects such as the Mexican bean beetle, milquerid bag, and the two-spotted spider mite (tsyo-
Spotted 5pider + n1tO) J: It is also effective for eel type 2, and only about 500 plums of raw silk is sufficient. Furthermore, J: da, △-28086 compound f,, 11 mosquite 1 arvae
) is also effective, and the usage m is 10).

One important property of this A-28086 compound is its anticoccidial activity. For example, as a result of breeding experiments,
Any compound of A-28086 improved weight gain when added to the feed of chicks (U11 chickens) in small amounts as low as 0.003%, and even lower doses as low as 0.005% showed that
Mortality of chicks attacked by coccidiosis pathogens is prevented and the number of IL1 damage is reduced.

Tables 8 to 10 show the results of the efficacy tests for Factor Δ in chick groups fed with various Eimeria pathogens.

89-190- For the prevention or treatment of coccidiosis in poultry, A.
Preferably, one of the -28086 compounds is administered orally to the bird daily, in a form that is non-toxic and yet effective against Coccidia protozoa. There are many ways to give the A-28086 compound to birds, but the simplest method is to give it together with a physiologically acceptable carrier, and a particularly preferred method is to give it together with the crow's feed. Also, A-2
Although various factors must be considered in determining the appropriate dosage of Compound 8086, the rate of administration is usually in the range of 0.003 to 0.04% of the drug-free feed, preferably 0.04% of the drug-free diet.
It will be in the range of 0.005-0.02%.

An important property of Δ-28086-28086 is its ability to improve animal feed utilization efficiency.

For example, A-28086 compounds improve feed utilization efficiency in developmental mammals.

As we have previously discussed, the efficiency of drinking water use in reptiles is greater than that of acetate or butyrate by stimulating the distributed biota within the animal's rumen. Rather, it is enhanced by manipulating J: to produce propionone-1. Feed utilization efficiency is detected by observing the production and concentration of propione-1~ compounds in the rumen using the following method.

First, rumen secretions are obtained from male calves in which a transverse canal (ristula) is surgically expanded into the lumen. These calves are raised on a diet rich in grains.

A sample of the rumen fluid '31 is percolated through four layers of cheesecloth and the filtrate is collected. The particulate matter left in the cheesecloth has enough physiological tfA to return it to the original rumen fluid stage.
Suspend in m solution and leached again.

Journal or Pulley Science, Volume 38, Volume 12
According to Cheno et al., pages 25-1230 (1955), the buffer solution used in this case has the following composition.

One litter 11q/I! .

93- NazHPOa 0.31
6KH2PO40,152 Na HCO32,260 K (40,375 Na C10,375 M0 SO4,0,112 Ca CjZ2 ・2H200,050Fe S
Oa ・7H200,008Mn SOa ・
H200,0047n SOa・7H200,
004Cu SO4・5H200,002 COCβ2・61-120 0.001
The two filtrates are mixed and allowed to stand, allowing suspended solids to float to the surface. Separate the clear layer and add the same buffer (1:1)
and then adjust the pH to between 6.8 and 7.0. The diluted rumen fluid (10 g) was added to the above feed 40 g.
n+o, 1 ma of soy protein addition, and the test compound to a 25-capacity flask. - Use four replicate flasks for each treatment. Additionally, two sets of four flasks are used as 94-controls, as well as an O-hour control and a 16-hour culture control. All test flasks are incubated at 38°C for 16 hours. IHy
2, measure 01-1 of each flask and add 25% metaphosphoric acid (2πj1) to each flask. After each sample is precipitated several times, the supernatant is analyzed by gas chromatography to determine probionate, acetate, and butyrate compounds. A compound in which an increase in the production of propione-1 to more than the control was observed is significant as an active compound.

Test compound results are compared to Control 1-Roll results using an If metric. Table 11 shows the ratio of the concentration of volatile fatty acids (VFA) in the treated flask to the concentration in the Con 1-roll flask.

Carbohydrate utilization efficiency is determined by the adhesion (f
islulas) performed on animals equipped with <i
n vivo) tests will further elucidate the results.

The test results shown in Table 12 each have a weight of approximately 10
Adhesion of 0C) ehs was performed on a fully-attached adult black cow. Two black cows were fed a normal diet and four in each treatment group were fed the same diet II supplemented with A-28086 factor A.

1 for each 100fffi of rumen fluid taken out.
0% metaphosphoric acid (100 g) was added, sample r1 was allowed to stand, and then the transparent liquid was analyzed by gas chroma 1 to graph method to measure propio 289 degrees. The result in table 12 is 14
Average percentage increase in propion flt in the rumen averaged over 5 times during the daily treatment period
It is. The control at this time was approximately 20 mole percent propionic acid.

Additionally, in vivo studies have shown evidence that A-28086 factor A enhances feed utilization efficiency in sheep as well. These were carried out over a period of 56 days or more, and the results were
The results are summarized in Table 3.

・ff12 -2 Δ-28086 Prisoner A Increased (mc+ per day) more than control %25
17.4 100, 54.0 98-Table 13 △-28086-Inori-1 Compounds are antiviral agents. A-28086 is effective against Maryland virus type B, polio virus, C○E virus, herpes virus, and Semiriki virus 1 to 1. This was confirmed by an in vitro plaque inhibition test similar to that described by Siminoff [Applied Microbiology, Vol. 9 (1), pp. 66-72 (1961)]. A-28086 congenital virus is also effective against infectious gastroenteritis virus, Newcastle disease virus, and infectious bovine foramen bronchitis virus, which was also confirmed in similar tissue culture studies. .

The fact that A-28OB6-Factor-D compounds have inhibitory activity against both viruses and anaerobic bacteria indicates that these compounds can be used for treatment of enteritis in pigs, cattle, and sheep. Or it is used for prevention purposes.

The A-28086-Factor-0 compounds are also effective in treating enterotoxiasis in reptiles.

Next, the △-28086-factor compounds according to the present invention! ■One of the important f4 i'irs is anti-coccidiosis activity. In vitro experiments demonstrated that the Δ-28086 factor has anticoccidial activity against Imelia tenella. The usage is described below [for details], see Experimental Parasitology, Vol. 34, 189-19.
6 pages (1973) Nori, R., McDougald and R.B.
, see Galloway's report].

Cultures of host cells are grown in Leighton tubes (L cighton tubes).
ube), plastic opening or other suitable 4
-C is prepared by a simple method using TI8 culture containers.

Growth in any suitable medium (e.g., Eagle's Minimum Requirement Medium, Armu or Bank's lactalbumin hydrolyzate in balanced saline, Medium 199) for 2 to 30 minutes usually produces a suitable TLI layer. This makes infection treatment and drug treatment easier. Primary cultures of kidney cells were used in these studies.

Eimeria Tenera has the power to live.
The oocysts are collected from the stool of chicks infected with P. 101-tenella and cleaned and sterilized prior to use.

The oocysts are suspended and germinated by aerating the suspension or by gently infiltrating it in a bladed shaker or other suitable device while keeping it at room temperature. During the germination process, potassium dichromate, sulfuric acid or other chemicals can be used to prevent bacterial and mold growth.

Infectious extrusions are completely ejected from the ascus by mechanically breaking the outer wall of the ascus and treating with lipsin and bile salts to activate the extrusions that have emerged from the ascus. let

Infect the cell culture by introducing viable explants into the cell culture in the container. A test compound can then be introduced into it. Test compounds are preferably introduced using 10% dimedylformamide in saline. Suppression at the end of the study is for the asexual stage. Ninety-six hours after infection, cultures were fixed, stained, and observed under a microscope to determine the end point of the infection. Shizon 1 as the second generation (sclliz
Ont) exists or not, and whether or not toxicity to a single cell layer is clear, the results can be validated141 (△)
, recorded as inactive (N) or III cytotoxic (C).

Table 14 summarizes the anti-coccidial activity of Δ-28086 coccidiosis as determined by the test described above.

Table 14 Antibiotic Δ-28086 Prisoner D5
CO,03A I C0,02A O,2CO,01±A -α-CO+A engineering *0.001- N-Note (1):
Concentration of one Δ-28086 prisoner (mCg/π2).

*Mark: Results of two tests.

Another feature of the A-28086 factor compound is that it can enhance the function of the rumen of ruminant animals and increase the efficiency of feed utilization. Main nutritional components in adult animal feed (
The mechanism of utilization of carbohydrates is well known. Microorganisms in the animal's rumen break down carbohydrates into monosaccharides, which are converted into pyruvate compounds. Pyruvate compounds are converted into volatile fatty acids (VF) by the metabolic action of microorganisms.
acetate-1 to butyrate-2 or propionate, known as・Auriel Press, Upon Tyne (1
9A > ■1) See description of Astragalus on pages 408-410)
.

Regarding the relative effectiveness of using VFA, see Matsuklov (
Foodstaffs (June 19, 1971) p. 19), Nisklund et al.・Luminants Volume 2 (1971) 622 and 625
There are reports of pages.

Acetate-1 and butyrate compounds are also utilized, but
The efficiency of propionate compounds is more human.

In addition, animals with very low levels of propionone-1 compounds may develop Ken's disease.

Therefore, useful compounds stimulate the production of higher parent propionate from carbohydrates in the animal body, increasing the efficiency of carbohydrate utilization and reducing the prevalence of ketosis.

The activity of a useful compound can be determined by observing its influence on the formation and concentration of propionate compounds in the rumen using the same procedure as for factor Δ. .

Statistically compare the results of the test compound with those of the control compound. Table 15 shows the ratio of volatile fat M FJ in flasks treated with Factor A-28086 to volatile f/l fatty acid concentration in control flasks.

105 - Table 106 - The compounds of the present invention exhibit typical efficacy in increasing propione-1~ compounds and therefore this compound is reacted at a rate of about 0.05 to 5.0 mq/Ita/day. 8 When administered casually to animals, feed utilization efficiency can be increased. The compound of the present invention is about 0.1-2.5n+a/k
The best results are obtained by administering at the rate of 0/day. A preferred method for administering the compound of the present invention is to mix it with animal feed; !
For example, it can be administered in the form of tablets, trenches, large pills, or capsules! i77 can be done. These various dosage forms can be formulated using methods well known in veterinary pharmacology.
It can be formulated into a drug. The individual dosage unit forms should be such that they contain a compound of the invention at a rate directly related to the daily dosage of the animal to be treated.

The compounds according to the invention can be utilized by feeding fattening livestock a feed composition consisting of livestock feed and 300 g of A-28086 per ton thereof.

Known in this field - (J: sea urchin, cattle breeding n1it
It is different from other feeds such as runaway feed. Cattle feeding! -1
includes forage such as cottonseed, corn silage, etc. In addition, cows feed at a higher rate (often 15
~25%) of non-protein nitrogen sources. Usually this non-protein nitrogen source is urea.

As mentioned above, the Δ-28086 compound of the present invention is an antiviral agent and is also effective against anaerobic bacteria, especially CI ostridium berfringens.
ringens). Therefore, it is useful for treating and preventing enteritis in chickens, pigs, cattle, sheep, etc. Compound A-28086 is also useful for treating intestinal toxicity in valiant animals.

Next, specific embodiments of the present invention will be described with reference to Examples.

Example 1 (Δ) Streptomysis aureofaciens 7,
(3, aureofaciens) NRRL575
Cultures of Mycis aureofaciens NRRI-5758 were prepared in stages 1 to 1 according to the following procedure.
.

Ash---J and 1 Agar 20 (] Dex 1 Herin 10g Hydrolyzed by intoxication Uin 2Q Beef extract 2g Fermented yeast Nikis 2g Add distilled water to make 11!.

1' That is, Streptomysis aureofaciens NRR 5758 was added to an agar slant medium having the composition shown in -.
was inoculated, and the inoculated jΔ soil 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 the spores were scattered using a sterilized platinum loop. The thus obtained live serum suspension of spores and mycelia pieces was freeze-dried to produce six pellets.

1 CC of freeze-dried PETZ 1-1 was inoculated into 50 plates of a growth medium having the following composition.

109- Ash 1112r type can (a) Grape sugar 20 pcs.
Bean flour 15 Corn Steebly force -10 Ca CO32 Add tap water and bring to 1℃.

The above-inoculated growth medium contained in a 25'O Erlenmeyer flask was cultured at 30°C for 24 hours with circular shaking at a speed of 25 Orpm on a shaker with a circular motion of 2 inches in diameter. did.

(B) Streptomysis aureofaciens NR
To prepare a tank-intoxicated inoculum of A-28086 using RL5758, 10 τβ of the growth culture cultured as described above was added to 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°C flask and incubated at 30°C for 24 hours at 250 rpm on a 2 inch diameter arc motion orbital shaker.
It was cultured in

This second growth culture solution was sterilized at 1°C with the following composition. Production medium 100/! It was used to inoculate.

Ingredients Skewers (o/1
Tapioca dextrin 60.0 Enzymatically hydrolyzed casein 8.0 Molasses
15. OM(l SOa ・7H20
0.5 Ca CO3 2.0 Refined soybean oil 5.0182 yen Make 12 with water. .

This medium was heated to 120°C for 30°C at a pressure of 15-20 Bonds.
I)l-1 after pressure sterilization for minutes was 6.7.

The production medium adjusted to this temperature and inoculated with bacteria was fermented for 10 days at a temperature of 29° C. in a 165-liter fermentation tank. During this time, sterile air was aerated through the fermentation solution at a rate of 0.4 volume of culture solution per minute. The culture solution was stirred at a rate of 25 Orpm using an ordinary stirrer.

Example 2 Example 1 except that a flask culture medium with the following composition was used.
Δ-28086 antibiotic was produced according to the method of .

Thoughts-m-” [l-yu”] rattan Lm grapes sugar 1
0 edible molasses 20 pep 1~n
5Ca CO32 Add tap water to 1J! , make it.

Example 3 Sl-1 Noptomysis aureofaciens NRR
Isolation of A-28086 Antibiotic Complex Produced by L575B Fermentation Liquor 132 Obtained by the Method Described in Example 1
The remaining part is filtered with a filter aid (Hyflo Super Cell, a type of diatomaceous earth, manufactured by John Zuman Building Manufacturing Company).
A filtrate 97A 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 into a 5007β foil. This concentrated ethyl acetate extract was added with an excess of petroleum ether (Skelly 3 oveF: approx. 10
1! , ) was added, and the resulting waste material was precipitated and separated. The filtrate after 11 minutes was evaporated under reduced pressure and A
6.9 g of -28086 antibiotic conjugate was obtained.

Next, the A-28086 antibiotic complex contained in the bacterial cell part is extracted by extracting the filtered bacterial body twice with about half the amount of methanol (6
2°C and 59°C). After combining these two methanol hb extracts, methanol was concentrated and removed under reduced pressure. Approximately 101 after this concentration! , an aqueous layer remained. The aqueous layer was adjusted to about 7.5 liters with dilute sodium hydroxide solution. The solution thus obtained was extracted twice (9°C and 10°C) with approximately the same amount of ethyl acetate. The ethyl acetate extracts were combined and concentrated into approximately 400 cod skewers. 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 compound content of A-28086 antibiotics obtained from the bacterial cells in the filtrate is 2.
It was 0.6 CI.

113-Example 4 Isolation of Individual Factors A and B in A-28086 A-28086 antibiotic conjugate (wJ made as described in Example 3) 235CI was dissolved in benzene of approximately 80 cod. did. Silica gel column chromatography was applied to this benzene solution (9×1300I11,8/,
Matheson grade 62 silica gel). The column was eluted with benzene-ethyl acetate mixtures in various proportions. , the elution behavior was followed by thin layer chromatography.

Using benzene-ethyl acetate (90:10) as the solvent system, factor B eluted first and was isolated as one of the individual factors.

Factor 13 (43mc+) was crystallized from acetone-water.

mp150-153℃.

When elution was continued with a benzene-ethyl acetate mixture while gradually increasing the proportion of ethyl acetate, the factor was eluted. The various fractions containing factor Δ were combined under reduced pressure in v:J
Dissolve the residue left behind in about 150 pieces of acetone, and add about 150 pieces of fishing rod to this acetone solution. Add 1N to the solution thus obtained.
The concentration was adjusted to IIH3 by adding hydrochloric acid. The mixed solution with an acid concentration of 171 was stirred for about 111 hours. However, a precipitate forms between the yi. This precipitate was separated by filtration to obtain approximately 150 yxE
It was dissolved in water and recrystallized by adding 60πβ of Honsuri. B1. The formed crystals were vacuum dried overnight and the factor A was about 6.6Q to 19. Partial evaporation of 7L71~ from the crystal filtrate yielded a secondary II! of factor A! Approximately 1.2 q of 1 inch was cut by 1 wa.

Example 5 Al? of A-28086 factor △? Preparation of Der Nisder Derivatives - Antibiotic Δ-28086 Factor A7.4 (I Pyridine 1
50 τβ of acetic anhydride was added to this solution. This solution was thoroughly diluted (111) and then left at room temperature overnight. 200 g 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 19 solid was thus dissolved in 100[tau][beta] of aluminum, and the solution of 19 was then evaporated to dryness under vacuum (this operation was repeated three times). The IC residue obtained in this manner was crystallized from 100 drops of acetone and 50 drops of water, giving Δ-2808
Alledel ester derivatives of Factor 6 A 6.14. (I is 1
! ? Ta. Melting point: 100-103°C.

Examples 6-9 Reaction of propionic anhydride to factor in the presence of pyridine according to the method of Example 5 and the antibiotic Δ-28086
Ladle out the propionyl ester derivative of factor Δ. F.A.D.
Point j: 96-98°C.

Following the method of Example 5, Factor A was reacted with n-butyric anhydride in the presence of pyridine to obtain the butyryl-Nisder derivative to the antibiotic Factor A-28086. Melting point 79-8
1°C0 According to the method of Example 5, a caproyl ester derivative of the antibiotic Δ-28086 factor Δ was prepared by reacting factor A with caproic anhydride in the presence of pyridine. Melting point 163-167°C.

Following the method of Example 5, Factor A was reacted with valeric anhydride in the presence of pyridine to form the antibiotic Δ-28086 Factor A.
An n-valeryl ester derivative of was prepared. Melting point 17
3-175℃0 Example 10 Preparation of Δ-28086 factor Δ no s 1 ~ RI salt: Antibiotic y1Δ-28086 convict Δ500 m(I was dissolved in acemine 5071β, IC6 was added to this solution, water 50πβ was added, Furthermore, add 5N sodium hydroxide solution to
1l-1 was set 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. Remove the residue 1~
378,111 g of the Δ-28086 prisoner Δnos1-lium salt was obtained by precipitation from a solution of water. Melting point 120-12
3°C6 Examples 11-15 Using the method of Example 10-C1 antibiotic Δ-28086
From factor A (500 mg) and saturated barium hydroxide solution, barium salt 3 of anti-A1] product A-28086 factor Δ
69m (I was prepared. Melting point 188-190°C.

-117- Using the method of Example 10, 363 mg of Δ-28086 factor A potassium J-rich was prepared from antibiotic A-28086 factor (500 mg) and 5N potassium hydroxide solution. Melting point 165-167°C.

Using the method of Example 10, 540 mg of factor A cesium salt of A-28086 was obtained from 500 mg of antibiotic A-28086 prisoner Δ500 mg of cesium hydroxide. Melting point 190~
210℃.

By the method of Example 10, antibiotic A-2808 was prepared from antibiotic A-28086 factor B and 5N sthorium hydroxide.
The sodium salt of Factor 6 B was prepared.

Example 16 Streptomysis aureofaciens (3, au
Shake Flask Fermentation of Δ-28086 Using S. reofaciens) N RRL 8092 A culture of Streptomysis aureofaciens NRRL 8092 was prepared on an agar slant with the following composition.

戊-111卤一 i工1m118- に21-]PO4,2 MCl SO4・71-12 0 0
．． 25NHaNO32 Ca COa 2.5
Fe SOa ・71-120 0.
001Mn C/! 2 ・71-120
0.001Zn SOa ・7 l-1200
,001 grape sugar
10 cold weather
Add 20 deionized water and go to U12.

pl-1 (regulatory zezu) 7.7? J' Aneil, this slope Jt'? Jl! Streptomysis aureofaciens NRRIJ'1
092 was inoculated, and the columnar medium was cultured at 30°C for about 7 [-1]. A suspension of spores and hyphae was prepared from the slant culture by rubbing it with sterilized platinum while protecting the slant culture with sterile beef wipes. The resulting suspension was freeze-dried to prepare six pellets.

One of the freeze-dried pellets thus prepared was used to inoculate growth medium 50711β having the following composition.

8-111 Rikiichi Fan''' - Grape
sugar 20 soy beans
Flour 15 corn
Suedeve Liquor 10 CaC○32 Add tap water to make 1 nin.

pl-( was adjusted to 6.5 with dilute 1-1a○1".

The inoculated growth medium in 250πβ Erlenmeyer flasks was incubated at 30° C. for 48 hours at a speed of 25 Orpm on a 2-inch arc motion orbital shaker.

Next, 50πg of a fermentation medium having the following composition was inoculated with the medium in which the above bacterial cells had grown (0.5yn1., i percent).

Ingredients p-tapioca deni 1-strin* 60. Casein hydrolyzed with Oa enzyme※※6. Casein hydrolyzate by Of+enzyme※※※2.0aCa CO32,O(I M!II SO7+ ・7H200,5(] Waste sugar
Honey 15.0g Refined soybean oil 5.0 Add tap water and 1!
pH (unadjusted) 6.6 *5taley 1) exlrin # 11 (A,
E, 5taleyCo, (Deca Tools, Illinois, USA)). ※※A+nt)cr El-IC(Am
l+er laL+oratories (US 13i
1 (manufactured by Junikou, Wisconsin). ※※※NZ
Am1ne A(SI+eNield Cll0
IIICal Co.

(Made in Norway, New York, USA).

Example 17 Streptomysis A-leofaciens NRRL8
Example 16 as a shake flask fermentation method of Nnee A-28086 from tank using Δ-28086 using 092
The method described in the introduction was also applied in the case of tank fermentation. Growth medium 1 in which bacteria were cultured to make a large amount of inoculated bacteria
0ffβ was used to inoculate the next stage growth medium 400τβ with the same composition as the first growth medium. 2! This second stage culture in a 121-volume Erlenmeyer flask was placed on a shaker rotating in a 2 inch arc and incubated at 30°C for 24 hours at 25 Orpm. Secondary growth medium 8 in which this bacterium was cultured
00π℃ is a sterile fermentation medium with the following composition: 100℃
It was used to inoculate.

Tadashi --- Ichi Rikiichi Scolding Tapioca Dextrin * 60. Casein hydrolyzed with OO/ffi enzyme※※6.0 (1/λ casein hydrolyzed product※※※2.Og/ACaC○3
2.0(1/jZMgSO4・71-
120 0. '5 g/A molasses 15.00/6 Refined soybean oil 5.0 mg/, +2 Add tap water and bring to 1°C.

*5taley Dextrin #l 1 (A,
E, 5ta-lcy Co (Dekatool, Illinois, USA).

※※AmberEI-IC (Amber (-abo
Ratories (located in June, Wisconsin, USA). ※※※NZ Am1ne A (Sh
effield Chemical Co.

(Made in Norwich, New York, USA).

122- After autoclaving at 121°C for 30 minutes at a pressure of 15-20 Bonds, the pH of this medium was 6.8±0°1.

The production medium inoculated with bacteria was fermented at 28±1°C for 10-12 days in a fermentation tank at 165°C. The fermentation medium also contains 0.00% of the culture solution per minute. Sterile air was vented at a rate of 1/1 volume. This culture solution was stirred at a ratio of 300 ppm using a Sotong stirrer.

Example 18 A-280 was produced according to the method of Example 17 except using a shake flask or tank production medium with the following composition:
86 antibiotics were produced.

Ingredient Amount (()/
1 tapioca dextrin* 30.0 grapes
Sugar 15.0 Enzymatically hydrolyzed casein※※ 3.0 Enzymatic casein thawed product※※
*1.0 yeast extract 2.5C
a CO32.0 M(] SOL ・7H201.0 Waste molasses 15.0 Refined soybean oil
5.0 is /I! .

Add tap water to make 111.

*5taley Dextrin No. 11 (A
, E.

Manufactured by Staley Co., Decatur, Illinois, USA). ※※A amberE@C(A amber
manufactured by L Laboratories (located in June, Rice Consin, USA). ※※※NZ Δm1ne △
(Shcffield Chemical Co, (
(located in Norwich, New York, USA) ￥J) 6 Example 17
As described above, the M of the medium after sterilization by the pressure method was 6°4.

Example 19 Streptomysis aureofaciens NRRL8
Isolation of the A-,28086 antibiotic conjugate produced by 092 Total fermentation broth 6 obtained by the method described in Example 17
0 liter of dilute HCl was added to adjust the concentration to I)l-(3).The resulting solution was treated with filter aid (Hv-flo 3).
upper-cel, a type of diatomaceous earth, Johns-M
an-ville Products Corp.). 301 separated mycelial masses! , and 1.56 kg of Na HCOa was added to the extract.
was added while stirring. After separating this extract, the mycelia were further extracted again with methanol at 30°C. Both methanol extracts were combined and concentrated in vacuo to remove methanol. Residual aqueous solution approximately 71! , with diluted HC1
Adjusted to H7.5. The solution thus obtained was extracted twice with ethyl acetate γλ. The ethyl acetate extracts were combined and concentrated in vacuo to give an oily residue. This oily residue was dissolved in 1,500 ml of Aletone, and 1,500 ml of water was added to the acetone solution. The solution created in this way is @+ H
The mixture was adjusted to 1-13 with Cl and stirred for 1 hour. The resulting precipitate was filtered and then dissolved in 1,500 g of Aretone, and 400 yxe of water was added to this solution.

The resulting solution was left for 16 hours to allow crystals to precipitate out. When the formed crystals were filtered and vacuum dried,
A crude crystalline product 74 containing A-28086 particles A and D and other crystalline impurities was obtained.

The crude crystalline product 40(+) was dissolved in approximately 2501 β of benzene. The benzene solution was then subjected to silica gel column chromatography (9-×120-cm column using Grace-O Avidson grade 62 silica gel). The column was sequentially eluted with 4 OIl of each of the following solvents.

1) Benzene 2) Benzene: Ethyl acetate (9: 1) 3)
(4:1)4)
(7:3)5)
(1:1) 6) Ethyl acetate 7) Methanol Fractions of 1°C were collected from these. 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 is benzene:ethyl acetate (4:
1) was eluted. A-28086 particle B was eluted with benzene:ethyl acetate (7:126-3). Δ-28086 factors A and D were eluted in the fraction (119 to 156) mixed with benzene:ethyl acetate (7:3 and 1:1). The parts were combined and evaporated to dryness under vacuum. Thus 1
The washed residue was dissolved in 500 g of acetone. 500 g of water was added to this acetic acid solution, and the solution was adjusted to pl-13 with diluted 1" C1 and stirred for 1 hour. The formed precipitate was separated by filtration and mixed with 500πβ of acetone and 1 part of water.
Crystallized from ROmx.

The crystals thus formed were filtered and dried under vacuum.A-
20.1 g of a mixture of 28086 factors A and D were obtained.

Example 20 Separation of simple substances of factors Δa3 and D and crystalline mixture 1 of factors A and D of A-28086 obtained in Example 18
8.80 was dissolved in 50 ml of benzene. This benzine solution was transferred to silica gel column Chroma 1 using a graph method (7-X10
0-awl column: E, Merck grade 60 silica gel, finer than 230 mesh (ASTM)) was applied. The column was eluted sequentially with the following solvents at a flow rate of 90° per hour.

1) Benzene 12L 2) Benzene:ethyl acetate (9:1) mixture 12℃ 3) Benzene:ethyl acetate ('l:1) 1 compound 12℃ /1) Benzene:ethyl acetate (7:3) U compound 12° C. 5) Methanol 1 (1) Bioautograph method with B. subtilis was carried out with HE cellulose cellulose (Merck armstaclt cellulose, on aluminum support plate) to monitor the elution progress. The solvent system used was as follows. Water: Methanol: Aton (1
2:3:1), the solution is first brought to pH 10.5 with N Ha OI-1 and then adjusted to r1H7.5 with HCI.

The samples were collected at 1 to 2°C until activity was detected, and thereafter, 200 samples were collected at a time. The portions containing only factor A-28086 were combined and vacuum distilled to obtain a residue.

This residue was crystallized from acetone-water (1:1). Separate the crystals and vacuum dry to obtain crystalline Δ-28086-2.
A total of 140 8086 factors When a part containing A-28086 factor mixed with a slight amount of A-28086 factor A is processed in the same manner, crystalline A-2808 containing a roughly small amount of A-28086 factor A is obtained.
150II1g of 6-factor was obtained.

A portion containing only A-28086 convict Δ was treated in the same manner to obtain 4.7 g of crystalline A-28086 factor.

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 .

[-1-L1fit±Ll Beef extract 2.00 Dextrin
20.00 yeast extract
2.00 Enzymatic casein thawing product 4.00129
- Co C12・6H200,02 Seiten 20. ○1 with deionized water
℃.

p)-1 was adjusted to 7.0 with KOH.

In this case, the slope planted with bacteria was cultured 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 antibiotics were produced.

Ingredients and (a/
jll tapioca dextrin * 80 Enzyme hydrolyzed casein * * 15.0 Waste sugar
Dense 15.0 Calcium carbonate 2.0 Ammonium sulfate
1.0 Magnesium Sulfate
0.5 refined soybean oil 4.6*3t
aley[)extrin#11 (A, E,
5taleyCO0 (located in Decatur, Illinois, USA)
manufactured by). ※※１３０− △mber E l−I C＜ A amber l
aborator! (manufactured by 0S (located in Juniko, Wisconsin, USA)).

Example 23 A Δ-28086 antibiotic was produced according to the method of Example 21, except that a medium with the following composition was used as the intermediate growth medium for the third stage.

Feeling quantity (q
/ff1)-cererose 20.0
]-Nsdeep Liquor 10.0 Ground Soybean 15. Oca C0132,0 yeast
2.0 to 11 vegetable molasses 5.0 Example 24 Production of chick crow feed N''I improved with Δ-28086 for the purpose of suppressing coccidiosis; raising chicks so that the number of chicks increases rapidly A well-balanced, high-performance feed suitable for the following 41 combinations is created.

Composition Ingredients %Yu Pond Higiwayu Yellow 1 to Corn 50 1,00
0. Soybean flour (finely crushed soybean pods removed and extracted with solution A:, contains 50% protein) 31.09 G2
1.8・Animal fat (beef tallow) 6.5
130・Dried fishmeal (contains soluble matter, protein content 160%) 5.0 100・
Soluble substances in distillation residue of corn whiskey 4,0 80・Dibasic phosphate (for feed) 1.8 313
・Calcium carbonate 0.8 1G・
Vitamin premix (consisting of vitamins △, D, E, and B10, choline, niacin, pantothenic acid, riboflavin, biotin, and bukkake as a bulking agent). 0.5 10・None (multi-salt premix (Mn SO4, Zn ○, Kl, "e SOa
, Ca CO3). 0.24・2-amino-
4-Hydroxybutyric acid (hydroxylated analogue of medionine) 0.1 2・A
-28086 Factor A O,010,2 These materials are mixed according to standard feed J'11 compounding techniques. There are no restrictions on the amount of water administered. J4 raised on this diet are protected from coccidiosis infection, increase in body mass, and compete against coccidiosis-free chicks raised on a similar diet without the drug.

Example 25 Preparation of A-28086 improved beef cattle feed: A balanced high performance beef cattle feed is prepared as follows.

Blend Ingredients %-Pond/Finely Ground Tomato/Umoro]shi 67.8 135 (i・1
~ Ground Corn Cob Powder 10 200133- - Dry coarse powder of purple alfalfa (75 white matter content > 17%) 5100 - Crude powder of dehulled soybean (extracted with solvent, protein content > 50%) 9.9956 199
．． 912・ Sweet cane molasses
5 100,0・Urea
0.6 12.0-A-28086 factor A
O,00440,0118・Diphosphate lime (
(for feed) 0.5 10.0・Carbonate lime 0.5 10.
0. Sodium chloride 0.3 6.0.
Premix containing trace amounts of inorganic salts 0.03 0.6・
Vitamin A (13 and D2 premix* 0.07 1.4・
Vitamin premix※※0.05 1.0・Lime propionate 0.15 3.0※Content per pound: 2. ○oo, ooo international units of vitamin Δ, 227,200 international units of vitamin D2 and 1
Soybean feed supplemented with 385.7% oil.

134- ※※1- When making corn whiskey, the grains are dried by adding 110 soluble matter containing 20,000 international units of d-α-tocof ■lilu acetate per pound of grains.

The mixed fodder (", compressed to a small base. At a rate of 15 bonds per animal [an average of 1'!), this feed will feed 100 animals per day. Approximately 30Qm per
It is necessary to calculate the Δ-28086 factor Δ of g as (B).

Example 26 Δ-28086 Prisoner A [? Preparation of Delicate Sprue Derivatives: Dissolve antibiotic A-28086 in pyridine.

To this solution is added 81 tons of acetic anhydride.

Mix this solution thoroughly and leave it at room temperature for 2 hours.
Ru. Then water is added in excess and the mixture is allowed to stand at room temperature. The resulting precipitate is filtered off, washed with water and dried. The solid thus obtained was dissolved in acetone and evaporated to dryness under vacuum to obtain an acetyl ester derivative of A28086.

Examples 27-30 According to the method of Example 26, Δ-2, in the presence of pyridine
Propionyl ester derivatives of antibiotic Δ-28086 were prepared by reacting 80F36 with propionic anhydride.

A-28 in the presence of pyridine according to the method of Example 26
By reacting 086 koriko with anhydrous n-butyric acid,
An n-acetic acid ester derivative of antibiotic A-28086 was produced.

Δ-28 in the presence of pyridine by the method of Example 26
086 factor was reacted with caproic anhydride, and antibiotic Δ
An n-caproyl ester derivative of -28086 was produced.

A-2 in the presence of pyridine according to the method of Example 26
By reacting 8086 confectionery with valeric anhydride, the antibiotic Δ~
A leaf valeryl ester derivative containing 28086 factors was produced.

Example 31 Preparation of Δ-28086 Rinosu salt: Antibiotic A-28086 Rinosu is dissolved in acetone. Add equal amounts of water to the solution and add enough 5N sodium hydroxide to bring the rlH of the solution to about 11. The resulting solution is stirred for about 1 hour and then extracted with ethyl acetate. Evaporating the ethyl acetate under vacuum yields the thorium salt of the Δ-28086 factor.

Examples 32-34 A-28086 factor 5N using the method of Example 31
The potassium salt of antibiotic A-28086 factor was prepared from potassium hydroxide.

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

A-28086 factor 1N using the method of Example 31
Hydroxide 1? A cesium salt of the antibiotic Δ-28086 factor was prepared from Cium.

Example 35 Production of chicken feed containing the A-28086 factor aimed at suppressing coccidiosis - Achieving a balance suitable for raising chickens so that their weight increases rapidly High-performance feed is prepared using the following formulation.

Shi-Color-L-LL LL Yellow Corn Flour 50 1,000 Minced Soybeans (solvent extracted, skin removed, finely ground, protein content 50%) 31.09 B
21.8 Animal fat (beef tallow) 6.5
130 Dried fish powder (contains soluble matter, protein content 60%) 5.0 100
Soluble in distillation residue of corn whiskey /1.0 80 Calcium diphosphate (for feed) 1.8 36 Calcium carbonate 0.8 16 Vitamin premix (vitamins A, D, E, and Bt2, choline, Niacin, pantothenic acid, riboflavin, and 138-pyrotin are listed.

The thickening agent is glucose). 0.510 Inorganic salts (Mn So, 1, Zno, K 11Fc SO, +, CaC03) 0.2 42
-Amino-4-hydroxybutyric acid (hydroxylated analogue of methionine) 0.1 2A
-28086 factor [) 0,01 0,2
These substances are mixed under the heading f1) -C according to the feed formulation technique. There are no restrictions on the amount of water administered. Single chicks fed this diet are well protected from coccidiosis infection, and their weight gain is comparable to that of chicks fed a similar diet without the drug, which has never been affected by coccidiosis.

Example 36 Feeding beef cattle containing A-28086 convict! Production of fl A balanced high grain feed for beef cattle is produced as follows.

10-%-pound finely ground corn 67.8 1356 ground corn cob 10 200 dried coarse powder of alfalfa (17% protein content) 5 100 coarse dehulled soybean powder (solvent extracted, Protein 50%) 9,9956 19
9.912 Sweet cane molasses
5 100 urea
0,6 12Δ-28086 factor [) 0,0044 0.088 Calcium diphosphate (for feed) 0.5 10.0
Calcium carbonate 0.5 10.0 Sodium chloride 0.3 6.0 Trace inorganic salts 0.03 0.6 Vitamin A and D2* 0.07 1.4 Vitamin E**
0.05 1.0 Lime propionate
0,15 3,0 *Include ffi per bond
:2.000.000 international units of vitamin A, 227.
Soybean feed 3B5.70゜ with 200 international levels of vitamin D2 and 1% oil added *※20,000 international units of d-α-tocopheryl acetate per 1 bond of grain during the production of corn whiskey ! Dry the grains by adding soluble substances including:

The mixed feed is compressed into small pieces. 15 per animal
If we take pounds of feed as a percentage of the average daily intake, this feed is approximately 300,111 (of) A-280 per animal.
This means that it supplies 86 causes.

[Brief explanation of drawings]

The drawings are all infrared absorption spectra of the antibiotic A-28086 of the present invention, and Figure 1 shows the antibiotic A-28086.
-28086 factor A1 Figure 2 is antibiotic A-28086
Factor B, Figure 3 is an acetyl ester derivative of antibiotic A-28086 factor A, Figure 4 is antibiotic Δ-28086
Propionyl ester M conductor of factor A, Fig. 5 shows the butyl-Riesdel derivative of antibiotic A-28086 factor A, Fig. 6 shows valeryl ni 1 of antibiotic A-28086 factor A.
/11- Stell derivative, Figure 7 shows antibiotic Δ-28086 factor octacaproyl ester derivative, Figure 8 shows antibiotic A-28
086 factor. Patent Applicant: Eli Lilly & Company Agent Patent Attorney: 142- 142 - Continuation of Page 1 Priority Claim April 21, 1975 ■ United States (US )■
569740 ■April 21, 1975■United States (US)■569719 (l Author: Marvin Martin Hoehn, 4975 East 79th Street, Indianapolis, Indiana, United States of America) Author: Walter Mitsuo Nakatsukasa, Indiana, United States of America No. 2118 Crossman Drive, Indianapolis, Procedural Amendment, Kao. ■, Indication of the case 1982 Patent Application No. 067673 ``52 Name of Invention Animal Growth Promoter 3 Person Who Makes Amendment Relationship to the Case Patent Applicant Name: Eli Lilly & Company 4 Agents Toki Cano @-?

Claims (1)

[Claims] , 1. Antibiotic A consisting of factor Δ, factor B and factor
-28086 complex, the factor A, the factor B1, or any combination of these factors as essential ingredients.