FIELD OF THE INVENTION 
The present invention relates to a new antibiotic 5057B substance and a 
process for production thereof. 
DESCRIPTION OF THE PRIOR ART 
The present inventors previously found that an actinomycete Streptomyces 
sp. 5057 strain belonging to the genus Streptomyces (FERM BP-62) produces 
a substance having an antibacterial activity in its culture broth and 
isolated an antibiotic 5057(A) from the culture broth (refer to the 
specification of Japanese Patent Publication No. 8641/1979). 
The present inventors, in the course of further researches, have found that 
a new substance different from the 5057A substance is present in said 
culture broth and isolated the same, which was designated as the 5057B 
substance. 
The 5057B substance, in view of its physicochemical and biological 
properties, is an antibiotic belonging to the antibiotic group of 
polyether series. Among antibiotics of polyether series, there are 
heretofore known, as those showing ultraviolet absorption in the vicinity 
of 290 nm, lysocellin (The Journal of Antibiotics, Vol. 28, Pages 118-121, 
1975) and the 5057A substance (Japanese Patent Publication No. 8641/1979); 
however, the 5057B substance is a new antibiotic which differs from the 
above-mentioned known antibiotics in the melting point, specific rotation, 
infrared absorption spectrum, and the Rf value on silica gel thin layer 
chromatography.

DISCLOSURE OF THE INVENTION 
The present invention relates to a new antibiotic 5057B substance having a 
potent antibacterial activity, especially against gram-positive bacteria, 
and a process for production thereof comprising cultivating a 
microoragnism, belonging to the genus Streptomyces, which has the ability 
to produce the 5057B substance and then recovering the 5057B substance 
from the culture broth. 
The strain Streptomyces sp. 5057, belonging to the genus Streptomyces, 
employed in the production of the 5057B substance was deposited on May 1, 
1981 with Fermentation Research Institute, Agency of Industrial Science 
and Technology (No. 1-3, Yatabechohigashi 1-chome, Tsukuba District, 
Ibaragi Prefecture, Japan) and accorded the accession number FERM BP-62. 
Microbiological properties of the strain is described in detail in the 
specification of Japanese Patent Publication No. 8641/1979. 
In order to obtain the 5057B substance according to the present invention, 
ordinary methods for cultivation of actinomycetes can be employed, but a 
cultivation with aeration-agitation is suited for the industrial 
production. The cultivation temperature of 25.degree.-35.degree. C. is 
usual, but the temperature of 30.degree. C. is preferable. As a culture 
medium, there can be used one customarily employed for cultivation of 
microoragnisms belonging to the genus Streptomyces, for example, one 
containing carbon sources such as glucose, starch, glycerol, dextrin, 
sucrose, and animal or vegetable oils and nitrogen sources such as soybean 
meal, corn steep liquor, wheat embryo, and ammonia. Further, if necessary, 
inorganic salts such as calcium carbonate, sodium chloride, potassium 
chloride, and phosphates can be added; it is also possible to suitably add 
organic or inorganic salts having an action to assist the growth of the 
microoragnism and encourage the production of the 5057B substance. The 
accumulation of the 5057B substance produced reaches the maximum usually 
4-8 days after the start of the cultivation in both cases of shaking and 
tank cultures. 
The 5057B substance is recovered from the culture broth by utilizing its 
physiochemical properties; since the substance is lipo-soluble and acidic, 
there can be used, in a suitable combination, extraction with a variety of 
organic solvents, chromatography on a variety of activated adsorbents, and 
other methods. For example, an isolation method consisting of a 
combination of extraction and chromatography is carried out as follows: 
The culture broth, after addition of a filter aid such as diatomaceous 
earth and Radiolite 700 and the like, is filtered, and the filtrate and 
the microbial cells are each extracted with suitable solvents such as 
ethyl acetate and acetone. The cell extract and the filtrate extract are 
combined. Evaporation of the solvent from the combined extracts leaves, as 
the residue, crude crystals of a mixture of the 5057B and 5057A 
substances. The mixture is subjected to, for example, chromatography to 
isolate the 5057B substance from the mixture. The eluate is concentrated 
under reduced pressure and then the residue treated, in a suitable 
solvent, for example ethyl acetate, with a dilute hydrochloric acid 
solution and then with a dilute sodium carbonate solution and so on to 
afford crystals of sodium salt of the 5057B substance. Recrystallization 
of the crystals from a suitable solvent such as n-hexane-ethyl acetate 
gives pure sodium salt of the 5057B substance. 
Properties of the sodium salt of the 5057B substance thus obtained are as 
follows: 
(1) Colorless needles (the 5057B substance itself is acidic) 
(2) Melting point: 143.degree.-145.degree. C. 
(3) Elementary analysis (Found %): C, 59.96; H, 9.03; O, 27.60; Na, 3.41. 
(4) Specific rotation: [.alpha.].sub.D.sup.25 =+5.5.degree. (C 1, 
CHCl.sub.3) 
(5) Ultraviolet absorption spectrum: The absorption spectrum taken in a 
0.25% methanol solution is shown in FIG. 1. Maximum absorption: 
.lambda..sub.max.sup.MeOH (E.sub.1 cm.sup.1%) 290 nm (0.88) 
(6) Characteristic absorption (cm.sup.-1) in infrared absorption spectrum 
(taken with the potassium bromide tablet): 3480, 3000, 2970, 2812, 1719, 
1645, 1595, 1465, 1385, 1160, 1100, 1035, 980 The infrared absorption 
spectrum is shown in FIG. 2. 
(7) Nuclear magnetic resonance spectrum (taken in CDCl.sub.3) Proton NMR 
spectrum is shown in FIG. 3. 
(8) Solubility: soluble in methanol, ethanol, ethyl acetate, chloroform, 
ether, acetone, and benzene and so on; insoluble in water 
(9) Color reaction: positive in the 2,4-dinitrophenylhydrazine reaction; 
positive in the ninhydrin reaction and the vanillinesulfuric acid 
reaction; colors with I.sub.2 gas 
(10) Thin layer chromatography silica gel: Kieselgel GF.sub.254 
manufactured by Merck Co. 
______________________________________ 
Solvent system Rf value 
______________________________________ 
chloroform-methanol (20:1) 
0.30 
ethyl acetate-methanol (20:1) 
0.64 
n-hexane-ethyl acetate (1:2) 
0.62 
benzene-ethyl acetate (1:1) 
0.37 
______________________________________ 
(11) The antibacterial spectrum is shown in the following table: 
______________________________________ 
Minimum inhibitory 
concentration 
Test microrganism (mc.sup.g /ml) 
Medium 
______________________________________ 
Staphylococcus aureus FDA 209 PJC-1 
0.5 a 
Staphylococcus aureus 
resistant to penicillins, strepto- 
0.5 a 
mycin, kanamycin, chloramphenicol, 
and tetracyclines 
Bacillus subtilis ATCC-6636 
0.5 a 
Sarcina lutea NIHJ 2 a 
Micrococcus luteus ATCC-398 
0.5 a 
Mycobacterium smegmatis ATCC-607 
5 a 
Mycobacterium tuberculosis H.sub.37 R.sub.v 
10 b 
Escherichia coli NIHJ JC-2 
100 a 
Escherichia coli 
resistant to streptomycin, 
100 a 
kanamycin, chloramphenicol, and 
tetracyclines 
Klebsiella pneumoniae GN-6445 
100 a 
Proteus vulgaris GN-75 100 a 
Pseudomonas aeruginosa IFO-3756 
100 c 
Penicillium citrinum ATCC-9849 
100 c 
Aspergillus fumigatus NI-5561 
100 c 
Alternaria kikuchiana CBS 
100 c 
Ophiobolus miyabeanus ITO 
50 c 
Nocardia asteroides 5 d 
Candida albicans 50 d 
______________________________________ 
In the table, the sign a means the heart infusion agar medium, the sign b 
the Kirchner's semifluid agar medium, the sign c the potato-sucrose agar 
medium, and the sign d the Sabouraud's 3% glucose agar medium. As can be 
seen from the antibacterial spectrum, the 5057B substance exhibits a 
potent antibacterial activity especially against gram-positive 
microorganisms. 
(12) Acute toxicity test Acute toxicity tests were performed with mice. As 
a result, the LD.sub.50 value was 50 mg/kg in the case of the 
intraperitoneal administration and 500 mg/kg or more in the case of the 
oral adminstration. 
Melting points, specific rotations and Rf values on silica gel thin layer 
chromatography of the 5057A and 5057B substances are shown in the 
following table: 
______________________________________ 
5057A substance 
5057B substance 
(as the Na salt) 
(as the Na salt) 
______________________________________ 
Melting point 
133-135.degree. C. 
143-145.degree. C. 
Specific rotation 
+1.0 (C 1, MeOH) 
+5.5 (C 1, CHCl.sub.3) 
Rf value (1) 
0.56 0.62 
Rf value (2) 
0.28 0.37 
______________________________________ 
The solvent systems used for the silica gel chromatography were 
n-hexane-ethyl acetate (1:2) and benzene-ethyl acetate (1:1) in (1) and 
(2), respectively. 
The 5057B substance is useful as an antibacterial agent since it has an 
antibacterial activity. 
THE BEST MODE OF CARRYING OUT THE INVENTION 
The Streptomyces sp. 5057 strain (FERM-P No. 62) was inoculated into 1 l of 
a medium (pH 6.0) composed of soluble starch 6.0%, soybean meal 2.5%, beer 
yeast 0.5%, potassium primary phosphate 0.5%, ammonium sulfate 0.3%, and 
calcium carbonate 0.3% and cultivation was carried out at 30.degree. C. 
for 48 hours. The culture broth was inoculated into 100 ml of a medium 
having the same composition as set forth above and cultivation was carried 
out with stirring under aeration in a 200-l tank at 30.degree. C. for 96 
hours. The rate of aeration was 100 l/min and the rotation rate of the 250 
rpm. 
The culture broth, after addition of a filter aid (Radiolite 700: 
registered trademark of Showa Chemical Industry Co.) was filtered to 
seperate the filtrate and cells. The filtrate was extracted with 40 l of 
ethyl acetate and the cells with 30 l of acetone. The acetone extract of 
the cells, concentrated under reduced pressure to remove the acetone, was 
extracted with 20 l of ethyl acetate. The extract thus obtained was 
combined with the foregoing extract of the filtrate and the mixture 
concentrated under reduced pressure. The residue was adsorbed on a column 
in which 200 g of activated alumina had been packed with the use of ethyl 
acetate and a mixture of ethyl acetate-ethanol (1:1) passed through the 
column. Active fractions were concentrated to dryness under reduced 
pressure to give a mixture of the 5057B and 5057A substances in the state 
of crude crystals. The mixture thus obtained was applied onto a column in 
which 500 g of silica gel had been packed with the use of n-hexane-ethyl 
acetate (1:2) and the same solvent passed through the column. Fractions 
obtained were subjected to silica gel thin layer chromatography, which was 
developed with n-hexane-ethyl acetate (1:2). Only elute fractions 
containing the 5057B substance were collected and concentrated under 
reduced pressure. The 5057A substance was eluted after the 5057B substance 
had been eluted. The residue, dissolved in ethyl acetate, was shaken 
together with dilute hydrochloric acid and then the ethyl acetate layer 
shaken together with a dilute sodium carbonate solution. The ethyl acetate 
layer was then concentrated under reduced pressure. Crystals formed were 
recrystallized from n-hexane-ethyl acetate to give 0.85 g colorless 
needles of sodium salt of the 5057B substance. 
BRIEF DESCRIPTION OF THE DRAWINGS 
FIG. 1 shows ultraviolet absorption spectrum of sodium salt of the 5057B 
substance taken in a 0.25% methanol solution, FIG. 2 infrared absorption 
spectrum of sodium salt of the 5057B substance, and FIG. 3 proton NMR 
spectrum of sodium salt of the 5057B substance.