ANTIBIOTIC 6270, PROCESS FOR ITS PRODUCTION, AND ITS USE AS AN ANTICOCCIDIOSIS AGENT AND A FEED ADDITIVE

Abstract: An antibiotic 6270 having the following physicochemical properties and a salt thereof: (1) Colorless needle-like crystals; acidic substance (2) Melting point: 115 - 118°C (3) Elementary analysis (Found %): C: 62.72%, H: 9.49%; O: 27.78% (4) Specific rotation: [α] 20 / D - 11.5° (C 1.0, methanol) (5) Ultraviolet absorption spectrum: The maximum absorption bands not observed at 210 nm or more, as measured in the methanol solution (6) Characteristic absorption (cm⁻¹) in the infrared absorp­ tion spectrum (taken with the potassium bromide tablet): 3470, 2890, 2945, 2980, 1735, 1460, 1380, 1315, 1165, 1102, 1075, 987, 980 (7) Solubility: Soluble in methanol ethanol, ethyl acetate, chloroform, ether, acetone, and benzene; insoluble in water (8) Color reactions: Positive in the vanilline-sulfuric acid reaction and the Dragendorff reaction; negative in the ninhydrin reaction; colors with I₂ gas

Description: ANTIBIOTIC 6270, PROCESS FOR ITS PRODUCTION, AND ITS USE AS AN ANTICOCCIDIOSIS AGENT AND A FEED ADDITIVE The present invention relates to a novel antibiotic 6270, a process for its production, and its use as an anticoccidiosis agent or as a feed additive to accelerate the growth of domestic animals or fowls. The present inventors have isolated a number of microorganisms from various soils for the purpose of searching new antibiotics and studied the antibiotics produced by the isolated microorganisms. As a result, they have found that when a microorganism belonging to the genus Nocardiopsis isolated from soil collected in Abashiri city, Hokkaido, Japan, is cultured in a proper culture medium, an antibiotic having a high antibacterial activity against gram positive bacteria is accumulated in the culture medium. This antibiotic was isolated and examined for the physicochemical properties and biological properties, whereby it has been confirmed to be a new antibiotic and named an antibiotic 6270. The present invention is based on this discovery, and provides the new antibiotic 6270 and a process for its production hL0k crises culturing an antibiotic 6270-producing microorganism belonging to the genus Nocardiopsis and isolating the antibiotic 6270 from the culture product. Further, the present invention provides an anticoccidiosis agent which contains the antibiotic 6270 as the active ingredient, and a method for preventing and curing coccidiosis of domestic fowls and animals which comprises administering an effective amount of the antibiotic 6270 to the domestic fowls and animals. Furthermore, the present invention provides a growth accelerating and feed efficiency increasing agent for domestic animals and fowls, which comprises the antibiotic 6270 as the effective ingredient, and a method for accelerating the growth of domestic animals and fowls and increasing the feed efficiency thereof, which comprises administering an effective amount of the antibiotic 6270 to the animals and fowls. Accordingly, the present invention provides a feed of domestic animals and fowls, which contains an effective amount of the antibiotic 6270. The effective amount in this respect is usually from 0.5 to 200 ppm,-preferably from 1 to 100 ppm. Now, the present invention will be described in detail with reference to the preferred embodiments. In the accompanying drawings, Figure 1 is the infrared absorption spectrum of the antibiotic 6270 as measured by means of potassium bromide tablet. Figure 2 is the 1H-NMR spectrum of the antibiotic 6270 as measured in heavy chloroform by using TMS as internal standard. Figure 3 is the 13C-NMR spectrum of the antibiotic 6270 as measured in heavy chloroform by using TMS as internal standard. The microorganism which may be employed for the production of the antibiotic 6270, may be any microorganism belonging to genus Nocardioosis so long as it is capable of producing the antibiotic 6270. However, it is preferred to employ Nocardiosis sp. 6270 strain isolated afresh by the present inventors (deposited in Fermentation Research Institute of Japan as FERM BP-717). The Nocardiosis sp. 6270 strain used in the present invention has branched mycelia, and aerial mycelia extending in air are fragmented to constitute long spore chains. From the hydrolyzate of the whole cells, meso-diaminopimeric acid, and galactose and ribose as sugar components are detected. However, arabinose and madurose are not detected. Further, mycolic acid is not detected. The menaquinone composition comprises MK-9 (H6) and (H8) as the main components and MK-9 (H4) was detected as a trace component. The width of substratal mycelium is about 0.5 pm. From the foregoing characteristics, the 6270 strain is determined to be a strain belonging to genus Nocardiopsis among Actinomyces. The Nocardiopsis sp. 6270 strain has the following microbiological properties. I. Morphological characteristics The substratal mycelia are well developed and branched, whereby the width of each mycelium is about 0.4 u=. Aerial mycelia grow well in some culture media such as a sucrose-nitrate agar culture medium (Difco. Czapeck's Solution Agar), a starch agar culture medium (ISP-4, Difco-Inorganic Salt Starch Agar), an oatmeal agar culture medium (ISP-8) or a glucose-asparagine agar culture medium. The aerial mycelia are straight or curved, and they constitute a cobweb as a whole. When matured, the aerial mycelia have a width of from 0.4 to 0.6 err;, Upon expiration of 10 days from the initiation of the cultivation, the aerial mycelia undergo fragmentation and form long spore chains each comprising at least 10 spores. The spore chains are not spiral. The surface of the spores are smooth, and the shape is cylindrical. The size is about 0.4 x 1.2 Vm. No sporangium, sclerotium or synnema was observed. II. Cultural characteristics The tests were conducted in accordance with the test method reported by E.B. Shirling et al. (International Journal of Systematic Bacteriology, 16, 313 - 340 (1966)). Additionally, known culture media and test methods were employed. The color was determined under the standard light source of xenon lump by using Color Harmony Manual, 4th edition, 1958 as the color standards. When the corresponding color tab was found, it was shown by the common names first, and then the color tab code was shown in parenthesis. When the same color appeared repeatedly, only the color tab code was indicated. Unless otherwise specified, the following data represent the growth condition as cultured for three weeks at 280C on a agar plate culture medium. Item (a) indicates the growth; item (b) indicates the state of aerial mycelia; item (c) indicates a soluble pignent; and item (d) presents other characteristics. (1) Sucrose-nitrate culture medium (Difco-Czapeck's Solution Agar) a: Good, near gray (2ba) b: White, powdery c: None (2) Glucose-asparagine culture medium a: Good, light amber (3ic) to beige (3ge) b: Near gray (3ba) c: None (3) Glycerol-asparagine culture medium a: No good, 2ba b: Slightly forms c: None (4) Starch agar culture medium a: Excellent, 3ic to Shale (3ca) b: Powdery, white c: None d: Hydrolysis of starch: negative (5) Tyrosine aear culture media a: No good, 3ge b: None c: None (6) Nutrient agar medium (Difco.Bacto Nutrient Agar) a: No good, light wheat (2ea) b: None c: None (7) Yeas-malt extract agar culture medium a: Excellent, cracks, topaz (3ne) b: White c: None (8) Oatmeal culture medium a: Good, shell (2ca) b: 2ba c: None III. Physiological characteristics (1) Growth temperature range (Slanted starch agar culture medium, temperature gradient incubator): 6.5 - 380C (No growth observed below 50C or above 39.5 C) Optimum temperature: 270C - 380C (2) Liquefaction of glucose-peptone-gelatin stab culture: Positive (3) Hydrolysis of starch: Negative (4) Coagulation and peptonization skim milk: No coagulation, and substantial peptonization (370C) (5) Formation of melanine: ISP-tyrosine agar culture medium: Negative ISP-triptone-yeast extract broth culture medium: Negative ISP-peptone-yeast-iron agar culture medium: Negative (6) NaCl torelance: No growth at 4% or higher (7) Solubility of xanthin, hypoxanthin, adenin: Negative IV. Carbon source utilization (Pridham and Gottlieb basal medium) Positive: D-glucose, L-arabinose, D-xylose, salicin Weakly positive: D-fructose, sucrose Negative: Inositol, L-rhamnose, raffinose, D-mannitol The 6270 strain has been identified to be a strain belonging to Nocardiopsis Meyer 1976 among Actinomyces, since among the above-mentioned various characteristics, it forms long spore chains on the aerial mycelia, and the whole cell hydrolizate contains meso-diaminopimeric acid and galactose, but does not contain arabinose and madurose, and further no mycolic acid is detected. In carrying out the process of the present invention, the antibiotic 6270-producing microorganism may be ,cultured in accordance with the conventional methods for cultivation of known actinomyces. However, from the industrial viewpoint, it is advantageous to employ a method of cultivation with aeration-agitation. As the culture medium for the production, there may be employed materials which are co"=nnly used for the cultivation of actinomyces. Metaly, varicus carbon sources, nitrogen sources and organic or inorganic salts, as well as a defoaming agent, if necessary, may be used in a suitable combination. Fer insta3ce, as the carbon sources, there may be used glucose, stareh, glycerol, dextrin, sucrose and animal or vegetable oils. As the nitrogen sources, there may be employed soybean meal, corn steep liquor, wheat embryo, and asmonia. Further, if necessary, inorganic salts such as calcium carbonate, sodium chloride, potassium chloride, and phosphates may be added. It is also possible to add organic or inorganic salts having an actioa to assist the growth of the microorganism and thereby facilitate the production of the antibiotic 6270. The temperature for the cultivation is usually within a range of from 25 to 350C, preferably around 300C. The cultivation time may vary depending upon the various conditions. However, usually the accueul-atict of the produced antibiotic 6270 reaches to the maximum within from 72 to 148 hours. The antibiotic 6270 may be isolated from the culture medium and purified by utilizing its physicochemical properties by means of conventional methods such as a method of utilizing the difference in the solubility from impurities, a method of utilizing the difference in the adsorption to ion exchange resins or various adsorbing agents, a method of extracting with an organic solvent immiscible with water, or a proper combination of various means such as precipitation, removal of impurities, dialysis, drying and recrystallization. For instance, the antibiotic 6270 produced in the culture medium may preferably be recovered as follows. After addition of a filter aid such as diatomaceous earth or Radiolite 700, the culture broth is filtered, and the filtrate and the microbial cells are respectively extracted with suitable solvents such as ethyl acetate and acetone. Then, the cell extract and the filtrate extract are combined. The solvent is distilled off from this extract solution, whereby the antibiotic 6270 is obtained as crude crystals. In order to isolate the antibiotic 6270 from the crude crystals, the crude crystals are subjected to, for example, chromatography. The eluate is concentrated under reduced pressure, and the residue is dissolved in a suitable solvent such as ethyl acetate and then treated with a dilute hydrochloric acid. The solvent layer is concentrated, whereby the antibiotic 6270 crystallizes as free acid. In order to obtain a sodium salt, the abovementioned solvent layer treated with a dilute hdyrochloric acid solution, is treated with a dilute sodium carbonate solution, and then the solvent layer is concentrated, whereby the antibiotic 6270 is obtainable in the form of a sodium salt. The free acid-type or sodium salt-type antibiotic 6270 thus obtained can be recrystallized foa a suitable solvent such as n-hexaneethylacetate to obtain pure crystals. The free acid-type antibiotic 6270 thus obtained has the following properties: (1) Colorless needle-like crystals; acidic substance (2) Melting point: 115 - 1180C (3) Elementary analysis (Found %): C: 62.72%, H: 9.49%, O: 27.78% (4) Specific rotation: [&alpha;]D20-11.5 (C 1.0, methanol) (5) Ultraviolet absorption spectrum: The maximum absorption bands not observed at 210 nm or more, as measured in the methanol solution (6) Characteristic absorption (cm 1) in the infrared absorption spectrum (taken with the potassium bromide tablet): 3470, 2980, 2945, 2890, 1735, 1460, 1380, 1315, 1165, 1102, 1075, 987, 980 (The absorption spectrum is shown in Figure 1.) (7) Solubility: Soluble in methanol ethanol, ethyl acetate, chloroform, ether, acetone, and benzene; insoluble in water (8) Color reactions: Positive in the vanilline-sulfuric acid reaction and the Dragendorff reaction; negative in the ninhydrin reaction; colors with 12 gas (9) Thin layer chromatography (by means of Kieselgel GF254, manufactured by Merck Co.): Solvent system Rf value Chloroform-methanol (20:1) 0.49 n-Hexane-ethyl acetate (1:1) 0.08 Benzene-ethyl acetate (1:1) 0.04 Benzene-acetone (1:1) 0.53 Chloroform-acetone (2:1) 0.36 n-Hexane-ethyl acetate (1:3) 0.13 Ethyl acetate 0.22 (10) H-NMR spectrum: The presence of three methoxy groups was observed at bppm 3.39 (3H), 3.49 (6H), as measured at 100 MHz in heavy chloroform by using TMS as internal standard (The spectrum is shown in Figure 2.) (11) 13C-NMR spectrum: the results of the measurement at 25 MHz in heavy chloroform by using TMS as internal standard, are shown in Figure 3. According to this results, the total number of carbon atoms is 44. (12) The antibacterial spectrum is shown in Table 1. Table 1 EMI12.1 <tb> <SEP> Minimum <tb> <SEP> Test <SEP> microorganism <SEP> inhibitory <SEP> Culture <tb> <SEP> concentra- <SEP> medium <tb> <SEP> tion <SEP> (mcg/ml: <SEP> <tb> Bacillus <SEP> cereus <SEP> IAM-1729 <SEP> 1.56 <SEP> a <tb> Bacillus <SEP> circulans <SEP> IFO <SEP> 3329 <SEP> 1.56 <SEP> a <tb> Bacillus <SEP> subtilis <SEP> PCI <SEP> 219 <SEP> 6.25 <SEP> a <tb> Micrococcus <SEP> flavus <SEP> IFO <SEP> 3242 <SEP> 1.56 <SEP> a <tb> Sarcina <SEP> lutea <SEP> NIHJ <SEP> 3.13 <SEP> a <tb> Staphylococcus <SEP> aureus <SEP> FDA209P <SEP> JC-l <SEP> 6.25 <SEP> a <tb> Staphylococcus <SEP> aureus <SEP> (resistant <SEP> 6.25 <SEP> a <tb> to <SEP> penicillin, <SEP> streptomycin, <tb> chloramphenicol, <SEP> tetracycline <SEP> and <tb> kanamycin) <tb> Escherichia <SEP> coli <SEP> NIHJ <SEP> JC-2 <SEP> > 100 <SEP> a <tb> Escherichia <SEP> coli <SEP> (resistant <SEP> to <SEP> > 100 <SEP> a <tb> streptomycin, <SEP> kanamycin, <tb> Chloramphenicol, <SEP> and <SEP> tetracycline) <tb> Klebsiella <SEP> pneumonia <SEP> PCI <SEP> 602 <SEP> > 100 <SEP> a <tb> Proteus <SEP> vulgaris <SEP> OX-19 <SEP> > 100 <SEP> a <tb> Pseudomonas <SEP> aeruginosa <SEP> IFO-3756 <SEP> > 100 <SEP> a <tb> Mycobacterium <SEP> smegmatis <SEP> ATCC <SEP> 607 <SEP> 1.56 <SEP> b <tb> Mycobacterium <SEP> avium <SEP> LFO <SEP> 3153 <SEP> 1.56 <SEP> b <tb> Mycobacterium <SEP> phlei <SEP> NIHJ <SEP> 3.13 <SEP> b <tb> Penicillium <SEP> chrysogenum <SEP> Q-176 <SEP> > 100 <SEP> c <tb> Aspergillus <SEP> flavus <SEP> ATCC <SEP> 9643 <SEP> > 100 <SEP> c <tb> Cochliobolus <SEP> miyabeanus <SEP> 12.5 <SEP> c <tb> Alternaria <SEP> mari <SEP> # <SEP> 50 <SEP> c <tb> Candida <SEP> albicans <SEP> YU <SEP> 1200 <SEP> > 100 <SEP> c <tb> Saccharomyces <SEP> cerevisiae <SEP> Strain <SEP> 77 <SEP> > 100 <SEP> c <tb> Notes: a: Nutrient agar culture medium b: Glycerol nutrient agar culture medium c: Potato-sucrose agar culture medium From the foregoing physicochemical and biological characteristics, this substance has been confirmed to belong to a general class of antibiotics called polyethers. This substance is assumed to have three methoxy groups in a molecule from the 1H-NMR spectrum. As known antibiotics having three methoxy groups in one molecule, among the polyether antibiotics, there may be mentioned C20-12 (Japanese Unexamined Patent Publication No. 53919/ 1983), A-204B (Handbook of Microbiology 410, vol. 3, CRC Press), T-42082 (Japanese Unexamined Patent Publication No. 79730/1976), T-40517 (Japanese Unexamined Patent Publication No. 10581/1975), 38295 (Japanese Unexamined Patent Publication No. 125793/1976), No. 6016 (Japanese Unexamined Patent Publication No. 84576/1979), X-14868C (Japanese Unexamined Patent Publication No. 120696/1981), CP-47433 (Japanese Unexamined Patent Publication No. 154108/1982), 47434 (Japanese Unexamined Patent Publication No. 154187/1982) and LL-C23024B B (Japanese Unexamined Patent Publication No. 78598/1983). However, the 6270 antibiotic differs from these known substances in the characteristics such as the melting point, specific rotation, elementary analytical values or infrared absorption spectrum, and is apparently a new antibiotic. The antibiotic 6270 has particularly strong antibacterial effects against gram-positive bacteria, and is useful as an antibacterial agent. Further, it has anticoccidiosis, acaricidal and antiviral activities at a low concentration, and it is useful as anticoccidiosis agent of domestic fowls such as chicken, quail, duck, goose and turkey, and domestic animals such as cattle, swine, rabbit, buffalo, goat and sheep, as an agent for treating diarrhea of domestic animals, or as an agent to increase feed efficiency or to accelerate the growth of domestic animals and fowls. Now, the present invention will be described in further detail with reference to Examples. However, it should be under stood that the present invention is by no means restricted to these specific Examples. EXAMPLE 1 Nocardiopsis sp. 6270 strain (FERM BP-717) was inoculated to 1 liter of a culture medium (pH 6.0) comprising 6.0% of glucose, 2.0% of soybean meal, 1.0% of torula yeast and 0.5% of calcium carbonate, and cultivated at 300C for 48 hours. This culture broth was inoculated to 100 liters of a culture medium having the same composition as above, and cultured at 300C for 96 hours under stirring and aeration in a tank having a capacity of 200 liters. The rate of aeration was 100 liters per minute, and the rotation of the stirrer was 250 rpm. After addition of a filter aid (Radiolite 700, trademark) this culture broth was filtered to separate the filtrate and cells. Then, the filtrate was extracted with 40 liters of ethyl acetate, and the cells were extracted with 30 liters of acetone. The acetone extract of the cells was concentrated under reduced pressure to remove acetone, and extracted with 20 liters of ethyl acetate. This extract was combined with the extract from the filtrate, and the combined solution was concentrated under reduced pressure. The reside was adsorbed on a column cf 750 a of silica gel (Wako gel C-200, tradename) packed with cnloroform, and a mitre of chloroform and methanol (100 : 1) was passed through the column. The active fraction was concentrated under reduced pressure to dryness, whereby an oily residue was obtained. Then, the residue was dissolved in a small amount of acetone, and the solution was adsorbed on a column of Sephadex LH-20 filled with acetone, and eluted with acetone. The active fraction thereby obtained was concentrated to obtain crude crystals of the antibiotic 6270. The crystals were dissolved in ethyl acetate, and shaked with dilute hydrochloric acid. The ethyl acetate layer was concentrated under reduced pressure. The formed crystals are recrystallized from n-hexane-ethylacetate, whereby 23.4 g of free acid of the antibiotic 6270 was obtained as colorless needle-like crystals. The free acid of the antibiotic 6270 thus obtained exhibited the abovementioned physicochemical and biological characteristics. test Example 1: Antinoccidiesis activity Tested agent apd feed: The antibiotic 6270 was uniformly mixed with a perfect combination feed for chicks (manufactured by Oriental Yeast Co.) to obtain a predetermined concnetration. The feed thus obtained was freely taken by chikens from twe days before the inoculation of oocyst to the end of the test (-eight days after the infection). As a comparative agent, Monensin was used which is known to be an anticoccidiosis agent. Chikens usea: Chickens used in this test were healthy cocks of egg-laying fowl (Shever Starcross) which were 7 days old (9 days old when infected) and had been bred under the conditions of perfect prevention of coccidiosis infection. Every group had five chickens. Oocyst inoculated and the quantity of inoculation: The oocyst used for the infection was a sensitive strain of Eimeria tenella. Every chicken was inoculated to the group with full group cocysts (3 x 104), orally by using metal zonde. Judgement of effect: The effect of the agents was determined by the Anticoccidial Index (ACI) which was calculated by the following formula: ACI = (Relative increase in weight + Survival rate) - (Oocyst value + Disease value) 120 or less: Not effective as an anticoccidiosis agent. 160 or less: Slightly effective as an anticoccidiosis agent. 160-180: Moderately effective as an anticoccidiosis agent. 180 or higher: Extremely effective as an anticoccidiosis agent. (i) Reiative increase in weight At the end of the test, the increase in weight (i.e the weight at the end of the test - the weight at the time of infection) of each test group was measured and the relative increase in weight was calculated based on the weight of the control group (100) which were bred with the anticoccidiosis agent free feed and not inoculated. (ii) Oocyst index The number of oocyst in the caecum was counted on 8 days after the inoculation by homogenizing the intestinal canal. The oocyst index was defined as follows: The number of oocyst found in the intestinal canal The oocyst index 0.0 - 0.1 x 106 0 0.1 - 1.0 x 106 1 1.0 - 5.0 x 106 10 5.0 - 11.0 x lo6 20 > 11.0 x 106 40 (iii) Disease index of the intestinal canal The chickens tested were anatomized at the end of the test (8 days after the fection), and the intestinal canal was examined with the naked eye to determine the disease index. T e disease index was defined as follows and the disease value was defined ten times as many as the value of the disease index. 0, (-' The caecum is quite normal. If bleeding spot is found, (-) is changed to (+). 1, (+; The caecum is normal in shape. The content therein is slightly fluid and yellowish. Slight swelling is found partly on a mucous membrane of the caecum which becomes whitish. 2, (+c) The caecum is generally normal in shape. Swelling is found on the whole surface of a mucous membrane. No bleeding is found in the content. Mucus is slightly yellowish and faded. A few white spot-like necroses or bleeding spots are found in a mucous membrane. 3, (+++)The caecum is clearly withered and changed in shape, and is a little longer than the rectum. The content is quite abnormal and is often filled with coagulated blood or white-gray, cheese-like degenerated matter. The wall of the caecum is clearly swelled and easily broken and sometimes bleeding spots still remain. The disease reaches a basis of the caecum but not the rectum. 4, (++++) Withering and deformation of the caecum are remarkable. The caecum looks like a sausage in shape and is not longer than the rectum. The disease reaches almost one third or fourth of the rectum. Groups 1 to 3 were fed with the feed containing a predetermined concentration of the antibiotic 6270 from two days prior to the inoculation of oocyst. Group 4 was fed with the feed containing the antibiotic Monensin as a comparative agent. Group 5 is the infected non-treated control group inoculated with oocyst and fed with the basic feed containing no anticoccidiosis agent. Group 6 is a non-infected, non-treated control group. Table 2 EMI20.1 <SEP> Degres <SEP> of <SEP> bloody <SEP> excrement <SEP> Average <SEP> Desenso <SEP> index <SEP> of <SEP> the <tb> <SEP> Weight <SEP> Survival <SEP> number <SEP> of <SEP> Occysi <SEP> Intestinal <SEP> canal <SEP> Desense <SEP> A.C.I. <tb> Group <SEP> Antibiotic <SEP> Dose <SEP> increnso <SEP> rate <SEP> anocyst <SEP> in <SEP> the <SEP> inedx <SEP> value <tb> <SEP> 4 <SEP> days <SEP> 5 <SEP> days <SEP> 6 <SEP> days <SEP> 7 <SEP> days <SEP> 8 <SEP> days <SEP> (%) <SEP> intestinal <SEP> ++++ <SEP> +++ <SEP> ++ <SEP> + <SEP> <SEP> (ppm <SEP> ) <SEP> (%) <SEP> canal <tb> 1 <SEP> 6270 <SEP> 8.3 <SEP> 95.0 <SEP> + <SEP> +++ <SEP> +++ <SEP> + <SEP> - <SEP> 100 <SEP> 2.52 <SEP> x <SEP> 108 <SEP> 10 <SEP> 8 <SEP> 5 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 30 <SEP> 155.5 <tb> 2 <SEP> 6270 <SEP> 12.5 <SEP> 96.6 <SEP> - <SEP> ++ <SEP> + <SEP> - <SEP> - <SEP> 100 <SEP> 3.40 <SEP> x <SEP> 108 <SEP> 10 <SEP> 0 <SEP> 0 <SEP> 2 <SEP> 3 <SEP> 0 <SEP> 14 <SEP> 174.6 <tb> 3 <SEP> 6270 <SEP> 25 <SEP> 103.0 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> 100 <SEP> 1.4 <SEP> x <SEP> 103 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 3 <SEP> 2 <SEP> 0 <SEP> 137.0 <tb> 4 <SEP> Monensis <SEP> 80 <SEP> 101.4 <SEP> + <SEP> ++ <SEP> ++ <SEP> + <SEP> - <SEP> 100 <SEP> 7.03 <SEP> x <SEP> 106 <SEP> 20 <SEP> 0 <SEP> 1 <SEP> 4 <SEP> 0 <SEP> 0 <SEP> 22 <SEP> 159.4 <tb> 5 <SEP> Infected, <SEP> non- <SEP> 5 <SEP> 67.7 <SEP> ++ <SEP> ++++ <SEP> ++++ <SEP> + <SEP> - <SEP> 100 <SEP> 7.27 <SEP> x <SEP> 106 <SEP> 30 <SEP> 5 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 40 <SEP> 107.7 <tb> <SEP> treated <SEP> group <tb> 6 <SEP> Non-infected, <SEP> 0 <SEP> 100.0 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> 100 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 300.0 <tb> <SEP> non-trated <tb> <SEP> group <tb> *Degres of bloody excrement: - : No bloody excrement + : Less then 10% ++ : 10 30% +++ : 30-50% ++++ : More than 50% From these test results, it is evident that the antibiotic 6270 has an anticoccidiosis activity. Test Example 2: Feed efficiency test on swine Pigs used: Land race species Basic feed: a) From the initiation of the test to four weeks later 60% of grain (corn, wheat, barley), 15% of soybean cake, 15% of animal-based feed (skimmilk powder, fish powder), and 10% of others (enzyme, calcium carbonate, potassium phosphate, sodium chloride, etc.) b) From 4 to 12 weeks 78% of grain (corn, miro, wheat), 13% of soybean cake, 5% fish powder and 4% of others (calcium carbonate, calcium phosphate, sodium chloride, etc.) Method: Fifteen young pigs of 35 days old in an average were divided into three groups, each group consisting of five pigs, so that the average weight would be substantially equal. The antibiotic 6270 was added to the basic feed in an amount of 0, 12.5 and 25 ppm, respectively. And then the three groups of pigs were fed with the feeds, respecitvely, for 12 weeks. Then, the body weight and the feed consumption were measured. Further, the overall number of days in which the pigs sufferred from diarrhea during the period of four weeks from the initiation of the test, were counted. The results are shown in Table 3. Table 3 EMI22.1 <SEP> Amount <SEP> of <SEP> Average <SEP> weight <SEP> Average <SEP> weight <SEP> Average <SEP> amount <SEP> Feed <SEP> * <SEP> Improvement <SEP> of <SEP> ** <SEP> Overall <SEP> number <SEP> of <SEP> days <tb> Group <SEP> antibiotic <SEP> 6270 <SEP> at <SEP> the <SEP> initiation <SEP> increase <SEP> during <SEP> of <SEP> concentrated <SEP> demand <SEP> the <SEP> feed <SEP> demand <SEP> in <SEP> which <SEP> asch <SEP> group <tb> <SEP> added <SEP> to <SEP> the <SEP> of <SEP> the <SEP> test <SEP> the <SEP> period <SEP> feed <SEP> ingested <SEP> inedx <SEP> inedx <SEP> sufferred <SEP> from <SEP> diarrhos <tb> <SEP> feed <SEP> (ppm) <SEP> (kg) <SEP> (kg) <SEP> during <SEP> the <SEP> test <SEP> (%) <SEP> Overall <SEP> number <SEP> of <SEP> days <tb> <SEP> period <SEP> (Kg) <SEP> during <SEP> the <SEP> test <SEP> for <tb> <SEP> each <SEP> group <tb> 1 <SEP> 12.5 <SEP> 8.20 <SEP> 45.82 <SEP> 106.9 <SEP> 2.333 <SEP> 5.0 <SEP> 13/140 <tb> 2 <SEP> 25 <SEP> 8.18 <SEP> 48.30 <SEP> 110.1 <SEP> 2.280 <SEP> 7.2 <SEP> 5/140 <tb> 3 <SEP> 0 <SEP> 8.23 <SEP> 43.73 <SEP> 107.4 <SEP> 2.456 <SEP> - <SEP> 48/140 <tb> * Feed demand index = ####### ###### ## #### ######## ###### ### #### ###### ** Improvement in the feed demand index (%) = (1- #### ###### ##### #### ## ########## ### ##### ) x 100 The feed demand index was improved by from 5 to 8% by feeding the feed containing the antibiotic 6270. The weight increase was improved by from 5 to 11%. Further, it was observed that diarrhea of pigs was substantially reduced. Test Example 3: Feed efficiency of ruminant Steers used: Holstein Concentrated feed: 71.5% of grain (corn, miro, barley), 11.5% of chaff and bran (corn gluten feed, wheat bran, rice bran oil cake), 5.5% of vegetable oil cake (soybean cake, linseed oil cake), and 11.5% of others (alphalpha meal, molasses, calcium phosphate, sodium chloride) Method: Fifteen castrated steers of 8 months old were divided into three groups, each consisting of five steers, so that the average body weight will-be substantially equal. The antibiotic 6270 was added to the basic concentrated feed in an amount of 0, 7.5 and 15 ppm, respectively. The three groups of steers were fed with the feeds, respectively. Further, dried rice straw was fed as crude feed in an amount of 1 kg per steer. The results are shown in Table 4. Table 4 EMI24.1 <SEP> Amount <SEP> of <SEP> Average <SEP> weight <SEP> Average <SEP> weight <SEP> Average <SEP> amount <SEP> Concentrated <SEP> * <SEP> Improvement <SEP> of <SEP> ** <tb> Group <SEP> antibiotic <SEP> 6270 <SEP> at <SEP> the <SEP> initiation <SEP> increase <SEP> during <SEP> of <SEP> concentrated <SEP> feed <SEP> demand <SEP> the <SEP> concentrated <tb> <SEP> added <SEP> to <SEP> the <SEP> of <SEP> the <SEP> test <SEP> the <SEP> period <SEP> feed <SEP> ingested <SEP> inedx <SEP> feed <SEP> demend <SEP> inedx <tb> <SEP> feed <SEP> (ppm) <SEP> (kg) <SEP> (kg) <SEP> during <SEP> the <SEP> test <SEP> (%) <tb> <SEP> period <SEP> (Kg) <tb> 1 <SEP> 7.5 <SEP> 320 <SEP> 308 <SEP> 2310 <SEP> 7.500 <SEP> 9.2 <tb> 2 <SEP> 15 <SEP> 318 <SEP> 310 <SEP> 2360 <SEP> 7.613 <SEP> 7.8 <tb> 3 <SEP> 0 <SEP> 323 <SEP> 281 <SEP> 2320 <SEP> 8.256 <SEP> * Concentrated feed = ####### ###### ## #### ######## ###### ### #### ###### ** Improvement of the = #### ##### ##### ##### ## ######### ### ##### ) x 100 The concentrated feed demand index was improved by from 7 to 10% by feeding the feed containing the antibiotic 6270. The weight increase was improved by from 9 to 11%.

Claims: CLAIMS: 1. An antibiotic 6270 having the following physicochemical properties and a salt thereof: (1) Colorless needle-like crystals; acidic substance (2) Melting point: 115 - 1180C (3) Elementary analysis (Found %): C: 62.72%, H: 9.49%; 0: 27.78% (4) Specific rotation: 50 - 11.5 (C 1.0, methanol) (4) (5) Ultraviolet absorption spectrum: The maximum absorption bands not observed at 210 nm or more, as measured in the methanol solution (6) Characteristic absorption (cm 1) in the infrared absorption spectrum (taken with the potassium bromide tablet): 3470, 2980, 2945, 2890, 1735, 1460, 1380, 1315, 116S, 1102, 1075, 987, 980 (7) Solubility: Soluble in methanol ethanol, ethyl acetate, chloroform, ether, acetone, and benzene; insoluble in water (8) Color reactions: Positive in the vanilline-sulfuric acid reaction and the Dragendorff reaction; negative in the ninhydrin reaction; colors with 12 gas (9) Thin layer chromatography (by means of Kieselgel GF254, manufactured by Merck Co.): Solvent system Rf value Chloroform-methanol (20:1) 0.49 n-Hexane-ethyl acetate (1:1) 0.08 Benzene-ethyl acetate (1:1) 0.04 Benzene-acetone (1:1) 0.53 Chloroform-acetone (2:1) 0.36 n-Hexane-ethyl acetate (13) 0.13 Ethyl acetate 0.22 (10) H-NMR spectrum: The presence of three methoxy groups was observed at bppm 3.39 (3H), 3.49 (6H), as measured at 100 MHz in heavy chloroform by using TMS as internal standard. 2. A process for producing the antibiotic 6270 as defined in Claim 1, which comprises culturing an antibiotic 6270-producing microorganism belonging to the genus Nocardiopsis and isolating the antibiotic 6270 from the culture product. 3. The process according to Claim 2, wherein the microorganism is FERM BP-717. 4. An anticoccidiosis agent comprising the antibiotic 6270 as defined in Claim 1, as an effective ingredient. 5. A method for prevention and cure of coccidiosis of domestic fowls and animals which comprises administering an effective amount of the antibiotic 6270 as defined in Claim 1 to the domestic fowls and animals. 6. A growth accelerating and feed efficiency increasing agent for domestic animals and fowls comprising the antibiotic 6270 as defined in Claim 1 as an effective ingredient. 7. A method for accelerating the growth of domestic animals and fowls and increasing the feed efficiency thereof which comprises administering an effective amount of the antibiotic 6270 as defined in Claim 1 to the animals and fowls. 8. A feed of domestic fowls and animals comprising the antibiotic 6270 as defined in Claim I in an amount effective against coccidiosis of the fowls and animals. 9. The feed of Claim 9, wherein the antibiotic 6270 is contained in a concentration of about 0.5 to 200 ppm. 10. A feed of domestic animals or fowls comprising the antibiotic 6270 as defined in Claim 1 in an amount effective in accelerating the growth of the animals or fowls and the increase in feed efficiency. 11. The feed of Claim 11, wherein the antibiotic 6270 is contained in a concentration of about 0.5 to 200 ppm. 12. The microorganism Nocardiopsis sp. 6270, FERM BP-717.

Inventors: IWAGAYA, YASUO  KAKEN PHARMACEUTICAL CO. LTD., KUSAKABE, YOKO KAKEN PHARMACEUTICAL CO. LTD., SEINO, AKIO KAKEN PHARMACEUTICAL CO. LTD., TAKAHASHI, NOBUKO KAKEN PHARMACEUTICAL CO. LTD., Iwagaya, Yasuo Kaken Pharmaceutical Co. Ltd.
Applicants: KAKEN PHARMACEUTICAL CO., LTD.
Application year: 1985
Main classification: C07G11
IPC codes: C12P1, A61P33, A23K1, C12P17, A61P31, C07D493, C12R1, A61K35, A61P3, C07G11, A61K31