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United States Patent 0 Fatented Apr 1 3,244,592 United States Patent 0 Fatented Apr. 5, 1966 1 2 Starch agar.-—Elevated growth. Vegetative mycelium, 3,244,592 ASCOMYCIN AND PRQCESS FQR 1T§ cream colored. Aerial mycelium, mouse-gray, powdery. PRGDUCTEGN The surface of the growth, mosaic of gray and black. Tadashi Arai, 1-71 Nogata-machi, Nalranaku, No soluble pigment. Tokyo-to, Japan Yeast extract nutrient agar.-—Growth, yellowish-brown, N0 Drawing. Filed May 1, 1963, Ser. No. 277,111 wrinkled, with cracks on its surface. Aerial mycelium, Claims priority, application Japan, .l‘une 9, 1962, poor, white, grows on the surroundings of colony. No 37/215,253 soluble pigment.‘ 4 Claims. (Cl. 167-65) Potato plug.—Growth, ?at, spread. Aerial mycelium, This invention relates to a new and useful substance white, cottony. The color of the plug is not changed. called ascomycin, and to its production. More particu Carrot plug.—Growth, cream-colored, spotted, with larly it relates to. processes for its production by fermenta~ subsided center. Aerial mycelium, abundant, white, cot tion and methods for its recovery and puri?cation. The tony. No color change of the plug. invention embraces this compound in dilute solutions, Litmus milk.-Thin surface growth, pale-cream colored. as crude concentrates and as puri?ed solids. Ascomycin Aerial mycelium, white. No soluble pigment. Coagu is an effective inhibitor of. ?lamentous fungi, e.g. Penicil lated from seventh day, digested gradually. lium chlysogenum; at very low concentrations, e.g. about Gelatin.—No pigment formation. strong lysis. one part per million in a nutrient agar, and does not Blood' again-Colony, grayish-brown, round shaped, inhibit various Gram-positive and Gram-negative bac with subsided center. No aerial mycelium. No soluble teria even at much. higher concentrations. pigment. Strong hemolysis. There is now‘provided, according to the present inven Cellulose medium.—Grows in synthetic culture solu tion, a new antifungal substance identi?ed as ascomycin tion‘ containing cellulose as a sole carbon source. which is produced by a process which comprises cultivat Starch hydr0lysis.-—Strong. ing a strain of Streptomyces hygroscop‘icus var. arcomy Nitrate reduction.—-Negative. ceticus, and preferably A.T.C.C. 14891 or an isolate, Sugar utilization using Pridham’s synthetic media con mutant, descendant or variant thereof, in an aqueous taining various sugars is as follows: carbohydrate solution containing an organic nitrogenous Glucose ++, Starch ++, Xylose +, Dextrin —|—+, nutrient under submerged aerobic conditions, e.g. at about Arabinose +, Mannitol +»—|-, Rhamnose ++, Sorbitol 26° C., for. at least twelve hours until substantial activity i, Fructose ++, Dulcitol —, Galactose ++, lnositol ——, against ?lamentous fungi, e.g. Aspergillus niger, is im 30 Saccharose ++, Adonitol -|—, Maltose ++, Salicin —, parted to said. solution and then, if desired, recovering Lactose +, Sodium acetate -|—, Ra?inose +, Sodium said ascomycin. citrate -—, lnulin —, Sodium succinate +. There is further provided, according to the present in In several media, aerial mycelia have a tendency to be vention, a process of- producing an antifungal agent, wetted. identi?ed as. ascomycin, which comprises cultivating The above-described morphological characters and Streptomyces hygroscopicus var. ascomyceticus under sub ?ndings on the identi?cation media are compared with merged aerobic conditions in an aqueous carbohydrate those described in Bergey’s Manual (Williams and Wil solution containing a nitrogenous nutrient for at least kins, 7th edition, 1957), Pridham’s Classi?cation Stan twelve hours and then, if desired, recovering the so-pro dard (Applied Microbiology, volume 6, pages 52-79, duced. ascomycin. from. the fermentation broth. 1958) and Waksman’s book (Actinomycetes, 2 volumes, ‘ Theworganisrn producing the ascomycin of the present 1961), and the classi?cation of this strain is investigated invention was isolated- from a sample of soil obtained in as follows: SannomiyaCity, Japan, and is a new variety, designated It is characteristic of this strain‘that its aerial mycelia ascomyceticus, of the-species Streptomyces hygroscopic-us. form spirals and it produces no speci?c soluble pigment A culture of the living organism, given the laboratory on any culture media including synthetic ones. designationKK 317, has. been deposited inthe American When only the colors of aerial mycelia and vegetative Type Culture Collection, Washington, D. C., and added mycelia on-synthetic'media are taken as important points, to its permanent collection or" micro-organisms as the resembling species, which have gray or black aerial A.T.C.C. 14891. mycelia and yellow or greenish yellow-brown vegetative With the color tones as de?ned by Maerz and Paulis ' mycelia, are found in the above-described classi?cation Dictionary of. Color, McGraw-Hill (1950 edition), the books. Streptomyces fungicidicus, Streptomyces albutr, representative strain of S. hygroscopicus var. ascomy Streptomyces niveus, Streptomyces platensis, Strepto‘myces ceticus has the following, characteristics: humz'dus, Streptomyces cacaoi, Streptomyces pseudogrise ]‘t/I0rphol0gy.—Aerial mycelia form 3-5 tight spirals on us, Streptomyces halstedii, Streptomyces rutgersensis and synthetic medium and on glucose-.asparagine agar. Spores 55 Sr'reptomyces hygroscopicus have resembling points. are ellipsoidal or small cylindrical, 1.6-2.0 x 1.0-1.2,u in Among these species, Streptomyces albus forms white size, and have smooth surface under electron-microscope. aerial mycelium on glucose asparagine agar, S. macro The growth on the identi?cation media for streptomyces spol'eus does not form aerial mycelium on the same species is. as follows: medium, and Streptomyces niveus forms cream-pink Glucose-Czapek medium.-—Growth, pale-lemon color aerial mycelia. Thus they are clearly different from to brownish, with rugged ‘surface (13L6). Aerial myceli the strain of the present invention. um, at ?rst‘white, later mouse-gray (7C7). Surface of the Streptomyces fungicidicus is different in that it pro colony turns to- black when touched with loop. Very duces yellow soluble pigment in certain media, and pro small amount of greenish-brown soluble pigment is pro duces dark brown pigment in potato plug. Streptomyces duced. pseudogriseus forms grayish brown aerial mycelia on Glucose - asparagine agar.-—Colony, pale yellowish green, round. Aerial mycelium, abundant, gray. Soluble synthetic media and differs from the present strain in its pigment, yellowish, scarce. color. Streptomyces halstedii and Streptomyces rutger Nutrient agar.—-Growth, pale greenish-brown, flat. The sensz‘s show different growth on potato plug but their surface iscovered with grayish aerial mycelia, like velvet. dark black color is characteristic. In this point they No soluble pigment. are differentiated from the strain of the present invention. 3,244,592 3 4 Thus, this strain has much resemblance to Streptomyces tion. Iron-hydroxamate reaction is positive-like. Nin platensl's, Streptomyces humidus, Streptomyces cacaoi hydrin, biuret, Molisch, Fehling, iron-perchlorate, 2,4 and Streptomyces hygroscopicus. Among them, Strep dinitrophenol and hydrazine reactions are all negative. It tomyces platensis and Streptomyces humidus belong to is stable when it is heated at 100° C. for one hour. The the Streptomyces hygroscopicus group, according to the ultraviolet absorption in ethanol, indicates only end-ab idea of the species-group in the modern classi?cation of sorption. Streptomyces. This is discussed in the paper of Tresner Ascomycin forms a single antibacterial spot of Rf 1.0 and Baccus (Applied Microbiology, vol. 4, pages 243— when it is paper-chromatographed using solvent systems 250, 1956) and also described in Waksman’s book. such as water-saturated butanol; 80% Phenol; butanol Thus, this strain is considered most properly to be 10 methanol-water (2:111); benzene-methanol (3:1); buta long to Streptomyces hygroscopicus when a hygroscopic nol-ethanol-water (221:1); 50% acetone, pyridine-buta tendency of aerial mycelium is also taken in considera nol-water (4:6:3). ‘ ' ' tion. The comparison of various characteristics of this The in vitro antibiotic activity of ascomycin is as strain and the standard strain of Slreptomyces hygro follows: scopicus also showed good resemblance. A (fermentation broth containing ascomycin is pre Gone. for Gone. for Test organism complete incomplete Culture, - pared by inoculating spores or mycelia of the ascomycin inhibition inhibition hours producing organism into a suitable medium and then (meg/ml.) (meg/ml.) cultivating under aerobic conditions. For the production of ascomycin, cultivation on a solid medium is possible, 20 Staphylococcus aureus 2091:‘ __________________ __ 24 but for production in large quantity cultivation in a Bacillus subtr'lz's (PC1219) 24 Escherichia coli (F1) ____ _- 24 liquid medium is preferable. The temperature of the Pseudomonas aelruginosa. 24 cultivation may be varied over a wide range, 20-35“ C., Salmonella enteritidis on nutrient agar at; 27° C_____ 24 within which the organism may grow but a temperature Candida krusel ____________ -_ 24 of about 27° C. and a neutral pH, i.e. pH 6.0—8.0, are Candida paralcrusei. _ 2A Candida nlbz'cans __________ __ 24 preferred. In the submerged aerobic fermentation of Cryptococcus neoformans_.__ 24 the organism for the production of ascomycin, the medi Trichophyton interdz'gz'tale__-_ 96 Trichophyton mentagrl» um contains as the source of carbon a commercially
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