Ether-soluble "oils" of specific gravity > 1 were produced extracellularly in yields of over 16 gm./liter fermentation mixture by strains of Ustilago zeae growing in shaken flasks on medium containing cerelose, urea, and sugar beet molasses. The bulk of the oily material was shown to be a glycoside of mannose and erythritol, and in addition, itaconic acid and dianthrone were shown to be present. Yields of itaconic acid as determined by a bromine–iodine method at pH 1.2 (Friedkin) reached values of over 15 gm./liter but such values were considerably higher than those indicated by quantitative isolation of this acid. One hundred and eighty isolates of Ustilago were grown on medium with and without calcium carbonate and some 45 isolates produced extracellular oily material, 98 produced ustilagic acid, and 50 produced both crystals and oil. Ether-soluble substances from freeze-dried fermentation mixtures of different isolates ranged from 1 to 12 gm./liter, while methanol-soluble substances from ether-extracted freeze-dried fermentation mixtures ranged from 1 to 45 gm./liter.

The antibiotic activity of Ustilago zeae (PRL 119) cultures is associated mainly with the culture solids and it has been shown that a mixture of glucolipids, called ustilagic acid and produced in good yields by the fungus, is responsible for part, if not all, of the observed activity. The antibiotic activity of ustilagic acid corresponds to that originally attributed to ustizeain B and therefore these two are considered to be identical. All samples of ustilagic acid obtained showed antibiotic activity and it has not been possible by paper chromatography to separate an active principle from the acid. Recrystallization of ustilagic acid did not alter its antibiotic properties. Alkaline hydrolysis of the acid destroyed its activity. Heating the culture mixture for up to an hour at temperatures less than 100° C. did not alter materially the antibiotic properties of the mixture or of the acid extracted therefrom. The antifungal spectrum is broad and the antibacterial spectrum includes more Gram-positive than Gram-negative organisms. Preliminary tests with rats indicated that ustilagic acid is nontoxic when administered orally up to at least 5% of the diet, and with mice, orally up to at least 1% of the diet or intraperitoneally in amounts up to 1.5 gm. per kgm. of body weight.

A water-soluble compound formed by Ustilago sp. (PRL 627) in aerobic, submerged culture has been identified as D-mannopyranosyl-1-meso-erythritol. The extracellular "oil" produced at the same time by this fungus contains D-mannose, meso-erythritol, acetic acid, and a number of saturated and unsaturated fatty acids, probably as a mixture of D-mannosido-meso-erythritol residues to which the various acids are joined by ester linkages.

T h e neetllelilie crystals ~i~l~i c h form in aerated submergecl c u l t~~r r s of the corn sn111t Uslilngo ecae (PIZL-119) \\;ere f o~~n c l t o be a 1)-glucolipid, m.p. 14G-i0C., [a]:+ 7' (pyriclinc), \~fhich contained one carbosyl group, two ester groups, a t least two terminal methyl groups, a n d two 11-glucose resitlues per ri~ole. On t h e basis of elementary a~ialyses, neutralization equivalent, a n d molecular iveight estimations, the molecular formllla of the material isolated \\,as approsirnately CjiHG?-aOli. T h e substance \\.as not shown t o be chemically honlogeneous.Conditions ~~s e d in c u l t~~r i n g the fungus ill stirred fermcntors to produce t h e crystalline nietabolic product, which was termed "~~s t i l a g i c acid", are describetl, and a procedure for the isolation of the material is given.Haskins ( I ) has shown that tlie corn smut Lirtilago zeae (PRL-119) can clissimilatc carbolij drates rapidly in aerated s~~brncrged cultures n-ith the procluction of a substa~ice which p~-ecipitatecl in the culture mixt~lre in the form of long neecllelike crystals. Since the c u l t~~r e mixture clisplayecl PI-onouncecl antibiotic activity ( I ) , our interest in the chemical nature of the substance was aroused.T h e conclitions usecl for the cultivatio~i of the fungus ancl the production of the material investigatecl in this work are descr-ibecl in the esperimc~ltal section. -4 culture n~eclium containing D-glucose, urea, corn steep liquor, and mineral salts buffered by calcium carbonate was usecl. T h e fungus was grown sirccessfully in 30-liter stainless steel fcrmentors.Tests sho\\~ecl the crystalline material which was cleposited in tlie culture meclium to be reaclily soluble in methanol, pyridine, 2,3-b~~tanecliol, ancl 1,2-PI-opanecliol, only spal-ingly soluble in ethanol, butanol, and acetone, and insoluble in water, glycerol, ethyl acetate, cliethyl ether, benzene, and petroleum ether. These remarlcable solubility 111-operties inclicatecl that the substance was moderately polar in che~kical nature and proviclecl means for its separation from the other culture solids.

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