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.

No abstract

A method is presented for the estimation of penicillin G in broths by chromatographic adsorption. The method is specific for penicillin G, samples require no preliminary purification, and only 100 to 200 units of penicillin are required for an analysis. The application of aqueous chromatography to resolution of mixtures of the other known penicillins is described.

In a study of factors affecting the formation of ustilagic acid by Ustilago zeae (PRL 119) in submerged culture, yields of the acid ranging from 5 to 23 mgm. per ml. were obtained in three to five days, depending upon the concentration of medium constituents and the rate of aeration. The media employed contained cerelose, urea, corn steep liquor, and inorganic salts. Yields of ustilagic acid were usually between 8 and 15 mgm. per ml., corresponding to 12 to 23% conversion of the available glucose, on a carbon basis. Yields of the acid increased with increasing rates of aeration. Urea gave slightly higher yields than did the ammonium salts tested. Corn steep liquor concentration was optimal at 0.06%. Yields of ustilagic acid increased with increasing cerelose concentration, but the rate of conversion of carbohydrate into the acid decreased. About the same yields of ustilagic acid were obtained in shaken 500-ml. flasks, in nine-liter, and 30-liter fermentors.

ABS'I'RACT T h e t w o D-glucose residues in the t w o main components o f thc ustilagic acid mixture are shown t o be present as 0-D-cellobiopyranoside units.The glucoustilic acid mixture (6) was completely methylatecl by standa.1-d procedures for methylation and the product was subjected to methanolysis followed by hydrolysis to yield a mixture of methyl derivatives of D-glucose and the ustilic acids (7). The presence in the hydrolyzate of only tri-0-and tetra-0-methyl derivatives of D-glucose was established by paper partition chromatography. Senlimicro amounts of the two fractions were obtainecl by preparative paper partition chromatography (13). Since the fractions crystallized readily and completely, there could be no doubt that each fraction contained only one component. The compounds were characterized as 2,3,Gtri-0-methyl-D-glucose and 2,3,4,6-tetra-0-methyl-D-glucose by the preparation of suitable derivatives. Quantitative estimations showed the glucose derivatives to be present, within experimental error, in equimolar amounts. The mixture of partially methylated ustilic acids isolated f~-om the hydrolyzate possessed the conlposition expected for a mixture of mono-0-methylustilic acid A and di-0-methylustilic acid B. Therefore, both the ustilagic acids A and B possessed disaccharide units which were derived either from D-cellobiose or D-maltose.The A and B components of the glucoustilic acid inixture must be present in the same proportion as are the ustilic acids A and B in the ustilic acid mixture. The ustilic acid mixture has been shown (7) to contain about 70% ustilic acid A and about 30% ustilic acid B. The molar rotations for the mixtures listed in Table I were calculated on this basis. Thus, the possible presence in these mixtures of a small amount of impurities (8) was disregarded.Acetylation of methyl glucoustilate mixture yieldecl a crystalline lnixture of the acetyl derivatives with molar rotation -25,100 in chloroform. Several recrystallizations of this substance gave pure methyl octa-0-acetylglucoustilate A with molar rotation -25,200 in chloroform. Since the lactol carbon atoms of glucose derivatives make a strong contribution to molar rotation (about f 25,000) (4), these rotations show conclusively that the corresponding anomeric denters in the glucoustilic acids A and B are of the same configuration:The molar rotations of acetylated glycosides of same configuration derived from a sugar and optically inactive alcohols are well-known to be remarkably constant in value. On this basis, a conlparison of the molar rotation of the Alanuscript