# Patent

## Title
Determination of the acid-base status of blood

## Abstract

This is a division, of application Ser. No. 304,393, filed Sept. 21, 1981 now U.S. Pat. No. 4,427,776.

## Background
Antibiotic U-64,815 can be differentiated from the known related antibiotics kijanimicin [A. K. Mallams, et al., J. Amer. Chem. Soc. 103, 3938, 3940 (1981)], antlermicins [K. Kobinata, el al., J. Antibiotics 33, 244 (1980); K. Isono, el al., ibid., 33, 772 (1980)] and tetrocarcins [F. Tomita, et al., J. Antibiotics 33, 668 (1980); T. Tamaoki, et al., ibid., 33, 946 (1980)] on the basis of observed .sup.13 C magnetic resonance spectra. Compound 12 disclosed in the J. A. C. S. article, appears to be identical to antibiotic U-64,815. Final resolution of this identity is in progress.

Antibiotic U-64,815 is producible in a fermentation under controlled conditions using a biologically pure culture of the new microorganism Streptomyces microspinus, NRRL 12524.

Antibiotic U-64,815 has moderate to excellent in vitro activity against all strains of Haemophilus influenzae tested. It is also active against various Gram-positive bacteria. Further, the base addition salts of antibiotic U-64,815 are also active against these bacteria. Thus, antibiotic U-64,815 and its salts can be used to disinfect washed and stacked food utensils contaminated with S. aureus. They can also be used as disinfectants on various dental and medical equipment contaminated with S. aureus. Still further, antibiotic U-64,815 and its salts can be used as a bacteriostatic rinse for laundered clothes, and for impregnating papers and fabrics; and, they are also useful for suppressing the growth of sensitive organisms in plate assays and other microbiological media.

Chemical and Physical Properties of Antibiotic U-64,815: O.sub.18 --.sub.25.

Ultraviolet Absorption Spectrum:

The UV spectrum of antibiotic U-64,815 is shown in FIG. 2 of the drawings. The solution of antibiotic U-64,815 in ethanol displayed absorption as follows: Solvent       .lambda. max ______________________________________ Ethanol       215 240 267 278 (sh) .1 N HCl      213 in Ethanol    261 ______________________________________

Infrared Absorption Spectrum:

Antibiotic U-64,815 has a characteristic infrared absorption spectrum in a mineral oil mull as shown in FIG. 1 of the drawings. Peaks are observed at the following wave lengths: Band                     Band Freq. Inten.    Type     Freq.   Inten. Type ______________________________________ 3480.0 40        SH*      1196.9  43     BRD 3442.3 40        BRD      1170.9  18     AVG 3280.0 56        SH*      1149.7  18     SH 3058.5 67        SH       1141.9  17     AVG 3025.7 56        AVG      1127.5  10     AVG 2956.2 0         BRD M    1095.6  8      AVG 2931.1 0         BRD M    1059.0  6      AVG 2870.4 3         BRD M    1014.6  10     AVG 2854.0 2         AVG M    1000.2  14     AVG 2726.7 70        BRD M    981.8   14     AVG 2661.1 71        AVG      936.5   51     SH 2617.7 76        SH       927.8   43     SHP 1760.2 16        AVG      914.3   56     AVG 1729.3 19        AVG      897.0   56     SHP 1629.0 31        AVG      865.1   37     AVG 1575.0 37        SH*      831.4   55     AVG 1544.2 10        AVG      800.5   70     AVG 1510.4 43        AVG      779.3   55     AVG 1455.4 9         AVG M    730.1   51     AVG 1413.9 37        AVG      704.1   62     AVG 1378.3 12        AVG M    667.4   57     SH 1366.7 20        AVG      656.8   54     AVG 1346.4 26        AVG      619.2   54     SH 1311.7 25        AVG      607.6   50     AVG 1250.9 26        AVG 1220.1 31        AVG ______________________________________ Band Freq.: Band Frequencies in Wavenumbers (CM.sup.-1) Inten.: Intensity in Percent Transmittance (% T) Date Type in Local Peak Region: BRD = Broad; AVG = Average; SHP = Sharp; SH = Shoulder Peak List Edited. *Indicates Peaks Added. M: Possible Interference from Mineral Oil.

Antibiotic U-64,815 has a characteristic infrared absorption spectrum when pressed into a KBr pellet. Peaks are observed at the following wave lengths: Band                     Band Freq. Inten.    Type     Freq.  Inten.  Type ______________________________________ 3480.0 32        SH*      1170.9 27      AVG 3441.4 30        BRD      1141.0 26      AVG 3259.1 52        SH       1126.5 18      AVG 3029.5 55        AVG      1096.6 14      AVG 2971.6 31        AVG      1058.0 11      AVG 2931.1 16        AVG      1014.6 16      AVG 2911.8 24        SH       999.2  20      AVG 2852.0 36        SHP      981.8  21      AVG 2799.0 66        SH       937.5  55      AVG 2662.0 67        AVG      927.8  50      AVG 1761.2 25        AVG      913.4  60      SH 1734.2 27        AVG      897.0  59      AVG 1631.0 33        AVG      865.1  46      AVG I577.9 41        SH       843.9  60      AVG 1545.1 18        SHP      831.4  59      AVG 1508.5 43        AVG      800.5  67      AVG 1451.6 28        AVG      778.3  59      AVG 1434.2 39        SH       732.0  59      AVG 1413.9 42        AVG      706.0  65      AVG 1384.0 31        AVG      667.4  60      SH I382.1 31        SH       657.8  59      AVG 1368.6 34        AVG      618.2  61      SH 1346.4 36        AVG      607.6  58      AVG 1310.7 36        AVG      507.3  60      AVG 1249.0 39        BRD      489.0  59      AVG 1235.5 40        BRD      430.0  52      AVG* 1221.0 37        AVG ______________________________________ Band Freq.: Band Frequencies in Wavenumbers (CM.sup.-1) Inten.: Intensity in Percent Transmittance (% T) Date Type in Local Peak Region: BRD = Broad; AVG = Average; SHP = Sharp; SH = Shoulder Peak List Edited. *Indicates Peaks Added.

.sup.13 C-Nuclear Magnetic Resonance (NMR) Spectrum:

The .sup.13 C-NMR spectrum of antibiotic U-64,815 is shown in FIG. 3 of the drawings. The .sup.13 C-NMR spectrum was observed on a Varian XL-200 Spectrometer (50mHz) on a solution (ca. 0.05 ml., ca. 40 mg/ml) of the sample of the antibiotic in deutero-methanol. The spectrum was calibrated against internal tetramethylsilane and frequencies were recorded in ppm downfield from tetramethylsilane.

Solubilities:

Antibiotic U-64,815 is soluble in methanol acetone, methylene chloride, ethanol and dimethylsulfoxide. It is insoluble in water and hexane.

Antimicrobial Spectrum of Antibiotic U-64,815:

Antibiotic U-64,815 is active against various Gram-positive bacteria and H. influenzae as shown in the following table.

Assay:

The antibacterial assay is a standard disc plate assay. Ten 6.35 mm absorbent discs are saturated with a 1000 .mu.g/ml solution prepared in methanol (solvent) and air-dried prior to spotting on organism-seeded agar plates. The results are as follows: Zone Size (mm) ______________________________________ Haemophilus influenzae UC 6482   10 (Ampicillin-susceptible Haemophilus influenzae UC 6483   9 (Ampicillin-resistant) Streptococcus pyogenes UC 152    13 Streptococcus pneumoniae UC 41     16 - outer ring 10 - inner ring Staphylococcus aureus UC 6685   8 (Multiple antibiotic-resistant) Staphylococcus aureus UC 6690   8 - light (Multiple antibiotic-resistant) N. gonorrhoeae    UC 3065   16 ______________________________________

"UC" is a registered trademark of The Upjohn Company Culture Collection. These cultures can be obtained from The Upjohn Company in Kalamazoo, Mich., upon request.

The microorganism used for the production of antibiotic U-64,815 is a biologically pure culture of Streptomyces microspinus, NRRL 12524.

A subculture of this microorganism can be obtained from the permanent collection of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill., U.S.A. Its accession number in this depository is NRRL 12524. It should be understood that the availability of the culture does not constitute a license to practice the subject invention in derogation of patent rights granted with the subject instrument by governmental action.

The microorganism of this invention was studied and characterized by Alma Dietz of The Upjohn Research Laboratories.

Streptomyces microspinus, Dietz sp. nov. ["microspinus" denotes the small spines found on the surface of the spores], appeared most similar on Ektachrome (Table 1) to Streptomyces impexus NRRL 3809, which is described in Goldstein and Prokop [Goldstein, A. W. and J. F. Prokop. 1972. Antibiotic effective against gram-positive bacteria and method of preparation. U.S. Pat. No. 3,651,217], and Streptomyes fungicidicus ATCC.RTM.21013, which is described in Shibata et al. [Shibata, M., E. Higashide, K. Hatano, K. Mizuno, M. Asai, and M. Muroi. 1974. Antibiotic enduracidin and process for the production thereof. U.S. Pat. No. 3,786,142]. All the cultures have open spiral chains of spores with spiny surfaces. S. impexus and S. fungicidicus have abundant chains of flexuous spore chains as well as open-spiral spore chains. The spines on spores of these cultures are more readily apparent than those on spores of Streptomyces microspinus. All cultures are melanin-negative. All have gray aerial growth when the surface color is matched with the NBS color chips [SRM 2106. ISCC-NBS Centroid Color Charts. Office of Standard Reference Material, Room B311, Chem. Building, National Bureau of Standards, Washington, D. C. 20234], but only Streptomyces microspinus belongs to the Gray series of Tresner and Backus [Tresner, H. D., and E. J. Backus. 1963. System of color wheels for streptomycete taxonomy. Appl. Microbiol. 11:335-338]. Streptomyces microspinus is further distinguished from S. impexus and S. fungicidicus by its growth on carbon compounds (Table 3) in the synthetic medium of Shirling and Gottlieb [Shirling, E. B., and D. Gottlieb. 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16:313-340] and by its general cultural characteristics (Table 4). The new culture grows well in the temperature range of 18.degree.-55.degree. C.; the other cultures do not grow at 45.degree.-55.degree. C. The new culture produces the antibiotic U-64815; S. impexus produces M-411 and S. fungicidicus produces Enduracidin. It can also be differentiated from cultures in the Gray spiral, melanin-negative, spiny spore group in Bergey's Manual, 8th ed. [Pridham, T. G., and H. D. Tresner. 1974. Part 17. Actinomycetes and related organisms. Family VII. Streptomycetaceae Waksman and Henrici 1943. Genus I. Streptomyces Waksman and Henrici 1943. Table 17.42h of the Gray series. Pages 782-785 in Buchanan and Gibbons, eds., Rergey's Manual of Determinative Bacteriology, 8th ed. The Williams and Wilkins Co., Baltimore]. Therefore, it is proposed that Streptomyces microspinus be considered a new species of the actinomycete genus Streptomyces. It is understood that in accordance with the Rules of Nomenclature of Bacteria [Lapage, S. P., P. H. A. Sneath, E. F. Lessel, V. B .D. Skerman, H. P. R. Seeliger, and W. A. Clark, ed. 1975. International code of nomenclature of bacteria, 1976 Revision. American Society for Microbiology, Wash., D.C.], this is the type strain and that, should another strain be found, the type strain would also be the type subspecies.

Streptomyces microspinus Dietz sp. nov. NRRL 12524.

Color Characteristics: Aerial mycelium prednominantly gray. Melanin-negative. The color pattern of the culture on Ektachrome is given in Table 1. Reference color characteristics are given in Table 2. The culture may be placed in the Gray (GY) color series of Tresner and Backus [Tresner, H. D., and E. J. Backus, Ibid.].

Microscopic Characteristics: Spore chains are predominantly open-spiral. Spores are oval, 1.1.times.0.76 .mu.m. Many spores have depressions like those of red blood cells; others have a double collapsed appearance which gives a ridged appearance. The surface of the spores is covered with blunt spines. Spore surface detail is best determined at magnifications of 10,000 to 20,000 with the scanning electron microscope. Fourteen day cover glass preparations were used for the microscopic examinations. sucrose and Maltose-tryptone agars and at 18.degree.-45.degree. C. on Bennett's agar. __________________________________________________________________________ Color Characteristics* on Ektachrome** Deter- S. microspinus S. impexus S. fungicidicus mina- NRRL 12524 NRRL 3809 ATCC 21013 Agar Medium tion Chip Color  Chip Color Chip Color __________________________________________________________________________ Bennett's S   233 pale gray 32 grayish 70 light yellowish orange pink     yellow R   72 dark orange 70 light 70 light yellow    orange   orange yellow   yellow Czapek's S   233 pale gray 32 grayish 9 pink white sucrose                 yellowish pink R   32 grayish 32 graysish 9 pink white yellowish yellowish pink      pink Maltose- S   233 pale gray 31 pale yellow 7 pale pink tryptone                pink R   72 dark orange 67 brilliant 68 strong yellow    orange   orange yellow   yellow Peptone- S   71 moderate 32 grayish 71 moderate iron          orange    yellowish orange yellow    pink     yellow R   68 strong 68 strong 68 strong orange    orange   orange yellow    yellow   yellow 0.1%   S   32 grayish 32 grayish 7 pale pink Tyrosine      yellowish yellowish pink      pink R   38 dark   38 dark  38 dark reddish   reddish  reddish orange    orange   orange Casein S   233 pale gray 32 grayish 10 pinkish starch                  yellowish gray R   93 yellowish 76 light 93 yellowish gray      yellowish gray brown __________________________________________________________________________ S = Surface R = Reverse *Growth on media in tubes was photographed after seven days incubation at 28.degree. C. Color was determined by comparison with NBS color chips [SP 440. Color: Universal Language and Dictionary of Name. U.S. Government Printing Office, Washington, D.C. 20402; SRM 2106. Ibid.]. **Dietz, A. 1954. Ektachrome transparencies as aids in actinomycete classification. Ann. N.Y. Acad. Sci. 60:152-154 and Dietz, A. and D.W. Thayer (ed.). 1980. Actinomycetes Taxonomy (Procedures for Studying Aerobid Actinomycetes with Emphasis on the Streptomycetes). SIM Special Publication Number 6. Soc. for Ind. Microbiol. Arlington, VA. __________________________________________________________________________ Reference Color Characteristics* Deter- S. microspinus S. impexus S. fungicidicus Agar  mina- NRRL 12524 NRRL 3809  ATCC 21013 Medium tion Chip Color Chip Color   Chip Color __________________________________________________________________________ Bennett's S   266 dark gray 90 grayish 90 grayish yellow     yellow R   58 moderate 90 grayish 70 light orange brown    yellow     yellow P   71 moderate -- --      -- -- orange yellow Czapek's S   45 light gray- 63 light   63 light brown- sucrose      ish red- brownish   ish gray dish brown gray R   93 yellowish 73 pale orange 73 pale orange gray     yellow     yellow P   -- --    -- --      -- -- Maltose- S   266 dark gray 93 yellowish 93 yellowish tryptone              gray       gray R   57 light brown 71 moderate 71 moderate orange     orange yellow     yellow P   87 moderate 87 moderate 87 moderate yellow   yellow     yellow Yeast S   23 dark red- 22 reddish 63 light brown- extract-     dish gray gray       ish gray malt  R   57 light brown 71 moderate 71 moderate extract               orange     orange (ISP-2)               yellow     yellow P   71 moderate 87 moderate 87 moderate orange   yellow     yellow yellow Oatmeal S   45 light gray- 63 light brown- 63 light brown- (ISP-3)      ish red- ish gray   ish gray dish brown R   29 moderate 71 moderate 71 moderate yellowish orange     orange pink     yellow     yellow P   30 dark yell- 87 moderate 87 moderate owish pink yellow     yellow Inorganic S   23 dark red- 63 light brown- 22 reddish Salts        dish gray ish gray   gray Starch R   73 pale orange 71 moderate 71 moderate (ISP-4)      yellow   orange     orange yellow     yellow P   79 light gray- 80 grayish 80 grayish ish yell- yellowish  yellowish owish    brown      brown brown Glycerol S   266 dark gray 93 yellow gray 93 yellow gray asparagine R   57 light brown 71 moderate 71 moderate (ISP-5)               orange     orange yellow     yellow P   -- --    87 moderate 87 moderate yellow     yellow __________________________________________________________________________ S = Surface R = Reverse P = Pigment *Color determination was made on growth on plates incubated 14 days at 28.degree. C. Color was determined by comparison with NBS color chips [SP 440, Ibid. and SRM 2106, Ibid.]- ______________________________________ Growth on Carbon Compounds in the Synthetic Medium of Shirling and gottlieb* Synthetic Medium S. microspinus S. impexus S. fungicidicus (ISP-9)    NRRL 12524 NRRL 3089  ATCC 21013 ______________________________________ Negative Control --         --         -- (No carbon cpd.) Positive Contro1 +          ++         + D-glucose) L-arabinose ++         ++         ++ Sucrose    --         .+-.       .+-. D-xy1ose   ++         ++         ++ Inosito1   ++         ++         ++ D-mannitol ++         ++         ++ D-fructose +          ++         ++ Rhamnose   ++         ++         ++ Raffinose  --         --         -- Cellulose  --         --         -- ______________________________________ ++ = Strong utilization + = Positive utilization .+-. = Doubtful utilization - = No utilization *Shirling, E.B. et al., Ibid. __________________________________________________________________________ Culture Characteristics - General Deter- mina- S. microspinus S. impexus S. fungicidicus Agar Medium tion NRRL 12524 NRRL 3809 ATCC 21013 __________________________________________________________________________ Agar Peptone- S    pale gray white center; gray center; iron                  pink-tan edge white edge R    tan      light yellow- yellow tan P    --       --       -- O    melanin  melanin  melanin negative negative negative Calcium S    gray     gray     gray maleate R    pale gray gray     gray-yellow P    --       --       -- O    malate not malate not malate not solubilized solubilized solubilized Glucose- S    gray-pink yellow vegeta- cream-gray asparagine            growth R    pink-tan deep yellow deep yellow P    pale pink --       -- Skim milk S    brown vegeta- gray center; trace gray tive growth cream edge R    tan      orange   orange P    tan      orange   orange O    casein not casein solu- casein solu- solubilized bilized  bilized Tyrosine S    pale gray gray center; cream center; cream edge gray-white edge R    orange-tan orange-yellow orange-tan P    orange-tan pale orange orange-tan O    tyrosine tyrosine tyrosine solubilized solubilized solubilized Xanthine S    pale gray on gray cream cream edge; tan vegetative center R    yellow-cream orange   cream P    pale yellow --       pale yellow O    xanthine xanthine not xanthine not solubilized solubilized solubilized Nutrient S    blue-gray gray center; cream starch                cream edge R    yellow cream yellow   yellow P    pale yellow pale yellow pale yellow O    starch solu- starch par- starch par- bilized  tially solu- tially solu- bilized  bilized Yeast   S    flavender-gray gray-cream gray with cream extract-                       edge malt    R    orange-tan yellow-orange yellow extract               tan P    pale orange- pale yellow pale yellow tan Peptone- S    light gray- light gray- colorless vegeta- yeast        pink     white    tive with very extract-                       slight trace iron                           gray-white (ISP-6) R    yellow-tan yellow-tan yellow-tan P    yellow-tan yellow-tan yellow-tan O    melanin  melanin  melanin negative negative negative Tyrosine S    steel gray mottled gray- light steel gray (ISP-7)               yellow with yellow exudate R    light gray yellow-tan light yellow-gray P    --       trace yellow- -- tan O    melanin  melanin  melanin negative negative negative Broth Synthetic S    trace white trace white slight white nitrate      aerial on aerial on aerial on surface  surface  surface pellicle pellicle pellicle P    --       --       -- O    fine growth trace growth slight growth throughout throughout throughout flocculent flocculent flocculent growth at base growth at base growth at base growth color- growth color- growth color- less     less     less nitrates not nitrates nitrates reduced  reduced  reduced Nutrient S    gray white white aerial white aerial nitrate      aerial on on surface on surface surface  pellicle pellicle pellicle P    --       --       -- O    flocculent flocculent flocculent yellowish yellowish yellowish bottom growth bottom growth bottom growth nitrates not nitrates nitrates reduced  reduced  reduced Litmus  S    trace gray- pale gray- pale gray- milk         white aerial white aerial white aerial on blue- on surface on surface gray surface pellicle pellicle ring     blue-gray-tan blue-gray-tan surface ring surface ring P    --       --       -- O    litmus reduced litmus reduced litmus reduced pH 7.7   pH 7.6   pH 7.4 Gelatin Plain   S    trace tan trace tan trace tan vegetative vegetative vegetative growth at growth at growth at surface  surface  surface P    trace olive trace yellow trace yellow O    liquefaction liquefaction liquefaction complete complete complete Nutrient S    tan vegetative tan vegetative tan vegetative growth at growth at growth at surface  surface  surface P    --       tan      yellow O    liquefaction liquefaction liquefaction complete complete complete __________________________________________________________________________ S = Surface (aerial growth unless otherwise noted) R = Reverse P = Pigment O = Other Characteristics

The compound of the invention process is produced when the elaborating organism is grown in an aqueous nutrient medium under submerged aerobic conditions. It is to be understood, also, that for the preparation of limited amounts surface cultures and bottles can be employed. The organism is grown in a nutrient medium containing a carbon source, for example, an assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include cornsteep liquor, yeast, autolyzed brewer's yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, fish meal, distillers' solids, animal peptone liquors, meat and bone scraps, and the like. Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium, manganese, cobalt, iron, and the like, need not be added to the fermentation medium since tap water and unpurified ingredients are used as components of the medium prior to sterilization of the medium.

Production of the compound by the invention process can be effected at any temperature conducive to satisfactory growth of the microorganism, for example, between about 18.degree. and 40.degree. C., and preferably between about 20.degree. and 28.degree. C. Ordinarily, optimum production of the compound is obtained in about 3 to 15 days. The medium normally remains alkaline during the fermentation. The final pH is dependent, in part, on the buffers present, if any, and in part on the initial pH of the culture medium.

When growth is carried out in large vessels and tanks, it is preferable to use the vegetative form, rather than the spore form, of the microorganism for inoculation to avoid a pronounced lag in the production of the compound and the attendant inefficient utilization of the equipment. Accordingly, it is desirable to produce a vegetative inoculum in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil, liquid N.sub.2 agar plug, or a slant culture. When a young, active vegetative inoculum has thus been secured, it is transferred aseptically to large vessels or tanks. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilized for the production of the compound, so long as a good growth of the microorganism is obtained.

A variety of procedures can be employed in the isolation and purification of the compound produced by the subject invention from fermentation beers. Isolation can be accomplished by extraction with solvents such as methylene chloride, acetone, butanol, ethyl acetate and the like; and silica gel chromatography can be used to purify crude preparations of the antibiotic.

In a preferred recovery process, the compound produced by the subject process is recovered from the culture medium by separation of the mycelia and undissolved solids by conventional means, such as by filtration or centrifugation and solvent extraction of the filtered broth. The filtrate can be extracted with a solvent for antibiotic U-64,815, for example, methylene chloride, and the extract evaporated under reduced pressure to an aqueous concentrate. This preparation can be purified by chromatography on silica gel. The solvent system used for the chromatography is methylene chloride:methanol (95:5) (v/v) and (90:10). Antibiotic U-64,815 is shown at R.sub.f =0.50-0.55 silica gel G plates in MeCl.sub.2 /MeOH 9:1 using standard bioautography on Bacillus subtilis.

The antibiotic of the subject invention also can be recovered from the fermentation broth by resin sorption on a resin comprising a non-ionic macro porous copolymer of styrene cross linked with divinyl-benzene. Suitable resins are Amberlite XAD-2; XAD-4, and XAD-7, according to the procedure disclosed in U.S. Pat. No. 3,515,717. (Amberlite resins are available from Rohm and Haas, Philadelphia, PA). The antibiotic can be eluted from said resins by using acetone.

Salts of antibiotic U-64,815 also can be formed with inorganic or organic bases. Such salts can be prepared, as for example, by suspending antibiotic U-64,815 in water, adding a dilute base until the pH of the suspension is about 10.0 to 11.0, and freeze-drying to provide a dried residue consisting of the U-64,815 salt. Antibiotic U-64,815 salts with inorganic cations which can be formed include the sodium, potassium, and calcium salts. Other salts of U-64,815, including those with inorganic bases such as primary, secondary, and tertiary monoamines as well as with polyamines, also can be formed using the above-described or other commonly employed procedures. Other valuable salts are obtained with therapeutically effective bases which impart additional therapeutic effects thereto. Such bases are, for example the purine bases such as theophyllin, theobromine, caffeine, or derivatives of such purine bases; antihistaminic bases which are capable of forming salts with weak acids, pyridine compounds such as nicotinic acid amide, isonicotinic acid hydrazide, and the like; phenylalkylamines such as adrenaline, ephedrine, and the like; choline, and others. Salts of U-64,815 can be used for the same biological purposes as the parent compound.

The following examples are illustrative of the process and product of the invention, but are not to be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

A. Fermentation

A biologically pure culture of Streptomyces microspinus, NRRL 12524, is used to inoculate 500-ml Erlenmeyer seed flasks containing 100 ml of sterile medium consisting of the following ingredients: g/liter ______________________________________ Cerelose          25 Pharmamedia*      25 Deionized water q.s. 1 liter ______________________________________ *Pharmamedia is an industrial grade of cottonseed flour produced by Traders Oil Co, Fort Worth, TX.

The seed medium post-sterilization pH is .about.6.5. The seed inoculum is grown for three days at 28.degree. C. on a Gump rotary shaker operating at 250 rpm and having a 21/2 inch stroke.

Seed inoculum (100 ml), prepared as described above, is used to inoculate 500-ml fermentation flasks (Erlenmeyer) containing 100 ml of sterile fermentation medium consisting of the following ingredients: g/liter ______________________________________ Cerelose          10 Bacto-Peptone (Difco) 5 Black Strap Molasses* 20 Tap Water         Balance ______________________________________ *Supplied by Knappen Milling Co., Asugusta, MI.

The pH is adjusted to 7.2 with NaOH before autoclaving.

The inoculated fermentation medium is incubated at a temperature of 28.degree. C. for 5 days on a Gump rotary shaker operating at 250 rpm and having a 21/2 inch stroke.

A typical five-day fermentation has the following titers of antibiotic in the fermentation broth: Day  Assay, BU/ml ______________________________________ 2    8.0 3    >16 4    >16 5    >16 ______________________________________

The assay is a Bacillus subtilis disc plate assay using 0.1 M potassium phosphate buffer, pH 5.0 as diluent.

B. Recovery

Whole beer, from a fermentation as described above, is filtered and adjusted to neutral pH with HCl and filtered with 5% diatomaceous earth (wt/v). The clear beer is then defatted with two half volumes of Skellysolve B (isomeric hexanes). The defatted beer is then extracted with two half volumes of methylene chloride and these extracts are combined and reduced in volume. ##STR1## Clear Beer (1/2" dipped disc bioassay) Whole Beer Clear Beer Organism    Zone (mm)  Zone (mm) ______________________________________ B. subtilis 29         29 M. luteus   25         24 C. perfringens 28         28 Skellysolve B 1st     2nd       Combined Organism Extract Extract   Extracts Spent Beer ______________________________________ B. subtilis 0       0         0       28 M. luteus 0       0         0       23 C. perfringens 0       0         0       27 ______________________________________ Me.sub.2 Cl.sub.2 1st     2nd       Combined Organism Extract Extract   Extracts Spent Beer ______________________________________ B. subtilis 27      19        27      21 S. lutea 23       0        21      16 C. perfringens 27      18        27      20 ______________________________________

C. Purification

A 21.5 g sample of a CH.sub.2 Cl.sub.2 extract, prepared as described above, is dissolved in a minimal volume of CH.sub.2 Cl.sub.2 and passed onto the head of a 5' column packed with 1400 g of Merck Silica Gel 60 slurried in CH.sub.2 Cl.sub.2. A flow rate of ca. 7 ml/min is established with a solvent of CH.sub.2 Cl.sub.2 /MeOH; 90/5; 20 ml fractions are collected. After 8.7 liters, the solvent is changed to CH.sub.2 Cl.sub.2 /MeOH; 90/10.

Fractions are analyzed by thin layer chromatography (TLC) using Analtech Silica Gel GF plates run in CH.sub.2 Cl.sub.2 /MeOH, 9/1. The component of interest is first detected by quenching under U. V. 254 nm light followed by spraying with a p-anisaldehyde reagent (EtOH:H.sub.2 SO.sub.4 :Acetic Acid:p-anisaldehyde, 90:5:1:5) and heating. U-64,815 runs in this system with R.sub.f =0.50-0.55 and develops a deep purple coloration when processed with this reagent.

Fractions 436-570 are pooled to give 4.25 g of a brown solid which has .about.32 bu/mg when assayed with 1/2" discs on B. subtilis by a dilution bioassay. (One bu is defined as the amount of antibiotic needed to produce a 20 mm zone.)

The 4.25 g solid is dissolved in a minimal volume of MeOH/H.sub.2 O, 80/20 and loaded onto the head of a 25 mm I. D. column packed with 42.5 g of Whatman LRP-1 reverse phase equilibrated with the solvent. A flow rate of 1.5 ml/min is established with MeOH/H.sub.2 O, 80/20 while 50 ml fractions are collected. After the seventh fraction, the solvent is charged to 100% MeOH. Fractions are analyzed in the identical manner as before and also with dipped discs plated against B. subtilis. The active fractions containing the best material as seen by TLC are pooled and reduced in volume to give 2.75 g of an off-white powder of essentially pure antibiotic U-64,815.

## Claims

1. A biologically pure culture of the microorganism Streptomyces microspinus, NRRL 12524, said culture being capable of producing the antibiotic U-64,815 in a recoverable quantity upon fermentation in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic substances.