Lucknomycin and process for producing same
The present invention relates to a new antifungal and antiprotozoal agent, hereinafter referred to as &#34;lucknomycin&#34;, and to a process for the preparation thereof. 
     Various antifungal antibiotics are already known but research is continuing throughout the world to discover new antifungal agents having various advantages over those already known. 
     We have now discovered a new antifungal agent, which we have called lucknomycin, which has been found to be effective against the growth of, in particular, Candida albicans, Candida krusei, Candida tropicalis and Aspergillus niger. It is also effective against Trichomonas vaginalis. 
     Lucknomycin has the following chemical and physical characteristics. 
     1. PHYSICAL CHARACTERISTICS: 
     1.1 Aspect: orange yellow powder 
     1.2 Elemental analysis: C 61  H 96  N 2  O 24  (molecular weight 1240): Calcd. : C, 59.03%; H 7.74%; N 2.26%. Found : C, 58.92%; H, 7.58%; N, 2.29%. 
     1.3 Melting point: 
     THE PRODUCT STARTS TO DECOMPOSE AT ABOUT 150° C. but, even at 300° C., it does not melt. 
     1.4 Solubility: 
     SOLUBLE IN PYRIDINE, DIMETHYLFORMAMIDE AND DIMETHYLACETAMIDE; LESS SOLUBLE IN 60% METHANOL, CONCENTRATED ETHANOL AND ISOPROPANOL; 
     INSOLUBLE IN BENZENE, ACETONE, WATER, CHLOROFORM, ABSOLUTE ETHANOL, CYCLOHEXANE, ETHYL ACETATE AND DIETHYL ETHER. 
     In sulfuric acid, the new antifungal agent gives an intense blue coloration. 
     
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1.5 Infrared absorption spectrum:                                         
taken from the spectrum in a KBr pellet (in cm.sup.-1)                    
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3400 (S)*       1465 (W)      1075 (S)                                    
3100 (W)***     1390 (W)      1045 (W)                                    
2930 (S)        1350 (W)      1010 (S)                                    
2860 (M)**      1325 (W)       995 (W)                                    
1715 (M)        1295 (W)       940 (W)                                    
1650 (M)        1250 (W)       890 (W)                                    
1600 (S)        1185 (S)       850 (M)                                    
1575 (W)        1140 (W)       835 (W)                                    
1545 (W)        1110 (M)       800 (W)                                    
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 Band intensities are indicated as &#34;S&#34;, &#34;M&#34; and &#34;W&#34; respectively          
 *S = strong absorption                                                   
 **M = medium absorption                                                  
 ***W = weak absorption                                                   
 
    
     1.6 Ultraviolet absorption spectrum: 
     TAKEN AT A CONCENTRATION OF 1 MG./100 ML. OF DIMETHYLFORMAMIDE 
     
         344 mμ (ε = 3.5 × 10.sup.4) 
    
     
         364 mμ (ε = 5.52 × 10.sup.4) 
    
     
         384 mμ (ε = 8.04 × 10.sup.4) 
    
     
         408 mμ (ε = 6.6 × 10.sup.4) 
    
     1.7 Optical rotation: 
     [α] D   25 ° C. = 187° to 194° (c. = 0.3% in dimethylformamide at 25° C). 
     2. CHEMICAL CHARACTERISTICS 
     Lucknomycin can be classified as an aromatic heptaene antibiotic because, by the retroaldolization reaction, it gives p-N-methylaminoacetophenone. 
     Methanolysis, attributes to Lucknomycin a substituent of the amino sugar type, identified with an authentic sample of mycosamine. 
     Lucknomycin does not contain a titratable carboxyl group. 
     3. BIOLOGICAL CHARACTERISTICS 
     3.1 Antifungal spectrum. 
     Lucknomycin is introduced at different dilutions into a peptonedextrose medium inoculated with the organism which is to be tested. After incubation for from 2 to 5 days at 28° C., the number of microgrammes of antibiotic per milliliter of medium is determined which must be used to obtain complete inhibition of growth (MIC) of the organism in question. In order to better point out the antifungal activity of Lucknomycin, it is compared with that of other known antifungal antibiotics. 
     
         ______________________________________                                    
                              MIC                                         
Microorganism    Product      (mcg./ml.)                                  
______________________________________                                    
Candida albicans 11651                                                    
                 Lucknomycin  0.1-0.2                                     
                 Nystatin     2.5                                         
                 Amphotericin 1.25                                        
                 Pimaricin    5                                           
Aspergillus niger                                                         
                 Lucknomycin  0.5                                         
                 Nystatin     5                                           
                 Amphotericin 0.6                                         
                 Pimaricin    2.5                                         
Trichophyton mentagrophytes                                               
                 Lucknomycin  25                                          
                 Nystatin     25                                          
Trichophyton acuminatum                                                   
                 Lucknomycin  12.5                                        
                 Nystatin     25                                          
Trichomoras vaginalis                                                     
                 Lucknomycin  &lt;0.05                                       
                 Nystatin     &gt;10                                         
                 Amphotericin B                                           
                              &gt;10                                         
                 Pimaricin    &gt;10                                         
                 Metronidazole                                            
                              0.5                                         
Candida krusei   Lucknomycin  0.2-0.4                                     
Candida tropicalis                                                        
                 Lucknomycin  0.1-0.2                                     
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     3.2 Antibacterial spectrum. 
     The experimental conditions are the same as for the determination of the fungal spectrum, except that the incubation time is one day at 37° C. 
     Lucknomycin does not exert an inhibiting activity, at a concentration of 10 mcg/ml, on the following bacteria: 
     Escherichia coli 
     Klebsiella pneumoniae 
     Lactobacillus acidophilus 
     Proteus mirabilis 
     Pseudomonas aeruginosa 
     Staphylococcus aureus 
     Streptococcus faecalis 
     3.3 Antiprotozoal activity. 
     A concentration of 0.5 mcg./ml. of the antibiotic kills 50% of individuals of a culture in full growth of Trichomonas vaginalis within a period of about 30 minutes. 
     3.4 Toxicity. 
     Lethal dose LD 50  in mice in mg./kg. body weight: 
     
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Mode of administration   LD.sub.50                                        
______________________________________                                    
intravenous               10-20                                           
intraperitoneal           5-10                                            
subcutaneous            &gt;800                                              
oral                    &gt;800                                              
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     4. PHARMACOLOGICAL TESTS IN VIVO. 
     4.1 Anti-Trichomonas vaginalis activity. 
     This test is made in mice on a subcutaneous Trichomonas vaginalis abscess. Subcutaneous injections at the level of the abscess of a suspension (or solution) of the following products at various concentrations are used to determine the ED 50 . 
     
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             ED.sub.50 on Trichomonas vaginalis                           
Product      (in mcg./ml.)                                                
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Lucknomycin  12                                                           
Candicidin   24                                                           
Trichomycin  43                                                           
Metronidazole                                                             
             250                                                          
Pimaricin    720                                                          
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     4.2 Anti-Candida albicans activity in gynecology. 
     The test is made in female rats on experimental Candida albicans vaginitis. Lucknomycin is compared with two reference products: Mycostatin (active principle = nystatin) and Canestene (active principle = clotrimazole). 
     
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                Number of                                                 
                cured rats/infested rats                                  
Active principle  after    after    after                                 
(in mg./tablet)   4 adm.*  8 adm.*  12 adm.*                              
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a)  lucknomycin  5 mg.    14/16  13/13  12/12                             
b)  clotrimazole 20 mg.   17/18  16/16  17/17                             
c)  nystatin     4 mg.     5/11   8/11   9/11                             
*adm. = administrations.                                                  
a)  galenic formula of the test tablet.                                   
lucknomycin  5 mg.                                                        
sodium borate                                                             
             50 mg.   *       colloidal silica                            
Aerosil R 972*                                                            
             0.8 mg.  **      glycerol palmitate                          
Precirol**   1 mg.            + stearate                                  
LSNa***      0.8 mg.  ***     sodium lauryl-sulfate                       
Avicel pH 102****                                                         
             52 mg.   ****    microcrystalline                            
Lactose FK q.s.p.                                                         
             200 mg.          cellulose                                   
b)  1/5 of a commercial tablet containing 100 mg.                         
    clotrimazole                                                          
c)  1/5 of a commercial tablet containing 20 mg.                          
    (100.000 units) nystatin.                                             
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     5. THERAPEUTIC USES. 
     Lucknomycin is an antifungal and antiprotozoal antibiotic which has the following uses: 
     treatment of candidiases which may be general or superficial on the skin (dermatology) or on the buccal, vaginal or intestinal mucous membranes; 
     treatment of infections of cavities due to Trichomonas vaginalis (trichomoniases) and more particularly the therapeutic use in gynecology; 
     veterinary use for the treatment of mycotic infections and 
     curative activity against prostatic hypertrophy. 
     It can also be used in agriculture to combat vegetal mycoses. 
     Lucknomycin may be administered orally, topically or intravaginally. The physician will indicate daily dosage. Thus for example in the treatment of vaginitis, lucknomycin may be administered intravaginally in a dosage of two vaginal tablets per day containing each 10 to 50 mg. lucknomycin per tablet of 800 to 900 mg. during two to three weeks. 
     
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5.1 Examples of formulation of tablets for use in gynecology.             
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Lucknomycin                10       mg.                                   
sodium bicarbonate         2.65     mg.                                   
tartaric acid              3.65     mg.                                   
Aerosil R 972              0.8      mg.                                   
Precirol                   1        mg.                                   
LSNa                       0.8      mg.                                   
Avicel pH 102              52       mg.                                   
Lactose FK q.s.p.          200      mg.                                   
or else                                                                   
Lucknomycin                10       mg.                                   
urea                       25       mg.                                   
Lactose SD                                                                
Avicel                     70       mg                                    
magnesium stearate                                                        
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     5.2 Examples of antifungal formulations for dermatological use. 
     Besides the usual carriers, appropriate amounts of an anti-inflammatory agent, a large spectrum antibiotic (or antiseptic) and/or an antihistamine may be added to the following formulations: 
     
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Ointment                                                                  
Lucknomycin                500        mg.                                 
dimethylacetamide          10         ml.                                 
vaseline 70%      q.s.p.    100       g.                                  
liquid paraffin                                                           
Cream                                                                     
Lucknomycin                500        mg.                                 
dimethylacetamide          10         g.                                  
mixture of glycerol mono-                                                 
 and distearate            12.68      g.                                  
Crodawax A22               0.55       g.                                  
Nipagin M*                 0.114      g.                                  
Nipasol**                  0.045      g.                                  
Tween 40***                0.5        g.                                  
aqua dist. q.s.p.          100        g.                                  
Anhydrous base                                                            
Lucknomycin                500        mg.                                 
dimethylacetamide          10         g.                                  
Tween 40                   0.5        g.                                  
bees wax                   3.5        g.                                  
stearyl alcohol            20         g.                                  
glycerol q.s.p.            100        g.                                  
Gel                                                                       
Lucknomycin                1000       mg.                                 
dimethylacetamide          98         g.                                  
glycerol                   98         g.                                  
nordihydroguaieretic                                                      
 acid                      0.2        g.                                  
citric acid                0.1        g.                                  
Carbopol 940****           2          g.                                  
Tween 40                   1          g.                                  
triethaolamine q.s.                                                       
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 *methyl hydroxybenzoate                                                  
 **propyl hydroxybenzoate                                                 
 ***polyoxyethylenesorbitan monopalmitate (ATLAS)                         
 ****acrylic acid polymer (GOODRICH)                                      
 
    
     6. DESCRIPTION of the MICROORGANISM. 
     The strain used for the production of lucknomycin has been subjected to an identification study by the Centraalbureau voor Schimmelcultures of Baarn (Holland) and has been deposited there under strain number CBS 101.74. The study showed that this microorganism clearly belongs to the species Streptomyces diastatochromogenes (Krains) Waksman and Henrici in its most essential characteristics. The only difference, the non-utilization of sucrose, is not of such importance as to constitute proof of a new species. S. bottropensis Waksman 1956, S. phaeophaciens Maeda et al., 1952 and also S. luteogriseus Schmitz et al., 1964, are also very close and may possibly be considered as synonyms as previously indicated by Hutter in 1967 for S. bottropensis and S. phaeophaciens. 
     For the purpose of studying its morphology and culture characteristics, the organism was cultivated (specimen spores in distilled water taken with a platinum loop from cultures in slant test tubes) in the media mentioned below for from 10 to 14 days at 28° ± 1° C. The well sporulated cultures in Petri dishes were observed directly with an optical microscope. The specimens for study under an electron microscope were obtained by carefully pressing copper grids coated with &#34;farmvar&#34; on the sporulated mycelium. Observations were also recorded with regard to the characteristics of the culture, particularly the colors of the aerial mycelium and of the mycelium in the substrate (Maers and Paul 1950) and with regard to any changes of coloration of the media. 
     
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6.1 Color:                                                                
             Observations                                                 
             Color of color of mycelium                                   
                                soluble                                   
Media        aerial mycelium                                              
                      in the substrate                                    
                                pigment                                   
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1.  Oatmeal-agar                                                          
             brownish-grey                                                
                      pale brown                                          
                                none                                      
2.  Mineral salt-                                                         
    starch-agar                                                           
             grey     pale yellow                                         
                                none                                      
3.  Asparagine-                                                           
    dextrose-agar                                                         
             grey     pale yellow                                         
                                none                                      
4.  Yeast-malt-agar                                                       
             grey     pale brown                                          
                                none                                      
5.  Czapek agar                                                           
             light grey                                                   
                      light grey                                          
                                none                                      
6.  Conn agar                                                             
             white to sandy                                               
                      pale brown                                          
                                none                                      
7.  Nutrient agar                                                         
             bronze-brown                                                 
                      fawn      brown                                     
8.  Tomato-                                                               
    oatmeal-agar                                                          
             brownish-grey                                                
                      pale brown                                          
                                none                                      
9.  Hickey-Tresner                                                        
    agar     olive green                                                  
                      amber yellow                                        
                                none                                      
10. Potato-agar                                                           
             white to slate                                               
                      maple sugar                                         
                                light                                     
             grey               brown                                     
11. Molasses-agar                                                         
             grey     dark brown                                          
                                none                                      
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     6.2 Microscopic characteristics. 
     Morphology of the spore chains: 
     Division spirals. The spirals are open with 4 to 6 turns. On maturity, the chains of spores have from 10 to 50 spores per chain. The sporophore branches have a sympodial arrangement. The spores are elliptical. The spore surface is smooth. 
     Color of colony: color of the aerial mass in various shades of greys on yeast-malt-agar, oatmeal-agar, glycerol-asparagine-agar and salt-starch-agar. 
     Reverse side of colony: no distinctive pigments (olive brown on yeast-malt-agar, yellowish-brown to brown with yellow edge on oatmeal-agar and greyish-yellow with some dark brown spots on glycerol-asparagine-agar and salt-starch-agar). 
     Color in medium: formation of melanoid pigments in peptone-yeast-iron-agar and tyrosine-agar. No pigment in yeast-malt-agar, oatmeal-agar, salt-starch-agar and glycerol-asparagine-agar. 
     
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6.3   Biochemical characteristics.                                        
Production of melamine    positive                                        
decomposition of tyrosine positive                                        
production of hydrogen sulfide                                            
                          positive                                        
proteolysis (gelatine)    positive                                        
reduction of nitrates     negative                                        
utilization of carbon                                                     
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 arabinose, rhamnose, glucose, galactose, fructose, mannose, lactose,     
 maltose, dextrose, glycerol, and salicine are completely utilized;       
 xylose, raffinose, starch, inositol, mannitol and sodium acetate are     
 partially utilized;                                                      
 sucrose, inulin, sorbitol and sodium citrate are not utilized.           
 
    
     Lucknomycin is obtained when the elaborating microorganism is grown in an aqueous nutrient medium under submerged aerobic conditions. For the preparation of limited amounts of the substance, surface cultures and bottles can be used. The organism is grown in a nutrient medium containing a carbon source, for example an assimilable carbohydrate, and a nitrogen source, for example an assimilable nitrogen compound or protein material. Preferred carbon sources include arabinose, rhamnose, glucose, galactose, fructose, mannose, lactose, maltose, dextrose, glycerol, salicine, xylose, raffinose, starch, inositol, mannitol and sodium acetate. Preferred nitrogen sources include ammonium sulfate, corn steep liquor, peptone, malt extract and cottonseed protein. Use may advantageously also be made of combinations of these carbon and nitrogen sources. If desired, traces of metals, for example, zinc, magnesium, manganese, cobalt, iron and the like, can but need not be added to the fermentation medium, since for the preparation of the culture medium, use is made of tap water and unpurified raw materials. 
     Lucknomycin is produced in the temperature range permitting satisfactory growth of the microorganism, namely, between 20° and 32° C. and preferably between 27° and 29° C. The optimum fermentation time may vary from 2 to 10 days. The culture medium should preferably have an initial pH value close to 7.0. The final pH depends upon the presence of buffers and is also preferably about 7.0 at the time sterilization is effected. 
     When fermentation is carried out in vessels of large dimensions, it is preferable to use the vegetative form, rather than the spore form, of the microorganism for inoculation to avoid any lag in the production of lucknomycin and to make better use of the fermentation equipment. For this reason, it is desirable to produce a vegetative inoculum in a nutrient culture broth by inoculating this broth with an aliquot from a soil or a slant culture. When a young and active vegetative inoculum has thus been obtained, it is transferred aseptically to larger vessels. The medium used for producing a vegetative inoculum may be identical to or different from the medium used for the large scale production of lucknomycin, provided that good growth of the microorganism is ensured. 
     The analytical data obtained with lucknomycin attribute to this compound the formula C 61  H 96  N 2  O 24 . Lucknomycin is soluble in pyridine, dimethylformamide, dimethylacetamide; it is less soluble in 60% methanol, ethanol and isopropanol and it is insoluble in benzene, acetone, water, chloroform, absolute ethanol, cyclohexane, ethyl acetate and ether. 
     Different processes may be used for isolating and purifying lucknomycin, for example solvent extraction, gel chromatography and liquid-liquid distribution in a Craig apparatus. Solvent extraction processes are preferred for industrial recovery because they are quicker and less expensive. 
     Preferred purification processes include those using &#34;Merckogel&#34; 500 or &#34;Sephadex&#34; LH 20. 
     In the preferred recovery process, lucknomycin is recovered from its culture medium by separation of the mycelium and undissolved solids by conventional means, such as by filtration or by centrifugation. The antibiotic is then removed from the filtered or centrifuged broth by extraction with normal butanol. After draining and filtration (or centrifugation), the mycelium is extracted with an alcohol (ethanol or methanol). This extract is concentrated and the insoluble matter is treated successively with hexane (or petroleum ether), chloroform and diethyl ether in order to eliminate traces of an antibacterial polypeptide substance which is of no interest. The insoluble material is then washed with acetone and dried in vacuo. Additional purification or a purification of the residues containing lucknomycin may be effected by gel chromatography, using, for example, dimethylformamide as eluent. The eluate from the chromatographic column is concentrated in vacuo at a temperature of at most 40° C. and the lucknomycin is precipitated with diethyl ether or a 1:1 mixture of diethyl ether and hexane and recovered by filtration or centrifugation. 
     The new compound of the invention can be used against the following microorganisms: Aspergillus niger, Candida albicans, Candida krusei, Candida tropicalis, Saccharomyces cerevisiae and Trichomonas vaginalis. It is moderately active against Trichophyton mentagrophytes and Trichophyton acuminatum. It is inactive against the following bacteria: Escherichia coli, Klebsiella pneumoniae, Lactobacillus acidophilus, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and Streptcoccus faecalis. 
    
    
     7. EXAMPLES. 
     The following Examples are given for the purpose of illustrating the present invention. 
     EXAMPLE 1 
     Fermentation of Streptomyces diastatochromogenes for the production of lucknomycin. 
     Germination stage 
     A lyophilized culture of S. diastatochromogenes var. Krains is added to a 500 ml. flask, capable of being shaken, containing 100 ml. of the following sterile medium: 
     
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bacto-beef extract*    3       g.                                         
bacto-peptone          5       g.                                         
dextrose               10      g.                                         
yeast extract*         5       g.                                         
tap water              1000    ml.                                        
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 (*Difco Laboratories, Detroit, Michigan, U.S.A.) in which the pH is      
 adjusted to 7.2 with an aqueous solution of sodium hydroxide.            
 
    
     The flask and its contents are incubated for 2 days at 28° ± 1° C. on a rotary shaker (250 r.p.m., stroke 2 inches (50.8 mm.)). 
     Inoculum Preparation Stage 
     A charge of inoculum of about 6 liters is prepared by the following method: 
     10 ml. of inoculum (from the germination stage) are transferred to each of a series of flasks, each containing 200 ml. of the following sterile medium: 
     
         ______________________________________                                    
glycerol               10      ml.                                        
L-asparagine**         1       g.                                         
dipotassium hydrogen phosphate***                                         
                       1       g.                                         
yeast extract*         0.5     g.                                         
tap water              1000    ml.                                        
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 *Difco Laboratories, Detroit, Michigan, U.S.A.                           
 **BDH Chemicals Ltd., Poole, Great Britain.                              
 ***J. T. Baker Chemicals Co., New Jersey, U.S.A.                         
 
    
     The flasks and their contents are incubated for 2 days at 28° ± 1° C. on a rotary shaker (250 r.p.m., stroke 2 inches (50.8 mm.)). The broths are combined and transferred aseptically to a sterile inoculum flask. 
     6 liters of inoculum are transferred aseptically to a 100-liters fermenter containing 60 liters of the following sterile medium: 
     
         ______________________________________                                    
groundnut flour         900    g.                                         
dextrose (cerelose)     900    g.                                         
sodium chloride         30     g.                                         
calcium carbonate       6      g.                                         
yeast extract           6      g.                                         
groundnut oil           60     ml.                                        
&#34;Rhodosil 410&#34; anti-foaming                                               
agent (Rhone-Poulenc, Paris,                                              
France) or some other appropriate                                         
anti-foaming agent      3      ml.                                        
tap water to make       60     liters                                     
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     The pH is adjusted to between 6.9 and 7.0 before sterilization and aerobic fermentation is carried out for 48 hours (until the volume of the cells represents about 10 to 15% of the total volume) under the following conditions: 
     
         ______________________________________                                    
temperature      28 ± 1° C.                                     
admission of sterile air                                                  
                 60 liters/minute                                         
pressure         0.49 kg./cm..sup.2 gauge                                 
agitation        300 r.p.m.                                               
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     Fermentation Stage 
     A charge of 30 liters of inoculum is transferred aseptically to a 400-liter fermenter containing the following medium: 
     
         ______________________________________                                    
flour                  6000    g.                                         
glucose syrup (containing                                                 
 60% of cerelose)      9000    g.                                         
sodium chloride        1500    g.                                         
calcium carbonate      300     g.                                         
yeast extract          300     g.                                         
groundnut oil          3       liters                                     
anti-foaming agent                                                        
(Rhodosil 410)         15      liters                                     
tap water              300     liters                                     
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     Fermentation is carried out at 28 ± 1° C., while stirring at 200 r.p.m., with the admission of air at a pressure of 0.49 kg/cm. 2  gauge and at the rate of 300 liters per minute. During the fermentation period of 5 days, samples were taken at different times from the fermentation broth and titrated for the production of antibiotic by the spectrophotometric method. The washed mycelium is extracted with ethanol and the optical density of the ethanol extract is measured at 383 mμ with a spectrophotometer. The calibrated curve of pure lucknomycin is plotted and the content of the samples is calculated by reference to this curve. 
     EXAMPLE 2 
     Production of Lucknomycin in Various Media. 
     Using the procedure described in Example 1, a lyophilized culture of S. diastatochromogenes var. Krains is subjected to germination. After shaking for 2 days at 28 ± 1° C., the cellular material is collected by centrifuging the fermentation mixture in a sterile flask. The solid material is washed by centrifuging twice with 100 ml. amounts of sterile distilled washing water and is then suspended in 100 ml. of sterile distilled water. The suspension is used as a 10% inoculum in various synthetic media for the fermentation and determination of the production of lucknomycin. The fermentation tests are carried out in flasks which are shaken and the spectrophotometric method of determination of lucknomycin described in Example 1 is used. The results of these tests are shown in the following Table: 
     
         ______________________________________                                    
                Shaking time                                              
                            Lucknomycin in                                
Medium (% by weight)                                                      
                (days)      micrograms/ml                                 
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groundnut flour: 2%                                                       
glycerol: 1%    4           30                                            
sodium chloride: 0.5%                                                     
calcium carbonate: 0.1%                                                   
groundnut flour: 2%                                                       
glycerol: 1%                                                              
sodium chloride: 0.5%                                                     
                4           91                                            
calcium carbonate: 0.1%                                                   
groundnut oil: 1%                                                         
soya flour: 2%                                                            
glycerol: 1%                                                              
sodium chloride: 0.5%                                                     
                4           71                                            
calcium carbonate: 0.1%                                                   
groundnut oil: 1%                                                         
groundnut flour: 2%                                                       
cerelose: 1.5%                                                            
sodium chloride: 0.5%                                                     
                4           160                                           
calcium carbonate: 0.1%                                                   
groundnut oil: 1%                                                         
groundnut flour: 2%                                                       
glucose syrup: 3%                                                         
sodium chloride: 0.5%                                                     
                4           212                                           
calcium carbonate: 0.1% -groundnut oil: 1%                                
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     EXAMPLE 3 
     Effect of Ammonium Sulfate and Organic Nitrogen on the Yield of Lucknomycin. 
     Experiments are carried out in 500 ml. conical flasks containing 100 ml. of the following medium, supplemented by organic nitrogen sources of various kinds or with ammonium sulfate at optimum levels: 
     
         ______________________________________                                    
groundnut flour 2% by weight/volume                                       
cerelose        1.5% by weight/volume                                     
sodium chloride 0.5% by weight/volume                                     
calcium carbonate                                                         
                0.1% by weight/volume                                     
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     A 10% inoculum as described in Example 2 is added and the flasks are shaken on a rotary shaker (250 r.p.m., stroke 2 inches (50.8 mm) at 28 ± 1° C. for 72 hours, the following titres of lucknomycin being obtained: 
     
         ______________________________________                                    
percentage of nitrogen                                                    
                     micrograms/ml. of                                    
 (weight/volume)     lucknomycin                                          
______________________________________                                    
0.1% ammonium sulfate                                                     
                     24                                                   
0.1% corn steep liquor                                                    
                     45                                                   
0.1% peptone         70                                                   
0.1% malt extract    100                                                  
0.5% cotton seed protein                                                  
                     100                                                  
control              91                                                   
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     EXAMPLE 4 
     Extraction and Purification of Lucknomycin. 
     After fermentation, the mycelium is recovered by draining and filtration (or centrifugation), washed with water and then extracted three times by trituration with alcohol (ethanol or methanol). It is found that 90% by weight of the antibiotic is contained in the mycelium and 10% in the liquid of the culture medium. The antibiotic can be recovered from the liquid of the culture medium by extraction with normal butanol. 
     The antibiotic extract is freed from solvent at a temperature below 50° C. in the absence of light. The residue of the alcoholic extracts is first extracted with hexane (or petroleum ether) and then treated with chloroform in order to extract an unwanted antibacterial polypeptide substance. This polypeptide antibiotic was found, in the course of previous investigations, to be of no particular interest, for which reason it is discarded. The residue is then treated with diethylether and dried in air. 
     Purification can also be effected by molecular filtration chromatography on &#34;Merkogel&#34; 500 or &#34;Sephadex&#34; LH-20, using dimethylformamide as eluent. The eluates are detected either with a Pye Unicam universal wire detector or with an ultraviolet absorptiometer at 460 mμ. 
     The fractions containing the antifungal substance are concentrated in vacuo (10 -1  mm.Hg.) at 40° C. and precipitated with diethyl ether. The precipitate is then centrifuged and washed three times with diethyl ether in order to eliminate all traces of dimethylformamide. 
     Lucknomycin can also be purified by countercurrent separation, using the solvent system comprising, by volume 35 parts pyridine, 65 parts ethyl acetate and 83 parts water in a Craig 100-tube countercurrent distribution machine, and subjecting it to 261 transfers. The optical densities of the bottom and top phases are measured; only one peak is observed. The fractions containing the antibiotic are combined and evaporated to dryness under reduced pressure. The resulting residue (golden yellow powder) is finally washed with a small amount of hot methanol. 
     Melting point: browns without melting starting from 150° C. (decomposition).