Antibiotic acanthomycin and process for preparing
BRIEF SUMMARY OF THE INVENTION 
     The antibiotic of the invention, acanthomycin, is obtained by culturing the novel microorgaism Streptomyces espinosus subsp. acanthus, NRRL 11081, in an aqueous nutrient medium under aerobic conditions. Acanthomycin and its base addition salts have the property of adversely affecting the growth of Gram-positive bacteria, for example, Staphylococcus aerueus and Streptococcus hemolyticus. Accordingly, acanthomycin and its base addition salts can be used alone or in combination with other antibiotic agents to prevent the growth of or reduce the number of bacteria, as disclosed above, in various environments. 
     DETAILED DESCRIPTION OF THE INVENTION 
     Chemical and Physical Properties of Acanthomycin 
     Empirical Formula: (C 90  H 139  N 19  O 42 ) n  ; Equivalent weight, 2,159. 
     Elemental Analysis: 
     Calcd.: C, 50.06; H, 6.48; N, 12.33; O, 31.12. Found: C, 49.81; H, 6.47; N, 12.36; O (by difference), 31.36. 
     Optical Rotation: [α] D   25 , -28.5° (c, 1, dimethylsulfoxide). 
     Solubilities: Acanthomycin free acid is soluble in dimethylformamide and dimethylsulfoxide. It is slightly soluble (ca. 1-2 mg/ml) in methanol and ethanol, and relatively insoluble in water, ether, halogenated hydrocarbon and saturated hydrocarbon solvents. Salts (ammonium and sodium) are soluble in water and lower alcohols. 
     Titration Data 
     Equivalent weight: 2,159 
     Titrant: KOH 
     Solvent: Dimethylformamide-60% aqueous ethanol (1:1). 
     Thin Layer Chromatography: On silica gel (Merck &amp; Co.) using chloroform-ethanol-water (2:3:1 v/v), R f  =0.52. 
     Infrared Absorption Spectra: Acanthomycin 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 expressed in reciprocal centimeters: 
     
         ______________________________________                                    
Band Frequency                                                            
(Wave Numbers)    Intensity                                               
______________________________________                                    
3280              S                                                       
3070              M                                                       
2960              S                                                       
2930              S                                                       
3860              S                                                       
2650              W                                                       
1723              M                                                       
1656              S, sh (sh = shoulder)                                   
1643              S                                                       
1540              S                                                       
1463              S                                                       
1415              M                                                       
1378              M                                                       
1341              M                                                       
1278              M                                                       
1268              M                                                       
1240              M                                                       
1223              M                                                       
1145              W, sh                                                   
1187              M                                                       
1098              W                                                       
1065              W                                                       
1033              W                                                       
1002              W                                                       
 960              W                                                       
 943              W                                                       
 920              W                                                       
 847              W                                                       
 723              W                                                       
 699              M                                                       
______________________________________                                    
 Key:                                                                     
 S = Strong, M = Medium, and W = Weak                                     
 
    
     Acanthomycin also has a characteristic infrared absorption spectrum when pressed in a KBr disc. Peaks are observed at the following wave lengths expressed in reciprocal centimeters: 
     
         ______________________________________                                    
Band Frequency                                                            
(Wave Numbers)        Intensity                                           
______________________________________                                    
3390                  S, sh                                               
3290                  S                                                   
3060                  M                                                   
2960                  M                                                   
2930                  M                                                   
2880                  M                                                   
2650                  W                                                   
1724                  M, sh                                               
1652                  S                                                   
1533                  S                                                   
1455                  M                                                   
1412                  M                                                   
1385                  M                                                   
1340                  M                                                   
1273                  M                                                   
1223                  M                                                   
1183                  M                                                   
1140                  M, sh                                               
1095                  W                                                   
1067                  W                                                   
1030                  W                                                   
1000                  W                                                   
 960                  W                                                   
 920                  W                                                   
 875                  W                                                   
 737                  W                                                   
 698                  M                                                   
______________________________________                                    
 
    
     Ultraviolet Absorption Spectrum: Acanthomycin has a characteristic ultraviolet absorption spectrum in methanol as shown in FIG. 2 of the drawings. λ max at 262 (a=16.81) in methanol. 
     BIOLOGICAL PROPERTIES OF ACANTHOMYCIN 
     Acanthomycin was tested for antibacterial activity in a standard tube assay using nutrient broth as the medium. The minimum inhibitory concentration in mcg/ml against S. aureus was 50, and against S. lutea it was 25. Additional testing on a standard disc (12.7 mm) plate assay gave the following results: 
     
         ______________________________________                                    
        Zone (mm)                                                         
Acanthomycin                                                              
          Strepto- Staphylo-              Sar-                            
mg/ml     coccus   coccus   Bacillus                                      
                                   Bacillus                               
                                          cina                            
Concentration                                                             
          pyogenes aureus   cereus subtilis                               
                                          lutea                           
______________________________________                                    
10        41.5     29.5     26     34     35                              
5         40       28.5     25     33     34                              
2.5       40       28       23     31.5   32                              
1.25      38.5     26.5     21     30     30                              
0.62      36.5     24.5     20     28     27                              
0.31      34       22.5     17     26     25                              
______________________________________                                    
 
    
     Acanthomycin was also tested in vivo in standard laboratory mice. S. pyogenese infected mice were protected by acanthomycin administered subcutaneously with a CD 50  of 0.23 (0.17-0.31) mg/kg. Against S. aureus infected mice the CD 50  was 1.0 mg/kg. 
     THE MICROORGANISM 
     The microorganism used for the production of acanthomycin is Streptomyces espinosus subsp. acanthus, NRRL 11081. A subculture of this microorganism is freely available from the permanent collection of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill., U.S.A. by request made thereto. 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. 
     An actinomycete culture, isolated from a soil sample from southeast Texas was characterized as Streptomyces espinosus sp. n. in U.S. Pat. No. 3,697,380. Four additional soil isolates from southeast Texas soils had the same color pattern on Ektachrome as the type culture. In expanded studies the isolates were found to have the same sporophore and spore types as well as general cultural characteristics of the type culture. Outstanding characteristics of these cultures are their gray-green aerial growth, short sporophores bearing round spiny spores, and thermoduric growth. Differences in cultural characteristics (see Tables) and antagonistic properties are not sufficient to warrant variety designation of these new isolates. Three of these new isolates, which are disclosed in U.S. Pat. No. 3,833,475, have the accession numbers NRRL 5729, NRRL 5730 and NRRL 5731. The remaining isolate is described herein as Stroptomyces espinosus subsp. acanthus, NRRL 11081. 
     Methods for culture characterization were those cited by Dietz [Dietz, A. 1967. Streptomyces steffisburgensis sp. n. J. Bacteriol. 94: 2,022-2,026.] and in part those cited by Shirling and Gottlieb [Shirling, E. B., and D. Gottlieb. 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16: 313-340.]. The cultures were also grown on Hickey-Tresner agar [Hickey, R. J. and H. D. Tresner. 1952. A cobalt-containing medium for sporulation of Streptomyces species. J. Bacteriol. 64: 891-892.] modified [Pepticase (enzymatic digest of casein) substituted for N-Z Amine A]. 
     Color characteristics: Aerial growth gray-green. Melanin-negative. Appearance on Ektachrome [Dietz, A. 1954.  Ektachrome transparencies as aids in actinomycete classification. Ann. N.Y. Acad. Sci. 60: 152-154.] is given in Table 1. Reference color characteristics are given in Tables 2 and 3. The cultures may be placed in the Green (GN) color series of Tresner and Backus [Tresner, H. D., and E. J. Backus. 1963. System of color wheels for streptomycete taxonomy. Applied Microbiol. 11: 335-338.]. 
     Microscopic characteristics: Sporophores short, straight to flexuous, to open spiral to spiral (RF, RA, S) in the sense of Pridham et al. [Pridham, T. G., C. W. Hesseltine, and R. G. Benedict. 1958. A guide for the classification of streptomycetes according to selected groups. Placement of strains in morphological sections. Applied Microbiol. 6: 52-79.]. Spores mostly spherical with many showing a distinct linkage. Spore surface thorny to spiny in the sense of Dietz and Mathews [Dietz, A., and J. Mathews. 1971. Classification of Streptomyces spore surfaces into five groups. Applied Microbiol. 21: 527-533.]. Some spines show a transition to hairy. Spines are profuse and show markings when observed on spores treated by the carbon replica method of Dietz and Mathews [Dietz, A., and J. Mathews. 1962. Taxonomy by carbon replication. I. An examination of Streptomyces hygroscopicus. Applied Microbiol. 10: 258-263.]. 
     Cultural and biochemical characteristics: Cultural and biochemical characteristics are cited in Table 4. 
     Carbon utilization: Growth of the cultures on carbon compounds was determined in the synthetic medium of Pridham and Gottlieb [Pridham, T. G., and D. Gottlieb. 1948.  The utilization of carbon compounds by some Actinomycetales as an aid for species determination. J. Bacteriol. 56: 107-114.], Table 5 and in the synthetic medium of Shirling and Gottlieb, op. cit., 313-340, Table 6. 
     Temperature: Growth was fair at 18° C. and 55° C.; good at 24° C., and heavy at 28°-37° C. At 45° C. growth was fair (vegative) in 24 hours and heavy (good sporulation) in 72 hours. The agar media used were Bennett&#39;s, Czapek&#39;s sucrose, maltose-tryptone, and Hickey-Tresner (modified). 
     Antibiotic-producing properties: S. espinosus NRRL 5729, NRRL 5730, and NRRL 5731 produce the antibiotic lincomycin. S. espinosus subsp. acanthus, NRRL 11081, produces acanthomycin. 
     
                                           TABLE 1                                 
__________________________________________________________________________
Appearance of Streptomyces espinosus Cultures on Ektachrome*              
        De-                                                               
        ter-                                                              
        mi-                                                               
Agar    na-                                                               
           NRRL    NRRL    NRRL    NRRL    NRRL                           
Medium  tion                                                              
           3890    5729    5730    5731    11081                          
__________________________________________________________________________
Bennett&#39;s                                                                 
        S  Gray-green                                                     
                   Gray-green                                             
                           White to gray-                                 
                                   Gray-green                             
                                           Gray-green                     
                           green                                          
        R  Pale yellow-tan                                                
                   Pale yellow-tan                                        
                           Pale yellow                                    
                                   Pale yellow-tan                        
                                           Pale yellow                    
Czapek&#39;s                                                                  
        S  Gray-green                                                     
                   Gray-green                                             
                           Gray-green                                     
                                   Gray-green                             
                                           Gray-green                     
sucrose R  Pale gray                                                      
                   Pale gray                                              
                           Pale gray                                      
                                   Pale gray                              
Maltose-                                                                  
        S  Gray-green                                                     
                   Gray-green                                             
                           Gray-green                                     
                                   Gray-green                             
                                           Gray-green                     
tryptone                                                                  
        R  Yellow-tan to                                                  
                   Yellow-tan to                                          
                           Yellow  Yellow-tan                             
                                           Pale yellow-                   
           olive   olive                   tan                            
Peptone-iron                                                              
        S  White   White   White   White   White                          
        R  Yellow  Yellow  Yellow  Yellow-tan                             
                                           Yellow                         
0.1% Tyrosine                                                             
        S  Colorless                                                      
                   Colorless                                              
                           Colorless                                      
                                   Gray-green                             
                                           Gray-green                     
        R  Red     Red     Red     Red     Red                            
Casein starch                                                             
        S  Gray-green                                                     
                   Gray-green                                             
                           Gray-green                                     
                                   Gray-green                             
                                           Gray-green                     
        R  Pale gray-green                                                
                   Pale gray-green                                        
                           Pale gray-green                                
                                   Pale gray-green                        
                                           Pale gray-                     
                                           green                          
__________________________________________________________________________
 S = Surface                                                              
 R = Reverse                                                              
 *Dietz, A. 1954. Ektachrome transparencies as aids in actinomycete       
 classification. Ann. N.Y. Acad. Sci. 60: 152-154.                        
 
    
     
                       TABLE 2                                                     
______________________________________                                    
Reference Color Characteristics of Streptomyces espinosus                 
Cultures                                                                  
______________________________________                                    
        De-                                                               
        ter-         Color                                                
        mi-           Harmony Manual, 3rd ed., 1948*                      
Agar      na-    NRRL    NRRL  NRRL  NRRL  NRRL                           
Medium    tion   3890    5729  5730  5731  11081                          
______________________________________                                    
Bennett&#39;s S      a       a     a     241/2fe                              
                                           241/2ih                        
                 241/2fe 241/2ih                                          
                               241/2ih                                    
                 11/2ge  1ge                                              
          R      11/2ca  11/2ca                                           
                               11/2gc                                     
                                     2ec   2ea                            
                 2fb     2fb         11/2ec                               
                                           2ic                            
                 2ec     2ec                                              
          P      --      --    --    --    --                             
Czapek&#39;s  S      1 li    1 li  2ig   2ig   2ig                            
sucrose          2ig     2ig                                              
          R      1ec     1ec   2fe   1ec   2fe                            
                 2ig     2ig                                              
          P      --      --    --    --    &#39;                              
Maltose-  S      1ig     241/2fe                                          
                               3ba   241/2fe                              
                                           241/2ih                        
tryptone         11/2ge  1ih   241/2ih                                    
                 1ih                                                      
          R      2ge     2gc   2ic   11/2ie                               
                                           2ie                            
                 2ec     11/2ge                                           
                 2gc                                                      
                 11/2ge                                                   
          P      --      --    --    --    --                             
Hickey-   S      1ig     1ig   3ba   241/2ih                              
                                           241/2ih                        
Tresner                        241/2ih                                    
(modified)                                                                
          R      2gc     2ec   2gc   2gc   2ec                            
          P      --      --    --    --    --                             
Yeast extract-                                                            
          S      241/2ih 241/2ih                                          
                               241/2ih                                    
                                     241/2ih                              
                                           241/2ih                        
malt extract     2ih     2ih                                              
(ISP-2)                                                                   
          R      2le     2gc   2le   2gc   2le                            
                 2gc     2ie                                              
          P      --      --    --    --    --                             
Oatmeal   S      1ig     1ig   241/2fe                                    
                                     241/2ih                              
                                           241/2fe                        
(ISP-3)          11/2ig  2fe                                              
          R      1ec     1ec   2ec   2ec   2ec                            
                 11/2ge  2ec                                              
          P      --      --    --    --    --                             
Inorganic-salts                                                           
          S      241/2ih 241/2ih                                          
                               241/2ih                                    
                                     241/2ih                              
                                           241/2ih                        
starch (ISP-4)   3ih     1ih                                              
          R      2ca     2gc   2gc   2ec   2gc                            
                 2gc                                                      
          P      2ec     2ec   2ec   2ig   2ec                            
Glycerol- S      1ig     a     3ba   11/2ig                               
                                           241/2ih                        
asparagine       2ih     2fe   241/2fe                                    
(ISP-5)                                                                   
          R      1ec     2gc   2ge   2ge   2ig                            
                 2ge     2ge                                              
          P      2ge     --    --    --    --                             
______________________________________                                    
        De-                                                               
        ter-                                                              
        mi-          NBS Circular 553, 1955**                             
Agar      Na     NRRL     NRRL NRRL  NRRL  NRRL                           
Medium    tion   3890    5729  5730  5731  11081                          
______________________________________                                    
Bennett&#39;s S      263gm   263gm 263gm 122gm 122m                           
                 122gm   122m  122m        127g                           
                 109gm   127g  127g                                       
                         109gm                                            
          R       89gm    89gm 102g   90gm  86gm                          
                  87g     87g  105gm  90gm  87gm                          
                  89m     89m         93m                                 
                  90gm    90gm                                            
          P      --      --    --    --    --                             
Czapek&#39;s  S      110gm   110gm 110g  110g  110g                           
sucrose          110g    112m  112m  112m  112m                           
          R      112m    112m   94g  121m   94g                           
                 121m    121m  112gm 122g  112gm                          
                 122g    122g                                             
                 110g    110g                                             
          P      112m    112m  --    --    --                             
Maltose-  S      109gm   122gm --    122gm 122m                           
tryptone         110g    112m  122m        127g                           
                 109gm   113g  127g                                       
                 112m                                                     
                 113g                                                     
          R       90gm    90gm  87gm 106gm  91gm                          
                  90gm   109gm              94g                           
                  90gm                     106g                           
                 109gm                                                    
          P      --      --    --    --    --                             
Hickey-   S      109gm   109gm --    122m  122m                           
Tresner          110g    110g  127g  127g  127g                           
(modified)                                                                
          R       90gm    90gm  90gm  90gm  90gm                          
          P      --      --    --    --    --                             
Yeast extract-                                                            
          S      122m    122m  122m  122m  122m                           
malt extract     127g    127g  127g  127g  127g                           
(ISP-2)          122m    122m                                             
                 113g    113g                                             
          R       88gm    90gm  88gm  90gm  88gm                          
                  94g     91gm  94g         94g                           
                  90gm    94g                                             
          P      --      --    --    --    --                             
Oatmeal   S      109gm   109gm 122gm 122gm 122gm                          
(ISP-3)          110g    110g                                             
                          94g                                             
                         112gm                                            
          R      121m    121m   90gm  90gm  90gm                          
                 109gm    gm                                              
          P      --      --    --    --    --                             
Inorganic-salts                                                           
          S      122m    122m  122m  122m  122m                           
starch (ISP-4)   113g    112m                                             
                 265m    113g                                             
          R       89gm    90gm  90gm  90gm  90gm                          
                  90gm                                                    
          P       90gm    90gm  90gm 110g   90gm                          
                                     112m                                 
Glycerol- S      109gm   263gm --    --    122m                           
asparagine       110g     94g  122gm       127g                           
(ISP-5)          112m    112gm                                            
                 113g                                                     
          R      121m     90gm  94m   94m  110g                           
                 122g     94gm 109gm 109gm 112m                           
                  94m    109gm                                            
                 109gm                                                    
          P      --      --    --    --    --                             
______________________________________                                    
 S = Surface                                                              
 R = Reverse                                                              
 P = Pigment                                                              
 m = matte                                                                
 g = glossy                                                               
 gm = glossy or matte surface of chip                                     
 *Jacobson, E., W.C. Granville, and C.E. Foss. 1948. Color harmony manual,
 3rd ed. Container Corporation of America, Chicago, Illinois.             
 **Kelly, K.L., and D.B. Judd. 1955. The ISCCNBS method of designating    
 colors and a dictionary of color names. U.S. Dept. Comm. Circ. 553.      
 
    
     
                       TABLE 3                                                     
______________________________________                                    
 Color Code for TABLE 2                                                   
Color Harmony Manual,                                                     
                  NBS Circular 553,                                       
3rd ed., 1948*    1955**                                                  
Color                 Color                                               
chip  Color name      chip    Color name                                  
______________________________________                                    
a     White           263gm   White                                       
1ec   Light citron gray, putty                                            
                      121m    Pale yellow green                           
                      122g    Grayish yellow green                        
1ge   Citron gray     109gm   Light grayish olive                         
1ig   Olive gray      109gm   Light grayish olive                         
                      110g    Grayish olive                               
1ih   Olive gray      112m    Light olive gray                            
                      113g    Olive gray                                  
                      127g    Grayish olive green                         
1 li  Light olive drab                                                    
                      110gm   Grayish olive                               
11/2 ca                                                                   
      Cream            89gm   Pale yellow                                 
11/2ec                                                                    
      Biscuit, ecru, oatmeal,                                             
                       90gm   Grayish yellow                              
      sand             93m    Yellowish gray                              
11/2 gc                                                                   
      Dusty yellow    102g    Moderate greenish                           
                              yellow                                      
                      105gm   Grayish greenish                            
                              yellow                                      
11/2 ge                                                                   
      Light olive gray                                                    
                      109gm   Light grayish olive                         
11/2 ie                                                                   
      Light olive     106gm   Light olive                                 
11/2 ig                                                                   
      Olive gray      --      --                                          
2ca   Light ivory eggshell                                                
                       89gm   Pale yellow                                 
2ea   Light wheat, light maize                                            
                       86gm   Light yellow                                
2ec   Biscuit, ecru, oatmeal,                                             
                       90gm   Grayish yellow                              
      sand                                                                
2fb   Bamboo, buff, straw,                                                
                       87g    Moderate yellow                             
      wheat            89m    Pale yellow                                 
2fe   Covert gray      94g    Light olive brown                           
                      122gm   Light olive gray                            
2gc   Bamboo, chamois  90gm   Grayish yellow                              
2ge   Covert tan, griege                                                  
                       94m    Light olive brown                           
                      109gm   Light grayish olive                         
2ic   Honey gold, light gold                                              
                       87gm   Moderate yellow                             
2ie   Light mustard tan                                                   
                       91gm   Dark grayish yellow                         
                       94g    Light olive brown                           
                      106g    Light olive                                 
2ig   Slate tan       110g    Grayish olive                               
                      112m    Light olive gray                            
2ih   Dark covert gray                                                    
                      112m    Light olive gray                            
                      113g    Olive gray                                  
2le   Mustard, old gold                                                   
                       88gm   Dark yellow                                 
                       94g    Light olive brown                           
3ba   Pearl, shell tint                                                   
                      --      --                                          
3ih   Beige gray, mouse                                                   
                      113g    Olive gray                                  
                      265m    Medium gray                                 
241/2 fe                                                                  
      Light mistletoe gray                                                
                      122gm   Grayish yellow green                        
241/2 ih                                                                  
      Mistletoe gray  122m    Grayish yellow green                        
                      127g    Grayish olive green                         
______________________________________                                    
 *Jacobson, E., W.C. Granville, and C.E. Foss. 1948. Color harmony manual,
 3rd ed. Container Corporation of America, Chicago, Illinois.             
 **Kelly, K.L., and D.B. Judd. 1955. The ISCCNBS method of designating    
 colors and a dictionary of color names. U.S. Dept. Comm. Circ. 553.      
 
    
     
                                           TABLE 4                                 
__________________________________________________________________________
Cultural and Biochemical Characteristics of Streptomyces espinosus        
Cultures                                                                  
        De-                                                               
        ter-                                                              
        mi-                                                               
        na-                                                               
           NRRL     NRRL     NRRL     NRRL      NRRL                      
Medium  tion                                                              
           3890     5729     5730     5731      11081                     
__________________________________________________________________________
Agar                                                                      
Peptone-iron                                                              
        S  Gray-white to                                                  
                    Gray-white to                                         
                             Trace gray-                                  
                                      Gray-green-                         
                                               Gray-green-                
           gray-green                                                     
                    gray-green                                            
                             green-white                                  
                                      white    white                      
        R  Yellow   Yellow   Yellow   Yellow   Yellow                     
        O  Melanin negative                                               
                    Melanin negative                                      
                             Melanin negative                             
                                      Melanin negative                    
                                               Melanin negative           
Calcium S  Fair to good                                                   
                    Trace gray-                                           
                             Trace gray-                                  
                                      Trace gray-                         
                                               Trace gray-                
malate     gray-green                                                     
                    green    green    green    green                      
        R  Pale gray                                                      
                    Pale gray                                             
                             Pale gray                                    
                                      Pale gray                           
                                               Pale gray                  
        O  Malate not                                                     
                    Malate not                                            
                             Malate not                                   
                                      Malate not                          
                                               Malate not                 
           solubilized                                                    
                    solubilized                                           
                             solubilized                                  
                                      solubilized                         
                                               solubilized                
Glucose-                                                                  
        S  Gray-white to                                                  
                    Gray-white to                                         
                             Fair gray-                                   
                                      Fair gray-                          
                                               Fair gray-                 
asparagine gray-yellow                                                    
                    trace green                                           
                             white    white    white                      
        R  Light yellow                                                   
                    Cream    Cream    Cream    Cream                      
           to cream                                                       
Skim milk                                                                 
        S  White on edge to                                               
                    White on edge                                         
                             White on edge                                
                                      White on edge                       
                                               White on edge              
           light gray-                                                    
                    to gray-green-                                        
           green yellow                                                   
                    pink                                                  
        R  Deep yellow to                                                 
                    Yellow-tan                                            
                             Yellow-tan                                   
                                      Yellow-tan                          
                                               Yellow-tan                 
           yellow-tan                                                     
        O  Yellow to yellow-                                              
                    Yellow-tan                                            
                             Yellow-tan                                   
                                      Yellow-tan                          
                                               Yellow-tan                 
           tan pigment                                                    
                    pigment  pigment  pigment  pigment                    
           Casein solu-                                                   
                    Casein solu-                                          
                             Casein solu-                                 
                                      Casein solu-                        
                                               Casein solu-               
           bilized under                                                  
                    bilized under                                         
                             bilized under                                
                                      bilized under                       
                                               bilized under              
           growth to                                                      
                    growth to                                             
                             growth   growth   growth                     
           completely                                                     
                    completely                                            
Tyrosine                                                                  
        S  Good to heavy                                                  
                    Good to heavy                                         
                             Good gray-green                              
                                      Good gray-green                     
                                               Good gray-green            
           gray-green                                                     
                    gray-green                                            
        R  Red-tan to                                                     
                    Red-tan to                                            
                             Red-tan  Red-tan  Red-tan                    
           red-brown                                                      
                    red-brown                                             
        O  Red-tan to red-                                                
                    Red-tan to red-                                       
                             Red-tan pigment                              
                                      Red-tan pigment                     
                                               Red-tan pigment            
           brown pigment                                                  
                    brown pigment                                         
           Tyrosine solu-                                                 
                    Tyrosine solu-                                        
                             Tyrosine solu-                               
                                      Tyrosine solu-                      
                                               Tyrosine solu-             
           bilized  bilized  bilized  bilized  bilized                    
Xanthine                                                                  
        S  Good gray-green                                                
                    Good gray-green                                       
                             Good gray-green                              
                                      Good gray-green                     
                                               Good gray-green            
           to good only                                                   
           on periphery                                                   
        R  Pale yellow                                                    
                    Pale yellow-                                          
                             Cream    Cream    Cream                      
           to cream green to cream                                        
        O  Xanthine not                                                   
                    Xanthine not                                          
                             Xanthine not                                 
                                      Xanthine not                        
                                               Xanthine not               
           solubilized                                                    
                    solubilized                                           
                             solubilized                                  
                                      solubilized                         
                                               solubilized                
Nutrient                                                                  
        S  Good gray-green                                                
                    Good gray-green                                       
                             Good gray-green                              
                                      Good gray-green                     
                                               Good gray-green            
starch  R  Cream-olive                                                    
                    Cream-olive                                           
                             Cream-olive                                  
                                      Cream-olive                         
                                               Cream-olive                
        O  Starch   Starch   Starch   Starch   Starch                     
           hydrolyzed                                                     
                    hydrolyzed                                            
                             hydrolyzed                                   
                                      hydrolyzed                          
                                               hydrolyzed                 
Yeast extract-                                                            
        S  Good to heavy                                                  
                    Good gray-green                                       
                             Good gray-green                              
                                      Good gray-green                     
                                               Good gray-green            
malt extract                                                              
           gray-green                                                     
        R  Pale yellow-tan                                                
                    Cream-yellow-tan                                      
                             Cream-yellow-tan                             
                                      Cream-yellow-tan                    
                                               Cream-yellow-tan           
           to cream-                                                      
           yellow-tan                                                     
Bennett&#39;s                                                                 
        S  Cream to heavy                                                 
                    Gray-green-white                                      
                             Trace gray-cream                             
                                      Cream    Gray-green-white           
           gray-green                                                     
                    to heavy gray-                                        
                    green                                                 
        R  Yellow to olive                                                
                    Cream to olive                                        
                             Yellow   Yellow   Yellow-orange              
Czapek&#39;s                                                                  
        S  Gray-green                                                     
                    Gray-green                                            
                             Gray-green                                   
                                      Gray-green                          
                                               Gray-green                 
sucrose R  Gray-green                                                     
                    Gray-green                                            
                             Gray-green                                   
                                      Gray-green                          
                                               Gray-green                 
Maltose-                                                                  
        S  Heavy gray-green                                               
                    Good to heavy                                         
                             Gray-green-                                  
                                      Pale gray-green                     
                                               Gray-green                 
tryptone            gray-green                                            
                             white             white                      
        R  Olive to orange-                                               
                    Olive to cream                                        
                             Yellow   Yellow-olive                        
                                               Yellow-orange              
           yellow                                                         
Hickey-Tresner                                                            
        S  Heavy gray-green                                               
                    Heavy gray-green                                      
                             Gray-green-white                             
                                      Gray-green                          
                                               Heavy gray-green           
(modified)                                                                
        R  Olive-orange                                                   
                    Cream    Deep yellow-tan                              
                                      Yellow-olive                        
                                               Olive-orange               
Peptone-yeast                                                             
        S  Cream to pale                                                  
                    Pale pink to                                          
                             Cream to pale                                
                                      Cream with trace                    
                                               Cream to pale              
extract-iron                                                              
           pink     gray-green                                            
                             pink     gray-green to                       
                                               pink                       
(ISP-6)                               pale pink                           
        O  Melanin negative                                               
                    Melanin negative                                      
                             Melanin negative                             
                                      Melanin negative                    
                                               Melanin negative           
Tyrosine                                                                  
        S  Gray-green-white                                               
                    Gray-green-white                                      
                             Gray-green                                   
                                      Gray-green                          
                                               Gray-green                 
(ISP-7)    to gray-green                                                  
                    to gray-green                                         
        R  Pale yellow-                                                   
                    Pale yellow-                                          
                             Gray-cream                                   
                                      Gray-cream                          
                                               Gray-green                 
           green to tan                                                   
                    green to gray-                                        
                    cream                                                 
        O  Melanin-negative                                               
                    Melanin-negative                                      
                             Melanin-negative                             
                                      Melanin-negative                    
                                               Melanin-negative           
Gelatin                                                                   
Plain   O  Colorless vege-                                                
                    Colorless vege-                                       
                             Gray-white                                   
                                      Gray-white                          
                                               Gray-green-white           
           tative growth                                                  
                    tative growth                                         
                             aerial growth;                               
                                      aerial growth;                      
                                               aerial growth;             
           to gray-white                                                  
                    to gray-white                                         
                             liquefaction                                 
                                      liquefaction                        
                                               liquefaction               
           aerial growth;                                                 
                    aerial growth;                                        
                             complete complete complete                   
           liquefaction                                                   
                    liquefaction                                          
           1/2 to   1/2 to                                                
           complete complete                                              
Nutrient                                                                  
        O  White to gray-                                                 
                    White to gray-                                        
                             Gray white                                   
                                      Trace gray-white                    
                                               Gray-white                 
           white aerial                                                   
                    white aerial                                          
                             aerial growth;                               
                                      aerial growth;                      
                                               aerial growth;             
           growth;  growth;  liquefaction                                 
                                      liquefaction                        
                                               liquefaction               
           liquefaction                                                   
                    liquefaction                                          
                             complete complete complete                   
           1/3 to   1/3 to                                                
           complete complete                                              
Broth                                                                     
Synthetic                                                                 
        O  White to pink-                                                 
                    Trace white to                                        
                             Trace white                                  
                                      Trace white                         
                                               Trace white                
nitrate    cream aerial                                                   
                    pink-cream                                            
                             aerial growth                                
                                      aerial growth                       
                                               aerial growth              
           growth on sur-                                                 
                    aerial growth                                         
                             on surface                                   
                                      on surface                          
                                               on surface                 
           face pellicle;                                                 
                    on surface                                            
                             pellicle;                                    
                                      pellicle;                           
                                               pellicle;                  
           growth through-                                                
                    pellicle;                                             
                             growth through-                              
                                      trace growth                        
                                               growth through-            
           out medium;                                                    
                    growth through-                                       
                             out medium;                                  
                                      throughout                          
                                               out medium;                
           nitrate not                                                    
                    out medium;                                           
                             nitrate not                                  
                                      medium; nitrate                     
                                               nitrate not                
           reduced to                                                     
                    nitrate not                                           
                             reduced to                                   
                                      not reduced to                      
                                               reduced to                 
           nitrite  reduced to                                            
                             nitrite  nitrite  nitrite                    
                    nitrite                                               
Nutrient                                                                  
        O  Gray-green-white                                               
                    Gray-green-white                                      
                             Gray-green-white                             
                                      Trace white                         
                                               Gray-green-white           
nitrate    aerial growth                                                  
                    aerial growth                                         
                             aerial growth                                
                                      aerial growth                       
                                               aerial growth              
           on surface                                                     
                    on surface                                            
                             on surface                                   
                                      on surface                          
                                               on surface                 
           pellicle;                                                      
                    pellicle;                                             
                             pellicle;                                    
                                      pellicle;                           
                                               pellicle;                  
           flocculent                                                     
                    flocculent                                            
                             flocculent                                   
                                      compact to                          
                                               flocculent                 
           bottom growth;                                                 
                    bottom growth;                                        
                             bottom growth;                               
                                      flocculent                          
                                               bottom growth;             
           nitrate test:                                                  
                    nitrate not                                           
                             nitrate not                                  
                                      bottom growth;                      
                                               nitrate not                
           neither  reduced to                                            
                             reduced to                                   
                                      nitrate not                         
                                               reduced to                 
           nitrate nor                                                    
                    nitrate  nitrite  reduced to                          
                                               nitrite                    
           nitrite                    nitrite                             
           present or                                                     
           nitrate not                                                    
           reduced to                                                     
           nitrite                                                        
Litmus milk                                                               
        O  Cream to green                                                 
                    Pink-tan to                                           
                             Tan aerial                                   
                                      Green-white                         
                                               Tan aerial                 
           aerial growth                                                  
                    gray-green                                            
                             growth on                                    
                                      aerial growth                       
                                               growth on                  
           on yellow to                                                   
                    aerial growth                                         
                             surface ring;                                
                                      on surface                          
                                               surface ring;              
           tan surface                                                    
                    on surface                                            
                             litmus reduced;                              
                                      ring; partial                       
                                               partial                    
           ring; litmus                                                   
                    ring; litmus                                          
                             peptonization                                
                                      reduction;                          
                                               reduction;                 
           reduced; reduced; pH 7.3   partial  partial                    
           peptonization;                                                 
                    peptonization;    peptonization;                      
                                               peptonization;             
           pH 7.3   pH 7.3            pH 7.1   pH 7.4                     
__________________________________________________________________________
 S = Surface                                                              
 R = Reverse                                                              
 P = Pigment                                                              
 O = Other Characteristics                                                
 
    
     
                       TABLE 5                                                     
______________________________________                                    
Utilization Of Carbon Compounds By                                        
Streptomyces espinosus Cultures In The Synthetic                          
Medium Of Pridham And Gottlieb*                                           
         NRRL   NRRL     NRRL    NRRL  NRRL                               
         3890   5729     5730    5731  11081                              
______________________________________                                    
  CONTROL    (+)**    (-)**  (+)   (+)   (+)                              
 1. D-Xylose                                                              
           +        +        +     +     +                                
 2. L-Arabinose                                                           
           +        +        +     +     +                                
 3. Rhamnose                                                              
           +        +        +     +     +                                
 4. D-Fructose                                                            
           +        (+),+    +     +     +                                
 5. D-Galactose                                                           
           +        +        +     +     +                                
 6. D-Glucose                                                             
           +        +        +     +     +                                
 7. D-Mannose                                                             
           +        +        +     +     +                                
 8. Maltose                                                               
           +        +        +     +     +                                
 9. Sucrose                                                               
           (+)      (-),(+)  (+)   (+)   (+)                              
10. Lactose                                                               
           +        +        +     +     +                                
11. Cellobiose                                                            
           +        +        +     +     +                                
12. Raffinose                                                             
           (+)      (-)      (+)   (+)   (+)                              
13. Dextrin                                                               
           +        +        +     +     +                                
14. Inulin (+),(-)  (-)      (+)   (+)   (+)                              
15. Soluble starch                                                        
           +        +        +     +     +                                
16. Glycerol                                                              
           +        +        +     +     +                                
17. Ducitol                                                               
           (+),(-)  (-)      (+)   (+)   (+)                              
18. D-Mannitol                                                            
           +        +        +     +     +                                
19. D-Sorbitol                                                            
           +   ,(-) (-)      +     +     +                                
20. Inositol                                                              
           +        (+)      +     +     +                                
21. Salicin                                                               
           (+)      (+)      +     (+)   (+)                              
22. Phenol (+),(-)  (-)      (+)   (+)   (+)                              
23. Cresol -        -        -     -     -                                
24. Na Formate                                                            
           (+),-    (-)-     (+)   (+)   (+)                              
  CONTROL    (+)**    (-)**  (+)   (+)   (+)                              
25. Na Oxalate                                                            
           (+),(-)  (+),(-)  (+)   (+)   (+)                              
26. Na Tartrate                                                           
           (+),(-)  (-),-    (+)   (+)   (+)                              
27. Na Salicylate                                                         
           -        -        -     -     -                                
28. Na Acetate                                                            
           (+)      (+)      (+)   (+)   (+)                              
29. Na Citrate                                                            
           +        (+)      +     (+)   +                                
30. Na Succinate                                                          
           (+),(-)  (-)      (+)   (+)   (+)                              
______________________________________                                    
 + = Good utilization                                                     
 (+) = Poor utilization                                                   
 (-) = Doubtful utilization                                               
 - = No growth                                                            
 *Pridham, T.G., and D. Gottlieb. 1948. The utilization of carbon compound
 by some Actinomycetales as an aid for species determination. J. Bacteriol
 56: 107-114.                                                             
 **Results from different studies                                         
 
    
     
                       TABLE 6                                                     
______________________________________                                    
Utilization Of Carbon Compounds By                                        
Streptomyces espinosus Cultures In The Synthetic                          
Medium Of Shirling And Gottlieb*                                          
           NRRL  NRRL    NRRL    NRRL  NRRL                               
           3890  5729    5730    5731  11081                              
______________________________________                                    
CONTROL                                                                   
Negative-basal                                                            
             ±**  -,±**                                             
                             ±  ±  ±                             
medium                                                                    
Positive-basal                                                            
             +       +       +     +     +                                
medium plus                                                               
D-glucose                                                                 
Carbon compounds                                                          
L-Arabinose  ±,++ ±,+  ±  ±  ±                             
Sucrose      -       ±,-  -     -     -                                
D-Xylose     ++      ±,++ ++    ++    ++                               
Inositol     ++      ±,+  ++    ++    ++                               
D-Mannitol   ++      ++      ++    ++    ++                               
D-Fructose   ++      ±,+  ++    ++    ++                               
Rhamnose     ++      ++      ++    ++    ++                               
Raffinose    -       -       -     -     -                                
Cellulose    +,++    ±,-  +     +     +                                
______________________________________                                    
 ++ Strong utilization                                                    
 ± Utilization doubtful                                                
 + Positive                                                               
 - Utilization negative                                                   
 *Shirling, E.B., and D. Gottlieb. 1966. Methods for characterization of  
 Streptomyces species. Int. J. Syst. Bacteriol. 16: 313-340.              
 **Results from different studies                                         
 
    
     The new compound of the invention 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 carbohydrate, and a nitrogen 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&#39;s yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, fish meal, distillers&#39; 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 of the invention can be effected at any temperature conductive to satisfactory growth of the microorganism, for example, between about 18° and 40° C., and preferably between about 20° and 28° C. Ordinarily, optimum production of the compound is obtained in about 3 to 15 days. The medium normally remains neutral 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 new 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 stock, an agar plug stored above liquid N 2 , 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 new compound, so long as good growth of the microorganism is obtained. 
     A variety of procedures can be employed in the isolation and purification of the compound of the subject invention, for example, solvent extraction, partition chromatography, silica gel chromatography, liquid-liquid distribution in a Craig apparatus, adsorption on resins, and crystallization from solvents. 
     In a preferred recovery process the compound of the subject invention is recovered from the culture medium by separation of the mycelia and undissolved solids by conventional means, such as by filtration or centrifugation. The antibiotic is recovered from the filtered or centrifuged broth by extraction with a suitable solvent for acanthomycin, for example, water-immiscible alcohols like 1-butanol (preferred). The solvent extract containing the desired antibiotic is concentrated and then subjected to purification procedures as disclosed infra. 
     Before disclosing purification procedures, there is first disclosed alternate recovery procedures. Acanthomycin can be recovered from the fermentation whole beer by filtration followed by adsorption of the antibiotic on a non-ionic resin. Suitable non-ionic resins include resins comprising a non-ionic macro porous copolymer of styrene crosslinked with divinylbenzene. Non-ionic resins of this type, marketed under the trade names Amberlite XAD-2 and XAD-4, are disclosed in U.S. Pat. No. 3,515,717. 
     Further, activated carbon, alumina and related adsorbants can be used to recover acanthomycin from filtered fermentation beer. 
     Since acanthomycin is an acidic compound, anion exchangers, for example, Dowex-1; Dowex-2; Dowex 21K (Dow Chemical Co., USA) or Amberlite IRA-900; -IRA-904; -IR-45 (Rohm and Haas Co., USA) can be used to recover the antibiotic from filtered fermentation beer. The antibiotic is adsorbed onto the resin and then eluted by the use of an aqueous solution of inorganic acids, salts like ammonium chloride and bases like ammonium hydroxide, dilute sodium hydroxide, and the like. 
     After recovery of acanthomycin from the fermentation beer, the recovery preparation is then subjected to purification procedures which will ultimately yield a purified crystalline preparation of acanthomycin. It should be recognized, however, that any of the crude preparations containing acanthomycin, including the fermentation beer, can be used for purposes well known to those skilled in the antibiotic art. For example, such crude preparations can be used as feed for animals. Where relatively pure preparations of acanthomycin are desired, the crude preparaions of acanthomycin can be purified by the following procedures, or obvious equivalents thereof. 
     The crude preparation of acanthomycin is first mixed with a suitable solvent, for example, ethyl acetate, and the like, to precipitate a material containing acanthomycin. This material is isolated by filtration and then distributed between water and a suitable solvent for acanthomycin, for example, 1-butanol. The solvent phase containing acanthomycin, is separated out, then mixed with water. The pH of the mixture is adjusted to a basic pH, for example, about 7.9, with concentrated base, for example, ammonium hydroxide. The resulting solvent and aqueous phases are separated and then the aqueous phase is extracted with a solvent for acanthomycin. The solvent extract containing acanthomycin is then subjected to chromatographic procedures on a suitable resin, for example, Amberlite IRA-904 anion exchanger in the chloride form. This resin is a product of Rohm and Haas Company, USA. The chromatographic resin column is eluted with aqueous ammonium chloride. 
     Active fractions from the above chromatographic procedure are then subjected to another chromatographic procedure to separate acanthomycin from ammonium chloride. The resin used in this procedure is a non-ionic resin known as Amberlite XAD-7, again a product of Rohm and Haas Co. This resin is eluted with a methanol-water mixture to give fractions containing acanthomycin. The active fractions are combined and then concentrated to an aqueous and freeze-dried to give a relatively pure preparation of acanthomycin. 
     A highly purified preparation of acanthomycin can be obtained by subjecting the above-described freeze-dried preparation to chromatography using DEAE-Sephadex (A-25) supplied by Pharmacia Fine Chem., Uppsala, Sweden. The preparation containing acanthomycin is dissolved in ammonium chloride and this solution is then passed over a column containing the DEAE-Sephadex (A-25). Fractions active against B. subtilis and S. pyogenes are pooled. Acanthomycin is isolated from the resulting three pools by use of chromatographic procedures using a non-ionic resin, for example, Amberlite XAD-7. 
     In processing the pools over the Amberlite XAD-7 resin, the resin is eluted with a methanol-water mixture to give acanthomycin. The ammonium salt of acanthomycin is made by adjusting the pH of these fractions to about 7.5 with ammonium hydroxide. 
     Acanthomycin is active against S. aureus and can be used to disinfect washed and stacked food utensils contaminated with these bacteria; it can also be used as a disinfectant on various dental and medical equipment contaminated with S. aureus. Further, acanthomycin 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. Since acanthomycin is active against B. subtilis, it can be used in petroleum product storage to control this microorganism which is a known slime and corrosion producer in petroleum products storage. 
     It is to be understood that the microbiological process disclosed herein, though described in detail with reference to Streptomyces espinosus subsp. acanthus, NRRL11081, is not limited to this particular microorganism deposit. It is intended that any microorganism meeting the cultural characteristics disclosed herein, or substantial equivalents thereof, wherever deposited in the world, is a part of the subject microbiological process. Further, it is intended that this invention include strains or mutants of the said microorganism which can be produced by procedures well known in the art, for example, by subjecting the novel microorganism to x-ray or ultraviolet radiation, nitrogen mustard, phage exposure, and the like. 
     Since acanthomycin is an acidic compound, it forms salts with both inorganic and organic bases. For example, the sodium, potassium, ammonium, calcium, and the like, salts can be made by neutralizing a solution of acanthomycin in aqueous methanol. Salts of acanthomycin with organic bases (primary, secondary and tertiary amines) for example, pyridine, piperidine, and the like, can be made by mixing of equal molar amounts of acanthomycin and the desired organic base. 
    
    
     The following examples are illustrative of the process and products of the subject 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. 
     EXAMPLE 1 
     Part A. Fermentation 
     An agar slant of Streptomyces espinosus subsp. acanthus, NRRL 11081, is used to inoculate a series of 500-ml Erlenmeyer flasks, each containing 100 ml of sterile preseed medium consisting of the following ingredients: 
     
         ______________________________________                                    
Glucose monohydrate    25 g/liter                                         
Pharmamedia*           25 g/liter                                         
Tap water q.s.         1 liter                                            
______________________________________                                    
 *Pharmamedia is an industrial grade of cottonseed flour produced by      
 Traders Oil Mill Company, Fort Worth, Texas.                             
 
    
     The preseed medium presterilization pH is 7.2. The preseed inoculum is grown for three days at 28° C. on a Gump rotary shaker operating at 250 r.p.m. and having a 21/2 inch stroke. 
     Preseed inoculum (300 ml), prepared as described above, is used to inoculate a seed tank containing 20 liters of sterile seed medium consisting of the following ingredients: 
     
         ______________________________________                                    
Glucose monohydrate    25 g/liter                                         
Pharmamedia            25 g/liter                                         
Tap water              Balance                                            
______________________________________                                    
 
    
     The inoculated seed medium is incubated at a temperature of 28° C. for 2 days while being agitated at a rate of 400 r.p.m. and aerated at a rate of 10 standard liters per minute with a back pressure of 10 psig. 
     After 2 days incubation, the seed medium is used to inoculate (the inoculation rate is 5 liters of seed inoculum per 100 liters of fermentation medium) a 250 liter fermentation tank containing sterile fermentation medium consisting of the following ingredients: 
     
         ______________________________________                                    
B and F Distillers Solubles*                                              
                       40 g/liter                                         
NaCl                    5 g/liter                                         
NaNO.sub.3             10 g/liter                                         
Ucon**                  2 ml/liter                                        
Tap water q.s.         Balance                                            
pH - 7.0 (presterilization)                                               
______________________________________                                    
 *Distillers residue supplied by BrownForman Distillers Corp., P.O. Box   
 1080, Louisville, KY 40201.                                              
 **a synthetic defoamer supplied by Union Carbide, N.Y., N.Y.             
 
    
     The fermentation tank is incubated at a temperature of 28° C., with agitation of 200 r.p.m. and aeration at 150 standard liters per minute with back pressure at 10 psig. Harvest is usually after 3 to 12 days of fermentation. 
     The fermentation can be monitored by a disc-plate assay using B. subtilis as the test organism and deionized H 2  O as diluent. A unit volume (0.08 ml) of solution containing the substance to be assayed is placed on a 12.7 mm paper disc which is then placed on an agar plate seeded with the assay organism. The agar plate is then incubated for 16 to 18 hours at 32° to 37° C. A biounit (BU) is defined as the concentration of the antibiotic which gives a 20 mm zone of inhibition under the standard assay conditions. Thus, if for example a fermentation beer has to be diluted 1/100 to give a 20 mm zone of inhibition, the potency of such beer is 100 BU/ml. 
     Part B. Recovery 
     Whole fermentation beer (ca. 5,000 l), obtained as described above, is adjusted to pH 2.75 with 6 N aqueous hydrochloric acid. The adjusted whole beer is then filtered using filter aid (diatomaceous earth). The mycelial cake is washed with 1,000 l of water. The washed filter cake is discarded. The aqueous wash is combined with the clear filtrate and the resulting solution (5,750 l) is extracted with 1,600 l of 1-butanol. The spent beer is discarded. The butanolic extract is concentrated to a volume of ca. 8 l (4.5 kg). This material is designated preparation ADA-117A. 
     Part C. Purification 
     (No. 1) Precipitation 
     One liter of preparation ADA-117A, isolated as described in the recovery section is poured into 10 l of ethyl acetate under stirring. The mixture is stirred at room temperature for one hour. The precipitated material containing acanthomycin is isolated by filtration and treated as described, infra under distribution. 
     (No. 2) Distribution 
     The precipitate is distributed between 2.5 l of water and 2.5 l of 1-butanol. The butanol phase (ADA-31C) is mixed with 7 l of water. The pH of the mixture is adjusted to 7.9 with concentrated ammonium hydroxide. The two phases are separated and designated Butanol-1 and Aqueous-1. Aqueous-1 is then extracted 3 times with 2.5 l portions of 1-butanol each time at pH 7.9 (adjusted with ammonium hydroxide). The three butanolic extracts (designated Butanol-2, Butanol-3-and Butanol-4) are combined with Butanol-1 and the combined extract is kept as ADA-31D. 
     Testing for bioactivities showed the following: 
     
         ______________________________________                                    
       Zone (mm)                                                          
         S.        B.       S.     B.     S.                              
Preparation                                                               
         pyogenes  cereus   aureus subtilis                               
                                          lutea                           
______________________________________                                    
ADA-31C  36        18       20     23     27                              
ADA-31D  36        16       24     26.5   27                              
______________________________________                                    
 
    
     Preparation ADA-31D containing most of the bioactivity was treated as described below. 
     (No. 3) Amberlite IRA-904 Chromatography 
     The column is prepared from 2 liters of Amberlite IRA-904 anion exchanger in the chloride form. (Product of Rohm and Haas, USA). 
     The butanolic extract (ADA-31D), obtained above, is mixed with one half of its volume of methanol and one half of its volume of water and this solution is passed through the column at a rate of 50-70 ml/minute. The spent is collected in three equal fractions designated &#34;Spent-1,&#34; &#34;Spent-2&#34; and &#34;Spent-3&#34; which are found bioinactive. 
     The column is washed with 6 l of water. This eluate, kept as &#34;Aqueous,&#34; is found bioinactive. 
     The column is then eluted with 2% aqueous ammonium chloride. Fractions of 20 ml are collected and combined in 500 ml fractions and tested. Results follow. Zone size in mm. 
     
         ______________________________________                                    
         S.        B.       S.     B.     S.                              
Fraction No.                                                              
         pyogenes  cereus   aureus subtilis                               
                                          lutea                           
______________________________________                                    
1         0        0        0      0      0                               
2         0        0        0      0      0                               
3        35        17       25     26.5   25                              
4        39        21       27.5   30     29                              
5        40        22       28.5   31     30                              
6        39        21       28     29.5   29.5                            
7        39        20.5     27.5   29.5   28.5                            
8        38        20       27     29     28.5                            
9        37        20       27     28.5   28.5                            
10       --        18       25     28     28                              
11       --        16.5     24     27.5   27.5                            
12       --        16       23     26.5   26.5                            
13       --        15.5     23     25.5   25                              
14       --        15.5     22.5   24.5   24.5                            
15       --        traces   21     22.5   23                              
16       --        0        18     18     17                              
17       --        0        0      0      0                               
18       --        --       --     --     --                              
______________________________________                                    
 
    
     Fractions 3-18 are combined, adjusted to pH 5.5 with 6 N aqueous hydrochloric acid and this solution is used as starting material for the chromatography described below. 
     (No. 4) Amberlite XAD-7 Chromatography 
     Separation of Acanthomycin from Ammonium Chloride 
     The column is prepared from 2 l of Amberlite XAD-7 (Rohm and Haas Co., USA). 
     The solution described above containing acanthomycin in mixture with ammonium chloride (8 l, pH 5.5) is passed over the column at a rate of 80-100 ml/minute. The spent is collected in two equal fractions designated &#34;Spent-1&#34; and &#34;Spent-2&#34;. 
     The column is washed with 4 l of water (flow rate 80-100 ml/minute). Four equal fractions (Aqueous-1; -2; -3; -4) are collected. 
     The column is then eluted with methanol-water (70:30 v/v). Fractions of 20 ml are collected. Testing shows the following. Zone size in mm. 
     
         ______________________________________                                    
          S.       S.       B.     B.    S.                               
Fraction  pyrogenes                                                       
                   aureus   cereus subtilis                               
                                         lutea                            
______________________________________                                    
Start-Material                                                            
          35       24.5     19     27.5  26                               
Spent-1   0        0        0      0     0                                
Spent-2   0        0        0      0     0                                
Aqueous-1 0        0        0      0     0                                
Aqueous-2 0        0        0      0     0                                
Aqueous-3 0        0        0      0     0                                
Aqueous-4 0        0        0      0     0                                
MeOH-Water                                                                
 10       0        0        0      0     0                                
 20       0        0        0      0     0                                
.         .        .        .      .     .                                
 80       0        0        0      0     0                                
 90       42       29       23.5   35.5  37                               
100       40       28.5     24     33    35                               
110       30       19.5     traces 22.5  24                               
120       24       traces   0      17.5  17                               
130       21.5     0        0      traces                                 
                                         traces                           
140       18       0        0      0     0                                
150       15.5     0        0      0     0                                
160       16       0        0      0     0                                
170       15       0        0      0     0                                
180       traces   0        0      0     0                                
190       0        0        0      0     0                                
200       0        0        0      0     0                                
.         .        .        .      .     .                                
250       0        0        0      0     0                                
______________________________________                                    
 
    
     Fractions 85-125 are combined. The solution is concentrated to an aqueous and freeze-dried to give preparation ADA-34.1, 3.7 g containing acanthomycin. 
     Using 5.5 l of preparation ADA-117A, obtained as described in Part B., supra, as the starting material, and using the above-described purification procedure (precipitation, distribution, IRA-904 and XAD-7 chromatographies) a total of 21.71 g of material of highly active acanthomycin is obtained (Prep. ADA-113.4). 
     Highly purified acanthomycin, obtained as described above, is purified by DEAE-Sephadex chromatography as shown below. 
     (No. 5) DEAE-Sephadex Chromatography 
     Eight hundred g of DEAE-Sephadex (A-25) (Pharmacia Fine Chem., Uppsala, Sweden) is slurried in 0.05 N ammonium chloride solution adjusted to pH 8.5 with ammonium hydroxide. The mixture is allowed to stand at room temperature for 12 hours. The swelled resin is then poured into a column. The column is then washed with 25 l of solvent. 
     The starting preparation, ADA-113.4 (20 g), obtained as described above, is dissolved in 200 ml of the 0.05 N ammonium chloride (pH 8.5) solvent and this solution is added on the top of the column. The column is then eluted with the same solvent. Fractions are tested for bioactivity against B. subtilis and S. pyogenes. Results follow. 
     
         ______________________________________                                    
                 Zone (mm)                                                
Fraction No.                                                              
           Volume (1)  B. subtilis S. pyogenes                            
______________________________________                                    
1          2.0         0           0                                      
2          2.0         0           0                                      
3          8.5         0           0                                      
4          12.0        0           0                                      
5          4.0         0           0                                      
6          4.0         0           0                                      
7          3.0         0           0                                      
8          12.0        0           0                                      
9          4.0         15          24                                     
10         3.5         17          28                                     
11         9.0         20          28                                     
12         7.5         21          28.5                                   
13         8.5         21          30                                     
14         6.0         23          30.5                                   
15         9.0         22.5        31.5                                   
16         4.0         22.5        31.5                                   
17         3.0         21          30.5                                   
18         12.0        20.5        30.5                                   
19         4.0         20.5        30                                     
20         4.0         20          30                                     
21         12.0        20          30                                     
22         9.0         20          31                                     
23         3.0         21          31                                     
24         12.0        19          29.5                                   
25         4.0         19          29.5                                   
26         4.0         18          29.5                                   
27         9.0         19          29                                     
28         12.0        --          --                                     
29         4.0         18          28                                     
30         4.0         18          27.5                                   
31         12.0        17          28                                     
32         12.0        17          27                                     
33         12.0        17.5        26                                     
34         12.0        17          26                                     
35         10.0        17          25                                     
36         12.0        17          25                                     
37         7.0         15          24                                     
38         12.0        15          23.5                                   
39         12.0        traces      23.5                                   
40         12.0        0           24                                     
41         7.0         0           25                                     
42         12.0        0           --                                     
43         12.0        0           --                                     
______________________________________                                    
 
    
     The following pools are made: 
     Pool I: Fractions 9-12 ca. 24 l 
     Pool II: Fractions 13-20 ca. 50.5 l 
     Pool III: Fractions 21-43 ca. 214 l 
     Acanthomycin is isolated from the above pools by Amberlite XAD-7 chromatographies as described below. 
     (A) Isolation of Acanthomycin from Pool I 
     Amberlite XAD-7 Chromatography 
     The column is prepared from 1 l of Amberlite XAD-7. Pool I is adjusted to pH 3.0 with 6 N aqueous hydrochloric acid and passed over the column with a flow rate of 25 ml/minute. The spent is found bioinactive and is discarded. The column is then washed with 3 l of water. The aqueous wash is found bioinactive and is also discarded. The column is then eluted with methanol-water (70:30 v/v). Fractions of 20 ml are collected. Testing shows the following. 
     
         ______________________________________                                    
             Zone (mm)                                                    
Fraction No.   B. subtilis  S. pyogenes                                   
______________________________________                                    
10             0            0                                             
20             0            0                                             
30             0            0                                             
40             0            0                                             
50             20.5         23.5                                          
60             25           29.5                                          
70             26           32.5                                          
 80            27           34                                            
 90            26.5         34.5                                          
100            26           34.5                                          
110            25.5         33.5                                          
120            25.5         32                                            
130            24           31                                            
140            23           31                                            
150            21.5         28.5                                          
160            20           27.5                                          
170            18           24.5                                          
180            16           22                                            
190            0            18                                            
200            0            16                                            
210            0            0                                             
.              .            .                                             
400            0            0                                             
______________________________________                                    
 
    
     Fractions 46-200 are combined, adjusted to pH 7.5 with ammonium hydroxide, concentrated to an aqueous solution and freeze-dried to give preparation ADA-44.1, 550 mg (acanthomycin ammonium salt). 
     (B) Isolation of Acanthomycin from Pool II 
     Amberlite XAD-7 Chromatography 
     The column is prepared from 2 l of Amberlite XAD-7. Pool II is adjusted to pH 3.0 with 6 N aqueous hydrochloric acid. A precipitate is formed which is separated by filtration and dried (ADA-45). This material yields crystalline acanthomycin, as described infra. The filtrate is passed over the column at a rate of 50 ml/minute. The spent solution is found bioinactive and is discarded. The column is then washed with 6 l of water. The aqueous wash is found bioinactive and is also discarded. The column is then washed with 6 l of water. The aqueous wash is found bioinactive and is also discarded. The column is then eluted with methanol-water (70:30 v/v). Fractions (20 ml) are collected. Testing shows the following. 
     
         ______________________________________                                    
             Zone(mm)                                                     
Fraction No.   B. subtilis  S. pyogenes                                   
______________________________________                                    
 10            0            0                                             
 20            0            0                                             
.              .            .                                             
140            0            traces                                        
150            0            15                                            
160            0            16                                            
170            0            16                                            
180            0            18                                            
190            16           22                                            
200            19           25                                            
210            22           29                                            
220            24           33.5                                          
230            27           33.5                                          
240            27.5         36                                            
250            28.5         38                                            
260            28           37.5                                          
270            28           38.5                                          
280            27           34.5                                          
290            27           33.5                                          
300            26           33                                            
310            25.5         32                                            
320            24           31                                            
330            23.5         29                                            
340            23           29.5                                          
350            22           29                                            
360            21           29                                            
370            20.5         27.5                                          
380            21           28                                            
390            20           27                                            
400            19.5         26                                            
410            19.5         26                                            
420            19           25                                            
430            19           24                                            
440            18           23                                            
450            17           23.5                                          
460            17           23                                            
470            17           23                                            
480            16           22                                            
______________________________________                                    
 
    
     The column is then eluted with methanol-0.05 N ammonium hydroxide. Fractions of 20 ml are collected. Results follow: 
     
         ______________________________________                                    
             Zone (mm)                                                    
Fraction No.   B. subtilis  S. pyogenes                                   
______________________________________                                    
10             15.5         21.5                                          
20             15           21                                            
30             15           21                                            
40             16           20.5                                          
50             16           19.5                                          
60             15           17.5                                          
70             15           19                                            
80             15           20.5                                          
90             17           23.5                                          
100            20           27.5                                          
110            25           33                                            
120            26           34.5                                          
130            20           25.5                                          
140            0            traces                                        
______________________________________                                    
 
    
     The following combinations are made: 
     I: Fractions (Methanol-water 70:30); 190-219 
     II: Fractions (Methanol-water 70:30); 220-399 
     III: Fractions (Methanol-water 70:30); 440-483 
     IV: Fractions (Methanol-0.05 NH 4  OH 70:30); 100-140 
     The combined solutions are concentrated to dryness to give the following: 
     I: Acanthomycin, prep. ADA-46.3; 70 mg 
     II: Acanthomycin, prep. ADA-46.4; 450 mg 
     III: Acanthomycin, prep. ADA-46.5; 50 mg 
     IV: Acanthomycin ammonium salt, prep. ADA-46.6; 250 mg 
     As mentioned above, a precipitate, formed by acidification of pool II, is isolated by filtration and kept as ADA-45. Trituration of this material with methanol, followed by concentration of the methanolic solution to dryness, yields a crystalline residue. Trituration of the residue with ether affords colorless crystalline acanthomycin which is isolated by filtration and dried (Prep. ADA-46.1, 420 mg). 
     (C) Isolation of Acanthomycin from Pool III 
     Amberlite XAD-7 Chromatography 
     The column is prepared from 3 l of Amberlite XAD-7. 
     Starting material, Pool III, prepared as described above, is adjusted to pH 3.0 with 6 N aqueous hydrochloric acid and this solution is passed over the column at a flow rate of 75 ml/minute. The spent is found bioinactive and is discarded. The column is washed with 9 l of water. The aqueous wash is found bioinactive and is discarded. The column is eluted with methanol-water (70:30 v/v). Fractions (20 ml) are collected. Testing results follow. 
     
         ______________________________________                                    
             Zone (mm)                                                    
Fraction No.   B. subtilis  S. pyogenes                                   
______________________________________                                    
 10            0            0                                             
 20            0            0                                             
.              .            .                                             
120            0            0                                             
130            0            18                                            
140            0            20                                            
150            traces       23                                            
160            16           27                                            
170            17           30                                            
180            19           31.5                                          
190            21           33.5                                          
200            22           34                                            
210            24           35                                            
220            25.5         36.5                                          
230            26           37                                            
240            27           38                                            
250            27           39                                            
260            27           38                                            
270            27           39                                            
280            27           39                                            
290            27           39                                            
300            27           39                                            
310            27           38                                            
320            28           37.5                                          
330            27.5         37                                            
340            27           37                                            
350            26           37.5                                          
360            26           37                                            
370            25           37                                            
380            24           36                                            
390            24           36                                            
400            24           34.5                                          
420            24           34                                            
440            23           33                                            
460            22.5         32                                            
480            22           32                                            
500            22           31.5                                          
520            21.5         31                                            
540            21           30                                            
560            20.5         31                                            
580            21           31                                            
600            21           30.5                                          
620            21           31                                            
640            21           30                                            
660            20.5         31                                            
680            21           31                                            
700            21           31                                            
720            21           31.5                                          
740            21           31                                            
760            21           31                                            
780            20.5         30.5                                          
800            20           30.5                                          
820            19.5         30                                            
840            19.5         30                                            
860            19           29.5                                          
880            18           29                                            
900            18           28                                            
920            18           28                                            
940            18           28                                            
______________________________________                                    
 
    
     The column is then eluted with methanol-0.05 N ammonium hydroxide. Fractions (20 ml) are collected and tested. Results follow. 
     
         ______________________________________                                    
             Zone (mm)                                                    
Fraction No.   B. subtilis  S. pyogenes                                   
______________________________________                                    
10             19           29                                            
20             18           28.5                                          
30             18           28                                            
40             18           28                                            
50             18           27                                            
60             17           26                                            
70             16           26                                            
80             16           26                                            
90             16           26                                            
100            16.5         26                                            
110            16           25.5                                          
120            22           31                                            
130            28           38                                            
140            30.5         41                                            
150            28           38                                            
160            17.5         28.5                                          
170            0            20.5                                          
180            0            20.5                                          
190            0            20.5                                          
200            0            20                                            
220            0            18                                            
240            0            17.5                                          
260            0            17                                            
280            0            17                                            
300            0            18                                            
320            0            16.5                                          
340            0            16                                            
360            0            16.5                                          
380            0            17                                            
400            0            17.5                                          
420            0            16                                            
440            0            0                                             
.              .            .                                             
500            0            0                                             
______________________________________                                    
 
    
     The following combinations are made: 
     Pool IA: Fractions 170-400 (methanol-water 70:30) 
     Pool IIA: Fractions 401-950 (methanol-water 70:30) and 10-100 (methanol-0.05 N NH 4  OH) 
     Pool IIIA: Fractions 115-170 (methanol-0.05 N NH 4  OH) 
     Pool IA is concentrated to dryness. The residue obtained is triturated with 50 ml of methanol. The colorless insoluble crystalline acanthomycin is isolated by filtration and dried (ADA-49.1, 210 mg). The filtrate is mixed with ether-Skellysolve B (isomeric hexanes) (1:1) to yield additional amorphous acanthomycin (ADA-49.2, 500 mg). 
     Pool IIA, similarly, yields crystalline acanthomycin, prep. ADA-49.3, 220 mg and amorphous acanthomycin, prep. ADA-49.4, 300 mg. 
     Pool IIIA is concentrated to an aqueous solution and then freeze-dried to give acanthomycin ammonium salt (prep. ADA-49.5, 800 mg).