Fermentation process for lipstatin and method of extracting lipstatin from a fermentation broth

Abstract

The present invention provides a fermentation process for producing lipstatin comprising the steps of: a) preparing a fermentation medium containing a lipstatin-producing microorganism comprising an oil and an assimilable carbon source, wherein the wt/wt ratio of oil and assimilable carbon source is regulated to achieve an optimal lipstatin biosynthesis by the microorganism; and b) feeding the fermentation medium with an emulsifier, wherein the emulsifier provides an optimal viscosity for the fermentation medium and optimal pH during the fermentation to permit fermentation for lipstatin production. The disclosed process also provides a process for extracting a lipstatin from a fermentation broth.

1 - 25 . (canceled)  
     
     
         26 . A process for separating lipstatin from a fermentation broth, comprising the steps of: 
 (a) extracting the lipstatin from a fermentation broth with a first extraction solvent;    (b) concentrating the first extraction solvent;    (c) extracting the lipstatin from the first concentrated extraction solvent with a second extraction solvent;    (d) concentrating the second extraction solvent;    (e) adding a third extraction solvent;    (f) washing the lipstatin in the third extraction solvent with a fourth extraction solvent; and    (g) separating the washed lipstatin from the third extraction solvent.    
     
     
         27 . The process for extracting lipstatin from a fermentation broth as in  claim 26 , wherein the separating step (g) comprises: 
 (h) concentrating the third extraction solvent;    (i) adding a fifth extraction solvent;    (j) extracting the lipstatin from the fifth extraction with a fourth extraction solvent;    (k) concentrating the fourth extraction solvent;    (l) diluting the extraction solvent with a lower alkyl alcohol; and    (m) applying the diluted lower alkyl alcohol solvent to an anion-exchanger to obtain lipstatin.    
     
     
         28 . The process of extracting the lipstatin from a fermentation broth as in  claim 26 , after the step (f) and before step (g), further comprising the steps of: 
 (n) extracting lipstatin from the fourth extraction solvent into a third extraction solvent;    (o) combining the washed third extraction solvent of step (f) with third extraction solvent of step (n); and    (p) concentrating the combined third extraction solvents of step (O).    
     
     
         29 . The process of extracting the lipstatin from a fermentation broth as in  claim 27 , after the step (k) and before step (l), further comprising the steps of: 
 (q) extracting lipstatin into the fifth extraction solvent; and    (r) concentrating the extracted fifth extraction solvent.    
     
     
         30 . The process of  claim 26 , wherein the first extraction solvent is a water immiscible solvent.  
     
     
         31 . The process of  claim 26 , wherein the first extraction solvent is selected from the group consisting of ethyl acetate, i-butyl acetate, butyl acetate and methyl ethyl ketone.  
     
     
         32 . The process of  claim 26 , wherein the first extraction solvent is i-butyl acetate.  
     
     
         33 . The process of  claim 26 , wherein the first extraction is done at a pH about 2 to about 10.5.  
     
     
         34 . The process of  claim 26 , wherein the first extraction is done at a pH about 5 to about 8.  
     
     
         35 . The process of  claim 26 , wherein the first extraction is done at a pH about 6 to about 7.  
     
     
         36 . The process of  claim 26 , wherein the concentrating step of the first extraction solvent is carried out under reduced pressure.  
     
     
         37 . The process of  claim 26 , wherein the concentrating step of the first extraction solvent is carried out at a maximum temperature of about 80° C.  
     
     
         38 . The process of  claim 26 , wherein the second extraction solvent is a lower alkyl alcohol.  
     
     
         39 . The process of  claim 26 , wherein the second extraction solvent is selected from the group consisting of methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, i-butanol, tert-butanol, acetonitrile, and acetic acid.  
     
     
         40 . The process of  claim 26 , wherein the second extraction solvent is methanol.  
     
     
         41 . The process of  claim 26 , wherein the concentrating step of the second extraction solvent is carried out under reduced pressure.  
     
     
         42 . The process of  claim 26 , wherein the concentrating step of the second extraction solvent is carried out at a maximum temperature of about 80° C.  
     
     
         43 . The process of one of claims  26  and  27 , wherein the third extraction solvent is acetonitrile.  
     
     
         44 . The process of one of claims  26  and  27 , wherein the fourth extraction solvent serves to wash lipstatin present in the third extraction solvent.  
     
     
         45 . The process of one of claims  26  and  27 , wherein the fourth extraction solvent is a hydrocarbon solvent.  
     
     
         46 . The process of one of claims  26  and  27 , wherein the fourth extraction solvent is selected from the group consisting of pentane, hexane, cyclohexane and heptane.  
     
     
         47 . The process of one of claims  26  and  27 , wherein the fourth extraction solvent is hexane.  
     
     
         48 . The process of one of claims  26  and  27 , wherein the fourth extraction solvent is heptane.  
     
     
         49 . The process of one of claims  26  and  27 , wherein the fourth extraction solvent serves to extract lipstatin from the fifth extraction solvent.  
     
     
         50 . The process of one of claims  27 ,  28  and  29 , wherein the fifth extraction solvent is a lower alkyl alcohol.  
     
     
         51 . The process of one of claims  27 ,  28  and  29 , wherein the fifth extraction solvent is a mixture of lower alkyl alcohol and water.  
     
     
         52 . The process of  claim 51 , wherein the lower alkyl alcohol is methanol.  
     
     
         53 . The process of  claim 52 , wherein the volume to volume ratio of methanol to water is about 70:15.  
     
     
         54 . The process of  claim 27 , wherein the lower alkyl alcohol is methanol.  
     
     
         55 . The process of one of claims  27 ,  28  and  29 , wherein the extracting step is carried out under reduced pressure.  
     
     
         56 . The process of one of claims  27 ,  28  and  29 , wherein the extracting step is carried out at a temperature of less than about 80° C.  
     
     
         57 . The process of  claim 27 , wherein the anion-exchanger is an anion-exchanger resin.  
     
     
         58 . The process of  claim 56 , wherein the anion-exchanger resin is Amberlite™ IRA.  
     
     
         59 - 63 . (canceled)