Purifying process for phosphatidylserine

Abstract

The present invention relates to a purifying process for phosphatidylserine, the latter being prepared by trans-phosphatidylation of phosphatidylcholine with serine in presence of the enzyme D-phospholipase and containing as impurities hydrophilic compounds, proteins and inorganic salts.

1. Purifying process for phosphatidylserine having formula (I) 
       
         
           
           
               
               
           
         
       
       where R 1  and R 2 , identical or different, are a C 10 -C 30  acyl group; X is OH or OM, where M is chosen from the group of alkali metals, alkaline-earth metals, ammonium and alkyl ammonium, 
       and where the serine portion is in D, L or racemic form, comprising the extraction of said phosphatidylserines from a solution in a hydrocarbon solvent chosen among aromatic and aliphatic hydrocarbon solvents, with a mixture of water and an alcohol solvent. 
     
     
       2. Process according to  claim 1 , in which said hydrocarbon solvent is selected from the group consisting of toluene, xylene, n-heptane, n-hexane and cyclohexane. 
     
     
       3. Process according to  claim 1 , in which said hydrocarbon solvent is used in an amount between 4 and 30 liters/kg of phosphatidylserine to be purified. 
     
     
       4. Process according to  claim 3 , in which said hydrocarbon solvent is used in an amount between 6 and 12 liters/kg of phosphatidylserine to be purified. 
     
     
       5. Process according to  claim 1 , in which said alcohol solvent is chosen among alcohols containing 1 to 5 carbon atoms. 
     
     
       6. Process according to  claim 1 , in which said alcohol solvent is chosen among secondary and tertiary alcohols. 
     
     
       7. Process according to  claim 1 , in which said alcohol solvent is isopropanol. 
     
     
       8. Process according to  claim 1 , in which said polar organic solvent is used in an amount between 0.2 and 2 liters/kg of hydrocarbon solvent used. 
     
     
       9. Process according to  claim 8 , in which said polar organic solvent is used in an amount between 0.3 and 1.2 liters/kg of hydrocarbon solvent used. 
     
     
       10. Process according to  claim 1 , in which the amount of water used is between 0.2 and 5 liters/kg of hydrocarbon solvent used. 
     
     
       11. Process according to  claim 10 , in which the amount of water used is between 0.3 and 1 liter/kg of hydrocarbon solvent used. 
     
     
       12. Process according to  claim 1 , in which said extraction is carried out at a temperature between 0 and 70° C. 
     
     
       13. Process according to  claim 12 , in which said extraction is carried out at a temperature between 20 and 30° C. 
     
     
       14. Process according to  claim 1 , in which said phosphatidylserines having formula (I) are prepared by trans-phosphatidylation of phosphatidylcholines of natural or synthetic origin. 
     
     
       15. Process according to  claim 1 , in which the said serine portion is in L form. 
     
     
       16. A purifying process for phosphatidylserine having formula (I) 
       
         
           
           
               
               
           
         
         where R 1  and R 2 , identical or different, are C 10 -C 30  acyl groups; 
         X is OH or OM, where M is chosen from the group consisting of alkali-earth metals, ammonium and alkyl ammonium; and 
         where the serine portion is in D, L or racemic form; which comprises stirring said phosphatidylserine in a mixture comprising water, an alcohol solvent, and a hydrocarbon solvent selected from the group consisting of aromatic and aliphatic hydrocarbon solvents. 
       
     
     
       17. The process of  claim 16 , in which said hydrogen solvent is selected from the group consisting of toluene, xylene, n-heptane, n-hexane and cyclohexane. 
     
     
       18. The process of  claim 16 , in which said hydrogen solvent is used in an amount between 4 and 30 liters per kilogram of phosphatidylserine to be purified. 
     
     
       19. The process of  claim 18 , in which said hydrogen solvent is used in an amount between 6 and 12 liters per kilogram of phosphatidylserine to be purified. 
     
     
       20. The process of  claim 16 , in which said alcohol solvent in an alcohol containing 1 to 5 carbon atoms. 
     
     
       21. The process of  claim 16 , in which said alcohol solvent is selected from the group consisting of secondary and tertiary alcohols. 
     
     
       22. The process of  claim 16 , in which said alcohol is isopropanol. 
     
     
       23. The process of  claim 16 , in which said polar organic solvent is used in an amount between 0.2 and 2 liters per kilogram of hydrocarbon solvent used. 
     
     
       24. The process of  claim 23 , in which said polar organic solvent is used in an amount between 0.3 and 1.2 liters per kilogram of hydrocarbon solvent used. 
     
     
       25. The process of  claim 16 , in which the amount of water used is between 0.2 and 5 liters per kilogram of hydrocarbon solvent used. 
     
     
       26. The process of  claim 25 , in which the amount of water used is between 0.3 and 1 liters per kilogram of hydrocarbon solvent used. 
     
     
       27. The process of  claim 16 , in which said stirring is carried out at a temperature between 0 and 70 ° C. 
     
     
       28. The process of  claim 27 , in which said stirring is carried out at a temperature between 20 and 30 ° C. 
     
     
       29. The process of  claim 16 , in which said phosphatidylserine is prepared by transphatidylation of phosphatidylcholines of natural or synthetic origin. 
     
     
       30. The process of  claim 16 , in which said serine portion is in L form. 
     
     
       31. The process of  claim 1 , further comprising precipitation of phosphatidylserine with acetone, after separation of the solvent phases. 
     
     
       32. The process of  claim 16 , further comprising precipitation of phosphatidylserine with acetone, after separation of the solvent phases. 
     
     
       33. Purifying process for phosphatidylserine having formula (I) 
       
         
           
           
               
               
           
         
       
       where R 1  and R 2 , identical or different, are a C 10 -C 30  acyl group; X is OH or OM, where M is chosen from the group of alkali metals, alkaline-earth metals, ammonium and alkyl ammonium, and where the serine portion is in D, L or racemic form, which consists of:
 a) stirring said phosphatidylserine in a mixture of water, an hydrocarbon solvent chosen among aromatic and aliphatic hydrocarbon solvents, and an alcohol solvent, selected from the group consisting of secondary alcohols containing 3 to 5 carbon atoms;   b) settling the mixture; and   c) isolating phosphatidylserine after separation of the hydrocarbon phase and the aqueous phase;   wherein said phosphatidylserine is prepared by transphosphatidylation of phosphatidylcholine of natural or synthetic origin with serine in the presence of D-phospholipase, and further wherein said purifying process removes the phospholipase and serine which remain in the aqueous phase.   
     
     
       34. Process according to claim 33, in which said hydrocarbon solvent is selected from the group consisting of toluene, n-heptane, n-hexane and cyclohexane. 
     
     
       35. Process according to claim 33, in which said hydrocarbon solvent is used in an amount between 4 and 30 liters/kg of phosphatidylserine to be purified. 
     
     
       36. Process according to claim 35, in which said hydrocarbon solvent is used in an amount between 6 and 12 liters/kg of phosphatidylserine to be purified. 
     
     
       37. Process according to claim 33, in which said hydrocarbon solvent is n-heptane. 
     
     
       38. Process according to claim 33, in which said alcohol solvent is isopropanol. 
     
     
       39. Process according to claim 33, in which said alcohol solvent is used in an amount between 0.2 and 2 liters/kg of hydrocarbon solvent used. 
     
     
       40. Process according to claim 35, in which said alcohol solvent is used in an amount between 0.3 and 1.2 liters/kg of hydrocarbon solvent used. 
     
     
       41. Process according to claim 33, in which the amount of water used is between 0.2 and 5 liters/kg of hydrocarbon solvent used. 
     
     
       42. Process according to claim 41, in which the amount of water used is between 0.3 and 1 liter/kg of hydrocarbon solvent used. 
     
     
       43. Process according to claim 33, in which said purification process is carried out at a temperature between 0 and 70° C. 
     
     
       44. Process according to claim 43, in which said purification process is carried out at a temperature between 20 and 30° C. 
     
     
       45. Process according to claim 33, wherein isolation of said phosphatidylserine includes precipitation with acetone. 
     
     
       46. A purifying process according to claim 33 which consists of:
 a) stirring a solution of phosphatidylserine in a hydrocarbon solution chosen among aromatic and aliphatic hydrocarbon solvents with a mixture of water, and an alcohol solvent selected from the group consisting of secondary alcohols containing 3 to 5 carbon atoms;   b) settling the mixture; and   c) isolating phosphatidylserine after separation of the hydrocarbon phase and the aqueous phase;   wherein said solution in a hydrocarbon solvent of phosphatidylserine is prepared by transphosphatidylation of phosphatidylcholine of natural or synthetic origin with serine in the presence of D-phospholipase in a two-phase system, and further wherein said purifying process removes the phospholipase and serine which remain in the aqueous phase.   
     
     
       47. The process of claim 46, in which said hydrocarbon solvent is selected from the group consisting of toluene, n-heptane, n-hexane and cyclohexane. 
     
     
       48. The process of claim 46, in which said hydrocarbon solvent is used in an amount between 4 and 30 liters per kilogram of phosphatidylserine to be purified. 
     
     
       49. The process of claim 48, in which said hydrocarbon solvent is used in an amount between 6 and 12 liters per kilogram of phosphatidylserine to be purified. 
     
     
       50. The process of claim 46, in which said hydrocarbon solvent is n-heptane. 
     
     
       51. The process of claim 46, in which said alcohol solvent is isopropanol. 
     
     
       52. The process of claim 46, in which said alcohol solvent is used in an amount between 0.2 and 2 liters per kilogram of hydrocarbon solvent used. 
     
     
       53. The process of claim 52, in which said alcohol solvent is used in an amount between 0.3 and 1.2 liters per kilogram of hydrocarbon solvent used. 
     
     
       54. The process of claim 46, in which the amount of water used is between 0.2 and 5 liters per kilogram of hydrocarbon solvent used. 
     
     
       55. The process of claim 54, in which the amount of water used is between 0.3 and 1 liters per kilogram of hydrocarbon solvent used. 
     
     
       56. The process of claim 46, in which said purification process is carried out at a temperature between 0 and 70° C. 
     
     
       57. The process of claim 56, in which said purification process is carried out at a temperature between 20 and 30° C. 
     
     
       58. The process of claim 46, wherein isolation of said phosphatidylserine includes precipitation with acetone.