Method for obtaining a TGF-beta enriched protein fraction in activated form, protein fraction and therapeutic applications
The invention concerns a method for obtaining a highly enriched TGF-beta protein fraction in activated form, from a liquid solution rich in proteins said to be soluble in the aqueous phase of milk and/or of whey, said method comprising the following steps; a) adjusting soluble proteins purified at a concentration between 5 and 30 g/liter of solution; b) precipitating part of the whey proteins by acidic treatment of the solution thus obtained to a pH ranging between 4 and 5.5 and at a temperature ranging between 55° C. and 68° C.; c) carrying out a microfiltration of the treated solution by diafiltration, so as to obtain respectively a microfiltration retentate and a microfiltrate; d) recuperating the microfiltration retentate containing the protein fraction highly enriched in TGF-beta; e) drying the microfiltration retentate which has been subjected to diafiltration to obtain a powder highly enriched in TGF-beta.
1. A method for obtaining a milk aqueous phase protein fraction enriched in TGFβ in activated form from a solution rich in milk aqueous phase soluble proteins, said method comprising the steps of:
a) adjusting the protein concentration of said solution rich in milk aqueous phase soluble proteins to a concentration between 5 and 30 g/liter of solution;
b) precipitating a part of said milk aqueous phase by acid treatment of said solution at a pH ranging between 4 and 5.5, at a temperature ranging between 55° C. and 68° C., and for a period of time between 30 seconds to 10 minutes, so as to obtain a precipitated suspension;
c) carrying out microfiltration on the precipitated suspension, so as to obtain a microfiltration retentate;
d) carrying out diafiltration on the microfiltration retentate, so as to obtain a diafiltrated microfiltration retentate;
e) recovering said diafiltrated microfiltration retentate comprising said milk aqueous phase protein fraction enriched in TGFβ; and
f) drying said diafiltrated microfiltration retentate, to obtain a powder enriched in TGFβ.
2. The method according to claim 1 , wherein said precipitation step b) is performed at a pH between 4 and 5.
3. The method according to claim 1 , wherein said precipitation step b) is performed at a temperature of about 63° C.
4. The method according to claim 1 , wherein at the end of step b), said liquid solution is cooled down in a time period ranging from 1 to 20 minutes.
5. The method according to claim 1 , wherein said microfiltration of step c) is performed using a membrane having an average pore size between 0.8 and 1.6 μm and step c) is performed in an equipment leading to a uniform transmembrane pressure.
6. The method according to claim 1 , wherein said diafiltration of step d) is performed using osmosed or demineralized filtrated water.
7. The method according to claim 1 , wherein said microfiltration and diafiltration are carried out at a pH between 4 and 5.5.
8. The method according to claim 1 , wherein said precipitating step b) is performed at a pH of about 4.6.
9. The method according to claim 1 , wherein the duration of said precipitating of step b) is between 30 seconds and 5 minutes.
10. The method according to claim 1 , wherein the duration of said precipitating of step b) is about 2 minutes.
11. The method according to claim 1 , wherein said microfiltration and diafiltration are carried out at a pH of about 4.6.
12. The method according to claim 1 , wherein said solution rich in milk aqueous phase soluble proteins is treated before step a) by performing the following steps:
a) carrying out cross-flow microfiltration of said solution rich in milk aqueous soluble proteins on a microfiltration membrane having an average pore size of 0.1 μm and said cross-flow microfiltration is performed on an equipment which will obtain a uniform transmembrane pressure so as to obtain a microfiltrate, and then
b) concentrating the microfiltrate obtained in step a) by ultrafiltration and diafiltration so as to obtain a concentrated retentate.
13. The method of claim 12 , wherein said solution rich in milk aqueous phase soluble proteins is skimmed milk.