Methods for extracting keratin proteins
Described herein are methods to produce keratin protein-based biomaterials, the parameters required to achieve improved extraction, the parameters required to improve isolation, the parameters of lyophilization and the grinding process to achieve consistent particulate sizes of protein materials.
1. A method for extracting keratin proteins comprising:
(a) treating a keratin protein source with an oxidizing or reducing agent to solubilize keratin proteins;
(b) treating the keratin protein source with an extraction agent to extract the solubilized keratin proteins,
wherein the extraction agent comprises:
(i) an aqueous solution of a denaturing agent if the treating in step (a) comprises mixing with the oxidizing agent; or
(ii) a base if the treating in step (a) comprises mixing with the reducing agent, and
(c) separating the soluble proteins from the keratin protein source by high speed centrifugation to produce a clarified soluble keratin protein solution;
(d) dialyzing the clarified soluble keratin protein solution under conditions of applied transmembrane pressure to fractionate the soluble keratin proteins; and
(e) lyophilizing the fractionated soluble keratin protein solution into a keratin protein cake.
2. The method of claim 1 , wherein said keratin protein source is selected from the group consisting of hair, wool, fur, horns, hooves, beaks, feathers, and scales.
3. The method of claim 1 , wherein said high speed centrifugation is performed at 5,000 rpm or higher.
4. The method of claim 1 , wherein said dialyzing is performed at a temperature from about 4° C. to about 20° C.
5. The method of claim 1 , wherein said method comprises treating with the oxidizing reagent at step (a).
6. The method of claim 5 , wherein said oxidizing reagent comprises peracetic acid.
7. The method of claim 5 wherein said clarified protein solution is substantially free of the oxidizing reagent.
8. The method of claim 1 , wherein said keratin protein is keratose.
9. The method of claim 1 , wherein said method comprises treating with the reducing agent at step (a).
10. The method of claim 9 , wherein said clarified protein solution is substantially free of the reducing agent.
11. The method of claim 9 , wherein said clarified protein solution comprises at least 90% or more kerateine.
12. The method of claim 1 , wherein said keratin protein is kerateine.
13. The method of claim 1 , further comprising:
(f) grinding the keratin protein cake.
14. The method of claim 13 , further comprising:
(g) deionizing the ground keratin protein cake.
15. The method of claim 1 , wherein the pressure at the step of dialyzing is between 10 psi and 70 psi.
16. The method of claim 15 , wherein the pressure is between about 30 psi to about 40 psi.
17. The method of claim 15 , wherein the pressure is between about 50 psi to about 70 psi.
18. The method of claim 16 , wherein the dialyzing employs a 100 kDa molecular weight membrane.
19. The method of claim 18 , wherein permeate of the dialyzing is retained and the permeate is subjected to a further step of dialysis employing a 5 kDa molecular weight membrane.
20. The method of claim 18 , wherein said keratin protein comprises 95% or more alpha-keratose.
21. The method of claim 19 , wherein said keratin protein comprises 95% or more gamma-keratose.
22. The method of claim 18 , wherein said keratin protein comprises 95% or more alpha-kerateine.
23. The method of claim 19 , wherein keratin protein comprises 95% or more gamma-kerateine.