Extraction of proteins by a two solvent method

• US 8,551,336 B2
• Filed: 11/12/2012
• Issued: 10/08/2013
• Est. Priority Date: 04/06/2010
• Status: Expired due to Fees

• Current Assignee: Heliae Development, LLC (PT Perusahaan Perkebunan London Sumatra Indonesia Tbk)
• Original Assignee: Heliae Development, LLC (PT Perusahaan Perkebunan London Sumatra Indonesia Tbk)
• Inventors: Kale, Aniket
• Primary Examiner(s): DRODGE, JOSEPH W
• Application Number:
  US13/674,631
• Publication Number:
  US 20130072661A1
• Time in Patent Office: 330 Days
• Field of Search: 210/633, 210/634, 210/639, 210/743, 210/749, 210/774, 210/806, 210/651, 210/770, 210/787, 210/804, 435/134, 435/262, 435/267, 435/271, 435/272, 443/07, 446/05, 47/14, 554/12, 554/14, 554/20, 208/15, 208/49, 208/87, 208/133, 208/142, 203/39, 530/370
• US Class Current: 210/634
• CPC Class Codes: C11B 1/10 by extracting
• CPC Class Codes: C11B 1/108 after-treatment, e.g. of miscellae
• CPC Class Codes: C11C 3/003 by esterification of fatty acids with alcohols C11C3/02 takes precedence
• CPC Class Codes: C12N 1/06 Lysis of microorganisms

Abstract

A method for separating proteins from plant material, in particular, intact algal cells, using an amphipathic solvent set and a hydrophobic solvent set. Some embodiments include dewatering intact algal cells and then extracting proteins from the algal cells. The methods provide for single and multistep extraction processes which allow for efficient separation of algal proteins from a wet algal biomass. These proteins are high value products which can be used as renewable sources of food and food additives. Neutral lipids remaining in the algal biomass after extraction of proteins can be used to generate renewable fuels.

Claims

1. A method of separating proteins, polar lipids, and neutral lipids from intact algal cells, comprising:

providing a wet algal biomass comprising intact algal cells which comprise proteins that are soluble in a mixture of an amphipathic solvent and water, polar lipids and neutral lipids;

dewatering the wet algal biomass by removing extracellular water to increase the solid content of the wet algal biomass to between 5% and 50% to generate a partially dewatered wet algal biomass;

mixing the partially dewatered wet algal biomass with a first amphipathic solvent set in a ratio of about one part first amphipathic solvent set to about one part wet algal biomass by weight, and a first hydrophobic solvent set to generate a first extraction mixture comprising a first heavier phase and a first lighter phase, wherein the first heavier phase comprises the first amphipathic solvent set, neutral lipids, proteins that are soluble in a mixture of an amphipathic solvent and water, and partially dewatered wet algal biomass and the first lighter phase comprises the first hydrophobic solvent set and polar lipids;

separating the first lighter phase of the extraction mixture from the first heavier phase of the extraction mixture;

separating the polar lipids from the first lighter phase;

separating the partially dewatered wet algal biomass from the first amphipathic solvent set in the first heavier phase of the extraction mixture to generate a separated partially dewatered wet algal biomass comprising neutral lipids and a protein mixture comprising proteins that are soluble in a mixture of an amphipathic solvent and water, water and the first amphipathic solvent set;

separating the proteins that are soluble in a mixture of an amphipathic solvent and water from at least a portion of the water and first amphipathic solvent set of the protein mixture, wherein the protein mixture has a higher concentration of proteins that are soluble in a mixture of an amphipathic solvent and water than the separated partially dewatered wet algal biomass;

mixing the separated partially dewatered algal biomass with a second amphipathic solvent set in a ratio of about one part second amphipathic solvent set to about one part separated partially dewatered wet algal biomass by weight, and a second hydrophobic solvent set to generate a second extraction mixture comprising a second heavier phase and a second lighter phase, wherein the second heavier phase comprises the second amphipathic solvent set and separated partially dewatered wet algal biomass and the second lighter phase comprises the second hydrophobic solvent set and the neutral lipids;

separating the second heavier phase from the second lighter phase; and

separating the neutral lipids from the second lighter phase, wherein the first lighter phase has a higher concentration of polar lipids than the second lighter phase, and the second lighter phase has a higher concentration of neutral lipids than the first lighter phase.

2. The method of claim 1, wherein the dewatering of the wet algal biomass includes centrifuging, filtering, settling or float fractionating.

3. The method of claim 1, wherein at least one of the first and second amphipathic solvent set is acetone, methanol, ethanol, isopropanol, butanone, dimethyl ether, propionaldehyde, 2-propanol, acetonitrile, t-butyl alcohol, 1-propanol, water, heavy water (D₂O), ethylene glycol, glycerin or a combination thereof.

4. The method of claim 1, wherein at least one of the first and second hydrophobic solvent set is selected from the group consisting of propane, butane, pentane, butene, propene, naphtha, an alkane, hexane, pentane, heptane, octane, an ester, ethyl acetate, butyl acetate, a ketone, methyl ethyl ketone, methyl isobutyl ketone, an aromatic, toluene, benzene, cyclohexane, tetrahydrofuran, a haloalkane, chloroform, trichloroethylene, an ether, diethyl ether, diesel, jet fuel, gasoline and mixtures thereof.

5. The method of claim 1, wherein the second heavier phase is separated from the second lighter phase by decanting, membrane filtration or centrifugation.

6. The method of claim 1, wherein the second lighter phase is heated prior to separating the neutral lipids from the second lighter phase.

7. The method of claim 1, wherein the second hydrophobic solvent set is removed from the second lighter phase by evaporation or distillation, thereby generating a neutral lipid fraction.

8. The method of claim 1, wherein at least one of the first and second amphipathic solvent set is evaporated.

9. The method of claim 1, wherein at least one of the first and second hydrophobic solvent set is recovered.

10. The method of claim 1, wherein at least one of the first and second hydrophobic solvent set is chilled and recovered.

11. The method of claim 1, wherein at least one of the first and second amphipathic solvent set is chilled and recovered.

12. The method of claim 1, wherein at least one of the first and second amphipathic solvent set is recovered.

13. The method of claim 1, wherein at least one of the first and second extraction mixture is heated.

14. The method of claim 13, wherein at least one of the first and second extraction mixture is heated with microwaves, water, steam, or hot oil or electricity.

15. The method of claim 13, wherein at least one of the first and second extraction mixture is heated at atmospheric pressure.

16. The method of claim 13, wherein at least one of the first and second extraction mixture is heated in a pressurized reactor.

17. The method of claim 16, wherein the pressurized reactor is a batch or a continuous reactor.

18. A method of separating proteins, polar lipids, and neutral lipids from intact algal cells, comprising:

providing a wet algal biomass comprising intact algal cells which comprise proteins that are soluble in a mixture of an amphipathic solvent and water, polar lipids and neutral lipids;

mixing the wet algal biomass with a first amphipathic solvent set in a ratio of about one part first amphipathic solvent set to about one part wet algal biomass by weight, and a first hydrophobic solvent set to generate a first extraction mixture comprising a first heavier phase and a first lighter phase, wherein the first heavier phase comprises the first amphipathic solvent set, neutral lipids, proteins that are soluble in a mixture of an amphipathic solvent and water, and wet algal biomass and the first lighter phase comprises the first hydrophobic solvent set and the polar lipids;

separating the first lighter phase of the extraction mixture from the first heavier phase of the extraction mixture;

separating the polar lipids from the first lighter phase;

separating the wet algal biomass from the first heavier phase of the extraction mixture to generate a separated wet algal biomass comprising neutral lipids and a protein mixture comprising proteins that are soluble in a mixture of an amphipathic solvent and water, water and the first amphipathic solvent set;

separating the proteins that are soluble in a mixture of an amphipathic solvent and water from at least a portion of the water and first amphipathic solvent set of the protein mixture, wherein the protein mixture has a higher concentration of proteins that are soluble in a mixture of an amphipathic solvent and water than the separated wet algal biomass;

mixing the separated algal biomass with a second amphipathic solvent set and a second hydrophobic solvent set in a ratio of about one part amphipathic solvent set to about one part separated wet algal biomass to generate a second extraction mixture comprising a second heavier phase and a second lighter phase, wherein the second heavier phase comprises the second amphipathic solvent set and separated wet algal biomass and the second lighter phase comprises the second hydrophobic solvent set and the neutral lipids;

separating the second heavier phase from the second lighter phase; and

separating the neutral lipids from the second lighter phase to generate a neutral lipids fraction, wherein the first lighter phase has a higher concentration of polar lipids than the second lighter phase, and the second lighter phase has a higher concentration of neutral lipids than the first lighter phase.