Eukaryotic microorganisms for producing lipids and antioxidants

• US 8,921,069 B2
• Filed: 03/22/2012
• Issued: 12/30/2014
• Est. Priority Date: 06/07/2005
• Status: Active Grant

• Current Assignee: DSM Nutritional Products AG (DSM-Firmenich AG)
• Original Assignee: DSM Nutritional Products AG (DSM-Firmenich AG)
• Inventors: Radianingtyas, Helia, Barrow, Colin James, Windust, Anthony James, Burja, Adam M.
• Primary Examiner(s): Shen, Bin
• Application Number:
  US13/427,562
• Publication Number:
  US 20130344546A1
• Time in Patent Office: 1,013 Days
• Field of Search: 435/41
• US Class Current: 435/41
• CPC Class Codes: A21D 8/04 treating dough with microorganisms or enzymes
• CPC Class Codes: A23K 10/18 of live microorganisms
• CPC Class Codes: A23K 20/158 Fatty acids; Fats; Products containing oils or fats
• CPC Class Codes: A23L 29/065 Microorganisms addition of bacteria for nutritional purposes A23L33/135
• CPC Class Codes: A23L 33/115 Fatty acids or derivatives thereof; Fats or oils
• CPC Class Codes: A23L 33/12 Fatty acids or derivatives thereof
• CPC Class Codes: A23L 33/135 Bacteria or derivatives thereof, e.g. probiotics
• CPC Class Codes: A23P 10/30 Encapsulation of particles, e.g. foodstuff additives
• CPC Class Codes: A23V 2002/00 Food compositions, function of food ingredients or processes for food or foodstuffs
• CPC Class Codes: A23V 2200/02 Antioxidant
• CPC Class Codes: A23V 2200/3262 having an effect on blood cholesterol
• CPC Class Codes: A23V 2200/328 having effect on glycaemic control and diabetes
• CPC Class Codes: A23V 2200/332 Promoters of weight control and weight loss
• CPC Class Codes: A61K 31/20 having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
• CPC Class Codes: A61K 9/4875 Compounds of unknown constitution, e.g. material from plants or animals oils, fats, waxes, shellac A61K9/4858
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/02 Nutrients, e.g. vitamins, minerals
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/10 : for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 39/06 : Free radical scavengers or antioxidants
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: C12N 1/00 : Microorganisms, e.g. protozoa; Compositions thereof medicinal preparations containing material from protozoa, bacteria or viruses A61K35/66, from algae A61K36/02, from fungi A61K36/06; preparing medicinal bacterial antigen or antibody compositions, e.g. bacterial vaccines, A61K39/00; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
• CPC Class Codes: C12P 7/6427 : Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12R 2001/00 : Microorganisms ; Processes using microorganisms

Abstract

Disclosed are compositions and methods related to eukaryotic microorganisms that can produce unsaturated fatty acids which can be purified and used.

Claims

1. A method of increasing biomass production of an isolated eukaryotic microorganism, the method comprising:

(i) culturing in a heterotrophic medium an isolated eukaryotic microorganism having an 18S sequence, wherein the 18S sequence has at least 97% identity to the sequence set forth in SEQ ID NO;

1; and

(ii) increasing agitation of the culture to 100 to 1000 rpm, increasing the concentration of dO2 in the culture to between 1 and 10%, increasing the temperature of the culture to 4 to 30°

C., or a combination thereof, wherein biomass production is increased.

2. The method of claim 1, further comprising isolating the biomass.

3. The method of claim 2, wherein isolating the biomass comprises harvesting the eukaryotic microorganism.

4. The method of claim 1, wherein the isolated eukaryotic microorganism has ATCC accession number PTA-6245.

5. The method of claim 1, wherein culturing decreases docosahexaenoic acid (DHA) production.

6. The method of claim 1, wherein the heterotrophic medium comprise a carbon source in an amount of from 1 g L⁻

1 to 60 g L<sup>−

1</sup>.

7. The method of claim 1, wherein the heterotrophic medium comprise a carbon source in an amount of from 1 g L⁻

1 to 200 g L<sup>−

1</sup>.

8. The method of claim 7, wherein the harvested eukaryotic microorganism is ruptured or permeabilized.

9. The method of claim 7, wherein the harvested eukaryotic microorganism is ruptured or permeabilized by sonication, liquid-shear disruption methods, bead milling, pressing under high pressure, freeze-thawing, or enzymatic digestion of the cell wall.

10. A method of increasing fatty acid production of an isolated eukaryotic microorganism, the method comprising:

(i) culturing in a heterotrophic medium an isolated eukaryotic microorganism having an 18S sequence, wherein the 18S sequence has at least 97% identity to the sequence set forth in SEQ ID NO;

1; and

(ii) increasing the agitation of the culture to 100 to 1000 rpm, increasing the concentration of dO2 in the culture to between 1 and 10%, increasing the temperature of the culture to 4 to 30°

C., or a combination thereof, wherein fatty acid production is increased.

11. The method of claim 10, further comprising isolating the fatty acids.

12. The method of claim 10, wherein the isolated eukaryotic microorganism has ATCC accession number PTA-6245.

13. The method of claim 10, wherein culturing decreases DHA production.

14. The method of claim 10, wherein the heterotrophic medium comprise a carbon source in an amount of from 1 g L⁻

1 to 60 g L<sup>−

1</sup>.

15. The method of claim 10, wherein the heterotrophic medium comprise a carbon source in an amount of from 1 g L⁻

1 to 200 g L<sup>−

1</sup>.

16. A method of increasing lipid production of an isolated eukaryotic microorganism, the method comprising:

(i) culturing in a heterotrophic medium an isolated eukaryotic microorganism having an 18S sequence, wherein the 18S sequence has at least 97% identity to the sequence set forth in SEQ ID NO;

1; and

(ii) increasing the agitation of the culture to 100 to 1000 rpm, increasing the concentration of dO2 in the culture to between 1 and 10%, increasing the temperature of the culture to 4 to 30°

C., or a combination thereof, wherein lipid production is increased.

17. The method of claim 16, further comprising isolating the lipid.

18. The method of claim 16, wherein the isolated eukaryotic microorganism has ATCC accession number PTA-6245.

19. The method of claim 16, wherein culturing decreases DHA production.

20. The method of claim 16, wherein the lipid is isolated with a solvent.

21. The method of claim 20, wherein the solvent comprises chloroform, hexane, methanol, ethanol, or supercritical CO₂.

22. The method of claim 21, further comprising hydrolyzing the lipid, wherein hydrolyzing the lipid composition converts triglycerides to free fatty acids or esters of fatty acids.

23. The method of claim 20, further comprising removing the solvent.

24. The method of claim 16, wherein the heterotrophic medium comprise a carbon source in an amount of from 1 g L⁻

1 to 60 g L<sup>−

1</sup>.

25. The method of claim 16, wherein the heterotrophic medium comprise a carbon source in an amount of from 1 g L⁻

1 to 200 g L<sup>−

1</sup>.

26. A method of decreasing biomass production of an isolated eukaryotic microorganism, the method comprising:

(i) culturing in a heterotrophic medium an isolated eukaryotic microorganism having an 18S sequence, wherein the 18S sequence has at least 97% identity to the sequence set forth in SEQ ID NO;

1; and

(ii) decreasing agitation of the culture to 100 to 1000 rpm, decreasing the concentration of dO2 in the culture to between 1 and 10%, decreasing the temperature of the culture to 4 to 30°

C., or a combination thereof, wherein biomass production is decreased.

27. The method of claim 26, further comprising isolating the biomass.

28. The method of claim 27, wherein isolating the biomass comprises harvesting the eukaryotic microorganism.

29. The method of claim 26, wherein the isolated eukaryotic microorganism has ATCC accession number PTA-6245.

30. The method of claim 27, wherein culturing increases DHA production.

31. The method of claim 26, wherein the heterotrophic medium comprise a carbon source in an amount of from 1 g L⁻

1 to 60 g L<sup>−

1</sup>.

32. The method of claim 26, wherein the heterotrophic medium comprise a carbon source in an amount of from 1 g L⁻

1 to 200 g L<sup>−

1</sup>.

33. The method of claim 32, wherein the harvested eukaryotic microorganism is ruptured or permeabilized.

34. The method of claim 32, wherein the harvested eukaryotic microorganism is ruptured or permeabilized by sonication, liquid-shear disruption methods, bead milling, pressing under high pressure, freeze-thawing, or enzymatic digestion of the cell wall.