Production of docosahexaenoic acid by dinoflagellates

• US 5,407,957 A
• Filed: 02/13/1990
• Issued: 04/18/1995
• Est. Priority Date: 02/13/1990
• Status: Expired due to Term

• Current Assignee: Martek Biosciences Corporation (DSM-Firmenich AG)
• Original Assignee: Martek Biosciences Corporation (DSM-Firmenich AG)
• Inventors: Kyle, David J., Sicotte, Valerie J., Reeb, Sue E.
• Primary Examiner(s): Robinson, Douglas W.
• Assistant Examiner(s): Weber, Jon P.
• Application Number:
  US07/479,135
• Time in Patent Office: 1,890 Days
• Field of Search: 435/134, 435/136, 560/205, 560/190, 560/191, 426/33, 426/601, 514/549, 514/552, 514/547
• US Class Current: 514/547
• CPC Class Codes: A23D 9/00 Other edible oils or fats, e.g. shortenings, cooking oils
• CPC Class Codes: A23K 20/158 Fatty acids; Fats; Products containing oils or fats
• CPC Class Codes: A23K 50/80 for aquatic animals, e.g. fish, crustaceans or molluscs
• CPC Class Codes: A23L 33/115 Fatty acids or derivatives thereof; Fats or oils
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/02 Nutrients, e.g. vitamins, minerals
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C11B 1/10 by extracting
• CPC Class Codes: C12N 1/125 Unicellular algae isolates
• CPC Class Codes: C12P 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12R 2001/89 Algae ; Processes using algae
• CPC Class Codes: Y10S 426/801 Pediatric
• CPC Class Codes: Y10S 426/805 Pet food for dog, cat, bird, or fish
• CPC Class Codes: Y10S 435/946 using algae

Abstract

This invention relates to a process for producing a single cell edible oil containing DHA, to the oil itself and to uses for the oil. Marine microorganisms are cultivated in fermentors and induced to produce the single cell oil which subsequently is recovered by extraction with solvents.

Claims

1. A method of producing a single cell edible oil containing at least about 20% docosahexaenoic acid (DHA) in triglyceride form comprising:

cultivating heterotrophic microalgae of the class Dinophyceae capable of producing said single cell oil in an aerated fermentor containing a nutrient solution having a limiting nitrogen source and an oxygen level of at least about 10% of air saturation level and continuing cultivation to achieve a cell density of at least about 10 grams biomass per liter of nutrient solution,

wherein the concentration of the nitrogen source in the nutrient solution is limited sufficiently to induce said microalgae to produce the single cell oil at a concentration of at least about 2 grams per liter of nutrient solution, and

recovering said single cell oil.

2. The method of claim 1, wherein the microalgae is of the genus Crypthecodinium.

3. The method of claim 2, wherein said microalgae comprises Crypthecodinium cohnii.

4. The method of claim 3, wherein said nutrient solution comprises seawater.

5. The method of claim 4, wherein said seawater comprises an artificial seawater.

6. The method of claim 5, wherein said nutrient solution comprises a one-quarter strength solution of artificial seawater.

7. The method of claim 4, wherein said nutrient solution further comprises a reduced carbon source.

8. The method of claim 7, wherein said reduced carbon source comprises glucose.

9. The method of claim 4, wherein said limiting nutrient comprises an organic nitrogen source.

10. The method of claim 9, wherein said nitrogen source comprises yeast extract.

11. The process of claim 10, wherein the ratio of said glucose to said yeast extract is from about 2 to 4 parts glucose to 1 part yeast extract.

12. The method of claim 11, further comprising maintaining a dissolved oxygen content at a range of from about 10% to about 50% of air saturation.

13. The method of claim 12, wherein said range is from about 20-30%.

14. The method of claim 13, wherein said fermentor is a stirred tank fermentor having a turbine.

15. The method of claim 14, wherein said turbine agitates said nutrient solution, thereby providing oxygen to said microalgae.

16. The method of claim 1, wherein said aerated fermentor is aerated at a rate of 1 volume of air per volume of fermentor per minute.

17. The method of claim 1, wherein said nutrient solution comprises ¹³ C-glucose or ¹⁴ C-glucose.

18. The method of claim 1, wherein the microalgae is cultivated to a density of at least about 20 g/liter of nutrient solution.

25. A single cell-edible oil comprising at least 20% docosahexaenoic acid in triglyceride form, said oil produced according to the method of claim 1 or the method of claim 19.

19. A method for the production of a single-cell-edible oil containing at least 20% DHA in triglyceride form and lacking a fishy taste comprising:

a) adding to a fermentor initially containing a nutrient solution comprising about one-quarter strength artificial seawater, 1-2% glucose and 0.4-0.8% yeast extract, about 0.5-1.0 g dry wt/liter (about 10⁵ cells/ml) of C. cohnii,

b) cultivating said C. cohnii at a temperature of from about 15°

C. to about 34°

C. and a pH of from about 5.0 to 9.0,

c) incrementally adding glucose and yeast extract to said nutrient solution for about 56 hours,

d) adding additional glucose to said nutrient solution for about 16 additional hours to induce said C. cohnii to produce a single-cell edible oil,

e) maintaining a dissolved oxygen content of about 20-30% of air saturation level throughout said cultivation,

f) harvesting said C. cohnii after about 60 to 90 hours, and

g) recovering said single-cell edible oil.

20. The process of claim 19, further comprising extracting said single cell edible oil from said C.cohnii.

21. The process of claim 20, wherein said extraction is by treatment with a solvent such that said oil is extracted into said solvent.

22. The process of claim 21, wherein said solvent comprises hexane.

23. The process of claim 22, wherein said hexane is applied to said C. cohnii at a rate of about 4 liters per kilogram of dry biomass of said C. cohnii and is mixed with said biomass in a stirred reaction vessel for about 2 hours at about 50°

C.

24. The process of claim 23, further comprising filtering said hexane containing extracted oil from said biomass and then removing said hexane by distillation from said oil.