Process for the Heterotrophic Production of Microbial Products with High Concentrations of Omega-3 Highly Unsaturated Fatty Acids

• US 20070082384A1
• Filed: 12/08/2006
• Published: 04/12/2007
• Est. Priority Date: 10/16/1992
• Status: Active Grant

• Current Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Original Assignee: Martek Biosciences Corporation (DSM-Firmenich AG)
• Inventors: Barclay, William
• Granted Patent: US 8,129,172 B2
• Time in Patent Office: Days
• US Class Current: 435/134
• CPC Class Codes: A23K 10/16 Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions A23K10/12 takes precedence
• CPC Class Codes: A23K 20/158 Fatty acids; Fats; Products containing oils or fats
• CPC Class Codes: A23K 40/20 by moulding, e.g. making cakes or briquettes
• CPC Class Codes: A23K 40/25 by extrusion
• CPC Class Codes: A23K 50/00 Feeding-stuffs specially adapted for particular animals
• CPC Class Codes: A23K 50/10 for ruminants
• CPC Class Codes: A23K 50/75 for poultry
• CPC Class Codes: A23K 50/80 for aquatic animals, e.g. fish, crustaceans or molluscs
• CPC Class Codes: A23L 11/07 Soya beans, e.g. oil-extracted soya bean flakes A23L11/30 takes precedence
• CPC Class Codes: A23L 13/00 Meat products; Meat meal; Preparation or treatment thereof
• CPC Class Codes: A23L 13/43 Addition of vegetable fats or oils; Addition of non-meat animal fats or oils; Addition of fatty acids
• CPC Class Codes: A23L 13/50 Poultry products, e.g. poultry sausages
• CPC Class Codes: A23L 15/20 Addition of proteins, e.g. hydrolysates, fats, carbohydrates, natural plant hydrocolloids; Addition of animal or vegetable substances containing proteins, fats, or carbohydrates
• CPC Class Codes: A23L 17/00 Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
• CPC Class Codes: A23L 17/40 Shell-fish
• CPC Class Codes: A23L 17/65 Addition of, or treatment with, microorganisms or enzymes
• CPC Class Codes: A23L 19/09 Mashed or comminuted products, e.g. pulp, purée, sauce, or products made therefrom, e.g. snacks A23L19/01, A23L19/10, A23L27/63 and A23L21/00 take precedence
• CPC Class Codes: A23L 25/30 Mashed or comminuted products, e.g. pulp, pastes, meal, powders; Products made therefrom, e.g. blocks, flakes, snacks; Liquid or semi-liquid products A23L25/10 and A23L25/40 take precedence
• CPC Class Codes: A23L 29/065 Microorganisms addition of bacteria for nutritional purposes A23L33/135
• CPC Class Codes: A23L 33/12 Fatty acids or derivatives thereof
• 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 31/202 : having three or more double bonds, e.g. linolenic eicosanoids, e.g. leukotrienes A61K31/557
• CPC Class Codes: C11B 1/10 : by extracting
• CPC Class Codes: C12N 1/10 : Protozoa; Culture media therefor
• CPC Class Codes: C12N 1/12 : Unicellular algae; Culture media therefor as new plants A01H13/00
• CPC Class Codes: C12N 1/14 : Fungi culture of mushrooms A01G18/00; as new plants A01H15/00; Culture media therefor
• CPC Class Codes: C12P 7/6409 : Fatty acids
• CPC Class Codes: C12P 7/6432 : Eicosapentaenoic acids [EPA]
• CPC Class Codes: C12P 7/6463 : obtained from glyceride producing microorganisms, e.g. single cell oil
• 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: Y02A 40/818 : Alternative feeds for fish, e.g. in aquacultures
• CPC Class Codes: Y02E 50/10 : Biofuels, e.g. bio-diesel
• CPC Class Codes: Y10S 426/805 : Pet food for dog, cat, bird, or fish
• CPC Class Codes: Y10S 426/807 : Poultry or ruminant feed
• CPC Class Codes: Y10S 435/911 : using fungi
• CPC Class Codes: Y10S 435/946 : using algae

Abstract

Disclosed is a process for growing the microflora Thraustochytrium, Schizochytrium, and mixtures thereof, which includes the growing of the microflora in fermentation medium containing non-chloride containing sodium salts, in particular sodium sulfate. In a preferred embodiment of the present invention, the process produces microflora having a cell aggregate size useful for the production of food products for use in aquaculture. Further disclosed is a food product which includes Thraustochytrium, Schizochytrium, and mixtures thereof, and a component selected from flaxseed, rapeseed, soybean and avocado meal. Such a food product includes a balance of long chain and short chain omega-3 highly unsaturated fatty acids.

Claims

1. A process for producing lipids comprising docosahexaenoic acid (DHA), comprising culturing a microorganism in a culture medium in a fermenter, the culture medium comprising less than about 120 mg of chloride per liter of the culture medium, whereby lipids comprising DHA are produced, and recovering lipids.

2. The process of claim 1, wherein the microorganism produces lipids having at least about 94% by weight of total omega-3 fatty acids in the lipids is DHA.

3. The process of claim 1, wherein the step of recovering lipids comprises extracting the lipids.

4. The process of claim 1, wherein at least about 49% by weight of the total lipids is omega-3 fatty acids.

5. The process of claim 4, wherein at least about 64.5% by weight of the total omega-3 fatty acids is DHA.

6. The process of claim 4, wherein at least about 86% by weight of the total omega-3 fatty acids is DHA.

7. The process of claim 4, wherein at least about 94% by weight of the total omega-3 fatty acids is DHA.

8. The process of claim 4, wherein the ratio of EPA to DHA in the total lipid is from about 1:

1 to about 1;

30.

9. The process of claim 1, wherein the ratio of DHA to EPA in the total lipid is about 7.07 or less.

10. The process of claim 1, wherein the ratio of DPA to DHA in the total lipid is at least about 1:

12.

11. The process of claim 1, wherein the total lipid comprises about 5% or less arachidonic acid.

12. The process of claim 1, wherein the culture medium contains between about 60 mg and about 120 mg of chloride per liter of culture medium.

13. The process of claim 1, wherein the culture medium contains at least about 1 g/l of sodium.

14. The process of claim 13, wherein the concentration of the sodium is between about 1 g/l and about 50 g/l.

15. The process of claim 1, wherein the culture medium comprises a source of carbon.

16. The process of claim 1, wherein the culture medium comprises a source of nitrogen.

17. The process of claim 1, wherein the culture medium comprises a source of micronutrients.

18. The process of claim 1, wherein the culture medium is at a temperature from about 5°

C. to about 48°

C.

19. The process of claim 1, wherein the culture medium is at a pH from about pH 5.0 to about pH 11.0.

20. The process of claim 1, wherein the culture medium comprises a source of carbon, a source of nitrogen, and a source of micronutrients, and wherein the culture medium is at a temperature from about 5°

C. to about 48°

C. and a pH from about pH 5.0 to about pH 11.0.

21. The process of claim 1, wherein the microorganism is obtained from a marine or inland saline environment.

22. The process of claim 1, wherein the microorganism can grow at a salinity level which results in a conductivity of from about 5 mmho/cm to about 40 mmho/cm.

23. The process of claim 1, wherein the microorganism can grow in 60% seawater or 60% artificial seawater.

24. The process of claim 1, wherein the microorganism is a member of the order Thraustochytriales.

25. The process of claim 24, wherein the microorganism is selected from the group consisting of a microorganism of the genus Thraustochytrium and a microorganism of the genus Schizochytrium.

26. The process of claim 1, wherein the microorganism replicates by successive bipartition.

27. The of claim 1, wherein the microorganism replicates by forming sporangia and releasing zoospores.

28. The process of claim 1, wherein the microorganism has all of the identifying characteristics of an organism selected from the group consisting of ATCC Nos. 20888, 20889, 20890, 20891, and 20892, and mutants thereof, wherein the mutants have an omega-3 highly unsaturated fatty acid content of at least about 0.5% dry weight.

29. The process of claim 1, comprising culturing the microorganism at a temperature from about 5°

C. to about 48°

C. and a pH from about pH 5.0 to about pH 11.0, wherein at least about 64.5% by weight of omega-3 fatty acids in the total fatty acids is DHA.

30. The process of claim 1, comprising culturing the microorganism at a temperature from about 5°

C. to about 48°

C. and a pH from about pH 5.0 to about pH 11.0, wherein at least about 86% by weight of omega-3 fatty acids in the total fatty acids is DHA.

31. The process of claim 1, comprising culturing the microorganism at a temperature from about 5°

C. to about 48°

C. and a pH from about pH 5.0 to about pH 11.0, wherein at least about 94% by weight of omega-3 fatty acids in the total fatty acids is DHA.

32. The process of claim 1, comprising culturing the microorganism at a temperature from about 5°

C. to about 48°

C. and a pH from about pH 5.0 to about pH 11.0, wherein the culture medium comprises between about 60 mg and about 120 mg of chloride per liter of culture medium, and wherein at least about 64.5% by weight of omega-3 fatty acids in the total fatty acids is DHA.

33. The process of claim 1, comprising culturing the microorganism at a temperature from about 5°

C. to about 48°

C. and a pH from about pH 5.0 to about pH 11.0, wherein the culture medium comprises between about 60 mg and about 120 mg of chloride per liter of culture medium, and wherein at least about 86% by weight of omega-3 fatty acids in the total fatty acids is DHA.

34. The process of claim 1, comprising culturing the microorganism at a temperature from about 5°

C. to about 48°

C. and a pH from about pH 5.0 to about pH 11.0, wherein the culture medium comprises between about 60 mg and about 120 mg of chloride per liter of culture medium, and wherein at least about 94% by weight of omega-3 fatty acids in the total fatty acids is DHA.

35. The process of claim 1, wherein the microorganism is from an environment where sodium chloride is the primary source of sodium.

36. The process of claim 1, wherein the microorganism has an exponential growth rate of at least about 5.5 doublings per day at 25°

C. in artificial seawater.

37. A culture medium comprising a microorganism and less than about 120 mg of chloride per liter of the culture medium, wherein the microorganism produces lipids comprising DHA.

38. The culture medium of claim 37, wherein at least about 94% by weight of total omega-3 fatty acids in the lipids is DHA.

39. The culture medium of claim 37, wherein at least about 49% by weight of the total lipids are omega-3 fatty acids.

40. The culture medium of claim 39, wherein at least about 64.5% by weight of the total omega-3 fatty acids is DHA.

41. The culture medium of claim 39, wherein at least about 86% by weight of omega-3 fatty acids is DHA.

42. The culture medium of claim 37, wherein a ratio of EPA to DHA in the total lipid is from about 1:

1 to about 1;

30.

43. The culture medium of claim 37, wherein the ratio of DHA to EPA in the total lipid is about 7.07 or less.

44. The culture medium of claim 37, wherein a ratio of DPA to DHA in the total lipid is at least about 1:

12.

45. The culture medium of claim 37, wherein total lipid comprises about 5% or less arachidonic acid.

46. The culture medium of claim 37, wherein the culture medium comprises between about 60 mg and about 120 mg of chloride per liter of culture medium.

47. The culture medium of claim 37, wherein the culture medium comprises at least about 1 g/l of sodium.

48. The culture medium of claim 47, wherein the concentration of the sodium is between about 1 g/l and about 50 g/l.

49. The culture medium of claim 37, wherein the culture medium further comprises a source of carbon.

50. The culture medium of claim 37, wherein the culture medium further comprises a source of nitrogen.

51. The culture medium of claim 37, wherein the culture medium further comprises a source of micronutrients.

52. The culture medium of claim 37, wherein the culture medium is at a temperature from about 5°

C. to about 48°

C.

53. The culture medium of claim 37, wherein the culture medium is at a pH from about pH 5.0 to about pH 11.0.

54. The culture medium of claim 37, wherein the culture medium comprises a source of carbon, a source of nitrogen, and a source of micronutrients, and wherein the culture medium is at a temperature from about 5°

C. to about 48°

C.

55. The culture medium of claim 37, wherein the microorganism is obtained from a marine or inland saline environment.

56. The culture medium of claim 37, wherein the microorganism can grow at a salinity level which results in a conductivity of from about 5 mmho/cm to about 40 mmho/cm.

57. The culture medium of claim 37, wherein the microorganism can grow in 60% seawater or 60% artificial seawater.

58. The culture medium of claim 37, wherein the microorganism is a member of the order Thraustochytriales.

59. The culture medium of claim 37, wherein the microorganism is selected from the group consisting of a microorganism of the genus Thraustochytrium and a microorganism of the genus Schizochytrium.

60. The culture medium of claim 37, wherein the microorganism replicates by successive bipartition.

61. The culture medium of claim 37, wherein the microorganism replicates by forming sporangia and releasing zoospores.

62. The culture medium of claim 37, wherein the microorganism has all of the identifying characteristics of an organism selected from the group consisting of ATCC Nos. 20888, 20889 20890, 20891, and 20892, and mutants thereof, wherein the mutants have an omega-3 highly unsaturated fatty acid content of at least about 0.5% dry weight.

63. The culture medium of claim 37, wherein the culture medium is at a temperature from about 5°

C. to about 48°

C., and wherein the microorganism produces at least about 64.5% by weight of omega-3 fatty acids as DHA.

64. The culture medium of claim 37, wherein the culture medium is at a temperature from about 5°

C. to about 48°

C., and wherein the microorganism produces at least about 86% by weight of omega-3 fatty acids as DHA.

65. The culture medium of claim 37, wherein the culture medium is at a temperature from about 5°

C. to about 48°

C., and wherein the microorganism produces at least about 94% by weight of omega-3 fatty acids as DHA.

66. The culture medium of claim 37, wherein the culture medium is at a temperature from about 5°

C. to about 48°

C. and a pH from about pH 5.0 to about pH 11.0, wherein the culture medium comprises between about 60 mg and about 120 mg of chloride per liter of culture medium, and wherein the microorganism produces at least about 64.5% by weight of omega-3 fatty acids as DHA.

67. The culture medium of claim 37, wherein the culture medium is at a temperature from about 5°

C. to about 48°

C. and a pH from about pH 5.0 to about pH 11.0, wherein the culture medium comprises between about 60 mg and about 120 mg of chloride per liter of culture medium, and wherein the microorganism produces at least about 86% by weight of omega-3 fatty acids as DHA.

68. The culture medium of claim 37, wherein the culture medium is at a temperature from about 5°

C. to about 48°

C. and a pH from about pH 5.0 to about pH 11.0, wherein the culture medium comprises between about 60 mg and about 120 mg of chloride per liter of culture medium, and wherein the microorganism produces at least about 94% by weight of omega-3 fatty acids as DHA.

69. The culture medium of claim 37, wherein the microorganism is from an environment where sodium chloride is the primary source of sodium.

70. The culture medium of claim 37, wherein the microorganism has an exponential growth rate of at least about 5.5 doublings per day at 25°

C.

71. A culture medium comprising a microorganism and less than about 3 g/l of chloride and at least about 2.4 g/L of sodium, wherein the microorganism produces lipids comprising DHA.

72. A culture medium comprising a microorganism, less than about 3 g/l of chloride and at least about 2.4 g/L of sodium, wherein the microorganism produces at least about 49% of the total lipids as omega-3 fatty acids, and wherein the microorganism produces lipids comprising DHA.

73. A culture medium comprising a microorganism and less than about 3 g/l of chloride and at least about 2.4 g/L of sodium, wherein the microorganism produces lipids comprising DHA, and wherein the ratio of DHA to EPA in the total lipid is about 7.07 or less.