PROCESSES FOR OBTAINING MICROBIAL OIL FROM MICROBIAL CELLS

Abstract

Disclosed herein are processes for obtaining a microbial oil comprising one or more polyunsaturated fatty acids (PUFAs) from one or more microbial cells by lysing the cells to form a lysed cell composition, treating the lysed cell composition to form an oil-containing emulsion and then recovering the oil from the oil-containing emulsion. Further disclosed herein is microbial oil comprising one or more PUFAs that is recovered from microbial cells by at least one process described herein.

Claims

1. A process for obtaining a microbial oil comprising one or more polyunsaturated fatty acids from one or more microbial cells, wherein the process comprises: (a) lysing the cells comprising the microbial oil to form a lysed cell composition; (b) treating the lysed cell composition to form an oil-containing emulsion; (c) separating the oil-containing emulsion from the lysed cell composition; (d) demulsifying the oil-containing emulsion; and (e) recovering the oil.

2. The process of claim 1, wherein at least one of (a) or (b) further comprises raising the pH of the cells or the lysed cell composition.

3. The process of claim 1 or claim 2, wherein at least one of (a) or (b) further comprises raising the pH to about 7 or above.

4. The process of any preceding claim, wherein at least one of (a) or (b) further comprises raising the pH from about 7 to about 11.

5. The process of any preceding claim, wherein (a) further comprises controlling the pH of the cells.

6. The process of claim 5, wherein controlling the pH of the cells comprises adding a base.

7. The process of any preceding claim, wherein at least one of (a) or (b) further comprises agitating the cells or the lysed cell composition.

8. The process of any preceding claim, wherein at least one of (a) or (b) further comprises heating the cells or the lysed cell composition to at least 50 C.

9. The process of any preceding claim, wherein at least one of (a) or (b) further comprises heating the cells or the lysed cell composition from about 50 C. to about 100 C.

10. The process of any preceding claim, wherein (a) further comprises adding an enzyme in an amount of from about 0.05% to about 20% by weight of the cell broth.

11. The process of any preceding claim, wherein (b) further comprises adding a salt in an amount of from about 0.05% to about 20% by weight of the cell broth.

12. The process of any of claim 11, wherein the salt is selected from the group consisting of alkali metal salts, alkali earth metal salts, sulfate salts, and combinations thereof.

13. The process of any preceding claim, wherein the cells of (a) are unwashed.

14. The process of any preceding claim, wherein the cells of (a) are contained in a fermentation broth.

15. The process of any preceding claim, wherein (c) further comprises heating the oil-containing emulsion and the lysed cell composition to at least 50 C.

16. The process of any preceding claim, wherein (c) further comprises heating the oil-containing emulsion and the lysed cell composition from about 50 C. to about 100 C.

17. The process of any preceding claim, wherein (c) further comprises centrifuging.

18. The process of any preceding claim, wherein (d) further comprises heating the oil-containing emulsion to at least 50 C.

19. The process of any preceding claim, wherein (d) further comprises heating the oil-containing emulsion from about 50 C. to about 100 C.

20. The process of any preceding claim, wherein (d) further comprises adding an acid.

21. The process of claim 20, wherein the acid comprises citric acid.

22. The process of any preceding claim, wherein (d) further comprises adding a base.

23. The process of claim 22, wherein the base comprises sodium hydroxide.

24. The process of any preceding claim, wherein the polyunsaturated fatty acid is selected from an omega-3 fatty acid, an omega-6 fatty acid, and mixtures thereof.

25. The process of any preceding claim, wherein the polyunsaturated fatty acid is selected from docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), arachidonic acid (ARA), gamma-linolenic acid (GLA), dihomo-gamma-linolenic acid (DGLA), stearidonic acid (SDA), and mixtures thereof.

26. The process of claim 25, wherein the polyunsaturated fatty acid is docosahexaenoic acid (DHA).

27. The process of claim 25, wherein the polyunsaturated fatty acid is arachidonic acid (ARA).

28. The process of any preceding claim, wherein the microbial cells are algae, yeast, fungi, protest, or bacteria cells.

29. The process of any preceding claim, wherein the microbial cells are from the genus Mortierella, genus Crypthecodinium, or order Thraustochytriales.

30. The process of claim 29, wherein the microbial cells are from the order Thraustochytriales.

31. The process of claim 29, wherein the microbial cells are from the genus Thraustochytrium, Schizochytrium, or mixtures thereof.

32. The process of claim 29, wherein the microbial cells are from Mortierella Alpina.

33. The process of any preceding claim, wherein the lysed cell composition comprises liquid, cell debris, and microbial oil.

34. The process of any preceding claim, wherein an organic solvent is not used to obtain the oil from the cells.

35. The process of any preceding claim, wherein (e) further comprises centrifuging the oil.

36. The process of any preceding claim, wherein (e) further comprises refining the oil.

37. The process of any preceding claim, wherein the oil comprises at least 30% by weight arachidonic acid.

38. The process of any preceding claim, wherein the oil comprises at least 30% by weight docosahexaenoic acid.

39. The process of any preceding claim, wherein the oil has an anisidine value of less than about 50.

40. The process of any preceding claim, wherein the oil has a phosphorus content of about 8 ppm or less.

41. The process of any preceding claim, wherein the oil has a peroxide value of less than about 5 meq/kg.

42. An oil obtained by the process of any preceding claim.

43. The process of any preceding claim, wherein (a) and (b) are combined together to form a one-step lysing and treating step.

44. A process for obtaining a microbial oil comprising one or more polyunsaturated fatty acids from one or more microbial cells, wherein the process comprises: (a) lysing the cells comprising the microbial oil to form a lysed cell composition; (b) treating the lysed cell composition to form an oil-containing emulsion; (c) separating the oil-containing emulsion from the lysed cell composition; and (d) demulsifying the oil-containing emulsion; and (e) recovering the oil, wherein at least one of (a) and (b) comprises raising the pH of the cells or lysed cell composition, agitating the cells or lysed cell composition, and heating the cells or lysed cell composition.

45. The process of claim 44, wherein (a) further comprises adding an enzyme in an amount of from about 0.05% to about 20% by weight of the cell broth.

46. The process of claim 44 or claim 45, wherein (b) further comprises adding a salt in an amount of from about 0.05% to about 20% by weight of the cell broth.

47. The process of any one of claims 44-46, wherein (c) further comprises heating the oil-containing emulsion and the lysed cell composition to at least 50 C.

48. The process of any one of claims 44-47, wherein (c) further comprises centrifuging.

49. The process of any one of claims 44-48, wherein (d) further comprises at least one of heating to at least 50 C., adding an acid, and adding a base.

50. The process of any one of claims 44-49, wherein (e) further comprises centrifuging the oil.

51. The process of any one of claims 44-50, wherein (e) further comprises refining the oil.

Description

EXAMPLES

[0159] As used herein, the extraction yield is the amount, by weight, of lipid in the oil-containing emulsion expressed as a percentage of the amount, by weight, of lipid in the cell broth. The refining yield is the amount, by weight, of lipid in the refined oil expressed as a percentage of the amount, by weight, of lipid in the oil-containing emulsion. The total yield is the amount, by weight, of lipid in the refined oil expressed as a percentage of the amount, by weight, of lipid in the cell broth.

Example 1

[0160] An unwashed cell broth (75.9 kg) containing microbial cells (Schizochytrium sp.) was pasteurized at 60 C. for 1 hour. The cells were lysed by adding 6N NaOH to pH adjust the broth to 7.5 and Alcalase enzyme (available from Novozymes (Franklinton, N.C.)) was added in an amount of 0.5% based on the weight of the cell broth, agitated at a speed of 184 RPM, heated to 60 C. and held for 2 hours. While maintaining the agitation, the pH of the lysed cell composition was adjusted to 10.5 by adding 6N NaOH. NaCl in an amount of 2% by weight of the cell broth was added and heated to 90 C. and held for 19 hours. The pH of the lysed cell composition was adjusted to 8.2 by adding 6N NaOH, agitated at 150 RPM, and held for 4.5 hours. The pH of the lysed cell composition was adjusted to 8.3 by adding 6N NaOH, agitated at 150 RPM, and held for 1 hour. An oil-containing emulsion was formed and separated from the lysed cell composition by heating the oil-containing emulsion and lysed cell composition to 80 C. and then centrifuging (Alfa Laval Disc Stack Centrifuge, LAPX 404/Clara 20). The extraction yield was 87.2%. The recovered oil-containing emulsion had an AV of 12.3 and contained 1.24% free fatty acids. The oil-containing emulsion was placed in a 20L tank with nitrogen blanketing and was heated to 50-55 C. and 50% citric acid was added in an amount of 0.2% by weight of the emulsion and held for 15 minutes. The oil-containing emulsion was further heated to 60-65 C. and NaOH was added in an amount of 12.5% by weight of the free fatty acid composition and weight of the oil-containing emulsion, and held for 30 minutes to refine the emulsion. The oil was separated from the emulsion by centrifuging. The refining yield was 86.2%. The total yield was 75%. The refined oil had an AV of 10.

TABLE-US-00001 TABLE 1 Comparison of oil-containing emulsion and refined oil Emulsion Refined oil Moisture 17.5% 0.1% Fat 80.5% 98.1%

Example 2

[0161] An unwashed cell broth (78.3 kg) containing microbial cells (Schizochytrium sp.) was pasteurized at 60 C. for 1 hour. The cells were lysed by adding 6N NaOH to pH adjust the broth to 7.5 and Alcalase enzyme (available from Novozymes (Franklinton, N.C.)) was added in an amount of 0.5% based on weight of the cell broth, agitated at a speed of 184 RPM, heated to 60 C. and held for 2 hours with pH controlled at 7.5 with 6N NaOH. While maintaining the agitation, the pH of the lysed cell composition was adjusted to 9.7 by adding 6N NaOH. NaCl in an amount of 2% by weight of the cell broth was added and heated to 90 C. and held for 18 hours. While maintaining the agitation, the pH of the lysed cell composition was adjusted to 8.3 by adding 6N NaOH and held for 4.5 hours. An oil-containing emulsion was formed and separated from the lysed cell composition by heating the oil-containing emulsion and lysed cell composition to 80 C. and then centrifuging (Alfa Laval Disc Stack Centrifuge, LAPX 404/Clara 20). The extraction yield was 86.3%. The oil-containing emulsion had an AV of 12.9 and contained 1.02% free fatty acids. The oil-containing emulsion was placed in a 20L tank with nitrogen blanketing and was heated to 50-55 C. and 50% citric acid was added in an amount of 0.2% by weight of the emulsion and held for 15 minutes. The oil-containing emulsion was further heated to 60-65 C. and NaOH was added in an amount of 12.5% by weight of the free fatty acid composition and weight of the oil-containing emulsion, and held for 30 minutes to refine the emulsion. The oil was separated from the emulsion by centrifuging. The refining yield was 98.9%. The total yield was 85.3%. The recovered oil had an AV of 13.5 and contained 0.5% free fatty acids.

Example 3

[0162] An unwashed cell broth (86.5 kg) containing microbial cells (Schizochytrium sp.) was pasteurized at 60 C. for 1 hour. The cells were lysed by adding 6N NaOH to pH adjust the broth to 7.5 and Alcalase enzyme (available from Novozymes (Franklinton, N.C.)) was added in an amount of 0.5% based on weight of the cell broth, agitated at a speed of 185 RPM, heated to 60 C. and held for 2 hours with pH controlled at 7.5 with 6N NaOH. While maintaining the agitation, the pH of the lysed cell composition was adjusted to 9.9 by adding 6N NaOH. NaCl in an amount of 2% by weight of the cell broth was added and heated to 90 C. and held for 18 hours. While maintaining the agitation, the pH of the lysed cell composition was adjusted to 8.5 by adding 6N NaOH and held for 4 hours. An oil-containing emulsion was formed and separated from the lysed cell composition by heating the oil-containing emulsion and lysed cell composition to 80 C. and then centrifuging (Alfa Laval Disc Stack Centrifuge, LAPX 404/Clara 20). The extraction yield was 94.4%. The oil-containing emulsion had an AV of 4.5 and contained 1.88% free fatty acids. The oil-containing emulsion was placed in a 20L tank with nitrogen blanketing and was heated to 50-55 C. and 50% citric acid was added in an amount of 0.2% by weight emulsion and held for 15 minutes. The oil-containing emulsion was further heated to 60-65 C. and NaOH was added in an amount of 12.5% by weight of the free fatty acid composition and weight of the oil-containing emulsion, and held for 30 minutes to refine the emulsion. The oil was separated from the emulsion by centrifuging. The refining yield was 91.5%. The total yield was 86.4%. The refined oil had an AV of 13.5 and contained 0.5% free fatty acids.

Example 4

[0163] An unwashed cell broth (82.6 kg) containing microbial cells (Schizochytrium sp.) was pasteurized at 60 C. for 1 hour. The cells were lysed by adding 6N NaOH to pH adjust the broth to 7.5 and Alcalase enzyme (available from Novozymes (Franklinton, N.C.)) was added in an amount of 0.5% based on weight of the cell broth, agitated at a speed of 185 RPM, heated to 60 C. and held for 2 hours with pH controlled at 7.5 with 6N NaOH. While maintaining the agitation, the pH of the lysed cell composition was adjusted to 10.2 by adding 6N NaOH. NaCl in an amount of 2% by weight of the cell broth was added and heated to 90 C. and held for 4 hours. While maintaining the agitation, the pH of the lysed cell composition was adjusted to 8.3 by adding 6N NaOH and held for 11 hours. While maintaining the agitation, the pH of the lysed cell composition was adjusted to 7.8 by adding 6N NaOH and held for 7 hours. An oil-containing emulsion was formed and separated from the lysed cell composition by heating the oil-containing emulsion and lysed cell composition to 80 C. and then centrifuging (Alfa Laval Disc Stack Centrifuge, LAPX 404/Clara 20). The extraction yield was 99.3%. The oil-containing emulsion had an AV of 4. and contained 0.67% free fatty acids. The oil-containing emulsion was placed in a 20L tank with nitrogen blanketing and was heated to 50-55 C. and 50% citric acid was added in an amount of 0.2% by weight of the emulsion and held for 15 minutes. The oil-containing emulsion was further heated to 60-65 C. and NaOH was added in an amount of 12.5% by weight of the free fatty acid composition and weight of the oil-containing emulsion, and held for 30 minutes to refine the emulsion. The oil was separated from the emulsion by centrifuging. The refining yield was 92.7%. The total yield was 92.3%. The refined oil had an AV of 2.8 and contained 0.54% free fatty acids.

Example 5

[0164] An unwashed cell broth (88.5 kg) containing microbial cells (Schizochytrium sp.) was pasteurized at 60 C. for 1 hour. The cells were lysed by adding 6N NaOH to pH adjust the broth to 7.5 and Alcalase enzyme (available from Novozymes (Franklinton, N.C.)) was added in an amount of 0.5% based on weight of the cell broth, agitated at a speed of 183 RPM, heated to 60 C. and held for 2 hours with pH controlled at 7.5 with 6N NaOH. While maintaining the agitation, the pH of the lysed cell composition was adjusted to 10.2 by adding 6N NaOH. NaCl in an amount of 2% by weight of the cell broth was added and heated to 90 C. and held for 22 hours. An oil-containing emulsion was formed and separated from the lysed cell composition by heating the oil-containing emulsion and lysed cell composition to 80 C. and then centrifuging (Alfa Laval Disc Stack Centrifuge, LAPX 404/Clara 20). The extraction yield was 94%. The oil-containing emulsion contained 2.1% free fatty acids. The oil-containing emulsion was placed in a 20L tank with nitrogen blanketing and was heated to 50-55 C. and 50% citric acid was added in an amount of 0.2% by weight of the emulsion and held for 15 minutes. The oil-containing emulsion was further heated to 60-65 C. and NaOH was added in an amount of 12.5% by weight of the free fatty acid composition and weight of the oil-containing emulsion, and held for 30 minutes to refine the emulsion. The oil was separated from the emulsion by centrifuging. The refining yield was 94.3%. The total yield was 87%. The refined oil contained 0.62% free fatty acids.

Example 6

[0165] An unwashed cell broth (84.9 kg) containing microbial cells (Schizochytrium sp.) was pasteurized at 60 C. for 1 hour. The cells were lysed by adding 6N NaOH to pH adjust the broth to 7.5 and Alcalase enzyme (available from Novozymes (Franklinton, N.C.)) was added in an amount of 0.5% based on weight of the cell broth, agitated at a speed of 184 RPM, heated to 60 C. and held for 2 hours. While maintaining the agitation, the pH of the lysed cell composition was adjusted to 10.5 by adding 6N NaOH. NaCl in an amount of 2% by weight of the cell broth was added and heated to 90 C. and held for 22 hours. An oil-containing emulsion was formed and separated from the lysed cell composition by heating the oil-containing emulsion and lysed cell composition to 80 C. and then centrifuging (Alfa Laval Disc Stack Centrifuge, LAPX 404/Clara 20). The extraction yield was 93.9%. The oil-containing emulsion contained 1.5% free fatty acids. The oil-containing emulsion was placed in a 20L tank with nitrogen blanketing and was heated to 50-55 C. and 50% citric acid was added in an amount of 0.2% by weight of the emulsion and held for 15 minutes. The oil-containing emulsion was further heated to 60-65 C. and NaOH was added in an amount of 12.5% by weight of the free fatty acid composition and weight of the oil-containing emulsion, and held for 30 minutes to refine the emulsion. The oil was separated from the emulsion by centrifuging. The refining yield was 89.8%. The total yield was 84.3%.

TABLE-US-00002 TABLE 2 Comparison of oil-containing emulsion and refined oil Emulsion Refined oil Moisture 24.8% 1% Fat 71.2% 95.8%

Example 7

[0166] An unwashed cell broth (77.2 kg) containing microbial cells (Schizochytrium sp.) was pasteurized at 60 C. for 1 hour. The cells were mechanically lysed. 6N NaOH was added to the lysed cell composition to pH adjust the composition to 9.5, NaCl in an amount of 2% by weight of the cell broth was added, agitated at 184 RPM, heated to 90 C. and held for 25 hours. An oil-containing emulsion was formed and separated from the lysed cell composition by heating the oil-containing emulsion and lysed cell composition to 80 C. and then centrifuging (Alfa Laval Disc Stack Centrifuge, LAPX 404/Clara 20). The extraction yield was 91.1%. The oil-containing emulsion contained 1.7% free fatty acids. The oil-containing emulsion was heated to 50-55 C. and held for 8 hours and 15 minutes. The oil was separated from the emulsion by centrifuging. The refining yield was 83.7%. The total yield was 76.2%.

Example 8

[0167] An unwashed cell broth (74.8 kg) containing microbial cells (Schizochytrium sp.) was pasteurized at 60 C. for 1 hour. The cells were lysed by adding 6N NaOH to pH adjust the broth to 7.5 and Alcalase enzyme (available from Novozymes (Franklinton, N.C.)) was added in an amount of 0.5% based on weight of the cell broth, agitated at a speed of 184 RPM, heated to 60 C. and held for 2 hours. While maintaining the agitation, the pH of the lysed cell composition was adjusted to 10.5 by adding 6N NaOH. NaCl in an amount of 2% by weight of the cell broth was added and heated to 90 C. and held for 23 hours. An oil-containing emulsion was formed and separated from the lysed cell composition by heating the oil-containing emulsion and lysed cell composition to 80 C. and then centrifuging (Alfa Laval Disc Stack Centrifuge, LAPX 404/Clara 20). The extraction yield was 82.8%. The oil-containing emulsion contained 1.1% free fatty acids. The oil-containing emulsion was placed in a 20L tank with nitrogen blanketing and was heated to 50-55 C. and 50% citric acid was added in an amount of 0.2% by weight of the emulsion and held for 15 minutes. The oil-containing emulsion was further heated to 60-65 C. and NaOH was added in an amount of 12.5% by weight of the free fatty acid composition and weight of the oil-containing emulsion, and held for 30 minutes to refine the emulsion. The oil was separated from the emulsion by centrifuging. The refining yield was 94.9%. The total yield was 78.5%.

Example 9

[0168] An unwashed cell broth (83.7 kg) containing microbial cells (Schizochytrium sp.) was pasteurized at 60 C. for 1 hour. The cells were lysed by adding 6N NaOH to pH adjust the broth to 7.5 and Alcalase enzyme (available from Novozymes (Franklinton, N.C.)) in an amount of 0.5% based on weight of the cell broth, agitated at a speed of 184 RPM, heated to 60 C. and held for 2 hours. While maintaining the agitation, the pH of the lysed cell composition was adjusted to 10.5 by adding 6N NaOH. NaCl in an amount of 2% by weight of the cell broth was added and heated to 90 C. and held for 22 hours. An oil-containing emulsion was formed and separated from the lysed cell composition by heating the oil-containing emulsion and lysed cell composition to 80 C. and then centrifuging (Alfa Laval Disc Stack Centrifuge, LAPX 404/Clara 20). The extraction yield was 90.9%. The oil-containing emulsion contained 1.7% free fatty acids. The oil-containing emulsion was placed in a 20L tank with nitrogen blanketing and was heated to 50-55 C. and 50% citric acid was added in an amount of 0.3% by weight of the emulsion and held for 15 minutes. The oil-containing emulsion was further heated to 60-65 C. and NaOH was added in an amount of 12.5% by weight of the free fatty acid composition and weight of the oil-containing, and held for 30 minutes to refine the emulsion. The oil was separated from the emulsion by centrifuging. The refining yield was 78.2%. The total yield was 78.2%.

Example 10

[0169] An unwashed cell broth (86.3 kg) containing microbial cells (Schizochytrium sp.) was pasteurized at 60 C. for 1 hour. The cells were lysed by adding 6N NaOH to pH adjust the broth to 7.5 and Alcalase enzyme (available from Novozymes (Franklinton, N.C.)) was added in an amount of 0.5% based on weight of the cell broth, agitated at a speed of 185 RPM, heated to 60 C. and held for 2 hours with pH controlled at 7.5 with 6N NaOH. While maintaining the agitation, the pH of the lysed cell composition was adjusted to 10.5 by adding 6N NaOH. NaCl in an amount of 2% by weight of the cell broth was added and heated to 90 C. and held for 2 hours. An oil-containing emulsion was formed and separated from the lysed cell composition by heating the oil-containing emulsion and lysed cell composition to 80 C. and then centrifuging (Alfa Laval Disc Stack Centrifuge, LAPX 404/Clara 20). The extraction yield was 96.9%. The oil-containing emulsion had an AV of 1.5, a PV of <0.1 meg/kg and contained 0.87% free fatty acids. The oil-containing emulsion was placed in a 20L tank with nitrogen blanketing and was heated to 50-55 C. and 50% citric acid was added in an amount of 0.3% by weight of the emulsion and held for 15 minutes. The oil-containing emulsion was further heated to 60-65 C. and NaOH was added in an amount of 12.5% by weight of the free fatty acid composition and weight of the oil-containing emulsion, and held for 30 minutes to refine the emulsion. The oil was separated from the emulsion by centrifuging. The total yield was 92.1%. The refined oil had an AV of 5.2, a PV of 0.52 meg/kg and contained 0.24% free fatty acids.

TABLE-US-00003 TABLE 3 Comparison of oil-containing emulsion and refined oil Emulsion Refined oil Moisture 23.8% 0.2% Fat 71.9% 100.4% Free Fatty Acid 0.9% 0.2%

Example 11

[0170] The extraction yields and coalescence times for the examples provided herein are compared as to a current process (two examples).

TABLE-US-00004 TABLE 4 Coalescence Times Process Extraction Yield Time (hr) Example 1 87.2% 20 Example 2 86.3% 22.5 Example 3 94.4% 22 Example 4 99.3% 22 Example 5 93.4% 22 Example 6 93.9% 22 Example 7 91.1% 25 Example 8 82.8% 23 Example 9 90.1% 22.6 Standard #1 91.3% 67 Standard #2 90.2% 43.5