Processes for obtaining microbial oil from microbial cells

Abstract

Processes are disclosed for obtaining a lipid from a cell by lysing the cell to form a lysed cell composition, adding an acid to lower the pH of the lysed cell composition, and demulsify the lysed cell composition, and obtaining the lipid from the demulsified lysed cell composition. Also disclosed are lipids prepared by the processes of the present invention wherein the lipids have a particular anisidine value, peroxide value, and/or phosphorus content.

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) demulsifying the lysed cell composition by adding an acid to form a demulsified lysed cell composition; (c) separating the oil from the demulsified lysed cell composition; and (d) recovering the oil; wherein (b) comprises lowering the pH of the lysed cell composition to a pH of from about 0.5 to 6.0.

2. The process of claim 1, wherein at least one of (a) or (b) further comprises heating the cells or the composition to at least 70? C.

3. The process of claim 2, wherein at least one of (a) or (b) further comprises heating the cells or the composition to from about 70? C. to about 100? C.

4. The process of claim 1, wherein (b) further comprises adding an emulsifier to the lysed cell composition.

5. The process of claim 1, wherein (b) further comprises adding an acid in an amount of from about 0.5% to about 20%, by weight, lysed cell composition.

6. The process of claim 1, wherein (b) further comprises adding a salt in an amount of from about 0.05% to about 20%, by weight, of the lysed cell composition.

7. The process of claim 1, wherein (b) further comprises agitating the lysed cell composition.

8. The process of claim 1, wherein (a) further comprises agitating the cells.

9. The process according to claim 1, wherein the acid is selected from sulfuric, phosphoric, hydrochloric, hydrobromic, hydroiodic, hypochlorous, chlorous, chloric, perchloric, fluorosulfuric nitric, fluoroantimonic, fluoroboric, hexafluorophosphoric, chromic, boric, acetic, citric, formic, and combinations thereof.

10. The process of claim 9, wherein the acid is sulfuric.

11. The process of claim 1, wherein the cells of (a) are unwashed.

12. The process of claim 1, wherein the cells of (a) are contained in a fermentation broth.

13. The process of claim 1, wherein (c) further comprises centrifuging the demulsified lysed cell composition.

14. The process of claim 1, wherein the polyunsaturated fatty acid is selected from an omega-3 fatty acid, an omega-6 fatty acid, and mixtures thereof.

15. The process of claim 1, 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.

16. The process of claim 15, wherein the polyunsaturated fatty acid is docosahexaenoic acid (DHA).

17. The process of claim 15, wherein the polyunsaturated fatty acid is arachidonic acid (ARA).

18. The process of claim 1, wherein the microbial cells are algae, yeast, fungi, protist, or bacteria cells.

19. The process of claim 1, wherein the microbial cells are from the genus Mortierella, genus Crypthecodinium, or order Thraustochytriales.

20. The process of claim 18, wherein the microbial cells are from the order Thraustochytriales.

21. The process of claim 19, wherein the microbial cells are from the genus Thraustochytrium, Schizochytrium, or mixtures thereof.

22. The process of claim 18, wherein the microbial cells are from Mortierella Alpine.

23. The process of claim 1, wherein the lysed cell composition comprises liquid, cell debris, and microbial oil.

24. The process of claim 1, wherein an organic solvent is not used to obtain the oil from the cells.

25. The process of claim 1, wherein the mean particle size of the demulsified lysed cell composition is 25 microns or less.

26. The process of claim 4, wherein the emulsifier is an ionic emulsifier.

27. The process of claim 26, wherein the ionic emulsifier is an anionic emulsifier selected from an anionic sulfate emulsifier, an anionic sulfonate emulsifier, an anionic phosphate emulsifier, an anionic carboxylate emulsifier, and combinations thereof.

28. The process of claim 27, wherein the ionic emulsifier is an anionic sulfate emulsifier selected from ammonium lauryl sulfate, sodium dodecyl sulfate, sodium laureth sulfate, sodium lauryl ether sulfate, sodium myreth sulfate, and combinations thereof.

29. The process of claim 4, wherein the emulsifier is added in an amount of from 0.2% to 10% by weight of the lysed cell composition.

30. The process of claim 6, wherein the salt is selected from the group consisting of alkali metal salts, alkali earth metal salts, sulfate salts, and combinations thereof.

31. The process of claim 1, wherein the oil of (d) is a crude oil.

32. The process of claim 31, wherein (d) further comprises refining the crude oil to obtain a refined oil.

33. The process of claim 1, wherein the oil comprises at least 30% by weight arachidonic acid.

34. The process of claim 1, wherein the oil comprises at least 30% by weight docosahexaenoic acid.

35. The process of claim 1, wherein the oil has an anisidine value of less than about 50.

36. The process of claim 1, wherein the oil has a phosphorus content of about 8 ppm or less.

37. The process of claim 1, wherein the oil has a peroxide value of less than about 5 meq/kg.

38. The process according to claim 1 wherein (a) and (b) are combined together to form a one-step lysing and demulsifying step.

39. The process according to claim 1, wherein (c) further comprises raising the pH of the demulsified lysed cell composition.

40. 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 by adding an acid to form a demulsified lysed cell composition having a pH from about 0.5 to 6; (b) separating the oil from the demulsified lysed cell composition; and (c) recovering the oil; wherein said demulsified lysed cell composition is obtained during said lysing.

Description

EXAMPLES

Example 1

(1) A cell broth (300 g) containing microbial cells (Schizochytrium sp.) was pasteurized at 60? C. for 1 hour. The unwashed cell broth was agitated at a speed of 180 RPM and heated to 60? C. The cells were lysed by adding a 50 wt % NaOH solution to pH adjust the broth to 7-7.5 and Alcalase? 2.4 FG (available from Novozymes (Franklinton, N.C.)) in an amount of 0.5% based on broth weight. While maintaining the agitation, the pH of the lysed cell composition was adjusted to 4 by adding a 98% H.sub.2SO.sub.4 by weight solution. Solid NaCl in an amount of 2% by weight of the lysed cell composition was added and the composition heated to 90? C. and held for 20 hours. The pH of the composition was adjusted to 7.5-8.5 by adding a 50 wt % NaOH solution and then centrifuged (Thermo Scientific Sorvell ST40R Centrifuge) at 8000 RPM for 5 minutes to provide a crude oil, which yielded 91.7% DHA (by weight DHA).

Example 2

(2) A cell broth (300 g) containing microbial cells (Schizochytrium sp.) was pasteurized at 60? C. for 1 hour. A demulsified lysed cell composition was formed by adding a 20% H.sub.2SO.sub.4 by weight solution to adjust the pH of the cell broth to 2 and then heating the broth to 90? C. and holding for 45 hours. The microbial oil was separated from the demulsified lysed cell composition by adding a 50 wt % NaOH solution to adjust the pH of the composition to 7.5-8.5 and then centrifuging (Thermo Scientific Sorvell ST40R Centrifuge) the composition at 8000 RPM for 5 minutes to provide a crude oil, which yielded 51.62% DHA (by DHA weight).

Example 3

(3) A cell broth (300 g) containing microbial cells (Schizochytrium sp.) was pasteurized at 60? C. for 1 hour. A demulsified lysed cell composition was formed by adding: a 20% H.sub.2SO.sub.4 by weight solution to adjust the pH of the cell broth to 2, solid NaCl in an amount of 2% by weight of the broth; and heating the broth 90? C. and holding for 18 hours. The microbial oil was separated from the demulsified lysed cell composition by centrifuging (Thermo Scientific Sorvell ST40R Centrifuge) the composition at 8000 RPM for 5 minutes to provide a crude oil, which yielded 38.82% DHA (by DHA weight).

Example 4

(4) A cell broth (300 g) containing microbial cells (Schizochytrium sp.) was pasteurized at 60? C. for 1 hour. A demulsified lysed cell composition was formed by adding 2%, by weight broth, of a 98% H.sub.2SO.sub.4 by weight solution and heating the broth to 90? C. for 4 hour. The microbial oil was separated from the demulsified lysed cell composition by adding a 50 wt % NaOH solution to adjust the pH of the composition to 7-8.5, holding for 21 hours at 90? C., and centrifuging (Thermo Scientific Sorvell ST40R Centrifuge) the composition at 8000 RPM for 5 minutes to provide a crude oil, which yielded 72% DHA (by weight DHA).

(5) The crude oil had an Anisidine Value (AV) of 14.5.

Example 5

(6) A cell broth (300 g) containing microbial cells (Schizochytrium sp.) was pasteurized at 60? C. for 1 hour. The unwashed cell broth was agitated at a speed of 180 RPM and heated to 60? C. The cells were lysed by adding: 50 wt % NaOH to pH adjust the broth to 7-7.5, and Alcalase 2.4 FG (available from Novozymes (Franklinton, N.C.) in an amount of 0.5% based on broth weight. While maintaining the agitation, the pH of the lysed cell composition was adjusted to 4 by adding a 98% H.sub.2SO.sub.4 by weight solution, and then heating the composition to 90? C. After 45 hours at 90? C., the composition was centrifuged (Thermo Scientific Sorvell ST40R Centrifuge) at 8000 RPM for 5 minutes to provide a crude oil, which yielded 81% DHA (by DHA weight). The crude oil has an AV of 14.5.

Example 6

(7) A cell broth (300 g) containing microbial cells (Schizochytrium sp.) was pasteurized at 60? C. for 1 hour. A demulsified lysed cell composition was formed by adding 2.5%, by weight broth, of a 98% H.sub.2SO.sub.4 by weight solution, and then heating the broth to 90? C. for 5 hours. The microbial oil was separated from the demulsified lysed cell composition by adding a 50 wt % NaOH solution to adjust the pH of the composition to 7-8.5 and, after a few hours at 90? C., centrifuging (Thermo Scientific Sorvell ST40R Centrifuge) the composition at 8000 RPM for 5 minutes to provide a crude oil, which yielded 92.5% DHA (by DHA weight).

Example 7

(8) A cell broth (350 g) containing microbial cells (Schizochytrium sp.) was pasteurized at 60? C. for 1 hour. A demulsified lysed cell composition was formed by adding 2%, by weight broth, of a 98% H.sub.2SO.sub.4 by weight solution, and then heating the broth to 90? C. for 5-6 hours. The microbial oil was separated from the demulsified lysed cell composition by adding a 50 wt % NaOH solution to adjust the pH to 7-8.5 and, after a few hours at 90? C., centrifuging (Thermo Scientific Sorvell ST40R Centrifuge) the composition at 8000 RPM for 5 minutes to provide a crude oil, which yielded 82.63% DHA (by DHA weight).

Example 8

(9) A cell broth (350 g) containing microbial cells (Schizochytrium sp.) was pasteurized at 60? C. for 1 hour. A demulsified lysed cell composition was formed by adding 1%, by weight broth, of a 98% H.sub.2SO.sub.4 by weight solution, and heating the broth to 90? C. for 5-10 hours. The microbial oil was separated from the demulsified lysed cell composition by adding a 50 wt % NaOH solution to adjust the pH to 7-8.5 and centrifuging (Thermo Scientific Sorvell ST40R Centrifuge) the composition at 8000 RPM for 5 minutes to provide a crude oil, which yielded 42% DHA (by DHA weight).

Example 9

(10) A cell broth (350 g) containing microbial cells (Schizochytrium sp.) was pasteurized at 60? C. for 1 hour. A demulsified lysed cell composition was formed by adding 3%, by weight broth, of a 98% H.sub.2SO.sub.4 by weight solution and heating the broth to 90? C. for 1-5 hours. The microbial oil was separated from the demulsified lysed cell composition by adding a 50 wt % NaOH solution to adjust the pH to 7-8.5 and centrifuging (Thermo Scientific Sorvell ST40R Centrifuge) the composition at 8000 RPM for 5 minutes to provide a crude oil, which yielded 88% DHA (by DHA weight).

Example 10

(11) A cell broth (350 g) containing microbial cells (Schizochytrium sp.) was pasteurized at 60? C. for 1 hour. A demulsified lysed cell composition was formed by adding 4%, by weight broth, of a 70 wt % nitric acid solution and heating the broth to 90? C. for 1 hour. The microbial oil was separated from the demulsified lysed cell composition by adding a 50 wt % NaOH solution to adjust the pH to 7-8.5 and centrifuging (Thermo Scientific Sorvell ST40R Centrifuge) the composition at 8000 RPM for 5 minutes to provide a crude oil, which yielded 82% DHA (by DHA weight).

Example 11

(12) A cell broth (350 g) containing microbial cells (Schizochytrium sp.) was pasteurized at 60? C. for 1 hour. A demulsified lysed cell composition was formed by adding 2%, by weight broth, of a 70 wt % nitric acid solution and heating the broth to 90? C. for 5-6 hours. The microbial oil was separated from the demulsified lysed cell composition by adding a 50 wt % NaOH solution to adjust the pH to 7-8.5 and centrifuging (Thermo Scientific Sorvell ST40R Centrifuge) the composition at 8000 RPM for 5 minutes to provide a crude oil, which yielded 58.5% DHA (by DHA weight).

Example 12

(13) A cell broth (350 g) containing microbial cells (Schizochytrium sp.) was pasteurized at 60? C. for 1 hour. A demulsified lysed cell composition was formed by adding 2.5%, by weight broth, of a 70% nitric acid solution and heating the broth to 90? C. for 5 hours. The microbial oil was separated from the demulsified lysed cell composition by adding a 50 wt % NaOH solution to adjust the pH to 7-8.5 and, after a few hours at 90? C., centrifuging (Thermo Scientific Sorvell ST40R Centrifuge) the composition at 8000 RPM for 5 minutes to provide a crude oil, which yielded 74% DHA (by DHA weight).

Example 13

(14) A cell broth (350 g) containing microbial cells (Schizochytrium sp.) was pasteurized at 60? C. for 1 hour. A demulsified lysed cell composition was formed by adding 4%, by weight broth, of phosphoric acid and heating the broth to 90? C. for 5 hours. The microbial oil was separated from the demulsified lysed cell composition by adding a 50 wt % NaOH solution to adjust the pH to 7-8.5 and, after a few hours at 90? C., centrifuging (Thermo Scientific Sorvell ST40R Centrifuge) the composition at 8000 RPM for 5 minutes to recover the crude oil. A 45 g sample of this composition yielded 1.0 mL of crude oil.

Example 14

(15) A cell broth (350 g) containing microbial cells (Schizochytrium sp.) was pasteurized at 60? C. for 1 hour. A demulsified lysed cell composition was formed by adding 8%, by weight broth, of phosphoric acid and heating the broth to 90? C. for 3 hours. The microbial oil was separated from the demulsified lysed cell composition by adding a 50 wt % NaOH solution to adjust the pH to 7-8.5 and, after a few hours at 90? C., centrifuging (Thermo Scientific Sorvell ST40R Centrifuge) the composition at 8000 RPM for 5 minutes to recover the crude oil. A 45 g sample of this composition yielded 0.5 mL of crude oil.

Example 15

(16) A cell broth (2000 L) containing microbial cells (Schizochytrium sp.) was placed in a treatment tank agitating at 190 rpm. A demulsified lysed cell composition was formed by adding 4%, by weight broth, of a 98% H.sub.2SO.sub.4 by weight solution and heating the broth to 90-95? C. for 1 hour. The microbial oil was separated from the demulsified lysed cell composition by cooling the composition to 75? C., adding a 50 wt % NaOH solution to adjust the pH to 7-8.5, mixing the composition overnight, and centrifuging the composition in line at 1754 RPM at 8 L/min to provide a crude oil, which yielded 93% DHA (by DHA weight) and a crude oil with a Peroxide Value (PV) of 0.69 meq, an AV of 23.9, and a free fatty acid (FFA) content of 1.04.

Example 16

(17) A cell broth (550 L) containing microbial cells (Schizochytrium sp.) was placed in a treatment tank agitating at 193 rpm. The cell broth was heated to 60? C., the pH adjusted to 7.3-7.5 by adding a 50 wt. % NaOH solution, and alcalase was added in an amount of 0.5% by weight broth. After 2 hours, the pH of the lysed cell composition was adjusted to 4 by adding a 98% H.sub.2SO.sub.4 by weight solution, and heated to 90? C. while maintaining the agitation. After 20 hours at 90? C., the microbial oil was separated from the demulsified lysed cell composition by cooling the composition to 75? C., adjusting the pH to 8.5 by adding a 50 wt. % NaOH solution, holding the composition overnight, and centrifuging the composition in line at 1754 RPM at 8 L/min to provide a crude oil, which yielded 84% DHA (by DHA weight) and a crude oil with a PV of 0 meq, an AV of 7.3, and a FFA content of 0.17.

Example 17

(18) A cell broth (2500-3000 Liters) containing microbial cells (Mortierella) was placed in a decanting tank for several hours to settle. Once settled, 50-60% of the biomass free liquid was pumped off for discard. The concentrated unwashed pasteurized cell broth was then passed through a high shear mixer (Siemens ULTRA-Turrax UTL 1000/10 at 60 Hz) that was in line to the treatment tank. A demulsified lysed cell composition was formed by adding 3%, by weight broth, of a 98% H.sub.2SO.sub.4 by weight solution to adjust the pH to 0.9 to 1.5 and the broth heated to 94? C. for 20-24 hours or until a 1 mL in a 10 mL dilution of broth in deionized water yields a particle size of 25 ?m or less. The demulsified lysed cell composition was diluted with deoxygenated water to a solids content of 10% (number generated using Omnimark Uwave moisture analyzer) and reheated to 85? C. if the water addition caused the temperature to drop below 85? C. The microbial oil was separated from the demulsified lysed cell composition by adding a 50% NaOH solution to adjust the pH to 7.8-8.2, allowing the composition to mix for 30 minutes or until centrifugation of a sample showed a complete absence of an emulsion layer, and centrifuging (Seital SR1010) the composition in line at 1750 RPM and at a flow rate of 7 liters/min for 7-10 hours to recover the crude oil.

(19) Three separate batches of cell broth were each independently subjected to the above process.

(20) The Batch 1 crude oil yielded 86.6% ARA (by ARA weight) and a dried, filtered crude oil with an AV of 26.1 and a PV of 0.39 meq.

(21) The Batch 2 crude oil yielded 87.6% ARA (by ARA weight) and a dried, filtered crude oil with an AV of 14 and a PV of 0.31meq.

(22) The Batch 3 crude oil yielded 85% ARA (by ARA weight) and a dried filtered crude oil with an AV of 24.7 and a PV of 0.51 meq.

Example 18

(23) A cell broth (1000 mL) containing microbial cells (Mortierella) was pasteurized at 70? C. for 1 hour. A demulsified lysed cell composition was formed by adding 4%, by weight broth, nitric acid (70% solution) to adjust the pH to 0.7-1.0 and heating the broth to 90? C. while agitating at a speed of 200 rpm for 30 hours. The microbial oil was separated from the demulsified lysed cell composition by adding 50% w/w solution of NaOH to adjust the pH 8.0-8.2 and centrifuging (Thermo Sorvall ST 40R) the composition at 8,000 g for 5 minutes to provide a crude oil, which yielded 74.13% ARA (by ARA weight) and a crude oil with an AV of 27.9.

Example 19

(24) A cell broth (1000 mL) containing microbial cells (Mortierella) was pasteurized at 70? C. for 1 hour. A demulsified lysed cell composition was formed by adding 15%, by weight broth, phosphoric acid (85% solution) to adjust the pH to 0.7 to 1.5 and heating the composition to 90? C. while agitating at a speed of 200 rpm for 50 hours. The microbial oil was separated from the demulsified lysed cell composition by adding 50% w/w solution of NaOH to adjust the pH 8.0-8.2 and centrifuging (Thermo Sorvall ST 40R) the composition at 8,000 g for 5 minutes to provide a crude oil, which yielded 42.61% ARA (by ARA weight). The crude oil had an AV of 16.1.

Example 20

(25) A cell broth (1000 mL) containing microbial cells (Mortierella) was pasteurized at 70? C. for 1 hour. A demulsified lysed cell composition was formed by adding 6%, by weight broth, hydrochloric acid (32% solution) to adjust the pH to 0.7-1.5 and heating the broth to 90? C. while agitating at a speed of 200 rpm for 50 hours. The microbial oil was separated from the demulsified lysed cell composition by adding 50% w/w solution of NaOH to adjust the pH of the composition to 8.0-8.2 and centrifuging (Thermo Sorvall ST 40R) the composition at 8,000 g for 5 minutes to provide a crude oil, which yielded 72.61% ARA (by ARA weight). The crude oil had an AV of 11.1.

Example 21

(26) A cell broth (300 g) containing microbial cells (Schizochytrium sp.) was pasteurized at 60? C. for 1 hour. A demulsified lysed cell composition was formed by: adding a 12.5 wt % NaOH solution to the cell broth to adjust the pH to 11, adding 2% NaCl, by weight broth, and heating the composition to 90? C. After 25 hours at 90? C., the microbial oil was separated from the demulsified lysed cell composition by centrifuging (Thermo Scientific Sorvell ST40R Centrifuge) the composition at 8000 RPM for 5 minutes to provide crude oil, which yielded 71% DHA (by DHA weight).

Example 22

(27) An unwashed cell broth (2129.8 kg) containing microbial cells (Mortierella) was pasteurized at 70? C. for 1 hour and then passed through a high shear mixer (Siemens ULTRA-Turrax UTL 1000/10 at 60 Hz) that was in line to the treatment tank. A demulsified lysed cell composition was formed by: adding 3.5%, by weight broth, of a 98% sulfuric acid solution to adjust the pH to 0.07 to 1.5; heating to 90? C.; and holding for 28 hours while agitating at a speed of 200 RPM. The microbial oil was separated from the demulsified lysed cell composition by neutralizing the composition and adjusting the pH to 8.0 by addition of 50% NaOH and 2%, by weight composition, NaCl, and then centrifuging (Seital SR1010) the composition to provide a crude oil, which yielded 77.4% ARA (by ARA Weight) and a crude oil with an AV of 12.4, a FFA content of 2.27%, and a PV of 0.13 meq.

Example 23

(28) A washed cell broth (750 g) containing microbial cells (Crypthecodinium cohnii) was pasteurized at 60? C. for 1 hour. The cells were mechanically lysed by passing the cell broth through a homogenizer (Microfluidizer M110) twice at 12,000 PSI. The lysed cell composition was placed in a round flask and demulsified by adding a 4%, by weight composition, sulfuric acid (98% pure) solution, heating to 90? C. while agitating at 200 RPM, and holding for 28 hours. The microbial oil was separated from the demulsified lysed cell composition by neutralizing the composition and adjusting the pH to 8.0 by addition of a 50% NaOH solution, and then centrifuging (Thermo Sorvall ST 40R) the composition at 8000 g for 5 minutes to provide a crude oil, which yielded 45% DHA (by DHA weight)(based on FAME analysis) and a crude oil with an AV of 12.35 and a PV of 1.09 meq.

Example 24

(29) An unwashed cell broth (750 g) containing microbial cells (Schizochytrium sp.) was pasteurized at 60? C. for 1 hour and placed in a 1 L flask agitating at 250 RPM. The temperature was raised to 60? C. to decrease mix time due to viscosity. A demulsified lysed cell composition was formed by: adding 4%, by weight broth, of a 50% NaOH to adjust the pH to 12.62; heating to 90? C.; and allowing to react for 26 hours until the pH dropped to 7.75. The microbial oil was separated from the demulsified lysed cell composition by centrifuging (Thermo Sorvall ST 40R) the composition at 8000 g for 5 minutes to provide a crude oil, which yielded 88% DHA (by DHA weight) (based on FAME analysis).

Example 25

(30) An unwashed cell broth (750 g) containing microbial cells (Schizochytrium sp.) was pasteurized at 60? C. for 1 hour and placed in a 1 L flask agitating at 250 RPM. The temperature was raised to 60? C. to decrease mix time due to viscosity. A demulsified lysed cell composition was formed by: adding 4%, by weight broth, of a 50% NaOH solution to adjust the pH to 12.93; heating to 90? C.; and allowing to react for 26 hours until the pH dropped to 7.95. The microbial oil was separated from the demulsified lysed cell composition by centrifuging (Thermo Sorvall ST) the composition at 8000 g for 5 minutes to provide a crude oil, which yielded 82.5% DHA (by DHA weight) (based on FAME analysis).

Example 26

(31) An unwashed cell broth (750 g) containing microbial cells (Schizochytrium sp.) was pasteurized at 60? C. for 1 hour and placed in a 1 L flask agitating at 250 RPM. The temperature was raised to 60? C. to decrease mix time due to viscosity and the pH raised to 7.5. A demulsified lysed cell composition was formed by: adding 3.5%%, by weight broth, of a 50% NaOH solution to adjust the pH to 12.28; heating to 90? C.; and allowing to react for 32 hours until the pH dropped to 7.88. The microbial oil was separated from the demulsified lysed cell composition by centrifuging (Thermo Sorvall ST 40R) the composition at 8000 g for 5 minutes to provide a crude oil, which yielded 88% DHA (by DHA weight) (based on FAME Analysis).

Example 27

(32) An unwashed cell broth (750 g) containing microbial cells (Schizochytrium sp.) was pasteurized at 60? C. for 1 hour and placed in a 1 L flask agitating at 250 RPM. The temperature was raised to 60? C. to decrease mix time due to viscosity. A demulsified lysed cell composition was formed by: adding 4.0%, by weight broth, of a 50% NaOH solution to adjust to a pH of 12.87; heating to 90? C.; and allowing to react for 36 hours until the pH dropped to 8.15. The microbial oil was separated from the demulsified lysed cell composition by centrifuging (Thermo Sorvall ST 40R) the composition at 8000 g for 5 minutes to provide a crude oil, which yielded 84% DHA (by DHA weight) (based on FAME analysis).

Comparative Example 1

(33) Cell broth (300 g) containing microbial cells (Schizochytrium sp.) was pasteurized at 60? C. for 1 hour. The unwashed cell broth was agitated at a speed of 180 RPM and heated to 60? C. The cells were lysed by adding 50 wt % NaOH solution to pH adjust the cells to 7-7.5 and Alcalase? 2.4 FG (available from, Novazymes (Franklinton, N.C.)) in an amount of 0.5% based on broth weight. While maintaining the agitation, the lysed cell composition was demulsified by simultaneously adding 12.5 wt % NaOH solution to adjust the pH to 10-11 and CaCl.sub.2 in an amount of 2%, by weight composition, and then heating the composition to 90? C. and holding for 68 hours. The microbial oil was separated from the demulsified lysed cell composition by centrifuging (Thermo Scientific Sorvell ST40R Centrifuge) the composition at 8000 RPM for 5 minutes to provide crude oil, which yielded 62.5% DHA (by DHA weight) and a crude oil with an AV of 43.8.