Alcalase 2.4 fg

Example 8

A cell broth (300 g) containing microbial cells (Schizochytrium sp.) was pasteurized at 60° C. for 1 hour. The 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 unwashed 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 10-11 by adding a 12.5 wt % NaOH solution. Simultaneously, SDS powder was added in an amount of 1% by weight of the lysed cell composition and the composition heated to 80-90° C. for 1.5 hours. After the hold time, 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 a few hours. The lysed cell composition was centrifuged (Thermo Scientific Sorvell ST40R Centrifuge) at 8000 RPM for 5 minutes to provide a crude oil.

Example 10

A cell broth (300 g) containing microbial cells (Schizochytrium sp.) was pasteurized at 60° C. for 1 hour. The 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 unwashed 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 10-11 by adding a 12.5 wt % NaOH solution. Simultaneously, SDS powder was added in an amount of 1% by weight of the lysed cell composition and the composition heated to 90° C. After 2.5 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 92% DHA (by DHA weight).

Example 6

An unwashed cell broth (500-700 kg) containing microbial cells (Schizochytrium sp.) was pasteurized at 60° C. for 1 hour. The cells were lysed by adding a 50 wt % NaOH solution to pH adjust to 7.2 to 7.5 and 0.5%, by weight broth, Alcalase® 2.4 FG (available from Novozymes (Franklinton, N.C.)) and agitating for 8 hours at 55-60° with pH control at 7.2-7.5. The lysed cell composition was demulsified by: continuing to agitate the composition at 221 RPM with narrow blade hydrofoil impellers and either water or steam for heating; adding 50 wt. % NaOH to adjust the pH to 10.5; adding 2%, by weight composition, NaCl; and heating to 90° C. After a few hours at 90° C., the microbial oil was separated from the demulsified lysed cell composition by adding 50 wt. % NaOH to adjust the pH to neutral (6.5 to 8.5) and centrifuging (Seital SR 1010 (Seital srl, Italy)) the composition at 1750 RPM to provide a crude oil, which yielded (on average) 85.28% DHA (by DHA weight).

Example 9

A cell broth (300 g) containing microbial cells (Schizochytrium sp.) was pasteurized at 60° C. for 1 hour. The 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 unwashed 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 10-11 by adding a 12.5 wt % NaOH solution. Simultaneously, SDS powder was added in an amount of 0.5% by weight of the lysed cell composition and the composition heated to 80-90° C. for 2 hours. After the hold time, 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 18 hours. The lysed cell composition was centrifuged (Thermo Scientific Sorvell ST40R Centrifuge) at 8000 RPM for 5 minutes to yield 80% crude oil, which yielded 85% DHA (by DHA weight).

Example 5

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% H₂SO₄ 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 8

An unwashed cell broth (500-700 kg) containing microbial cells (Schizochytrium sp.) was pasteurized at 60° C. for 1 hour. The cells were lysed by adding a 50 wt % NaOH solution to pII adjust to 7.2 to 7.5 and 0.5%, by weight broth, Alcalase® 2.4 FG (available from Novozymes (Franklinton, N.C.)) and agitating for 2 hours at 55-60°. The lysed cell composition was demulsified by: continuing to agitate the composition at 221 RPM with narrow blade hydrofoil impellers; adding 50 wt. % NaOH to adjust the pH to 10.5; adding 2%, by weight composition, NaCl; and heating to 90° C. After a few hours at 90° C., 50 wt. % NaOH was added to the composition to readjust the pH to 9-10.5 and the composition held at 90° C. for few hours with agitation. The microbial oil was separated from the demulsified lysed cell composition by centrifuging (Seital SR 1010 (Seital srl, Italy)) the composition at 1750 RPM to provide a crude oil, which yielded (on average) 86.57% DHA (by DHA weight).

Example 7

An unwashed cell broth (500-700 kg) containing microbial cells (Schizochytrium sp.) was pasteurized at 60° C. for 1 hour. The cells were lysed by adding a 50 wt % NaOH solution to pH adjust to 7.2 to 7.5 and 0.5%, by weight broth, Alcalase® 2.4 FG (available from Novozymes (Franklinton, N.C.)) and agitating for 2 hours at 55-60° with pH control. The broth was treated with a second dose of 0.5%, by weight broth, Alcalase® and held under agitation for an additional 2 hours with pH control. The lysed cell composition was demulsified by: continuing to agitate the composition at 221 RPM with narrow blade hydrofoil impellers and either water or steam for heating; adding 50 wt. % NaOH to adjust the pH to 10.5; adding 2%, by weight composition, NaCl; and heating to 90° C. After a few hours at 90° C., the microbial oil was separated from the demulsified lysed cell composition by adding 50 wt. % NaOH to adjust the pH to neutral (6.5 to 8.5) and centrifuging (Seital SR 1010 (Seital srl, Italy)) the composition at 1750 RPM to provide a crude oil, which yielded (on average) 81.12% DHA (by DHA weight).