METHOD OF ISOLATING LIPIDS FROM A LIPIDS CONTAINING BIOMASS

Abstract

The present invention relates to a method of isolated polyunsaturated fatty acids containing lipids from a lipids containing biomass.

Claims

1-15. (canceled)

16. A method of isolating a polyunsaturated fatty acids (PUFAs) containing lipid from a biomass, comprising the following steps: a) providing a demulsified PUFAs containing composition comprising a heavy phase and a light phase, characterized in that the ratio of oil to total dry matter (TDM) in the suspension is at least 0.5; b) separating the oil containing light phase from the water, salt, remaining oil and cell debris containing heavy phase by mechanical means.

17. The method of claim 16, wherein the composition of step a) has a TDM content of 20 to 60 wt.-%.

18. The method of claim 16, wherein the preparation of the demulsified PUFAs containing composition comprises the following steps: a) providing a suspension of a biomass comprising cells which contain a PUFAs containing lipid; b) lysing the cells of the biomass at least in part; c) concentrating the suspension to a total dry matter (TDM) content of 20 to 60 wt.-%, if the suspension has a lower TDM content; d) adjusting the suspension to a temperature of 20° C. to 100° C.; e) keeping the temperature in the range of step d) for at least 0.5 hours, while adding in a total of 7.5 to 25 moles of base equivalent to 10 kg of total dry matter in the suspension; f) adjusting the composition of step e) to a pH of 5.0 to 8.5, and incubating for 0.1 to 6 hours; wherein oil is added to the suspension sometime after step b), so that the ratio of oil to total dry matter (TDM) reaches a value of at least 0.5.

19. The method of claim 18, wherein the oil which is added to the suspension is a microbial oil, an oil of a marine organism, a vegetable oil, or a mixture thereof.

20. The method of claim 16, wherein the preparation of the demulsified PUFAs containing composition comprises the following steps: a) providing a suspension of a biomass comprising cells which contain a PUFAs containing lipid; b) lysing the cells of the biomass at least in part; c) concentrating the suspension to a total dry matter (TDM) content of 20 to 60 wt.-%, if the suspension has a lower TDM content; d) adjusting the suspension to a temperature of 60° C. to 100° C.; e) keeping the temperature in the range of step d) while adding in a total of 12 to 17 moles of base equivalent to 10 kg of total dry matter in the suspension; f) adjusting the composition of step e) to a pH of 5.0 to 6.5, and incubating for 0.5 to 4 hours; wherein oil is added to the suspension after step (f), so that the ratio of oil to total dry matter (TDM) reaches a value of 0.5 to 0.7; and wherein the oil is selected from the group consisting of: an oil of Stramanopiles; fish oil, canola oil, soya oil, or a mixture thereof.

21. The method of claim 16, wherein the preparation of the demulsified PUFAs containing composition comprises the following steps: a) fermenting PUFAs producing cells, until the ratio of oil to total dry matter (TDM) in the fermentation broth reaches a value of at least 0.5; b) lysing the cells of the biomass at least in part; c) concentrating the suspension to a total dry matter (TDM) content of 20 to 60 wt.-%, if the suspension has a lower TDM content; d) adjusting the suspension to a temperature of 20° C. to 100° C.; e) keeping the temperature in the range of step d) for at least 0.5 hours, while adding in a total of 7.5 to 25 moles of base equivalent to 10 kg of total dry matter as contained in the suspension; f) adjusting a pH value of 5.0 to 8.5, and incubating for 0.1 to 6 hours.

22. The method of claim 16, wherein the preparation of the demulsified PUFAs containing composition comprises the following steps: a) fermenting PUFAs producing cells, until the ratio of oil to total dry matter (TDM) in the fermentation broth reaches a value of 0.5 to 0.7; b) lysing the cells of the biomass at least in part; c) concentrating the suspension to a total dry matter (TDM) content of 20 to 60 wt.-%, if the suspension has a lower TDM content; d) adjusting the suspension to a temperature of 70 to 90° C.; e) keeping the temperature in the range of step d) for 10 to 24 hours, while adding in a total of 12 to 17 moles of base equivalent to 10 kg of total dry matter as contained in the suspension; f) adjusting a pH value of 5.0 to 6.5, and incubating for 0.5 to 4 hours.

23. The method of claim 19, wherein the pH in step (e) is kept below 11.5.

24. The method of claim 16, wherein the base is selected from hydroxides and/or carbonates and/or bicarbonates.

25. The method of claim 18, wherein the suspension as provided in step a) has a total dry matter content of 30 to 55 wt.-%.

26. The method of claim 19, wherein: the pH in step (e) is kept in the range of 9.0 to 10.5; wherein, in step e), the base used is selected from the group consisting of: a sodium hydroxide, lithium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, magnesium carbonate, lithium bicarbonate, sodium bicarbonate, and potassium bicarbonate; and the suspension as provided in step a) has a total dry matter content of 30 to 55 wt.-%.

27. The method of claim 16, wherein, to lyse cells, salts are added in an amount of less than 0.1 g/l fermentation broth.

28. The method of claim 16, wherein to lyse cells, organic solvents are added in an amount of less than 0.1 g/l fermentation broth.

29. The method of claim 16, wherein the suspension is provided as a fermentation broth with a biomass density of at least 80 g/l.

30. The method of claim 16, wherein the cells which contain a PUFAs containing lipid are selected from algae, fungi, protists, bacteria, microalgae, plant cells, and mixtures thereof.

31. The method of 16, wherein the cells that contain a PUFAs containing lipid are from the genus Schizochytrium.

32. A lipid comprising a mixture of a microbial oil, an oil from a marine organism, and a vegetable oil.

33. The lipid of claim 32, comprising 5 to 95 wt.-%, microbial oil; and 5 to 95% of marine organism oil, or vegetable oil.

34. The lipid of claim 32, comprising 30 to 70 wt.-% microbial oil and 30 to 70 wt.-% of marine organism oil or vegetable oil.

35. The lipid of claim 32 wherein the lipid has at least one of the following characteristics: a) a peroxide value of less than 0.5; b) an anisidine value of less than 10; c) a TOTOX value of less than 15; d) a content of free fatty acids of less than 3 wt.-%; e) a content of moisture and impurities of less than 1 wt.-%; f) a content of omega-3 fatty acids of at least 10 wt.-%; g) a DHA and EPA each in an amount of at least 8 wt.; h) an amount of organic solvents of less than 0.5 wt.-%; i) an amount of chlorides of less than 0.05 wt; j) a content of crude fat of more than 95 wt.-%.

Description

WORKING EXAMPLES

Example 1: Preparation of the Suspension for Use in the Demulsification Tests

[0143] An unwashed cell broth containing microbial cells (Schizochytrium sp.) at a biomass density of over 100 g/l was heated to 60° C. in an agitated vessel. After heating up the suspension, the pH was adjusted to 7.5 by using caustic soda (50 wt.-% NaOH solution), before an alcalase (Alcalase® 2.4 FG (Novozymes)) was added in liquid form in an amount of 0.5 wt.-% (by weight broth). Stirring was continued for 3 hours at 60° C. After that, the lysed cell mixture was transferred into a rotary evaporator and heated to a temperature of 80° C. at a reduced pressure of 400 mbar. The mixture was concentrated in the rotary evaporator, until a total dry matter content of about 35 wt.-% was reached.

[0144] As the next step, demulsification of the concentrated suspension was carried out. All experiments were carried out with about one liter of enzymatically treated and subsequently concentrated fermentation broth using a stirring vessel BIOSTAT® B-DCU-Quad 2L (Sartorius, Germany). Demulsification was carried out for 24 hours at a temperature of 90° C. The suspension was stirred with 300 rpm. 1.5 mol of caustic soda per kg TDM was added continually over a period of 3 hours. After 24 hours, the demulsified compositions were neutralized to either pH 5.5 or pH 7.5. After neutralization, the TDM content and oil content were determined to find out the ratio of oil to TDM. Subsequently different amounts of oil were added to investigate the influence of the oil to TDM ratio on the final oil yield. As oil either Schizochytrium oil as obtained by the method of the invention before or vegetable oil (canola oil or soya oil) were added. About two hours after the optional addition of oil, a 50 g sample of the homogenized suspension was taken and separation of the cell debris was carried out by centrifugation at 13.500 g. Subsequently the amount of EPA and DHA in the supernatant was determined.

TABLE-US-00001 TABLE 1 Influence of the TDM to oil ratio on the oil yield Initial Initial Added Total Oil to Recovery Recovery mass oil oil oil TDM of oil at of oil at Yield at Yield at [g] [g] [g] [g] ratio pH 7.5 pH 5.5 pH 7.5 pH 5.5 1004 205.1 0 205.1 0.54 193.0 201.0 94.1% 98.0% 1000 204.3 21.5 225.8 0.57 214.8 221.1 94.6% 97.7% 1009 206.1 48.5 254.6 0.61 243.8 250.7 94.8% 98.1% 1002 204.7 119 323.7 0.70 313.3 319.8 94.9% 98.1% 1007 205.7 0 205.7 0.44 192.1 198.0 93.4% 96.2% 1011 206.5 30.1 236.6 0.49 223.9 228.8 93.8% 96.2% 1008 205.9 0 205.9 0.44 193.6 197.8 94.0% 96.0% 1012 206.7 107.2 313.9 0.60 302.3 308.5 94.4% 97.4%

[0145] The experimental data show that when the ratio of oil to TDM exceeds the value of 0.5, the overall oil yield is increased significantly. It does not play a role here, whether this value is reached by fermenting the cells, until the ratio of oil to TDM exceeds this value or by recycling Schizochytrium oil during the demulsification, before the separation of the oil containing light phase from the aqueous phase takes place. Further it can be seen that when after the demulsification a pH value of 5.5 is adjusted, the oil yield is much higher in comparison to adjusting a pH value of 7.5.

TABLE-US-00002 TABLE 2 Influence of the TDM to oil ratio on the oil yield; addition of canola oil Initial Initial Added Total Oil to Recovery mass oil oil oil TDM of oil at Yield at [g] [g] [g] [g] ratio pH 7.5 pH 7.5 1008 134.1 0 134.1 0.42 125.7 93.7% 1008 134.1 171.2 305.3 0.60 290.4 95.1%

[0146] The experimental data show that the effect which was reached with recycling Schizochytrium oil can also be realized by adding vegetable oils, in particular canola oil.

TABLE-US-00003 TABLE 3 Influence of the TDM to oil ratio on the oil yield; addition of soy oil Initial Initial Added Total Oil to Recovery mass oil oil oil TDM of oil at Yield at [g] [g] [g] [g] ratio pH 7.5 pH 7.5 1004 133.9 0 133.9 0.42 125.3 93.6% 1000 133.9 173.2 305.3 0.60 291.7 95.0%

[0147] The experimental data show that the effect which was reached with recycling Schizochytrium oil can also be realized by adding soy oil.