METHOD FOR ISOLATING LIPIDS FROM LIPID-CONTAINING CELLS

Abstract

The current invention relates to a method for isolating polyunsaturated fatty acids containing lipids from lipid-containing cells.

Claims

1-17. (canceled)

18. A method for obtaining a polyunsaturated fatty acids (PUFAs)-containing lipid, comprising the following steps: a) providing a suspension of a biomass comprising cells which contain a PUFAs-containing lipid; b) heating the suspension of paragraph (a) to a temperature of between 50 C. and 70 C., adding a cell wall-degrading enzyme to the suspension, and, if necessary, adjusting the suspension to a pH value at which the enzyme is active; c) maintaining the temperature and pH in the ranges specified in paragraph (b) for at least one hour; d) concentrating the suspension obtained in step (c) by evaporation of water at a temperature not higher than 100 C., until a total dry matter content of 30 to 60 wt.-% is reached; e) adjusting the suspension obtained in step (d) to a temperature of 80 C. to 100 C., and adding an alkalizing agent, to adjust the pH to 9.5 to 11.5; maintaining the temperature in the ranges specified in paragraph (e) and the pH value in the range of 9.0 to 11.5, for at least 10 hours, thereby allowing de-emulsification of the suspension.

19. The method of claim 18, wherein: in step b), the suspension of paragraph (a) is heated to a temperature of between 55 C. and 65 C.; in step c), the temperature and pH in the ranges specified in paragraph (b) are maintained for two to four hours; in step d), the suspension obtained in step (c) is concentrated by evaporation of water at a temperature of 70 C. to 100 C., until a total dry matter content of 35 to 55 wt.-%, is reached; in step e, the suspension obtained in step (d) is adjusted to a temperature of 85 C. to 95 C., and an alkalizing agent is added to adjust the pH to a value of 10.0-11.0; the temperature in the ranges in paragraph (e) and a pH value in the range of 9.0 to 11.0, are maintained for 15 to 40 hours.

20. The method of claim 19, wherein, in step e), the pH is adjusted to a value of 10.3 to 10.7 and the temperature and pH are maintained for a period of 20 to 36 hours.

21. The method of claim 18, further comprising harvesting the PUFAs-containing oil.

22. The method of claim 21, wherein harvesting of the PUFAs-containing oil comprises neutralizing the demulsified suspension and subsequently separating the resulting oil-containing light phase from the water, salt and cell debris-containing heavy phase.

23. The method of claim 22, wherein neutralization is realized by adding an acid to the demulsified suspension to adjust it to a pH of 5.5 to 8.5, and wherein separation of the oil-containing light phase from the water, salt and cell debris-containing heavy phase is realized by mechanical means at a temperature of 60-90 C.

24. The method of claim 23, wherein neutralization is realized by adding an acid to the demulsified suspension to adjust it to a pH of 7.0-8.0, and wherein separation of the oil-containing light phase from the water, salt and cell debris-containing heavy phase is realized by mechanical means at a temperature of 70-80 C.

25. The method of claim 21, wherein less than 1 wt.-% of organic solvents and/or less than 1 wt.-% of chloride are used in the method.

26. The method of claim 25, wherein less than 0.1 wt.-%, of organic solvents and/or less than 0.1 wt.-%, of chloride are used in the method.

27. The method of claim 21, comprising as a further step the conversion of the water, salt, residual oil and cell debris-containing heavy phase into a dried biomass by drying the biomass to a total dry matter content of more than 90 wt.-%,

28. The method of claim 27, wherein conversion into a dried biomass is carried out by concentration of the heavy phase to a dry matter content of 30-50 wt.-% and subsequent spray granulation using a fluidized bed granulator.

29. The method of claim 21, wherein the suspension is provided as a fermentation broth with a biomass density of at least 80 g/l.

30. The method of claim 29, 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 claim 30, wherein the cell-wall degrading enzyme is selected from the group consisting of: proteases, cellulases, hemicellulases, chitinases, pectinases, sucrases, maltases, lactases, alpha-glucosidases, beta-glucosidases, amylases, lysozymes, neuraminidases, galactosidases, alpha-mannosidases, glucuronidases, hyaluronidases, pullulanases, glucocerebrosidases, galactosylceramidases, acetylgalactosaminidases, fucosidases, hexosaminidases, iduronidases, maltases-glucoamylases, beta-glucanases, mannanases, and combinations thereof.

32. The method of claim 31, wherein the suspension is provided as a fermentation broth with a biomass density of at least 100 g/l, and the cells which contain a PUFAs-containing lipid are Thraustochytrids.

33. A PUFAs-containing biomass comprising less than 0.5 wt.-%, of a non-polar organic solvent and less than 0.5 wt.-%, of chloride, wherein the biomass contains cells and/or cell debris of the genus Schizochytrium.

34. The PUFAs-containing biomass of claim 33, comprising a mixture of DHA and EPA, wherein the content of DHA is at least 10 wt.-% relative to the total amount of lipid present.

35. The biomass of claim 33, wherein the biomass is a delipidated biomass.

36. A PUFAs-containing aqueous suspension, containing a biomass, wherein: the content of non-polar organic solvents is less than 1 wt.-% and the content of chloride is less than 1 wt.-%; the biomass contains cells and/or cell debris of the family of Thraustochytrids; and the aqueous suspension comprises a total dry matter content of 20 to 60 wt.-%.

37. The PUFAs-containing aqueous suspension of claim 36, wherein the content of non-polar organic solvents is less than 0.1 wt.-% and wherein the biomass contains cells and/or cell debris of the genus Schizochytrium.

Description

WORKING EXAMPLES

Example 1

[0129] 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 forced circulation evaporator (obtained from GEA, Germany) and heated to a temperature of 85 C. The mixture was concentrated in the forced circulation evaporator, until a total dry matter content of about 30 wt.-% was reached. The concentrated lysed cell mixture was transferred into a new vessel, heated up to 90 C. under low shear agitation, while adjusting the pH to 10.5 by adding caustic soda, Low shear agitation was continued for about 30 hours, while keeping the temperature at 90 C. and the pH above 9.0 by adding caustic soda.

[0130] After that the resulting demuisified mixture was neutralized by adding sulfuric acid to adjust a pH of 7.5. Phase separation into a light phase, containing the oil, and a heavy phase, containing water, cell-debris, residual oil and salts, was carried out mechanically by using a disc stack separator (Alfa Laval Disc Stack Centrifuge, LAPX 404/Clara 20).

[0131] After separation of the crude oil, the remaining cell debris were resuspended in the aqueous phase, concentrated and dried by spraygranulation.

[0132] Due to the efficient demuisification, more than 90 wt.-% of the oil could be separated from the biomass without the addition of organic solvents or sodium chloride.

[0133] The remaining heavy phase was converted into a solid biomass by concentrating via evaporation to a total dry matter of 45 wt.-% at a temperature of about 90 C. and subsequent drying via spray granulation in a fluidized bed spray granulator. The resulting biomass exhibits a high bulk density of more than 530 kg/m3, a high energy value of about 4000 kcal/kg and very good handling properties, in particular a flowabiiity of 4. Comparable biomasses originating from Schizochytria as available on the market exhibit all a much worse flowability of 6 and a much lower bulk density of between 325 to 500 kg/m3.