PRODUCTION OF LIPIDS CONTAINING POLY-U N SAT U RATE D FATTY ACIDS
FIELD OF THE INVENTION
The invention relates to recovery of lipids containing poly-unsatu rated fatty acids.
BACKGROUND OF THE INVENTION The worldwide market for lipids containing polyunsaturated fatty acids (PUFA) such as omega-3 (n-3) oils as a healthy food ingredient is increasing rapidly, in the areas of adult supplements, infant formula and as food ingredients. Such lipids may be produced by cultivation of algal cells, followed by cell cracking (or lysis) (WO 01/53512, WO 01/54510, US 2006/0286205). It is known that after the cell lysis, quality and stability of the poly-unsaturated lipid may deteriorate due to oxidation of the unsaturated olefinic bonds. It is further known that metallic ions may catalyze the oxidative reaction, and chelators may be added to reduce the speed of the oxidative damage.
WO 01/53512 (Omegatech) describes a method for extracting lipids from microor- ganisms by lysing cells and removing water soluble materials by washing until a non- emulsified lipid is obtained.
US 2006/0286205 (to Martek) describes methods for producing a product comprising a long chain polyunsaturated fatty acid by hydrolyzing biomass and emulsifying the hy- drolyzed biomass. US 2004/0170728 (to Kraft) describes the use of antioxidant compositions containing a siderophore such as rhodotorulic acid and an organic acid such as lactobionic acid to retard oxidation of lipids such as fish oil.
SUMMARY OF THE INVENTION
The invention provides an improved process for recovery of lipids containing poly- unsaturated fatty acids (PUFA) from a culture broth of algae with intracellular PUFA lipids.
More particularly, the invention leads to a PUFA product with a low level of free or total metal ions (particularly iron and copper ions) and good oxidation stability, and this can be achieved in a simple process with few separation steps without the need for a siderophore. Fewer separation steps may lead to less loss of antioxidant and hence a lower need for addition of antioxidant to achieve good oxidation stability.
Accordingly, the invention provides a process for producing a lipid, comprising: a) cultivating microbial cells so as to produce intracellular lipid comprising a polyunsaturated fatty acyl group, b) lysing the microbial cells in the culture broth so as to release the lipid, c) separating the lysate by gravitational force to collect a fraction which is a lipid- containing emulsion, d) adding a metal chelating agent during or after the lysis or the separation..
DETAILED DESCRIPTION OF THE INVENTION
Lipids containing poly-unsaturated fatty acid (PUFA)
The PUFA-containing lipids produced in the process of the invention may comprise mono-, di- or triglycerides comprising a poly-unsaturated fatty acyl group. The polyunsaturated fatty acids may include omega-3 or omega-6 fatty acid such as docosa- 4:7:10:13:16:19-hexaenoic acid (DHA), eicosapentaenoic acid (EPA) and docosapentaenoic acid (DPA).
Cultivation of microbial cells
The microbial cells may be Eukaryota; stramenopiles; Labyήnthulida;
Thraustoch ytriidae; Schizochytrium or Thraustochytrium, e.g. S. limacinum. They may be fungi or algae. The microbial cells are cultivated so as to produce intracellular lipid. This may be done as described in EP 823475 (Agency of Industrial Science and Technology et al.), JP
1 1-318484A (Sagami Chem. Res. Cent, et al.), WO 01/53512 (Omegatech), US
2006/0286205 (to Martek), and WO 01/54510 (Omegatech).
The microorganism may form structured triglycerides mainly with one poly- unsaturated fatty acyl group in the sn2 position and saturated or mono-unsaturated acyl (e.g. palmitoyl) in the sn1 and/or the sn3 positions, or it may form an unstructured triglyceride with an essentially random distribution.
Lysis of microbial cells The culture broth after cultivation is treated so as to lyse the microbial cells and release the lipid. This may be done by bead milling, homogenization, treatment with a cell-wall degrading enzyme, addition of an alkali or heating.
The cell-wall degrading enzyme may include protease, alginate lyase, pectinase, phospholipase or a carbohydrate hydrolase such as a cellulase, hemicellulase. Homogenization is advantageously done at high temperature (above 450C) and high pressure (e.g., 500-1000 bars). Two or more runs through the homogenizer may be used in order to break a large percentage of the cells. Gravitational separation
The separation by gravitational force conveniently includes centrifugation where the lipid-containing emulsion is collected as the top phase. Continuous centrifugation may be advantageous. The separation may include one or more additional cycles, where each cycle includes adding water to the lipid emulsion, agitating, and separating by centrifugation.
The emulsion collected after the first centrifugation may particularly be an oil-in- water emulsion. The emulsion collected after the last cycle may be a water-in-oil emulsion and/or may have a water content of 0.1-10 %, particularly 1-10 % by weight.
Metal-chelating agent
The metal chelator may include EDTA (ethylene-diamine tetra-acetic acid), an aldo- bionic acid (such as lactobionic acid), phosphoric acid, gluconic acid, citric acid, transferrin
(e.g. produced recombinantly or isolated from milk) or another protein chelator. The metal chelator may be added up to as much as can nutritionally be responsible to add, e.g. in an amount of 10-10,000 ppm by weight, particularly 100-5,000 ppm.
Optional oxygen scavenger
An oxygen scavenger may be added in addition to the metal chelator. The oxygen scavenger may comprise laccase, superoxide dismutase or an enzymatic system comprising glucose oxidase, catalase and glucose, e.g. as described in WO9631 133, US3920521 -A, EP649603 or EP934702. Alternatively, the oxygen scavenger may comprise an anti-oxidant such as cocoa, tocopherol or resveratrol, e.g. in an amount of 0.1-1 % by weight.
Optional triglyceride hydrolysis
It may be desirable to include hydrolysis of triglycerides to diglycerides with a higher PUFA content and free fatty acids with a lower PUFA content, followed by separation to re- move free fatty acid. This may be done after the lipid separation and before the chelator addition.
The hydrolysis may advantageously be done by incubation with a lipase which preferentially hydrolyzes saturated fatty acids rather than polyunsaturated fatty acids, e.g. the lipase from Candida rugosa. In the case of structured lipid, the hydrolysis may advanta- geously be done with a 1 ,3-specific lipase so as to hydrolyze saturated fatty acids in the sn1 and sn3 positions and thereby increase the PUFA content of the resulting lipid. The 1 ,3- specific lipase may be derived from Thermomyces lanuginosus (EP305216), Rhizomucor miehei, Aspergillus niger, Mucorjavanicus, Rhizopus delemar, or Rhizopus arrhizus.
The free fatty acid may be removed by converting it to a methyl ester and distillation. Oxidation stability
The oxidative stability of PUFA lipid can be evaluated by determination of peroxide value, e.g. by AOCS Official method Cd 8b-90 (Peroxide Value, Acetic Acid - lsooctane Method) or as described in R. Abuzaytoun et al., J. Agric. Food Chem., 54 (21 ), 8253 -8260, 2006.
EXAMPLES
Example 1 : Recovery of PUFA-containing lipid from algal culture broth
PUFA-containing lipid was produced and recovered by the following process:
Cultivation of cells Cells of Schizochytrium sp. were cultivated to obtain a culture broth. Approx. 50 %
(by weight) of the mature cells was lipids. 25-30 % of the lipid content was DHA (22:6(n-3)), and 50-60 % was palmitic acid (16:0). The lipid in the algae was present mainly as lipid vesicles with triglycerides (3 fatty acids pr molecule), and as sterol esters (1 fatty acid) and phospholipids (2 fatty acids) in the cell and organelle membranes. Pre lysis
A 750 L pressure tank was heated to 75-8O0C with steam and flushed with nitrogen.
The pressure was kept at 1 atm + 0.2 bars after flushing.
Lysis of cells
The culture broth was run through a bead mill with 0.6 mm SiLi beads (Zirconium Oxide), Flow was 200 L/h, after which the fluid was heated in a 1 -2 m2 heat exchanger to 750C, and transferred to the pressure tank.
The pH in the pressure tank was regulated to pH 10 with 45% KOH, as soon as the fluid level reached the pH sensor in the tank.
Pre separation A Westfalia SB-7 centrifuge was heated to 750C with steam, and jerrycans were prepared for product reception by nitrogen-flushing.
Separation
After 1.5-4.0 hours in the pressure tank, the alkaline liquid still over 7O0C was run through the SB-7 oil centrifuge (200 L/h, 4.0-4.4 bars counter pressure), and the lipid emul- sion was recovered as the top phase. The lipid emulsion was collected in 25 L jerrycans.
In separate experiments, it was found that very often water should be added in order to get a good separation; 1 to 2 volumes of water to 1 volume alkaline lysed culture broth, especially if the culture broth is rich in biomass.