Background of the Invention Phospholipids containing poly-unsaturated fatty acids (PUFA) supply the organism with important building blocks which improve membrane fluidity, an essential property for the function of biological membranes.

Studies conducted with PUFA containing phospholipids have shown that these biomaterials have many important physiological roles. They are high- energy, basic, structural, and functional elements of all biological membranes such as cells, blood corpuscles, lipoproteins, and the surfactant. Furthermore, they are indispensable for cellular differentiation, proliferation, and regeneration, maintaining and promoting the biological activity of many membrane-bound proteins and receptors. PUFA-containing phospholipids also play a decisive role in the activity and activation of numerous membrane-located enzymes, such as sodium-potassium-ATPase, adenylate cyclase, and lipoprotein lipase, are important for the transport of molecules through membranes and control membrane-dependent metabolic processes between the intracellular and intercellular space. Moreover, some PUFAs, such as linoleic acid, are precursors of the cytoprotective prostaglandins and other eicosanoids.

Due to all of their properties, many health benefits have been attributed to the consumption of fatty acids and in particular PUFA. For example, it has been reported that PUFA of the type omega-3 and omega-6 may be effective in the treatment and prevention of cardiovascular disease (CVD) [Din et al.

2004 BMJ 2004; 328 : 30-5; Hirafuji et al. J Pharmacol Sci. 2003; 92 (4): 308- 16], immune disorders and inflammation [Heller et al. Drugs 1998; 55: 487- 96; Gil Biomed Pharmacother. 2002; 56 (8): 388-96], , renal disorders [Donadio et al. Semin Nephrol. 2004; 24 (3): 225-43; Das et al. Prostaglandins Leukot Essent Fatty Acids. 2001; 65 (4): 197-203], allergies [Mickleborough et al. Am J Respir Crit Care Med. 2003; 168 (10): 1181-9; Simopoulos J Am Coll Nutr.

2002; 21 (6): 495-505; Arm et al. Allergy Proc. 1994; 15 (3): 129-34], diabetes [Stene et al. Am J Clin Nutr. 2003; 78 (6): 1128-34; Mori et al. Free Radic Biol Med. 2003; 35 (7): 772-81; Simopoulos Am J Clin Nutr. 1999; 70 (3 Suppl): 560S- 569S], and even cancer [Larsson et al. Am J Clin Nutr. 2004; 79 (6): 935-45; Astorg Cancer Causes Control. 2004; 15 (4): 367-86].

Besides its benefits with regards to CVD, diabetes and cancer, DHA is also important for enhancement of brain function, and in particular for brain development in infants. Nutritional studies, investigating the importance of DHA in the brain, found that low levels of DHA are associated with depression, memory loss, dementia and visual problems [Kalmijn et al.

Neurology 2004; 62 (2): 275-280; Conquer et al. Lipids 2000; 35 (12): 1305-1312; Logan Altern Med Rev. 2003; 8 (4): 410-25; Cho et al. Am J Clin Nutr. 2001; 73 (2): 209-18. Fugh-Berman et al. Psychosom Med. 1999; 61 (5): 712-28]. All studies showed a dramatic improvement in the elderly brain function as blood levels of DHA increased [Naliwaiko et al. Nutr Neurosci. 2004; 7 (2): 91- 9; Frasure-Smith et al. Biol Psychiatry. 2004; 55 (9): 891-6; Moriguchi et al. J Neurochem. 2003; 87 (2): 297-309; Horrocks et al. Pharmacol Res. 1999; 40 (3): 211-25].

The human body does not synthesize DHA in sufficient amounts. Therefore it is necessary to obtain it from the diet. DHA is initially obtained through the placenta, then from breast milk, and later from sources like fish, red meats, animal organ meats and eggs. These types of fatty acids are naturally occurring mainly in fish and algae, where they are randomly distributed on the s7 1, S7X-2, and sn-3 positions of the glycerol backbone of triglycerides. In particular, tuna, salmon and sardines are rich sources.

Furthermore, the ability to enzymatically produce omega-6 and omega-3 products of linoleic and alpha-alpha linolenic acid declines with age. Thus, as human beings age, there is an increased need to acquire DHA directly from diet or supplements.

Because DHA is important for signal transmission in the brain, eye and nervous system, many consumers concerned with maintaining mental acuity seek for a pure, safe way to supplement their DHA levels. Until recently, the primary source of DHA dietary supplements has been fish oils.

In light of the important physiological roles of phospholipids containing PUFA for human health, and the scarce availability of said compounds in the organism, there is a demand for dietary supplementation of PUFA-containing phospholipids.

Many PUFA-containing agents suffer from stability and quality problems due to the high degree of oxidation of the polyunsaturated fatty acids. These problems require the incorporation of antioxidants as well as the utilization of special measures which attempts to reduce this oxidation. The utilization of phospholipids as carriers of PUFA may result in enhanced stability of such products due to the anti-oxidative properties of phospholipids.

PUFA-containing phospholipids may be prepared by various ways, mainly by (i) enzymatic esterification and transesterification of phospholipids, (ii) chemical synthesis of phospholipids, or (iii) enzymatic transphosphatidylation of phospholipids.

One example was reported by Hosokawa et al. [Hosokawa, M. et al. (1995) J. A » a. 0il Chem. Soc. 72: 1287], wherein phospholipids containing PUFA at the si2-1 position were prepared by lipozyme-catalyzed acidolysis of phosphatidylcholine (PC) with a mixture of eicosapentenoic acid (EPA) and docosahexaenoic acid (DHA) in hexane media. In addition PC containing PUFA at the sn-2 position was prepared by PLA2-mediated condensation of lyso-PC and PUFA in glycerol as a reaction medium. The addition of a small amount of formamide (as a water-mimic) to the reaction mixture gave best results, with 60% yield. In contrast to this process, our enzymatic reactions are preformed without an organic solvent but in the DHA-FFA itself. We don't have to add water mimic since the water bounded to the enzymatic preparation is sufficiently for the reaction and do not cause the hydrolytic reaction to proceed to a great extent (up to 5% Lysophospholipids, i. e. the hydrolytic product, from overall phospholipids).

In the same reference Hosokawa describes the transphosphatidylation of the omega-3 containing phosphatidylcholine to phosphatidylethanol amine and phosphatidyl serine. The transphosphatidylation occurs in biphasic system of ethylacetate and buffer. At the end, the recovered phospholipids was purified by TLC. This method is valid however could not be used for the production of large quantities.

Another example of enzymatic esterification of long PUFA and lyso-PC was reported [Lilja-Hallberg, M. and Harrod, M. (1994) Biocatalysis, 9,195-207].

Phosphatidylcholine was synthesized from lyso-PC and long PUFA using immobilized PLA2 (immobilized on polymeric carrier Deloxan). The esterification was preformed using the fatty acids as main solvent and isooctane or ethanol at low concentrations (7-45%) as additional solvents. The best yield of PC (22%) was found in the isooctane system when its concentrations were below 7% and the reaction times were as long as 9 days.

The long reaction, combined with the heating at 45OC, make these conditions very problematic with respect to the stability of omega-3 towards oxidation.

The diminished stability, together with the use of organic solvents, such as isooctane, makes this process less convenient for the production of PC in large scale.

Mutua and Akoh [Mutua, L. N. and Akoh, C. C. (1993) JAOCS, 70 (2): 125] modified phospholipids through lipase-catalyzed transesterification in order to incorporate n-3 PUFA. The phospholipid modification was carried out in organic media with lipase from Mucor miehei and PLA2 as biocatalysts. The phosphatidylcholine was initially hydrolyzed and then synthesized with omega-3 fatty acids. The enzyme that gave the best incorporation was non- immobilized lipase from Mucor miehei (17. 7 mol%) followed by non- immobilized PLA2 (17.2 mol%). With the PLA2, the yield of PC was not satisfactory.

Synthesis of phosphatidylethanolamine (PE), which contains highly unsaturated fatty acids specifically in the su-2 position, was performed with porcine PLA2 by Hosokawa et al [Masashi Hosokawa et al. (1995) I2ternationzal JourTZal of Food Science a7ad Technology, 29,721-725.). PE was synthesized from lysophosphatidylethanolamine and highly unsaturated fatty acids (HUFA), utilizing glycerol as a solvent, and resulting in a yield of 27% up to 94.5% of son-2 EPA-containing PE. Reactions were terminated by addition of chloroform : methanol: water mixture and the products recovered from the chloroform layer were separated on silica columns.

WO 91/00918 reports a method for the preparation of a phospholipid with carboxylic acid residue in the 2-position and a phospholipid with an omega-3 acid residue in the 2-position. The preparation is through esterification of the lyso phospholipids with an omega-3 fatty acid in microemulsion of organic solvent (like isooctane or heptane), in the presence of 0.1-2% of water. Apart from the lyso-phospholipid, the surface-active component comprises at least one nonionic or anionic surface active component. After a 24-hour reaction, the phospholipid fraction was obtained with a yield of 7%, of which more than 90% contained omega-3 fatty acid residues.

WO 91/03564 discloses a process whereby phospholipids and fatty acid (or ester) are treated with suitable lipase to obtain at least 5-20% exchange of the fatty acid. The process is obtained by using a lipase immobilized on a particulate macroporous carrier. The immobilized enzyme has water content prior to contact with the phospholipids in the range of 5-15% (by weight). The process is carried out in organic solvent such as petroleum ether or heptane.

Egger et al. studied PLA2 synthesis and the hydrolysis of PC in a water controlled organic medium, using PC and oleic acid as reactants [Egger, D. et al. (1997) Biochimica et Biophysica Acta 1343, 76-84]. The best yield in the synthetic reaction was 60%, at a water activity of 0.11 and an oleic concentration of 1.8M, however the synthesis was in the absence of omega-3 and omega-6.

There are several reports on the chemical synthesis of phospholipids.

Existing chemical methods for 1,2-diacyl PC synthesis rely primarily on acylation of lysoPC with fatty acyl chlorides [Baer, E. and Buchnead, D.

(1959) Can. J. Biochem. Physiol. 37,953-959] and acid anhydrides [Pugh, E. and Kates, M. (1975) J. Lipid Res. 16,392-394]. The reagents are usually used in large excess. Further, the use of fatty acyl chlorides may be accompanied by the formation of significant amounts of several side products [Aneja, R. and Chadha, J. S. (1971) Biochim. Biophys. Acta 239,84-91].

Acylation with anhydrides require relatively vigorous conditions, e. g. 48 hours at 80OC [Robles, E. C. and van DenBerg, D. (1969) Biochim. Biophys. Acta 187, 520-526] and the yield most often is unsatisfactory. Furthermore, one equivalent of the acyl substituent in the anhydride is wasted since fatty acyl carboxylate is the leaving group of the acylating agent. Methods which avoid the use of such extreme conditions have also been published and involve the use of catalysts like p- dimethylaminopyridine [Chhitar M. et al. (1977) Proc. Natl. Acad. Sci. USA 74 (10), 4315-4319] or 4-pyrrolidinopyridine [Mason, J. T. et al. (1981) Analytical Biochemistry 113,96-101]. Yields are excellent and the reaction is convenient to carry out. The drawback of using pyrrolidine is a slow rate of acylation, unless a large excess of anhydride is used.

Nicholas et al. report a method for synthesis of mixed acid phosphatidylcholine that relies on a silver-ion catalyzed acylation of lysoPC with 2-pyridinethiol fatty acid esters [Nicholas, A. W. et al. (1983) Lipids, 18 (6), 1983]. Although this method appears to be applicable for a variety of diacyl PC, its feasibility for using in product designed for food grade process appears to be very poor since it utilizes hazardous chemicals such as 2- pyridethiol and phosgene.

Werner and Benson reported a method for preparation of unsaturated phosphatidylcholine, which can be carried out on moderate or small scale under mild conditions and uses twice the theoretical amount of fatty acid [Werner, T. G. and Benson, A. A. (1977) Journal of Lipid Research, 18,548].

The main disadvantage of this process is that it is not applicable to large scale industry process for obtaining phospholipids with unsaturated fatty acids.

Lindberg et al. reported a new synthesis of phospholipids [Lindberg, J. (2002) J. Org. Chem. 67,194-199) starting from enantiomerically pure (S)-glycidol.

Direct phosphorylation and subsequent opening of the epoxide produced dibenzyl-protected lysoalkyl phosphatidic acid with 67% yield. DCC and DMAP promoted esterification of the lysophospholipid with palmitic acid and subsequent debenzylation of the phosphate produced l-o-alkyl-2-O-acyl- phosphatidic acid in 40% overall yield from (S) -glycidol. All the reactions were in the absence of omegap-3 and omega-6 fatty acids. The insertion of omega-3 and 6 is the main barrier in the esterification of fatty acids with phospholipids.

Haider et al. have also described chemical synthesis of PC bearing icosadienoyl group at the 1-position, with very long chain PUFAs [Haider, S. et al. (1998) Chemistry Letters, 175]. The synthesis was performed in the presence of ethanol-free chloroform at room temperature, using a synthetic phosphatidyl choline, prepared through carbon chain elongation of linoleic acid via malonic ester synthesis, preparation of lyso-phosphatidylcholine via lipase catalyzed mono-acylation of 2-O-methoxyethoxymethylglycerol and phosphodiester synthesis, and finally DCC-mediated esterification.

It is important to mention that PS is the major acidic phospholipid component in the membranes of the brain. It has been the subject of numerous human clinical trials of memory loss, mood, cognitive performance and learning abilities. Many of the studies show that PS can be helpful for those with age-related memory impairment, and that it can even help optimizing the cognition in those with no cognitive impairment [Sakai et al. J Nutr Sci Vitaminol 1996; 42: 47-54; Heiss et al. Dementia 1994; 5: 88-98; Kidd (1996) id ibid. ; Crook et al. Psychopharmacol Bull 1992; 28: 61-66].

Dietary PS is efficiently and rapidly absorbed in the intestine, is taken up into the blood, and readily crosses the blood-brain barrier to reach the nerve cells of the brain.

PS can be extracted from bovine brain or from plants, or it can be produced from soybean lecithin using biocatalysis. The main difference between the two sources is the type of fatty acids attached to positions 1 and 2 on the phospholipid skeleton. Long-chain polyunsaturated n-3 type fatty acids are characteristic of marine fat and occur pervasively in the phospholipids of marine species.

Phosphatidylserine can be made by using the transphosphatidylation reaction with phospholipases D (PLDs), by which the head group of phospholipids can be readily modified. Thus, phosphatidylserine can be produced from phosphatidylcholine or any other phospholipid mixture and serine by catalysis with PLD.

US 5,965, 413 describes a process for the production of phosphatidylserine having a long chain unsaturated fatty acid in its side chain. In this process a natural lecithin containing long chain unsaturated fatty acid side chain is used as starting material. The transphosphatidylation was performed in the presence of serine, PLD and ethyl acetate as a solvent. Hosokawa et al. describe a method for PLD-mediated transphosphatidylation of squid lecithin with L-serine, in the preparation of DHA acid-containing phosphatidylserine, in which the synthesis is conducted in a biphasic system of organic solvent and 0.2M acetate buffer [Hosokawa M. et al. (2000) J. Agric. Food Chem. 48, 4550-4554]. This transphosphatidylation process was performed on very low scale, The biphasic reaction system consists of 2.5 ml of organic solvent, 30 mg squid lecithin in addition to 0.8 unit of PLD dissolved in 1 ml acetate buffer containing 3.4M L-serine.

The present invention provides improved and more cost-effective methods for the production of omega-3/omega-6 enriched glycerophospholipids.

Thus, it is an object of the present invention to provide an improved enzymatic interesterification process for the enrichment of phospholipids with omega-3 and omega-6 fatty acids. The interesterification includes the processes of transesterification of lecithin with omega-3 and 6 fatty acid and esterification process.

It is another object of the present invention to provide chemical methods for the production of omega-3 and omega-6 enriched phosphatidylcholine, phosphatidylinositol, phosphatidylserine and phosphatidylethanolamine.

It is a further object of the present invention to provide a method for the production of stabilized phosphatidylserine preparations enriched with omega-3/omega-6 acid residues. In the method presented herein, the production is by transphosphatidylation of lecithin that contains omega-3 and omega-6 fatty acids by a simple, single step reaction, which can be easily performed on industrial scales.

These and other objects of the invention will become apparent as the description proceeds.

Summary of the Invention The present invention provides various methods for the preparation of glycerophospholipids enriched with omega-3 and/or omega-6. Said methods are essentially methods of enzymatic transesterification and esterification of glycerophospholipids, chemical synthesis, and enzymatic production of phosphatidylserine, in the presence of immobilized PLD.

Thus, in a first aspect, the present invention provides a method for the production of a glycerophospholipid enriched with omega-3 and/or omega-6 fatty acids through enzymatic transesterification, comprising the steps of : a) incubating said glycerophospholipid with an omega-3 and/or omega-6 fatty acid source in the presence of an immobilized phospholipase which can catalyze transesterification at the sn-1 and/or sn-2 positions of the glycerol moiety, for a suitable period of time to give a glycerophospholipid enriched with said omega-3 and/or omega-6 fatty acids at the sn-1 and/or sn-2 positions; b) removing and filtering the upper layer which contains the said enriched glycerophospholipid, in order to separate the glycerophospholipid from the enzyme; and c) optionally de-oiling the filtrate to remove excess FFA; In one embodiment of this method, said glycerophospholipid is any one of phosphatidylcholine, phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylethanolamine (PE).

In another aspect the invention provides a method for the production of a glycerophospholipid enriched with omega-3 and/or omega-6 fatty acids through enzymatic esterification, comprising the steps of : a) incubating said glycerophospholipid in an aqueous medium or in an organic solvent with an immobilized phospholipase, which is sn-l or S7z-2 regio-specific, to give the corresponding lyso-phospholipid; b) incubating said lysophospholipid with an omega-3 and/or omega-6 fatty acid source in the presence of an immobilized phospholipase which can catalyze esterification at the s7T-l and/or sn-2 positions of the glycerol moiety, for a suitable period of time to give a glycerophospholipid enriched with said omega-3 and/or omega-6 fatty acids at the sn-1 and/or sn-2 positions; c) removing and filtering the upper layer which contains the said enriched glycerophospholipid, in order to eliminate remaining enzyme; and d) optionally de-oiling the filtrate with acetone to remove excess neutral phospholipids.

In one particular embodiment of the above method, said glycerophospholipid is any one of phosphatidylcholine, phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine.

In the above-described process, the termination of the reaction is by filtering out the enzyme, and adding acetone for the deoiling process, further to which the phospholipids are precipitated and filtered out. This is much less harmful than the process described for example by Hosokawa et al. (1995) id ibid., wherein the final step involves washes with a mixture of chloroform, methanol and water.

The immobilized enzyme used in the above-described methods may be any one of PLAT or PLA2, and the reaction is carried out in aqueous media. When the enzyme is not immobilized, the reaction is carried out in an organic solvent.

In a further aspect the present invention provides chemical methods for the synthesis of enriched glycerophospholipids. In one method, the chemical synthesis of a glycerophospholipid enriched with omega-3 and/or omega-6 fatty acid starts from its corresponding lyso-glycerophospholipid, and comprises the steps of : a) dissolving said lyso-glycerophospholipid with said omega-3 and/or omega-6 fatty acid source in a suitable organic solvent, preferably dichloromethane; b) incubating the mixture obtained in step (a) with a coupling reagent for a suitable period of time while stirring; c) filtering the product, preferably with CeliteR.

In a second method, the chemical synthesis of a glycerophospholipid enriched with omega-3 and/or omega-6 fatty acids starts from its corresponding lyso- glycerophospholipid, and differs in the reactive moieties used in the synthetic procedure, which comprises the steps of : a) incubating a mixture of said lyso-glycerophospholipid and said omega-3 and/or omega-6 fatty acid source under acidic conditions, for example in the presence of naphthalene beta sulphonic acid, wherein said mixture is optionally dissolved in an organic solvent, for a suitable period of time while stirring; b) extracting the phospholipids with a suitable organic solvent; and c) evaporating the solvent.

Said enriched glycerophospholipid obtained further to the above-described chemical methods is any one of phosphatidylcholine and phosphatidylinositol.

Thus, the present invention also provides phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and phosphatidylinositol preparations obtained through said chemical methods.

Most importantly, for all of the above-described methods (transesterification, esterification and chemical methods), the omega-3 source may be docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and alpha linolenic acid in the form of a free fatty acid, an ethyl ester of any one of said fatty acids, or a triglyceride comprising thereof. Likewise, the omega-6 source may be any one of gamma linoleic acid (GLA), arachidonic acid (ARA), and linoleic acid in the form of a free fatty acid, an ethyl ester of any one of said fatty acids, or a triglyceride comprising thereof.

In a yet further aspect the present invention provides a method for the production of a phosphatidylserine preparation enriched with omega-3 and/or omega-6 fatty acids, comprising the steps of : a) incubating an aqueous mixture of L-serine with lecithin which is rich with omega-3 or omega-6 fatty acid residues in the presence of phospholipase D, for a suitable period of time to give phosphatidylserine; b) removing and filtering the upper layer which contains the phosphatidylserine; c) washing the filtrate with water to remove excess serine; d) washing the resulting phosphatidylserine with ethanol to remove any traces of phospholipase ; and e) drying the washed phosphatidylserine wherein the resulting phosphatidylserine is enriched with omega-3 or omega-6 fatty acid residues which are covalently bound to the phospholipid backbone.

Said lecithin may be derived from a marine animal, like for example krill, or it may also be obtained through any one of the above-described methods.

In one embodiment of the method of producing enriched phosphatidylserine, said omega-3 or omega-6 fatty acids are selected from the group consisting of EPA, DHA, GLA, arachidonic acid alpha linolenic acid and linoleic acid.

In another embodiment of this method, said phospholipase is immobilized on an insoluble matrix and is optionally surfactant coated.

The present invention thus further provides: - a phosphatidylserine (PS) preparation enriched with omega-3 or omega-6 acyl moieties prepared by any one of the methods of transesterification, esterification, utilizing PLD, or through the chemical methods as described above.

- a phosphatidylcholine (PC) preparation enriched with omega-3 or omega-6 acyl moieties prepared by any one of the methods of transesterification, esterification, or through chemical methods as described above.

- a phosphatidylinositol (PI) preparation enriched with omega-3 or omega-6 acyl moieties prepared by any one of the method of transesterification, esterification, or through chemical methods as described above.

- a phosphatidylethanolamine (PE) preparation enriched with omega-3 or omega-6 acyl moieties prepared by the methods of transesterification, esterification, or through the chemical methods as described above.

Any of these enriched glycerophospholipid preparations may further comprise at least one additional functional ingredient and/or at least one non- functional nutritionally acceptable ingredient. For the PS, PI and PE preparations, said additional functional ingredient may be, for example, lecithin.

Any one of the enriched glycerophospholipid preparations of the invention may be used as nutraceutical foods and/or drug additives.

The present invention further provides a food article comprising at least one of : the phosphatidylserine preparation of the invention, the phosphatidylcholine preparation of the invention, the phosphatidylinositol preparation of the invention and the phosphatidylethanolamine of the invention.

In an even further aspect, the present invention provides a pharmaceutical composition comprising as active agent at least one of the omega-3/omega-6 enriched glycerophospholipids presented by the invention, or specifically, PS, PC, PI or PE as prepared by any one of the methods described herein, respectively.

Another particular aspect of the present invention is a capsule, containing any one of the PS preparation of the invention, the PC preparation of the invention, the PE preparation of the invention or the PI preparation of the invention, or any combination thereof. Said capsule is preferably, but not limited to a gelatin capsule.

Due to its known properties in brain function, as previously mentioned, the stabilized phosphatidylserine preparation of the present invention may be used as an enhancer of cognitive performance and learning ability, and in preventing memory loss, particularly age-related memory loss.

Detailed Description of the Invention The present invention provides various methods for the preparation of glycerophospholipids enriched with omega-3 and/or omega-6. Said methods are essentially methods of enzymatic transesterification and esterification of glycerophospholipids, chemical synthesis, and enzymatic production of phosphatidylserine, in the presence of immobilized PLD.

The present inventors have developed synthetic pathways that enable the industrial production of the aforementioned phospholipids, which possess unique nutritional and clinical benefits.

The synthetic pathways described herein may be divided into three main categories: 1. Enzymatic esterification and transesterification of phospholipids with omega-3 and/or omega-6 fatty acids utilizing PLA1 or PLA2 enzymes, accordingly.

2. Chemical esterification of phospholipids with omega-3 and omega-6 acyl donors.

3. Enzymatic transphosphatidylation of phospholipids with PLD.

Thus, the present invention provides an improved enzymatic interesterification processes for the enrichment of phospholipids with omega 3 and 6 fatty acids. The interesterification includes a process of transesterification of lecithin with omega-3 and 6 fatty acids and an esterification process. In the latter, lecithin is converted to lyso lecithin by Plat or PLA2 and the resulting lyso-lecithin product then reacts with omega- 3 and/or omega-6 fatty acids. Both enzymatic processes utilize enzymes that are modified and immobilized by the AMIET technology (Activation Modification Immobilization Enzyme Technology), described in US 6,605, 452.

Thus, in a first aspect, the present invention provides a method for the production of a glycerophospholipid enriched with omega-3 and/or omega-6 fatty acids through enzymatic transesterification, comprising the steps of : a) incubating said glycerophospholipid with an omega-3 and/or omega-6 fatty acid source in the presence of an immobilized phospholipase which can catalyze transesterification at the s7l-l and/or sn-2 positions of the glycerol moiety, for a suitable period of time to give a glycerophospholipid enriched with said omega-3 and/or omega-6 fatty acids at the 571-1 and/or srz-2 positions ; b) removing and filtering the upper layer which contains the said enriched glycerophospholipid, in order to separate the glycerophospholipid from the enzyme; and c) optionally de-oiling the filtrate to remove excess FFA; In one embodiment of this method, said glycerophospholipid is any one of phosphatidylcholine, phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylethanolamine (PE).

In another aspect the invention provides a method for the production of a glycerophospholipid enriched with omega-3 and/or omega-6 fatty acids through enzymatic esterification, comprising the steps of : a) incubating said glycerophospholipid in an aqueous medium or in an organic solvent with an immobilized phospholipase, which is son-1 or sit-2 regio-specific, to give the corresponding lyso-phospholipid; b) incubating said lysophospholipid with an omega-3 and/or omega-6 fatty acid source in the presence of an immobilized phospholipase which can catalyze esterification at the erz-1 and/or sn-2 positions of the glycerol moiety, for a suitable period of time to give a glycerophospholipid enriched with said omega-3 and/or omega-6 fatty acids at the s7z-1 and/or S71-2 positions; c) removing and filtering the upper layer which contains the said enriched glycerophospholipid, in order to eliminate remaining enzyme; and d) optionally de-oiling the filtrate with acetone to remove excess FFA.

In one particular embodiment of the above method, said glycerophospholipid is any one of phosphatidylcholine, phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine.

In the above-described process, the termination of the reaction is by filtering out the enzyme, and adding acetone for the deoiling process, further to which the phospholipids deposit and are filtered out. This is much less harmful than the process described for example by Hosokawa et al. (1995) id ibid., wherein the final step involves washes with a mixture of chloroform, methanol and water.

The immobilized enzyme used in the above-described methods may be any one of PLA1 or PLA2, and the reaction is carried out in aqueous media. When the enzyme is not immobilized, the reaction is carried out in an organic solvent.

Most importantly, the enzyme utilized in this method may be re-cycled, which reflects significant reduction in the cost of the reaction and consequently also of the final product, thus making these methods much more cost-effective than what is currently available in the market.

The present invention also provides two different chemical processes: one using DCC/DMAP and the other based on esterification with naphthalene- beta sulphonic acid.

In a further aspect the present invention provides chemical methods for the synthesis of enriched glycerophospholipids. In one method, the chemical synthesis of a glycerophospholipid enriched with omega-3 and/or omega-6 fatty acid starts from its corresponding lyso-glycerophospholipid, and comprises the steps of : a) dissolving said lyso-glycerophospholipid with said omega-3 and/or omega-6 fatty acid source in a suitable organic solvent, preferably dichloromethane; b) incubating the mixture obtained in step (a) with a coupling agent for a suitable period of time while stirring; c) filtering the product, preferably with CeliteR.

In one specific embodiment, said coupling agent may be any one of N, N- dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP) and diisohexylcarbodiiamide.

In a second method, the chemical synthesis of a glycerophospholipid enriched with omega-3 and/or omega-6 fatty acids starts from its corresponding lyso- glycerophospholipid, and differs in the reactive moieties used in the synthetic procedure, which comprises the steps of : a) incubating a mixture of said lyso-glycerophospholipid and said omega-3 and/or omega-6 fatty acid source under acidic conditions, for example in the presence of naphthalene beta sulphonic acid, wherein said mixture is optionally dissolved in an organic solvent, for a suitable period of time while stirring; b) extracting the phospholipids with a suitable organic solvent, for example ethylacetate; and c) evaporating the solvent.

Said enriched glycerophospholipid obtained further to the above-described chemical methods is any one of phosphatidylcholine and phosphatidylinositol.

Thus, the present invention also provides phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and phosphatidylinositol preparations obtained through said chemical methods.

Most importantly, for all of the above-described methods (transesterification, esterification and chemical methods), the omega-3 source may be docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and alpha linolenic acid in the form of a free fatty acid, an ethyl ester of any one of said fatty acids, or a triglyceride comprising thereof. Likewise, the omega-6 source may be any one of gamma linoleic acid (GLA), arachidonic acid (ARA), and linoleic acid in the form of a free fatty acid, an ethyl ester of any one of said fatty acids, or a triglyceride comprising thereof.

Thus, it is a further object of the present invention to provide improved chemical interesterification processes for the enrichment of phospholipids with omega 3 and 6 fatty acids. The methods comprise the enzymatic hydrolysis of soy lecithin and esterification of the lysolecithin with omega 3 and 6 fatty acids by two alternative chemical ways. One involves the esterification with DCC and DMAP, wherein 10% DCC and 50% phospholipids are preferably utilized. The second esterification is performed with naphthalene-beta-sulphonic acid, at 135-145°C for about 5-6 hours in a vacuum, under pressure of about 1 mm Hg. The lecithin obtained by the two ways can be further transformed to phosphatidylserine enriched with omega- 3 or omega-6 by the process described in applicant's co-pending PCT application claiming priority from IL158552.

It is important to mentioned that in contrast to processes described by others [see e. g. Haider et al. (1998) id ibid.] the procedure presented herein involves the hydrolysis of natural occurring lecithin and esterification with solvents that are less toxic than chloroform. Thus, the process described in the present invention (after the complete removal of all reagents and solvents) is more suitable for use in the generation of products with food grade quality.

Human brain PS is characterized by about 20-30% PS containing omega-3 fatty acyls, preferably at the son-2 position of the glycerol moiety, and mainly DHA or EPA. As mentioned above, phospholipids, and PS in particular, are responsible for membrane structure and physical properties. One of the major physical properties governed by phospholipids is the fluidity of these membranes. Omega-3 fatty acids, DHA and EPA in particular, also have a crucial role in membrane fluidity in light of their unique 3D structure.

Therefore, PS with omega-3 fatty acyl moieties, DHA and EPA in particular, has unique bio-functionality which cannot stem from just the basic phospholipid skeleton of this phospholipid.

The present inventors have developed synthetic pathways that enable the industrial production of the aforementioned phospholipids, which possess unique nutritional and clinical benefits. The synthetic pathways described herein may thus be divided into three main categories: 1. Enzymatic esterification and transesterification of phospholipids with omega-3 and/or omega-6 fatty acids utilizing PLA1 or PLA2 enzymes, accordingly.

2. Chemical esterification of phospholipids with omega-3 and omega-6 acyl donors.

3. Enzymatic transphosphatidylation of phospholipids with PLD.

Polyunsaturated fatty acids are known to be bioactive compounds. Because those fatty acids are very unstable, enzymatic conversion of PUFA's under mild conditions is worthwhile.

Among the enzymes that can be utilized for esterifications and/or transesterifications are the lipases and the two types of phospholipases- PLA1 and PLA2.

The production of glycerophospholipid preparations that are suitable as food ingredients or nutraceuticals is a major advantage over obtaining said glycerophospholipids from animal sources. Considering the risks involved with prion diseases, particularly bovine spongiform encephalopathy (BSE), as well as other disadvantages associated with ingredients obtained from animal sources, glycerophospholipid supplements (PS, PC, etc. ) derived from animal sources are to be avoided. Furthermore, PS originating from plant lecithin is characterized by low levels of omega-3 fatty acids, with almost no DHA and EPA. Thus, the only viable way for the population to be safely supplied with said dietary supplements is through their industrial synthesis from safe raw materials.

It is therefore desirable to provide a PS ingredient with a fatty acid composition that mimics the fatty acid composition of the human brain PS. Applicant's co-pending PCT Application No. IL2004/000895 describes the synthesis of stabilized phosphatidylserine preparations. This process has now been further developed for the preparation of omega-3 and omega-6 containing phosphatidylserine.

Thus, in a further aspect, the present invention provides a process for the preparation of a stable phosphatidylserine composition of matter, comprising the steps of : (a) incubating an aqueous mixture of L-serine and optionally appropriate organic solvents with lecithin which is rich in omega-3 and/or omega-6 fatty acid in the presence of a phospholipase D (PLD), for a suitable period of time to give phosphatidylserine; (b) removing the upper layer which contains the phosphatidylserine; (c) obtaining the phosphatidylserine from said removed upper layer by standard means; (d) washing the phosphatidylserine obtained in step (c) with an appropriate aqueous solution to remove excess L-serine; (e) optionally washing the phosphatidylserine obtained in step (d) with a suitable organic solvent, preferably ethanol at an elevated temperature; and (f) drying the phosphatidylserine obtained in step (e).

The resulting phosphatidylserine is enriched with omega-3 or omega-6 fatty acid residues, which are covalently attached to the phospholipid backbone.

Omega-3 and omega-6 may be obtained from a variety of phospholipid sources, such as marine animals and egg yolks. One important source of omega-3 PUFA is the krill.

In another embodiment the method of the invention may employ a PLD which is immobilized on a suitable rigid matrix. The immobilized enzymatic preparation can be filtered off the reaction medium at the end of the reaction.

An advantage of this immobilized enzyme preparation is that it can be re- used in many further reaction batches. Matrix-immobilized, preferably surfactant-coated phospholipases can be prepared according to the methods described in WO00/56869, fully incorporated herein by reference.

A further advantage of the method of the invention is that the resulting phosphatidylserine preparations are stable to decomposition during prolonged storage, or in different nutritional, nutraceutical or pharmaceutical applications.

The content of omega-3 or omega-6 fatty acid residues in the preparations produced by the method of the invention may vary, and is preferably from about 10 to about 60-70% of the total acid moieties content.

According to the present invention it is possible to control the position of the omega-3 fatty acid either by choosing the raw lecithin that contains the desired fatty acid on the beta position (which is preferable) or by conducting the hydrolysis of the lecithin by specific phospholipase e. g. PLA1 or PLA2.

In a further embodiment, the invention relates to omega-3/omega-6-enriched PS preparations, particularly as produced by the method of the invention.

One clear advantage provided by the present invention is that it makes possible to control the position of the insertion of omega-3 and 6 fatty acid either by choosing the raw lecithin that contains the desired fatty acid on the beta position (which is preferable) for a transphosphatidylation or by conducting the hydrolysis of the lecithin by specific phospholipase e. g. PLA or PLA2 and then esterify the lyso product with omega 3 or 6 fatty acid.

The present invention thus further provides: - a phosphatidylserine (PS) preparation enriched with omega-3 or omega-6 acyl moieties prepared by any one of the methods of transesterification, esterification, and utilizing PLD.

- a phosphatidylcholine (PC) preparation enriched with omega-3 or omega-6 acyl moieties prepared by any one of the methods of transesterification, esterification, or through chemical methods as described above.

- a phosphatidylinositol (PI) preparation enriched with omega-3 or omega-6 acyl moieties prepared by any one of the method of transesterification, esterification, or through chemical methods as described above.

- a phosphatidylethanolamine (PE) preparation enriched with omega-3 or omega-6 acyl moieties prepared by the methods of transesterification or esterification.

Any of these enriched glycerophospholipid preparations may further comprise at least one additional functional ingredient and/or at least one non- functional nutritionally acceptable ingredient. For the PS, PI and PE preparations, said additional functional ingredient may be, for example, lecithin.

Any one of the enriched glycerophospholipid preparations of the invention may be used as nutraceutical foods and/or drug additives.

The present invention further provides a food article comprising at least one of : the phosphatidylserine preparation of the invention, the phosphatidylcholine preparation of the invention, the phosphatidylinositol preparation of the invention and the phosphatidylethanolamine of the invention.

In an even further aspect, the present invention provides a pharmaceutical composition comprising as active agent at least one of the omega-3/omega-6 enriched glycerophospholipids presented by the invention, or specifically, PS, PC, PI or PE as prepared by any one of the methods described herein, respectively.

Another particular aspect of the present invention is a capsule, containing any one of the PS preparation of the invention, the PC preparation of the invention, the PE preparation of the invention or the PI preparation of the invention, or any combination thereof. Said capsule is preferably, but not limited to a gelatin capsule.

Due to its known properties in brain function, as previously mentioned, the stabilized phosphatidylserine preparation of the present invention may be used as an enhancer of cognitive performance and learning ability, and in preventing memory loss, particularly age-related memory loss.

Examples 1. Analytical Procedures: l. a. Analytical method for the detection of 1 and 2 lyso phosphatidylcholine as described [JAOCS, 78, 10 (2001)]. l. b. HPLC method for analyzing phospholipids The HPLC analysis were carried out with Merck Hitachi D7000-IF instrument consisting of an Autosampler L7200 and a Polymer Laboratories LTD-PL-ELS 1000 detector. PC, PS, PA, PE, Lyso PC, and Lyso PS were separated on Lichrospher Si 60 511m column. l. c. Fatty acid analysis of the phospholipids In order to analyze the fatty acid composition of the phospholipids, internal standard (C17 phosphatidylcholine) was added to the sample. The phospholipids were applied to 20X20 TLC plate and developed in 80: 20: 2 isohexane: ether: formic acid system. The TLC plates were sprayed with primuline solution (0.01% in 60: 40 acetone: water), placed under UV lamp and the desire bands marked and scrabbed. Second internal standard had been added to the silica that consists of C21-Methyl ester. The silica and lipids were subjected to acidic hydrolysis by adding 1 ml of toluene and 2 ml of 2% H2SO4 in methanol. The acidic hydrolysis was for 16 h in a 50C. At the end of the hydrolysis, 5 ml of 5% NaCl (in DDW) was added, followed by 2 ml of isohexane. The tube had being vortex, spined down, and the upper phase (organic with isohexane and fatty acids) transferred to another tube, and an additional extraction with 2 ml of isohexane is performed.

To the combined upper phases of the phospholipids hydrolysis, 3 ml of 2% potassium carbonate (KHC03 in DDW) was added; the tube vortexed and spun down for phase separations. The upper phase was then transferred through an ammonium sulfate column (pre-washed with iso-hexane) and an additional 2 ml of iso-hexane were used to wash the column. The tube was dried under nitrogen, and then resuspended for Gas Chromatography analysis. A model GC-HP gas chromatograph was employed, equipped with fused silica capillary 007 Carbowax 20M column, 25 meters, 0. 25mm I. D. and 0.1 11 Film thickness.

Example 1-Enzyme preparations - Materials : L-Serine : CAS N. 56-45-1 (Degussa).

Lecithin: krill (high concentrations of long-chain PUFA).

Calcium chloride: CALCIOL (MarschalITM, Rhodiafood).

Acetic Acid: CAS N 64-19-7 (Acetex Chimie).

Sodium Hydroxide: CAS N. 1310-73-2 (Sigma Chemical Co.) MCT Crodamol GTCC: Manufacture by Croda.

TitriplexR III (ethylenedinitrilotetraacetic acid disodium salt dihydrate) (Merck KgaA) Hexane (Sigma-Aldrich).

Dicyclohexylcarbodiimide (DCC) (Acros) Dimethylaminopyridine (DMAP) (Acros) Naphtalene beta sulphonic acid (Sigma) PC (70%) - (Phospholipids GmbH) - Immobilization of the enzymes Matrix-immobilized, preferably surfactant-coated phospholipases and lipases can be prepared according to the methods described in WO00/56869, fully incorporated herein by reference.

Briefly, the crude enzyme (300mg/l protein) is dissolved in 1L tris buffer, pH 6.5 containing 4 g insoluble inorganic or organic matrix (Celite, silica gel, alumina, polypropylene or ion-exchange resin). The solution is stirred vigorously with a magnetic stirrer for 30 minutes at 25°C. In the case of surfactant-coated immobilized enzyme preparations, sorbitan mono-stearate is added drop-wise to the stirred enzyme solution. All enzyme preparations (i. e. both the surfactant-coated immobilized lipases and the immobilized- crude lipases) are sonicated for 10 minutes and then stirred for 8 hours at 25°C. The formed precipitate is collected by either filtration or centrifugation (12,000 rpm, 4°C), followed by overnight freezing at-20°C and lyophilization.

Example 2 Transesterification reactions with immobilized phospholipase (PLA1 from Aspergillus 71iger) were carried out over fixed time periods, usually for 16 hours, at 45 OC in a rotary shaker in 50 ml Erlenmeyer flask containing 32.5 gr mixture of soy PC (70%) from Phospholipids GmbH and DHA-FFA (70%, Croda) in the ratio of 1: 5.5 and 5 gr of immobilized phospholipase. At the end of the reaction, after 48 hours, the enzyme was filtered out and recycled in similar process and the phospholipids were subjected to hydrolysis to methyl esters for GC analysis (results summarized in Table 1). In case that the phospholipids fraction is needed, it is possible to obtain that fraction by adding an acetone and filter the solid that consists mainly of phospholipids.

In contrast to other systems described (e. g. W091/03564) this process our process is conducted in solvent free system and the water content of the immobilized enzyme is low, resulting in much less hydrolysis during the synthetic reaction.

In this enzymatic process the inventors obtained 3-fold higher incorporation of omega-3 fatty acids in the phospholipids, when compared to the yield obtained by others [see e. g. , Mutua, L. N. and Akoh, C. C. (1993) id ibid.].

Moreover, the present procedure utilizes immobilized enzyme, which may be re-used. Both factors have a great impact on the final cost and stability of the end product.

Table 1: Fatty acid content of the phosphatidylcholine before and after the transesterification reaction. C16: 0 C18 : 0 C18 : 1 C18: 2 C18: 3 C20: 5 C22: 6 Before 13 5 12 62 6 After 5 2 10 10 4 10 59 As can be seen from Table 1,70% of the fatty acid on the phosphatidylcholine are omega-3 fatty acid Calculating the percentage of saturated vs. unsaturated fatty acids before and after the reaction (without taking into account the omega-3 fatty acid), shows that the percentage of saturated fatty acids increased to 25% while there was a decrease in the unsaturated fatty acid. Those changes in the identity of the fatty acid can indicate that the insertion of omega-3 was preferable at the second position (since most of the unsaturated fatty acids are located on the second position of the phospholipids, whereas the saturated fatty acids are located at the Ist position).

Example 3-Enzymatic esterification of phosphatidylcholine -Preparation of Lysophosphatidylcholine Lyso-phosphatidylcholine can be obtained by hydrolysis with PLA2 in Tris buffer or water followed by extraction of the lyso product. 0.2 g of PLA2 were added to 2 g of PC, in 100 ml buffer Tris pH=8 and lOmM CaCl2. After 16 hours of reaction, ethyl acetate was added in order to extract the neutral lipids as well as the phospholipids. After evaporation of the organic solvent, the residue was dissolved in a mixture of ethyl acetate and 2-propanol (95: 5), and all neutral lipids were extracted from the mixture leaving the 2- lysophosphatidylcholine as the main component.

An optional process for concentrating the phospholipids is to add acetone to the reaction mixture and filter the phospholipids.

Esterification of lyso-phosphatidylcholine was conducted under the same conditions as described for the transesterification of phosphatidylcholine.

Example 4-Chemical esterification of phosphatidylcholine a. Esterificatio7 catalyzed by DCC aux DMAP Both reagents, DCC (Dicyclohexylcarbodiimide) and DMAP (dimethylaminopyridine) were dried for 16 hours at room temperature. 1 g of lyso PC (47%) and 0.8 gr of DHA-FFA (70%) were combined with 0.5 gr DMAP and 0.4 g DCC in 10 ml dichloromethane. The reaction was stirred for 16 hours at room temperature, filtered through CeliteTM and analyzed for fatty acid distribution of the phosphatidylcholine product (Table 2). The conversions were almost 100% as no lyso-phosphatidylcholine was left after the reaction was ended.

Table 2: Fatty acid distribution of the phosphatidylcholine obtained by esterification of lyso-phosphatidylcholine and omega-3 fatty acids C16: 0 C18: 0 C18 : 1 C18 : 2 C18 : 3 C20: 5 C22: 6 Before* 13 5 12 62 6 After 3 2 9 28 2 7 49 * the percentage of the fatty acid before the reaction, related to the fatty acid distribution in the phosphatidylcholine starting material (before the hydrolysis into the lyso product).

As can be seen from Table 2, almost 60% of the fatty acid content on the phosphatidylcholine is omega-3. In order to be able to predict and determine the reaction product configuration, it is necessary to start the synthesis with the appropriate lyso isomer (1 or 2 lyso). b. Clzemvcal esterificatiom of phosphatidylcholine using acidic conditio7is.

The main advantage in acidic catalytic esterifications is the fact that the equilibrium is much more suppressed and the reaction proceeds mainly to the products. In a typical procedure, 3.5 g of deoiled lysophosphatidylcholine were combined with 7 g of DHA-FFA and 0.7 g of naphthalene beta-sulphonic acid under vacuum of 0.1 mm Hg at 90 °C degree. The reaction was followed by the HPLC and the formation of the phosphatidylcholine was almost complete. At the end of the reaction, after 5 hours, the reaction was extracted with hexane and water, and the hexane layer dried with magnesium sulphate and evaporated. The product had been analyzed for its omega-3 content by hydrolysis and methylation of the fatty acid. The results are summarized in Table 3.

Table 3 C16 : 0 C18: 0 C18: 1 C18: 2 C18 : 3 C20: 5 C22 : 6 Before* 13 5 12 62 6 After 4 2 9 14 0. 5 10 60 * the percentage of the fatty acid before the reaction, related to the fatty acid distribution in the phosphatidylcholine starting material (before the hydrolysis to the lyso product.

As can be seen in Table 3,70% of the fatty acid contains omega 3 fatty acids.

By calculation of the saturated and unsaturated fatty acid (without taking into account the omega-3 fatty acid) before and after the reaction, it is clear that the percentage of each fatty acid did not change. This result is expected since the chemical catalysts have no preferences to any of the positions in the phospholipids backbone.

Example 5-Transphosphatidylation of phosphatidylcholine All of the products described before can be transformed to phosphatidylserine by Phospholipase D in the presence of serine. In a typical reaction 380 g of L- Serine were placed in a 2 liter reactor filled with 1140 ml phosphate buffer pH 5.6 containing 200mM CaCl2. After complete dissolution of the serine, 80gr of marine lecithin were added.

The mixture was stirred at 40°C for 1 hour, to homogeneously disperse the phospholipid in the aqueous phase.

2.1 g of enzyme (Phospholipase D) were added to the aqueous dispersion. The reaction mixture was stirred for 24 hours. The upper layer consisting of the phospholipid dispersion was removed from the reactor. The dispersion was washed four times with water to remove the excess serine.

The phosphatidylserine was washed with ethanol to remove traces of enzyme.

The obtained ethanol cake of phosphatidylserine was dissolved in 1L hexane and stirred for 1 hour. Serine precipitated as a white solid. The mixture was filtered and the hexane was evaporated to obtain phosphatidylserine. For further concentration of the phosphatidylserine obtained, acetone was added and the phospholipids fraction filtered out from the solution containing mainly triglycerides. The composition of the phosphatidylserine and omega-3 before and after deoiling appear in Table 4. Final weight was 30 g.

Table 4: Omega-3 Fatty Acid Analysis of the Phosphatidylserine- product % DHA on % EPA on Total DHA Total EPA % PS the PS the PS (%) (%) Before 20 15.4 27.4 9 15 deoiling After 40 15.4 27.4 15 26 deoiling Example 6: Large scale reaction A large scale reaction of transphosphatidylation of phospholipids with PLD, for producing phosphatidylserine enriched with omega-3 or omega-6 was performed. The same reaction as described in Example 5 was performed in large scale, in a 5 liter reactor containing 600 g of DHA-enriched lecithin stirred with 1.2 Kg of L-serine dissolved in 3.5 liter buffer. 10 g of PLD enzyme (1500 U) were added to facilitate the transphosphatidylation reaction. The final phosphatidylserine concentration, after deoiling, was 38%.

The process described in the present invention is a one phase system which may be performed in large scale, in 5 liter reactors containing 600 g of lecithin, and thus it is much more advantageous than other systems previously described [e. g. Hosokawa M. et al. (2000) id ibid.