PHOSPHOLIPID COMPOSITIONS AND THEIR PREPARATION

Abstract

The invention provides improved processes for extracting and preparing polar lipids (in particular, desirable phospholipids) from krill and other biological sources. The inventors have discovered processes through which it is possible to extract phospholipids to give high phospholipid content and a reduction of undesired components.

Claims

1. A composition comprising a mixture of phospholipid compounds of formula (I): ##STR00002## wherein: R.sub.1 and R.sub.2 are each independently selected from a fatty acid moiety of formula COC.sub.nH.sub.m, a fatty acid moiety of formula CH.sub.2C.sub.nH.sub.m, and H; R.sub.1 and R.sub.2 include omega-3 fatty acid moieties, such that at least 30% by weight of the phospholipid compounds is composed of omega-3 fatty acid moieties; at least 90% by weight of total omega-3 fatty acid moieties are at position R.sub.2; R.sub.1 and R.sub.2 are not both H in a phospholipid compound, and R.sub.1 or R.sub.2 is H in less than 3% by weight of the compounds of formula (I); R.sub.3 is selected from H, a choline moiety, an ethanolamine moiety, a N-acetylethanolamine moiety, an inositol moiety, and a serine moiety; and R.sub.3 is a choline moiety in at least 85% by number of the compounds of formula (I). and wherein the composition also has one or more of the following properties: (a) at least 85% by weight of the composition consists of phospholipid compounds of formula (I), optionally wherein the composition is substantially free from acetone; (b) the weight ratio of C16:0/C14:0 fatty acid moieties in the mixture is between 10:1 and 18:1 and/or the weight ratio of C18:4 n3/C18:3 n3 fatty acid moieties is between 1:1 and 3:2; (c) the composition includes less than 300 g astaxanthins per gram of phospholipid; (d) the composition comprises less than 0.01% by weight trimethylamine N-oxide; (e) the composition comprises less than 0.01% by weight homarine; (f) the composition includes less than 5% by weight water; (g) the composition has less than about 0.03% by weight PUFA polymers (h) the mixture includes both phospholipids where R.sub.1 is a fatty acid moiety of formula COC.sub.nH.sub.m and phospholipids where R.sub.1 is a fatty acid moiety of formula CH.sub.2C.sub.nH.sub.m; (i) the mixture includes both phospholipids where R.sub.1 is an omega-3 fatty acid moiety and phospholipids where R.sub.2 is an omega-3 fatty acid moiety; (j) the composition includes less than 5% by weight sphingomyelin; (k) the composition is free from chloroform and hexane; and/or (l) less than 0.9% by weight of phospholipids in the composition is formed of compounds where R.sub.1 or R.sub.2 is H, or more than 1.1% by weight of phospholipids in the composition is formed of compounds where R.sub.1 or R.sub.2 is H.

2. The composition of claim 1, wherein at least 85% by weight of the composition consists of phospholipid compounds of formula (I) and the composition is substantially free from acetone.

3. The composition of claim 1, having 2 or more of properties (a) to (l).

4. The composition of claim 3, having 3 or more of properties (a) to (l).

5. The composition of claim 1, wherein less than 0.9% by weight of phospholipids in the composition is formed of compounds where R.sub.1 or R.sub.2 is H.

6. The composition of claim 1, having at least properties (h) and (i).

7. The composition of claim 6, having at least properties (a), (c), (d), (e), (g) and (j).

8. The composition of claim 7, having at least properties (a), (b), (c), (d), (e), (g) and (j).

9. The composition of claim 6, having at least properties (a), (c), (d), (e), (f), (h), (j), and (k).

10. The composition of claim 9, having properties (a), (b) (c), (d), (e), (f), (h), (j), and (k), and wherein: with reference to property (h) fatty acid moieties of formula CH.sub.2C.sub.nC.sub.m are either saturated or mono-unsaturated, and not polyunsaturated; the compounds of formula (I) include C16:0, C14:0, C18:4 n3 and C18:3 n3 fatty acid moieties at R.sub.1 and/or R.sub.2; the amount of water is less than 2% by weight; the composition has less than 1% by weight free fatty acids; the composition has less than 0.005% by weight trimethylamine; the composition is free from canthaxanthin and flavonoid; and, optionally, wherein the phospholipids of formula (I) include EPA and DHA moieties in a molar ratio (EPA:DHA) from 1.8:1 to 2.2:1.

11. (canceled)

12. A composition comprising (1) a pharmaceutically-acceptable solvent and (2) a mixture of phospholipid compounds of formula (I), as defined in claim 1, and further having one or more of properties (b) to (1) as defined in claim 1, wherein the phospholipid compounds of formula (I) are dissolved, suspended or emulsified in the pharmaceutically-acceptable solvent, and wherein the composition is liquid when at 20 C.

13-26. (canceled)

Description

MODES FOR CARRYING OUT THE INVENTION

Example 1

[0166] This example describes the extraction of oil from a wet material. A coagulum from krill comprising about 70% water, 15% lipids and about 15% other dry matter, mainly proteins, was obtained as described in reference 16. This material was subjected to an extraction procedure as follows. 3500 grams of pure ethanol was added to 1004 grams of the coagulum and stirred for 45 minutes. The mixture was then filtered through a filter paper applying vacuum on the receiving flask to obtain 3854 gram of filtrate. 1179 gram of the filtrate was subjected to evaporation on a rotary evaporator and the obtained dry matter was washed 4 times with a 60% solution of ethanol and finally the solvent was evaporated in a rotary evaporator. The obtained oil, 23.7 gram, was solid at room temperature and comprised 76.8% phospholipids. Water is removed by freeze drying.

[0167] The content of EPA was 200 mg/gram and the content of DHA 87 mg/gram oil. The composition of the phospholipid fraction was as follows:

TABLE-US-00001 Phospholipid Weight-% Mol-% MW [g/mol] PC 71.97 93.03 790.0 1-LPC 0.24 0.45 534.5 2-LPC 0.73 1.39 534.5 PI *) *) 907.0 LPI *) *) 629.5 PS-Na *) *) 833.0 LPS *) *) 555.5 SPH *) *) 812.0 PE 3.37 4.47 770.0 LPE *) *) 492.5 APE *) *) 1032.0 PG *) *) 820.0 DPG *) *) 774.0 PA *) *) 746.0 LPA *) *) 468.5 Other 0.53 0.66 812.0 Sum 76.83 100.00 Phosphorus 3.03

Example 2

[0168] This example describes an alternative method for extraction of oil from the krill wet material, starting from a frozen paste from krill, which was subjected to an extraction procedure as described below. Unlike example 1, all steps were performed under a nitrogen atmosphere.

[0169] The paste comprises about 65% water (assessed via dry matter), 17% lipids (about equal weights of phospholipids and neutral lipids), and about 18% other dry matter, mainly proteins. Within the lipids, the proportions of certain fatty acids by weight were as follows: C16:0 about 15-17%; C14:0 about 6-10%; C18:3 n3 about 1.4-3.1%; and C18:4 n3 about 3.5-7%. 100 kg of the frozen coagulum (20 C.) was added to a vessel. Based on the water content of the coagulum, 350 kg of pure ethanol (99.8% w/w, room temp) was then added to the vessel, giving a final ethanol concentration in the liquid phase of about 84% w/w (350 kg ethanol in 415 kg liquid solvents). Ethanol was added to give near to the desired final concentration, and then water content was checked by Karl Fischer titration and extra ethanol was added to give the correct final amount.

[0170] The mixture was stirred in the vessel for 45 minutes, with gentle heating if required. Four final temperatures were studied in separate batches, namely a) 2 C., b) 10 C., c) 15 C. and d) 20 C. After stirring was complete, the mixtures were allowed to settle, and they each included a red-coloured liquid phase and a wet slurry which contained shell fragments and other insoluble materials. To remove the liquid phase from the slurry the mixtures were decanted, and the liquid material was put through a coarse filter and then serial-filtered through a 75 m and 5 m cartridge filter to obtain a) 345 kg, b) 366 kg, c) 372 kg or d) 374 kg of filtrate, with residual material remaining in the filtration cake. Smaller cartridge filters (e.g. 1.2 m) have also been used.

[0171] The filtrates were then subjected to a sequence of washes. Firstly, de-ionized water was added to give 60% w/w ethanol solutions (a: 137 kg water; b: 149 kg; c: 152 kg; d: 155 kg) and the mixtures were stirred for 10-15 minutes and left to settle for 12-24 h at room temperature (15-20 C.) in vessels having a valve at the base. The bottom phase was isolated by draining the bottom phase through the valve, to give between 5.4-9.0 kg of a lipid-rich fraction. The lipid-rich fraction was re-washed 2 to 5 times with 60% w/w ethanol at room temperature to give a final material which contained about 80% by weight phospholipids and 20% neutral lipids. In even the first wash, 85% of TMAO was removed, and the further washes led to material with undetectable TMAO (less than 1 mgN/100 g i.e. at least 20-fold lower than reported in Table X of reference 18).

[0172] This lipid-rich material was treated at least once by cold acetone precipitation. Three parts w/w acetone were added and the lipid rich material was dissolved by gentle heating and slow stirring. The stirring was stopped and the mixture was cooled to 4 C. for precipitation. When the precipitation was complete, the upper solvent phase was removed. This cold precipitation procedure was performed three times in total, after first re-dissolving in fresh acetone each time.

[0173] The precipitate was then subjected to evaporation and freeze-drying to remove residual acetone and water. Batch c (i.e. extracted at 15 C., then washed 360% EtOH before cold acetone precipitation) provided 1.9 kg of solid material (an orange wax) consisting of 98% phospholipids/1.7% neutral lipids with a water content of 3%. The content of EPA was 19.2 g/100 g and the content of DHA was 11.0 g/100 g solid material. The composition of the phospholipid fraction measured by .sup.31P NMR was as follows:

TABLE-US-00002 Phospholipid Weight-% Mol-% MW [g/mol] PC 82.59 89.03 790.0 1-LPC *) *) 534.5 2-LPC 0.12 0.19 534.5 PI 0.47 0.44 907.0 LPI *) *) 629.5 PS-Na *) *) 833.0 LPS *) *) 555.5 SPH *) *) 812.0 PE 8.25 9.13 770.0 LPE *) *) 492.5 APE 0.59 0.49 1032.0 PG *) *) 820.0 DPG *) *) 774.0 PA *) *) 746.0 LPA *) *) 468.5 Other 0.69 0.73 812.0 Sum 92.72 100.00 Phosphorus 3.64 *)= not observed, no signal assignment

[0174] Thus, based on total weight of the material analysed by NMR, nearly 93% of the final material was phospholipid. After compensating for residual water (about 3%), residual organic solvent, and salts/minerals present after ignition, the overall purity was 98%. Thus this process provides phospholipids with higher purity than seen using Example 1.

[0175] Further analysis of lipid composition was performed by HPLC, and results are shown below (grams per 100 g of oil):

TABLE-US-00003 Parameter Results Lipid composition.sup.(1) Triacylglycerol <0.5 Diacylglycerol <0.5 Monoacylglycerol <1 Free fatty acids <0.5 Cholesterol <0.5 Cholesterol ester <0.5 Phosphatidylethanolamine 7.7 Phosphatidylinositol <1 Phosphatidylserine <1 Phosphatidylcholine 92 Lyso-phosphatidylcholine <0.5 Total polar lipids 99.4 Total neutral lipids <0.5 Total sum lipids 99.6 Fatty acid composition.sup.(2) Sum saturated fatty acids 17.8 Sum monoenic fatty acids 9.1 Sum PUFA (n-6) fatty acids 1.2 Sum PUFA (n-3) fatty acids 34.4 Sum total PUFA fatty acids 35.8 Sum fatty acids total 62.7 Cholesterol 0.31 weight % Astaxanthin/esters <2 mg/kg Water content 3% .sup.(1)Calculated based on techniques in refs. 7, 9 & 10. .sup.(2)Calculated as fatty acid methyl esters, by AOCS Ce 1b-89.

[0176] Looking at specific fatty acids, proportions were as follows, measured across several batches:

TABLE-US-00004 18:4/ 16/14 C18:3 C18:4 18:3 C14:0 C16:0 Ratio n-3 n-3 Ratio Wet paste 6-10% 15-17% 2-2.5 1.4-3.1% .sup.3.5-7% 2-3 Final 1.0-1.5% 15-17% 12-16 1.0-2.5% 1.0-2.5% 1-1.5 material

[0177] The purified phospholipids included both ether-linked and ester-linked fatty acids, but 10% or fewer were ether-linked. NMR showed ether-linked fatty acid moieties at position sn1 but not at sn2, and ether-linked fatty acids were either fully saturated or were monounsaturated. Where a phospholipid was a phosphatidylcholine, about 10% of the molecules included ether-linked fatty acids; where a phospholipid was a phosphatidylethanolamine (with or without N-acetylation), about 40% of the molecules included ether-linked fatty acids. PUFAs were seen only with ester linkages. 30-40% by weight of fatty acids in the purified phospholipids were omega-3, and these were distributed at the sn1 and sn2 positions (mainly at sn2). Most of the omega-3 fatty acids were EPA and/or DHA, with about 2 more EPA than DHA.

[0178] The phosphatidylethanolamine content using this process was higher than seen when using the method of Example 1 (about 2 higher).

[0179] The lysophosphatidylcholine content (0.2-0.4 mol %) is very low in the purified phospholipids, when compared both to the amount observed using the method of Example 1 (about 1%) and in the starting wet material (about 1.2-1.4 mol %). No molecules were detected where fatty acid chains had been lost at both the sn1 and sn2 positions. Lyso-phosphatidylethanolamine (with or without N-acetylation) and lyso-phosphatidylinositol also were not seen.

[0180] Levels of astaxanthins were much lower in the purified phospholipids when compared to the material obtained in Example 1. This reduction was even visible due to the weaker red colour.

[0181] Amino acids, TMAO and homarine were all below LOQ by standard analytical methods.

[0182] Thus very pure krill phospholipids can be achieved by a process using extraction in 84% ethanol, followed by washing in 60% ethanol, and then multiple steps of cold-acetone precipitation.

Example 3

[0183] Rather than being subjected to cold acetone precipitation, the washed lipid-rich material produced during example 2 (80% phospholipid, 20% neutral lipid) was precipitated using ethyl acetate. In initial testing, the material was thoroughly mixed with 3 parts of ethyl acetate at room temperature and then placed at 4 C., 11 C. or 20 C. No precipitation was seen at 4 C., but there was some phase separation at 11 C. and precipitation was observed at 20 C.

[0184] Further washed lipid-rich material was mixed with 2, 3 or 5 parts of ethyl acetate and placed at 20 C. to achieve precipitation. With 3 parts of solvent the phospholipid yield was 32%, but with 5 parts of solvent the yield was 66%. Re-precipitation of this material gave results as follows:

TABLE-US-00005 Precipitation Yield Phospholipid Neutral lipid Phospholipid yield 1 65.6% 96.1% 3.9% 81.6% 2 95.4% 99% 1% 62.6%

[0185] Thus phospholipids can be effectively purified from the washed krill extract using repeated steps of precipitation with 5 volumes of EtOAc at 20 C.

PUFA polymerisation

[0186] Purified krill phospholipids were exposed to air at 70 C. to investigate polymerisation of PUFAs. After 12 hours of treatment the region in a .sup.31P NMR spectrum at 0.95 to 1.15 ppm showed a peak with a shoulder to the right. At samples taken during the treatment the peak was higher and the shoulder was less prominent, and in the starting material the peak was sharp with no shoulder. In all cases, however, the integrated area of this spectral region was constant. The shoulder is a pseudomarker of polymerization, either inter- or intra-molecular, and thus represents the conversion of PUFA chains (the main peak) into various polymers. The absence of oxygen during processing of the krill material (e.g. by performing the process under nitrogen) means that this oxidative polymerisation does not occur.

[0187] It will be understood that the invention is described above by way of example only and modifications may be made while remaining within the scope and spirit of the invention.

REFERENCES

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