TRIGLYCERIDE ENRICHMENT

Abstract: The present invention relates to a method for the separation of triglyceride containing edible oils into fractions that are enriched in polyunsaturated triglycerides and fractions that are enriched in saturated triglycerides. The invention is particularly applicable to the separation of triglyceride containing oils derived from fish. The method comprises (a) solidifying the edible oil by use of low temperature and (b) extracting the solidified edible oil with a polar organic solvent at a temperature of -50 DEG C or below; wherein the extract comprises the polyunsaturated enriched oil and the residue comprise the saturated triglyceride enriched solidified oil. The solidification step is preferably carried out by mixing the oil with liquid nitrogen and the extraction step is preferably carried out with acetone.

Description: TRIGLYCERIDE ENRICHMENT. The present invention relates to a method for the separation of triglyceride containing oils, particularly edible oils, into fractions that are enriched in polyunsaturated triglycerides and fractions that are enriched in saturated triglycerides, and to apparatus for carrying it out. This invention is particularly applicable to the separation of triglyceride containing oils derived from fish. The triglyceride (TG) composition of fish oils is diverse. This is in part a consequence of their wide ranging fatty acid composition which incorporates short chain saturated acids such as tetradecanoic acid and the long chain polyunsaturated fatty acids (PUFAs) cis-5»8,11,14, 17-eicosapentanoic acid (EPA) and cis-4,7.10,13.l6,19-docosahexanoic acid (DHA) . The composition of virtually all fish oils can be described by reference to eight major fatty acids (see Gunstone et al; The Lipid Handbook, Chapman and Hall, 1986, p. 130); these being 14:0, 16:0, 16:1, 18:1, 20:1, 22:1, 20:5 (n-3) and 22:6 (n-3) . An additional thirteen acids are often present above a level of 0.3# and include 15:0, 16:2, 16:3. 16:4, 18:0, 18:2, 18:3 (n-3). 18:4 (n-3), 20:0, 20:4 (n-6 and n-3) , 21:5 and 22:5 (n-3) . Furthermore, trace quantities of the odd numbered fatty acids 17:0 and 17-1 are often detected (Ackman R G; Advances in Fish Science and Technology edited by J.J. Connell, Fishing News Books Ltd, England, 9δ0, pp 86-103), while methyl-branch fatty acids are also present as minor components (Ratnayake, W.M.N. , B. Olsson and R.G. Ackman, Lipids 24:630 (I989)). The triglyceride distribution of fish oils has been studied by Ag + -HPLC (Laakso et al, Lipids (1989) 2 : p630 ) and reverse phase chromatography (Laakso and Christie, J. Am. Oil. Chem. Soc.(1991) 68: 213), whereby it has been shown that they contain triglycerides with only saturated fatty acid moieties present in the molecule and those where each fatty acid contains at least five double bonds with a wide range of variation of fish oil triglyceride composition. The increased interest in both the fatty acid composition and triglyceride distribution of fish oils is a direct result of the association of high fish containing diets and the consumption of fish oils with the prevention and treatment of a variety of human diseases. The influences of the n-3 acids EPA and DHA, which are regarded as being the nutritionally important components of the fish oils, have been observed in relation to beneficial effects upon occurrence of cardiovascular disease, inflammatory diseases and cancer. A wide range of fish oil supplements containing approximately 18# EPA and 12# DHA are readily available at retail outlets (Ackman, R.G., Chemistry in Industry (1988) pl39) . but supplements containing higher percentages of EPA and DHA are, however, generally derived from ethyl or methyl esters. The present inventors have now provided a method for the production of triglyceride fish oils enriched with respect to the polyunsaturated fatty acids themselves, a preferred trigyceride form from the point of view of food supplementation. In GB 2069520 there is described a method for separating components of fatty mixtures whereby the mixtures, particularly purified oils, are solidified by cooling rapidly, the resultant solid is heated to a temperature higher than the freezing point of a desired component fraction whereby that melts, the melted fraction is eparated and the process repeated as necessary. This patent teaches that the method can be applied to fish oils, but specifies that the heating step be carried out for a short time, particularly by intimately contacting the solid mixture with a polar solvent. GB 2069520 teaches that a 10 minute contact time with acetone at -60°C is the preferred method of melting the desired components whereby about a 3 % yield of 20:4, 20:5; 22:5 and 22:6 oils is provided. All compounds investigated were of defined acid type with presumed discrete melting points. None of the examples given in the aforesaid document relate to triglycerides, compounds having three fatty acid moieties per molecule with the melting point being determined by the individual acids and their poisition therein. As stated above, there are at least 8 major fatty acids in any oil and these can be at any one of the three positions, thus providing great scope for variation. It will thus be seen that there is no discrete 'fish oil triglyceride' with a specific melting point, and that the difference between melting points for such oils overlaps greatly in many cases. Using the method of GB 2069520 to separate fish oil triglycerides, the present inventors have found that even after heating the solidified mixture for up to an hour, only 1 to 1.5% yield of enriched oil could be obtained. They have concluded that this method is unsatisfactory for commercial scale preparation of the desired enriched oils due to the inability of convenient solvent heating agents to separate triglycerides when used in this way. Furthermore, GB 2069520 specifies formation of fine crystals when the solidified fatty components are stirred, whereas the present inventors find that with triglyceride oils are amorphous solids when frozen rapidly, but are part liquid at -30°C. With a view to enabling commercially useful separation they have studied extraction of the oil at low temperatures using polar solvents and found that this can provide suitable yields. In a first aspect the present invention provides a method for separating an oil comprising triglycerides into polyunsaturated triglyceride enriched and saturated triglyceride enriched oil fractions comprising: (a) solidifying the oil by freezing it to form discrete bodies (b) extracting the solidified oil with a polar organic solvent at a temperature of ~30°C or below, for a period of at least 1 hour, preferably 5 or more, but most preferably 24 to 72 hours; (c) separating the solution obtained from the residue of the bodies and (d) removing the solvent from the solution to leave enriched extract oil, wherein the extract comprises the polyunsaturate enriched oil and the residue comprises the saturated triglyceride enriched oil. The solidification step (a) is preferably rapid such that the oil does not crystallise, preferably such that an amorphous solid is formed and substantial transfer of components from the oil is avoided. It is particularly preferred that this solidification step results in the formation of bodies, eg. spheroids or rods, of 2 cm or less, preferably 0.5cm or less, in diameter and most preferably about 2mm in diameter. The preferred method of solidifying the oil is by contact with liquid nitrogen, thus providing the rapid cooling and lack of product transfer that is desired. The preferred means of contact is by directing oil droplets or streams (eg. from a spray of edible oil) of diameter suitable for producing a desired solidified body diameter, into contact with the liquid nitrogen, preferably to settle therein. The precise desired dimensions of the solidified oil product of step (a) will depend upon the desired product of step (b) and conditions used to extract the solidified product of (a) . This particularly depends upon the oil to be separated, the polarity of the extracting solvent, its temperature and the time over which extraction is carried out. Most preferably the method is applied to the separation of fish oils but other suitable oils will occur to a person skilled in the art or may be determined by simple bench experimentation using this method. The solvent used for step (b) may be any polar solvent, but if the final product has application as a food or medicament additive such solvent should be easily separable from the extract and be non-harmful by ingestion. Thus while halogenated hydrocarbons and aldehydes may be usable for research purposes and for production of non-food or medicament additive extracts, the preferred solvent will be a physiologically acceptable and/or easily separated solvent. Because the extraction is carried out at -30°C or below the use of solvents such as ethanol is not practicable thus preferred solvents for extractions where food or medicament additive use is envisaged are ketones such as acetone or esters such as ethyl acetate; most preferably the solvent is acetone. Where the extraction is to be carried out on bodies having the preferred diameter range it is found that extraction with acetone is preferably carried out at between -50°C and -70°C, most preferably at about -6θ°C for 2mm diameter bodies. Optimally under such conditions extraction is carried for between 24 and 72 hours, most preferably for about 42 hours. It should be noted that, contrary to the teaching that just melting the components is applicable in the presence of polar solvents, the use of extraction times above 72 hours tends to increase the amount of saturated oil in the extraction product, and thus is undesirable. It will be realised that such upper time limit will vary from oil to oil and the desired end use. Obviously longer extracting times will increase overall yield of product. If the temperature of the acetone is as low as -85°C the poly -unsaturated extract is found to be very pure with regard to the desired n-3 acids but the yield is low. Conversely temperatures of above -50°C may be used but they result in increasing amounts of saturated triglycerides being extracted into the solvent. Similarly, increasing the time of extraction step (b) , while increasing the yield of desired acids will also increase saturate yield in the extract, thus for a given dimension of solidified oils the person skilled in the art will need to determine temperature and time of extraction required for the desired purity and yield. Means for determining these will occur to those skilled in the art but will conveniently involve small scale bench trials. The method of the invention provides an oil extract, preferably an edible fish oil extract, that is enriched in unsaturated triglycerides, while preferred forms provide such oils enriched in n-3 triglycerides. Thus a second aspect of the invention provides an unsaturated triglyceride enriched edible oil obtainable by the method of the invention. The preferred n-3 triglyceride enriched oils are characterised by being liquids at low temperatures, particularly preferred oils being liquids at about ~30°C. The method of the invention further provides an extracted edible oil, preferably a fish oil, which is enriched in saturated triglycerides, that being useful for use in oil containing foodstuffs eg. such as margarines and other items where hydrogenated triglycerides might usually be used. Thus a third aspect of the invention provides a saturated triglyceride enriched edible oil obtainable by the method of the invention. The method of the invention is simple and avoids chemical modification of the triglycerides. Large quantities of crude fish oil, with no prior refinement other than having been pressed from the fish and centrifuged, can be enriched relatively quickly giving a polyunsaturate product free of all particulate material. This is contrasted by the method of GB 2069520 wherein the examples refer to use of distilled oils or discrete fatty acid mixtures. Furthermore, the saturated triglyceride rich residue contains a reduced proportion of PUFAs and as such is of direct use to other processing industries where oils with a reduced PUFA content are required. The enriched polyunsaturated triglyceride rich oils obtained by the preferred form of the method give a relatively consistent Ag + -HPLC profile indicating that specific triglycerides are extracted from the solidified lipid by the polar solvent. Rapid solidification produces an amorphous solid, rather than a crystalline material, which is at liquid nitrogen temperature when mixed with the cooled acetone, and the structure of the solid probably resembles that of triglycerides in the liquid state (Larsson, Fette Seifen Anstrichmittel. (1972) 2 - Pl36) . During storage at -60°C the triglyceride bodies remain in discrete, hard form. The exact process which the biphasic system undergoes is unclear, but selective dissolution of the PUFA containing triglycerides by the polar solvent is most likely. The solubility of fatty acids, containing up to three double bonds, using various solvents at a variety of temperatures has been studied in detail (Brown and Kolb (1955) Prog. Chem. Fats. Lipids 3 P57) but only linoleic and linolenic acids show significant solubility at -6θ°C in acetone. Free fatty acids are concentrated by the method but this is of little consequence when the original oil contains only low quantities of these molecules; they will, however, have to be removed from oils where their concentration is higher. The present method is conveniently applied to crude fish oils, eg of about 15% triglyceride content without further processing. The production of significant quantities of a high quality enriched oil will provide a material which can be used for structural or oxidative studies and, in addition, could be used to further investigate the physiological effects of the consumption of such highly unsaturated n-3 acids. In a fourth aspect of the present invention there is provided an apparatus for separating an oil comprising triglycerides into polyunsaturated triglyceride enriched and saturated triglyceride enriched oil fractions comprising (a) an insulated container having cooling means sufficient to to freeze oil comprising triglycerides (b) a means for supplying oil comprising triglycerides in the form of discrete bodies or streams to the insulated container; (c) a means for supplying a polar organic solvent to the container such that it is in contact with oil bodies formed therein; (d) a means for maintaining the temperature of the solvent and oil bodies therein below -30°C for a period of 1 hour or more; and (e) a means for separating the solvent from the oil bodies Preferably the means for supplying the oil is such that continuous streams of oil or streams of droplets of oil are formed by it such that on cooling in the container the form spheroids or rods of desired diameter. Preferably the container comprises a means for admitting liquid nitrogen prior to supply of the oil, such that on entering the liquid nitrogen the oil is solidified. Preferably a means is provided for removing the liquid nitrogen while retaining frozen oil bodies in the container, eg, in the form of a drainage outlet, a tipping mechanism or a pipe attached to a pump; each having a mesh for prevention of passage of the oil bodies across its opening. In preferred embodiments the apparatus will feed the polar solvent from the container after the 1 or more hours extraction to a chamber where the solvent will be evaporated, with or without application of vacuum, and further purification steps will also be provided. Further apparatus features such as devices for addition of antioxidants to the residue provided on evaporation of the polar solvent, or for adding solvent to the extraction residue in the insulated container for removal of saturate enriched oil may also be provided. The method and products of the invention will now be illustrated by way of example only by reference to the following Figures and Examples; further embodiments falling within the scope of this invention being derivable by the person skilled in the art by application of their knowledge of the art of edible oil separations. FIGURES Fig. 1A to IF: show Ag + -HPLC results for the original (pressed and centrifuged only) and polyunsaturate enriched fish oil triglycerides prepared from acetone extracts made by the method of the invention. 1A, original sardine oil; IB, enriched sardine oil; 1C, original Chilean fish oil; ID, enriched Chilean fish oil; IE, original Danish fish oil; IF, enriched Danish fish oil. S = saturated, M = monounsaturated, TO = triolein, TL = trilinolenin, TA = triarachidonin. MATERIALS AND METHODS Chemicals: Hexane, methanol and acetonitrile were obtained from Rathburn Chemicals Ltd (Walkerburn, Scotland) . Hipersolv 1,2-dichloroethane, used for the HPLC, and Aristar acetic acid were from BDH Chemicals Ltd (Poole, England). The acetone used for the enrichment was a general purpose reagent from the same company. Diethylether, chloroform and benzene were Pronalys AR from May and Baker (Dagenham, England). Sodium sulphate, copper sulphate, orthophosphoric acid and sodium hydroxide were AnalaR grade chemicals from BDH Chemicals Ltd (Poole, England). Lipid standards were purchased from Sigma Chemical Co Ltd (Poole, England). Fish Oils: Sardine (Sardina pilchardus) oil and a refined stabilised fish body oil were provided by Isaac Spencer and Co. Fleetwood (1920) Ltd (Fleetwood, England). Menhaden (Brevoαrtia spp) oil was from Zapata Haynie Corporation (Reedville, Virginia). A white fish oil was provided by United Fish Products Ltd (Aberdeen, Scotland) while a commercial grade fish oil was supplied by HBP Ltd (Fraserburgh, Scotland). Sun f n' Sea cod liver oil (S and S) (Cupar Ltd, Blackburn, England) was purchased from a local retailer. A Chilean fish oil, South African anchovy (Engraulis capensis) oil and a Danish fish oil were taken from samples held at Torry Research Station. The fatty acid distribution of the original and enriched fish oils was determined as described by Moffat et al (J. High Res. Chromatogr. (1991) -!---.322). The triglyceride distribution of the original and enriched fish oils was determined by Ag + -HPLC using a 1,2-dichloro -ethane, acetone, acetone/acetonitrile (75:25, v/v) ternary gradient system. The gradient was designed to give a shorter analysis time and improved resolution of the PUFA containing triglycerides. This is summarised in Table 1. The original and extracted polyunsaturated triglyceride enriched oils were further analysed by high performance thin layer chromatography (TLC) , to assess the effect of the enrichment process on free fatty acids, using LHP-K linear high performance TLC plates (20 x 10 cm, Whatman International Ltd Maidstone, England). EXAMPLE 1: Separation of oils: Each fish oil (200 g) was run down a finely drawn out pasteur pipette. The small droplets thus produced fell into a conical flask containing, and surrounded by, liquid nitrogen. The oil droplets solidified instantaneously thus producing very small beads of fish oil of about 1 or 2mm diameter thus having a high surface area to volume ratio; these sank to the bottom of the flask. Once all the oil was converted into small beads, the liquid nitrogen was allowed to boil off and the beads were poured into acetone (1800 mL) at -6θ°C. The mixture was stored at -6θ°C for 42 hours with intermittent mixing, after which the solid was separated from the supernatant by. filtration at -6θ°C, using a glass sinter funnel. The acetone was removed by rotary evaporation (water bath 4θ°C) with final traces of solvent being removed using a high vacuum pump. The antioxidant 2,6-di-tert-butyl-4-methylphenol (BHT) was added to the resultant oil at a final concentration of 0.012. EXAMPLES 2 to 10: Polyunsaturated triglyceride enriched products. The method of Example 1 was applied to the oils shown in Table 2. The yields of polyunsaturate enriched fish oil varied from 26.0% for a sardine oil to 2.8% for the Danish fish oil (Table 2). Fish oils which gave the higher yields (greater than 15 ) were characterised by a low concentration of the monoenoic acids eicosenoic acid (20:1) and docosenoic acid (22:1) (Table 3)- These were the sardine, South African anchovy, Chilean and Menhaden fish oils. Similar trends were observed for the changes in the fatty acid composition of the fish oils following enrichment. The saturated fatty acids comprised less than 1.2% of the total, corresponding to less than one-fifth that of the original oils. Palmitic acid (16:0) was significantly reduced while octadecanoic acid (18:0) was absent from all the polyunsaturate enriched fish oils (Table 3) • The concentration of palmitoleic acid (16:1) increased by a factor of 1.1 to 1.3 while the percentage octadecenoic acid (18:1) decreased slightly except for the Danish fish oil. The concentration of 20:1 and 22:1 decreased except for the sardine oil where 22:1 showed a small increase. All fatty acids containing two or more double bonds were present at a higher concentration in the enriched oils. Significant increases were observed in the concentration of EPA and DHA. The minimum combined percentage of these two fatty acids (4l.7%) was observed for the Menhaden fish oil while a maximum of 57•4% was noted for the enriched South African anchovy oil. The degree of enrichment was more significant in the oils that gave the lowest yields (Table 2) . The refined stabilised fish body oil was enriched, with respect to EPA and DHA, by a factor of, 2.52 but the yield was 6.3%. The highest concentration of the six n-3 acids (66.0 ), which included 18:3, 18:4, 20:4, 20:5, 22:5 and 22:6, was observed for the enriched Chilean fish oil but the enriched South African anchovy oil contained the highest concentration (76.2%) of fatty acids with two or more double bonds. The original sardine oil gave a typical Ag * -HPLC profile (Fig. 1A) which was conveniently divided into three regions. The first region (0 to 17 min) comprised triglycerides containing only saturated fatty acids (3«0 min) together with those comprising one (5«0 min), two (8.0 min) or three (11.5 min) monoenoic acids. A small proportion of dienoic acids was present in peaks eluting after 11.5 min. These triglycerides were readily identified by reference to standard triglycerides and previous Ag + -HPLC fractionation of fish oils. Triglycerides eluted between 17 and 30 minutes (region 2) contained increasing proportions of PUFAs with the progressive introduction of more highly unsaturated fatty acids including EPA and DHA. The third region (30 to 60 min) contained very highly unsaturated triglycerides with equivalent retention times to trilinolenin (TL) and triarachidonin (TA; Fig. 1A) . The most highly unsaturated triglycerides, those with greater than 13 double bonds per triglyceride, were present at very low concentrations and were eluted after 44 minutes. The enrichment process radically altered the Ag + -HPLC profile for the sardine oil (Fig. IB) . Almost all the triglycerides from the first region, those containing saturated, monoenoic and a small proportion of dienoic acids, were left in the saturate rich oil fraction by the treatment. In contrast, the concentration of the most highly unsaturated triglycerides, those eluted after 44 minutes, was greatly increased, there being a series of discrete peaks evident on the HPLC chromatogram. In addition to the PUFA containing triglycerides, the process was shown, by high performance TLC, to concentrate the free fatty acids. This was qualified by the presence of a broad peak at 20.3 minutes in the Ag + -HPLC chromatogram (Fig. IB). The relative concentration of triglycerides in the second region (17 to 30 min. ) was considerably reduced although the triglycerides eluted between 26 and 30 minutes were of approximately the same peak intensity. The most intense peaks were those with retention times approximating to the standard triarachidonin which contains 12 double bonds per triglyceride. The apparent percentage of triglycerides eluted after 30 minutes was 24.9% and 79• % for the original and polyunsaturate enriched oils respectively. Due to the variation in detector response associated with longer chain length and degree of unsaturation these figures do not directly reflect a degree of enrichment. The Chilean fish oil gave a similar triglyceride pattern (Fig. IC) to the sardine oil (Fig. 1A) when analysed by Ag + -HPLC. The split peak with maxima at 4.5 and 5«0 minutes corresponds to alpha or β- substitution respectively of the glycerol moiety by the monoenoic acid. There was no equivalent peak to the one observed at 17-4 minutes in the sardine oil, but thereafter the Ag + -HPLC profile of the Chilean oil varied only with respect to peak intensities. The enrichment process again left all the triglycerides which were eluted in the first region of the Ag + -HPLC profile (Fig. ID) in the saturate enriched fraction, although there was a peak at 10.4 minutes which was more intense relative to a peak with an equivalent retention time present in the original oil. This sharp, gaussian peak was typical of all the enriched fish oils. The changes in the relative intensities of the peaks present after 17 minutes showed similar trends to the sardine oil. The most significant area of variation was between 43 and 49 minutes. The Chilean fish oil gave a significant peak at 43-5 minutes while the sardine oil gave a peak of low amplitude in this region which was coincident with a shoulder on the Chilean fish oil peak. Thereafter, the peak shapes and distribution were quite distinct until after 49 minutes when both oils gave four similarly shaped peaks. The Chilean fish oil gave a significantly lower yield of poly -unsaturate fraction (Table 2) yet there were no stark differences in either the triglyceride profile of the original oils or the two enriched products. The Danish fish oil gave the lowest yield of polyunsaturate of all the fish oils on enrichment. The Ag * -HPLC profile (Fig. IE) was distinct from the other two oils with respect to the triglyceride pattern after 17 minutes. Prior to this time the profile was typical with trisaturated and monoenoic substituted triglycerides. The peak intensities of the early eluting triglycerides in the second region (17 to 30 min) of the Ag + -HPLC profile were greater for the Danish fish oil than for the sardine or Chilean fish oils. Furthermore, the peak at 26.0 minutes was more significant than for the other two oils. Thus, the triglyceride composition of the Danish oil was quite different. The third region (30 to 60 min) contained less than 10% of the detected triglycerides; there were no very highly unsaturated triglycerides (greater than 44 min) detected. A low yield was, therefore, expected from this oil. The enriched Danish fish oil gave a Ag + -HPLC profile similar to those obtained for the sardine and Chilean fish oils with the exception of the presence of a peak at 8.0 minutes. This arises from triglycerides which contain one saturated and two monoenoic acids per glycerol moiety. The relative concentration of triglycerides in the second region (17 to 30 min) was reduced and there was only a slight change in peak intensities for triglycerides eluted between 30 and 35 minutes. The relative increase in the concentration of triglycerides with retention times equivalent to TL and TA in the polyunsaturated fraction was far greater for the Danish fish oil than for the other two oils. Although the peaks were not as discrete, it was apparent that the enrichment process extracted quite specific triglycerides, those that were highly unsaturated. Such observations were reinforced by the Ag + -HPLC profiles obtained for the other enriched oils. The presence of very highly unsaturated triglycerides (eluted after 44 minutes. Fig. IF) again typified the results for all the oils. Equivalent triglycerides were not identified in the Ag + -HPLC profile of the original Danish fish oil (Fig. IE) but it was apparent that such triglycerides were present in all the original fish oils even though their concentration was extremely low. TABLE 1 Ternary Gradient Conditions for Elution of Triglycerides from oils. The period from 60.0 to 80.0 minutes was required to recondition the column. A is 1,2-dichloromethane; B is acetone and C is acetone/acetonitrile (75: , v/v) . TABLE 2 Percentage Yield Polyunsaturated fraction as % of initial oil and Degree of Enrichment for that fraction. EXAMPLE 3: Comparative yield of enriched oil with length of extraction. In order to demonstrate the ineffectuality of the melting method of GB 2069520 as compared to the extraction method of the present invention, the relationship between the length of time the solidified oil is contacted with polar solvent and the yield of enriched oil product was determined. Crude sardine oil was used obtained only by pressing fish and centrifuging the expressed oil; this containing about 75% by weight of triglycerides. In order that the effects of varying surface area were eliminated 2mm diameter bodies were frozen using liquid nitrogen; it should be noted that these bodies do not crumble to give rise to crystals as do the purer fatty acids disclosed in GB 2069520. The yield of enriched product after contact with acetone at -6θ°C for a period sufficient to achieve melting of products (which are often liquid at -30°C) and periods beyond that suitable for extraction and the composition of the enriched oil determined. The product was obtrained by evaporation of the acetone as described above. TABLE : YIELD OF ENRICHED OIL OBTAINED WITH TIME. Thus from Table 3 it can clearly be seen that melting coupled with separation from unmelted components alone cannot provide suitable enrichment of this oil with commercially acceptable yield as compared to the potential available. It is necessary to extract the oil with the polar solvent, at low temperature, for a period sufficient to remove desired products by dissolution, while this temperature keeps the saturate solubility relatively low. TABLE 4: FATTY ACID CONCENTRATIONS (WEIGHT X) . TA R TF- * Fatty acid composition (% Nominated Acids) and percentage fatty acids with specific number of double bonds from the original (0) and enriched (E) Fish Oils. In Table 5 the percentage of the saturated (Sat) , monenoic (Mono) , dienoic (Di) , trienoic (Tri), tetraenoic (Tet), pentaenoic (Pent) and hexaenoic (Hex) acids were summed to assess the overall double bond distribution in the triglycerides. Tr is trace (<0.3%). the percentages include various isomers where appropriate (20:4, n-6 and n-3) . Annotation b indicates the n~3 isomer alpha-linolenic acid: (c_is.-9,12,15-octadecanoic acid). Annotation c indicates that the fraction includes n-3 isomers of 18:3. 18:4, 20:4, 20:5. 22:5 and 22:6.

Claims: CLAIMS 1. A method for separating an oil comprising triglycerides into polyunsaturated triglyceride enriched and saturated triglyceride enriched oil fractions comprising: (a) solidifying the oil by freezing to form discrete bodies; (b) extracting the solidified oil bodies with a polar organic solvent at a temperature of -30°C or below, for at least 1 hour; (c) separating the solution obtained from the residue of the bodies; and (d) removing the solvent from the solution to leave enriched extract oil; wherein the extract oil comprises the polyunsaturate enriched oil and the residue comprise the saturated triglyceride enriched oil. 2. A method as claimed in claim 1 wherein the period is at least 5 hours. 3. A method as claimed in claim 2 wherein the period is from between 24 to 72 hours. 4. A method as claimed in any one of claims 1 to 3 wherein the solidification step (a) is rapid wherein crystallisation of the oil substantially does not take place. 5. A method as claimed in any one of claims 1 to 4 wherein substantial transfer of components from the oil during step (a) is avoided. 6. A method as claimed in any one of claims 1 to 5 wherein the bodies are less than 2.0cm in diameter. . A method as claimed in claim 6 wherein the bodies are about 2mm in diameter. 8. A method as claimed in claim 7 wherein the bodies are about 1mm in diameter. 9. A method as claimed in any one of the aforesaid claims wherein the solidification step (a) is carried out by contacting the oil with liquid nitrogen. 10. A method as claimed in claim 9 wherein the contact is made by directing oil droplets or streams into contact with the liquid nitrogen. 11. A method as claimed in any one of the preceding claims wherein the solvent used for step (b) is easily separable from the extract and/or is non-harmful by ingestion, 12. A method as claimed in claim 11 wherein the solvent used for step (b) comprises a ketone or an organic acid ester. 13. A method as claimed in claim 12 wherein the solvent is acetone. 14. A method as claimed in any one of the preceding claims wherein step (b) is carried out at between ~50°C and ~70°C. 15. A method as claimed in claim 14 wherein step (b) is carried out at about -60°C. 16. A method as claimed in any one of the preceding claims wherein the product of step (a) is extracted with the polar solvent for between 24 and 72 hours. 17- A method as claimed in claim 16 wherein the extraction is carried out for about 42 hours. 18. A method as claimed in any one of claims 1 to 17 wherein the oil is an edible fish oil. 19. An unsaturated triglyceride enriched oil obtainable by a method as claimed in any one of claims 1 to 18 as the solvent extraction solvent fraction. 20. An oil as claimed in claim 19 characterised in that it is enriched in n-3 triglycerides. 21. An oil as claimed in claim 19 or 20 characterised in that it is a liquid at about -30°C. 22. An saturated triglyceride enriched oil obtainable by a method as claimed in any one of claims 1 to 18 as the solvent extraction residue fraction. 23. An apparatus for separating an oil comprising triglycerides into polyunsaturated triglyceride enriched and saturated triglyceride enriched oil fractions comprising (a) an insulated container having cooling means sufficient to to freeze oil comprising triglycerides (b) a means for supplying oil comprising triglycerides in the form of discrete bodies or streams to the insulated container; (c) a means for supplying a polar organic solvent to the container such that it is in contact with oil bodies formed therein; (d) a means for maintaining the temperature of the solvent and oil bodies therein below -30°C for a period of 1 hour or more; and (e) a means for separating the solvent from the oil bodies 24. An apparatus as claimed in claim 23 wherein the means for supplying the oil is such that continuous streams of oil or streams of droplets of oil are formed by it such that on cooling in the container they form spheroids or rods of desired diameter. 25- An apparatus as claimed in claim 23 or 24 wherein the cooling means comprises, in part at least, a means for admitting liquid nitrogen to the container prior to supply of the oil, such that on entering the liquid nitrogen the oil is solidified. 26. An apparatus as claimed in any one of claims 23 to 25 wherein a means is provided for removing the liquid nitrogen while retaining frozen oil bodies in the container. 27. An apparatus as claimed in any one of claims 23 to 26 wherein a chamber is provided for receiving polar solvent from the 1 or more hours maintenance at ~30°C or less; that chamber being adapted for the evaporation of the solvent with or without application of vacuum. 28. A method as claimed in claim 1 substantially as described in Example 1. 29. An oil as claimed in claim 19, 20 or 21 substantially as described in any of Examples 1 to 10. 30. An oil as claimed in claim 29 being the solvent extraction residue of an initial oil after solvent extraction of polyunsaturate enriched oil as described in any one of the Examples 1 to 10.

Inventors: HARDY ROY, MCGILL ALISTER SMITH, MOFFAT COLIN FORBES, HARDY, ROY, MCGILL, ALISTER, SMITH, MOFFAT, COLIN, FORBES
Applicants: HARDY ROY, MCGILL ALISTER SMITH, MINI AGRICULTURE & FISHERIES, MOFFAT COLIN FORBES, THE MINISTER OF AGRICULTURE, FISHERIES AND FOOD IN
Application year: 1993
Main classification: C11B7
IPC codes: C11B7