WO2009020406A1

WO2009020406A1 - Methods of making lipid substances, lipid substances made thereby and uses thereof

Info

Publication number: WO2009020406A1
Application number: PCT/NZ2008/000200
Authority: WO
Inventors: Keith Raymond Morgan
Original assignee: Granate Seed Limited; Roxdale Foods Limited
Priority date: 2007-08-07
Filing date: 2008-08-07
Publication date: 2009-02-12

Abstract

Methods of enriching lipid substances, particularly fats and oils, in polyunsaturated fatty acids (PUFAs), particularly omega-3 fatty acids are described, along with methods of producing glycerides from FFAs, particuarly from PUFAs, methods of producing lipid substances comprising glycerides enriched in PUFAs and oils or fats enriched in PUFAs or derivatives thereof.

Description

Methods of Making Lipid Substances, Lipid Substances Made Thereby and Uses

Thereof

FIELD

The present invention relates to methods of enriching lipid substances, particularly fats and oils, in polyunsaturated fatty acids (PUFAs), particularly omega-3 fatty acids. The invention also relates to methods of producing glycerides from FFAs, particuarly from PUFAs. The invention further relates to methods of producing lipid substances comprising glycerides enriched in PUFAs. The invention also relates to oils or fats enriched in PUFAs or derivatives thereof.

BACKGROUND

Fats and oils are triacylglycerides (TAG), that is esters of free fatty acids (FFAs) with glycerol. They are important macronutrients for animals, including the human animal, where they contribute about 30 % of the calorie intake. The FFAs that form fats and oils have a wide variety of structures but all are carboxylic acids and most contain linear carbon chains capped by a terminal methyl group.

Four major categories of FFA are considered important in the human diet, saturated, monounsaturated, and the polyunsaturated omega-6 and omega-3 FFAs. Here, the term omega-n refers to monounsaturated or polyunsaturated FFA, where the first double bond is at the n^th carbon-carbon bond from the terminal methyl group. Saturated FFAs contain no double bonds whereas monounsaturated contain one usually at the omega-9 position. Both saturated and monounsaturated fatty acids are able to be synthesised in vivo.

Two categories of FFA, omega-6 and omega-3, are essential and cannot be synthesised by the human animal and must therefore be supplied by diet. Omega-6 is found mostly in vegetable oils as well as fats from animals. The average modern western diet includes plenty of omega-6 from these sources.

Omega-3's are much less common in the average western diet. There are several different types of omega-3 that are important for the human animal, and other mammals: the long chain omega-3's dodecahexenoic acid (DHA, G22:6n-3) and eicosapentenoic acid (EPA, C20:5n-3), and the short chain omega-3 alpha-linolenic acid (ALA, C18:3n-3). Although the human animal can convert ALA to EPA and DHA, the method for doing this is considered to be inefficient, and it is thought that optimally DHA and EPA should be provided in the human diet from other sources, namely fish.

Some studies suggest that low intakes of DHA and EPA are risk factors in heart disease, mental health and impaired brain performance. In addition, EPA is an anti-inflammatory. It may therefor be considered useful to provide omega-3 supplements to humans.

Levels of EPA and DHA in fish oil are reasonably low, typically below 20w%, and levels of ALA are very low, typically below 1w% and therefore it may be considered useful to have enriched sources of these lipids.

Various methods are known for enriching fish oil so that the oil contains greater quantitites of long chain omega-3 FFA. Winterising, that is cooling, fish oil can cause precipitation of saturated triglycerides. The remainder of the oil is therefore enriched in PUFAs. This technique has the disadvantage that saturated FFA residues can be on the same glycerol residue as the long chain omega-3 FFA and thus purity and the degree of enrichment is reasonably low.

Molecular fractional distillation can also be used to enrich the fish oil itself in DHA and EPA, but yields of the enriched oil are usually very low, since only a small fraction of the oil contains triacylglycerides where all three FFA residues are either DHA or EPA.

Other methods may involve only the enrichment of the FFA itself, after the FFA has been isolated from the triacylglyceride.

Fish oil FFAs or esters of the FFAs can be crystallised in the presence of urea. The PUFAs including omega-3 preferentially form an inclusion complex with the urea. A disadvantage of this process is that water immiscible organic solvents such as hexane are required to liberate the FFA from the inclusion complex.

Various FFA fractions can be recovered by fractional crystallisation, but the yields are not high and require water immiscible organic solvents and low temperatures of less than approximately -40 ⁰C especially for crystallisation of the PUFA fractions.

Fractional distillation of the ethyl ester of the FFA may also be used. to purify FFA derived from fish oil, but there is some indication that the ester is not as effective a source of PUFA as the triacylglceride. Methods for recombining FFA to reform glycerides (for example tri-, di- and mono- glycerides) are known. One method uses Upases but yields are often low arid reaction times can be as long as three days. Another method uses high temperatures and a catalyst such as a sodium or potassium soap to convert mixtures of FFAs and glycerol into glycerides but such a method is not suitable for PUFAs which tend to degrade under these reaction conditions.

OBJECT It is an object of this invention to provide improved methods of enriching lipid substances, particularly oils or fats, in PUFAs, particularly omega-3 fatty acids, as well as lipid substances enriched in such PUFAs. It is a further or alternative object to provide improved methods of forming glycerides from FFAs, particularly PUFAs, as well as glycerides produced by such methods. It is a further or alternative object to provide improved methods of producing lipid substances comprising glycerides enriched in PUFAs. It is a further or alternative object to at least provide the public with a useful choice of any one of the foregoing.

STATEMENT OF INVENTION In a first broad aspect the present invention provides a method for enriching lipid substances in PUFAs the method comprising at least the steps: a. Reacting a lipid substance with alkali in the presence of water and a water miscible organic solvent to create a liquid containing solids; b. Removing the solids from the liquid; c. Recovering FFAs enriched in PUFAs from the liquid.

Preferably the lipid substance is a fat or oil derived from vegetables or animals. More preferably, the lipid substance is fat or oil from fish and the method is for enriching the lipid substance in omega-3 fatty acids.

Preferably the alkali is sodium hydroxide. Preferably the water miscible organic solvent is ethanol.

Preferably the ratio of water to water miscibie organic solvent is between approximately 2.0 and approximately 0.05. Preferably the ratio of water to water miscible organic solvent is between approximately 0.7 and approximately 0.1 , more preferably between approximately 0.3 and approximately 0.1. Preferably the solids are removed by filtration.

In one embodiment step a. includes: i. Mixing an aqueous solution of the alkali with the water miscible organic solvent to create an alkali and water miscible organic solvent mix; ii. Adding the lipid substance to the alkali and water miscible organic solvent mix iii. Stirring the lipid substance and alkali and water miscible organic solvent mix for an initial period so that an emulsion forms containing small droplets of the lipid substance in the solvent mix.

In regards to i. the alkali is alternatively mixed directly with the water and water miscible organic solvent. In a further embodiment, in step i. an aqueous solution of the alkali is mixed with water and a water miscible organic solvent.

Preferably stirring for an initial period occurs for approximately 10 minutes. More preferably the mix is further stirred for approximately 1 minute approximately every half an hour for approximately 2 hours.

Preferably the reaction of step a. is performed for a period of greater than approximately 4 hours.

In one embodiment, the method is conducted at a temperature of approximately 20⁰C or below.

In one embodiment, after step a) the temperature of the liquid containing solids is reduced. Preferably, the temperature of the liquid containing solids is reduced to approximately 5°C or less.

Preferably step c. includes: i. neutralising the liquid with an acid, preferably HCI, H₂SO₄, or H₃PO₄; and ii. recovering a water immiscible fraction containing FFAs.

In one particular embodiment, step c. includes: iii. neutralising the liquid with an acid, preferably HCI, H₂SO₄, or H₃PO₄; iv. removing the water miscible organic solvent from the liquid; v. washing the FFAs with water to form two immiscible fractions; vi. recovering the water immiscible fraction containing FFAs.

Preferably, the water miscible organic solvent is removed or recovered from the liquid by evaporation, condensation and/or ultrafiltration.

Preferably the solids removed during the method are washed with additional water miscible organic solvent to recover any additional or residual PUFA. In one embodiment the solids are washed or mixed with the water miscible organic solvent or a. mixture of water and water miscible organic solvent, the solids removed, and the remaining liquid recycled through the method steps mentioned above.

In a related aspect the method of the first aspect further includes a first step of hydrolysing the lipid substance by one or more of high temperature hydrolysis with an oxide catalyst and use of an enzyme such as a lipase. - ^{• ■}

In a second broad aspect the invention provides a method for enrichment of FFAs in

PUFAs the method comprising at least the steps: a. neutralising FFAs with alkali in a water miscible organic solvent to create a liquid containing solids; b. Removing the solids from the liquid; c. Recovering FFAs enriched in PUFAs from the liquid.

Preferably the alkali is dissolved in a water miscible organic solvent and/or water. Preferably the FFAs are dissolved in a water miscible organic solvent.

Preferably step a. involves adding the alkali and the FFAs separately and slowly to a water miscible organic solvent and gently stirring the mixture. Preferably the solids are removed following complete addition and mixing of the FFAs, alkali and water miscible organic solvent.

Preferably the total quantity of water miscible organic solvent used in the method contains less than or equal to approximately 15% water or less than or equal to approximately 10% water.

Preferably the method is conducted at a temperature such that the liquid containing solids is at a temperature of approximately 15°C or less. In a third broad aspect, the invention provides a method for producing glycerides from FFAs the method comprising at least reacting FFAs with glycerol at temperatures of approximately 140⁰C or above and under a partial vacuum.

Preferably the reaction occurs under a partial vacuum at a pressure of approximately 5OkPa or below. In one embodiment the reaction occurs under a partial vacuum at a pressure of approximately 1OkPa or below.

Preferably the reaction occurs at temperatures of approximately 16O⁰C or above. In one embodiment the reaction occurs at a temperature of approximately 180⁰C or above.

Preferably, steps are taken to minimise or eliminate oxygen from the reaction.

Preferably, the FFAs and glycerol are rapidly stirred to break up the glycerol into small droplets, with any glycerol that evaporates being condensed and added back to the reaction.

Preferably the FFAs are PUFAs, more preferably omega-3 fatty acids.

Preferably the glycerides are mono- (MAG), di- (DAG) and/or tri-acylglycerides (TAG).

Preferably the method includes a means to control the ratio of TAG/DAG/MAG formed. Preferably the ratio of TAG/DAG/MAG is adjusted by altering the mol ratio of FFAs to glycerol where mol ratios of FFA/glucerol greater than approximately 2 favour formation of MAG and mol ratios of FFA/glycerol less than approximately 2 favour formation of TAG.

In a fourth broad aspect the invention provides a method for producing lipid substances comprising glycerides enriched in PUFAs the method comprising at least the steps: a. hydrolysing a starting lipid substance including a level of glycerides comprising fatty acids to produce FFAs and glycerol; b. increasing the PUFA content of the FFAs to form FFAs enriched in PUFAs; c. reacting the FFAs enriched in PUFAs with glycerol to form glycerides.

Preferably steps a. and b. employ a method of the first aspect of the invention. ■^{" ■}

Preferably step c. employs a method of the third aspect of the invention. In a fifth broad aspect the invention provides lipid substances produced by a method of any one of the methods of the first to fourth aspects of the invention.

In. a sixth broad aspect the invention provides omega-3 compositions comprising at least approximately 40% of the long chain FFA, that is EPA and DHA, and at least approximately 20% ALA.

In one embodiment, the omega-3 compositions comprise at least approximately 60% of the long chain FFA, that is EPA and DHA.

The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

PREFERRED EMBODIMENT(S)

The following is a description of the present invention, including preferred embodiments thereof, given in general terms. The invention is further elucidated from the disclosure given under the section "Examples" which provides experimental data supporting the invention and specific examples thereof.

In general terms the invention provides methods for enriching lipid substances in PUFAs, and methods for forming glycerides from FFAs, particularly PUFAs. It also provides methods for producing lipid substances comprising glycerides enriched in PUFAs the methods involving enrichment of a starting lipid substance in PUFAs followed by formation of glycerides therefrom.

As used herein a "lipid substance" is any substance or composition comprising at least lipids, which lipids include a level of PUFAs and/or derivatives thereof. Generally a "lipid substance" will comprise a heterogenous mix of different lipids including PUFAs or derivatives thereof. Most preferably the lipid substance includes a level of omega-3 fatty acids or derivatives thereof. A "derivative" of a fatty acid should be taken to include for example esters of the fatty acid and glycerides such as triacylglycerides, diacylglycerides and monoacylglycerides. A "lipid substance" includes fats and oils such as those derived from animals and vegetables. Most preferably the "lipid substance" is an oil or fat obtained from fish or algae, particularly those that produce long chain omega-3 fatty acids. Alternatively a lipid substance is FFAs derived from an oil or fat.

As used herein the terms "enrich", "enriched" and the like are used to refer to an increase in the proportion of a particular type of lipid or component relative to other lipids or components. The terms should not be taken to imply the particular lipid or component is completely isolated or purified from other lipids or components.

First Embodiment

A first embodiment of the invention is a method for enriching lipid substances in PUFAs, particularly omega-3s, by reacting a lipid substance, or FFAs derived from the lipid substance, with alkali in the presence of water and a water miscible organic solvent, removing the solids that form, and recovering the enriched PUFA fraction from the liquid fraction.

In one embodiment the method comprises at least the steps: a. Reacting a lipid substance with alkali in the presence of water and a water miscible organic solvent to create a liquid containing solids; b. Removing the solids from the liquid; c. Recovering FFAs enriched in PUFAs from the liquid.

The alkali used in the invention is preferably sodium hydroxide. However, any other appropriate alkali may be used; for example, potassium hydroxide.

Most preferably, the water miscible organic solvent is ethanol. However, the inventor contemplates the use of other water miscible organic solvents such as isopropanol for example.

In one embodiment, the ratio of water to water miscible organic solvent is between approximately 2.0 and approximately 0.05 (w/w). In one preferred embodiment the ratio of water to water miscible organic solvent is between approximately 0.7 to approximately 0.1 (w/w), more preferably between approximately 0.3 to approximately 0.1 (w/w). In a particular embodiment the ratio of water to water miscible organic solvent is approximately 0.1 or approximately 0.2 (w/w). The "ratio of water to water miscible solvent" is the ratio of the total water present in the reaction to organic solvent present in the reaction. In a preferred form of the invention the solids are removed by filtration, with the enriched PUFAs contained in the supernatant. Alternative means of removing solids may be used, for example centrifugation. S

The alkali, water, water miscible organic solvent and lipid may be combined or mixed in any order. In one particular embodiment of the invention the alkali is first dissolved in a water and water miscible organic solvent mixture and then the lipid substance- is added. In an alternative embodiment, an aqueous solution of the alkali is mixed with. the water0 . miscible organic solvent and then the lipid substance is added. In a further alternative embodiment, an aqueous solution of the alkali is mixed with the water miscible organic solvent and water and then the lipid substance is added. The mixture is preferably stirred so that an emulsion forms containing small droplets (for example, droplets of approximately 2mm in diameter) of the lipid substance in the water miscible organic5 solvent. The mixture is preferably stirred for approximately 10 min. It can also be beneficial, for example, to completely stir the mixture for approximately 1min every 0,5h for the next 2h, preferably stirring gently or being careful not to stir so vigorously as to break up the solid particles into a finer emulsion. In a preferred embodiment, the entire reaction is performed for a time greater than approximately 4h. 0

In one embodiment, the method is conducted at a temperature of approximately 20⁰C or below. In a preferred embodiment, after the lipid substance is reacted with alkali in the presence of water and a water miscible organic solvent, the temperature of the resultant liquid containing solids is reduced. Preferably, the temperature of the liquid containing5 solids is reduced to approximately 5°C or less. This may aid in obtaining a higher purity product.

The inventor contemplates the use of a catalyst to speed hydrolysis during the reaction. In addition, other components could be added to the reaction mix where appropriate. For0 example catalytic enzymes such as lipases that hydrolyse lipids, or oxides such as calcium oxides could be used.

A number of methods may be used to recover the FFA's from, the supernatant or liquid fraction. In one embodiment, it is preferable to neutralise the supernatant with an acid, for5 example HCI, H₂SO₄, or H₃PO₄. The solvent may be removed (or optionally recovered), by evaporation and condensation, ultrafiltration or any other known method. Alternatively, the solvent may be removed in part or in full before neutralisation, or the solvent may not be removed at all.

In one embodiment, after neutralisation and removal of the solvent (if any) the FFA can be washed with water to remove salt and glycerol, and the FFAs which are non-miscible with water and lighter than water, can be recovered by any method of separating two immiscible liquids.

In a preferred method of the invention the solids removed during the method are washed with additional water miscible organic solvent to recover any additional or residual PUFA present therein. The FFAs enriched in PUFA can be recovered from the water miscible organic solvent as described above, or preferably the FFA in the water miscible organic solvent can be cycled through the process again. The next batch would use the method as described but some of the solvent would be supplied from the water miscible organic solvent used to wash the solids of the previous batch.

Examples of the method of this embodiment of the invention are provided herein after; examples 1 , 3, 4, 5, 6 and 7a.

In a modified form of the first embodiment of the invention the inventor contemplates that the first step in the method could include the lipid substance being hydrolysed using - techniques such as high temperature hydrolysis with an oxide catalyst, by the use of enzymes such as lipases or other appropriate methods rather than by the direct use of alkali. The FFAs formed from this reaction could then be processed through the steps mentioned herein before (i.e. mixing with proportions of alkali, water and a water miscible solvent and the solids formed removed leaving a supernatant enriched in PUFAs).

The method of this embodiment of the invention may have advantages over conventional methods in its simplicity, its high recovery of PUFAs from triglycerides and the relatively high increase in PUFA content observed after application of the method.

Second Embodiment

^•The invention also provides a method, for increasing the PUFA content of FFAs by reacting FFAs with alkali, in a water miscible organic solvent, together with optional amounts of additional water, removing the solids, preferably by filtration, and recovering the enriched FFA's from the supernatant. Preferably the method is for further enrichment of FFAs enriched in PUFAs recovered from the method of the first embodiment of the invention.

Preferably, in this method the alkali (which is dissolved in a water miscible organic solvent and/or water) and the FFA (which optionally may be dissolved in a water miscible organic solvent), are added separately and slowly to a water miscible organic solvent. The mixture is stirred until all the components are added. Preferably the stirring is slow to allow slow cyrstalisation to occur. After complete addition and mixing of the- individual compositions the solids that form are separated and the FFAs enriched in PUFAs recovered from the supernatant.

In this method of further enrichment, the total quantity of water miscible organic solvent used in the reaction preferably contains approximately less than or equal to 15% water, more preferably less than or equal to. approximately 10% water.

Preferably the alkali (dissolved in a water miscible organic solvent) and the FFAs (optionally dissolved in a water miscible organic solvent) are added to a water miscible organic solvent at a temperature of approximately 15°C or less and the reaction is continued at that temperature.

The solids that are separated during this method may preferably be added to a lipid substance followed by processing through the method of the first embodiment of the invention. Alternatively, the solids could, be added to FFAs and recycled through the method of this second embodiment. This allows for the reprocessing of any PUFAs, particularly any omega-3s, that may have been trapped in the solid phase.

The second embodiment of the invention is exemplified in Examples 2, 6, and 7b herein after.

The method of this embodiment of the invention may have advantages over conventional methods in its simplicity, its high recovery of PUFAs and the relatively high increase in PUFA content observed after application of the method.

Third Embodiment In a third embodiment of the invention there is provided a process to produce glycerides, particularly monoacylglycerides (MAG), diacylglycerides (DAG), and triacylglycerides (TAG) mixtures of various compositions by reacting FFAs with glycerol at temperatures at or above approximately 14O⁰C, under a partial vacuum. Preferably, the FFAs are the FFAs enriched in PUFAs obtained from the methods of the first or second embodiments of the invention.

In one embodiment, the reaction occurs under a partial vacuum at a pressure of approximately 50 kPa or below. By way of example, the reaction may occur under a partial vacuum at a pressure of approximately 10kPa or below.

In one embodiment the reaction occurs at temperatures of approximately 160°C. or above. By way of example, the reaction occurs at a temperature of approximately; 18O⁰C or above.

In a preferred embodiment, the reaction occurs under a partial vacuum at a pressure of approximately 50 kPa or below and at temperatures of approximately 160⁰C or above.

In a preferred embodiment, steps are taken to minimise or eliminate oxygen from the reaction. Such steps can include applying the partial pressure under an inert gas such as nitrogen or argon.

Preferably, the reaction mixture is stirred rapidly (or otherwise treated) so that the glycerol is broken up into small droplets in the mixture. Preferably, any glycerol that evaporates is- condensed and added back into the reaction mixture. In one embodiment, ultrasound is used to break up the glycerol into small droplets in the mixture. Skilled persons may readily appreciate alternative means to achieve this result. .

Preferably the ratio of TAG/DAG/MAG formed is adjusted by altering the mol ratio of FFAs to glycerol, .with mol ratios of FFA/glycerol greater than approximately 2 favouring formation of MAG while ratios less than 2 favouring formation of TAG.

The third embodiment of the invention is exemplified in Examples 9 to 16 herein after.

The methods of this embodiment of the invention may have one or more of the following advantages: simple to perform; result in high yields without decomposition of the PUFAs and without lengthy reaction times.

Fourth Embodiment A method of a fourth embodiment of the invention involves producing lipid substances comprising glycerides (particularly TAGs) enriched in PUFAs, by first hydrolysing a starting lipid substance which includes glycerides comprising FFAs to produce FFA and glycerol, increasing the PUFA content of the FFA, and then reacting the FFAs enriched in PUFAs with glycerol to form glycerides, which are a mixture of mono-, di- or tri- acylglycerides.

Hydrolyses of the lipid may be by any method and enriching the PUFA content of the FFA may be by any method, but preferably it is by the methods of the first or second embodiments of the invention.

Reformation of the glycerides from the FFA containing the enriched PUFA content may be by any method, but preferably it is by the method described in the third embodiment of the invention.

The method of this embodiment of the invention may have advantages over current methods as those methods may not give a high recovery of the PUFA since the saturated and unsaturated FFAs are mostly on the same triglycerides as the PUFAs.

The fourth embodiment of the invention can be exemplified- by Examples 1 , 2, 3, 4, 5, 6, or 7 combined with any one of Examples 9, 10, 11 , 12, 13, 14, 15, and 16 herein after.

Fifth Embodiment

In a fifth embodiment the invention relates to novel omega-3 compositions formed from FFAs enriched in omega-3s, where the combined EPA and DHA content of the oil is at least approximately 40% and at least approximately 20% is ALA. Such compositions are exemplified in Example 16 herein after.

In one embodiment, the omega-3 composition has a combined EPA and DHA content of at least approximately 60%.

These compositions may find use as functional oils in processed foods and as health supplements. They could be coated, encapsulated or microencapsulated to hinder oxidation. Encapsulation could include gel encapsulation to form a dietary pill or microencapsulation to form a product that could be used in processed foods.

Preferably, the compositions are produced by a method of the invention. The FFAs enriched in PUFAs obtained from the methods of the invention may be further purified by known methods such as distillation, fractional cyrstallisation or formation of a urea complex. It will be appreciated they may be further purified using a method of the invention. Further, the mixtures of DAG, MAG and TAG obtained by a method of the invention may be further purified into their individual components by molecular distillation for example.

It should be appreciated that salts of the FFAs obtained by the methods of the invention may be formed as desired for certain applications. For example, for ruminant feed it is preferred that the calcium salt of the FFA be formed.

EXAMPLES

Measurement of PUFA content of FFA's GC and preparation of methyl ester

To the FFA or triglycerides (5 - 10 mg) was added toluene (0.25 ml) followed by 1% H₂SO₄ in MeOH (0.5ml). The mixture was flushed with Ar and subsequently heated at 50 0C for 1 h in a sealed tube. Salt solution (NaCI 5 %, 1 ml) was then added and the fatty acid methyl acids FAME extracted into pet ether (1 ml). The upper phase was transferred to a second tube and washed with sodium bicarbonate (2 %, 1 ml). This mixture was centrifuged and the upper FAME phase used for the determination of the individual FFA components by gas chromatography. The long chain omega-3 content is reported. Long chain means that the carbon backbone of the FFA must be > 18.

UV-spectroscopy method

The following method was developed for determining the total amount of PUFA in a sample of FFA. Approximately 50 μL of FFA is added to exactly 50 ml of ethanol in an A grade volumetric flask. A Hamilton pipette is used to transfer accurately 100 μL of the FFA/ethanol solution to a tube and exactly 4 ml of water is added followed by a drop of HCI (5 μL, 0.1 M). The adsorption at 200 nm is recorded soon afterwards. The blank for the determination is 100 μL of ethanol in exactly 4 ml of water and HCI (5 μL, 0.1 M). The ^' measurement is compared to a standard with a similar PUFA content where the PUFA content of the standard has been determined by GC as described above Solvents

Unless otherwise stated the ethanol used in the experiments was food-grade with an ethanol content of about 95 % and a water content of about 5 %.

Determination of FFA content of an oil (MAG, DAG or TAG or mixtures thereof) by pH titration

About 0.5g of the oil was added to ethanol (10ml) and water (10ml) with stirring. The solution was titrated with NaOH (0.1mol/l) and the pH monitored. The amount of FFA present in the mixture was determined from the end point of the titration.

Isolation of FFA of Codliver and Safflower oil

The oil (-100 g) was reacted with 10 % mol excess of NaOH (50 w% in water) for 1 h. The mixture was neutralised with HCI and then washed with water. The oily FFA layer was separated and retained.

Example 1

NaOH (30.3 g, 50w% in water) was mixed with ethanol (300 g) below 20 ⁰C. Hoki oil was added and the mixture was stirred rapidly to form an oil in ethanol emulsion, where the size of the emulsion droplets were about 1 - 2 mm across. After about 5 min the droplets had started to solidify and after about 10 min, most of the reaction mixture had solidified and the stirring was stopped. Every half hour for the next two hours the mixture was - stirred gently (to help prevent formation of a finer emulsion) and then left for 8 h. During this time the mixture was kept below 2O⁰C. Thin layer chromatography (TLC) showed no TAG left in the reaction mixture and only a very small amount of DAG. The solids produced were porous and readily filtered under vacuum. After filtering, the ethanol was evaporated from the supernatant and the oily residue acidified with HCI (22 ml, 37 %) in water (200 ml) and the yellow oily FFA was separated using a separating funnel, after having been washed several times with water. Analysis indicated that the omega-3 content of the FFA was about 65 %, and the overall recovery of the omega-3 FFA found in the original oil was about 78 %.

In contrast if the reaction was at a temperature of 3O⁰C then the colour of the oil was an ^' orange-brown showing that higher temperatures give a more coloured product.

Example 2

Solution a) was formed from NaOH (0.65 g) dissolved in ethanol (4.7g). Solution b) was formed from purified FFA (5 g) from example 1 dissolved in ethanol (2.5 g). Solution a) and b) were separately pumped, using a Biochem Valve micro-diaphragm pump over a period of 2 h, in about equal molar portions, to a gently (to help prevent formation of a finer emulsion) stirred solution of ethanol ( 5 g) and water (0.2 ml), at 10⁰C. Solids started to form after about 10min of reaction. The solids were filtered and the supernatant was acidified as per Example 1, to recover the FFA. Omega-3 content of the purified FFA was determined by GC to be 76 %, with a recovery of the omega-3 content of about 65 %.

Example 3

Solids (30 g) were filtered from a reaction similar to that described in Example 2, that is NaOH (9.5g, 2% excess) in ethanol (70ml) was added dropwise to FFA (65.2g) in ethanol (50ml) at room temperature. The solids were recovered and mixed with hot ethanol (70 ml) at 60°C and the mixture was cooled to 2⁰C and left for 2 h. Filtering the solids yielded another 5.1 g of purified FFA from the supernatant having a PUFA content of about 75 %, as estimated by UV spectroscopy.

Example 4

Hoki liver oil (15Og) was stirred with ethanol (30Og) for 10min. NaOH (43.95g, 50w% in water) was added and the mixture stirred rapidly for 5 min, to give a clear solution. The temperature of the solution was kept below about 20⁰C. After about 10 min. curds started to form. The mixture was left another 5h to complete the reaction. The solids that formed were filtered on a 55um nylon mesh in a basket centrifuge. Yield of wet solids was 215g having an ethanol content of 36%. The supernatant was neutralised with HCI (22 ml, 37%) and the oily layer separated after washing several times with water. Based on UV- spectroscopy results and the yield of the FFA it was estimated that the recovery of PUFA was at least 80% and that the PUFA content was at least 63%.

Example 5

Hoki liver Oil (100 g) was mixed with ethanol (30Og) for 10 min. 3Og NaOH (50 w% in water) was added and the mixture left for 4 h to react. The mixture was cooled at -16°C for 16h. This solids were separated on a basket centrifuge. The yield of solid was 127.9 g having a liquid content is 39.8 %. The yield of supernatant was 273 g. Analysis by GC gave the long chain omega-3 content as 46%.

Example 6 An enriched FFA (11.5 g) fraction isolated by the method described in example 4 was mixed with ethanol (12 g) and NaOH (3.35 g, 50w% in water) and the ethanol evaporated. An additional aliquot of ethanol (20 g) was added and the mixture heated to 7O⁰C. The solution was kept at -15 ⁰C overnight. The solids were filtered on 55 um mesh. The yield of wet solids was 12g having a liquid content of 43%. The supernatant was neutralised with HCI and the oily layer was separated' from the water layer and washed several times with water. The yeild of enriched FFA was 3.Og and the amount of long chain omega-3 ^' was determined by GC to be 78 %.

Example 7 a) Hoki oil (100 g) was mixed with a NaOH solution (30.3 g, 50 w% in water) in ethanol (300 g) at 20⁰C for 15min. The oily droplets that formed solidified after about 5 min. After 15 min the temperature of the mixture was raised to 30⁰C to increase the reaction rate and the reaction continued for another 5 h. After this time a TLC showed that only a small amount of TAG, and DAG was present in the reaction mixture. The reaction mixture was subsequently cooled to ambient temperature over 2h. The solids were filtered in a basket centrifuge and the solids and supernatant retained. Yield of solid was 134.7Og having a liquid content of 41 %. The FFA were isolated from the supernatant by neutralising with HCI and washing with water to yield 22.37 g of FFA with an omega-3 content of about 60 % as determined by UV spectroscopy.

b) NaOH (3.1g) was dissolved in ethanol (25g) and added drop-wise with gentle stirring to an ethanol (25g) solution of the FFA that were isolated in a). After addition of all the

NaOH the mixture was filtered, and the solids washed with an additional aliquot of ethanol (10 ml). The FFA were recovered from the supernatant by neutralising with HCI and washing with water to yield 7.9g of FFA having a PUFA content of about 75% as determined by UV spectroscopy.

c) Solids from a) were soaked in ethanol (10Og) and the liquid was recovered on a basket centrifuge. Sodium hydroxide pellets (14.25g) were dissolved in ethanol (200ml) and water (1Og)^' and the liquid recovered from the basket centrifuge was added. The solids isolated in b) were also added to the reaction mixture. To the reaction mixture was added Hoki oil (100 g) and then the reaction was continued as described for the method in a) above. TLC indicates that little TAG remained after reaction for 16h at ambient temperatures. Solids were removed on a basket centrifuge. The yield of solids was ^•134.1g having a liquid content of 40%. The FFA were isolated from the supernatant by neutralising with HCI and wasing with water. The yield of FFA was 49.73g having an omega-3 content of about 59%. This was almost twice the yield of example a) due to recycling of the omega-3 FFA that were not recovered in example a) and example b). Example 8

■Purification of mixture of MAG, DAG and TAG. A mixture of MAG, DAG and TAG was formed by stirring and heating at 61⁰C a mixture of linseed oil (51 g), a lipase (Lipozyme TLJM from Novo Nordisk) (2g) and adding drop-wise glycerol (1 ml) over a period of 1 h. The reaction mixture was then reacted for a further 15h at 61⁰C. ¹³C NMR analysis of the reaction mixture indicated that the ratio of MAG:DAG:TAG:glycerol was about 25:27:33:35. The reaction mixture was mixed with water to remove glycerol and the top layer separated. The top layer was then mixed with ethanokwater in a ratio of approximately 4:1 by volume (20ml) and set aside to separate. Two layers formed. The top MAG-enriched layer was recovered using a separating funnel. The ratio of MAG/DAG was shown by NMR to be about 9/1 with only small amounts of TAG detected.

Example 9

Safflower FFA were isolated from safflower oil by hydrolysis with alkaline sodium hydroxide, followed by neutralisation with HCI, washing the free FFA with water, before separating the FFA from the water phase. The FFAs (0.826g) were reacted with 1/3 mol ratio of glycerol (91 mg) at 210°C, with rapid stirring and under a partial vacuum of about

1OkPa. After 0.5h mostly MAG had formed but after further reaction for 2.5 h mostly TAG had formed. After 3h reaction most of the product appeared to be TAG with some DAG and only a small amount of MAG detected. A titration showed that 10% FFA remained unreacted.

Example 10

Safflower FFA (0.5 g), isolated by the method described in Example¹9, and 1/3 glycerol (250 mg) were heated at 17O⁰C, under a partial vacuum of about 10 kPa. After a 2h reaction TLC showed that mostly MAG had formed.

Example 11

High vacuum was applied to a mixture of safflower FFA (1g), isolated by the method described in Example 9, and glycerol (111mg) in a round bottom flask. The flask was sealed and heated for 1h at 22O⁰C, which evaporates water from the reaction mixture. Vacuum was reapplied, the system sealed and the reaction continued at 220⁰C for a further 1h. Very little water was released after the second hour and TLC shows mostly TAG and very little DAG and no MAG is formed. A pH titration shows that some FFA (10%) remains in the reaction mixture.

Example 12 Codliver oil FFA isolated from codliver oil by hydrolysis with alkaline sodium hydroxide, followed by neutralisation with HCI, wasing the free FFA with water and separating the FFA from the water phase. The FFAs were placed in a round bottom flask and distilled under high vacuum (0.1 Torr) on a silicon oil bath. A first fraction containing mainly C16 FFA's was collected at 142°C. A second fraction containing mostly C16 FFA's was collected at 15O⁰C and a third fraction containing mostly EPA and DHA was collected at 158 to 164°C. The third fraction was retained and used in Examples 13 to 17.

Example 13

The EPA and DHA enriched fraction from example 12 (1g) and glycerol (92 mg) were heated after application of a vacuum from a water aspirator. After 1 h reaction at 220⁰C TLC showed that mostly TAG had formed with some DAG and very little MAG. The vacuum was reapplied for a few seconds and the reaction continued at 220⁰C for another 1h. Very little water was released after this time and TLC shows mostly TAG, very little DAG and no MAG. A pH titration shows that some FFA (5%) remain in the reaction mixture.

Example 14

The EPA and DHA enriched fraction from example 12 (1g) and glycerol (50mg) were heated at 16O⁰C for 6 h, under vacuum supplied by a water aspirator. TLC showed TAG, DAG and MAG had formed but there was also a large proportion of unreacted FFA in the reaction mixture.

Example 15

The EPA and DHA enriched fraction from example 12 (1g) and glycerol (85mg) heated under argon at 220°C for 2h. The water given off by the reaction was collected away from the reaction mixture. A pH titration of the final mixture showed that only 10% of the FFA remained after this time.

Example 16

The EPA and DHA enriched fraction from example 12 (25g) and distilled linseed FFA (0.25g) were reacted with glycerol (52 mg) under partial vacuum at a temperature of 190°C for 4h. The reaction was shown by TLC to have formed about 70% TAG, with the remainder mostly unreacted FFA.

Example 17 A calcium salt of enriched FFA was formed. Ca(OH)₂ (25 mg) is dispersed in water (5 ml) and enriched codliver FFA (200 mg) added. White stringy precipitate forms which was removed by filtration.

The invention has been described herein, with reference to certain preferred embodiments, in order to enable the reader to practice the invention without undue experimentation. However, a person having ordinary skill in the art will readily recognise that many of the components and parameters may be varied or modified to- a certain extent or substituted for known equivalents without departing from the scope of the invention. It should be appreciated that such modifications and equivalents are herein incorporated as if individually set forth.

Titles, headings, or the like are provided to enhance the reader's comprehension of this document, and should not be read as limiting the scope of the invention.

The entire disclosures of all applications, patents and publications, cited above and below, if any, are hereby incorporated by reference.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in any country in the world.

Throughout this specification and any claims which follow, unless the context requires otherwise, the words "comprise", "comprising" and the like, are to be construed in an inclusive sense as opposed to an exclusive sense, that is to say, in the sense of "including, but not limited to".

Claims

CLAIMS:

1. A method for enriching lipid substances in PUFAs the method comprising at least the steps: a. Reacting a lipid substance with alkali in the presence of water and a water

. miscible organic solvent to create a liquid containing solids; b. Removing the solids from the liquid; c. Recovering FFAs enriched in PUFAs from the liquid.

2. A method as claimed in claim 1 wherein the lipid substance is fat or oil derived from vegetables or animals.

3. A method as claimed in claim 2 wherein the lipid substance is fat or oil from fish and the method is for enriching the lipid substance in omega-3 "fatty acids.

4. A method as claimed in any one of claims 1 to 3 wherein the alkali is sodium hydroxide.

5. A method as claimed in any one of claims 1 to 4 wherein the water miscible organic solvent is ethanol.

6. A method as claimed in any one of claims 1 to 5 wherein the ratio of water to water miscible organic solvent is between approximately 2.0 and approximately 0.05 (w/w).

7. A method as claimed in any one of claims 1 to 6 wherein the solids are removed by filtration.

8. A method as claimed in any one of claims 1 to 7 wherein step a. includes: i. Mixing an aqueous solution of the alkali with the water miscible organic solvent to create an alkali and water miscible organic solvent mix; ii. Adding the lipid substance to the alkali and water miscible organic solvent mix; iii. Stirring the lipid substance and alkali and water miscible organic solvent mix for an initial period so that an emulsion forms containing small droplets of the lipid substance in the solvent mix.

9. A method as claimed in claim 8 wherein in i. an alkali is mixed directly with the water and water miscible organic solvent.

10. A method as claimed in claim 8 wherein in i. an aqueous solution of the alkali is mixed with water and a water miscible organic solvent.

11. A method as claimed in any one of claims 8 to 10 wherein in iii. stirring for an initial period occurs for approximately 10 minutes.

12. A method as claimed in claim 11 wherein the mix is further stirred for approximately 1 minute approximately every half an hour for approximately 2 hours.

13. A method as claimed in any one of claims 1 to 12 wherein the reaction of step a. is performed for a period of greater than approximately 4 hours.

14. A method as claimed in any one of claims 1 to 13 wherein the method is conducted at a temperature of approximately 2O⁰C or less.

15. A method as claimed in any one of claims 1 to 14 wherein after step a) the temperature of the liquid containing solids is reduced, preferably to approximately 5°C or less.

16. A method as claimed in any one of claims 1 to 15 wherein step c. includes: i. neutralising the liquid with an acid, preferably HCI, H2SO4, or H3PO4; and, ii. recovering the water immiscible fraction containing FFAs.

17. A method as claimed in claim 16 wherein step c. includes: i. neutralising the liquid with an acid, preferably HCI, H2SO4, or H3PO4; ii. removing the water miscible organic solvent from the liquid; iii. washing the FFAs with water to form two immiscible fractions; and, iv. recovering the water immiscible fraction containing FFAs.

18. A method as claimed in claim 16 or 17 wherein the water miscible organic solvent is removed or recovered from the liquid by evaporation, condensation and/or ultrafiltration.

19. A method as claimed in any one of claims 16 to 18 wherein the solids removed during the method are washed with additional water miscible organic solvent to recover any additional or residual PUFA.

20. A method as claimed in any one of claims 1 to 19 wherein the method further includes a first step of hydrolysing the lipid substance by one or more of high temperature hydrolysis with an oxide catalyst and use of an enzyme such as a lipase.

21. A method for enrichment of FFAs in PUFAs the method comprising at least the steps: a. neutralising FFAs with alkali in a water miscible organic solvent to create a liquid containing solids; b. Removing the solids from the liquid; c. Recovering FFAs enriched in PUFAs from the liquid.

22. A method as claimed in claim 21 wherein the alkali is dissolved in a water miscible organic solvent and/or water.

23. A method as claimed in claim 21 or 22 wherein the FFAs are dissolved in a water miscible organic solvent.

24. A method as claimed in any one of claims 21 to 23 wherein step a. involves adding the alkali and the FFAs separately and slowly to a water miscible organic solvent . and gently stirring the mixture.

25. A method as claimed in any one of claims 21 to 24 wherein the solids are removed following complete addition and mixing of the FFAs, alkali and water miscible organic solvent.

26. A method as claimed in any one of claims 21 to 25 wherein the total quantity of water miscible organic solvent used in the method contains approximately 15% water or less.

27. A method as claimed in any one of claims 21 to 26 wherein the method is conducted at a temperature such that the mix is at or below approximately 15°C.

28. A method for producing glycerides from FFAs the method comprising at least reacting FFAs with glycerol at temperatures at or above approximately 140⁰C₁ under a partial vacuum.

29. A method as claimed in claim 28 wherein the reaction occurs under a partial vacuum at a pressure of approximately 5OkPa or less.

30. A method as claimed in claims 28 or 29 wherein the reaction occurs at a temperature of approximately 160⁰C or above.

31. A method as claimed any one of claims 28 to 30 wherein steps are taken to minimise or eliminate oxygen from the reaction.

32. A method as claimed in any one of claims 28 to 31 wherein the FFAs and glycerol are rapidly stirred to break up the glycerol into small droplets, with any glycerol that evaporates being condensed and added back to the reaction.

33. A method as claimed in any one of claims 28 to 32 wherein the FFAs are PUFAs, more preferably omega-3 fatty acids.

34. A method as claimed in any one of claims 28 to 33 wherein the glycerides are mono- (MAG), di- (DAG) and/or tri-acylglycerides (TAG).

35. A method as claimed in any one of claims 28 to 34 wherein the method includes a means to control the ratio of TAG/DAG/MAG formed.

36. A method as claimed in claim 35 wherein the ratio of TAG/DAG/MAG is adjusted by altering the mol ratio of FFAs to glycerol where mol ratios of FFA/glycerol greater than approximately 2 favour formation of MAG and mol ratios of FFA/glycerpl less than approximately 2 favour formation of TAG.

37. A method for producing lipid substances comprising glycerides enriched in PUFAs the method comprising at least the steps: a. hydrolysing a starting lipid substance including a level of glycerides comprising fatty acids to produce FFAs and glycerol; b. increasing the PUFA content of the FFAs to form FFAs enriched in PUFAs; c. reacting the FFAs enriched in PUFAs with glycerol to form glycerides.

38. A method as claimed in claim 37 wherein steps a. and b. employ a method as claimed in any one of claims 1 to 20.

39. A method as claimed in claim 37 or 38 wherein step c. employs a method as claimed in any one of claims 28 to 36.

40. A lipid substance produced by a method of any one of claims 1 to 39.

41. Omega-3 compositions comprising at least approximately 40% of the long chain FFA, that is EPA and DHA, and at least approximately 20% ALA.