WO2015024055A1

WO2015024055A1 - Separation of omega-3 fatty acids

Info

Publication number: WO2015024055A1
Application number: PCT/AU2014/000825
Authority: WO
Inventors: Colin James Barrow; Taiwo Olusesan AKANBI
Original assignee: Deakin University
Priority date: 2013-08-20
Filing date: 2014-08-20
Publication date: 2015-02-26

Abstract

The present disclosure provides for the use of a pancreatic lipase for separating n-3 docosapentaenoic acid (DPA) from other omega-3 fatty acids present in an omega-3 containing oil and to uses of n-3 DPA enriched by the method.

Description

SEPARATION OF OMEGA-3 FATTY ACIDS

Related Applications and Incorporation by Reference

The present application claims priority from Australian provisional Patent Application No. 2013903147 entitled "Method for separating omega-3 fatty acids" filed 20 August 2013, the entire contents of which are herein incorporated by reference.

All documents cited or referenced herein, and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference in their entirety.

Field of the Invention

Background of the Invention

Omega-3 fatty acids are particularly useful in pharmaceutical and/or nutritional supplement products. Omega-3 fatty acids may regulate plasma lipid levels, cardiovascular and immune functions, insulin action, neuronal development and visual function. Omega-3 fatty acids have also been shown to have beneficial effects on the risk factors associated with cardiovascular disease, including hypertension and hypertriglyceridemia, and on the coagulation factor VII phospholipid complex activity. Omega-3 fatty acids may also lower serum triglycerides, increase serum HDL cholesterol, lower systolic and diastolic blood pressure and/or pulse rate. Furthermore, omega-3 fatty acids are generally well tolerated when consumed by subjects.

However, humans are unable to de novo synthesise omega-3 fatty acids and poorly convert short-chain omega-3 fatty acids into c/^'s-5,8,1 1 ,14,1 7-eicosapentaenoic acid (EPA), and c/^'s-4,7,10,13,16,19-docosahexaenoic acid (DHA). Therefore consumption of omega-3 rich foods such as fatty fish is important as a source of these compounds. The growing nutritional supplement, food and pharmaceutical markets have a high demand for more concentrated omega-3 oils than those present in fish and therefore methods for the production of high quality omega-3 concentrates are industrially important.

Because of the complex fatty acid compositions of fish oils, it is difficult to prepare highly purified compositions of particular omega-3 fatty acids using a single concentration technique. Normally, a combination of purification and/or concentration techniques are required, most often techniques that combine separation according to unsaturation (e.g. urea fractionation) with separation according to carbon chain length (e.g. molecular/short path distillation and/or supercritical fluid extraction or chromatographic separation). Conventional techniques have the disadvantage of giving concentrates with low yields of omega-3 fatty acids compared to the amounts in the starting oil and environmentally unfriendly fractional distillation and urea complexation techniques can result in partial oxidation and polymerisation of these oxidatively unstable omega-3 fats (Shahidi & Wanasundara (1998) Adv Exp Med Biol 434:135- 60).

Lipases can have positional or fatty acid specificities and have been investigated for ability to cleave some fatty acids from the glycerol backbone. They carry out hydrolysis or synthetic reactions on the ester bonds of triglycerides in living beings. Lipases offer particular advantages in that operating conditions are milder. Moreover, it is not necessary to eliminate impurities of any sort (whether acid or alkaline), simplifying phase separation.

Pancreatic lipase, also known as triacylglycerol acylhydrolase (EC 3.1 .1 .3), is an important enzyme for the digestion of dietary fats (Arroyo R et al (1996) Lipids 31 (1 1 ):1 133- 1 139). It cleaves dietary triacylglycerol (TAG) derivatives into monoacylglycerols (MAGs) and free fatty acids (FFAs). The amino acid sequence of pancreatic lipase has been reported to be conserved among animals such as human, canine, porcine and rat (Kirchgessner TG et al (1987) Journal of Biological Chemistry 262(18):8463-8466).

Since positional selective lipases have been reported to be applicable in the production of omega-3 fatty acids such as DHA, it is possible that the selectivity of pancreatic lipase may be useful in studies aimed at concentrating omega-3 fatty acids. Several studies have also shown that pancreatic lipases can be successfully immobilized for repeated reuse, an important criteria for cost effective application of these enzymes. However, investigation into the relative selectivity of pancreatic lipase to a broad range of polyunsaturated fatty acids from diverse sources is lacking.

Summary of the invention

In work leading up the present disclosure, the inventors examined the hydrolysis activity of a pancreatic lipase on omega-3 polyunsaturated fatty acids in seal, anchovy and canola oil which are oil sources rich in omega-3 fatty acids. Determining the relative selectivity towards different omega-3 fatty acids is complicated by the differing positional distribution of each on the acyl glycerol backbone for fish and seal oils.

However, the present inventors found that a pancreatic porcine lipase was able to discriminate against polyunsaturated fatty acids regardless of chain length, number of double bonds and the position of the glycerol backbone. In particular, omega-3 fatty acids a-linolenic acid (ALA) and docosapentaenoic acid (DPA) were found to be better substrates for the lipase compared to stearidonic acid (STA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Accordingly, the pancreatic lipase was postulated to be useful for facilitating the separation of omega-3 polyunsaturated fatty acids, and in particular, facilitating separation of omega-3 DPA (hereinafter n-3 DPA) from other omega-3 fatty acids such as EPA and DHA (hereinafter n-3 EPA and n-3 DHA respectively).

The ability to separate n-3 DPA form other omega-3 fatty acids has advantages, particularly where it is desirable to obtain an enriched source of n-3 DPA. For example, the separated n-3 DPA is thus an enriched source of n-3 DPA which can be utilised in pharmaceutical and non-pharmaceutical applications. For example, the separated n-3 DPA can be utilised in dietary supplements in subjects, particularly for vegetarians or those having low red meat diets, n-3 DPA has been found to have particular advantages that distinguish this fatty acid over other omega-3 fatty acids such as n-3 EPA and n-3 DHA, such as, for example in endothelial cell migration ability which may be important in wound healing processes, as well as inhibition of platelet aggregation, and enhanced cardiovascular benefits.

In one embodiment, the present disclosure provides for the use of a pancreatic lipase for separating n-3 docosapentaenoic acid (DPA) from other omega-3 fatty acids present in an omega-3 containing oil. Preferably, the omega-3 containing oil is one which comprises n-3 DPA.

In a particular example, the present disclosure provides for the use of a pancreatic lipase for separating n-3 docosapentaenoic acid (DPA) from n-3 eicosapentaenoic acid (EPA) and n-3 docosahexaenoic acid (DHA). In another example, the present disclosure also provides for the use of a pancreatic lipase for separating n-2 DPA from n-3 EPA, n-3 DHA and n-3 stearidonic acid (STA).

In one example, the separated n-3 DPA is in free fatty acid form.

In another embodiment, the present disclosure provides a method for separating n-3 DPA from other omega-3 fatty acids present in an omega-3 containing oil, the method comprising:

(i) combining the oil with a lipase that preferentially hydrolyses n-3 DPA glycerides; (ii) allowing hydrolysis by the lipase for a time and temperature sufficient to hydrolyse at least a portion of the glycerides; and

(iii) extracting the n-3 DPA as free fatty acids in the aqueous fraction formed following hydrolysis.

In one example, the n-3 DPA glycerides are triglycerides.

By "preferentially hydrolyses" it is meant that the lipase hydrolyses an ester linkage of an omega-3 fatty acid that is sensitive to the activity of the lipase. The term is also understood to refer to omega-3 fatty acid glycerides (e.g. triglycerides) that are hydrolysed more rapidly by the lipase compared to other omega-3 fatty acid substrates. In one example, it is meant that the lipase hydrolyses n-3 DPA ester linkages more rapidly compared with hydrolysis of ester linkages of n-3 EPA and/or n-3 DHA.

The omega-3 containing oil according to the present disclosure may be native oil or a synthetic oil. The native oil may be derived from animal or non-animal oils. In some examples the oil is derived from at least one oil chosen from marine oil, single cell oil, algal oil, plant- based oil, microbial oil, mammalian oil and combinations thereof. In one example, the mammalian oil is seal oil. In another example, the marine oil is a fish oil, sardine oil, mackerel oil, herring oil, cod oil, tuna oil, saury oil, anchovy oil, krill oil, or other lipid composition derived from fish. In one example, the oil is not whale oil. Plant-based oils include for example, flaxseed oil, canola oil, mustard seed oil, and soybean oil. Single cell/microbial oils include for example, products by Martek, Nutrinov and Nagase & Co. Single cell oils are often defined as oils derived from microbial cells and which are destined for human consumption (see e.g. Wynn and Ratledge, "Microbial oils: production, processing and markets for specialty long chain omega-3 polyunsaturated fatty acids" pg 43-76 in Breivik (Ed.) Long-Chain Omega-3 Specialty Oils, the Oily Press, P.J. Barnes and Associates, Bridgewater UK, 2007). The oil according to the present disclosure may be chosen from among one or more glyceride esters. The term "glyceride" as used herein refers to mono-, di- or triesters; i.e. one or more monoglycerides, diglycerides, triglyceride or mixtures of these.

In a further example, the oil is rich in omega-3 polyunsaturated fatty acids. In another example the oil comprises n-3 DPA glycerides and/or phospholipids. In a further example the oil comprises n-3 DPA triglycerides.

In another example, the oil mixture is selected from seal oil, anchovy oil, AHIFLOWER™ oil, fish oil or algal oil.

In another example, the oil is one in which n-3 DPA is enriched at sn-2.

The lipase according to the present disclosure is one which preferentially hydrolyses n- 3 DPA glycerides. In particular, the lipase preferentially hydrolyses n-3 DPA triglycerides or diglycerides. In one example the lipase is a pancreatic lipase. In a further example it is a porcine pancreatic lipase. Any type of porcine pancreatic lipase may be employed in the method of present disclosure, for instance a lipase selected from a commercial industrial porcine pancreatic lipase (PPL), an enzyme preparation containing it, such as for example Sigma's Pancreatin, or a pancreatic lipase extracted directly from pig pancreas. In one example, the lipase comprises the sequence shown in Figure 1 , or a sequence at least 80%, 85%, 87%, 90%, 92%, 95%, 97%, 98%, or 99% identical thereto. It will be appreciated by persons skilled in the art that activities of different lipases can vary and thus need to be standardised according to methods known in the art and as described for example in Akanbi T et al (2013) Food Chemistry 138:615-620. The degree of hydrolysis should be obtained as quickly as possible in order to minimise acyl migration and inactivation of the lipase enzyme. Typically, a concentration of lipase is selected which results in a degree of hydrolysis of n-3 DPA glycerides of between about 1 5 to 80%, about 25 to 75%, about 30 to 70%, about 45 to 65%, about 50 to 60%, about 50 to 55% or about 50 to 52%. In another example, the degree of hydrolysis is about 50 + 5%. It will be appreciated that the degree of hydrolysis needs to be balanced against the concentration of lipase used since excess lipase load has been reported to be a major cause of undesirable acyl migration of fatty acids during hydrolysis (Fernandez-Lafuente (201 0) Journal of Molecular Catalysis B: Enzymatic 62(3- 4):197-212). Importance of the proportionate use of lipases for fish oil hydrolysis has been previously reported (Yan L et al (2010) Applied Biochemistry and Biotechnology 162(3):757- 765. Accordingly, in some examples, the lipase concentration may be 100, 200, 400, 600, 800, 1000, 1200, 1500, 3000, 5000, 7000, 8000, 9000 or 10000 U/g of oil.

In one example, the hydrolysis is carried out at a pH which does not cause substantial denaturation of lipase secondary structure, in particular a-helical content. In one example, the hydrolysis is carried out at neutral or alkaline conditions. In another example, the hydrolysis is carried out at a pH between about 4.5 and 12, a pH between about 5 and 8, a pH between about 6 and 7.5, a pH between about 6.2 and 7.2, or a pH at or about 7. In examples where pancreatic lipase is used it is preferable that the pH is between about 7 and about 8.

The pH at which the hydrolysis reaction is carried out may be obtained through the addition of a base. Suitable bases include the following: NaOH, KOH, NH₄OH, Na₂C0₃, NaHC0₃, K₂C0₃, KHC0₃ or a combination of these.

The lipase according to the present disclosure may be free or immobilised. The lipase may be immobilised by attachment to an organic or inorganic support. In one example, the lipase is adsorbed on an organic or inorganic support. In one example, the lipase is used in immobilized form by chemical attachment of the enzyme to an inorganic solid that acts as a support, allowing for its easy recovery at the end of the process and its subsequent reuse. In another example, the lipase is present in free or attached form in a proportion of 1 /1000 with respect to the weight of the oil used, making it possible to convert 1 mole of oil in 24 hours per gram of lipase. Covalent immobilisation of pig pancreatic lipase has been previously reported Bautista FM et al (1999) Journal of Chemical Technology and Biotechnology 72(3) :249-254.

According to the method of present disclosure, the hydrolysis reaction is carried out for a period of time sufficient to hydrolyse at least a portion of the n-3 DPA glycerides. In one example, the period of time is such that about 30% to 80% of the glycerides present in the oil mixture are hydrolysed. In another example, the period of time is such that about 35 to 70%, about 40-65%, about 45-60%, about 47-55%, or about 50-55% of the n-3 DPA glycerides are hydrolysed.

Suitable temperatures at which the hydrolysis reaction can occur, include, but are not limited to from about ambient (e.g. about 20^°C) to about ^"Ι ΟΟΌ, from about 35 °C to about 80°C, or from about 37^°C to about 50^°C, or from about 37^°C to about 45^°C. In certain examples, the reaction time can be adjusted by varying the temperature. In examples where pancreatic lipase is used, it is preferred that the temperature is between about 37^°C and about 40^°C.

In another example where the lipase is not immobilised, the lipase may be recovered by methods known in the art including centrifugation, or chromatography.

The method of the present disclosure may also incorporate the additional use of other lipases. For example, one or more additional lipases may be included which do not hydrolyse n-3 DPA glycerides. Such additional lipase may be employed prior to the use of PPL to facilitate removal of saturated and monounsaturated fatty acids present in the oil mixture. Examples of such lipases include Rhizomucor miehei lipase (RmL), Thermomyces lanuginosus lipase (TL 100L), and Candida Antarctic lipase (Cal B).

In another example, the one or more additional lipase(s) may be employed concurrently with the PPL. In certain examples, hydrolysed products prior to the addition of PPL may be removed according to known methods.

Accordingly, in another example, the method of the present disclosure further comprises combining the oil with a lipase that does not preferentially hydrolyse n-3 DPA glycerides contained in the oil.

The present disclosure also provides a method for enriching n-3 DPA from other omega-3 glycerides present in an omega-3 containing oil or an oil comprising omega-3 and omega-6 glycerides, the method comprising:

(i) optionally separating the omega-3 and omega-6 glycerides in the oil;

(ii) optionally combining the oil with a lipase that does not preferentially hydrolyse n-3 DPA glycerides contained in the oil;

(iii) combining the oil with a lipase that preferentially hydrolyses n-3 DPA glycerides; (iv) allowing hydrolysis by the lipase according to (iii) for a time and temperature sufficient to hydrolyse at least a portion of the glycerides;

(v) extracting the n-3 DPA as free fatty acids in the aqueous fraction formed following hydrolysis; and

(vi) optionally concentrating the n-3 DPA fatty acids.

In certain examples, the hydrolysis reaction may be carried out either in an agitated- tank reactor or in a flow reactor. In another example, the method of the present disclosure is not used in biodiesel production.

In yet another example, the method may comprise esterification of free fatty acids with an alcohol(s), in particular n- and iso-alcohols. The esterification may be carried out simultaneously with the hydrolysis reaction.

As a result of the hydrolysis reaction n-3 DPA is primarily converted to its free fatty acid form whilst n-3 EPA and n-3 DHA remain on the glyceride backbone. Without wishing to be bound by theory, following the hydrolysis reaction a phase separation occurs where the n-3 EPA and n-3 DHA which are resistant to hydrolysis by the lipase, are retained in the organic fraction whilst hydrolysed n-3 DPA as free fatty acids form is in the aqueous fraction. These phases can be separated for example by solvent extraction according to methods known in the art based on pH adjustment.

The degree of hydrolysis can be determined by methods known to those skilled in the art. In one example, the degree of hydrolysis can be determined using capillary chromatography, for example iatroscan.

In one example, free fatty acids in the aqueous fraction may be extracted with a solvent such as hexane after adjusting the pH to acidic conditions. Such methods have previously been described (Shimada et al (1998) Journal of the American Oil Chemists' Society 75:1581 - 1586).

Short chain fatty acids (typically comprising aliphatic tails of two to six carbons) can be removed by methods known to persons skilled in the art. For example, short chain fatty acids can be enzymatically removed as free fatty acids prior to DPA hydrolysis. Alternatively, the short chain free fatty acids and n-3 DPA as free fatty acid can be removed after the hydrolysis reaction and then separated using industrial methods such as for example, fractional distillation or urea complexation.

In one example, the method further comprises extracting and concentrating the n-3 EPA and n-3 DHA containing fraction. Such methods are known in the art. For example, distillation methods such as fractional distillation or thin film distillation, urea complexation or chromatographic methods can be used. Further, n-3 EPA can be further separated from n-3 DHA using known methods for example chromatographic methods, distillation methods, enzymatic methods, chemical methods, low-temperature crystallisation and supercritical fluid extraction, or by methods described in for example US 6846942, US 4792418, WO 2004043894. In another example, n-3 EPA and n-3 DHA may be separated by use of the Thermomyces lanuginosus lipase as described in Akanbi T et al (2013) Food Chemistry 138:615-620. n-3 DPA free fatty acids may be further separated from other omega-3 free fatty acids by methods known in the art. For example, urea complexation may be used to separate n-3 DPA free fatty acids from the other free fatty acids in the free fatty acid fraction as described in for example Hidajat K et al ( 995; J Chromatogr A 702:21 5; Gamez-Meza N et al (2003) Food Research International 36:721 -727). In another example, chromatographic separation may be used e.g. gas chromatography.

Accordingly, the method further comprises separating n-3 DPA free fatty acids from other omega-3 free fatty acids contained in the aqueous free fatty acid fraction.

In another embodiment, the present disclosure provides an oil composition enriched in n-3 DPA free fatty acids obtained according to a method of the present disclosure.

The n-3 DPA free fatty acids may be further modified as desired for intended use as a pharmaceutical, food supplement, or cosmetic. In one example, the n-3 DPA may be retained in free fatty acid form. In one example, the n-3 DPA free fatty acids may be converted to a methyl ester. In another example, n-3 DPA free fatty acids may be derivatised for example by chemical modification, conjugates, salts thereof or mixtures of any of the foregoing. Such derivatives include alkyl ester, ethyl ester, methyl ester, propyl ester, or butyl ester. In another example, the n-3 DPA free fatty acids may be converted to acylglycerol forms. In another example, the n-3 DPA free fatty acids can be prepared in the form of ethyl-DPA, lithium-DPA, mono-, di-, or triglyceride DPA or any other ester or salt of DPA. n-3 DPA may also be in the form of a 2-substituted derivative or other derivative which slows down its rate of oxidation but does not otherwise change its biological action to any substantial degree.

In one example, the oil composition enriched in n-3 DPA free fatty acids comprises at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8% by weight.

The present disclosure also provides a pharmaceutical composition comprising an oil composition enriched in n-3 DPA free fatty acids and/or derivatised n-3 DPA free fatty acids as described herein, together with a pharmaceutically acceptable carrier or excipient.

The enriched n-3 DPA oil compositions may be utilised in a number of pharmaceutical and non-pharmaceutical applications as described herein. In one example, the enriched n-3 DPA compositions may be used in a weight loss supplement for treating or preventing obesity in a subject. The term "obesity" as used herein is understood to mean a subject whose body mass index (BMI) is above the recommended range (i.e. above 25).

The present disclosure also provides a food additive comprising an enriched n-3 DPA oil composition or a pharmaceutical composition as described herein. In one example, the food additive is added to a solid food. In one example, the food additive is added to liquid food. In one example, the food additive is added to animal feed. In one example, the enriched n-3 DPA oil composition is used as a food additive in a food selected from a functional food, nutrient- supplementing food, formula suitable for feeding infants or premature infants, baby foods, foods for expectant or nursing mothers, and geriatric foods. In one example, the enriched n-3 DPA composition is combined with carnitine and/or fibrates.

The present disclosure also provides an enriched n-3 DPA oil composition or pharmaceutical composition as described herein for use in treating or preventing hypertriglyceridemia or a disorder associated with hypertriglyceridemia in a subject.

The present disclosure also provides a method of treating or preventing a disorder selected from hypertriglyceridemia, a cardiovascular-related disease or disorder, obesity, or platelet aggregation disorder in a subject, comprising administering to a subject in need thereof an oil composition, a pharmaceutical composition, or a food additive according to the present disclosure.

In one example, the enriched n-3 DPA oil composition, a pharmaceutical composition is administered to a subject from one to about four times per day.

The present disclosure also provides for the use of an oil composition enriched in n-3 DPA free fatty acids and/or derivatised n-3 DPA free fatty acids in the manufacture of a medicament for treating or preventing a disorder selected from hypertriglyceridemia, a cardiovascular-related disease or disorder, obesity, or platelet aggregation disorder in a subject.

The present disclosure also provides an enriched n-3 DPA oil composition or pharmaceutical composition as described herein for use in promoting wound healing in a subject in need thereof.

The present disclosure also provides an enriched n-3 DPA oil composition as described herein, in the manufacture of a medicament for promoting wound healing in a subject in need thereof.

The present disclosure also provides use of an enriched n-3 DPA composition or pharmaceutical composition as described herein in a cosmetic formulation. In one example, the cosmetic formulation is a topical formulation. In one example, the topical formulation is a moisturising cream or lotion, bar soap, lipstick, shampoo or therapeutic skin preparation for dryness, eczema and psoriasis.

It will be understood that the purified n-3 DPA or pharmaceutical composition according to the present disclosure is administered to a subject in a therapeutically effective amount.

In some embodiments it may be necessary to remove omega-6 fatty acids if present in the oil mixture. If required, omega-6 and other saturated and monounsaturated fatty acids can be removed using reverse phase high performance liquid chromatography (HPLC). This can be performed following the hydrolysis reaction.

Detailed Description

General

The use of numerical values in the various quantitative values specified in this disclosure, unless expressly stated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word "about". Also, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values recited as well as any ranges that can be formed by such values.

The term "about" is intended to mean nearly the same as a referenced number or value. As used herein, the term "about" should be generally understood to encompass + 10% of a specified amount or value.

The singular form "a" or "the" includes the plural unless the context dictates otherwise.

The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally- equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

Any example herein shall be taken to apply mutatis mutandis to any other example unless specifically stated otherwise. Key to Sequence Listing

SEQ ID No:1 : refers to the sequence of porcine pancreatic lipase. Selected Definitions

The term "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The term "n-3 DPA" or "docosapentaenoic acid" as used herein is intended to refer to the omega-3 (ω3 or n-3) fatty acid and depending on the context can include the free fatty acid form or the natural forms, being the triglyceride form, or the phospholipid form. Docosapentaenoic acid is also known as clupanodonic acid and hence reference to one term includes reference to the other.

The term "disorder associated with hypertriglyceridemia" is intended to refer to a disorder cause by elevation in plasma or serum triglyceride levels above fasting levels and typically refers to high blood levels of triglycerides. High triglyceride levels are typically in the range of about 200 to about 499 mg/dl. Very high triglyceride levels are typically >500 mg/dl. Baseline triglycerides are typically measured when the subject is in a fasting state, that is, the subject has fasted for a period of between 8 and 12 hours. Non-limiting examples of such disorders include atherosclerosis, cardiovascular disease, acute pancreatitis, xanthomas, lipemia reinalis, and hepatosplenomegaly.

The term "cardiovascular-related disease or disorder" as used herein refers to any disease or disorder of the heart or blood vessels (i.e. arteries and veins) and any symptom thereof. Non-limiting examples of cardiovascular-related disease and disorders include hypertriglyceridemia, hypercholesterolemia, mixed dyslipidemia, coronary heart disease, vascular disease, stroke, athersclerosis, arrhythmia, hypertension, myocardial infarction and other cardiovascular events.

The "subject" according to the present disclosure shall be taken to mean any subject, including a human or non-human subject. The non-human subject may include non-human primates, ungulate (bovines, porcines, ovines, caprines, equines, buffalo and bison), canine, feline, lagomorph (rabbits, hares and pikas), rodent (mouse, rat, guinea pig, hamster and gerbil), avian, and fish. In one example, the subject is a human. In one example, the subject consumes a traditional Western diet.

The term "fatty acid" as used herein refers to a molecule that is derived from a triglyceride or phospholipid and is comprised of a carboxylic acid with a long aliphatic tail (chain) which is either saturated or unsaturated. When not attached to other molecules, they are known as "free" fatty acids. Most naturally occurring fatty acids have a chain of an even number of carbon atoms, from 4 to 28. Short chain fatty acids (SCFA) are fatty acids with aliphatic tails of fewer than six carbons. Medium chain fatty acids (MCFA) are fatty acids with aliphatic tails of 6-12 carbons which can form medium chain triglycerides. Long chain fatty acids (LCFA) are fatty acids with aliphatic tails 13 to 21 carbons. Very long chain fatty acids (VLCFA) are fatty acids with aliphatic tails longer than 22 carbons. In one example, the fatty acid or the ester thereof can comprise at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20 carbon atoms. In some specific examples, the fatty acid or the ester thereof can contain 10, 1 1 , 12, 13, 14, 15, 1 6, 17, 18, 1 9, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, or 45 carbon atoms, where any of the stated values can form an upper or lower endpoint when appropriate. In other examples, the fatty acid or the ester thereof can comprise a mixture of fatty acids or the esters thereof having a range of carbon atoms. For example, the fatty acid or the ester thereof can comprise from 10 to 40, from 12 to 38, from 14 to 36, from 16 to 34, from 18 to 32, or from 20 to 30 carbon atoms.

The term "polyunsaturated fatty acids" or PUFAs as used herein are intended to refer to fatty acids that contain more than one double bond in their backbone. Unsaturated refers to the fact that the molecules contain less than the maximum amount of hydrogen. Polyunsaturated fatty acids may be divided into omega 3 and omega 6 type fatty acids. Omega 3 fatty acids have a double bond that is three carbons away from the methyl carbon. Examples of omega 3 polyunsaturated fatty acids include hexadecatrienoic acid (16:3 (n-3)), alpha-linolenic acid (18:3 (n-3)), stearidonic acid (18:4 (n-3)), eicosatrienoic acid (20:3 (n-3)), eicosatetraenoic acid (20:4 (n-3)), eicosapentaenoic acid (20:5 (n-3)), heneicosapentaenoic acid (21 :5 (n-3)), docasapentaenoic acid (22:5 (n-3)), docosahexaenoic acid (22:6 (n-3)), tetracosapentaenoic acid (24:5 (n-3)), tetracosahexaenoic acid (24:6 (n-3)).

The term "omega-3 fatty acid(s)" includes natural as well as synthetic omega-3 fatty acids, as well as pharmaceutically acceptable esters, free acids, triglycerides, derivatives or conjugates, precursors, salts or mixtures thereof (see for example US 2004/0254357 or US 624581 1 ). Examples of omega-3 fatty acid oils includes, but are no limited to omega-3 polyunsaturated fatty acids such as a-linolenic acid (ALA 18:3n-3), octadecatetraenoic acid (i.e. stearidonic acid, STA 18:4n-3), eisosatrienoic acid (ETS, 20:3n-3), eicosatetraenoic (ETA 20:4n-3), eicosapentaenoic acid (EPA 20:5n-3), heneicosapentaenoic acid (HPA 21 :5n-3), docosapentaenoic acid (DPA, clupanodonic acid 22:5n-3) and docosahexaenoic acid (DHA 22:6n-3); esters of omega-3 fatty acids with glycerol such as mono-, di- triglycerides and ester of the omega-3 fatty acids and a primary, secondary and/or tertiary alcohol such as for example fatty acid methyl esters and fatty acid ethyl esters.

The term "glyceride" (also known as acylglycerol) as used herein refers to a diglyceride, a triglyceride or combinations thereof. They are esters formed from glycerol and fatty acids. The glyceride in the oil can comprise a plurality of fatty acids saturated, unsaturated and even short chain carboxylic acids.

The term "triglyceride" as used herein refers to an ester derived from glycerol and three fatty acids. The triglycerides of the present disclosure may be saturated or unsaturated.

The term "sn- 1, sn-2, and sn-3' as used herein refers to the stereospecific numbering

(sri) system as known in the art.

As used herein, the term "therapeutically effective amount" shall be taken to mean a sufficient quantity of n-3 DPA to reduce, inhibit or prevent one or more symptoms of a clinical disorder associated with elevated triglyceride levels to a level that is below that observed and accepted as clinically diagnostic or clinically characteristic of that disorder. The term also means that the substance in question does not produce unacceptable toxicity to the subject or interaction with other components in the composition. The skilled artisan will be aware that such an amount will vary depending on, for example, the particular subject and/or the type or severity or level of disorder. Accordingly, this term is not to be construed to limit the composition of the disclosure to a specific quantity, e.g., weight or amount of n-3 DPA and/or derivative(s), rather the present disclosure encompasses any amount of n-3 DPA and/or derivative(s) sufficient to achieve the stated result in a subject.

As used herein, the term "treating" includes administering a therapeutically effective amount of n-3 DPA described herein sufficient to reduce or eliminate at least one symptom of a specified disorder. In one example, the treatment involves administering a therapeutically effective amount of n-3 DPA to reduce plasma triglyceride levels. In one example, the reduction is measured over a specific time period against a baseline level of fasting plasma triglycerides. In one example, the reduction in plasma triglyceride levels is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% compared to baseline. In one example, treatment also refers to prophylactic treatment.

As used herein, the terms "preventing", "prevent" or "prevention" include administering a therapeutically effective amount of n-3 DPA described herein sufficient to stop or hinder the development of at least one symptom of a specified disorder. In one example, the administration of n-3 DPA or derivative thereof prevents elevation of plasma triglycerides.

Description of the Figures

Figure 1 shows the sequence of porcine pancreatic triacyglycerol lipase. Figure 2 shows ¹³C-NMR profile of native (A) Canola oil (with different types of fatty acids defined as; S=Saturated; V=Vaccenic; 0=Oleic; L=Linoleic; Ln=Linolenic), (B) Anchovy oil and (C) Seal oil Figure 3 shows percentage hydrolysis and capillary chromatography (latroscan) profile of lipid classes from hydrolysed (a) anchovy and (b) seal oils using 10,000 units of porcine pancreatic lipase at 37 °C and pH 7.0.

Figure 4 shows fatty acid composition of canola oil before and after hydrolysis.

Figure 5 shows GC profile of (a) anchovy and (b) seal oil before and after hydrolysis.

Figure 6 shows chemical structures of ALA; C18:3n3 (A9,12,15-octadecatrienoic acid), STA; C18:4n3 (Δ6,9,12,1 5-octadecatetraenoic acid), EPA; C20:5n3 (Δ5.8.1 1 ,14,17- eicosapentaenoic acid), DPA; C22:5n3 (A7,10,13,16,19-docosapentaenoic acid) and DHA; C22:6n3 (A4,7,10,13,16,19-docosahexaenoic acid).

Figure 7 shows Progressive hydrolysis of (a) anchovy and (b) seal oils by porcine pancreatic lipase as measured using gas chromatography. EPA and DHA retention in the glycerol portions increased as DPA is progressively removed in the FFA portions.

Detailed Description of the Invention

Omeqa-3 containing oil

The oil according to the present disclosure can be any suitable oil composition that comprises omega-3 fatty acids and in particular comprises a docosapentaenoic acid (DPA). The oil may be derived from animal oil(s) and/or non-animal oil(s). In some embodiments of the present disclosure, the oil is a fatty acid oil mixture derived from at least one oil chosen from marine oil, single cell oil, algal oil, plant-based oil, microbial oil, and combinations thereof. Marine oils include, for example, fish oil, krill oil, and lipid compositions derived from fish. Plant- based oils include, for example, flaxseed oil, canola oil, mustard seed oil, and soybean oil. Single cell/microbial oils include, for example, products by Martek, Nutrinova, and Nagase & Co. Single cell oils are often defined as oils derived from microbial cells and which are destined for human consumption. See, e.g. , Wynn and Ratledge, "Microbial oils: production, processing and markets for specialty long-chain omega-3 polyunsatutrated fatty acids," pp. 43-76 in Breivik (Ed.), Long-Chain Omega-3 Specialty Oils, The Oily Press, P.J. Barnes & Associates, Bridgewater UK, 2007.

Additional oils include triglyceride vegetable oils, commonly known as long chain triglycerides such as castor oil, corn oil, cottonseed oil, olive oil, peanut oil, safflower oil, sesame oil, soybean oil, hydrogenated soybean oil, and hydrogenated vegetable oils; medium chain triglycerides such as those derived from coconut oil or palm seed oil, monoglycerides, diglycerides, and triglycerides. In addition to mixed glycerides, there are other oils such as esters of propylene glycol such as mixed diesters of caprylic/capric acids of propylene glycol, esters of saturated coconut and palm kernel oil-derived caprylic, linoleic, succinic, or capric fatty acids of propylene glycol.

The fatty acids of the oil may be esterified, such as alkyl esters, for example ethyl esters. In some embodiments, the fatty acids are in glyceride form, such as chosen from mono- , di-, and triglycerides. In other embodiments, the fatty acids are in free acid form.

Unsaturated fatty acids in the oil may be in cis and/or trans configuration. Examples of omega-3 fatty acids in all-c/s configuration include, but are not limited to, (all-Z)-9,12,1 5- octadecatrienoic acid (ALA), (all-Z)-6,9,12,15- octadecatetraenoic acid (STA), (all-Z)-1 1 ,1 ,17- eicosatrienoic acid (ETE), (all-Z)- 8,1 1 ,14,17-eicosatetraenoic acid (ETA), (all-Z)-,7,10,13, 16,19-docosapentaenoic acid (DPA), (all-Z)-5,8,1 1 ,14, 17-eicosapentaenoic acid (EPA), (all- Z)-4,7,10,13,1 6,1 9- docosahexaenoic acid (DHA), and (all-Z)-6,9, 12, 1 5, 18- heneicosapentaenoic acid (HPA). Examples of omega-6 fatty acids in all-cis configuration include, but are not limited to, (all-Z)-4,7,10,13,16-docosapentaenoic acid (osbond acid), (all- Z)-9, 12-octadecadienoic acid (linoleic acid), (all-Z)-5,8,1 1 ,14-eicosatetraenoic acid (AA), and (all-Z)-6,9,12- octadecatrienoic acid (GLA). Examples of monounsaturated fatty acids in cis configuration include, but are not limited to, (Z)-9-hexadecenoic acid (palmitoleic acid), (Z)-9- octadecenoic acid (oleic acid), (Z)-1 1 -octadecenoic acid (vaccenic acid), (Z)-9- eicosenoic acid (gadoleic acid), (Z)-1 1 -eicosenoic acid (gondoic acid), (Z)-1 1 - eicoesenoic acid, (Z)-1 1 - docosenoic acid (cetoleic acid), Z-13-docosenoic acid (erucic acid), and (R-(Z))-12-hydroxy-9- octadecenoic acid (ricinoleic acid).

Examples of fatty acid oils according to the present disclosure include, but are not limited to, the fatty acids defined in pharmacopoeias such as the European Pharmacopoeia Omega-3 Acid Ethyl Esters 60, the European Pharmacopoeia Fish Oil Rich in Omega-3 Acids Monograph, the USP Fish Oil Monograph, the European Pharmacopoeia Omega-3 Acid Triglycerides, the European Pharmacopoeia Omega-3-Acid Ethyl Esters 90, and the USP Omega-3-Acid Ethyl Esters monograph.

Commercial examples of fatty acid oils comprising different fatty acids include, but are not limited to: Incromega™ omega-3 marine oil concentrates such as Incromega™ TG7010 SR, Incromega™ E7010 SR, Incromega™ TG6015, Incromega™ EPA500TG SR, Incromega™ E400200 SR, Incromega™ E401 0, Incromega™ DHA700TG SR, Incromega™ DHA700E SR, Incromega™ DHA500TG SR, Incromega™ TG3322 SR, Incromega™ E3322 SR, Incromega™ TG3322, Incromega™ E3322, Incromega™ Trio TG/EE (Croda International PLC, Yorkshire, England); EPAX6000FA, EPAX5000TG, EPAX451 OTG, EPAX2050TG, EPAX5500EE, EPAX5500TG, EPAX5000EE, EPAX5000TG, EPAX6000EE, EPAX6000TG, EPAX6500EE, EPAX6500TG, EPAX1050TG, EPAX2050TG, EPAX6015TG/EE, EPAX4020TG, and EPAX4020EE (EPAX is a wholly-owned subsidiary of Trygg Pharma AS; Omacor®/Lovaza™/Zodin®/Seacor® finished pharmaceutical product, K85EE, AGP 1 03, K30EE, K50EE, and K70EE (Pronova BioPharma Norge AS); MEG-3® EPA/DHA fish oil concentrates (Ocean Nutrition Canada); DHA FNO "Functional Nutritional Oil" and DHA CL "Clear Liquid" (Lonza); Superba™ Krill Oil (Aker Biomarine); omega-3 products comprising DHA produced by Martek; Neptune krill oil (Neptune); cod- liver oil products and anti-reflux fish oil concentrate (TG) produced by Mellers; Lysi Omega-3 Fish oil; Seven Seas Triomega® Cod Liver Oil Blend (Seven Seas); Fri Flyt Omega-3 (Vesteralens); and Epadel (Mochida). Those commercial embodiments provide for various omega-3 fatty acids, combinations, and other components as a result of the transesterification process or method of preparation in order to obtain the omega-3 fatty acid(s) from various sources, such as marine, algae, microbial (single cell), and/or plant-based sources.

In at least one embodiment, the fatty acid oil comprises n-3 EPA, n-3 DHA and n-3

DPA. The fatty acid oil may further comprise at least one other fatty acid, for example a polyunsaturated fatty acid (PUFA) other than n-3 EPA and n-3 DHA. Examples of such PUFAs include, but are not limited to, other omega-3 fatty acids, such as C20-C22 omega-3 fatty acids other than n-3 EPA and n-3 DHA, and omega-6 fatty acids.

Hydrolysis

The hydrolysis step according to the present disclosure comprises the use of a lipase in liquid form to hydrolyse at least a portion of the n-3 DPA glyceride and provide a hydrolysed glyceride fraction and a free fatty acid fraction. In one example the lipase is a porcine pancreatic lipase obtained from a commercial supplier e.g. Sigma-Aldrich (Castle Hill, Australia). In another example, the lipase has a sequence homology of at least 80%, at least 85%, at least 90%, at least 95%, at least 97% and at least 99% homology to porcine pancreatic lipase as described in Figure 1 .

Prior to hydrolysis the oil can be optionally washed with water and/or a pH buffer such as pH 10 buffer (e.g. a potassium carbonate, potassium borate, potassium hydroxide buffer). An optional wash if performed can comprise one or more washes of the same of varying compositions and conditions. In one example, an oil composition is washed with 60°C water. In another example, an oil composition is twice washed with 60°C water. The quantity of wash solution (e.g., water, pH buffer, or other wash liquid), temperature, and duration of a wash can vary depending upon the starting oil composition and the desired outcome, such as, for example, purity, of a wash step. A pH buffer of buffer solution can comprise any suitable buffer and/or buffer solution for use with the specific oil composition. The specific pH of a buffer solution, if used, can vary and can range, in various examples, from about 7 to about 12. In one example, a pH 10 buffer comprises a potassium carbonate- potassium borate-potassium hydroxide buffer (0.05 M). After an optional washing step, the aqueous fraction of the wash can optionally be separated and removed from the oil composition.

Prior to the addition of the pancreatic lipase, it is important that the pH of the oil is brought to pH 7.2. The oil can then be contacted with an aqueous solution of porcine pancreatic lipase. Contacting of the oil composition and the lipase solution can be performed either separate from, combined with, or subsequent to an optional washing step. In one example, an oil mixture is twice washed with water, washed with a pH 10 buffer solution, and then contacted with an aqueous solution of the lipase enzyme. In another example, the aqueous solution of porcine pancreatic lipase is a phosphate buffered solution of pH about 7.2 (0.01 -0.1 M). The amount of liquid lipase used can vary. An aqueous solution of porcine pancreatic lipase can be prepared by, for example, mixing a quantity of the lipase enzyme with water. The amount of lipase enzyme mixed with water, and thus, the concentration of a resulting lipase solution, can vary.

The conditions for hydrolysis of the starting glyceride can vary, depending upon the desired extent of hydrolysis and the specific reaction components, provided that at least a portion of n-3 DPA on a glyceride are hydrolysed. Thus, the phrase, "at a time and temperature sufficient to hydrolyze at least a portion of the glyceride" can be selected by one of skill in the art, depending on the desired amount of hydrolysis and the desired final product, while monitoring the reaction by known analytical techniques. Thus, in general, longer reaction times, higher temperatures (within the limits of the lipase), and/or more amounts of lipase can be used for more complete hydrolysis. After contacting the oil with the lipase enzyme, the resulting mixture can, in various examples, be sealed in an inert or substantially inert atmosphere, such as, for example, nitrogen or argon atmosphere. The resulting mixture can also be optionally agitated for a period of time sufficient to allow a desired amount of hydrolysis. In one example, an oil/lipase mixture is vigorously agitated for a period of from about 48 to about 72 hours at about 37°C. Suitable temperatures at which the hydrolysis can occur include, but are not limited to, from about ambient to about 1 00°C, from about 35°C to about 80°C, or from about 40°C to about 50°C, preferably between ambient and 40^°C. In certain examples, the reaction time can be adjusted by varying the temperature. Thus, reaction times can vary from about 2 hours to about 72 hours or more, from about 24 hours to about 72 hours, from about 36 hours to about 72 hours, or from about 48 hours to 72 hours.

After hydrolysis, the hydrolyzed oil composition (comprising a glyceride fraction and free saturated fatty acid fraction) can optionally be washed with water one or more times. The aqueous portion of the mixture can then be separated from the non-aqueous portion, and the non-aqueous portion dried. Drying conditions can vary and the disclosed methods are not intended to be limited to any particular drying conditions. In one example, the non-aqueous portion is dried under vacuum at about 80°C.

In many examples, the hydrolysis step is conducted in water and in the absence of alcohol; thus the hydrolyzed product is a free polyunsaturated fatty acid and not a free polyunsaturated fatty ester. These processes are normally carried out under nitrogen and/or with added antioxidants such as citric acid, ascorbic acid or BHT. Determining the degree of hydrolysis

Methods for determining the degree of hydrolysis by the lipase of the present disclosure will be familiar to persons skilled in the art. In one example, capillary chromatography with flame ionisation detector may be used and the percent hydrolysis determined using appropriate software (e.g. SIC-480 II software) for multiple chromatogram processing by comparing the percentage peak areas of the unhydrolysed and hydrolysed triglycerides. Capillary chromatography standards to identify each lipid class can be purchased from a commercial supplier. Examples of such methodology are described in, for example Luddy FE et al (1964) Journal of the American Oil Chemists' Society 41 (10):693-696; Gamez-Meza N et al (2003) Food Research International '36:721 -727).

Processes for separation of hydrolysed and non-hydrolysed fractions

Hydrolysed free fatty acid can be removed from acyl glycerol by methods known in the art. For example, wipe film evaporation and/or short path distillation can be used on the combined oil to selectively remove the more volatile free fatty acids via distillation. Alternatively, water may be added to create an aqueous phase and the pH adjusted so that the free fatty acid exists primarily as unprotenated, charged, carboxylic acid dissolved in the aqueous phase. The organic phase (containing glycerides) and aqueous phase (containing free fatty acids) may be separated according to methods known in the art e.g. by allowing the mixture to stand for a sufficient amount of time to obtain two substantially transparent phases, by centrifugation, by membrane technology, or by other suitable means. By way of non-limiting example, after removal of the organic phase, the aqueous phase may be extracted with a displacement liquid, such as an organic solvent, resulting in formation of at least one extract. Examples of suitable displacement liquids include, but are not limited to, alkanes, alkenes, cycloalkanes, cycloalkenes, dienes, aromates, and halogenated solvents. Non-limiting mention may be made of specific examples, such as dichloromethane and other solvents containing one or more chlorine atoms and/or one or more of other halides, hexane, hexene, heptane, heptene, cyclohexane, cyclohexene, 1 ,7-octadiene, 1 ,5-cyclooctadiene, as well as other alkenes comprising one or more double bonds, such as alkenes comprising one, two, or even three double bonds, and oxygen- and nitrogen-containing solvents such as ketones and amides/amines. The aqueous phase may be extracted more than once, i.e., at least two successive extractions. The amount of displacement liquid for each extraction may range from about 0.1 to about 5 times by weight the amount of fatty acid that is dissolved in the aqueous phase.

Different displacement liquids and/or combinations of displacement liquids may be used according to the selectivity desired in concentrating n-3 DPA free fatty acids.

The aqueous phase and organic phase may be heated before they are separated. In such cases, the boiling point of the organic phase may be considered in determining the appropriate temperature. Generally speaking, the aqueous phase/organic phase mixture may be heated to a temperature ranging from about 30 °C to about 90°C. In some embodiments, the aqueous phase is heated after removing the organic phase, resulting in formation of at least one extract. For example, the aqueous phase may be heated to a temperature of at least 30 °C, such as a temperature ranging from about 30 °C to about 90 °C.

In such cases, heating may cause the release of a fatty acid oil concentrated in omega- 6 fatty acids and/or specific omega-3 fatty acids, such as C20-C22 omega-3 fatty acids other than n-3 DPA, from the aqueous phase. Heating should be done carefully in the absence of oxygen and at sufficiently mild conditions to avoid oxidation, isomerization and/or degradation of the polyunsaturated fatty acids.

The process according to the present disclosure may concentrate or enrich n-3 DPA omega-3 free fatty acid while reducing the concentration of other omega-3 fatty acids in the oil. The process may increase the ratio of n-3 DPA omega-3 to n-3 DHA and n-3 EPA. In some embodiments, for example, the process concentrates or enriches n-3 DPA while reducing the concentration of C20-C22 omega-3 fatty acids other than n-3 DPA. In some embodiments, the total concentration of C_2o-C₂2 omega-3 fatty acids other than n-3 DPA in the fatty acid fraction is less than 3% by weight, such as less than 2.5% by weight, such as less than 0.5% by weight. In another example, the n-3 DPA free fatty acid may be concentrated or enriched using an aqueous silver salt according to known methods and as described for example in WO 2012/038833.

The n-3 DPA free fatty acid fraction may be purified by using at least one purification process. The purification process may remove, for example, displacement liquid or lower-chain fatty acids or complexation compounds e.g. urea if urea complexation has been performed, cholesterol and/or vitamins. Such purification processes include, but are not limited to, short- path distillation, molecular distillation, supercritical fluid extraction, enzymatic separation processes, iodolactonization fractionation, and preparative chromatography.

The process presently disclosed may be repeated to further concentrate the n-3 DPA omega-3 free fatty acid. The fatty acid concentrate obtained from one or more concentration processes according to the present disclosure may comprise at least 80% of n-3 DPA omega-3 fatty acid, such as at least 90%, at least 95%, or even at least 98% of n-3 DPA omega-3 fatty acid.

The fatty acid concentration obtained according to the process of the present disclosure may also be treated by at least one conventional fractionation process such as short-path distillation, molecular distillation, iodolactonization fractionation, enzymatic fractionation processes, extraction, and/or chromatography. The free fatty acid concentrate thus obtained may comprise at least 80% of n-3 DPA omega-3 free fatty acid, such as at least 90%, at least 95%, or even at least 98% of n-3 DPA omega-3 free fatty acid. In one embodiment, for example, the at least one fractionation process produces a free fatty acid concentrate comprising at least 90% cis-7, 10, 13, 1 6, 19-docosapentaenoic acid (DPA), such as at least 95% n-3 DPA, or for example, at least 98% n-3 DPA.

The process presently disclosed may reduce the concentration of at least one environmental pollutant in the oil, such that the free fatty acid oil concentrate comprises a lower concentration of the at least one environmental pollutant than the oil mixture. Environmental pollutants include, but are not limited to, polychlorinated biphenyl (PCB) compounds, polychlorinated dibenzodioxin (PCDD) compounds, polychlorinated dibenzofuran (PCDF) compounds, brominated flame retardants like polybrominated diphenyl ethers (PBDE), tetrabromobisphenol A (TBBP-A) and hexabromocyclododecane (HBCD), and pesticides like DDT (2,2 bis-(p-chlorophenyl)- 1 ,1 ,1 -trichloroethane) and metabolites of DDT. The process presently disclosed may also reduce the concentration of total cholesterol (i.e., free and/or bound cholesterol) in the oil mixture, such that the fatty acid concentrate comprises a lower concentration of total cholesterol than the oil.

Other methods of separation known in the art may be employed as required. For example high performance liquid chromatography and silver resin chromatography have been used for the production of n-3 polyunsaturated fatty acid concentrates. Solvent choice for separation of fatty acid esters depends on the desired purity of eluted fractions and their use as well as production requirements. Higher purity fractions of n-3 EPA and n-3 DHA can be obtained using tetrahydrofuran (THF) or ethanol and water.

Distillation has been used for partial separation of mixtures of fatty acid esters. This method takes advantage of the differences in boiling point and molecular weight of fatty acids under reduced pressure. The technique requires high temperatures of approximately 250^°C. Short-path distillation or molecule distillation uses lower temperatures and short heating intervals. The most widely used distillation techniques is fractional distillation under reduced pressure (0.1 -1 mm HG). Even under these conditions moderately high temperatures are required sufficient to cause oxidation, polymerisation and isomerisation of double bonds of omega-3 polyunsaturated fatty acids.

Low temperature crystallisation may be used to separate fatty acids. The solubility of fats in organic solvents decreases with increasing mean molecular weight and increases with increasing unsaturation. At low temperature long chain fatty acids which have high melting point crystallise out and polyunsaturated fatty acids remain in the liquid form. Low temperature crystallisation process may be carried out in the absence of a solvent or in a selected solvent/solvent mixture. The commonly used solvents are methanol and acetone which have been employed to separate stearic and oleic fractions. It has been reported that use of different organic solvents affects the concentration of polyunsaturated fatty acid. Therefore proper choice of solvent is necessary to achieve optimum concentration yield of omega-3 polyunsaturated fatty acid.

Supercritical fluid extraction (SPE) is a relatively new process. The separation of polyunsaturated fatty acids by SPE is dependent on the molecular size of the components involved rather than their degree of unsaturation, therefore a prior concentration step is need to achieve a high concentration of polyunsaturated fatty acid in the final product.

Compositions

Any biologically acceptable dosage forms, and combinations thereof may be contemplated by the present disclosure. Examples of such dosage forms include, without limitation, chewable tablets, quick dissolve tablets, effervescent tablets, reconstitutable powders, elixirs, liquids, solutions, suspensions, emulsions, tablets, multi-layer tablets, bi-layer tablets, capsules, soft gelatin capsules, hard gelatin capsules, caplets, lozenges, chewable lozenges, beads, powders, granules, particles, microparticles, dispersible granules, cachets, douches, suppositories, creams, topicals, inhalants, aerosol inhalants, patches, particle inhalants, implants, depot implants, ingestibles, injectables, infusions, health bars, confections, cereals, cereal coatings, foods, nutritive foods, functional foods and combinations thereof. The preparations of the above dosage forms are well known to persons of ordinary skill in the art.

Pharmaceutical compositions useful in accordance with the methods of the present disclosure are orally deliverable. The terms "orally deliverable" or "oral administration" herein include any form of delivery of a therapeutic agent (e.g. n-3 DPA or a derivative thereof) or a composition thereof to a subject, wherein the agent or composition is placed in the mouth of the subject, whether or not the agent or composition is swallowed. Thus "oral administration" includes buccal and sublingual as well as oesophageal administration. In one example, the purified n-3 DPA or derivative thereof is present in a capsule, for example a soft gelatin capsule.

The pharmaceutical compositions according to the present disclosure are not limited with regard to their mode of use. Representative modes of use include foods, food additives, medicaments, weight supplements, additives for medicaments, and feedstuffs.

Examples of food compositions, besides general foods, are functional foods, nutrient- supplementing foods, formula suitable for feeding infants, baby foods, foods for expectant or nursing mothers, and geriatric foods. The composition may be added upon cooking such as soup, food to which oils and fat are used as heating medium such as doughnuts, oils and fat food such as butter, processed food to which oils and fat are added during processing such as cookies or food to which oils and fat are sprayed or applied upon completion of processing such as hard biscuits.

Furthermore the compositions of the present disclosure can be added to foods or drinks which do not normally contain oils or fat.

The definition of food also includes functional food. Functional foods and medicaments may be provided in processed form such enteral agent for promoting nutrition, powder, granule, troche, internal solution, suspension, emulsion, syrup, capsule and such.

The compositions according to the present disclosure can be formulated as one or more dosage units. The term "dose unit" and "dosage unit" herein refer to a portion of a composition that contains an amount of a therapeutic agent suitable for single administration to provide a therapeutic effect. Such dosage units may be administered one to a plurality (i.e. 1 to about 10, 1 to 8, 1 to 6, 1 to 4 or 1 to 2) of times per day, or as many times as needed to elicit a therapeutic response.

In one example, a composition of the present disclosure is administered to a subject over a period of about 1 to about 200 weeks, about 1 to about 100 weeks, about 1 to about 80 weeks, about 1 to about 50 weeks, about 1 to about 40 weeks, about 1 to about 20 weeks, about 1 to about 1 5 weeks, about 1 to about 12 weeks, about 1 to about 1 0 weeks, about 1 to about 5 weeks, about 1 to about 2 weeks, or about 1 week. In one example the compositions of the present disclosure comprise one or more antioxidants (e.g. tocopherol) or other impurities in an amount of not more than about 0.5%, or not more than 0.05%. In another example, the compositions of the present disclosure comprise about 0.05% to about 0.4% tocopherol, or about 0.4% tocopherol, or about 0.2% by weight tocopherol.

In one example, the compositions of the present disclosure include one or more additional excipients including, but not limited to gelatin, glycerol, polyol, sorbitol and water.

In one example, the n-3 DPA or derivative thereof is present in the composition in an amount of about 50 mg to about 5000 mg, about 75 mg to about 2500 mg, or about 100 mg to about 1000 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg, about 1000 mg, about 1025 mg, about 1050 mg, about 1 075 mg, about 1200 mg, about 1250 mg, about 1300 mg, about 1350 mg, about 1400 mg, about 1450 mg, about 1500 mg, about 1550 mg, about 1600 mg, about 1 650 mg, about 1700 mg, about 1750 mg, about 1800 mg, about 1850 mg, about 1900 mg, about 1950 mg, or about 2000 mg.

In one example the compositions of the present disclosure comprise about 300 mg to about 1 g of the composition in a capsule. In one example, the dosage form is a gel or liquid capsule and is packaged in blister packages of about 1 to about 20 capsules per sheet.

In one example, a composition of the present disclosure is administered to a subject once or twice per day. In another example, the composition is administered to a subject as 1 , 2, 3, or 4 capsules daily.

The composition may be administered to a subject in need thereof immediately before a meal, during consumption of the meal or shortly following the meal.

In another example, the composition of the present disclosure is formulated for topical application, for example in a cosmetic. Topical products that may incorporate n-3 DPA according to the present disclosure include moisturizing creams and lotions, bar soaps, lipsticks, shampoos and therapeutic skin preparations for dryness, eczema and psoriasis.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. Examples

Materials and Methods

Materials

Anchovy oil was provided by Ocean Nutrition Canada and seal oil was a gift from Professor Fereidoon Shahidi, Memorial University of Newfoundland, and canola oil was purchased from a local supermarket in Australia. Porcine pancreatic lipase was purchased from Sigma-Aldrich (Castle Hill, Australia). Capillary chromatography standards were purchased from Nu-Chek Prep (Elysian, MN, USA). All other chemicals were of analytical grade. ¹³C NMR spectroscopy

Quantitative ¹³C NMR spectra of the unhydrolysed oils were recorded under continuous ¹ H decoupling at 24 ^SC using a Brucker Avance 500 MHz. The spectra were collected on 0.5 g of the oil samples dissolved in 700 μΙ_ CDCI₃ (99.8% pure). In order to quantify the residue of each fatty acid at different positions, peak area ratios were analysed by integration and presented in percentages (Akanbi et al (2013) Food Chemistry 138:615-620).

Lipase-catalvsed hydrolysis of oil

Enzymatic hydrolysis was carried out following the method of Luddy et al., (1963) Journal of the America Oil Chemists' Society 41 (10):693-696 with some modifications. To a 5 mL flask containing 300 mg of oil was added 100 μΙ_ of 22% calcium chloride (CaCI₂) solution, 200 μΙ_ of 1 M tris(hydroxymethyl)aminomethane at pH 7.7 and 320 μΙ_ of 0.1 % bile salts solution. The mixture was gently flushed with nitrogen and warmed in a water bath at 37 ^SC for 5 min before the addition of 10,000 units of PPL. Hydrolysis was thereafter carried out in the dark at 37 ^SC under nitrogen with magnetic stirring at 200 rpm and aliquots of sample were withdrawn at different time intervals until 50(±5)% hydrolysis degree was attained. The glycerol and free fatty (FFA) portions of each sample was obtained and separated as previously reported (Akanbi et al (2013) Food Chemistry 138:615-620).

Hydrolysis degree and lipid class analysis

Capillary chromatography with flame ionisation detector (latroscan MK5, latron Laboratories Inc., Tokyo, Japan) was used to determine the degree of hydrolysis. Portions of both the unhydrolysed and hydrolysed oil were analysed by latroscan as previously reported (Akanbi et al (2013) Food Chemistry 138:615-620). Percent hydrolysis was determined using SIC-480 II software for multiple chromatogram processing, by comparing the percentage peak areas of the unhydrolysed and hydrolysed triacylglycerol (TAG). Capillary chromatography standards purchased from Nu-Chek Prep were used to identify each lipid class. Analysis of fatty acid composition by gas chromatography

Fatty acids in both the unhydrolysed and hydrolysed portions of the oils were converted to methyl esters and the resulting fatty acid methyl esters (FAMEs) were analysed by an Agilent 6890 gas chromatograph with flame ionisation detector (FID), as previously described (Akanbi et al (2013) Food Chemistry 138:615-620).

Statistical analysis

Unless otherwise stated, experiments were carried out in triplicates and data are presented as the mean ± standard deviation as calculated using Excel statistical software package (Microsoft Office Excel® 2010).

Example 1 Positional distribution of polyunsaturated fatty acids by ¹³C NMR spectroscopy

In order to determine the positional distribution of the major polyunsaturated fatty acids on the glycerol backbone, quantitative ¹³C NMR spectra of the native oils were recorded (Fig. 2) and positional distributions summarised for anchovy and seal oils in Table 1 . Peak assignments are distinct for fatty acid types and positions as previously reported (Akanbi et al (2013) Food Chemistry 138:615-620; Khallouki F et al (2008) Food Chemistry 1 10(1 ):57-61 ). ¹³C NMR spectrum of canola oil (Fig. 2A) shows that oleic acid is distributed in an approximately statistical 2:1 ratio for sn-1 ,3 versus sn-2, whereas linoleic and linolenic (signals overlapping) appear to be enriched at sn-2, with an approximate 1 :1 ratio between sn-1 ,3 and sn-2. For anchovy oil (Fig. 2B, Table 1 ) DHA and DPA are enriched at sn-2, STA, saturates and monounsaturates are approximately statistically distributed, while EPA is enriched as sn- 1 ,3. Seal oil has a different omega-3 fatty acid distribution pattern to anchovy oil. As shown (Fig. 2C, Table 1 ), monounsaturates are enriched at sn-2, saturates are statistically distributed, while STA, EPA, DPA and DHA are all highly enriched at the sn-3 position. These results for seal oil obtained using ¹³C NMR are consistent with previous findings using GC and TLC methods of analysis (Wanasundara UN et al (1997) Journal of Food Lipids 4(1 ):51 -64). Table 1 Molar percentages of fatty acids at the sn-1 ,3 and sn-2 glycerol positions of anchovy and seal oils

Position Fatty acid assignment Molar percentage (%)

Anchovy oil Seal oil sn-1 ,3 Saturated 23.32 19.67

Monounsaturated, Δ9 15.95 25.34

STA (C18:4), A6 6.45 3.03

EPA (C20:5), Δ5 14.49 6.78

DPA (C22:5), Δ7 2.13 4.15

DHA (C22:6), Δ4 4.25 7.98 sn-2 Saturated 12.96 10.06

Monounsaturated, Δ9 5.51 21 .23

STA (C18:4), A6 2.86 0.64

EPA (C20:5), Δ5 3.52 0.64

DPA (C22:5), Δ7 1 .93 ND^*

DHA (C22:6), Δ4 6.63 0.48

ND^* not detected Example 2 Positional versus fatty acid selective hydrolysis for canola, seal and anchovy oils.

Hydrolysis of anchovy oil with pancreatic porcine lipase, PPL (50% hydrolysis in 1 1 0 minutes; Figure 3A) is slower than hydrolysis of seal oil (52% hydrolysis in 70 minutes) (Figure 3B) and canola oil (53% hydrolysis in 25 minutes). The slower hydrolysis of fish oil is due to the presence of higher amount of partially PPL resistant n-3 PUFAs. Hydrolysis of TAG leads to a mixture of FFA, DAG and MAG, with DAG increasing initially and then decreasing after about 30% TAG hydrolysis, due to increased MAG formation (Heier C et al (2010) Biochimica et Biophysica Acta 9BBA)-Molecular and Cell Biology of Lipids 1801 (9):1063-1071 ). For canola oil all major fatty acids, including oleic linoleic and linolenic acid, were approximately equally hydrolysed after 53% hydrolysis, irrespective of their length, number of double bonds, or positional distribution (Fig. 4). For both anchovy and seal oils, shorter chain fatty acids were hydrolysed more rapidly by PPL than longer-chain fatty acids, irrespective of their positional distributions (Fig. 5). For both oils the major saturated and monounsaturated fatty acids, including gondoic acid (C20:1 n9), were preferentially hydrolysed and enriched in the FFA fraction. Importantly, DPA was highly enriched in the FFA fraction and so was selectively hydrolysed versus all other omega-3 fatty acids, including STA, EPA and DHA. The selectivity of PPL toward DPA hydrolysis is not due to positional selectivity, since DPA is primarily at sn- 1 ,3 position in seal oil and enriched at the sn-2 position in anchovy oil. Also, DHA was resistant to PPL hydrolysis even though it is primarily at sn-1 ,3 in seal oil and enriched at sn-2 in anchovy oil. EPA is enriched equally to DHA in anchovy oil, even though EPA is primarily at sn- 1 ,3 and DHA is primarily at sn-2, further indicating that the hydrolysis is fatty acid and not regiospecific.

Example 3 Why is DPA hydrolysed much more rapidly by PPL than is either EPA or DHA?

Previous studies indicate that the resistance of EPA and DHA to hydrolysis by PPL is at least partly due to the presence of a double bond at C4-5 for DHA and C5-6 for EPA, indicating that double bonds near the carbonyl inhibit PPL activity (Bottino NR et al (1967) Lipids 2(6):489-493; Lawson & Hughes (1988) Biochemical and Biophysical Research Communications 1 52(1 ):328-335). It was further argued that EPA and DHA at sn-2 position confers PPL resistance, although this was not seen in the present results (Lawson & Hughes supra). It was found that ALA and DPA were more readily hydrolysed than DHA, EPA and STA, which is consistent with double bonds at C4-5 (Δ4 DHA), C5-6 (Δ5 EPA) and C6-7 (Δ6 STA) conferring greater resistance to hydrolysis than double bonds at C7-8 (Δ7 DPA) and C9- 10 (Δ9 ALA), irrespective of carbon chain length or position on the acyl glycerol backbone (Fig. 6). This explanation is also consistent with the observed rapid hydrolysis of eicosatetraenoic acid (ΕΤΑ-Δ8; C20:4n3) from menhaden oil (Yang L et al (1990) Journal of Lipid Research 31 (1 ):137-147) and the observed resistance of gama-linolenic acid (018:3η6-Δ6) compared with the rapid hydrolysis of linoleic acid (018:2η6-Δ8) by PPL (Syed Rahmattullah et al (1994) Journal of the American Oil Chemists' Society 71 (6):569-573).

Example 4 Use of PPL to separate DPA from EPA and DHA.

The rapid hydrolysis of DPA compared with EPA and DHA indicates that PPL could be used to purify this fatty acid away from similar omega-3 fatty acids that are difficult to separate by other means. After approximately 50% anchovy (Fig 7A) or seal oil (Fig 7B) hydrolysis, DPA was primarily a free fatty acid (FFA), while EPA and DHA primarily remained on the glyceride backbone. The released DPA as FFA can be readily separated from the remaining glycerides by a weak base wash.

Currently DPA is produced commercially via synthetic elongation from alpha linolenic acid (ALA) or stearidonic acid (STA), which is an expensive and difficult process (Kuklev DV et al (2006) Chemistry and Physics of Lipids 144(2):172). Using PPL, perhaps after removal of abundant saturated and monounsaturated fatty acids using a lipase that doesn't hydrolyse DPA, it might be possible to enable the rapid isolation of DPA from seal oil or other DPA containing oil for commercial purposes. Multiple re-use of PPL would be required to make this process cost effective, requiring immobilization, which has been achieved successfully for PPL (Bagi K et al (1997) Enzyme and Microbial Technology 20(7) :531 -535; Bautista FM et al (1 999) Journal of Chemical Technology and Biotechnology 72(3):249-254; Kartal F et al (2009) Journal of Molecular Catalysis B: Enzymatic 57(1 ):55-61 ; Kilinc DA et al (2006) Preparative Biochemistry & Biotechnology 36(2):153-1 63). It should be possible to combine the use of multiple lipases, including PPL and Thermomyces lanuginosus lipase (TLL), which provides a partial separation of EPA and DHA (Akanbi et al (2013) Food Chemistry 138:615-620, to produce separate DPA, EPA and DHA concentrates.

Remarks

The selectivity of porcine pancreatic lipase for a broad range of polyunsaturated fatty acids was investigated via hydrolysis of canola, anchovy and seal oils. A combination of GC- FID and ¹³C NMR was used to show that PPL discriminates against some polyunsaturated fatty acids regardless of their chain lengths, number of double bonds and position on the glycerol backbone. PPL found ALA and DPA better substrates as it hydrolysed them more rapidly, while STA, EPA and DHA were highly discriminated against. Therefore, PPL enabled partial separation of DPA from EPA and DHA.

Claims

The claims defining the invention are as follows:

1 . Use of a pancreatic lipase for separating n-3 docosapentaenoic acid (DPA) from other omega-3 fatty acids present in an omega-3 containing oil.

2. Use according to claim 1 , wherein the other omega-3 fatty acids are n-3 eicosapentaenoic acid (EPA) and n-3 docosahexaenoic acid (DHA).

3. Use according to claim 1 or 2, wherein the separated n-3 docosapentaenoic acid (DPA) is in free fatty aid form.

4. A method for separating n-3 DPA from other omega-3 fatty acids present in an omega- 3 containing oil, the method comprising:

(i) combining the oil with a lipase that preferentially hydrolyses n-3 DPA glycerides; (ii) allowing hydrolysis by the lipase for a time and temperature sufficient to hydrolyse at least a portion of the glycerides in the oil; and

5. A method for enriching n-3 DPA from other omega-3 glycerides present in an omega-3 containing oil, or an oil comprising omega-3 and omega-6 glycerides, the method comprising:

(i) optionally separating the omega-3 and omega-6 glycerides in the oil;

(iii) combining the oil with a lipase that preferentially hydrolyses n-3 DPA glycerides;

(iv) allowing hydrolysis by the lipase according to (iii) for a time and temperature sufficient to hydrolyse at least a portion of the glycerides;

(vi) optionally concentrating the n-3 DPA fatty acids.

6. The method according to claim 4 or 5, wherein the lipase hydrolyses n-3 DPA glycerides more rapidly compared to n-3 EPA and/or n-3 DHA glycerides.

7. The use or method according to any preceding claim, wherein the oil is a native oil or a synthetic oil.

8. The use or method according to any preceding claim, wherein the oil is rich in omega-3 polyunsaturated fatty acids.

9. The use or method according to any preceding claim, wherein the oil comprises n-3 DPA.

10. The use or method according to claim 8 or 9, wherein the oil is selected from a marine

011, single cell oil, algal oil, plant-based oil, microbial oil, mammalian oil and combinations thereof.

1 1 . The use or method according to any preceding claim, wherein the oil is selected from seal oil, anchovy oil, AHIFLOWER™ oil, fish oil or algae oil.

12. The use or method according to any preceding claim, wherein the lipase is a pancreatic lipase.

13. The use or method according to claim 12, wherein the lipase is a porcine pancreatic lipase.

14. The method according to any one of claims 4 to 13, wherein the hydrolysis is carried out at a pH which does not cause substantial denaturation of lipase secondary structure.

15. The method according to claim 14, wherein the hydrolysis is carried out at a pH between about 7 and 8.

16. The use or method according to any preceding claim, wherein the lipase is free or immobilised.

17. The method according to any one of claims 4 to 16, wherein at least about 15 to 70% of the n-3 DPA glycerides in the oil are hydrolysed.

18. The method according to any one of claims 4 to 17 further comprising combining the oil with a lipase that does not preferentially hydrolyse n-3 DPA glycerides.

19. The method according to claim 18, wherein the lipase hydrolyses saturated and monounsaturated fatty acids present in the oil.

20. The method according to any one of claims 4 to 17, wherein the hydrolysis reaction may be carried out either in an agitated-tank reactor or in a flow reactor.

21 . The method according to any one of claims 4 to 17 further comprising esterification of free fatty acids with alcohol(s).

22. The method according to any one of claims 4 to 21 , further comprising extracting and/or concentrating the n-3 EPA and n-3 DHA containing fraction formed following hydrolysis.

23. The method according to any one of claims 4 to 21 further comprising separating n-3 DPA free fatty acids from other omega-3 free fatty acids contained in the aqueous free fatty acid fraction.

24. An oil composition enriched in n-3 DPA free fatty acids obtained according to a method of any one of claims 4 to 23.

25. An oil composition according to claim 24 wherein the n-3 DPA free fatty acids are modified or derivatised.

26. An enriched oil composition according to claim 24 or 25, wherein n-3 DPA free fatty acids comprise at least 10% by weight of the oil.

27. A pharmaceutical composition comprising an oil composition according to any one of claims 24 to 26 together with a pharmaceutically acceptable carrier or excipient.

28. An oil according to any one of claims 24 to 26, or pharmaceutical composition according to claim 27 for use in a weight loss supplement for treating or preventing obesity in a subject.

29. An oil according to any one of claims 24 to 26 or pharmaceutical composition according to claim 27 for use in treating or preventing hypertriglyceridemia or a disorder associated with hypertriglyceridemia in a subject.

30. An oil according to any one of claims 24 to 26 or pharmaceutical composition according to claim 27 for use in promoting wound healing in a subject in need thereof.

31 . A food additive comprising an oil composition according to any one of claims 24 to 26.

32. A food additive according to claim 31 selected from functional food, nutrient- supplementing food, formula suitable for feeding infants or premature infants, baby foods, foods for expectant or nursing mothers, geriatric foods and animal feed.

33. A method of treating or preventing a disorder selected from hypertriglyceridemia, a cardiovascular-related disease or disorder, obesity, or platelet aggregation disorder in a subject, comprising administering to a subject in need thereof an oil composition according to any one of claims 24 to 26, a pharmaceutical composition according to claim 27 or a food additive according to claim 31 or 32.

34. Use of an oil composition according to any one of claims 24 to 26, a pharmaceutical composition according to claim 27 in the manufacture of a medicament for treating or preventing a disorder selected from hypertriglyceridemia, a cardiovascular-related disease or disorder, obesity, or platelet aggregation disorder in a subject.

35. Use of an oil composition according to any one of claims 24 to 26, a pharmaceutical composition according to claim 27 in a cosmetic formulation.