NZ613916B2

NZ613916B2 - Artificial oil bodies

Info

Publication number: NZ613916B2
Application number: NZ613916A
Authority: NZ
Inventors: Thomas Boiteau; Leif Lundin; Zhiping Shen; Rajendranatha Chakrapani Wijesundera; Xinqing Xu
Original assignee: Commonwealth Scientific And Industrial Research Organisation
Priority date: 2011-02-07
Filing date: 2012-02-07
Publication date: 2015-11-03

Abstract

Disclosed is a method of producing artificial oil bodies, the method comprising i) obtaining an extract of a plant comprising oleosin, wherein the extract is plant meal, ii) at least partially purifying protein from the extract, wherein the protein comprises oleosin, and iii) mixing the protein comprising oleosin with surfactant and oil comprising polyunsaturated fatty acids with four or more double bonds to produce the artificial oil bodies. Also disclosed is a method of partially purifying oleosin from a plant extract, the method comprising 1) adjusting the pH of the extract to a pH of at least about 11.5, 2) separating 1) to produce a solid and liquid phase and selecting the liquid phase, 3) reducing the pH of the liquid phase to precipitate the proteins, 4) separating 3) to produce a solid and liquid phase and selecting the solid phase, and 5) dispersing the solid phase in a carrier, wherein the extract is plant meal. omprising oleosin with surfactant and oil comprising polyunsaturated fatty acids with four or more double bonds to produce the artificial oil bodies. Also disclosed is a method of partially purifying oleosin from a plant extract, the method comprising 1) adjusting the pH of the extract to a pH of at least about 11.5, 2) separating 1) to produce a solid and liquid phase and selecting the liquid phase, 3) reducing the pH of the liquid phase to precipitate the proteins, 4) separating 3) to produce a solid and liquid phase and selecting the solid phase, and 5) dispersing the solid phase in a carrier, wherein the extract is plant meal.

Description

ARTIFICIAL OIL BODIES FIELD OF THE INVENTION The present invention relates to artiﬁcial oil bodies comprising fatty acids, and methods of preparing said artiﬁcial oil bodies. The ial oil bodies can be used in a wide variety of products, and are particularly useful for producing oxidatively stable oil-in-water emulsions in the absence of antioxidants.

BACKGROUND OF THE ION Fat has a high caloriﬁc t and makes a major contribution to the human dietary energy intake. Certain types of fat such as saturated fats and trans fats have been implicated in a range of e conditions including cardiovascular e.

However, fat also plays a positive role in human health and nutrition, and ational dietary guidelines allow a certain proportion of dietary energy to be derived from fat as long as it is ‘good fat’. The ‘good fats’ are monounsaturated and polyunsaturated oils, and there is increasing interest in replacement of saturated fats in food products with healthier unsaturated oils. Moreover, for many years now, a ular group of polyunsaturated oils, namely long-chain omega-3 oils, have received considerable attention due to a ude of health beneﬁts including protection against cardiovascular disease which have been attributed to them. Spurred by the g list of health beneﬁts of omega—3 oils, the food industry worldwide is striving to produce food products fortiﬁed with omega-3 oils.

One of the major impediments to the expansion of omega—3 fortiﬁed foods and r healthy foods containing highly unsaturated oil is the extreme susceptibility of these oils to oxidative deterioration during storage. ion generates products some of which can have highly undesirable ﬂavour characteristics thereby reducing the palatability and shelf-life of the product. It is important therefore to adequately protect these oils from oxidation. Whilst microencapsulation and addition of anti—oxidants have successfully been used as strategies to control oxidation of highly unsaturated oils in food products, these logies have several drawbacks in relation to cost, naturalness, and general applicability.

Oil in plants and oilseeds occurs in the form of te deposits referred to as oil bodies (OBs) which are similar in structure irrespective of the plant species they are present in. A remarkable feature of natural CBS is their exceptional physical stability both inside the cells and in isolated preparations. In both situations, OBs occur as individual es, and do not aggregate or coalesce when they are d against one another even after prolonged storage; in viva due to desiccation, and in vitro after ﬂotation centrifugation (Tzen and Huang, 1992). The exceptional physical stability of OBS has been attributed to unique amphipathic proteins called oleosins occurring in the oil body surface (Huang, 1994). It has been suggested that the entire e of the oil body is covered by oleosin such that the compressed OBs never coalesce or ate in the cells of a mature seed (Tzen and Huang, 1992). The content of oleosin ranges from 1-4% of the total oil bodies; the greatest amount occurring in species in which the CBS are the st, e. g. rape/ canola (Huang, 1992; Tzen, 1993).

In addition to good physical stability, OBs within oilseeds have l tion against oxidation. Recent studies have shown that this ive stability is extended to aqueous dispersions oil body isolates. Fisk and co-workers (2008) showed intact OBs extracted from sunﬂower seed and dispersed in continuous aqueous phase were more resistant to oxidation than equivalent emulsions prepared from er oil and emulsiﬁer. More recently, similar results have been obtained for echium oil body isolates containing the highly unsaturated stearidonic acid (Gray et a1, 2010). Superior ive stability of aqueous dispersions of intact canola OBs over lent canola oil-in—water emulsions prepared using Tween40 as the emulsiﬁer has also been reported (Shen and Wijesundera, 2009).

Limited attempts have been made to reassemble OBs from their constituents.

Murphy and Cummins (1989) reported that incubation ofTAG extracted from rapeseed oil or puriﬁed triolein with rapeseed oil body membrane material resulted in the appearance of oil stable droplets of similar dimensions to native oil bodies. Later, Tzen and Huang (1992) sfully prepared physically stable artificial (reconstituted) oil bodies (AOBs) by combining a TAG mixture of triolein and trilinolein (1:2 molar) with dioleoyl phosphatidylcholine and oleosin isolated from wheat, rice, ed, soybean or jojoba. However, the oxidative stability of such artiﬁcial OBs does not seem to have been investigated.

There is a need for means to protect fatty acids from ion which do not rely on the use of added antioxidants.

SUMMARY OF THE INVENTION Oils containing high levels of polyunsaturated fatty acids with four or more double bonds, such as marine oils rich in (D3 fatty acids, are chemically distinct from oils produced in plants such as oilseeds. However, the present inventors were surprised to ﬁnd that n extracted from plant meal, such as commercial canola meal, can be used to construct artiﬁcial oil bodies with highly polyunsaturated oils, such as CD3 fatty acid rich marine and/or ﬁsh oils. The present inventors where also surprised to ﬁnd that the phospholipid component of oil bodies could be substituted with a wide variety of surfactants without ely affecting oxidative stability.

Thus, in a ﬁrst aspect the present ion provides an artiﬁcial oil body comprising oleosin, tant, and oil sing polyunsaturated fatty acids with three or more double bonds, more preferably four or more double bonds.

In an embodiment, the surfactant is phospholipid.

In an embodiment, at least 5%, at least 10%, at least 15%, at least 20% or at least 25%, of the polyunsaturated fatty acids have four or more double bonds.

In another aspect, the present invention provides an artiﬁcial oil body comprising oleosin, surfactant, and oil sing fatty acids, wherein at least some of the surfactant is not phospholipid.

Whilst the oleosin can be obtained from any source, in a preferred ment the oleosin is an oilseed oleosin.

The oil may also be obtained from any source. In a preferred ment, the oil is marine oil and/or fish oil.

In a r embodiment, at least 50%, at least 75%, or at least 90%, of the fatty acids are in the form of glycerides.

In an embodiment, at least 80%, at least 90%, or at least 95%, of the dry weight of artiﬁcial oil body is oil.

The oil bodies of the invention can be used as a carrier for, and hence may r comprise, one or more other molecules. Examples such other molecules include, but are not limited to, a probiotic, a preservative, a therapeutic agent, a diagnostic agent, a delivery agent or a food colouring agent.

The size ofthe oil body can be varied by manipulating the relative concentration of oil to oleosin with lower relative amounts of oleosin promoting larger sizes. In an embodiment, the oil body has a size of between about 0.1 and about 100 pm, or at least between about 0.5 and about 50 um, or at least between about 0.5 and about 10 um, or at least between about 0.5 and about 2 um.

In yet a further aspect, the present invention provides a composition comprising one or more artiﬁcial oil bodies of the invention, and a carrier. In an embodiment, the carrier is deionized water.

In an embodiment, the composition is an oil-in-water emulsion. In particular, an oil-in—water emulsion of the invention is more resistant to oxidation than an oil-in- water emulsion comprising fatty acids produced using as an emulsiﬁer such as Tween40 in the absence of oleosin.

The present inventors have found that the artiﬁcial oil bodies of the invention are generally resistant to oxidation Thus, in an embodiment, the artiﬁcial oil bodies and/or composition does not comprise a synthetic antioxidant. Furthermore, in a preferred embodiment the artiﬁcial oil bodies of the invention are oxidatively stable.

In an embodiment, the artiﬁcial oil body consists of oleosin, surfactant, and oil sing fatty acids such as polyunsaturated fatty acids with four or more double bonds.

Apart from being useful in the delivery of a therapeutic agent, artiﬁcial oil bodies of the invention can be used to treat or t diseases due to the well known beneﬁts of the fatty acids, particularly polyunsaturated fatty acids, of the oil bodies.

Thus, in a further aspect, the present invention provides a method of treating or preventing a condition which would beneﬁt from fatty acids, the method comprising stering to a subject with the condition one or more oil bodies of the invention and/or a composition of the invention.

Also provided is the use of one or more oil bodies of the invention and/or a ition of the invention for the manufacture of a ment for treating or preventing a condition which would beneﬁt from fatty acids.

Furthermore, provided is the use of one or more oil bodies of the ion and/or a composition of the ion as a medicament for treating or ting a condition which would beneﬁt from fatty acids.

In an ment of the above method and uses, the condition would beneﬁt from polyunsaturated fatty acids with four or more double bonds.

The oil bodies can be used in a wide y of products. Accordingly, in another aspect the present invention provides a product comprising one or more ial oil bodies, and/or a ition, of the invention. es of such products include, but are limited to, a food or feed product, a drink product, a personal care product, a pharmaceutical product or an industrial product.

In a preferred embodiment, the product is, or comprises, an oil-in-water emulsion.

Also provided is the use of one or more artiﬁcial oil bodies of the invention, and/or a composition of the invention, for the preparation of a product.

In a further aspect, the t invention es a method of preparing a feed, food or drink, the method comprising admixing one or more artiﬁcial oil bodies of the invention, and/or a composition of the invention, with one or more other edible ingredients.

In a r aspect, the present invention provides a method of producing artiﬁcial oil bodies, the method comprising i) obtaining n, surfactant, and oil comprising polyunsaturated fatty acids with four or more double bonds, and ii) mixing the oleosin, the surfactant and the oil to produce the artiﬁcial oil bodies.

In another aspect, the present invention provides a method of producing artiﬁcial oil bodies, the method comprising i) obtaining an extract of a plant comprising n, wherein the extract is plant meal, ii) at least lly purifying protein from the extract, wherein the protein comprises n, and iii) mixing the protein comprising oleosin with surfactant and oil comprising polyunsaturated fatty acids with four or more double bonds to e the artiﬁcial oil bodies.

In another aspect, the present invention provides a method of producing artiﬁcial oil bodies, the method comprising i) obtaining oleosin, surfactant, and oil comprising fatty acids, wherein at least some of the surfactant is not phospholipid, and ii) mixing the n, the surfactant and the oil to produce the artiﬁcial oil bodies.

In another , the present invention provides a method of producing artiﬁcial oil bodies, the method comprising 3O i) obtaining an extract of a plant comprising oleosin, wherein the extract is plant meal, ii) at least partially purifying protein from the extract, n the protein comprises oleosin, and iii) mixing the protein comprising oleosin with surfactant and oil to produce the artiﬁcial oil bodies, wherein at least some of the surfactant is not phospholipid.

In an embodiment, the surfactant is mixed and dissolved in the oil before mixing in the olesoin.

In a further embodiment, the method further comprises, iii) selecting artiﬁcial oil bodies.

The present inventors have also identiﬁed methods of obtaining oleosin for use in the artiﬁcial oil bodies of the invention. The present inventors were particularly surprised to find that a protein extract comprising a highly heterogeneous population of ns with relatively low ties of oleosin (for example about 10% of the total protein) could be used, with the natural afﬁnity of the oil, surfactant and oleosin seemingly being highly efﬁcient at forming the artiﬁcial oil bodies. This is particularly advantageous e it avoids the need to conduct extensive puriﬁcation procedures and provides a vely cheap source of the n. Thus, in a further embodiment, step i) comprises a) obtaining an t of a plant comprising oleosin, and b) at least partially purifying protein from the extract, wherein the protein ses oleosin.

Preferably, step b) comprises 1) ing the pH of the extract to a pH of at least about 11.5, 2) separating 1) to produce a solid and liquid phase and selecting the liquid phase, 3) reducing the pH of the liquid phase to precipitate the proteins, 4) ting 3) to produce a solid and liquid phase and selecting the solid phase, and ) dispersing the solid phase in a carrier.

The present invention also provides an artiﬁcial oil body produced by a method of the invention.

In a further aspect, the present invention provides a method of partially purifying oleosin from a plant extract, the method comprising 1) adjusting the pH of the extract to a pH of at least about 11.5, 2) separating l) to produce a solid and liquid phase and selecting the liquid phase, 3) reducing the pH of the liquid phase to precipitate the ns, 4) separating 3) to produce a solid and liquid phase and selecting the solid phase, and ) dispersing the solid phase in a carrier.

In an embodiment, step 3) comprises reducing the pH of the liquid phase to less than about 7, to about pH 4 to about 7, or about pH 5.5 to about 7.

Preferably, the method of the two above s does not use an organic solvent.

The extract can be from any plant such as plant meal. However, in a preferred embodiment the plant is an d.

In another , the present invention provides a method of partially purifying oleosin from a plant extract, the method comprising 1) adjusting the pH of the extract to a pH of at least about 11.5, 2) separating l) to produce a solid and liquid phase and selecting the liquid phase, 3) reducing the pH of the liquid phase to itate the proteins, 4) separating 3) to produce a solid and liquid phase and selecting the solid phase, and 5) dispersing the solid phase in a carrier, wherein the extract is plant meal.

The present inventors have also found that meal resulting from the tion of oils from plant material, such as the seeds of oilseeds, can be used as a source for oleosin. As an added beneﬁt of the claimed invention, following the partial puriﬁcation of the oleosin the remaining plant extract can be used as an animal feed. Thus, in a preferred embodiment the extract is meal produced following the extraction of oil.

The present invention also provides at least partially puriﬁed n produced by a method of the invention.

Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless speciﬁcally stated otherwise.

The present ion is not to be limited in scope by the speciﬁc embodiments bed herein, which are intended for the purpose of exempliﬁcation only.

Functionally—equivalent products, compositions and methods are y within the scope of the invention, as described herein.

Throughout this speciﬁcation, unless cally stated otherwise or the t 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 ity (ie. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

The invention is hereinafter described by way of the following non—limiting Examples and with reference to the accompanying ﬁgures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS Figure 1: Procedure for the extraction of oleosin from canola meal Figure 2: Electrophoretic anaylysis of oleosin extract from canola meal.

Figure 3: Effect of pH of the extracting medium on oleosin recoveries from canola meal.

Figure 4: Electrophoretic is of oleosin extracted at different pH values. Lane 2 and 3 — pH 10.5; Lane 4 and 5 — pH 11.0; Lane 6 and 7 —pH 11.5; Lane 8 and 9— pH 12.0.

Figure 5: Change in particle size (um) of D[3,2} and d (0.5) for Tween 40 and olesoin emulsions subjected to accelerated auto ion (60°C). Average data from ate measuremnts.

Figure 6: Electrophoretic analysis of e proteins of artiﬁcial tuna oil bodies.

Lanes: M — markers, Raw pH12 —pH12 soluble crude protein extract from canola meal, CPl - protein extracted from ial canola oil bodies made from canola oil and re- solubilised pH 6.5-precipitated canola meal protein, s - protein extracted from artiﬁcial canola oil bodies made from canola oil, re~solubi1ised pH 6.5-precipitated canola meal protein, and phospholipids.

Figure 7: Development of trans,trans,2,4,heptadiena1 during accelerated oxidation of tuna oil emulsions prepared with Tween40 and Oleosin extract, repectively. Average data from duplicate measuremnts. 7A — Oxidation at 60 °C; 7B — Oxidation at 40 °C.

Figure 8: ion of EPA and DHA in tuna oil emulsions made with Tween40 and Oleosin extract (respectively) during storage at 60°C. e data from duplicate measuremnts. 8A — Oxidation at 60 °C; 8B — Oxidation at 40 °C.

Figure 9: Peroxide value (PV) of oils extracted from tuna oil artiﬁcial oil body emulsions and equivalent Tween40 emulsions subjected to accelerated oxidation at 40 Figure 10: Development of trans, trans, 2,4—heptadienal during accelerated e (40° C, air) of tuna oil emulsions stabilised by oleosin, x and sodium caseinate, respectively.

Figure 11: Development of trans, trans, 2,4-heptadienal during accelerated storage (40° C, air) of tuna oil emulsions ed using oleosin in combination with monoacylglycerol (MAG), Tween40 (Tween), and sodium stearoyl lactylate (SSL), repectivley as the secondary surfactant.

KEY TO THE SEQUENCE LISTING SEQ ID NO 1: Brassica napus oleosin (CAA57545.1) SEQ ID NO 2: Brassica napus oleosin Sl—l (ACG69504. 1) SEQ ID NO 3: Brassica napus n SZ-l (ACG69503. 1) SEQ ID NO 4: Brassica napus oleosin S3—l (ACG69513. 1) SEQ ID NO 5: ca napus oleosin S4~l (ACG69507.1) SEQ ID NO 6: Brassica napus oleosin SS-l (ACG69511.1) SEQ ID NO 7: s hypogaea oleosin l (AAZ20276.1) SEQ ID NO 8: Arachis hypogaea oleosin 2 (AAU215001) SEQ ID NO 9: Arachis ea oleosin 3 (AAU21501.1) SEQ ID NO 10: Arachis hypogaea oleosin 5 (ABC96763.1) SEQ ID NO 11: Ricinus communis n 1 (EEF40948.1) 3O SEQ ID NO 12: Ricinus communis oleosin 2 (EEF51616.1) SEQ ID NO 13: Glycine max oleosin isoform a (P295302) SEQ ID NO 14: Glycine max oleosin isoform b (P295311) SEQ ID NO 15: Linum issz'mum oleosin low molecular weight isoform (ABB01622.1) SEQ ID NO 16: Linum usitaz‘issimum oleosin high molecular weight isoform (ABB01624. 1) SEQ ID NO 17: Helianthus annuus oleosin (CAA442241) SEQ ID NO 18: Zea mays oleosin (NP_001105338.1) SEQ ID NO 19: Brassica napus steroleosin (ABM30178. 1) SEQ ID NO 20: ca napus steroleosin SLOl-l 522. 1) SEQ ID NO 21: Brassica napus steroleosin SL02-1 (ACG69525.1) SEQ ID NO 22: Sesamum indicum steroleosin (AAL13315.1) SEQ ID NO 23: Zea mays steroleosin (NP_001152614.1) SEQ ID NO 24: Brassica napus in CLO-1 (ACG69529.1) SEQ ID NO 25: Brassica napus in CLO-3 (ACG69527.1) SEQ ID NO 26: Sesamum indicum caleosin (AAF13743.1) SEQ ID NO 27: Zea mays caleosin (NP_001151906.1) DETAILED DESCRIPTION OF THE ION General Techniques and ions Unless speciﬁcally deﬁned otherwise, all technical and scientiﬁc terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in lipid try, molecular genetics, chemistry, and biochemistry).

Unless otherwise ted, the recombinant protein, cell e, and logical techniques utilized in the t invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), TA. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and ED. Hames (editors), DNA g: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and FM. Ausubel et a1. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley— Interscience (198 8, including all updates until present).

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.

As used herein, the term about, unless stated to the contrary, refers to +/- 20%, more preferably +/- 10%, even more preferably +/— 5%, of the designated value.

Throughout this specification the word "comprise", or variations such as "comprises“ or "comprising", will be understood to imply the inclusion of a stated element, r 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.

Oils and Fatty Acids As used , the term "fatty acid" refers to a carboxylic acid with a long aliphatic tail of at least 8 carbon atoms in length, either saturated or unsaturated.

Typically, fatty acids have a carbon-carbon bonded chain of at least 12 s in length. Most lly occurring fatty acids have an even number of carbon atoms because their biosynthesis es e which has two carbon atoms. The fatty acids may be in a free state (non-esterified) or in an esteriﬁed form such as part of a TAG, DAG, MAG, acyl-CoA (thio-ester) bound, or other covalently bound form.

When covalently bound in an esterified form, the fatty acid is referred to herein as an "acyl" group. ted fatty acids do not contain any double bonds or other functional groups along the chain. The term "saturated" refers to hydrogen, in that all carbons (apart from the carboxylic acid [-COOH] group) contain as many hydrogens as possible. In other words, the omega (0)) end contains 3 hydrogens (CH3-) and each carbon within the chain contains 2 ens (-CH2—). Unsaturated fatty acids are of similar form to saturated fatty acids, except that one or more alkene functional groups exist along the chain, with each alkene substituting a singly-bonded ”-CH2-CH2—" part of the chain with a doubly-bonded "-CH=CH-" portion (that is, a carbon double bonded to another carbon). The two next carbon atoms in the chain that are bound to either side of the double bond can occur in a cis or trans conﬁguration. Apart from double bonds, the acyl chain of the fatty acid may have, triple bonds, acyl side chains such a methyl or ethyl groups, hydroxyl groups or other modiﬁcations known in the art.

Artiﬁcial oil bodies of the invention may include fatty acids such as, but not limited to, stearic acid, palmitic acid, oleic acid, linoleic acid, ct—linolenic acid, or a combination of two or more thereof.

As used herein, the terms "polyunsaturated fatty acid" or "PUFA" refer to a fatty acid which comprises at least 18 carbon atoms in its carbon chain and at least three, more preferably at least four, alkene groups (carbon-carbon double bonds). One aspect of the invention relates to artificial oil bodies sing polyunsaturated fatty acids (PUFAs) with four or more double bonds. Examples of such PUFAs include, but are 3O not limited to, stearidonic acid (SDA, 18:4A6,9,12,15, (03), arachidonic acid (ARA, :4A5,8,ll,14; CO6), eicosatetraenoic acid (ETA, 20:4A8,11,14,17, m3), eicosapentaenoic acid (EPA, 20:5A5,8,11,l4,17; m3), docosapentaenoic acid (DPA, 22:5A7,10,l3,16,19, (o3), hexaenoic acid (DHA, 22:6A4,7,10,13,16,19, (03), as well as mixtures of two or more thereof. The artiﬁcial oil bodies may further comprise other fatty acids such as those described above with, for example, two or less double bonds, triple bonds, side chains such as a methyl group, a hydroxyl group and/or an epoxide group.

The fatty acid may be in a free state sterified) or in an esteriﬁed form such as part of a ceride, diacylglyceride, monoacylglyceride, acyl-CoA bound or other bound form, or mixtures of two or more thereof. The fatty acid may be esterified as a phospholipid such as a phosphatidylcholine, atidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol or diphosphatidylglycerol forms.

The term "glyceride" as used herein encompasses lipids selected from the group consisting of triglycerides, erides, monoglycerides, phosphoglycerides and combinations thereof. More preferably, the glycerides are ed from the group consisting of triglycerides and diglycerides. Most ably, the glyceride is triglyceride oil.

"Triacylglycerol" or (TAG) is glyceride in which the glycerol is esteriﬁed with three fatty acids. In the Kennedy pathway of TAG synthesis, the precursor sn-glycerol- 3-phosphate is esterified by a fatty acid coenzyme A ester in a reaction catalysed by a glycerolphosphate acyltransferase at position sn-l to form lysophosphatidic acid (LPA), and this is in turn acylated by an acylglycerophosphate acyltransferase in position sn-Z to form phosphatidic acid. The phosphate group is removed by the enzyme phosphatidic phosphohydrolase, and the resultant 1,2—diacyl-sn-glycerol is acylated to form the triacyl—sn—glycerol.

In one embodiment, the glyceride (preferably, TAG) content of the oil comprises essentially the all (for e at least 75%, or at least 90%, or at least 95%) of the fatty acids.

The oil comprising fatty acids can be obtained from a wide variety of sources including micro-organisms including yeast, crusteceans, fish, animals and plants. The organism may or may not be genetically modiﬁed.

In an embodiment, seedoil is present in the cial oil bodies. As used herein, the term "seedoil" refers to a composition obtained from the seed/grain of a plant which 3O comprises at least 60% (w/w) lipid, or obtainable from the seed/grain if the seedoil is still present in the seed/grain. That is, seedoil includes seedoil which is present in the seed/grain or portion thereof, as well as seedoil which has been extracted from the seed/grain. The seedoil is preferably ted seedoil. Seedoil is typically a liquid at room ature. ably, the total fatty acid (TFA) content in the seedoil inantly (>50%) comprises fatty acids that are at least 16 carbons in length.

More preferably, at least 50% of the total fatty acids in the seedoil are C18 fatty acids for example, oleic acid, The fatty acids are typically in an esteriﬁed form such as for example, TAG, DAG, acyl-CoA or phospholipid. The fatty acids may be free fatty acids and/or in an esterified form. In an embodiment, seedoil in the artiﬁcial oil bodies is antially purified" or "puriﬁed" oil that has been separated from one or more other lipids (with the exception of phospholipids), nucleic acids, polypeptides (with the exception of oleosin), or other contaminating molecules with which it is associated in the seed or in a crude extract. It is preferred that the substantially puriﬁed seedoil is at least 60% free, more preferably at least 75% free, and more preferably, at least 90% free from other components with which it is associated in the seed or extract. Seedoil may further comprise tty acid molecules such as, but not limited to, sterols and ics. In an ment, the seedoil is canola oil (Brassica napus, Brassica rapa 53p), d oil (Brassz‘ca juncea), other Brassica oil (e. g., Brassz‘ca napobrassz’ca, Brassz‘ca camelz'na), sunﬂower oil (Helianthus amzus), linseed oil (Linum usitaz‘issz‘mum), soybean oil ne max), safﬂower oil (Carthamus tinctorius), corn oil (Zea mays), tobacco oil (Nicotiana tabacum), peanut oil (Arachis hypogaea), palm oil (Elaeis guineensz’s), seed oil (Gossypium hirsutum), coconut oil (Cocos nucifera), avocado oil (Persea amerz’cana), olive oil (01% europaea), cashew oil (Anacardz’um occidentale), macadamia oil (Macadamia intergrt‘ﬁilz’a), almond oil (Prunus amygdalus), oat seed oil (Avena sattva), rice oil (Oryza sativa or Oryza glaberrima), or Arabidopsz’s seed oil (Arabidopsis thaliana). Seedoil may be extracted from rain by any method known in the art. This typically involves extraction with nonpolar solvents such as diethyl ether, petroleum ether, chloroform/methanol or butanol mixtures, generally associated with ﬁrst ng of the seeds. Lipids associated with the starch in the grain may be extracted with water—saturated l.

The seedoil may be "de-gummed“ by methods known in the art to remove polysaccharides or treated in other ways to remove contaminants or improve purity, stability, or colour. The TAGS and other esters in the seedoil may be hydrolysed to release free fatty acids, or the seedoil hydrogenated, d chemically, or enzymatically as known in the art.

As used herein, “marine oil” is oil ed from an organism that lives in salt water, particularly marine microalgae, fish or crusteceans. Thus, an oil for use in the invention may be both a marine oil and a fish oil oil derived from a saltwater fish.

The oil comprising polyunsaturated fatty acids with four or more double bonds may be obtained from any source, such as oil ted from marine micro-organisms, crustaceans, fish or transgenic organisms, such a transgenic plants or yeast, comprising exogenous cleotides which enable the organisms to synthesize polyunsaturated fatty acids with four or more double bonds. Examples of marine micro-organisms from which the PUFAs may be obtained include, but are not limited to, species of Amphidinium, Amp/tor, Asterionella, phz‘a, Ceratz‘um, Chaefoceros, Chlorella, Chroomonas, Cochlodz‘nium, Crisphaera, Crypthecodz‘nium, Cryptomonas, Cycloz‘ella, Cyﬁndroz‘heca, Dum‘ella, Emilianz‘a, arz‘a, Glenodinium, lax, Gyrodinium, Haematococcus pluvialz's, Heteromastix, [sochrysia z’a, Monochrysis, Monodus, Moritella, Mortierella, Nannochloris, Nannochloropis, Navicula, Nitzschia, Odontella, Olisthodiscus, Pavlova, Peridz'nium, Phaeodactylum, Porphyrz'dz'um, Prorocentrum, pedz‘nella, Rhodella, Rhododomas, Schizochytrz‘um, Skelentonema, Stauroneis, Tetraselmis, Yhalassiosz‘ra, Thrautochytrium, or Ulkem'a. Examples of ﬁsh oils useful for the invention include, but are not limited to, tuna oil, bonito oil, sea bass oil, halibut oil, spearﬁsh oil, uda oil, cod oil, menhaden oil, sardine oil, pilchard oil, anchovy oil, capelin oil, Atlantic cod oil, Atlantic herring oil, Atlantic mackerel oil, Atlantic en oil, salmonids oil, shark oil, squid oil, octopus oil, krill oil, seal oil, whale oil, and the like, including mixtures and combinations thereof. es of transgenic organisms producing polyunsaturated fatty acids with four or more double bonds are described in, for example, W0 2005/103253, , W0 2010/023202, and .

As noted above, ques that are routinely practiced in the art can be used to extract and process oils produced by cells, plants, seeds, etc. For e, oil extraction may comprise degumming (or acid treatment), neutralization (or alkali treatment), water washing, bleaching, ﬁltering, deodorising, polishing and/or cooling (or winterization). Preferably the purifying comprises acid treatment and/or alkali treatment (degumming and neutralisation). Alternatively, purifying methods may comprise bleaching and/or deodorization, Preferably, however, the purifying will e ing and/or deodorization, and optimally in addition acid and alkali treatment.

Oleosin Oleosins are hydrophobic proteins present in the ne of oil bodies in the storage tissues of seeds (see, for example, Huang, 1996; Lin et al., 2005; o et al, 2007; Lui et a1, 2009; Shimada and Hara—Nishimura, 201 0), They are of low MW (15-26,000), and are abundant in oilseeds. Within each seed species, there are usually two or more oleosins of different MW. Each Oleosin molecule contains a relatively hydrophilic N-terminal domain (for example about 48 amino acid residues), a central totally hydrophobic domain (for example of about 70—80 amino acid residues) which is particularly rich in aliphatic amino acids such as alanine, glycine, leucine, cine and valine, and an amphipathic oc-helical domain (for example about of about 33 amino acid residues) at or near the inus. Generally, the central stretch of hydrophobic residues is inserted into the lipid core and the amphiphatic N—terminal and/or amphiphatic C-terminal are located at the surface of the oil , with vely charged residues embedded in a tant monolayer and the vely charged ones exposed to the exterior.

As used , the term "oleosin" encompasses caleosins which bind calcium, and steroleosins which bind sterols, as well as polyoleosin (Scott et al., 2010).

However, generally a large proportion, if not all, of the oleosins of artiﬁcial oil bodies of the invention will not be caleosins and/or steroleosins. Furthermore, the term "oleosin" as used herein refers to either a homogenous population of the same oleosin protein, or more lly a heterogenous, population of different oleosin proteins.

A substantial number of oleosin protein sequences, and nucleotide sequences encoding therefor, are known from a large number of different plant species. Examples include, but are not limited to, oleosins from Arabidposis, canola, com, rice, peanut, castor, soybean, ﬂax, grape, cabbage, cotton, sunﬂower, sorghum and barley.

Examples of some of these oleosins are provided in the ce g. Thus, in one embodiment, the oleosin comprises a sequence selected from: a) an amino acid sequence as provided in any one of SEQ ID NOS 1 to 27, b) an amino acid sequence which is at least 50% identical, more preferably at least 60%, more ably at least 70%, more preferably at least 80%, preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more ably at least 99%, identical to any one or more of SEQ ID NOS 1 to 27, and c) a biologically active fragment of a) or b).

In a further embodiment, the oleosin comprises a sequence selected from: a) an amino acid sequence as provided in any one of SEQ ID NOs 1 to 18, b) an amino acid sequence which is at least 50% identical, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, identical to any one or more of SEQ ID NOS 1 to 18, and c) a biologically active fragment of a) or b).

The % identity of a n is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation y=5, and a gap extension penalty=0.3. The query sequence is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More ably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the query ce is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the GAP analysis aligns the two sequences over their entire length.

As used herein, a "biologically active" fragment is a portion of an oleosin that can be used to prepare an ial oil body of the invention. Biologically active fragments can be any size as long as they maintain the deﬁned activity.

In a preferred embodiment, the oleosin is obtained from an oilseed such as, but not limited to, canola (Brassz‘ca spp.), soybean (Glycine max), sunﬂower (Heliamhus animus), oil palm (Elaez‘s guineez‘s), cottonseed (Gossypz'um spp.), groundnut (Arachis hypogaea), coconut (Cocus nucz‘fera), castor (Ricinus communis), safflower amus tinctorius), mustard (Brassica spp. and Sinapis alba), coriander (Coriandrum sativum), squash (Cucurbz‘ta ), linseed/ﬂax (Linum usitatissz‘mum), Brazil nut (Bartholletia excelsa), jojoba (Simmondsia chinensis) or maize (Zea mays).

The oleosin may be obtained from a natural, synthetic or recombinant source.

Advantageously, the oleosin can be obtained from a natural source such as a plant t, particularly the meal of an oilseed following oil extraction. Figure 1 shows an example of a procedure used to extract n from plant meal such as canola meal.

Approximately 50% of the canola meal protein has been reported to precipitate at pH 6.0-7.0 (Manamperi et al., 2010). Canola meal typically contains up to 40% protein derived mainly from seed ar protein. Commercial oil extraction processes of mechanical ing followed by solvent extraction disrupt the canola oil body structure thereby releasing oleosin to the mix of the vely more abundant cellular proteins. As the isoelectric point of canola n is 6.5, the protein precipitated at the pH range 6.0-7 .0 contains oleosin in addition to cellular protein.

The oleosin may also be obtained from a recombinant source, either plants with an exogenous ) encoding an oleosin ing in enhanced levels of oleosin production, or a recombinant organism comprising an exogenous gene(s) but do not naturally produce an oleosin, for example a recombinant yeast. The production of recombinant organisms producing, and/or with enhanced levels of, oleosin is well within the capability of those skilled in the art (see, for example, Roux et al., 2004; Abenes et a1., 1997; Bhatla et al., 2010).

Amino acid sequence mutants of the naturally ing oleosins can be prepared by ucing appropriate nucleotide s into a nucleic acid encoding the oleosin, or by in vitro synthesis of the desired protein. Such mutants include, for example, deletions, insertions or substitutions of es within the amino acid sequence. A combination of deletion, insertion and substitution can be made to arrive at the ﬁnal uct, provided that the ﬁnal protein product ses the desired characteristics. The production and identiﬁcation of functional mutants of naturally occurring oleosins is also well within the capability of those skilled in the art.

Also included within the scope of the ion are proteins which are differentially modiﬁed during or after synthesis, e.g., by biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.

Olesoins may be prepared using techniques known the art or using a method of the invention. In one embodiment, Olesoins are prepared in as a crude extract by 1) adjusting the pH of the extract to apH of at least about 11.5, 2) separating l) to e a solid and liquid phase and selecting the liquid phase, 3) reducing the pH of the liquid phase to precipitate the proteins, 4) separating 3) to produce a solid and liquid phase and selecting the solid phase, and ) dispersing the solid phase in a carrier.

In an alternate embodiment, ﬁltration methods are used to at least partially purify n from plant material.

Surfactants Artiﬁcial oil bodies of the invention comprise one or more tants.

The surfactant may be a non-ionic or anionic surfactant or a combination of two or more thereof. More ularly, the surfactant may be anionic, cationic, zwitterionic or a combination of two or more thereof. For example the surfactant may be selected from the group consisting of, but not limited to, phospholipid, monoglyceride, Perﬂuorooctanoate (PFOA or PFO), Perﬂuorooctanesulfonate (PFOS), Sodium dodecyl sulfate (SDS), ammonium lauryl sulfate, and other alkyl sulfate salts, sodium laureth sulphate, also known as sodium lauryl ether sulfate , Alkyl benzene sulfonate, Soaps, or fatty acid salts, Cetyl trimethylammonium bromide (CTAB), cyl trimethyl ammonium bromide, and other alkyltrimethylammonium salts, Cetylpyridinium chloride (CPC), Polyethoxylated tallow amine (POEA), Benzalkonium chloride (BAC), Benzethonium chloride (BZT), Dodecyl e, Cocamidopropyl betaine, Coco ampho glycinate, Alkyl poly(ethylene oxide), Alkylphenol poly(ethylene , Copolymers of poly(ethylene oxide) and ropylene oxide) (commercially called Poloxarners or Poloxamines), Alkyl polyglucosides, including Octyl glucoside, Decyl ide, Fatty alcohols, Cetyl alcohol CA), Poly(vinyl alcohol)(PVA), Oleyl alcohol, Cocamide MBA, cocamide DEA, Polysorbates such as Tween 20, Tweeen 40, Tween 80, and l dimethylamine oxide.

In one embodiment, the tant is a olipid. However, when the fatty acid is not a polyunsaturated fatty acid with at least four double bonds a preferred embodiment is that the artiﬁcial oil body comprises no olipid or phospholipid is present with at least one other surfactant (for example, the at least one other surfactant comprises at least 5%, or at least 25%, or at least 50%, or at least 75%, or at least 90%, of the total surfactant in the artificial oil body).

Phospholipids are a class of lipids and are a major component of all cell membranes as they can form lipid bilayers. Most phospholipids contain a diglyceride, a phosphate group, and a simple organic molecule such as choline. One exception to this rule is sphingomyelin, which is derived from sphingosine instead of glycerol.

Examples of phospholipids useful for the invention include, but are not limited to, phosphatidylethanolamine, phosphatidylcholine, in, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol and mixtures of two or more thereof.

Phospholipids for tion of oil bodies of the invention may be from s sources, for e, from seeds, and more typically from the oilseeds. These include lecithin and lyso—lecithin. The lecithin may, for example, be obtained from soy (soy in) or sunﬂower (sunﬂower lecithin). Additionally, phospholipids with a modiﬁed fatty acid composition can be used. Such phospholipids can be, for example, enzyme modified soy phospholipids. Enzyme modified phospholipids may be prepared, for example, from soy phospholipids (SLP; True Lecithin, Mie, Japan) by treatment with phospholipase A2 (Novo Industry, Bagsvaerd, Denmark). Additionally, phospholipids with a d fatty acid composition can be obtained from plants or plant seeds that have been genetically modified to produce phospholipids with modified fatty acid compositions. An example of a phospholipid with a modiﬁed fatty acid composition is lecithin with a modiﬁed fatty acid composition. Other methods that are well known in the art may also be used to modify the fatty acid compositions of phospholipids.

The surfactant layer surrounding the lipid core of the oil body y is a monolayer.

Any concentration of the surfactant can be used which allows the formation of the artiﬁcial oil bodies, Artiﬁcial Oil Bodies An used herein, the term icial oil body" refers to a structure, typically between about 0.1 and about 100 pm in size, sing oil surrounded by a surfactant layer with oleosins embedded in the surfactant layer. They are referred to as artiﬁcial because there is no evidence that the oil bodies of the ion exist in nature.

Nonetheless, an "artificial oil body" of the invention can also be referred to as an "oil body".

Typically, an artiﬁcial oil body of the invention comprises about 90% to about 98.5% glyceride, about 02% to about 5% surfactant, and about 0.5% to about 5% protein. For example, a typical artiﬁcial oil body of the invention comprises an oil/ surfactant/oleosin ratio of approximately 97 5/10/15. As shown herein, reducing the concentration of oleosin results in increased size of the oil body.

In an embodiment, at least 10%, more preferably at least 20%, more preferably at least 30%, of the fatty acids are polyunsaturated fatty acids with four or more double bonds. For example, Hi—DHA tuna oil produced by Numega has about 37% PUFAs, whereas algal oil DHASCO produced by Martek has about 61% PUFAs.

In another embodiment, at least 10%, more preferably at least 20% of the fatty acids are DHA, For example, Hi~DNA tuna oil produced by Numega has about 26% DHA, whereas algal oil DHASCO produced by Martek has about 40% DHA, The oil bodies can be obtained by ing a pre-emulsion by mixing oil, surfactant and oleosin with water (for example) using an tic stirrer operated at 800mm for 30 minutes. Advantageously, the oleosins can be ed as a protein extract from plants obtained using a method of the invention. In an embodiment, the oleosin is stored as a , for example as produced by freeze drying, before being used in the methods of the ion.

Artiﬁcial oil bodies of the ion are oxidatively stable. For example, less than 40%, more preferably less than 30%, more ably less than 20%, and even more preferably less than 15%, of the fatty acids, such as total concentration of EPA and DHA, in the artificial oil bodies are depleted when the oil bodies are stored at 40°C for 15 days. In another example, following storage at 40°C for 15 days, the oil bodies comprise less than 6, more preferably less than 5, and even more preferably less than 4 ug/ml trans,trans-2,4-heptadienal. In yet another example, less than 90 meq/kg oil, more preferably less than 50 meq/kg oil, more preferably less than 25 meq/kg oil, of hydroperoxides are produced after 5 days at 40°C.

Products Comprising Artiﬁcial Oil Bodies and Uses Thereof The oil bodies can be used in a wide variety of ts. Examples of such products include, but are limited to, a food or feed product, a drink product, a personal care product, a pharmaceutical product or an industrial t. ses for the production of these products are well known in the art.

In one preferred embodiment, the subject invention is ed to ts which may be ingested by animals and/or humans. Since, these compositions may be ingested they must be of food grade quality. The particular product and the particular form in which the oil bodies are applied, however, is not of critical importance and may be as desired. Examples of a food, feed or drink product, include, but are not limited to, a non-dairy substitutes, a non—dairy cheese, a non—dairy yoghurt, a margarine, a mayonnaise, a vinaigrette, an icing, cream, soup, ice creams (for example as a sation agent), a salad ng, amustard, a candy, bechamel sauce, chewing gum, a pudding, a baking product, a condiment, a juice (for example as a lly clouding agent), baby formula, a ﬂavour carrier, a texturing agent, ﬁsh food, d and livestock feed.

Examples of a personal care t include, but are not limited to, a soap, a cosmetic product, a skin cream, a facial cream, a tooth paste, a lipstick, a perfume, make-up, a foundation, a blusher, a mascara, an eyeshadow, a sunscreen lotion and a hair care product.

Examples of a pharmaceutical product include, but are not limited to, a therapeutic agent, a diagnostic agent and a delivery agent.

The therapeutic or diagnostic agent can be molecule that one wishes to deliver to a host. In one embodiment, the active ingredient may be a protein or peptide that has therapeutic or diagnostic value. Such peptides include antigens (for e formulations), antibodies or antibody-related molecules, cytokines, blood clotting factors and growth es.

Examples of an industrial product e, but are not limited to, a paint, a coating, a lubricant, a ﬁlm, a gel, a drilling ﬂuid, paper sizing, a latex, a building or road uction material, an ink, a dye, a wax, a polish and an agrochemical formulation.

Artiﬁcial oil bodies of the invention can be used to treat or prevent diseases due to the well knovm beneﬁts of the fatty acids in the oil bodies of the invention. Thus, in a further aspect, the t invention provides a method of treating or preventing a condition which would beneﬁt from fatty acids, in particular a condition which s from polyunsaturated fatty acids with four or more double bonds, the method comprising administering to a subject with the condition one or more oil bodies of the invention and/or a composition of the invention. es of such conditions include, but are not limited to, cardiac hmia’s, angioplasty, inﬂammation, asthma, psoriasis, osteoporosis, kidney stones, AIDS, multiple sclerosis, rheumatoid arthritis, IO Crohn’s disease, schizophrenia, cancer, foetal alcohol syndrome, attention deﬁcient hyperactivity disorder, cystic fibrosis, phenylketonuria, unipolar depression, aggressive hostility, adrenoleukodystophy, coronary heart disease, hypertension, diabetes, obesity, Alzheimer’s disease, chronic obstructive pulmonary disease, ulcerative s, restenosis after angioplasty, eczema high blood pressure, platelet aggregation, gastrointestinal bleeding, endometriosis, premenstrual syndrome, myalgic encephalomyelitis, chronic fatigue after viral infections or an ocular disease.

In an embodiment, the oil bodies or composition is provided by oral administration. Typically, the oil bodies are administered in an amount that is sufﬁcient to deliver at least 10 mg of fatty acid such as omega-3 PUFA per administration event. More preferably, at least 30 mg, even more preferably at least 50 mg and most preferably at 100 mg of of fatty acid such as 3 PUFA is delivered per stration event.

It will be understood that the present invention may suitably be employed to treat animals, such as mammals. Most ably, the present invention is used to treat .

In a red ment, the product is, or comprises, an oil—in—water emulsion. Such emulsions of the invention are physically and oxidatively , and do not coalesce on standing.

The oil bodies may be formulated into an on using techniques known in the art. Preferably, at least one additional ingredient is added to the oil body preparation. The additional ingredient may be added as a solution, suspension, 21 gel or solid and ties of the additional ingredient will depend on the formulation. The additional ingredient may upon formulation become associated with the oil bodies, remain suspended in solution, or form a suspension in which the oil bodies are dispersed. The ingredient may also penetrate the surfactant layer surrounding the oil body. Ingredients which may penetrate the oil body include oils, waxes and the colorant Nile Red. In a preferred embodiment, the additional ingredient is a liquid phase. In a further preferred embodiment the liquid phase is water.

Water may be added either directly or through moisture associated with another ient. The ﬁnal amount of water is not critical, as long as upon mixing of the ingredients, a stable emulsion is formed. Generally, the compositions will contain at least 1% of water and up to 99% water. Usually mixing will be required to provide an adequate emulsion and it may be necessary to apply heat or pressure.

In another preferred embodiment, the additional ingredient is an oil or a wax.

Oils or waxes may partition within the oil bodies and in this manner lipid soluble ingredients, such as lipid soluble Vitamins may be red to the oil body. Where oils or waxes comprise the added ingredient, the oil bodies may remain suspended in the lipophilic phase or double emulsions may be formed.

The ﬁnal compositions may be in solid or in liquid form or of any other desired viscosity. The on may be thickened using gelling agents such as cellulose and derivatives, Carbopol and derivatives, carob, carregeenans and derivatives, xanthane gum, sclerane gum, long chain alkanolamides, and bentone and derivatives, typically present in concentrations less than 2% by weight.

The emulsion may r comprise surfactants to wet, foam, penetrate, emulsify, solubilize and or se a selected material. For example anionic surfactants such as sodium t monoglyceride sulphonate, cationic surfactants, such as lauryl hyl um chloride, cetyl pyridinium chloride and trimethylammonium bromide, nonionic surfactants including ics, and polyethylene oxide condensates of alkyl s, and rionic surfactants such as derivatives of aliphatic quaternary ammonium, phosmomium and sulphonium compounds may all be added as required.

Chelating agents, e of binding metal ions, such as tartaric acid, EDTA, citric acid, alkali metal es, pyrophosphate salts or anionic polymeric polycarboxylates may be also included in the emulsion formulation as desired.

Generally, the emulsion formulations will be treated such that contamination by bacteria, fungi, mycoplasmas, Viruses and the like or undesired chemical reactions, such as oxidative reactions are prevented. In red embodiments this is accomplished by the addition of preservatives, for e sodium metabisulfite or other chemical additives or by irradiation, for example by ng radiation such as cobalt-60 or cesium-137 irradiation or by ultraviolet irradiation.

In addition, active agents may be added. For example, cosmetic compositions may be formulated as stable suspensions using the present emulsion formulation and vitamins and moisturizing agents may be included in skin creams. One particularly ageous way in which an active ingredient may be included in emulsions of the subject ion, is through construction of oleosin gene fusions as detailed in WO 96/21029. These fusion proteins are created by genetically linking the gene encoding oleosin to a gene encoding a peptide or protein of interest. Expression of the fusion gene, in for example an oilseed plant, results in synthesis of a fusion protein which is then used to produce artiﬁcial oil bodies of the invention. In principle any desired protein or peptide may be ed using this technology, including polar ﬁsh antifreeze peptides or a therapeutic protein may be produced as an oleosin fusion.

An emulsion with film forming properties may also be formulated. Such an emulsion when it is applied to a surface and dried forms a coating. An example of an emulsion where a coated oil body ﬁlm is applied is in ﬁsh food, where oil bodies (for example produced from lgae oil) may be applied to the ﬁsh food to enhance the dietary value. A ﬁlm forming on is particularly useful in ments of the present invention where lled release of an active ingredient is desirable such as in ry of pharmaceuticals or volatiles such as fragrances. The release time of the active agent from a ﬁlm of emulsion, which occurs during drying, depends, among other s, on the thickness of the ﬁlm. When a thicker coating is applied a longer drying time will result in a slower release of the active agent. In variant contemplated formulations, release of the agent occurs only when the ﬁlm is dry.

Other factors, such as the composition of the emulsion and the type and concentration of the active ingredient also determine the characteristics of release. For example, cosolvents, such as ethanol, may be included in the formulation and inﬂuence the e time. Release of an active ingredient is also desirable in food applications, where a nt entrapped in an emulsion is released during consumption. The release of the ﬂavorant, depending on the exact formulation of the emulsion, may elicit a sudden intense sensation or a more subtle blend of ﬂavours and essences.

The emulsion formulation may also be used in sprays and aerosols. Volatiles, such as l and fragrances may be included in these sprays. Emulsions of this type may also be sprayed onto the surface of dried food preparations such as potato chips and dried soup. The on might include a flavorant and add preservative value or assist in maintaining the appropriate moisture levels of the food.

The stability of the present on formulation may be exploited in formulations of acid emulsions. For example, the emulsion ation may be used in the preparation of a mayonnaise-like food product, which besides the oil body preparation comprises a vegetable oil, mustard, vinegar and egg yolk, if desired.

Pourable emulsions, such as salad dressings may be prepared by increasing the relative amount of vinegar and/or by the addition of water.

An example of an application where heat may be d without nt deleterious effects, is in the preparation of a savory sauce such as a bechamel sauce or in sweet sauces such as chocolate sauces. In these ations, the oil body preparation is employed as a frying substitute. To prepare a el sauce, to 1 part of the heated oil body preparation, 1 part (w/w) of ﬂour is added and stirred until a thick suspension is formed. At moderate heat milk is gradually added until a sauce with a desired viscosity is obtained.

The emulsion formulation may also be used as a butter substitute. In this application, small amounts of water are added to the oil body preparation, for example, less than 10% until a desired viscosity is obtained. Natural butter ﬂavours and thickeners may be added as desired. The butter substitute may be used on sweet corn, bread, in cake mixes or bread making. Salt, which contributes ﬂavour and acts as a preservative may be added typically to a level of about 2.5% (wt/vol). Colour agents, for example, extracts of annatto seed or carotene may be added to deepen the colour as desired. An age of this application is that the oil body based butter does not se enated fatty acids, which are used in the formulations of ines and the like to achieve a desirable consistency, but are also with associated with cardiovascular diseases.

Shortenings may be prepared to various degrees of stiffness, from a foam to a pourable shortening. In this ation, air is beaten into the emulsion ation and the emulsion formulation can be considered to be dispersed into the continuous phase, air. Shortenings may be applied to mixes where creaming and ﬂufflng are desired.

These mixes include icings, synthetic creams, ice creams and cake batter.

An imitation fruit juice may be prepared from artiﬁcial or natural ﬂavours and nutrients. Such imitation juices do not have the correct appearance and due to transparency appear to be weak or diluted. By adding a small amount, for example 0.1 to 1% (v/v) of the oil body preparation or an emulsion thereof clouding may occur to 3O give the juice a rich ance. Thus the present oil body preparation may be used as a clouding agent. In another application involving juices, the oil body preparation or an emulsion thereof may be added to juices with settleable solids, such as tomato juice.

Adding a small amount of the oil body ation, for e 0.1 to 1% (v/v), may decrease the rate of settling of the solids in the juice and assist in maintaining the rich appearance.

Topical applications of the oil body preparation of the present invention are also envisaged. In this embodiment the on is formulated as a dermatologically acceptable emulsion, which may for example be employed to rize facial and/or body skin, including nails and lips or may have properties to combat ageing of the skin, acne, pigmentation, hair loss, or promote hair removal or facilitate wound healing and/or restructuring of the skin tissue. The oil body ation represents preferably l—99% by weight of the ﬁnal composition.

The ic compositions of the present invention may comprise additional hydrocarbon compounds such as plant, animal, mineral or tic oils or waxes or mixes thereof. They comprise parafﬁn, petrolatum, perhydrosqualene, arara oil, almond oil, calphyllum oil, avocado oil, sesame oil, castor oil, jojoba oil, olive oil, or cereal germ oil.

Esters may be included such as esters of lanolic acid, oleic acid, lauric acid, stearic acid, myristic acid. It is also le to include alcohols for example, oleoyl alcohol, linoleyl alcohol or linolenyl alcohol, isostearyl alcohol or octyl dodecanol, alcohol or polyalcohol.

Further hydrocarbons which may be included are octanoates, decanoates, ricinoleates, caprylic/capric triglycerides or C10 to C22 fatty acid triglycerides.

Addition of these agents may result in the formation of double emulsions.

Hydrogenated oils, which are solid at 25°C, such as hydrogenated castor oil, palm oil or coconut oil, or hydrogenated tallow; mono- di- tri- or sucroglycerides; lanolins; and fatty acids which are solid at 25°C may also be included in the cosmetic formulations of the present ion. Among the waxes which may be included are animal waxes such as beeswax; plant waxes such as carnauba wax, candelilla wax, rry wax, Japan wax or waxes from cork ﬁbres or sugar cane; mineral waxes, for e paraffin wax, lignite wax, microcrystalline waxes or ozokerites and synthetic waxes.

Pigments may be included and may be White or coloured, inorganic or organic and/or paerlescent. These pigments se titanium dioxide, zinc oxide, ziriconium 3O dioxide, black, , red and brown iron , cerium dioxide, chromium oxide, ferric blue, carbon black, barium, strontium, calcium and aluminum lakes and mica coated with titanium oxide or with bismuth oxide.

Active ingredients commonly employed in skin creams, such as vitamins, for example as n A or C and alpha hydroxy acids, such as citric, glycolic, lactic and tartaric, into cosmetic and/or dermatological compositions may be included. For example, US 183 teaches that vitamin C or ascorbic acid promotes growth of connective tissue, particularly in the skin strengthens the skin against external aggressions such as from smoke and UV ion. Moisturizing agents which may be included in skin creams and cosmetics are for example mineral oil and urea.

Antioxidants such as the naturally occurring tocopherols and polyphenols, or butylated hydroxytoluene and hydroxyanisole may also be also added. Sunscreens such as octyl methoxycinnamate (Parsol MCX), 3- benzophenone (Uvinul M40) and butylmethoxydibenzoylmethane (Parsol 1789) may be employed to prepare a sun tanning lotion. Pharmaceutically active ients which may be used to formulate cosmetic compositions include for example antibiotics, fungicides and ant- atory agents.

The ﬁnal cosmetic product may be in the form of a free, poured or compacted powder (foundation, blusher or eyeshadow), a relatively greasy product such as lipstick, mascara, or an oil or lotion for the body or face.

The oil body preparation may also be used to serve as an orally acceptable carrier in toothpaste which may further comprise silicas, surfactants, chelating agents, a ﬂuoride, thickeners, sweeteners, nts, for e as oil of mint, enzymes and biocides.

An example of an industrial t which may be formulated is paint wherein the main resin, such as those based on silicone type nds, acrylic compounds, polyester, akyd, ﬂuorine, epoxy, polyurethane may be partly or entirely replaced by the oil body preparation of the present invention. Further additives such as pigments, dyes, glass ﬂakes, and aluminum ﬂakes, pigment dispersants, ners, levelling agents, hardening sts, hardening agents such as dioisocyanates, hardening catalysts, gelling inhibitors, ultraviolet absorbing agents, free radical quenching agents. etc. may be ated in paint compositions as required.

The oil body preparation may also be to formulate lubricants. For example, the oil body preparation may be used to partially or entirely e the lubricating oils such as animal oils, ble oils, petroleum lubricating oils, synthetic ating oils, or the lubricating grease such as lithium grease, urea grease and calcium grease. Other compositions employed in a lubricant formulation comprise antioxidants, detergent dispersants, oilness agents, on modifiers, viscosity index improvers, pour point depressants, solid ant material, rust inhibitors and antifoamers.

Waxes may also be prepared using the oil body preparation of the present invention. These comprise rinse-wax types, such as those providing a stable hydrophobic ﬁlm-ﬁnish onto automobiles and other protective coatings. Other compositions used in the preparation of a wax comprise surfactants, mineral oils, such as mixed inic and aromatic/naphtenic oils, perfumes, biocides, colouring agents which may be added in compatible amounts as desired.

EXAMPLES Example 1 ~ Materials and Methods Canola (Brassica napus) seed, oil, and meal were gifts by l Australia The meal was the residue after canola oil tion by the usual industrial process of mechanical ing followed by solvent tion. On arrival, the seeds were sieved to remove straw and other non—seed material and stored at 4°C until required. The seeds contained 38.0 % oil (information provided by Cargill, Ltd) and 25.2% proteins as determined by LECO® FP—ZOOO analysis. Canola oil was of reﬁned, bleached and deodorized grade, and had TBHQ (200 ppm) antioxidant added. Winterized tuna oil (HT303-4) was obtained from LYSI HF (Reykjavik, Iceland).

Extraction of oil canola bodies Natural oil bodies were extracted from canola seeds according to the method described of Tzen (1993) with the exception that the ﬁnal hexane washing step was omitted. In brief, the seeds were soaked in sodium phosphate buffer (pH 7.5) overnight and homogenized with the grinding medium (10 g dry seeds per 50 ml) for 40 seconds using a kitchen blender (Sunbeam lender, 650W). The grinding medium contained 0.6 M sucrose and 10 mM sodium phosphate buffer (pH 7.5). The homogenate was ﬁltered through three layers of cheesecloth. The e was placed in centrifuge tubes, an equivalent amount of ﬂotation medium (grinding medium containing 0.4 M instead of 0.6 M sucrose) was layered on top, and the tubes were centrifuged at 5,000 g for 60 min at 10°C in a swinging-bucket rotor (Beckman J6- HC).

The oil bodies collected at the top were resuspended in twice their volume of detergent washing solution containing een—20, 0.2 M sucrose, and 5 mM sodium phosphate buffer pH 7.5. The pension was placed at the bottom of centrifuge tubes and 10 mM sodium phosphate buffer pH 7.5 was layered on top in the ratio 1:1, and the tubes were centrifuged. The oil bodies on top were collected and resuspended in twice their volume of ionic elution buffer (grinding medium additionally containing 2 M NaCl). The suspension was placed at the bottom of centrifuge tubes, ﬂoating medium (grinding medium containing 2 M NaCl and 025 M instead of 0.6 M sucrose) was layered on top (ratio 1:1), and the tubes were centrifuged. The oil bodies on top were collected and resuspended in their volume of 9 M urea. The re-suspension was vigorously shaken at room temperature for 10 min, then placed at the bottom of centrifuge tubes, 10 mM sodium phosphate buffer pH 7.5 was d on top (ratio 1:1); and the tubes were centrifuged. The oil bodies on top were collected and resuspended in their volume of grinding medium. The resuspension was placed at the bottom of centrifuge tubes, ﬂotation medium was layered on top (ratio 1:1), and the tubes were centrifuged. The oil bodies on top were collected and ended with grinding medium to give a concentration of about 100 mg/ml.

Isolation of n from canola oil bodies Oleosin was extracted from the purified canola OBs dried to 14% (w/w) moisture content. The oil body preparation was redispersed in a solution of deionised water and ethanol (60% ethanol) and stirred for lit and the ethanol was removed using a rotary evaporator (BUCHI Rotavapor R-124). The emulsion was then centrifuged at 5,000 g for 30 min in a swinging-bucket rotor at 20°C (Beckman Jé-HC). The top layer ning water and traces of ethanol was removed and the proteins redispersed in deionised water at a concentration of 100 g/L. This aqueous n dispersion was stored at -18 °C until required, Protein characterisation The proteins isolated from canola oil bodies (COBs) were characterised according to their molecular weight by electrophoresis. The electrophoresis was performed using NuPAGE® gel 4-12% BT 1.0 gel (NuPAGE® invitrogen) according to supplier’s recommendations. In brief, the concentration of protein samples was adjusted to 1 mg/ml and 25 pl placed in eppendorf tubes. Then, 10p] of ® LDS sample buffer (4X) and Sul ofNuPAGE® reducing agent (10x) were respectively added and the tubes centrifuged for 3 seconds at 14,000 rpm at room temperature (Eppendorf Centrifuge 5415C) before being heated to 70°C for 10 s. Markers (Mark 12TM Unstained Standards) were ed the same way except that their concentrations were not adjusted. Finally, 10 ul amounts of both markers and samples were added to the gel already placed in an electrophoresis cell (Novex Mini-Cell, Invitrogen) containing NuPAGE® Running Buffer and anti-oxidant. The cell was connected to a generator (BIORAD Power Pac 300) set at 200 Volts and 400 milliampers for 35 min.

Once the electrophoresis was complete, the gel was washed 3 times in deionised water and placed in a plastic container with BlueTM SafeStain (Invitrogen) for 2 h under slow agitation, 42 rpm (RATEK Platform Mixer model 0M6). Afterwards, the gel was washed twice more with deionised water and placed in deionised water for 2 hours in order to remove non-protein bound stain, and photographed with a G:BOX (SYNGENE) and the GeneSnap 7.07 software (SYNGENE).

Extraction of oleosin from canola meal Oleosin was extracted from canola meal according to a modiﬁcation of the procedure of Ghodsvali et a1. . In brief, canola meal (1.0 kg) was soaked in deionised water (100 L) and the pH ed to 12.0 with aqueous sodium hydroxide (5%, w/w) and mixed for 10 minutes using an Ultraturax mixer. After standing for 10 minutes, the contents were mixed for a further 10 minutes. The resulting slurry was centrifuged at 4,000 g in a swinging-bucket rotor for 30 min at 20°C (Beckman J6- HC). The pH of the atant containing crude protein adjusted to pH 6.5 (isoelectric point of oleosin) when protein precipitation occurred. The stirring was continued for a further 30 min before centrifuging at 4,000 g when the supernatant and the precipitate were recovered. The precipitate was washed with pH 6.5 water using Ultraturax and centrifuged again for 30 min at 4,000 g. The washed precipitate was recovered as a paste and stored in the wet form at -18 °C until required.

Construction of artiﬁcial oil bodies In intial experiements ial oil body dispersions containing canola oil and tuna oil, respectively, were prepared using oleosin extracts variously obtained from canola meal, both in the presence and absence of phospholipid. First, a pre—emulsion was prepared by mixing oil and water containing the proteins using an automatic stirrer (RZR 2051 Control from Heidolph) operated at 800rpm for 30 s. When phospholipids were included, they were dissolved in the oil prior to emulsiﬁcation.

The weight ratios of the ingredients used resembled their ratios in natural canola OBS.

Thus, the preemulsions contained 1.5% of ns, 1% of phospholipids if required, and 10% of oil in order to achieve a particle size between 0.5 and 2am and oil droplets 3O well covered by proteins and phospholipids. The pre-emulsion was homogenized (RATEK Pilot Lab Homogenizer) three times at 500 bars.

In later experiments artiﬁcial oil body sions ning canola oil and tuna oil, respectively, were prepared using surfactants and oleosin extracts sly obtained from canola meal. When phospholipids or other surfactants were ed, they were mixed with the oil prior to emulsiﬁcation. First, the pH of the oleosin extract was adjusted to approximately 8.0 by dropwise addition of 40% aqueous sodium hydroxide while being subject to urax mixing. Oil (10 g), oleosin extract (1.5 g), tant (various) and deionized water (87.5 g) were placed in a glass beaker and warmed in a water bath (60 °C) before being mixed for 2 min using a Silverson mixer under a t of nitrogen to minimize oxidation. The mixture was homogenized (EmulsiFlex C5) twice at 1000 bar under nitrogen. The weight ratios of the ients used resembled their ratios in natural canola OBs. Thus, the preemulsions contained 1.5% of proteins, 1% of surfactant if required, and 10% of oil.

The oleosin extract from canola meal was “worked up” prior to emulsion preparation as follows. The stored extract was allowed to thaw and mixed using an Ultraturax mixer 40% (w/W) while adding sodium hydroxide dropwise until the pH reached 8.0.

Preparation of control emulsions using Tween40 emulsifier % (w/w) o/w emulsions were prepared with canola and tuna oils using Tween40 (6%, w/w) as the emulsiﬁer. Emulsification was med as above. al characterisation of artiﬁcial oil bodies The physical stability of the AOBs was studied over 12 days at 4°C, ambient temperature (approx. 20°C), 45°C and 60°C respectively, respectively. Particle size of the emulsions was measured with a MASTERSIZER 2000 (Malvern) using a Malvern Small Volume Sample Presentation Unit combined with a Hydro 2000g module. (particle R1 : 1,456 / absorption : 0,001 / sant R1 : 1,330 water) Light microscopy photos were obtained with a light microscope Olympus BH-2 linked to a Color View IIIu camera and sed using AnalySIS getIT 5.0 software.

Aggregation of particles was ed by treatment with sodium l sulfate (SDS). This was performed by mixing three volumes of the emulsion with one volume of a 10% (w/w) solution of SDS for 10 min using a magnetic stirrer at room temperature followed by reanalysis.

Oxidative stability of oil in water emulsions prepared from oil bodies The oxidative stability of the AOB dispersions were evaluated from the depletion of EPA/DHA, as well as the amount of trans, trans-2,4-heptadienal produced during storage under accelerated oxidation conditions. For the latter purpose samples (2 g) of the emulsions were placed headspace vials (10 ml) and sealed with silicone- lined aluminium caps: and heated at 40 °C or 60 °C inside a dark oven over 15 days.

Samples were withdrawn daily and immediately analyzed for 2,4-heptadiena1 by solid phase micro extraction and gas tography-mass spectrometry as below. ive volatile compounds generated during the accelerated oxidation of AOB emulsions were determined by headspace solid phase micro extraction (SPME) using a DVB/CAR/PDMS fiber, (50/30 um, Supelco, Sydney, Australia). The ﬁber was inserted into the sample headspace and the vials were incubated at 60 °C for 15 min and then withdrawn and transferred to the GC injector (operated in the splitless mode) and held for 7 min to desorb the extracted volatile compounds into the GC column. The entire series of events was performed using a Combi PAL Auto Injector (CTC Analytics, Zwingen, Switzerland). GC-MS was med using an Agilent Model 6890 GC and Model 5973 MSD (Palo Alto, CA) ﬁtted with a VOC fused silica capillary column (60 mm, 0.32mm id, 0.18 mm ﬁlm thickness, t, rne, VIC, Australia). The GC oven was programmed from 40 °C increasing to 220 °C at the rate of 22 ° C.min -l and held at that temperature for further 14 minutes. Helium was used as the carrier gas at a constant ﬂow rate of 2.0 mL min-1. The or was initially operated in the ess mode and then switched to the split mode (1 :20) 2 min after sample injection. The temperature of the injector and the MS or were both held at 230°C. The MS was operated in scan mode (29-350 amu). Data analyses were performed using Chemstation re and nds were identified by standards as well as reference to a library of spectra (Wiley 275). Volatile compounds in the emulsions were quantiﬁed by using ation curves established from their corresponding fresh emulsions added with different levels of volatile compound 2,4- heptadienal (trans, trans) standards.

Depletion of ALA a) EPA and DHA (tuna) during storage was measured by fatty acid analysis using capillary gas chromatography. For this purpose lipids were extracted from the stored emulsions using iso-propanol/hexane and converted to methyl esters by potassium hydroxide catalysed transesterifcation prior. The esters were analysed on a BPX-70 capillary column and detected by ﬂame ionisation detection. 3O Example 2 — Extraction of oleosin from canola meal Figure 2 shows electrophoresis results for the protein isolated from canola meal by alkaline extraction (pH 12) followed by precipitation at pH 6.5 and water washing.

The molecular weight of oleosin has been reported to in the range 18-20 kDa, and the above isolate contained significant amounts of protein in this molecular range confirming the presence of oleosin. Precipitation of protein at different pH values in the range 3.0 — 12.0, and subsequent electrophoresis showed that the maximum oleosin recovery was obtained at pH 6.5. Previous studies by other workers have focussed on isolation of oleosin from pre-extracted oil bodies after they have been destabilised by treatment of ethanol or mixtures of chloroform and ol. To the best of our knowledge, no attempts have been made previously to isolate oleosin from oilseed or meal by aqueous extraction. This could be because oleosins are considered insoluble in water regardless of the pH (Beisson et al., 2001). The apparent water bility could be the result of the tendency of oleosin to agglomerate on drying; and once this happens it is hard to dissolve or disperse oleosin in water.

Following the intial ments, optimisation of extraction of oleosin from canola meal was conducetd. The highest oleosin yield was obtained at pH 12.0 with signiﬁcantly lower recoveries obtained as the pH was reduced (Figure 3). pH 12.0 also furnished the purest oleosin extracts as shown by SDS-PAGE (Figure 4), Lower pH values tended to extract increasing amounts of two lower molecular weight components of approximately 12 and 5 kDa, Example 3 — Particle size of artiﬁcial oil bodies To eliminate any effects of particle size on the rate of oxidation, the le size of the two emulsions (Tween40 and oleosin) were matched as far as possible (Figure ). It was also important to ensure that no separation occurred and the particle size remained relatively constant during storage for the accelerated oxidation study. The droplet size of AOBS made with oleosin as well as that of the 0 on remained unchanged during e over 12 days at 60°C. The uniformity of t size during storage at elevated atures enabled accelerated oxidation studies based on ace analysis.

Clearly, the pH 12-soluble extract was primarily composed of cellular protein.

The fraction precipitated also would have contained relatively large amounts of non- oleosin protein. Interestingly, r, when AOBs were prepared using either the pH 12-soluble extract or the pH 6.5-precipitated protein re-solubilised at pH 12, there appeared to be a l selection of oleosin to form the oil body surface (Figure 6).

This will have the advantage of not requiring further puriﬁcation of the precipitated protein to enhance the oleosin t prior to preparation of AOBs.

Example 4 —— Oxidative stabiliﬂ The oxidative ities of aqueous dispersions of canola and tuna oil AOBs prepared with re-solubilised pH 6.5 precipitated protein both in the absence and presence of PL were compared with a equivalent emulsions prepared with the corresponding oil and Tween40 emulsiﬁer. For this purpose, the ons were stored at an elevated temperature (60°C) and the ion levels were measured by three different methods, i.e. primary oxidation products (hydroperoxides), secondary oxidation products (trans,trans-2,4-heptadienal), and depletion of unsaturated fatty acids (linolenic acid for canola and DHA for tuna oil). The hydroperoxide levels of the ACE emulsions were signiﬁcantly lower than that of the corresponding Tween40 on throughout the storage period tested, showing the greater oxidative stability of the ACE emulsions (data not shown).

The concentration of 2,4-heptadienal in the tuna oil emulsion made with the n extract was signiﬁcantly lower than that made with Tween40, showing the greater oxidative ity of the oleosin emulsion (Figure 7). This showed that antioxidant effect of canola oleosin is not limited to canola oil but is extended to ﬁsh oil such as tuna oil. Additional evidence in support of this was provided by fatty acid data. The EPA and DHA contents of the tuna oil on made with n extracted from canola meal depleted more slowly than those of the Tween40 emulsion (Figure 8), with greater than 90% of DHA remaining after 5 days at 60 °C ed to less than 70% for the Tween40 on.

Figure 9 shows the rate of development of eroxides (as represented by PV) during accelerated oxidation (40 °C, exposed to air) of the oleosin- and Tween 40- stabilised emulsions. When subjected to accelerated oxidation, the PV of the Tween 40-stabilised emulsion rose sharply without an apparent induction period reaching a value of 120 meq/kg oil after 10 days; the PV gradually declined beyond this point which can be explained as been due to the rate of formation of hydroperoxides falling below the rate of their degradation in to ary oxidation products such as aldehydes and ketones. The PV value of the oleosin-stabilised emulsion did not approach the PV maximum of the Tween 40-stabilised emulsion (120 meq/kg) even after 30 days accelerated at which point the experiment was terminated. The result demonstrates the superior oxidative stability of the oleosin-stabilized emulsion over the Tween 40-stabilized emulsion. 3O The ial oil bodies (AOBs) produced from tuna oil and n dispersed easily in water to form tuna -water emulsions. The emulsions did not separate when stored for 1 week at 4°C irrespective of the secondary surfactant used (phospholipid, monoglyceride or sodium stearoyl lactylate, SSL). In contrast, the emulsions produced from tuna oil and Isolexx (canola protein isolate) with and without phospholipid were unstable and separated within hours of preparation. At 40 °C, the sodium caseinate emulsion was the most stable showing no separation after 1 week.

The n-stabilsed emulsions containing phospholipid or SSL were practically unseparated after 1 week at 40 °C.

Example 5 — Emulsification and antioxidant efficacy of oleosin relative to other protein ingredients Comparisons were made with sodium ate, which is widely used as an emulsiﬁer as well as “Isolexx”, which is a canola protein e produced in Canada and being promoted as a food-grade emulsiﬁer. The following formulations were tested for physical stability and oxidative stability. 1. Tuna oil + oleosin + phospholipid 2. Tuna oil + Isolexx (canola—based protein emulsifier marketed by BioExx, Canada) 3. Tuna oil + lsolexx + phospholipid 4. Tuna oil + sodium ate Accelerated oxidation conditions (storage at 40 °C, d to air) were used to compare oxidative stability using trans,trans, ptadienal as a marker of oxidative degradation of tuna oil omega-3 fatty acids. Figure 10 shows the pment of trans,trans, ptadienal within emulsions during e. All emulsions with the exception of n (plus phospholipid) emulsion reached maximum oxidation within 18 days. The sodium caseinate and lsolexx emulsions reached heptadienal concentration of 3.0 ppm within 9-10 days, whereas the olesosin emulsion did not reach same level of oxidation until after 24 days demonstrating the superior antioxidant properties of oleosin compared with sodium caseinate and Isolexx.

Example 6 - Role of secondary surfactants on artiﬁcial oil body stability Based on previous work the presence of phospholipid in addition to oleosin was necessary to produce emulsions that did not coalesce on storage. The t inventors investigated the essentiality of phospholipid for stability of artiﬁcial emulsions by 3O preparing emulsions where phospholipid was replaced by Tween40, monoglyceride and SSL as below Phospholipid, Tween 40, MAG and SSL were used at the same level (1% w/w by weight of oil). a. Tuna oil + n and phospholipid b. Tuna oil + oleosin and Tween 40 0. Tuna oil + oleosin and monoglyceride d. Tuna oil + oleosin and sodium stearoyl lactylate (SSL) Accelerated oxidation conditions ge at 40 °C, d to air) were used to compare oxidative stability using trans,trans, 2,4-heptadienal as a marker of oxidative degradation of tuna oil omega—3 fatty acids. Figure 11 shows the development of trans,trans, 2,4—heptadienal within emulsions during storage. There was no cant difference in the rate of development of heptadienal between the different emulsions showing that replacement of phospholipid with Tween 40, monoglyceride or SSL did not adversely affect the oxidative stability of oleosin-based artiﬁcial oil bodies.

It will be appreciated by persons skilled in the art that numerous variations and/or modiﬁcations may be made to the invention as shown in the speciﬁc embodiments without departing from the spirit or scope of the invention as y described. The t embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

The present application claims priority from AU 2011900383 ﬁled 7 February 2011, the entire contents of which are orated herein by reference.

All publications discussed and/or referenced herein are incorporated herein in their entirety.

Any discussion of documents, acts, materials, s, es or the like which has been included in the present speciﬁcation is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the t invention as it existed before the priority date of each claim of this application.

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Claims

1. A method of producing artiﬁcial oil , the method comprising i) obtaining an extract of a plant comprising n, n the extract is plant meal, ii) at least partially purifying protein from the extract, wherein the protein comprises oleosin, and iii) mixing the protein comprising oleosin with surfactant and oil comprising polyunsaturated fatty acids with four or more double bonds to produce the artiﬁcial oil 10 bodies.

2. The method of claim 1, wherein the surfactant is phospholipid.

3. A method of producing artiﬁcial oil bodies, the method comprising 15 i) obtaining an extract of a plant comprising oleosin, wherein the extract is plant meal, ii) at least partially purifying protein from the extract, wherein the protein comprises oleosin, and iii) mixing the protein comprising n with tant and oil to produce the 20 artiﬁcial oil bodies, wherein at least some of the surfactant is not phospholipid.

4. The method of any one of claims 1 to 3, wherein step ii) comprises 1) adjusting the pH of the extract to a pH of at least about 11.5, 2) ting l) to produce a solid and liquid phase and selecting the liquid 25 phase, 3) reducing the pH of the liquid phase to precipitate the proteins, 4) ting 3) to produce a solid and liquid phase and selecting the solid phase, and 5) dispersing the solid phase in a carrier.

5. The method of claim 4, wherein step 3) ses reducing the pH of the liquid phase to less than about 7.

6. The method of any one of claims 1 to 5 which further comprises, 35 iv) selecting artiﬁcial oil bodies.

7. The method of any one of claims 1, 2, 4 to 6, wherein at least 5% of the polyunsaturated fatty acids have four or more double bonds.

8. The method of any one of claims 1, 2, 4 to 7, wherein at least 50% of the fatty acids are in the form of glycerides.

9. The method of any one of claims 1 to 8, n the oleosin is an oilseed oleosin.

10 10. The method of any one of claims 1 to 9, wherein the oil is marine oil and/or ﬁsh oil.

11. The method of any one of claims 1 to 10, wherein the artiﬁcial oil bodies have a size of between 0.1 and 100 6m.

12. The method of any one of claims 1 to 11 which does not use an organic solvent.

13. The method of any one of claims 1 to 12, wherein the plant is an oilseed. 2O

14. The method of any one of claims 1 to 13, wherein the extract is plant meal produced following the extraction of oil.

15. A method of lly purifying oleosin from a plant extract, the method comprising 25 1) adjusting the pH of the extract to a pH of at least about 11.5, 2) separating l) to produce a solid and liquid phase and selecting the liquid phase, 3) reducing the pH of the liquid phase to precipitate the proteins, 4) separating 3) to e a solid and liquid phase and selecting the solid 3O phase, and 5) dispersing the solid phase in a carrier, wherein the extract is plant meal.

16. The method of claim 15, wherein step 3) comprises reducing the pH of the 35 liquid phase to less than about 7.

17. The method of claim 15 or claim 16 which does not use an organic t.

18. The method of any one of claims 15 to 17, wherein the plant is an oilseed. 5

19. The method of any one of claims 15 to 18, wherein the extract is plant meal produced following the extraction of oil.