DHA-enriched polyunsaturated fatty acid composition

Abstract

Provided is a vegetable-based lipid composition that contains high levels of at least three different long-chain polyunsaturated fatty acids (typically as fatty acid esters).This composition typically contains DHA as the main long-chain polyunsaturated fatty acid.This composition can be obtained from a single source by conventional processing methods and has improved stability characteristics. [Selected Figure] Figure 1

Description

[Technical Field]

[0001] Embodiments disclosed herein relate to novel lipid compositions enriched with multiple polyunsaturated fatty acids that have multiple health benefits, including nutritional benefits, and are potentially obtained from a single source that is both scalable and sustainable, and also has improved oxidative stability. [Background technology]

[0002] Omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs) are widely recognized as important compounds for human and animal health. These fatty acids can be obtained from dietary sources or, to a lesser extent, by conversion of linoleic acid (LA, 18:2ω-6) or α-linolenic acid (ALA, 18:3ω-3) fatty acids, all of which are considered essential fatty acids in the human diet.

[0003] From a nutritional perspective, the most important omega-3 fatty acids are probably alpha-linolenic acid, eicosapentaenoic acid (EPA, 20:5n-3), and docosahexaenoic acid (DHA, 22:6n-3). DHA is a LC-PUFA and is important for brain and eye development. Consumption of omega-3 PUFAs may also help prevent coronary artery disease. Medical research clearly demonstrates that these fatty acids have beneficial health aspects, such as improved cardiovascular and immune function and reduced risk of cancer, diabetes, and hypertension. Clinical results have demonstrated that a weekly dietary intake of 5.5 g of omega-3 PUFAs may be associated with a 50% reduction in the risk of primary cardiac arrest. As a result, oils containing omega-3 PUFAs are in high demand for pharmaceutical and nutritional purposes.

[0004] In general, the oxidative stability of fatty acids decreases significantly as the number of carbon-carbon double bonds, or degree of unsaturation, increases. Unfortunately, ALA, EPA, and DHA are all polyunsaturated fats and prone to oxidation. EPA (with five carbon-carbon double bonds) is significantly more susceptible to oxidation than ALA, and DHA (with six carbon-carbon double bonds) is even more susceptible to oxidation than EPA. As a result, increasing the omega-3 content tends to shorten the shelf life of many products. These problems are particularly severe with oils containing significant amounts of EPA or DHA.

[0005] US2015 / 223483 discloses canola oil-based blends with improved oxidative stability, which is achieved by the addition of one or more additives.

[0006] US2011 / 0027443 discloses an oil and fat composition containing a specific blend of oleic acid, linoleic acid, alpha-linolenic acid, and LC-PUFA with an improved flavor profile. US2004 / 209953 discloses a nutritional product containing primarily monoglycerides and diglycerides of LC-PUFA. US5,130,061 describes the use of interesterification and distillation processes to extract DHA from crude oil. US9,040,730 describes the purification of lipid mixtures containing PUFA to reduce the amount of undesirable sterols in the composition. In all of these cases, fish oil or microbial oil is used as the raw material to obtain the specific blend.

[0007] International Patent Application No. WO2013 / 185184 discloses a process for producing ethyl esters of polyunsaturated fatty acids.

[0008] International Patent Application No. WO2015 / 089587 and U.S. Patent Application No. US2015 / 0166928 disclose plant lipid compositions containing a mixture of omega-3 and omega-6 fatty acids. Genetically modified canola is described in WO2017 / 218969 and WO2017 / 219006.

[0009] International Patent Applications WO2016 / 182452 and WO2014 / 105576 disclose compositions containing significant amounts of DHA and EPA.

[0010] The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. Summary of the Invention

[0011] According to a first aspect of the present invention there is provided a vegetable-based lipid composition comprising: (i) a first polyunsaturated fatty acid in an amount of about 50% to about 85% by weight of the total fatty acid content of the composition, the first polyunsaturated fatty acid having at least five unsaturations in its carbon chain, having at least 20 carbon atoms in its carbon chain, or being selected from the group consisting of DHA and EPA; (ii) ALA in an amount of about 10% to about 90% by weight of the first polyunsaturated fatty acid; (iii) a third polyunsaturated fatty acid in an amount of about 10% to about 70% by weight of the ALA; A vegetable-based lipid composition is provided, wherein the total amount of all other fatty acids in the composition is up to about 20% by weight of the total fatty acid content of the composition, and wherein each of the ALA, first and third polyunsaturated fatty acids is independently provided in the form of a fatty acid, a fatty acid salt, a fatty acid ester, or a salt of a fatty acid ester.

[0012] The lipid composition is referred to herein as the "composition of the present invention."

[0013] The present invention relates to lipid compositions containing high levels of a first polyunsaturated fatty acid together with at least two other polyunsaturated fatty acids, each of which may be in the form of a free fatty acid, a salt, an ester, or a salt of an ester.

[0014] These compositions have been found to be available from sustainable sources, such as plant sources. They have also been found to have improved storage stability profiles, evidenced by reduced oxidative degradation during storage. Many polyunsaturated fatty acids, including DHA in particular, are recognized as important compounds for human and animal health. These compositions can be used in feed, nutritional supplements, cosmetics, and other chemical compositions, and they may be useful as intermediates and active pharmaceutical ingredients.

[0015] The fatty acid level in the composition of the present invention can be determined using conventional methods known to those skilled in the art. Such methods include, for example, gas chromatography (GC) combined with a reference standard, according to the method disclosed in the Examples. In certain methods, fatty acids are converted to methyl esters or ethyl esters before GC analysis. Such techniques are described in the Examples. The peak position of the chromatogram can be used to identify each specific fatty acid, and the amount can be determined by integrating the area under each peak. As used herein, unless otherwise stated, the percentage of a specific fatty acid in a sample is determined by calculating the area under the curve of the chromatogram of that fatty acid as a percentage of the total area of ​​the fatty acids in the chromatogram. This essentially corresponds to weight percent (w / w). The identity of the fatty acid can be confirmed by GC-MS.

[0016] The term "polyunsaturated fatty acid" or alternatively "PUFA" refers to a fatty acid containing at least two carbon-carbon double bonds. The terms "long-chain polyunsaturated fatty acid" and "LC-PUFA" refer to a fatty acid containing at least 20 carbon atoms and at least two carbon-carbon double bonds in its carbon chain, and thus include VLC-PUFA. As used herein, the terms "very long-chain polyunsaturated fatty acid" and "VLC-PUFA" refer to a fatty acid containing at least 22 carbon atoms and at least three carbon-carbon double bonds in its carbon chain. Typically, the number of carbon atoms in the carbon chain of a fatty acid refers to an unbranched carbon chain. If the carbon chain is branched, the number of carbon atoms excludes the carbon atoms of the side groups.

[0017] The long-chain polyunsaturated fatty acids may be omega-3 ("omega-3") fatty acids, i.e., fatty acids having an unsaturation (carbon-carbon double bond) at the third carbon-carbon bond from the methyl end of the fatty acid. Alternatively, they may be omega-6 ("omega-6") fatty acids, i.e., fatty acids having an unsaturation (carbon-carbon double bond) at the sixth carbon-carbon bond from the methyl end of the fatty acid. While other unsaturation patterns may exist, the omega-6 and especially the omega-3 types are particularly relevant in the context of the present invention.

[0018] The composition of the present invention comprises at least three different polyunsaturated fatty acids.The first polyunsaturated fatty acid is the most abundant fatty acid present in the composition (by weight relative to the total fatty acid content of the composition).ALA is the second most abundant fatty acid present in the composition (by weight relative to the total fatty acid content of the composition).

[0019] The ALA and the first and third PUFA are each independently provided in the form of a fatty acid, a fatty acid salt, a fatty acid ester, or a salt of a fatty acid ester.

[0020] As used herein, the term "fatty acid" often refers to a carboxylic acid (or organic acid) with a long aliphatic tail, either saturated or unsaturated. Typically, fatty acids have a carbon-carbon bond chain at least 8 carbon atoms long, more specifically at least 12 carbon atoms long. Most naturally occurring fatty acids have an even number of carbon atoms because their biosynthesis involves acetate, which has two carbon atoms. Fatty acids can be in a free (unesterified) state, referred to herein as "free fatty acids," or in an esterified form, such as an alkyl ester, part of a triglyceride, part of a diacylglyceride, part of a monoacylglyceride, acyl-CoA (thioester) or other bond, or a mixture thereof. Fatty acids can be esterified as phospholipids, such as in the form of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, or diphosphatidylglycerol, but are preferably esterified as alkyl esters, particularly ethyl esters. For the avoidance of doubt, unless otherwise specified, the term "fatty acid" encompasses free fatty acids, fatty acid esters, and salts of any of these.

[0021] Unless otherwise specified, a quantitative value associated with a particular fatty acid refers to the amount of that fatty acid present (calculated on a weight basis) regardless of the form in which it exists (e.g., free acid or ester).

[0022] Each fatty acid in the composition can also be independently provided in the form of fatty acid salt, for example, alkali salt or alkaline earth salt.Specific salts that can be mentioned include lithium salt and calcium salt.Such salts have potential additional medicinal effects or provide improved processability.Similarly, fatty acid esters can be provided in the form of fatty acid ester salts.Any combination of fatty acids in the form of free fatty acid, salt, ester, or ester salt can be present in the composition of the present invention.This means, for example, that the first PUFA can be mainly present as ethyl ester, ALA can be mainly present as calcium salt of methyl ester, and the third PUFA can be mainly present as free fatty acid.

[0023] "Saturated fatty acids" do not contain any double bonds or other functional groups along the chain. The term "saturated" refers to hydrogen in that every carbon (except the carboxylic acid [-COOH] group) contains as many hydrogen atoms as possible. In other words, the omega (ω) end is bonded to three hydrogen atoms (CH3-), and each carbon in the chain is bonded to two hydrogen atoms (-CH2-). The term "total fatty acids" includes all forms of fatty acids, whether saturated or unsaturated, free acids, esters, and / or salts.

[0024] The term "about" as used herein when referring to a measurable value such as amount of compound, weight, time, temperature, etc. refers to a variation of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the stated amount.

[0025] The compositions of the present invention that may be mentioned include those that contain high concentrations of omega-3 fatty acids, many of which are so-called "essential fats" that are considered to be particularly important for human health.Omega-3 fatty acids can have beneficial effects on HDL cholesterol levels, and have been shown to support the development of the brain in young people and be beneficial to mental health.These fatty acids are generally considered to be precursors of eicosanoids that have anti-inflammatory properties.Specific compositions of the present invention that may be mentioned include those in which the total amount of omega-3 polyunsaturated fatty acids in lipid composition is at least about 80% by weight, for example, at least about 85% by weight, of the total fatty acid content of the composition.

[0026] Omega-6 fatty acids are also considered important for human health. In particular, certain omega-6 fatty acids are "essential fats" necessary for health, but the body cannot synthesize them. However, omega-6 fats have been shown to be precursors to more pro-inflammatory eicosanoids, and excessive production of these eicosanoids can increase inflammation and inflammatory diseases. Therefore, it may be desirable to minimize the amount of such fatty acids in lipid compositions. It is generally accepted that the ratio of omega-6 fatty acids to omega-3 fatty acids in a diet should be 4:1 or less. However, typical Western diets typically have a higher proportion of omega-6 fatty acids. The lipid compositions of the present invention advantageously contain small amounts of omega-6 fatty acids while simultaneously containing large amounts of more beneficial omega-3 fatty acids. In one embodiment, the total amount of omega-6 polyunsaturated fatty acids in the composition is at most about 10% by weight of the total fatty acid content of the composition. In another embodiment, the ratio of the total weight of omega-3 polyunsaturated fatty acids to the total weight of omega-6 polyunsaturated fatty acids in the composition is at least about 10:1. In a further embodiment, the ratio of total weight of omega-3 polyunsaturated fatty acids to total weight of omega-6 polyunsaturated fatty acids in the composition is at least about 20:1.

[0027] Lipid compositions containing long-chain polyunsaturated fatty acids are typically obtained from marine sources (e.g., fish, shellfish), algae, or plant sources (e.g., flax or echium). The starting organic matter is first processed to extract the oil contained therein (commonly referred to as "crude" oil). For example, in the case of plant seeds, the seeds are crushed to release the oil, which is then separated from the solids by filtration and / or decantation. Crude oil often contains levels of polyunsaturated fatty acids that are too low to be useful (e.g., nutritional products), and therefore requires concentration. When crude oil lacks one or more essential components, crude or concentrated oils from multiple sources (e.g., from fish and algae) are commonly blended together to obtain the desired composition. Alternatively, concentration can be achieved by processing crude oil to remove unwanted components (e.g., components that adversely affect the color, odor, or stability of the product, or unwanted fatty acids) while maximizing the level of the desired fatty acid components.

[0028] The composition of the present invention is advantageously obtainable from a single source. The use of a single source facilitates the efficient and economical processing of crude oil and the production of the lipid composition of the present invention. The phrase "obtainable from a single source" (or "obtained from a single source") means that the lipid composition is obtainable from one or more organisms of a single taxonomic classification. In certain embodiments, the lipid composition is not derived from multiple organisms of different taxonomic classes. For example, the lipid composition should not be a blend of oils obtained from a combination of fish and algae, or a combination of fish and plants.

[0029] Instead, the lipid compositions of the present invention (or the "crude" oil from which the compositions can be obtained by concentration techniques such as interesterification, distillation, and chromatography) are obtainable from a single population of organisms, e.g., a single plant or herbaceous source. For the avoidance of doubt, the phrase "obtainable from a single source" does not exclude the use of multiple organisms of the same species as the source of the lipid composition or "crude" oil, i.e., multiple fish, algal sources, plants, or plant seeds of the same species. The multiple organisms are preferably all of the same species, or from the same breeding line, or of the same plant species, or from the same production source or batch.

[0030] The first polyunsaturated fatty acid (i) has at least 5 unsaturations in its carbon chain, (ii) has at least 20 carbon atoms in its carbon chain, or (iii) is a PUFA selected from the group consisting of DHA and EPA.In certain embodiments, the first polyunsaturated fatty acid has at least 5 unsaturations in its carbon chain and at least 20 carbon atoms.The specific fatty acids that can be mentioned in this regard include DHA, docosapentaenoic acid ("DPA", 22:5n-3) and EPA.In yet another embodiment, the first polyunsaturated fatty acid is DHA.

[0031] References to docosahexaenoic acid and "DHA" and "EPA" in this context, unless otherwise specified, refer to the ω3 forms of docosahexaenoic acid and eicosapentaenoic acid, respectively, i.e., docosahexaenoic acid and eicosapentaenoic acid, which have an unsaturation (carbon-carbon double bond) in the third carbon-carbon bond from the methyl end of the fatty acid. Synonymous abbreviations for DHA include "22:6n-3" and "22:6ω-3." Synonymous abbreviations for EPA include "20:5n-3" and "20:5ω-3."

[0032] In certain lipid compositions of the present invention, the first polyunsaturated fatty acid (e.g., DHA) is present in an amount of at least about 55% by weight (e.g., at least about 60% by weight) of the total fatty acid content of the composition.In further embodiments, the first polyunsaturated fatty acid (e.g., DHA) is present in an amount of up to about 80% by weight (e.g., up to about 75% by weight) of the total fatty acid content of the composition.

[0033] Advantageously, it has been found that the lipid composition of the present invention contains high DHA level compared with the amount of EPA present.Therefore, in one embodiment, the weight ratio of docosahexaenoic acid to eicosapentaenoic acid in lipid composition is at least about 10:1.In another embodiment, the weight ratio of docosahexaenoic acid to eicosapentaenoic acid in lipid composition can be at least about 20:1, for example, more than about 30:1.

[0034] Preferably, the amount of EPA in the composition may be very low. In certain embodiments, the lipid composition contains up to about 3% by weight of EPA, more particularly up to about 2% by weight of the total fatty acid content of the composition.

[0035] The compositions of the present invention contain ALA in an amount of about 10% to about 90% by weight of the first polyunsaturated fatty acid. ALA is typically the second most abundant polyunsaturated fatty acid in the composition. In certain embodiments, ALA is present in an amount of about 10% to about 75% by weight (e.g., about 10% to about 60% by weight) of the first polyunsaturated fatty acid. ALA may also be present in an amount of about 15% to about 55% by weight of the first polyunsaturated fatty acid (e.g., DHA).

[0036] The third polyunsaturated fatty acid can be any polyunsaturated fatty acid disclosed herein, except that the first and third PUFAs are different and neither is ALA.In one embodiment, the third polyunsaturated fatty acid is a C:18-24 omega-3 polyunsaturated fatty acid containing at least three (e.g., at least four) unsaturations.Specific examples of such PUFAs that may be mentioned include DHA, EPA, eicosatetraenoic acid ("ETA", 20:4n-3), DPA, and stearadonic acid ("SDA", 18:4n-3).In yet another embodiment, the third polyunsaturated fatty acid is EPA or DPA.

[0037] The compositions of the present invention contain a third polyunsaturated fatty acid in an amount of about 10% to about 70% by weight of ALA. In one embodiment, the third polyunsaturated fatty acid is the third most abundant polyunsaturated fatty acid in the composition. In another embodiment, the third polyunsaturated fatty acid is present in an amount of about 10% to about 65% by weight of ALA.

[0038] The compositions of the present invention can also contain other polyunsaturated fatty acids (i.e., in addition to ALA and the first and third PUFAs). Such additional polyunsaturated fatty acids include those described above for the third PUFA (e.g., DHA, EPA, ETA, DPA, and / or SDA), as well as linoleic acid ("LA", 18:2n-6) and γ-linolenic acid ("GLA", 18:3n-6). In the compositions of the present invention, the total amount of all other fatty acids in the composition is up to about 20% by weight of the total fatty acid content of the composition. This means that the total amount of all fatty acids in the composition, excluding ALA and the first and third PUFAs, is about 20% by weight of the total fatty acid content of the composition. In a specific embodiment, the total amount of all other fatty acids in the composition is up to about 18% by weight (e.g., up to about 16% by weight) of the total fatty acid content of the composition.

[0039] The compositions of the present invention contain palmitic acid (16:0) in an amount of up to about 0.5% by weight of the total fatty acid content of the composition. In certain embodiments, the compositions contain palmitic acid in an amount of up to about 0.1% by weight, more particularly up to about 0.05% by weight of the total fatty acid content of the composition.

[0040] Preferences and options for a given aspect, feature, or embodiment of the present invention should be considered as disclosed in combination with any and all preferences and options for all other aspects, features, and embodiments of the present invention, unless the context indicates otherwise. For example, the specific amounts of ALA and the first and third PUFAs set forth in the previous paragraph are disclosed in all combinations.

[0041] Thus, particular lipid compositions that may be mentioned are: (i) DHA in an amount of about 50% to about 85% by weight of the total fatty acid content of the composition; (ii) ALA in an amount of about 10% to about 90% by weight of DHA; (iii) a third polyunsaturated fatty acid in an amount of about 10% to about 70% by weight of the ALA; The lipid composition wherein the total amount of all other fatty acids in the composition is up to about 20% by weight of the total fatty acid content of the composition, and wherein the DHA, ALA, and third polyunsaturated fatty acid are each independently provided in the form of a fatty acid, a fatty acid salt, a fatty acid ester, or a salt of a fatty acid ester.

[0042] Further particular lipid compositions that may be mentioned are: (i) DHA in an amount of about 55% to about 80% by weight of the total fatty acid content of the composition; (ii) ALA in an amount of about 10% to about 60% by weight of DHA; (iii) a third polyunsaturated fatty acid selected from ETA and DPA in an amount of about 10% to about 70% by weight of the ALA; The lipid composition wherein the total amount of all other fatty acids in the composition is up to about 20% by weight of the total fatty acid content of the composition, and wherein the DHA, ALA, and third polyunsaturated fatty acid are each independently provided in the form of a fatty acid, a fatty acid salt, a fatty acid ester, or a salt of a fatty acid ester.

[0043] In the composition of the present invention, ALA and the first and third PUFA can each independently exist in the form of fatty acid, fatty acid salt, fatty acid ester or the salt of fatty acid ester.In certain embodiments, these components are each in the same form, for example, they can all be in the form of fatty acid, all in the form of fatty acid salt, all in the form of fatty acid ester, or all in the form of the salt of fatty acid ester.When a component is in the form of fatty acid salt, ester or the salt of ester, the component can be in the form of the same salt, ester or the salt of ester.For example, ALA and the first and third PUFA can be provided in the form of fatty acid ethyl ester.

[0044] In certain embodiments, ALA and the first and third PUFAs are each independently provided in the form of a fatty acid ester salt, or most specifically, in the form of a fatty acid ester.Suitable fatty acid ester forms are known to those skilled in the art.For example, nutritionally acceptable and / or pharmaceutically acceptable fatty acid ester forms include fatty acid ethyl esters, methyl esters, phospholipids, monoglycerides, diglycerides, and triglycerides.Depending on the intended use of the lipid composition, different ester forms may be required.For example, triglycerides are particularly suitable for use in foods intended for human consumption, especially for infants, due in part to the taste and stability of these ester forms against heat treatment (which may be required for such foods).Therefore, in one embodiment, a food for human or animal consumption is provided, comprising the lipid composition of the present invention, in which ALA and the first and third polyunsaturated fatty acids are provided in the form of triglyceride esters.Ethyl esters are particularly suitable for use in dietary supplements, because these ester forms can be efficiently and easily produced and do not require conversion to triglyceride forms. Thus, in a further embodiment, the ALA and the first and third PUFAs are each independently provided in the form of a fatty acid ethyl ester or as part of a triglyceride.

[0045] Triglycerides are esters derived from glycerol and three fatty acids. Since the present invention relates to a blend of fatty acids, the fatty acid components in such triglycerides can be mixed in corresponding ratios. That is, a mixture of different triglyceride molecules can be present in the composition, but the overall fatty acid profile in the composition is as defined in the claims.

[0046] Alternatively, fatty acid components can be in the form of "free" fatty acid, i.e., the -COOH form of fatty acid.However, in certain compositions of the present invention, the composition contains a relatively low level of this form of fatty acid, because fatty acid has an unpleasant (often "soapy") taste, and is less stable than the fatty acid in esterified form.Free fatty acid is typically removed from oil and lipid compositions by alkali or physical refining, for example, according to the process discussed elsewhere herein.Therefore, in one embodiment, the total free fatty acid content in lipid composition is less than 5 wt% (for example, less than 3 wt%, particularly less than 2 wt%) of the total fatty acid content of the composition.

[0047] The fatty acid in the lipid composition of the present invention is typically linear (i.e., unbranched) chain fatty acid.The composition of the present invention that can be mentioned includes those that contain very low levels of branched-chain fatty acids and their esters, so that the composition is essentially free of branched-chain fatty acids and branched-chain fatty acid esters.The term "low level" means that the composition contains branched-chain fatty acids and fatty acid esters in an amount of at most about 0.1% by weight of the total fatty acids of the composition.

[0048] The lipid compositions of the present invention may also contain other components (e.g., other than fatty acids) that originate from the raw material and are not completely removed during the extraction and concentration process. The exact identity of these other components varies greatly depending on the raw material. Examples of such other components include plant sterols (i.e., plant sterols and plant stanols) present either as free sterols or as sterol esters (such as β-sitosterol, β-sitostanol, Δ5-avenasterol, campesterol, Δ5-stigmasterol, Δ7-stigmasterol, and Δ7-avenasterol, cholesterol, brassicasterol, carinasterol, campesterol, campestanol, and ebricol). Other examples include antioxidants such as tocopherol and tocotrienol. Accordingly, specific lipid compositions of the present invention that may be mentioned include those containing detectable amounts of one or more plant sterols (e.g., β-sitosterol). Such sterols may be present in amounts of at least about 0.01% by weight of the lipid composition, but typically less than about 1% by weight.

[0049] The compositions of the present invention are advantageously obtained from plant sources ("vegetable" sources). The term "vegetable-based" means that at least 70% by weight of the lipids present in the compositions of the present invention are obtained from vegetable sources. Vegetable sources include plant sources, particularly crops such as cereals. In at least one embodiment, lipids are obtained from seed oil crops such as Brassica napus or Brassica juncea. However, for the avoidance of doubt, it is not essential that the composition is obtained exclusively from such sources; that is, a portion (e.g., at most 30% by weight) of the lipids in the compositions of the present invention may be obtained from other sources, including marine (e.g., fish or shellfish) oils, algae oils, and combinations thereof. In one example, at least 80% by weight, for example at least 90% by weight, of the lipids present are obtained from vegetable sources. In certain compositions of the present invention, essentially all (i.e., at least 95%, at least 99%, or about 100%) of the lipids are obtained from vegetable sources.

[0050] In one embodiment, the compositions of the present invention (and the feed and pharmaceutical compositions defined herein below) are not of animal (e.g., marine) origin. That is, in such embodiments, the lipid composition does not contain any components derived from animals such as fish and crustaceans. Lipid compositions that do not contain animal-derived components are believed to be advantageous in terms of lipid content and stability profile that can be achieved using standard purification and / or concentration procedures.

[0051] There are many advantages to using plants as lipid or fatty acid sources. For example, marine oil sources are known to contain relatively high levels of contaminants not found in plant materials, such as mercury, PCBs, and fish allergens (e.g., parvalbumin). Historical overfishing has also depleted fish and crustacean (e.g., krill) resources, which are no longer sustainable. Therefore, the present invention provides a polyunsaturated fatty acid oil composition from a sustainable source that contains relatively low levels of undesirable contaminants.

[0052] In certain embodiments, the composition of the present invention is derived from plant.The plant from which oil is obtained is typically oilseed crops such as copra, cottonseed, flax, palm kernel, peanut, rapeseed, soybean and sunflower seed.Therefore, the composition that is obtained only from plant can be called "vegetable" oil or "vegetable lipid composition". Suitable plants from which the lipid compositions of the present invention can be obtained are known to those skilled in the art and include Brassica sp., Gossypium hirsutum, Unum usitatissimum, Helianthus sp., Carthamus tinctorius, Glycine max, Zea mays, Arabidopsis thaliana, Sorghum bicolor, Sorghum vulgare, Avena sativa, Trifolium sp., Elaesis guineenis, Nicotiana benthamiana, Hordeum vulgare, Lupinus angustifolius, Oryza sativa, Oryza glaberrima, Camelina sativa, or Crambe abyssinica. A particular plant source that may be mentioned in this regard is Brassica sp.

[0053] Suitable sources (including marine, algae, and plant sources) can be naturally occurring or can be genetically modified to improve the ability to produce long-chain polyunsaturated fatty acids.Examples of plant sources that are genetically modified for this purpose, i.e., derived from recombinant plant cells, are known to those skilled in the art and are disclosed in International Patent Application Nos. PCT / AU2013 / 000639 (published as WO2013 / 185184), PCT / AU2014 / 050433 (published as WO2015 / 089587), and PCT / AU2015 / 050340 (published as WO2015 / 196250).Genetically modified canola is described in WO2017 / 218969 and WO2017 / 219006.The disclosures of all publications mentioned herein are incorporated by reference in their entirety.

[0054] The lipid composition of the present invention can be obtained directly from naturally occurring sources (for example, animals, algae, and / or plants).However, the oils obtained from naturally occurring sources usually need to be processed to concentrate them.Suitable concentration process is illustrated in the examples.

[0055] Suitable sources of the lipid compositions of the present invention, or "crude" oils that can be blended or concentrated to produce these compositions, include marine species, algae, and plants. Processes for obtaining oils from marine sources are well known in the art.

[0056] As discussed above, plant sources (such as oilseed sources) are particularly suitable due to their low levels of certain contaminants and superior sustainability. Plants such as Brassica sp. (e.g., canola) produce seeds that can be processed to obtain oil.

[0057] Oil / Lipid Extraction The oil produced by plants and seeds can be extracted, processed, and analyzed using techniques routinely practiced in the art. Typically, plant seeds are cooked and pressed, and oil is extracted to produce crude oil. This oil can then be degummed, refined, bleached, and / or deodorized. The combination of degumming, refining, bleaching, and deodorization has been found to be particularly effective for preparing lipid mixtures rich in LC-PUFA. Thus, in one embodiment, the lipid composition is obtained from degummed, refined, bleached, and / or deodorized seed oil. However, it is not necessary to process the oil in this way, and suitable purification and concentration can be achieved without using these methods.

[0058] Generally, techniques for crushing seeds are known in the art. For example, oilseeds can be tempered by spraying them with water to raise the moisture content, e.g., to 8.5%, and then flaked using smooth rollers with a gap setting of 0.23 mm to 0.27 mm. Depending on the type of seed, water may not be added before crushing. Extraction can also be achieved using an extrusion process, which may or may not be used in place of flaking, or as an add-on process either before or after the screw press.

[0059] In one embodiment, the majority of the seed oil is released by crushing using a screw press. The solids discharged from the screw press are then extracted with a solvent, such as hexane, using a heat-traced column, after which the solvent is removed from the extracted oil. Alternatively, the crude oil produced by the press operation can be passed through a settling tank equipped with a slotted wire drain top to remove solids that developed in the oil during the press operation. This clarified oil can be passed through a plate and frame filter to remove any remaining fine solid particles. If desired, the oil recovered from the extraction process can be combined with the clarified oil to produce a blended crude oil. Once the solvent has been removed from the crude oil, the pressed and extracted portions are combined and subjected to conventional petroleum processing procedures.

[0060] Refining and Purification As used herein, the term "purified" when used in connection with the lipids or oils of the present invention typically means that the extracted lipid or oil has been subjected to one or more processing steps to increase the purity of the lipid / oil component. For example, the purification step may include one or more of degumming, deodorizing, bleaching, or drying the extracted oil. However, as used herein, the term "purified" does not include a transesterification process or another process that alters the fatty acid composition of the lipid or oil of the present invention so as to increase the LC-PUFA content as a percentage of the total fatty acid content. In other words, the fatty acid composition of the purified lipid or oil is essentially the same as the fatty acid composition of the unpurified lipid or oil.

[0061] Once extracted from the plant source, the vegetable oil may be refined (purified) using one or more of the following processes, particularly a combination of degumming, alkaline refining, bleaching, and deodorization. Suitable methods are known to those skilled in the art (e.g., those disclosed in WO2013 / 185184).

[0062] Degumming is the initial stage of oil refining, and its primary purpose is to remove most phospholipids from the oil. Typically, the addition of approximately 2% water containing phosphoric acid to crude oil at 70–80°C causes the separation of most phospholipids, along with traces of metals and pigments. The insoluble material removed is primarily a mixture of phospholipids and triacylglycerols. Degumming can be accomplished by adding concentrated phosphoric acid to crude seed oil to convert nonhydratable phospholipids to a hydratable form and chelate any rare metals present. The gum is separated from the seed oil by centrifugation.

[0063] Alkali refining, sometimes called neutralization, is a refining process for treating crude oil. It typically follows degumming and precedes bleaching. Following degumming, the seed oil can be treated by adding an amount of alkaline solution sufficient to titrate out all free fatty acids and phosphates and remove the soaps thus formed. Suitable alkaline materials include sodium hydroxide, potassium hydroxide, sodium carbonate, lithium hydroxide, calcium hydroxide, calcium carbonate, and ammonium hydroxide. Alkali refining is typically performed at room temperature to remove the free fatty acid fraction. The soaps are removed by centrifugation or extraction into a solvent, and the neutralized oil is washed with water. If necessary, any excess alkali in the oil can be neutralized with a suitable acid, such as hydrochloric acid or sulfuric acid.

[0064] Bleaching is a refining process in which oil is heated to 90-120°C for 10-30 minutes in the presence of bleaching earth (0.2-2.0%) and in the absence of oxygen, operating with nitrogen or steam, or under vacuum. Bleaching is designed to remove unwanted pigments (carotenoids, chlorophyll, etc.); the process also removes oxidation products, trace metals, sulfur compounds, and traces of soaps.

[0065] Deodorization is the treatment of oils and fats at high temperatures (e.g., about 180°C) and low pressures (0.1-1 mmHg). This is typically accomplished by introducing steam into the seed oil at a rate of about 0.1 ml / min / 100 ml of seed oil. After about 30 minutes of application, the seed oil is allowed to cool under vacuum. This treatment improves the color of the seed oil and removes most of the volatile or odorous compounds, including any remaining free fatty acids, monoacylglycerols, and oxidation products.

[0066] Winterization is a process sometimes used in commercial oil production to separate fats and oils into solid (stearin) and liquid (olein) fractions by crystallization at subambient temperatures. It was originally applied to cottonseed oil to produce a solids-free product. It is typically used to reduce the saturated fatty acid content of the oil.

[0067] Interesterification Crude oil typically contains the fatty acids of interest in the form of triacylglycerols (TAGs). Transesterification is a process that can be used to exchange fatty acids within and between TAGs, or to transfer a fatty acid to another alcohol to form an ester (such as an ethyl ester or a methyl ester). In embodiments of the present invention, transesterification is achieved using chemical means, typically involving a strong acid or base as a catalyst.

[0068] Sodium ethoxide (in ethanol) is an example of a strong base that can be used to form fatty acid ethyl esters by transesterification. This process can be carried out at ambient or elevated temperatures (e.g., up to about 80°C).

[0069] In other embodiments of the present invention, transesterification is accomplished using one or more enzymes, particularly lipases known to be useful for hydrolyzing ester bonds in glycerides, for example. The enzymes may be position-specific (sn-1 / 3 or sn-2 specific) for the fatty acids on the triacylglycerides, or lipases that prefer some fatty acids over others. Specific enzymes that may be mentioned include Lipozyme 435 (available from Novozymes A / S). This process is typically carried out at ambient temperature. This process is typically carried out in the presence of an excess of alcohol corresponding to the desired ester form (e.g., by using ethanol to form ethyl esters of fatty acids).

[0070] distillation Molecular distillation is an effective method for removing large amounts of highly volatile components, such as saturated fatty acids, from crude oil. Distillation is typically carried out under reduced pressure, e.g., less than about 1 mbar. Temperature and time can then be selected to achieve an approximately 50:50 split between the distillate and the residue after several hours (e.g., 1 to 10) of distillation time. Typical distillation temperatures used to produce the lipid compositions of the present invention range from 120°C to 180°C, particularly 145°C to 160°C.

[0071] Multiple distillations can be performed, with each distillation considered complete when an approximately 50:50 split between distillate and residue is achieved. Using sequential distillations reduces the overall yield, but optimal results may be achieved with two distillations.

[0072] Chromatography Chromatography is an effective method for separating various components of LC-PUFA mixtures. It can be used to increase the concentration of one or more preferred LC-PUFAs in the mixture. Chromatographic separation can be achieved under a variety of conditions, but typically involves the use of a fixed-bed chromatography system or a simulated moving-bed system.

[0073] Fixed-bed chromatography systems are based on the concept that a mixture containing components to be separated is permeated (usually together with an eluent) into a column containing a packing of porous material (stationary phase) that is highly permeable to fluids. The permeation rate of each component of the mixture depends on the physical properties of that component so that the component is continuously and selectively expelled from the column. Thus, some components tend to be strongly fixed to the stationary phase and will be slower, while other components tend to be weakly fixed and expel from the column after a while.

[0074] The simulated moving bed system is composed of several individual columns containing adsorbent, which are connected together in series and operated by periodically shifting the injection points of the mixture and eluent within the system, as well as the separated component collection points, so that the overall effect simulates the operation of a single column containing a moving bed of solid adsorbent. Thus, the simulated moving bed system is composed of a column containing a fixed bed of solid adsorbent through which an eluent passes, just like a conventional fixed bed system, but in the simulated moving bed system, operation is such that it simulates a continuous countercurrent moving bed.

[0075] The columns used in these processes typically contain silica (or modified silica) as the base of the stationary phase. The mobile phase (eluent) is typically a highly polar solvent mixture, often containing one or more protic solvents such as water, methanol, ethanol, and mixtures thereof. The flow rate of the eluent can be adjusted by those skilled in the art to optimize the efficiency of the separation process. For example, the products defined in the claims can be obtained using a relatively fast eluent flow rate. Using a slower flow rate can improve the separation of the PUFAs contained in the initial mixture, thus allowing for a higher initial PUFA concentration. Detection methods for LC-PUFAs are known to those skilled in the art and include UV-vis absorption and refractive index detection methods.

[0076] Thus, according to a second aspect of the present invention, there is provided a process for producing the lipid composition of the present invention, comprising providing a mixture of fatty acid ethyl esters and subjecting the mixture to a chromatographic separation process. The present invention also relates to the lipid composition obtained by such a process. Suitable chromatographic conditions include those described herein.

[0077] For example, a specific mobile phase that can be used in chromatographic separation is a mixture of methanol and water (for example, 88% methanol / water), but this can be changed during the separation process to improve efficiency (for example, to increase the methanol content).A specific stationary phase that can be used is a silica-based stationary phase, such as a Deltaprep C18 column.Analytical HPLC or other suitable techniques known to those skilled in the art can be performed on the obtained fractions to identify fractions that contain a sufficiently high concentration of the desired PUFA, and therefore contain the lipid composition of the present invention.

[0078] In the second embodiment of the present invention, the mixture of fatty acid ethyl esters is obtained by transesterification and distillation of vegetable-based lipid oil, for example, according to any one of the processes described hereinabove.Vegetable-based lipid oil can be obtained from any plant disclosed herein or otherwise known in the art, particularly oilseed.Before transesterification and distillation, the vegetable-based lipid oil can be optionally refined using degumming, alkali refining, bleaching, and / or deodorization.

[0079] Other concentration methods The lipid compositions of the present invention are useful as active pharmaceutical ingredients (APIs) or as precursors (or "intermediates") to APIs that can be obtained therefrom by further enrichment methods. Such compositions are further enriched with beneficial PUFAs such as DHA and / or ALA.

[0080] The concentration of polyunsaturated fatty acids in oils can be increased by various methods known in the art, such as freeze crystallization, complexation with urea, supercritical fluid extraction, and silver ion complexation. Complexation with urea is a simple and efficient method for reducing the level of saturated and monounsaturated fatty acids in oils. First, the TAGs in the oil are split into their constituent fatty acids, often in the form of fatty acid esters. These free fatty acids or fatty acid esters can then be mixed with an ethanolic solution of urea for complexation, as the fatty acid composition is usually not altered by processing. Saturated and monounsaturated fatty acids easily complex with urea and crystallize upon cooling, which can then be removed by filtration. This enriches the non-urea-complexed fraction with long-chain polyunsaturated fatty acids.

[0081] product The lipid composition of the present invention is a bulk oil, i.e., the lipid composition has been separated from the source (e.g., plant seed) from which some or all of the lipids were obtained.

[0082] The lipid composition of the present invention can be used as feed. That is, the composition of the present invention can be provided in an orally available form. For the purposes of the present invention, "feed" includes any food or preparation for human consumption that, when taken into the body, helps to nourish or build tissues, or provide energy, and / or maintain, restore, or support proper nutritional status or metabolic function. Feed includes, for example, nutritional compositions for infants and / or young children, such as infant formula. In the case of feed, fatty acids can also be provided in the form of triglycerides to minimize unpleasant taste and maximize stability.

[0083] The feed contains the lipid composition of the present invention, optionally together with a suitable carrier. The term "carrier" is used in its broadest sense and includes any component that may or may not have nutritional value. As those skilled in the art will understand, the carrier must be suitable for use in feed (or be used at a sufficiently low concentration) so as not to have a harmful effect on the organism that consumes the feed.

[0084] The feed composition may be in solid or liquid form. Additionally, the composition may contain edible macronutrients, proteins, carbohydrates, vitamins, and / or minerals in amounts desired for a particular application, as is well known in the art. The amounts of these components will vary depending on whether the composition is intended for use in normal individuals or individuals with special needs, such as those suffering from metabolic disorders.

[0085] Examples of suitable nutritious carriers include macronutrients such as edible fats (e.g., coconut oil, borage oil, fungal oil, Kuroshio oil, soybean oil, and mono- and diglycerides), carbohydrates (e.g., glucose, edible lactose, and hydrolyzed starch), and proteins (e.g., soybean protein, electrodialyzed whey, electrodialyzed skim milk, milk whey, or hydrolysates of these proteins).

[0086] Vitamins and minerals that may be added to the feeds disclosed herein include, for example, calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, and vitamins A, E, D, C, and B complex.

[0087] The lipid composition of the present invention can be used in pharmaceutical compositions. Such pharmaceutical compositions optionally contain the lipid composition of the present invention together with one or more pharmaceutically acceptable excipients, diluents, or carriers known to those skilled in the art. Suitable excipients, diluents, or carriers include phosphate-buffered saline, water, ethanol, polyols, wetting agents, or emulsions such as water / oil emulsions. The composition can be in the form of either a liquid or solid, including a solution, suspension, emulsion, oil, or powder. For example, the composition can be in the form of a tablet, capsule, encapsulated gel, ingestible liquid (including oil or solution) or powder, emulsion, or topical ointment or cream. The pharmaceutical composition can also be provided as an intravenous formulation.

[0088] Particular forms suitable for feed and for pharmaceutical compositions include liquid-containing capsules and encapsulated gels.

[0089] The lipid composition of the present invention can be mixed with other lipids or lipid mixtures (especially vegetable-based fatty acid esters and fatty acid ester mixtures) before use.

[0090] The lipid compositions of the present invention may be provided with one or more additional components selected from the group consisting of antioxidants (e.g., tocopherols (such as alpha-tocopherol or gamma-tocopherol) or tocotrienols), stabilizers, and surfactants. Both alpha-tocopherol and gamma-tocopherol are naturally occurring components of various plant seed oils, including canola oil.

[0091] For example, it may be desirable to include isotonicity agents such as sugars, sodium chloride, etc. In addition to such inert diluents, composition can also include adjuvants such as wetting agents, emulsifying agents and suspending agents, sweeteners, flavoring agents and fragrances.In addition to the lipid composition of the present invention, suspension can include suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan ester, microcrystalline cellulose, metahydroxyaluminum, bentonite, agar and tragacanth or the mixture of these substances.

[0092] Solid dosage forms such as tablets and capsules can be prepared using techniques well known in the art.For example, the fatty acid produced according to the method disclosed herein can be tableted with conventional tablet bases such as lactose, sucrose and cornstarch, combined with binders such as acacia, cornstarch or gelatin, disintegrants such as potato starch or alginic acid, and lubricants such as stearic acid or magnesium stearate.Capsules can be prepared by incorporating these excipients into gelatin capsules together with related lipid compositions and optionally one or more antioxidants.

[0093] Possible administration routes of the pharmaceutical composition of the present invention include, for example, enteral (e.g., oral and rectal) and parenteral.For example, liquid preparations can be administered orally or rectally.In addition, the homogeneous mixture can be completely dispersed in water and mixed with physiologically acceptable diluents, preservatives, buffers, or propellants under sterile conditions to form sprays or inhalants.

[0094] The lipid compositions of the present invention are indicated as pharmaceuticals. According to a further aspect of the present invention, there is provided a composition of the present invention comprising any of the pharmaceutical compositions described above for use as a pharmaceutical.

[0095] The lipid composition of the present invention can provide many advantages typically associated with long-chain polyunsaturated fatty acids.For example, the lipid composition of the present invention and the pharmaceutical composition described above can be used to treat or prevent cardiovascular disease, protect against death in cardiovascular disease patients, reduce overall serum cholesterol level, reduce hypertension, increase HDL:LDL ratio, reduce triglycerides, or reduce apolipoprotein B level, as can be determined by using tests well known to those skilled in the art.Therefore, the method of treating (or preventing) the diseases and conditions listed above using the lipid composition of the present invention is also disclosed.

[0096] As used herein, the terms "treatment," "treat," and "treating" refer to reversing, alleviating, or inhibiting the progression of a disease or disorder described herein, or delaying, eliminating, or reducing the incidence or onset of a disorder or disease as described herein compared to what would occur in the absence of the measures taken. As used herein, the terms "prevent," "prevention," and "preventing" refer to reducing the risk of acquiring or developing a given condition, or reducing or inhibiting the recurrence or occurrence of the condition in a subject who is not ill.

[0097] The typical dosage of specific fatty acid is 0.1mg-20g, taken 1-5 times a day (maximum 100g a day), and particularly ranges from about 10mg to about 1, 2, 5 or 10g a day (taken in one or more doses).As known in the art, it is desirable to have at least about 300mg / day of fatty acid, especially LC-PUFA.However, it is understood that any amount of fatty acid can be beneficial to the subject.

[0098] When used as a pharmaceutical composition, the dosage of the lipid composition to be administered to a patient will be determined by one of ordinary skill in the art and will depend on various factors, such as the patient's weight, the patient's age, the patient's overall health, the patient's past medical history, the patient's immune status, etc.

[0099] The composition of the present invention is a readily available composition, which may have an improved stability profile and may contain a mixture of fatty acids, the relative ratio of omega-3 and omega-6 fatty acids being particularly beneficial to human health.Stability can be evaluated using various methods known to those skilled in the art.Such methods include the Rancimat method, evaluation of propanal formation (particularly suitable for omega-3 fatty acids), evaluation of hexanal formation (particularly suitable for omega-6 fatty acids), the "peroxide value" method (e.g., using AOCS official method Cd8-53), and the "p-anisidine value" method (e.g., using AOCS official method Cd18-90).The examples show that the composition of the present invention is obtained from a starting mixture that does not show an improved stability profile compared to a reference blend (a reference blend that has a similar composition in terms of the main LC-PUFA but contains a significant amount of animal (fish)-derived lipids).

[0100] The compositions of the present invention may also have the advantage that they may be more effective, less toxic, longer acting, more potent, have fewer side effects, be more easily absorbed, and / or have a better pharmacokinetic profile (e.g., higher oral bioavailability and / or lower clearance) than lipid compositions known in the prior art, and / or may have other useful pharmacological, physical, or chemical properties. [Brief explanation of the drawings]

[0101] The invention is illustrated by the following examples. [Figure 1] Propanal release data (after interesterification, distillation, and chromatography) for canola oil and a reference oil are presented, demonstrating the improved stability of the canola oil described herein. [Figure 2] 1 shows propanal release data (after RBD refining, interesterification, distillation, and chromatography) for canola oil and a reference oil, demonstrating the improved stability of the canola oil described herein. [Example]

[0102] Example 1 - Extraction of DHA Canola Oil from Seeds Canola varieties disclosed in U.S. Patent Publication No. US2018 / 0016590A1 were grown as a summer crop. After harvesting, the seeds were stored at room temperature before being ground.

[0103] A Kern Kraft KK80 screw press was used to crush 272 kg of seeds to produce DHA oil. The expeller collar heater temperature was set to the maximum thermostat setting. The initial ambient and choke temperatures were 20°C, and the choke distance was set at 73.92 mm. Seeds were fed without stopping the expeller, with oil and meal continuously collected, until all seeds were crushed.

[0104] Auger rotation speed, meal and discharge oil temperatures were monitored throughout the press. Crushing time was 4 hours for 270 kg at a throughput rate of 67.5 kg / hr. A crude oil yield of 87.2 kg (32%) was obtained. After filtering to remove fines, the yield was 77.2 kg (28%).

[0105] Example 2 - Reference Blend Oil Pure fish oil contains low levels of ALA fatty acid and significantly high levels of EPA and DHA.The reference oil blend (referred to herein as "crude triglyceride reference blend oil" or similar) is designed to be as similar as possible in composition to the filtered DHA canola oil obtained in Example 1.This is done by (a) matching the total DHA level with that of DHA canola oil, and (b) matching the DHA / (ALA+EPA) ratio.This is achieved by blending DHA-rich (tuna) fish oil, ALA-rich (linseed oil) oil, and standard canola oil.The resulting reference blend oil also has the same total omega-3 content as DHA canola oil.

[0106] Example 3 - Fatty Acid Composition of Crude DHA Canola Oil and Reference Blends The filtered crude oil and the reference blend oil were analyzed for fatty acid composition, and the results are shown below. [Table 1]

[0107] Example 4 - Oil Stability Evaluation Rancimat stability studies were conducted using crude DHA canola oil and the reference blend described in Examples 1 and 2, respectively. The method involved testing approximately 2.5 g of test material using the standard procedure for a Metrohm 743 Rancimat at 90° C.

[0108] The table below summarizes the results obtained with these oils at 90° C. The experiment was performed in duplicate. [Table 2]

[0109] DHA canola oil consistently demonstrated inferior stability to the reference oil.

[0110] Example 5 - Enzymatic Interesterification of Crude Canola-DHA Oil The following enzymatic interesterification procedure was carried out on approximately 5 kg of crude triglyceride oil obtained in Example 1. Lipozyme 435 was obtained from Novozymes NS.

[0111] To a dry, nitrogen-flushed chemical reactor equipped with a mechanical stirrer was added 100% absolute ethanol (5.00 kg) and the crude triglyceride canola oil (5.00 kg) obtained in Example 1, and the mixture was stirred. To this mixture was added Lipozyme 435 (420 g), and the mixture was heated at 40° C. for 21 hours. The recorded values ​​of the samples taken from the mixture were 1 1 H NMR spectrum showed the reaction was complete.

[0112] The mixture was cooled to 20°C. The mixture was discharged from the reactor and filtered through a 4 μm polypropylene filter cloth on a 20 L Neutcher filter. The reactor was rinsed with ethanol (2 × 1.6 L) and petroleum spirits (2.5 L), which were used to sequentially wash the filter cake. Petroleum spirits (10 L) and water (4 L) were added to the resulting crude reaction mixture, and the mixture was thoroughly mixed in the reactor and then allowed to settle, after which two phases were formed.

[0113] The petroleum spirit layer was removed, and the aqueous layer was further extracted with petroleum spirit (2 x 10 L). The combined petroleum spirit layers were returned to the reactor and evaporated in vacuo to a small volume (approximately 10 L). The resulting concentrated solution was drained from the reactor, dried over anhydrous magnesium sulfate (approximately 1 kg), filtered, and concentrated in vacuo to give a yellow oil (yield: 99%).

[0114] Example 6 - Enzymatic interesterification of crude reference oil blend Enzymatic interesterification of the crude triglyceride reference blend oil (5.00 kg) obtained according to Example 2 was completed using the process described in the previous example. The product was obtained as a yellow oil.

[0115] Example 7 - Distillation of Interesterified Canola Oil Standard procedure for removing the more volatile components of fatty acid ethyl ester (FAEE) mixtures by vacuum distillation. Crude fatty acid ethyl esters (FAEEs) from crude canola-DHA (obtained in Example 5) were subjected to distillation under the following conditions: Distillative separation was achieved by passing the transesterified crude oil through a Pope 2-inch (50 mm) thin film still under vacuum, equipped with two 1000 ml collection flasks to collect the distillate and residue. The fatty acid composition of each was analyzed.

[0116] Vacuum was supplied by an Edwards3 rotary pump and vacuum was measured by an ebro vacuum gauge VM2000.

[0117] The oil was fed to the still at 4 ml / min with a Cole-Palmer Instrument Company easy-load II peristaltic pump, the still motor was set at 325 rpm, and the distillate was condensed using a water condenser until one of the receiver flasks was full.

[0118] Crude canola DHA FAEE was distilled under these conditions with the heater bands initially set at 147°C. The objective was to obtain a 50:50 split of distillate:residue. During the first 30-45 minutes of the experiment, the heater band temperature was increased to 154°C to increase the proportion of oil distilled, after which the still was allowed to equilibrate. After 30 minutes, the heater band temperature was adjusted to 149°C over 30 minutes. The remainder of the distillation was conducted at 149°C. The total distillation time was 350 minutes. A portion of the residue from the above distillation was again subjected to removal of more volatile components by distillation under standard conditions with the heater band temperature set at 149°C. The total distillation time was 95 minutes. [Table 3]

[0119] Example 8 - Distillation of FAEE from Transesterified Crude Reference Blend Crude fatty acid ethyl esters (FAEEs) from the reference crude blend (obtained in Example 6) were subjected to distillation under the same conditions as those set out in the previous examples.

[0120] The reference crude blend FAEE was distilled under these standard conditions with the heater bands initially set at 152°C. The objective was to obtain a 50:50 split of distillate:residue. After 20 minutes, the heater band temperature was set to 154°C to increase the distillate flow. After another hour, the heater band temperature was adjusted to 153°C, and then to 152°C for the next hour. For the final hour of distillation, the heater band temperature was set to 153°C. The total distillation time was 380 minutes. The residue from the above distillation was again subjected to removal of more volatile components by distillation under standard conditions. The objective was to obtain a 50:50 split of distillate:residue. The distillation was primarily carried out with the heater bands set at 150-151°C. The total distillation time was 195 minutes. [Table 4]

[0121] Example 9 - Chromatographic separation of FAEEs from canola Preparative HPLC The fatty acid ethyl esters (FAEEs) obtained in Example 7 (i.e., obtained from crude canola-DHA and processed using interesterification and distillation) were subjected to chromatographic separation under the following conditions: A preparative HPLC system equipped with a Waters Prep 4000 system, a Rheodyne injector with a 10 ml loop, a 300 x 40 mm Deltaprep C18 column, a Waters 2487 dual wavelength detector, and a chart recorder was equilibrated with an 88% methanol / water mobile phase at 70 ml / min. The detector was set at 215 nm and a full scale of 2.0 absorbance units, and the chart was run at 6 cm / hr.

[0122] 1.0 g of FAEE oil was dissolved in a minimum amount of 88% methanol / water and injected onto the column via a Rheodyne injector. Fractions of approximately 250 ml were collected when the solvent front appeared after approximately 7 minutes.

[0123] Analytical HPLC was performed on all fractions and the "symmetric" fractions, which contained mainly DHA, were combined (yield: 28%).

[0124] AnalyticalHPLC Sample analysis was performed using an HPLC system equipped with a Waters 600E pump controller, 717 autosampler, 2996 photodiode array detector, and 2414 refractive index detector. Analyses were performed on a 150 x 4.6 mm Alltima C18 column using an isocratic 90% methanol / water or 95% methanol / water mobile phase at 1.0 ml / min. Data collection and processing were performed with Waters Empower3 software.

[0125] Example 10 - Chromatographic separation of FAEEs from a reference blend The distilled fatty acid ethyl esters (FAEEs) of the reference blend (obtained in Example 8) were subjected to chromatographic separation under the same conditions as those given in the previous examples.

[0126] Analytical HPLC was performed on all fractions and the "symmetric" fractions, which contained primarily DHA, were combined.

[0127] Example 11 - Fatty acid composition analysis of concentrated oil The fatty acid compositions of the products obtained in Examples 9 and 10 were analyzed, and the results are shown below. [Table 5] [Table 6]

[0128] Example 12 - Oil Stability Evaluation Headspace GC-MS stability test Headspace analysis was performed on the above concentrated products to assess the amount of propanal released under specific conditions. Increasing levels of propanal release indicate a decrease in the stability of the test material.

[0129] SPME (Solid Phase Microextraction) A 65 μm PDMS / DVB StableFlex fiber (Supelco fiber kit 57284-u) was selected. The fibers were conditioned in a Triplus RSH conditioning station at 250°C for 10 min before use. Samples were incubated at 40°C for 1 minute before extraction. The Headspace vial was extracted for 1 minute. It is expected to be an excellent general method capable of capturing a wide range of volatile components.

[0130] GC method Thermo Scientific TRACE1310 GC Thermo Scientific TR-DIOXIN 5MS column, 0.25 mm ID, 30 m film, 0.1 μm split injection, 250°C, split 83, 1.2 ml He / min GC ramp: 5°C / min to 40°C, 1 min to 100°C, then 50°C / min to 300°C

[0131] A common MS-specific column was used, which showed excellent synergy for headspace analysis. A slow initial temperature ramp was employed to maximize volatile separation before ramping up to maintain column performance. A split injection was employed to avoid the need for cryogenic cooling of the inlet and improve column resolution.

[0132] Although peak overlap hindered separation of the standards, quantification was still possible. For the calibration results of three standards (0.1, 0.01, and 0.01%), the molecular ion m / z 56 was used to detect propanal. The base peak m / z 58 was used to detect hexanal.

[0133] MS method Thermo ScientificDFS High Resolution GC-MS Low resolution (1000), full scan 35-350 Da, 0.5 seconds / scan Standards: Standard dilutions of propanal and hexanal were made in the supplied DHA canola ethyl ester. These standard mixtures were then added to 20 ml headspace vials in a volume of 540 μl.

[0134] Full scanning was adopted, making it possible to monitor all development products rather than specific molecules.

[0135] Headspace Stability Results: The following table summarizes the results obtained for the canola oil obtained in Example 9 and the reference oil obtained in Example 10 over a time period of T = 0 to 5 days. The test samples were kept at ambient temperature on a light box and under fluorescent tube lighting during this period. The m / z 58 molecular ion was analyzed, and the mass chromatogram clearly shows the appearance of propanal at 1.37 minutes at room temperature. The evolution of propanal is quantified in the table below, and the data are shown in Figure 1. The DHA canola oil released substantially less propanal, demonstrating the improved stability of the canola oil compared to the reference. [Table 7]

[0136] DHA canola oil showed superior stability against oxidation compared to the reference oil.

[0137] Example 13 - Purification of DHA Canola Oil A portion of the canola oil obtained in Example 1 was refined before undergoing further concentration. The refining process included degumming, alkaline refining, bleaching, and deodorization.

[0138] Acid degumming Degumming is the removal of non-hydratable and hydratable phospholipids from the oil. The dried crude oil obtained in Example 1 was heated to 53±2°C and 0.2% of a 50% citric acid solution was added. After mixing for approximately 30 minutes, 2.0% of heated (53±2°C) soft water was added and mixed for approximately 30 minutes. During the holding period, the oil was heated to 67±3°C and then centrifuged.

[0139] Acid Pretreatment / Refining Refining involves the removal of free fatty acids after saponification with caustic to render them water soluble, followed by removal by centrifugation. An acid pretreatment step was used to continue hydration of the phosphatides. The degummed oil was heated to 65±5°C, and 0.1% of 85% phosphoric acid was added and mixed for a total of 30 minutes. After the acid addition and holding period, 20Be' (Baume, 14.4%, w / w) sodium hydroxide was added to neutralize the free fatty acids, adding a 0.05% (w / w) excess. The caustic and oil were then mixed for an additional 15 minutes. After heating the oil to 62±2°C with a 15-minute hold, the oil was centrifuged.

[0140] Trisyl silica treatment To further remove soap, a Trisyl silica treatment was performed to a level compatible with bleaching. The Trisyl pretreatment was combined with the bleaching step. The refined oil was heated to 68±5°C and treated with 0.3% Trisyl 300. The oil / Trisyl mixture was mixed for approximately 15 minutes before bleaching was continued.

[0141] bleaching The refined oil was treated with adsorbent clay to remove peroxides, phospholipids, color bodies, and traces of soap. An acid pretreatment step was used to continue hydration of the phosphatides. The Trisyl-pretreated oil was mixed with 0.2% (w / w) of a 50% citric acid solution. After mixing for 15 minutes, 2% (w / w) of Tonsil Supreme 126FF bleaching clay was added. The mixture was then heated to 90±2°C under vacuum and held for approximately 30 minutes. The oil was cooled to 60±2°C, vacuum broken with nitrogen, and filtered with the addition of 1.0 kg of filter aid. Pressure Vessel: 500 L Cherry-Burrell pressure vessel, all 316 stainless steel construction with steam or cooling water jacket, mixing impeller, and baffles, serial number E-227-94. Filter Press: A 24" Polypropylene Sperry Filter Press with a 4.8 cubic foot capacity filter, paper, and cloth supports was used.

[0142] Deodorization The bleached oil was sparged with steam at high temperature and low pressure to remove odorants, flavor compounds, and additional free fatty acids. Thermal bleaching at high temperatures also reduces color. Half of the bleached oil was deodorized with 1% sparge steam at 180 ± 2°C for 60 minutes, and the fatty acid composition (FAC) was monitored. Deodorization vessel (OD4): 400L Coppersmithing vacuum-rated vessel, steam or cooling water jacketed, all 316 stainless steel construction. A slight decrease in DHA levels was observed after a 60-minute hold at 180°C. Another test was then performed at 180°C for 30 minutes. The product was packaged under nitrogen in 20L plastic HOPE pails and stored in a cooler at 4°C.

[0143] Example 14 - Refining of the original reference oil blend A portion of the reference blend described in Example 2 was purified before being further concentrated. In the purification process, the reference blend was purified under the same conditions as those set forth in the previous example.

[0144] Example 15 - Fatty Acid Composition of RBD Crude DHA Canola Oil and Reference Blends The fatty acid composition of the filtered crude oil of RBD (Example 13) and the RBD reference blend oil (Example 14) was analyzed, and the results are shown below. [Table 8]

[0145] Reference to "RBD" in connection with the examples (and accompanying figures) means that the product in question was obtained directly or indirectly from the "refined" product of either Example 13 (in the case of canola oil) and Example 14 (in the case of the reference blend).

[0146] Example 16 - Enzymatic Interesterification of RBD Canola-DHA Oil Enzymatic interesterification of the crude triglyceride reference blend oil (5 kg) obtained according to Example 13 was completed using the process described in Example 5. The product was obtained as a yellow oil.

[0147] Example 17 - Enzymatic interesterification of RBD reference oil blend Enzymatic interesterification of the crude triglyceride reference blend oil (5 kg) obtained according to Example 14 was completed using the process described in Example 5. The product was obtained as a yellow oil.

[0148] Example 18 - Distillation of Interesterified RBD Canola Oil Standard procedure for removing the more volatile components of fatty acid ethyl ester (FAEE) mixtures by vacuum distillation. Fatty acid ethyl esters (FAEEs) from RBD canola-DHA (obtained in Example 16) were subjected to distillation under the following conditions. Distillative separation was achieved by passing the transesterified crude oil through a Pope 2-inch (50 mm) thin film still under vacuum, equipped with two 1000 ml collection flasks to collect the distillate and residue. The fatty acid composition of each was analyzed.

[0149] Vacuum was supplied by an Edwards3 rotary pump and vacuum was measured by an ebro vacuum gauge VM2000.

[0150] The oil was fed to the still at 4 ml / min with a Cole-Palmer Instrument Company easy-load II peristaltic pump, the still motor was set at 325 rpm, and the distillate was condensed using a water condenser until one of the receiver flasks was full.

[0151] The RBD canola-DHA FAEE was distilled under these conditions with the heater bands initially set at 152°C to give a 50:50 split of distillate:residue. A portion of the residue from this distillation was resubjected to removal of more volatile components by distillation under standard conditions with the heater band temperature set at 152°C. The total distillation time was approximately 90 minutes. [Table 9]

[0152] Example 19 - Distillation of FAEE from transesterified RBD reference blend Fatty acid ethyl esters (FAEEs) from the RBD reference blend (obtained in Example 17) were subjected to distillation under the same conditions as those set out in the previous examples.

[0153] The RBD Reference Blend FAEE was distilled under these standard conditions with the heater bands initially set at 152°C to obtain a 50:50 split of distillate:residue. The residue from this distillation was again subjected to removal of more volatile components by distillation under standard conditions. The objective was to obtain a 50:50 split of distillate:residue. The distillation was primarily carried out with the heater bands set at 152°C. The total distillation time was approximately 200 minutes. [Table 10]

[0154] Example 20 - Chromatographic separation of FAEEs from RBD canola The fatty acid ethyl esters (FAEEs) obtained in Example 18 (i.e., obtained from RBD canola-DHA and processed using interesterification and distillation) were subjected to chromatographic separation under the following conditions: A preparative HPLC system equipped with a Waters Prep 4000 system, a 10 ml loop Rheodyne injector, a 300 x 40 mm Deltaprep C18 column, a Waters 2487 dual wavelength detector, and a chart recorder was equilibrated with an 88% methanol / water mobile phase at 70 ml / min. The detector was set at 215 nm and a full scale of 2.0 absorbance units, and the chart was run at 6 cm / hr.

[0155] 1.0 g of FAEE oil was dissolved in a minimum amount of 88% methanol / water and injected onto the column via a Rheodyne injector. Fractions of approximately 250 ml were collected when the solvent front appeared after approximately 7 minutes.

[0156] Analytical HPLC was performed on all fractions and the "symmetric" fractions, which contained primarily DHA, were combined.

[0157] Example 21 - Chromatographic separation of FAEEs from RBD reference blend Distilled fatty acid ethyl esters (FAEEs) of the RBD reference blend (obtained in Example 19) were subjected to distillation under the same conditions as those set out in the previous examples.

[0158] Analytical HPLC was performed on all fractions and the "symmetric" fractions, which contained primarily DHA, were combined.

[0159] Example 22 - Fatty acid composition analysis of concentrated RBD oil The fatty acid compositions of the products obtained in Examples 20 and 21 were analyzed, and the results are shown below. [Table 11]

[0160] Example 24 - Oil Stability Evaluation Headspace analysis was performed on the concentrated products described in Examples 20 and 21 according to the method described in Example 12.

[0161] The following table summarizes the results obtained for the RBD canola oil obtained in Example 20 and the RBD reference oil obtained in Example 21 from T=0 to 3 days. The test samples were kept at ambient temperature on a light box and under fluorescent tube lighting during this period. The m / z 58 molecular ion was analyzed, and the mass chromatogram clearly shows the appearance of propanal at 1.37 minutes at room temperature. The evolution of propanal is quantified in the table below, and the data are shown in Figure 2. The DHA canola oil released substantially less propanal, demonstrating the improved stability of the canola oil compared to the reference. [Table 12]

[0162] DHA canola oil showed superior stability against oxidation compared to the reference oil.

Claims

1. A lipid composition comprising: (i) a first polyunsaturated fatty acid in an amount of 55% to 75% by weight of the total fatty acid content of the composition; a first polyunsaturated fatty acid that is the most abundant fatty acid in the composition and has at least five unsaturations in its carbon chain, or has at least 20 carbon atoms in its carbon chain, or is selected from the group consisting of DHA and EPA; (ii) ALA in an amount of 10% to 90% by weight of the first polyunsaturated fatty acid; (iii) the third most abundant polyunsaturated fatty acid in the composition, the third polyunsaturated fatty acid being in an amount of 10% to 70% by weight of the ALA; the total amount of all other fatty acids in the composition is up to 20% by weight of the total fatty acid content of the composition, and each of the ALA, the first and the third polyunsaturated fatty acids is independently provided in the form of a fatty acid, a fatty acid salt or a fatty acid ester, at least 70% by weight of the lipids in the lipid composition are obtained from one or more plants; The lipid composition is obtained by a process comprising transesterification and distillation of lipids obtained from one or more plants.

2. 2. The lipid composition of claim 1, wherein the first polyunsaturated fatty acid is present in an amount that is at least 60% by weight of the total fatty acid content of the composition.

3. 3. The lipid composition of claim 1, wherein the ALA is present in an amount of 10% to 60% by weight of the first polyunsaturated fatty acid.

4. 4. The lipid composition according to claim 1, wherein the third polyunsaturated fatty acid is present in an amount of 10% to 65% by weight of the ALA.

5. The first polyunsaturated fatty acid is The lipid composition according to any one of claims 1 to 4, wherein the lipid is docosahexaenoic acid (22:6n-3), docosapentaenoic acid (22:5n-3), or eicosapentaenoic acid (20:5n-3).

6. 6. The lipid composition of claim 1, wherein the third polyunsaturated fatty acid is a polyunsaturated fatty acid selected from the group consisting of C:18-24 omega-3 polyunsaturated fatty acids containing at least three unsaturations, docosahexaenoic acid (22:6n-3), eicosapentaenoic acid (20:5n-3), eicosatetraenoic acid (20:4n-3), docosapentaenoic acid (22:5n-3), and stearadonic acid (18:4n-3).

7. the ALA and the first and third polyunsaturated fatty acids each independently comprise: The lipid composition according to any one of claims 1 to 6, provided in the form of a fatty acid ester, in the form of a fatty acid ethyl ester or as part of a triglyceride.

8. The lipid composition according to any one of claims 1 to 7, wherein the lipid composition is obtained from a single source.

9. The one or more plants are oilseeds or are selected from the group consisting of Brassica sp., Gossypium hirsutum, Unum usitatissimum, Helianthus sp., Carthamus tinctorius, Glycine max, Zea mays, Arabidopsis thaliana, Sorghum bicolor, Sorghum vulgare, Avena sativa, Trifolium sp.

9. The lipid composition of claim 8, wherein the lipid composition is selected from the group consisting of: Elaesis guineenis, Nicotiana benthamiana, Hordeum vulgare, Lupinus angustifolius, Oryza sativa, Oryza glaberrima, Camelina sativa, or Crambe abyssinica.

10. The lipid composition of claim 9, wherein the one or more plants are from the Brassica sp.

11. The lipid composition of claim 9, wherein the one or more plants are from Brassica napus or Brassica juncea.

12. A lipid composition described in any one of claims 1, 9, 10, and 11, wherein the one or more plants have been genetically modified to enhance their ability to produce long-chain polyunsaturated fatty acids.

13. A tablet, capsule, encapsulated gel, ingestible liquid or powder, emulsion, or topical ointment or cream containing the lipid composition of any one of claims 1 to 9.

14. The lipid composition according to any one of claims 1 to 12, further comprising one or more additional components selected from the group consisting of antioxidants, stabilizers, and surfactants.

15. A dietary supplement composition comprising the lipid composition according to any one of claims 1 to 12 and 14.

16. 15. A food product for human or animal consumption comprising the lipid composition of any one of claims 1 to 12, 14, wherein the ALA and the first and third polyunsaturated fatty acids are provided in the form of triglyceride esters.

17. A lipid composition according to any one of claims 1 to 12, 14 for use in treating or preventing cardiovascular disease, protecting patients with cardiovascular disease from mortality, lowering overall serum cholesterol levels, lowering high blood pressure, increasing the HDL:LDL ratio, lowering triglycerides or lowering apolipoprotein B levels.

18. 18. A process for producing a lipid composition according to any one of claims 1 to 12, 14 and 17, comprising providing a mixture of fatty acid ethyl esters and subjecting said mixture to a chromatographic separation process, wherein said mixture of fatty acid ethyl esters is obtained by transesterification and distillation of said one or more plants.

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