DHA-rich polyunsaturated fatty acid composition

Abstract

Provided is a plant-based lipid composition that contains DHA, ALA, and oleic acid (typically as fatty acid ester) in a specific ratio.This composition also contains low levels of EPA and palmitic 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] The embodiments disclosed in this specification relate to novel lipid compositions fortified with docosahexaenoic acid. The compositions contain mixtures of polyunsaturated fatty acids that have numerous health benefits. The compositions provide nutritional benefits, are available from a single source, and have both scalability and sustainability. They also have improved stability against oxidation.

Background Art

[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 the 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 α-linolenic acid, eicosapentaenoic acid ("EPA", 20:5n-3), and docosahexaenoic acid ("DHA", 22:6n-3). DHA is an LC-PUFA and is important for brain and eye development. Intake of omega-3 PUFAs may also help prevent coronary artery disease. Medical research has clearly shown that these fatty acids have beneficial health aspects such as improvement of cardiovascular and immune functions or reduction of cancer, diabetes, and hypertension. Clinical results have demonstrated that a dietary intake of 5.5 g of omega-3 PUFAs per week 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] Generally, 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 tend to oxidize easily. EPA (with 5 carbon-carbon double bonds) is significantly more susceptible to oxidation than ALA, and DHA (with 6 carbon-carbon double bonds) is even more susceptible than EPA. As a result, increasing the omega-3 content tends to shorten the shelf life of many products. These problems become particularly severe with oils containing significant amounts of EPA or DHA.

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

[0006] US2011 / 0027443 discloses a lipid composition containing a specific blend of oleic acid, linoleic acid, alpha-linolenic acid, and LC-PUFAs having an improved flavor profile. US2004 / 209953 discloses nutritional products containing mainly monoglycerides and diglycerides of LC-PUFAs. US5,130,061 describes the use of transesterification and distillation processes for extracting DHA from crude oil. US9,040,730 describes the purification of lipid mixtures containing PUFAs 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 WO2013 / 185184 discloses a process for producing ethyl esters of polyunsaturated fatty acids.

[0008] International patent application WO2015 / 089587 and U.S. patent application US2015 / 0166928 disclose plant lipid compositions comprising a mixture of omega-3 and omega-6 fatty acids. Genetically modified canola is described in WO2017 / 218969 and WO2017 / 219006.

[0009] Any list or discussion of previously published literature in this specification should not necessarily be construed as an endorsement that such literature is part of the latest technology or common general knowledge. [Overview of the project]

[0010] According to a first aspect of the present invention, (i) docosahexaenoic acid (22:6n-3) in an amount of approximately 15% to approximately 35% by weight of the total fatty acid content of the composition, (ii) eicosapentaenoic acid (20:5n-3) in an amount of approximately 5% by weight of the total fatty acid content of the composition, (iii) α-linolenic acid (18:3n-3) in an amount of about 10% to about 20% by weight of the total fatty acid content of the composition, (iv) Oleic acid (18:1n-9) in an amount of about 20% to about 40% by weight of the total fatty acid content of the composition, (v) A plant-based lipid composition is provided comprising palmitic acid in an amount of up to approximately 1.5% by weight of the total fatty acid content of the composition, Components (i) to (v) 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.

[0011] The lipid composition is referred to herein as the "Composition of the Invention."

[0012] This invention relates to lipid compositions containing high levels of docosahexaenoic acid (DHA) and alpha-linolenic acid (ALA) simultaneously, in the form of free fatty acids, salts, esters, or salts of esters. These compositions have been found to be available from sustainable sources, such as plant sources. They have also been found to have an improved storage stability profile, demonstrated by reduced oxidative degradation during storage. In particular, DHA is recognized as an important compound for human and animal health. These compositions can be used in animal feed, nutritional supplements, cosmetics, and other chemical compositions. They may also be useful as intermediates and pharmaceutical active ingredients.

[0013] The fatty acid levels in the compositions 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 methods disclosed in the Examples. In certain methods, the fatty acids are converted to methyl or ethyl esters before GC analysis. Such techniques are described in the Examples. Each specific fatty acid can be identified using the peak position of the chromatogram, and the amount can be determined by integrating the area under each peak. Where used herein, unless otherwise stated, the percentage of a particular 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 ​​fatty acids in the chromatogram. This essentially corresponds to a weight percentage (w / w). The identity of the fatty acids can be confirmed by GC-MS.

[0014] In this context, references to docosahexaenoic acid and "DHA," unless otherwise specified, refer to the ω3 form of docosahexaenoic acid, i.e., docosahexaenoic acid having an unsaturated (carbon-carbon double bond) carbon-carbon bond at the third carbon-carbon bond from the methyl terminus of the fatty acid. Synonymous abbreviations include "22:6n-3" and "22:6ω-3".

[0015] More generally, the terms “polyunsaturated fatty acids” and “PUFAs” refer to fatty acids containing at least two carbon-carbon double bonds. The terms “long-chain polyunsaturated fatty acids” and “LC-PUFAs” refer to fatty acids whose carbon chain contains at least 20 carbon atoms and at least 2 carbon-carbon double bonds, and therefore include VLC-PUFAs. As used herein, the terms “very long-chain polyunsaturated fatty acids” and “VLC-PUFAs” refer to fatty acids whose carbon chain contains at least 22 carbon atoms and at least 3 carbon-carbon double bonds. Typically, the number of carbon atoms in a fatty acid's carbon chain 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.

[0016] Long-chain polyunsaturated fatty acids can be ω3 ("omega-3") fatty acids, i.e., fatty acids having an unsaturated (carbon-carbon double bond) carbon-carbon bond at the third carbon-carbon bond from the methyl terminus of the fatty acid. Alternatively, they can be ω6 ("omega-6") fatty acids, i.e., fatty acids having an unsaturated (carbon-carbon double bond) carbon-carbon bond at the sixth carbon-carbon bond from the methyl terminus of the fatty acid. While other unsaturation patterns may exist, the ω6 and especially the ω3 types are particularly relevant in the context of the present invention.

[0017] The composition of the present invention comprises at least two different polyunsaturated fatty acids, including DHA and alpha-linolenic acid (ALA, 18:3n-3). In one embodiment, either DHA or ALA is the most abundant fatty acid present in the composition (by weight relative to the total fatty acid content of the composition). In another embodiment, DHA and ALA are the two most abundant fatty acids present in the composition.

[0018] Components (i) to (v) in the composition of the present invention may each exist in the form of a fatty acid, a fatty acid salt, a fatty acid ester, or a salt of a fatty acid ester.

[0019] As used herein, the term “fatty acid” often refers to a carboxylic acid (or organic acid) having a long aliphatic tail, either saturated or unsaturated. Typically, fatty acids have a carbon-carbon bond chain with a length of at least eight carbon atoms, more specifically at least twelve carbon atoms. Most natural fatty acids have an even number of carbon atoms because their biosynthesis involves acetates with two carbon atoms. Fatty acids can be in a free state (unesterified), referred herein as “free fatty acids,” or in esterified forms such as alkyl esters, parts of triglycerides, parts of diacylglycerides, parts of monoacylglycerides, acyl-CoA (thioester) bonds or other bonding forms, or mixtures thereof. Fatty acids can be esterified as phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, or diphosphatidylglycerol, but are preferably esterified as alkyl esters, particularly as ethyl esters. To avoid misunderstanding, unless otherwise specified, the term “fatty acid” encompasses free fatty acids, fatty acid esters, and salts of any of these. Unless otherwise specified, quantitative values ​​related to a particular fatty acid refer 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).

[0020] Each fatty acid in the composition may also be provided in the form of a salt of the fatty acid, such as an alkali salt or alkaline earth salt. Specific salts that may be mentioned include lithium salts and calcium salts. Such salts may have potential additional pharmaceutically active properties or provide improved processability. Similarly, fatty acid esters may be provided in the form of a salt of the fatty acid ester. Any combination of fatty acids in the form of free fatty acids, salts, esters, or salts of esters may be present in the composition of the present invention. This means, for example, that DHA may exist mainly as an ethyl ester, ALA may exist mainly as a calcium salt of a methyl ester, and oleic acid may exist mainly as a free fatty acid.

[0021] A "saturated fatty acid" contains no double bonds or other functional groups along the chain. The term "saturated" refers to hydrogen in that all carbons (excluding the carboxylic acid [-COOH] group) contain as much hydrogen as possible. In other words, the omega (ω) end is bonded to three hydrogen atoms (CH 3- ), and each carbon within the chain is bonded to two hydrogen atoms (-CH 2- ). The term "total fatty acids" includes all forms of fatty acids, whether saturated or unsaturated, free acids, esters, and / or salts.

[0022] As used herein, the term "about" when referring to measurable values such as amounts, weights, times, temperatures, etc. refers to a variation of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the stated amount.

[0023] Compositions of the invention that may be mentioned include those containing high concentrations of omega-3 fatty acids, many of which are so-called "essential fats" that are considered particularly important for human health. Omega-3 fatty acids have been shown to have beneficial effects on HDL cholesterol levels, support the development of the brains of the young, and be helpful for mental health. These fatty acids are generally thought to be precursors of anti-inflammatory eicosanoids. Specific compositions of the invention that may be mentioned include those in which the total amount of omega-3 polyunsaturated fatty acids in the lipid composition is at least about 30% by weight, for example at least about 35% by weight, of the total fatty acid content of the composition. In certain embodiments, the total amount of omega-3 polyunsaturated fatty acids in the lipid composition is at least about 40% by weight of the total fatty acid content of the composition.

[0024] 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 of more pro-inflammatory eicosanoids, and when these eicosanoids are overproduced, they can increase inflammation and inflammatory diseases. Therefore, it may be desirable to minimize the amount of such fatty acids in the lipid composition. It is generally recognized that the ratio of omega-6 fatty acids to omega-3 fatty acids in the diet should be 4:1 or less. However, in a typical Western diet, the proportion of omega-6 fatty acids typically becomes high. The lipid composition of the present invention advantageously contains a relatively small amount of omega-6 fatty acids and, at the same time, a relatively large amount 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 8% 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 6:1. In a further 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 8:!

[0025] Omega-9 fatty acids are monounsaturated fats that the body can produce. Consuming foods rich in omega-9 fatty acids instead of other types of fats can have many beneficial health effects, including a decrease in plasma triglycerides and "bad" very low density lipoprotein (VLDL) cholesterol in diabetics, reducing inflammation, and improving insulin sensitivity. In one embodiment, the total amount of omega-3 and omega-9 polyunsaturated fatty acids in the lipid composition is at least about 50% by weight of the total fatty acid content of the composition, such as at least about 60% by weight or at least about 70% by weight. In a further embodiment, the ratio of the total weight of omega-3 and omega-9 polyunsaturated fatty acids to the total weight of omega-6 polyunsaturated fatty acids in the composition is at least about 5:1, such as at least about 10:1.

[0026] Lipid compositions containing long-chain polyunsaturated fatty acids are typically obtained from marine sources (e.g., fish, crustaceans), algal sources, or plant sources (e.g., flax or echium). The starting organic matter is first processed to extract the oil contained therein (commonly called "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. Since crude oil often contains levels of polyunsaturated fatty acids (e.g., nutritional products) that are too low to be useful, concentration is necessary. If the crude oil is deficient in one or more essential components, it is common to blend crude or concentrated oils from multiple sources (e.g., fish and algae) together to obtain the desired composition. Alternatively, concentration can be achieved by processing the crude oil while maximizing the level of the desired fatty acid components and removing unwanted components (e.g., components that adversely affect the color, odor, or stability of the product, or unwanted fatty acids).

[0027] The compositions of the present invention are advantageously available 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 compositions of the present invention. The phrase “available from a single source” (or “obtained from a single source”) means that the lipid composition is available from one or more organisms of a single taxonomic class. In certain embodiments, the lipid composition does not originate from multiple organisms across different taxonomic classes. For example, the lipid composition shall not be a blend of oils obtained from a combination of fish and algae, or a combination of fish and plants. Instead, the lipid compositions of the present invention (or the “crude” oils from which the compositions can be obtained by concentration techniques such as transesterification and distillation) are available from a single biological population, e.g., a single plant source or herbaceous plant source. To avoid misunderstanding, the phrase “available from a single source” does not exclude the use of multiple organisms of the same species as a source of the lipid composition or “crude” oil, i.e., multiple fish, algal resources, plants, or plant seeds of the same species. Preferably, the multiple organisms are all of the same species, or originate from the same breeding line, or belong to the same plant species, or to the same production resource or batch.

[0028] In the specific lipid composition of the present invention, DHA is present in an amount of about 20% to about 35% by weight (for example, about 22% to about 33% by weight) of the total fatty acid content of the composition.

[0029] The lipid composition of the present invention has been found to be advantageous in that it contains a high level of DHA compared to the amount of EPA present. Therefore, in one embodiment, the weight ratio of docosahexaenoic acid to eicosapentaenoic acid in the lipid composition is at least about 20:1. In a further embodiment, the weight ratio of docosahexaenoic acid to eicosapentaenoic acid in the lipid composition may be greater than about 25:1, for example, greater than about 30:1, and most specifically greater than about 35:1.

[0030] Preferably, the amount of EPA in the composition may be relatively small. The compositions of the present invention contain EPA at a maximum of about 5% by weight of the total fatty acid content of the composition. In certain embodiments, the composition contains EPA at a maximum of about 3% by weight of the total fatty acid content of the composition, more specifically at a maximum of about 1% by weight.

[0031] The composition of the present invention contains alpha-linolenic acid (ALA) in an amount of about 10% to about 20% by weight of the total fatty acid content of the composition. ALA is an essential fat important for the proper health of humans or animals. In certain embodiments, the composition contains ALA in an amount of about 12% to about 20% by weight, more specifically about 12% to about 18% by weight, of the total fatty acid content of the composition.

[0032] Oleic acid is a component of the composition of the present invention, present in an amount of about 20% to about 40% by weight of the total fatty acid content of the composition. Oleic acid is a monounsaturated fat commonly found in the human diet. The consumption of monounsaturated fats is associated with a decrease in low-density lipoprotein (LDL) cholesterol and possibly an increase in high-density lipoprotein (HDL) cholesterol. In one embodiment, oleic acid is present in an amount of about 20% to about 35% by weight of the total fatty acid content of the composition. In a further embodiment, oleic acid is present in an amount of about 22% to about 33% by weight of the total fatty acid content of the composition.

[0033] The composition of the present invention contains palmitic acid (16:0) at a maximum of about 1.5% by weight of the total fatty acid content of the composition. In certain embodiments, the composition contains palmitic acid at a maximum of about 1.0% by weight of the total fatty acid content of the composition, more specifically at a maximum of about 0.7% by weight.

[0034] The compositions of the present invention also contain eicosatetraenoic acid (ETA, 20:4n-3) in an amount of at least about 0.5% by weight of the total fatty acid content of the composition. In certain embodiments, the composition contains at least about 1.0% by weight, more specifically at least about 1.5% by weight, of the total fatty acid content of the composition.

[0035] Any preferences and choices regarding a given aspect, feature, or embodiment of the present invention should be considered to be disclosed in conjunction with any and all other preferences and choices regarding all other aspects, features, and embodiments of the present invention, unless otherwise specifically indicated in the context. For example, the specific amounts of DHA, EPA, ALA, oleic acid, and palmitic acid shown in the previous section are disclosed in all combinations.

[0036] Therefore, the specific lipid compositions that may be mentioned are (i) docosahexaenoic acid (22:6n-3) in an amount of approximately 20% to approximately 35% by weight of the total fatty acid content of the composition, (ii) Eicosapentaenoic acid (20:5n-3) in an amount of approximately 3% by weight of the total fatty acid content of the composition, (iii) α-linolenic acid (18:3n-3) in an amount of about 12% to about 20% by weight of the total fatty acid content of the composition, (iv) Oleic acid (18:1n-9) in an amount of about 20% to about 35% by weight of the total fatty acid content of the composition, (v) The composition comprises palmitic acid in an amount of approximately 1.0% by weight of the total fatty acid content, Components (i) to (v) 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.

[0037] In the compositions of the present invention, each of components (i) to (v) is a fatty acid that may exist independently in the form of a fatty acid, a fatty acid salt, a fatty acid ester, or a salt of a fatty acid ester. In certain embodiments, these components each take the same form, for example, they may all be in the form of fatty acids, all in the form of fatty acid salts, all in the form of fatty acid esters, or all in the form of salts of fatty acid esters. If a component is in the form of a fatty acid salt, ester, or salt of an ester, the component may be in the form of the same salt, ester, or salt of an ester. For example, each of components (i) to (v) may be provided in the form of an ethyl ester of a fatty acid.

[0038] In certain embodiments, components (i) to (v) are provided in the form of salts of fatty acid esters, or most specifically, in the form of fatty acid esters. Preferred forms of fatty acid esters are known to those skilled in the art. For example, nutritionally acceptable and / or pharmaceutically acceptable forms of fatty acid esters include ethyl esters, methyl esters, phospholipids, monoglycerides, diglycerides, and triglycerides of fatty acids. 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 infant consumption, partly due to the taste and stability of these ester forms against heat treatment (which may be required in such foods). Ethyl esters are particularly suitable for use in dietary supplements because these ester forms can be produced efficiently and easily and do not require conversion to triglyceride forms. Therefore, in further embodiments, components (i) to (v) are each provided independently in the form of fatty acid ethyl esters or as part of triglycerides.

[0039] 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 proportions. That is, a mixture of different triglyceride molecules may be present in the composition, but the overall fatty acid profile in the composition is as defined in the claims.

[0040] Alternatively, the fatty acid component may exist in the form of “free” fatty acids, i.e., the -COOH form of fatty acids. However, in certain compositions of the present invention, the compositions contain relatively low levels of fatty acids in this form because it has an unpleasant (often “soapy”) taste and is less stable than fatty acids in esterified form. Free fatty acids are typically removed from oil and lipid compositions by alkaline or physical purification, according to processes discussed elsewhere herein, for example. Thus, in one embodiment, the total free fatty acid content in the lipid composition is less than 5% by weight (e.g., less than 3% by weight, in particular less than 2% by weight) of the total fatty acid content of the composition.

[0041] The fatty acids in the lipid compositions of the present invention are typically linear (i.e., unbranched) chain fatty acids (such as DHA and ALA). Compositions of the present invention that may be referred to include those containing very low levels of branched chain fatty acids and their esters, such 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 amounts of up to about 0.1% by weight of the total fatty acids in the composition.

[0042] The lipid compositions of the present invention may also contain other components (e.g., non-fatty acids) derived from the raw materials that are not completely removed during the extraction and concentration processes. The exact identity of these other components varies considerably depending on the raw materials. Examples of such other components include plant sterols (i.e., plant sterols and plant stanols) that exist 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 evericol). Other examples include antioxidants such as tocopherols and tocotrienols. Therefore, certain lipid compositions of the present invention that may be mentioned include those containing one or more phytosterols (e.g., β-sitosterol) in detectable amounts. Such sterols may be present in at least about 0.01% by weight, but typically less than about 1% by weight, of the lipid composition.

[0043] The compositions of the present invention are advantageously available 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, in particular crops such as cereals. In at least one embodiment, the lipids are obtained from seed oil crops such as Brassica napus or Brassica juncea. However, to avoid misunderstanding, it is not essential that the compositions are obtained only from such sources, i.e., the proportion of lipids in the compositions of the present invention (e.g., up to 30% by weight) are obtained from other sources, including marine (e.g., fish or crustacean) oils, algal oils and combinations thereof. In one example, at least 80% by weight, e.g., at least 90% by weight of the lipids present are obtained from plant 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 plant sources.

[0044] In one embodiment, the compositions of the present invention (and the feed and pharmaceutical compositions defined herein below) are not of animal origin (e.g., marine animals). That is, in such embodiments, the lipid compositions do not contain any components derived from animals such as fish and crustaceans. Lipid compositions from which no animal components are obtained are considered advantageous in terms of lipid content and stability profiles that can be achieved by standard purification and / or concentration procedures.

[0045] Using plants as a source of lipids or fatty acids offers many advantages. For example, marine oil sources are known to contain relatively high levels of pollutants not found in plant materials (such as mercury, PCBs, and fish allergens (e.g., parvalbumin)). Historical overfishing has also depleted fish and crustacean (such as krill) resources, making them unsustainable. Therefore, the present invention provides a polyunsaturated fatty acid oil composition from a sustainable source that contains relatively low levels of undesirable pollutants.

[0046] In certain embodiments, the compositions of the present invention are derived from plants. The plants from which oils are obtained are typically oilseed crops such as copra, cottonseed, flax, palm kernel, peanut, rapeseed, soybean, and sunflower seed. Thus, compositions obtained solely from plants are sometimes referred to as “vegetable” oils or “vegetable lipid compositions.” Suitable plants from which the lipid composition of the present invention can be obtained are known to those skilled in the art and include Brassica sp., Gossypium hirsutum, Linum 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 specific plant source that can be mentioned in this regard is Brassica sp.

[0047] Suitable sources (including marine, algal, and plant sources) may be naturally occurring or may be genetically modified to enhance their ability to produce long-chain polyunsaturated fatty acids. Examples of plant sources 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 applications 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. All disclosures in publications mentioned herein are incorporated in their entirety by reference.

[0048] The lipid compositions of the present invention can be obtained directly from naturally occurring sources (e.g., animals, algae, and / or plants). However, typically, oils obtained from naturally occurring sources need to be processed to concentrate them. Preferred concentration processes are illustrated in the examples.

[0049] Suitable sources of the lipid compositions of the present invention, or "crude" oils that can be blended or concentrated to produce such compositions, include marine species, algae, and plants. Processes for obtaining oil from marine sources are well known in the art.

[0050] As discussed above, plant sources (such as oilseed sources) are particularly suitable because they have low levels of certain pollutants and are highly sustainable. Plants such as Brassica sp. (e.g., canola) produce seeds that can be processed to obtain oil.

[0051] Oil / lipid extraction Oils produced from plants and seeds can be extracted, processed, and analyzed using techniques routinely employed in the art. Typically, plant seeds are cooked, pressed, and the oil is extracted to produce crude oil. This oil can then be degummed, refined, bleached, and / or deodorized. A combination of degumming, refining, bleaching, and deodorization has been found to be particularly effective in preparing DHA-rich lipid mixtures. 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 adequate purification and concentration can be achieved without using these methods.

[0052] In general, techniques for grinding seeds are known in the art. For example, oilseeds can be tempered by spraying them with water to increase their moisture content to, for example, 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 grinding. Extraction can also be achieved using an extrusion process. The extrusion process may or may not be used as an alternative to flaking, and may be used as an add-on process either before or after screw pressing.

[0053] 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 trace column, and the solvent is subsequently removed from the extracted oil. Alternatively, the crude oil produced by the pressing operation can be passed through a sedimentation tank equipped with a slotted wire drain top to remove any solids that have formed in the oil during the pressing operation. This clarified oil can then be passed through plate and flame filters to remove any remaining fine solid particles. If necessary, the oil recovered in the extraction process can be combined with the clarified oil to produce a mixed crude oil. Once the solvent has been removed from the crude oil, the pressed and extracted portions are combined and subjected to the usual petroleum processing procedures.

[0054] Purification and refining As used herein, the term “purified” as used in relation to the lipids or oils of the present invention typically means that the extracted lipids or oils have been subjected to one or more processing steps to increase the purity of the lipid / oil components. For example, the purification step may include one or more of degumming, deodorizing, decolorizing, or drying of 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 lipids or oils of the present invention in order to increase the DHA content as a percentage of the total fatty acid content. In other words, the fatty acid composition of purified lipids or oils is essentially the same as that of unpurified lipids or oils.

[0055] Vegetable oils, once extracted from a plant source, can be purified (refined) using one or more of the following processes, particularly a combination of degumming, alkaline refining, bleaching, and deodorization. Preferred methods are known to those skilled in the art (e.g., disclosed in WO2013 / 185184).

[0056] Degumming is an initial stage in oil refining, primarily aimed at removing most phospholipids from the oil. Typically, adding about 2% water containing phosphoric acid to crude oil at 70-80°C separates most phospholipids, along with trace amounts of metals and pigments. The insoluble substances removed are mainly mixtures of phospholipids and triacylglycerols. Degumming can be carried out by adding concentrated phosphoric acid to crude seed oil, converting the inhydrateable phospholipids into a hydrateable form and chelating any rare metals present. The gum is then separated from the seed oil by centrifugation.

[0057] Alkaline refining is one of the refining processes for processing crude oil, and is sometimes called neutralization. It typically follows degumming and precedes bleaching. Following degumming, seed oil can be processed by adding enough alkaline solution to titrate all free fatty acids and phosphoric acid, thereby removing the soap thus formed. Suitable alkaline materials include sodium hydroxide, potassium hydroxide, sodium carbonate, lithium hydroxide, calcium hydroxide, calcium carbonate, and ammonium hydroxide. Alkaline refining is typically carried out at room temperature and removes the free fatty acid fraction. The soap is removed by centrifugation or extraction of the soap 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.

[0058] Bleaching is a refining process in which oil is heated to 90-120°C for 10-30 minutes by operating under nitrogen, steam, or vacuum in the presence of bleaching clay (0.2-2.0%) and in the absence of oxygen. Bleaching is designed to remove unwanted pigments (carotenoids, chlorophyll, etc.), and the process also removes oxidation products, trace metals, sulfur compounds, and trace amounts of soap.

[0059] Deodorization involves treating oils at high temperatures (e.g., approximately 180°C) and low pressures (0.1–1 mm Hg). This is typically achieved by introducing vapor into the seed oil at a rate of approximately 0.1 ml / min / 100 ml. Approximately 30 minutes after application, the seed oil is cooled under vacuum. This treatment improves the color of the seed oil and removes most of any residual free fatty acids, monoacylglycerols, and volatile substances or odor compounds, including oxidation products.

[0060] Dewaxing is a process sometimes used in commercial oil production to separate oils and fats into solid (stearin) and liquid (oleic) fractions through crystallization below ambient temperature. Originally applied to cottonseed oil, it produced a product free of solids. It is typically used to reduce the saturated fatty acid content of oils.

[0061] Transesterification Crude oil typically contains the fatty acid 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 fatty acids to another alcohol to form esters (such as ethyl esters or methyl esters). In embodiments of the present invention, transesterification is typically achieved using chemical means, including a strong acid or strong base as a catalyst. Sodium ethoxide (in ethanol) is an example of a strong base used to form fatty acid ethyl esters by transesterification. This process can be carried out at ambient temperature or at high temperatures (e.g., up to about 80°C).

[0062] distillation Molecular distillation is an effective method for removing large quantities of highly volatile components, such as saturated fatty acids, from crude oil. Distillation is typically carried out under reduced pressure, for example, less than about 1 mbar. The temperature and time can then be selected to achieve a ratio of about 50:50 between the distillate and the residue after several hours (e.g., 1 to 10) of distillation time. Typical distillation temperatures used for producing the lipid compositions of the present invention are in the range of 120°C to 180°C, particularly 145°C to 160°C.

[0063] Multiple distillations can be performed, and each distillation is considered complete when an approximately 50:50 split between the distillate and the residue is achieved. While continuous distillation may reduce the overall yield, two distillations may yield optimal results.

[0064] Accordingly, according to a second aspect of the present invention, a process for producing the lipid composition of the present invention is provided, which comprises providing a mixture of fatty acid ethyl esters, subjecting the mixture to a first molecular distillation step to obtain a first residue, and subjecting the first residue to a second molecular distillation step. The present invention also relates to a lipid composition obtained by such a distillation process. Preferred distillation conditions include those described herein. For example, specific distillation temperatures that can be used in the first and second molecular distillation steps are in the range of 120°C to 180°C, such as 145°C to 160°C. The temperature and time are typically selected to achieve a separation of about 50:50 between the distillate and the residue after a distillation time of about 1 hour to about 10 hours. Surprisingly, it has been found that the distillation of plant-based transesterified oils can be achieved at the same or slightly lower (e.g., 3°C lower) temperatures as transesterified oils of other origins (in particular those containing a significant amount (e.g., more than 30% by weight) of marine-derived oils) without impairing the efficiency of the separation process. In other words, while it's not necessary to increase the distillation time, it can actually be reduced while maintaining the same degree of separation (e.g., 50:50 separation by weight) within a reasonable timeframe.

[0065] In one embodiment, the crude oil is heated for 4 to 8 hours, for example, 4 to 6 hours, in a first distillation step. In the same or another embodiment, the crude oil is heated for 0.5 to 4 hours, for example, 1 to 2 hours, in a second distillation step.

[0066] In a second embodiment of the present invention, a mixture of fatty acid ethyl esters is obtained, for example, by transesterification of a plant-based lipid oil according to any one of the processes described above. The plant-based lipid oil can be obtained from any plant, in particular oilseeds, disclosed herein or known in the art.

[0067] Other concentration methods The lipid compositions of the present invention are useful as pharmaceutical active ingredients (APIs) or as precursors (or "intermediates") of APIs that can be obtained therefrom by further concentration. Such compositions can be further concentrated to beneficial PUFA levels such as DHA and / or ALA.

[0068] The concentration of polyunsaturated fatty acids in oil can be increased by various methods known in the art, such as freeze crystallization, complex formation using urea, supercritical fluid extraction, and silver ion complex formation. Complex formation with urea is a simple and efficient method for reducing the levels of saturated and monounsaturated fatty acids in oil. First, the TAG of oil is broken down into its 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 ethanol solution of urea for complex formation, as the fatty acid composition usually does not change upon processing. Saturated and monounsaturated fatty acids readily complex with urea, crystallize upon cooling, and can then be removed by filtration. This enhances the non-urea-complexed fraction with long-chain polyunsaturated fatty acids.

[0069] product The lipid composition of the present invention is bulk oil. That is, the lipid composition is obtained by separating some or all of the lipids from a raw material (e.g., plant seeds).

[0070] The lipid compositions of the present invention can be used as animal feed. That is, the compositions of the present invention can be provided in an orally usable form. For the purposes of the present invention, “feed” includes any food or preparation for consumption by humans or animals that, when taken into the body, helps to nourish or build tissues or supply energy, and / or maintain, restore or support adequate nutritional status or metabolic function. Feed includes, for example, nutritional compositions for infants and / or toddlers, such as infant formula. In the case of feed, fatty acids can be provided in the form of triglycerides to further minimize unpleasant tastes and maximize stability.

[0071] The feed comprises the lipid composition of the present invention together with a suitable carrier of choice. 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 the feed (or used at sufficiently low concentrations) so as not to have harmful effects on organisms that consume the feed.

[0072] Feed compositions may be in solid or liquid form. Additionally, as is well known in the art, the composition may contain amounts of edible macronutrients, proteins, carbohydrates, vitamins, and / or minerals desirable for a particular use. The amounts of these components will vary depending on whether the composition is intended for use in normal individuals or in individuals with special needs, such as those suffering from metabolic disorders.

[0073] Suitable nutritious carriers include major nutrients such as edible fats (e.g., coconut oil, borage oil, fungal oil, Kuroshio oil, soybean oil, and monoglycerides and diglycerides), carbohydrates (e.g., glucose, edible lactose, and hydrolyzed starch), and proteins (e.g., soy protein, electrodialyzed whey, electrodialyzed skim milk, milk whey, or hydrolyzed products of these proteins).

[0074] Vitamins and minerals that may be added to the feed 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.

[0075] The lipid composition of the present invention can be used in pharmaceutical compositions. Such a pharmaceutical composition may optionally comprise 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 may be in liquid or solid form, comprising a solution, suspension, emulsion, oil, or powder. For example, the composition may be in the form of tablets, capsules, encapsulated gels, ingestible liquids (including oil or solution) or powders, or topical ointments or creams. The pharmaceutical composition may also be provided as an intravenous formulation.

[0076] Specific forms suitable for feed and pharmaceutical compositions include liquid-containing capsules and encapsulated gels.

[0077] The lipid compositions of the present invention can be mixed with other lipids or lipid mixtures (particularly plant-based fatty acid esters and fatty acid ester mixtures) before use. 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 in various plant seed oils, including canola oil.

[0078] For example, it may be desirable to include isotonic agents such as sugars and sodium chloride. In addition to such inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances. In addition to the lipid composition of the present invention, the suspension may contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, metahydroxyaluminum, bentonite, agar, and tragacanth or mixtures thereof.

[0079] Solid dosage forms, such as tablets and capsules, can be prepared using techniques well known in the art. For example, fatty acids produced according to the methods disclosed herein can be tableted on conventional tablet bases such as lactose, sucrose, and corn starch, in combination with binders such as acacia, corn starch, 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 a gelatin capsule along with the relevant lipid composition and optionally one or more antioxidants.

[0080] Possible routes of administration for the pharmaceutical compositions of the present invention include, for example, enteral (e.g., oral and rectal) and parenteral administration. For example, liquid preparations may be administered orally or rectally. Additionally, a homogeneous mixture may be completely dispersed in water and mixed under sterile conditions with a physiologically acceptable diluent, preservative, buffer, or propellant to form a spray or inhalant.

[0081] The lipid compositions of the present invention are shown as pharmaceuticals. According to a further aspect of the present invention, compositions of the present invention comprising any of the above pharmaceutical compositions are provided for use as pharmaceuticals.

[0082] The lipid compositions of the present invention can offer many benefits typically associated with long-chain polyunsaturated fatty acids. For example, the lipid compositions of the present invention, and the pharmaceutical compositions described above, may be used in the treatment or prevention of cardiovascular disease, protection against death in patients with cardiovascular disease, reduction of overall serum cholesterol levels, reduction of hypertension, increase of HDL:LDL ratio, reduction of triglycerides, or reduction of apolipoprotein B levels, as can be determined by tests well known to those skilled in the art. Therefore, methods for treating (or preventing) the diseases and conditions listed above using the lipid compositions of the present invention are also disclosed.

[0083] As used herein, the terms “treatment,” “treat,” and “treating” mean reversing, mitigating, or inhibiting the progression of a disease or disorder as described herein, or delaying, eliminating, or reducing the incidence or onset of such disorders or diseases as described herein, compared to what would occur if no measures were taken. As used herein, the terms “prevent,” “prevention,” and “preventing” mean reducing the risk of acquiring or developing a given condition, or reducing or inhibiting recurrence or such condition in a non-disease subject.

[0084] Typical dosages of specific fatty acids range from 0.1 mg to 20 g, taken once to five times a day (maximum 100 g per day), particularly in the range of approximately 10 mg to 1, 2, 5, or 10 g per day (taken in single or multiple doses). As is known in the art, a minimum of approximately 300 mg / day of fatty acids, especially LC-PUFAs, is desirable. However, it will be understood that any amount of fatty acid may be beneficial to the subject.

[0085] When used as a pharmaceutical composition, the dosage of the lipid composition administered to the patient is determined by one of the usual techniques in the art and depends on various factors such as the patient's weight, age, overall health, medical history, and immune status.

[0086] The compositions of the present invention are readily available compositions that have an improved stability profile and may contain a mixture of fatty acids in which the relative ratios of omega-3, omega-6, and / or omega-9 fatty acids are 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 the AOCS formula Cd8-53), and the "p-anisidine value" method (e.g., using the AOCS formula Cd18-90). The examples show that the compositions of the present invention can be obtained from starting mixtures that do not exhibit an improved stability profile compared to a reference blend (a reference blend having a similar composition with respect to major LC-PUFAs but containing significant amounts of animal (fish) derived lipids).

[0087] The compositions of the present invention may also have advantages over lipid compositions known in the prior art, such as being more effective, less toxic, longer-acting, more potent, having fewer side effects, being more easily absorbed, and / or having a better pharmacokinetic profile (e.g., higher oral bioavailability and / or lower clearance), and / or having other useful pharmacological, physical, or chemical properties.

[0088] The present invention will be explained by the following examples. [Brief explanation of the drawing]

[0089] [Figure 1] The propanal release data for canola oil and the reference oil (after transesterification and distillation) are shown to demonstrate the improved stability of the canola oil described herein. [Figure 2] The propanal release data for canola oil and the reference oil (after RBD refining, transesterification, and distillation) are shown to demonstrate the improved stability of the canola oil described herein.

[0090] General method GC sample preparation and GC parameters Neat fatty acid ethyl ester was diluted to 0.25% (v / v) with 50:50 chloroform:methanol and 0.01% BHT. The ethyl ester solution was diluted to 2.5 mg / mL with chloroform:methanol.

[0091] Control checks were prepared as fatty acid methyl esters of canola oil, tuna oil, and 3x canola-DHA oil. These were analyzed batch by batch of samples to check GC performance and monitor DHA degradation due to GC system activity.

[0092] The methyl ester was prepared as follows. Neat oil was diluted to 0.33% (v / v) with 50:50 chloroform:methanol and 0.01% BHT (butylated hydroxytoluene). 50 μL of 0.05 N Meth-Prep II solution (a methanol solution of 0.2 N m-trifluoromethylphenyltrimethylammonium hydroxide) was added, the solution was vortexed, and incubated at 40°C for 30 minutes. The final solution corresponded to 0.25% (v / v) oil.

[0093] Ethyl ester (sample) and methyl ester (check) were investigated using a Shimadzu GC-2010 Plus with a flame ionization detector (FID) and segmented jetting using the parameters described below. Column: 30m BPX-70, inner diameter 0.32mm, film thickness 0.25pm. Injection volume: 0.5μL

[0094] The results were calculated as normalized areas (i.e., the area percentage after the fatty acid peak was identified, and the non-fatty acid peak was excluded from the sum of the areas of all peaks).

[0095] The identity of the ethyl ester peak was determined by comparing the chromatogram of fatty acid ethyl ester with the chromatogram of fatty acid methyl ester. While the relative elution order was nearly identical, the ethyl ester eluted later as a group compared to the fatty acid methyl ester. The elution order of fatty acid methyl ester had been previously identified using a reference standard and GC-MS. [Examples]

[0096] Example 1 - Extraction of DHA canola oil from seeds The canola variety disclosed in U.S. Patent Publication US2018 / 0016590A1 was grown as a summer crop. After harvesting the seeds, they were stored at room temperature before being ground.

[0097] DHA oil was produced by grinding 272 kg of seeds using a Kern Kraft KK80 screw press. The temperature of the expeller color heater was set to the maximum setting temperature of the thermostat. The initial ambient temperature and choke temperature were 20°C, and the choke distance was set to 73.92 mm. Seeds were fed while continuously collecting oil and coarse powder without stopping the expeller until all seeds were ground.

[0098] The auger rotation speed, coarse powder, and discharge oil temperature were monitored throughout the pressing process. The crushing time, which resulted in a throughput rate of 67.5 kg / hour, was 4 hours for 270 kg. A yield of 87.2 kg of crude oil (32%) was obtained. After filtration to remove fine powder, the yield was 77.2 kg (28%).

[0099] Example 2 - Reference Blend Oil Pure fish oil contains low levels of ALA fatty acids, as well as significantly higher levels of EPA and DHA. The reference oil blend (hereinafter referred to herein as the “crude triglyceride reference blend oil” or similar) was designed to be as similar in composition as possible to the filtered DHA canola oil obtained in Example 1. This was done by (a) matching the total level of DHA to that of DHA canola oil, and (b) matching the DHA / (ALA+EPA) ratio. This was achieved by blending DHA (tuna) rich fish oil, ALA (flaxseed oil) rich oil, and standard canola oil. The resulting reference blend oil also has a similar total omega-3 content to DHA canola oil.

[0100] Example 3 - Fatty acid composition of crude DHA canola oil and reference blend The fatty acid composition of filtered crude oil and the reference blend oil was analyzed. The results are shown below. [Table 1]

[0101] Example 4 - Evaluation of oil stability Rancimat stability studies were conducted using the crude DHA canola oil and reference blend described in Example 1 and Example 2, respectively. This method involved testing approximately 2.5 g of test material using the standard procedure for Metrohm743 Rancimat at 90°C.

[0102] The table below summarizes the results obtained from these oils at 90°C. The experiment was conducted twice. [Table 2]

[0103] DHA canola oil consistently showed inferior stability compared to the reference oil.

[0104] Example 5 - Chemical transesterification of crude canola-DHA oil In a dry nitrogen-flashed Buchi CR101 chemical reactor equipped with a mechanical stirrer, anhydrous ethanol (12.5 L) and crude triglyceride canola oil ("DHA canola oil") (5.00 kg) obtained in Example 1 were added, and the mixture was stirred.

[0105] Sodium ethoxide (150g) was added to the above mixture, and this was further rinsed in the reactor with anhydrous ethanol (2.5L), and stirring was continued at ambient temperature for 16 hours. The recorded samples taken from the mixture were 1 The 1H NMR spectrum indicated that the reaction was complete.

[0106] The transesterification procedure was carried out with 5.22 kg of crude canola-DHA oil. To the resulting crude reaction mixture, 10 L of petroleum spirit (PET spirit, boiling point 40-60°C) and 10 L of water were added and the mixture was carefully acidified to pH 7 with 10% hydrochloric acid (870 mL total required, Merck Universal Indicator strips, pH 0-14) while thoroughly mixing.

[0107] After the resulting mixture was left in the reactor, two phases formed. The petroleum spirit layer was removed, and the aqueous layer was further extracted with petroleum spirit (3 x 5 L). The combined petroleum spirit layer was returned to the reactor and evaporated in a vacuum to a small amount (approximately 10 L). The resulting concentrated solution was discharged from the reactor, dried with anhydrous magnesium sulfate (approximately 1 kg), filtered, and concentrated in a vacuum to obtain yellow oil (5.13 kg).

[0108] Example 6 - Chemical transesterification of reference crude oil blend. In a dry nitrogen-flushed Buchi CR101 chemical reactor equipped with a mechanical stirrer, anhydrous ethanol (12.5 L) and crude triglyceride reference blend oil (5.00 kg) obtained according to Example 2 were added, and the mixture was stirred.

[0109] Sodium ethoxide (150 g) was added to the above mixture, and this was further rinsed in the reactor with anhydrous ethanol (2.5 L), and stirring was continued at ambient temperature for 16 hours. The recorded sample1 The 1H NMR spectrum showed little to no reaction. Sodium ethoxide (57 g) was then added to the mixture, and stirring was continued.

[0110] Five hours later, the sample 1 The 1H NMR spectrum indicated that the reaction was 75% complete. Sodium ethoxide (60 mL of 21% ethanol solution) was then added to the mixture, and stirring was continued for 3 days, after which the reaction was complete.

[0111] Separation of Example Products from Chemical Transesterification of Reference Crude Blend The transesterification procedure was carried out with 5.17 kg of the reference crude blend oil. To the resulting crude reaction mixture, petroleum spirits (15 L) and water (3.3 L) were added, and the mixture was carefully acidified to pH 7 with 10% hydrochloric acid (a total of 910 mL was needed) while thoroughly mixing.

[0112] After the resulting mixture was left in the reactor, two phases formed. The petroleum spirit layer was removed, and the aqueous layer was further extracted with petroleum spirit (2 x 7.5 L). The combined petroleum spirit layer was returned to the reactor and evaporated in a vacuum to a small amount (approximately 10 L). The resulting concentrated solution was discharged from the reactor, dried with anhydrous magnesium sulfate (approximately 1 kg), filtered, and concentrated in a vacuum to obtain yellow oil (5.39 kg).

[0113] Example 7 - Analysis of the fatty acid composition of transesterified oil The fatty acid composition of the transesterification products obtained in Example 5 and Example 6 was analyzed. The results are shown below. [Table 3]

[0114] Example 8 - Distillation of transesterified canola oil Standard procedure for removing more volatile components from 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. Separation by distillation was achieved by passing the trans-esterified crude oil through a Pope 2-inch (50 mm) thin-film distiller under vacuum, equipped with two 1000 ml collection flasks for collecting the distillate and residue. The composition of each fatty acid was analyzed.

[0115] Vacuum was supplied by an Edwards 3 rotary pump and measured by an Ebro VM2000 vacuum gauge.

[0116] The oil was supplied to the still at 4 mL / min by a Cole-Palmer Instrument Company easy-load II peristaltic pump, the still motor was set to 325 rpm, and the distillate was condensed using a water condenser. Supply continued until one of the receiver flasks was full.

[0117] Crude canola DHA FAEE was distilled under these conditions with the heater band initially set to 147°C. The objective was to obtain a 50:50 distillate:residue ratio. During the first 30–45 minutes of the experiment, the heater band temperature was increased to 154°C to increase the proportion of distilled oil, after which the still was equilibrated. After 30 minutes, the heater band temperature was adjusted to 149°C over 30 minutes. The remaining distillation was performed at 149°C. The total distillation time was 350 minutes. A portion of the residue from the above distillation was again subjected to distillation under standard conditions with the heater band temperature set to 149°C to remove more volatile components. The total distillation time was 95 minutes. [Table 4]

[0118] Example 9 - Distillation of FAEE derived from the transesterified crude blend of the reference. Crude fatty acid ethyl esters (FAEEs) from the reference crude blend (obtained in Example 6) were subjected to distillation under the same conditions as shown in the previous example.

[0119] The reference crude blend FAEE was distilled under these standard conditions with the heater band initially set to 152°C. The objective was to obtain a 50:50 distillate:residue ratio. 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 last 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 subjected again to distillation under standard conditions to remove more volatile components. The objective was to obtain a 50:50 distillate:residue ratio. Distillation was mainly carried out with the heater band set to 150-151°C. The total distillation time was 195 minutes. [Table 5]

[0120] Example 10 - Analysis of the fatty acid composition of distilled oil The fatty acid composition of the double-distillation products obtained in Examples 8 and 9 was analyzed. The results are shown below. [Table 6]

[0121] Example 11 - Evaluation of oil stability Headspace GC-MS stability testing Headspace analysis was performed on the concentrated product described above to evaluate the amount of propanal released under specific conditions. An increase in the level of propanal release indicates a decrease in the stability of the test material.

[0122] SPME (Solid-Phase Microextraction) I selected the 65μm PDMS / DVB StableFlex fiber (Supelco fiber kit 57284-u). The fibers were conditioned for 10 minutes at 250°C in the Triplus RSH conditioning station before use. The sample was incubated at 40°C for 1 minute before extraction. Extracted from the headspace vial for 1 minute. This is expected to be an excellent general method capable of capturing a wide range of volatile components.

[0123] GC method Thermo Scientific TRACE1310 GC Thermo Scientific TR-DIOXIN 5MS column, inner diameter 0.25 mm, 30 m film, 0.1 μm Split injection 250℃ split 83, 1.2ml He / min GC lamp: 40°C for 1 minute, then 5°C / min to 100°C, then 50°C / min to 300°C

[0124] A general MS-specific column that exhibits excellent synergistic effects for headspace analysis was used. To maximize the separation of volatile substances before reaching maximum temperature, a slow initial temperature rise was employed to maintain column performance. Split injection was employed to avoid the need for cryogenic cooling of the injection port and to improve the column's separation ability.

[0125] Standard separation was hampered by peak overlap, but was still sufficient for quantification. Calibration results for three standards (0.1%, 0.01%, and 0.01%), with the molecular ion m / z 56 used for propanal detection, and the base peak at m / z 58 used for hexanal detection.

[0126] 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 prepared in the supplied DHA canola ethyl ester. These standard mixtures were then added to 20 ml headspace vials in a volume of 540 μl.

[0127] The adoption of full scanning made it possible to monitor all development products, rather than just specific molecules.

[0128] Headspace stability results: The following table summarizes the results obtained from double-distilled canola oil obtained in Example 8 and double-distilled reference oil obtained in Example 9 at T=0–4 days. Test samples were held at ambient temperature on a lightbox and under fluorescent lighting during this period. Analysis of m / z 58 molecular ions revealed that the mass chromatogram clearly shows the appearance of propanal at room temperature in 1.37 minutes. The development of propanal is quantified in the table below, and the data are shown in Figure 1. The DHA canola oil releases a substantially small amount of propanal, demonstrating improved stability of the canola oil compared to the reference. [Table 7]

[0129] DHA canola oil exhibits superior stability against oxidation compared to the reference oil.

[0130] Example 12 - 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.

[0131] Acid degumming Degumming is the process of removing non-hydrated and hydrated phospholipids from oil. The dried crude oil obtained in Example 1 was heated to 53±2°C, and a 0.2% 50% citric acid solution was added. After mixing for about 30 minutes, 2.0% heated (53±2°C) soft water was added and mixed for about 30 minutes. While holding, the oil was heated to 67±3°C and then centrifuged.

[0132] Acid pretreatment / purification Purification involves saponification with a corrosive agent to make the oil water-soluble, followed by the removal of free fatty acids and subsequent centrifugation. An acid pretreatment step was used to continue the hydration of the phosphatide. The defugummed oil was heated to 65±5°C, 0.1% of 85% phosphoric acid was added, and the mixture was mixed for a total of 30 minutes. After the addition of the acid and the holding time, 20 Be'(Baume; 14.4%, w / w) sodium hydroxide was added to neutralize the free fatty acids and the 0.05% (w / w) excess. The corrosive agent and oil were then mixed for a further 15 minutes. The oil was heated to 62±2°C while holding for 15 minutes, and then centrifuged.

[0133] Trisyl silica treatment To further remove soap, Trisyl silica treatment was performed to a level suitable for bleaching. The Trisyl pretreatment was combined with the bleaching step. The refined oil was heated to 68±5°C and treated with 0.3% Trisyl300. After mixing the oil / Trisyl for approximately 15 minutes, bleaching was continued.

[0134] bleaching The refined oil was treated with adsorbent clay to remove peroxides, phospholipids, color bodies, and trace amounts of soap. An acid pretreatment step was used to continue the hydration of phosphatides. Trisyl pretreated oil was mixed with a 0.2% (w / w) 50% citric acid solution. After mixing for 15 minutes, 2% (w / w) 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, the vacuum was broken with nitrogen, and it was filtered with 1.0 kg of filter aid added. Pressure vessel: 500L Cherry-Burrell pressure vessel, all 316 stainless steel construction with steam or cooling water jacket, impeller and baffle for mixing, manufacturing serial number E-227-94. Filter press: 24'' Polypropylene Sperry Filter Press, with a capacity of 4.8 cu ft filter, paper and cloth support material.

[0135] Deodorization The bleached oil was sprayed using steam at high temperature and low pressure to remove odor components, flavor components, and additional free fatty acids. Color fading also occurs with heat bleaching at high temperatures. Half of the bleached oil was deodorized with 1% spurged steam at 180±2°C for 60 minutes, and the fatty acid composition (FAC) was monitored. Deodorization container (OD4): 400L Coppersmithing vacuum-rated container, steam or cooling water jacket, all 316 stainless steel construction. A slight decrease in DHA levels was observed after holding at 180°C for 60 minutes. Another test was then performed by holding at 180°C for 30 minutes. The product was packed in 20L plastic HDPE pails under nitrogen and stored in a 4°C cooler.

[0136] Example 13 - Refinement of the original reference blend oil A portion of the reference blend described in Example 2 was purified before further concentration. In the purification process, the reference blend was purified under the same conditions as described in the previous example.

[0137] Example 14 - Fatty acid composition of RBD DHA canola oil and RBD reference blend The fatty acid composition of RBD DHA canola oil (from Example 12) and RBD reference blend oil (from Example 13) was analyzed. The results are shown below. [Table 8]

[0138] Any reference to “RBD” in the examples (and accompanying figures) means that the product in question was obtained directly or indirectly from either the “refined” product of Example 12 (for canola oil) or Example 13 (for the reference blend).

[0139] Example 15 - Chemical transesterification of RBD canola-DHA oil In a dry nitrogen-flashed Buchi CR101 chemical reactor equipped with a mechanical stirrer, anhydrous ethanol (12.5 L) and the purified ("RBD") triglyceride canola-DHA oil obtained in Example 12 (5.00 kg) were added, and the mixture was stirred.

[0140] Sodium ethoxide (150g) was added to the above mixture, and this was further rinsed in the reactor with anhydrous ethanol (2.5L), and stirring was continued at ambient temperature for 16 hours. The recorded samples taken from the mixture were 1 The 1H NMR spectrum indicated that the reaction was complete.

[0141] The transesterification procedure was carried out with approximately 5 kg of RBD canola-DHA oil. To the resulting crude reaction mixture, 10 L of petroleum spirit (PET spirit, boiling point 40-60°C) and 10 L of water were added and the mixture was carefully acidified to pH 7 with 10% hydrochloric acid (870 mL total required, Merck Universal Indicator strips, pH 0-14) while thoroughly mixing.

[0142] After the resulting mixture was left in the reactor, two phases formed. The petroleum spirit layer was removed, and the aqueous layer was further extracted with petroleum spirit (3 x 5 L). The combined petroleum spirit layer was returned to the reactor and evaporated in a vacuum to a small amount (approximately 10 L). The resulting concentrated solution was discharged from the reactor, dried with anhydrous magnesium sulfate (approximately 1 kg), filtered, and concentrated in a vacuum to obtain a pale yellow oil (yield: 99%).

[0143] Example 16 - Chemical transesterification of RBD reference blend oil In a dry nitrogen-flushed Buchi CR101 chemical reactor equipped with a mechanical stirrer, anhydrous ethanol (12.5 L) and RBD triglyceride reference blend oil (approximately 5 kg) obtained according to Example 13 were added, and the mixture was stirred.

[0144] Sodium ethoxide (150 g) was added to the above mixture, and this was further rinsed in the reactor with anhydrous ethanol (2.5 L), and stirring was continued at ambient temperature for 16 hours. The recorded sample 1 The 1H NMR spectrum showed little to no reaction. Sodium ethoxide (57 g) was then added to the mixture, and stirring was continued.

[0145] Five hours later, the sample 1The 1H NMR spectrum indicated that the reaction was 75% complete. Sodium ethoxide (60 mL of 21% ethanol solution) was then added to the mixture, and stirring was continued for 3 days, after which the reaction was complete.

[0146] Separation of example products from chemical transesterification of RBD reference blend The transesterification procedure was carried out with approximately 5 kg of RBD reference blend oil. To the resulting crude reaction mixture, petroleum spirits (15 L) and water (3.3 L) were added, and the mixture was carefully acidified to pH 7 with 10% hydrochloric acid (a total of 910 mL was needed) while thoroughly mixing.

[0147] After the resulting mixture was left in the reactor, two phases formed. The petroleum spirit layer was removed, and the aqueous layer was further extracted with petroleum spirit (2 x 7.5 L). The combined petroleum spirit layer was returned to the reactor and evaporated to a small amount (approximately 10 L) under vacuum. The resulting concentrated solution was discharged from the reactor, dried with anhydrous magnesium sulfate (approximately 1 kg), filtered, and concentrated under vacuum to obtain a pale yellow oil (yield: 99%).

[0148] Example 17 - Distillation of transesterified RBD canola oil Standard procedure for removing more volatile components from fatty acid ethyl ester (FAEE) mixtures by vacuum distillation. Fatty acid ethyl esters (FAEEs) from RBD canola-DHA (obtained in Example 15) were subjected to distillation under the following conditions. Separation by distillation was achieved by passing the transesterified oil through a Pope 2-inch (50 mm) thin-film distiller under vacuum, equipped with two 1000 ml collection flasks for collecting the distillate and residue. The composition of each fatty acid was analyzed.

[0149] Vacuum was supplied by an Edwards 3 rotary pump and measured by an Ebro VM2000 vacuum gauge.

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

[0151] RBD Canola-DHA FAEE was distilled under these conditions with the heater band initially set to 152°C, yielding a 50:50 distillate:residue ratio. A portion of the residue from this distillation was again subjected to distillation under standard conditions with the heater band temperature set to 152°C to remove more volatile components. The total distillation time was approximately 90 minutes. [Table 9]

[0152] Example 18 - Distillation of FAEE derived from a transesterified RBD reference blend Fatty acid ethyl esters (FAEEs) from the RBD reference blend (obtained in Example 16) were distilled under the same conditions as shown in the previous example.

[0153] The RBD reference blend FAEE was distilled under these standard conditions with the heater band initially set to 152°C to obtain a 50:50 distillate:residue ratio. The residue from this distillation was subjected to further distillation under standard conditions to remove more volatile components. The objective was to obtain a 50:50 distillate:residue ratio. Distillation was mainly carried out with the heater band set to 152°C. The total distillation time was approximately 200 minutes. [Table 10]

[0154] Example 19 - Analysis of the fatty acid composition of concentrated RBD oil The fatty acid composition of the products obtained in Example 17 and Example 18 was analyzed. The results are shown below. [Table 11]

[0155] Example 20 - Evaluation of oil stability Headspace analysis was performed on the concentrated products described in Examples 17 and 18 according to the method described in Example 11.

[0156] The following table summarizes the results obtained from RBD canola oil obtained in Example 17 and RBD reference oil obtained in Example 18 at T=0-3 days. Test samples were held at ambient temperature on a lightbox and under fluorescent illumination during this period. Analysis of m / z 58 molecular ions revealed that the mass chromatogram clearly shows the appearance of propanal at room temperature at 1.37 mins. The development of propanal is quantified in the table below, and the data is shown in Figure 2. The DHA canola oil releases a substantially small amount of propanal, demonstrating improved stability of the canola oil compared to the reference. [Table 12]

[0157] DHA canola oil showed superior stability against oxidation compared to the reference oil.

Claims

1. A plant-based lipid composition, (i) Docosahexaenoic acid (22:6n-3) in an amount of 15% to 35% by weight of the total fatty acid content of the composition, (ii) eicosapentaenoic acid (20:5n-3) in an amount of 0% to 5% by weight of the total fatty acid content of the composition, (iii) α-linolenic acid (18:3n-3) in an amount of 10% to 20% by weight of the total fatty acid content of the composition, (iv) Oleic acid (18:1n-9) in an amount of 20% to 40% by weight of the total fatty acid content of the composition, (v) comprising palmitic acid (16:0) in an amount of 0% to 1.5% by weight of the total fatty acid content of the composition, Components (i) to (v) 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. At least 90% by weight of the lipids present in the composition are derived from plant sources, and A lipid composition in which the plant source is the oilseed of Brassica sp.

2. The lipid composition according to claim 1, wherein 100% by weight of the lipids present in the composition are derived from the plant source.

3. The lipid composition according to claim 1 or 2, wherein docosahexaenoic acid is present in an amount of 20% to 35% by weight of the total fatty acid content of the composition.

4. The lipid composition according to claim 3, wherein docosahexaenoic acid is present in an amount of 22% to 33% by weight of the total fatty acid content of the composition.

5. The lipid composition according to any one of claims 1 to 4, wherein the eicosapentaenoic acid is present in an amount of 0% to 3% by weight of the total fatty acid content of the composition.

6. The lipid composition according to claim 5, wherein the eicosapentaenoic acid is present in an amount of 0% to 1% by weight of the total fatty acid content of the composition.

7. The lipid composition according to any one of claims 1 to 6, wherein the α-linolenic acid is present in an amount of 12% to 20% by weight of the total fatty acid content of the composition.

8. The lipid composition according to claim 7, wherein the α-linolenic acid is present in an amount of 12% to 18% by weight of the total fatty acid content of the composition.

9. The lipid composition according to any one of claims 1 to 8, wherein the oleic acid is present in an amount of 20% to 35% by weight of the total fatty acid content of the composition.

10. The lipid composition according to claim 9, wherein the oleic acid is present in an amount of 22% to 33% by weight of the total fatty acid content of the composition.

11. The lipid composition according to any one of claims 1 to 10, wherein the palmitic acid is present in an amount of 0% to 1.0% by weight of the total fatty acid content of the composition.

12. The lipid composition according to claim 11, wherein the palmitic acid is present in an amount of 0% to 0.7% by weight of the total fatty acid content of the composition.

13. The lipid composition according to any one of claims 1 to 12, wherein components (i) to (v) are each independently provided in the form of a fatty acid ester or a salt of a fatty acid ester.

14. The lipid composition according to claim 13, wherein components (i) to (v) are each independently provided in the form of a fatty acid ethyl ester or a triglyceride.

15. The lipid composition according to any one of claims 1 to 14, wherein the lipid composition is derived from a single source.

16. The lipid composition according to any one of claims 1 to 15, wherein the plant source comprises oilseeds of Brassica napus or Brassica juncea.

17. The lipid composition according to any one of claims 1 to 16, wherein the composition is provided in the form of a tablet, a capsule, an encapsulated gel, an ingestible liquid or powder, or a topical ointment or cream.

18. The lipid composition according to any one of claims 1 to 17, further comprising one or more additional components selected from the group consisting of antioxidants, stabilizers, and surfactants.

19. A lipid composition according to any one of claims 1 to 18, for use in the treatment or prevention of cardiovascular disease, protection against death in patients with cardiovascular disease, reduction of overall serum cholesterol levels, reduction of hypertension, increase of HDL:LDL ratio, reduction of triglycerides, or reduction of apolipoprotein B levels.

20. A process for producing a lipid composition according to any one of claims 1 to 16, comprising: providing a mixture of fatty acid ethyl esters; subjecting the mixture to a first molecular distillation step to obtain a first residue; and subjecting the first residue to a second molecular distillation step.

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