Non-therapeutical use of phosphatidylcholine and / or lysophosphatidylcholine, ingestible composition, and method of preparation

Abstract

The invention relates to the non-therapeutical use of phosphatidylcholine and / or lyso- phosphatidylcholine for increasing the nutrient retention of at least one Long-Chain Fatty Acid (LCFA) in an animal or a human, or to a use of phosphatidylcholine and / or lyso- phosphatidylcholine for increasing the thermal stability of at least one Long-Chain 5 Unsaturated Fatty Acid (LC-UFA). The invention further relates to an ingestible composition, and a method of preparation of such a composition.

Description

[0001] Non-therapeutical use of phosphatidylcholine and / or lyso- phosphatidylcholine, ingestible composition, and method of preparation

[0002] Technical field of the invention

[0003] The invention relates to the non-therapeutical use of phosphatidylcholine and / or lyso- phosphatidylcholine. The invention further relates to an ingestible composition, and a method of preparation of a composition.

[0004] Background of the invention

[0005] Long-Chain Fatty Acids (LCFA), and in particular Very Long-Chain Unsaturated Fatty Acids (VL-UFA), are a desirable component of the diet of mammals. The specific fatty acids, for which numerous health effects have been described in literature contain typically 20 to 24 carbon atoms, including Docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and Dihomo-y-linolenic acid (DGLA). These fatty acids have been reported as being associated with developmental benefits, a reduction of health risks, healthy aging, and general health and wellbeing. More specifically, such fatty acids have been reported as being associated with a healthy development in early life, with a healthy immune system, with a reduction of inflammation, with maintaining brain health, positively affecting metabolic health, and with a reduction in colon cancer risk.

[0006] A well-known LCFA fatty acid is DHA. Docosahexaenoic acid (DHA) is known to be important for cell signalling, gene expression, and the production of lipid mediators. Increased DHA levels enhance its presence in the membrane phospholipids of cells, leading to several effects. Firstly, cell membranes become more fluid, which affects the behaviour of various membrane proteins, including their organization into signalling platforms known as lipid rafts. This increased fluidity diminishes the transmission of inflammatory signals within cells, such as those from LPS or saturated fatty acids, resulting in reduced activation of pro-inflammatory transcription factors like NF-kappaB. These transcription factors regulate genes that encode cytokines, chemokines, adhesion molecules, inflammatory enzymes, and proteases. Consequently, the anti-inflammatory effects of these mediators can be extensive. Additionally, omega-3 PUFAs partially replace omega-6 PUFA arachidonic acid (ARA), which is typically the substrate for cyclooxygenases, lipoxygenases, and cytochrome P450 enzymes that produce eicosanoids, primarily responsible for inducing pro-inflammatory effects. A third effect of DHA in the membranes (of inflammatory cells) is that they can be released upon cellular activation. The free DHA can have further actions including that they are the substrate for the formation of highly active anti-inflammatory lipid mediators (e.g. resolvins, protectins, maresins) [1], Moreover, membrane DHA reduces EGFR signalling and by this (colon) cancer risk [2],

[0007] Another well-known LCFA fatty acid is eicosapentaenoic acid (EPA). The attachment of an EPA fatty acid residue to the phospholipids of the cell membrane prevents the conversion of ARA to eicosanoids, such as thromboxanes and prostaglandins, thus reducing the risk of inflammation. In addition, EPA is the precursor of series-3 of prostaglandins and thromboxanes. In higher concentrations, eicosanoids produced in small amounts show vasodilating, antithrombotic and antiatherosclerosis effects. They have the opposite effect (increased blood coagulability and proinflammatory effect) because leukotrienes (mainly LTB4 and other ARA derivatives) release lysosomal enzymes and generate free radicals. The transformation products of acids from the omega-3 family show a similar but much weaker effect, therefore, replacing ARA with EPA leads to leukotrienes and prostaglandins with much lower biological activity (having only 1-10% of LTB4 activity). Similarly, thromboxane produced from EPA through the transformation of omega-3 family acids shows a much weaker ability to aggregate blood platelets. In this way, acids from the omega-3 family inhibit inflammatory reactions, have an anti-aggregating effect and reduce excessive contractility of blood vessels [3],

[0008] Dihomo-y-linolenic acid (DGLA) has emerged as a significant molecule differentiating healthy and inflamed tissues. It’s positioned at a pivotal point of metabolic pathways leading to anti-inflammatory derivatives [4], Moreover, adrenic acid could represent a novel pro-resolving mediator effectively blocking the production of LTB4 by neutrophils, thereby attenuating ongoing inflammation. In addition, Adrenic acid enhances phagocytosis by macrophages, a mechanism that could be important for the efficient clearance of (cartilage) breakdown products in for example osteoarthritis joints resulting in enhanced resolution [5],

[0009] Phospholipids (PLs) are the main component of the lipid bilayer forming cellular membranes and organelle membranes, such as mitochondrial membranes. In mammalian cells, phosphatidylcholine (PC) is the most abundant PL. The equilibrium between saturated fatty acids and unsaturated fatty acids (UFAs) within membrane PLs affects a variety of membrane properties including the stability, membrane fluidity, bending rigidity and hydrophobic thickness, resulting in an effect on cell signaling and cell to cell communication. PC also serves as a very efficient source of choline with choline acting vitamin-like in metabolism serving amongst others as a methyl donor and as a precursor for acetylcholine

[0013] , Specific lipid mediators can rapidly be produced after UFAs with 20 carbon atoms or longer are released from cell membranes by phospholipases. [6, 7], After this, UFAs such as arachidonic acid (ARA) are converted by lipoxygenases (LO) and cyclooxygenases (COX) into proinflammatory lipid mediators such as prostaglandin (PG) and leukotriene (LT). These in turn promote vascular permeability, platelet aggregation, further leukocyte recruitment, and augmentation of inflammatory cytokines. During the inflammatory state a lipid mediator class switch occurs in which LO and COX begin to convert a second group of UFA (e.g., DPA, DGLA, DHA, EPA) into specialized proresolving lipid mediators (SPM). SPMs downregulate inflammatory processes, reducing cytokine production and promote clearance of debris and tissue repair [8],

[0010] Supplementation of UFAs from the second group was found to result in an increase in the production of SPMs [9, 10], However, the levels of SMPs are often not high enough, or results are conflicting, to modulated chronic inflammatory conditions (e.g., metabolic disease, neurodegenerative disease).

[0011] W02020236075A1 describes increasing the stability of Long-Chain Polyunsaturated Fatty Acids (LC-PUFA) using polar lipids, making handling and transport easier and improving the shelf-life of the product

[0012] W02009095435A1 describes isolated egg yolk lecithin, which can be used in triglyceride compositions compromising a long chain polyunsaturated fatty acid (LCPUFA) and as an ingredient in a fat mixture for an infant food formula.

[0013] WO2016120471 describes a method for the prophylaxis and / or treatment of a developmental disorder which is associated with preterm birth. The method involves the administration of a composition which comprises arachidonic acid (ARA), docosahexaenoic acid (DHA) and choline to a preterm infant. Also described is a method of determination of the ratio of arachidonic acid (ARA)-phosphatidylcholine (PC): docosahexaenic acid (DHA)-phosphatidylcholine (PC) in a plasma sample, which is mentioned to show an inverse correlation of the ratio of ARA-PC:DHA-PC in the plasma with the degree of severity of a developmental disorder which is associated with preterm birth.

[0014] Summary of the invention

[0015] In a first aspect, the present invention relates to a non-therapeutical use of phosphatidylcholine and / or lyso-phosphatidylcholine for increasing the nutrient retention of at least one Long-Chain Fatty Acid (LCFA) in an animal or a human. Nutrient retention refers to the extent a nutrient, for this patent a fatty acid, is taken up from the intestinal tract and ultimately incorporated into a specific mammal body tissue. Nutrient retention can be determined by analysing the change of the specific fatty acid in the body tissue resulting from a change in dietary inclusion and / or dietary form, and can be subdivided in the amounts found as free fatty acids, phospholipids (tissue cell membranes), and triglyceride (storage inside a tissue cell).

[0016] Non-medical use indicates that the use described herein is a use that is not aimed at medical treatment or therapy related to a specific disease or medical condition. Reference to a “non-therapeutical use” herein may, in alternative aspects and embodiments, refer to non-therapeutical methods characterised by the purpose of the uses described herein. Thus, the present invention provides non-therapeutic methods having the features of the uses, across all aspects and embodiments, described herein.

[0017] In a second aspect, the present invention provides an ingestible composition for increasing the nutrient retention of at least one Long-Chain Fatty Acid (LCFA) as described herein, wherein the composition comprises i) phosphatidylcholine and / or lyso- phosphatidylcholine; and ii) at least one triglyceride-bound LCFA with increased nutrient retention.

[0018] In a third aspect, the present invention relates to a use of phosphatidylcholine and / or lyso-phosphatidylcholine for increasing the thermal stability of at least one Long-Chain Unsaturated Fatty Acid (LC-UFA) in an ingestible composition.

[0019] Brief description of the drawings

[0020] Figures 1a)-1e) describe the % increase of DHA nutrient retention in brain tissues of mice dosed with a composition of the invention versus a control;

[0021] Figures 2a)-2e) show the effect on nutrient retention in adipose tissue of mice dosed with a composition of the invention versus control; and

[0022] Figure 3 shows a particle size distribution curve for a composition comprising HOSO / Lecithin / Algal (DHA) oil composition in comparison to lecithin.

[0023] Detailed description of invention Long-Chain Fatty Acids (LCFA) are fatty acids with 14-26 carbon atoms. The LCFA may be a free fatty acid or salt thereof, or bound to glycerol in mono-, di- and / or triglycerides or in glycolipids in oils or fats.

[0024] The term “LC-UFA” refers to Long-Chain unsaturated fatty acids (LC-UFA), having 14- 26 carbon atoms and at least one double bond. The LC-UFA may be a free fatty acid or salt thereof, or bound to glycerol in mono-, di- and / or triglycerides or glycolipids in oils or fats.

[0025] The term “LC-PUFA” refers to Long-Chain Poly-Unsaturated fatty acids (LC-PUFA), having 14-26 carbon atoms and multiple double bonds. The LC-PUFA may be a free fatty acid or salt thereof, or bound to glycerol in mono-, di- and / or triglycerides or in glycolipids. Long chain polyunsaturated fatty acids (LC-PUFA) are important components of membrane lipids and act as signalling molecules via various mechanisms, such as modulating gene expression and being precursors for eicosanoids and docosanoids.

[0026] Unsaturated fatty acids include n- or omega-3, 4, 5, 6, 7 or 9 (omega-3, omega-5, omega- 5, omega-6, omega-7, and omega-9) fatty acids of various chain lengths, having the first double bond in the indicated position counting from the omega-carbon (methyl-carbon). Unsaturated fatty acids include mono-, di-, tri-, tetra-, penta-, and hexa-unsaturated fatty acids. Long-chain fatty acids include fatty acids with 14 to 26 carbons, both even and odd-numbered (proof:C16:x, 18:x, 20:x, 22:x, 24:x), although even numbered fatty acids are predominant in nature.

[0027] Specific examples of such fatty acids include docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA), eicosaenoic acid, erucic acid, arachidonic acid, adrenic acid, nervonic acid, vaccenic acid, palmitoleic acid, pentadecanoic acid, alpha-linolenic acid, eicosatrienoic acid, gamma-linolenic acid (GLA), dihomo-gamma-linolenic acid (DGLA), eicosatetraenoic acid, and conjugated linoleic acid. The most well-known and important LC-PUFAs are eicosapentanoic (EPA, 20:5 n-3), docosahexaenoic (DHA, 22:6 n-3), and arachidonic acid (ARA, 20:4 n-6).

[0028] The inventors have identified a need to increase the nutrient retention of long chain fatty acids in animals and humans. Increased nutrient retention is in particular desirable for long chain UFA, more in particular the fatty acids DGLA, EPA and DHA. An increased level of beneficial LCFA in cell membranes (i.e. as phospholipid bound LCFA), as well as increased triglyceride bound LCFA, is believed to provide general health benefits in both humans and animals.

[0029] In particular, it is desirable to achieve this increased nutrient retention in an efficient way. Whereas it is known to raise the levels of certain fatty acids by simply adding more of that particular fatty acid to the diet, this has the disadvantage of requiring more of scarce omega-3 fatty acid sources such as fish oil, krill oil, algal oil or microbial oil. The invention provides a non-therapeutical use of phosphatidylcholine and / or lyso-phosphatidylcholine for increasing the nutrient retention of at least one Long-Chain Fatty Acid (LCFA) in an animal or a human.

[0030] It was found that the nutrient retention of specific LCFA’s is more strongly increased when phosphatidylcholine and / or lyso-phosphatidylcholine is administered in combination with the LCFA, when compared with a diet only containing the LCFA.

[0031] This allows for an efficient diet that allows for achieving desired increased levels of nutrient retention of specific LCFAs in an animal or a human, while maintaining a similar amount of administered LCFA in the diet. Alternatively, this effect also can be used to achieve the same tissue levels at lower levels of administered LCFAs in the diet. In another alternative, this effect would also allow even higher levels of nutrient retention by increasing the amount of dietary LCFA when administered in combination with phosphatidylcholine and / or lyso-phosphatidylcholine.

[0032] It is preferred if the use is for increasing the nutrient retention of LCFA as phospholipids. Phospholipids have a glycerol backbone containing a polar phosphate group and two apolar fatty acid residues. Either one or two of these fatty acids residues can be of the LCFA of interest.

[0033] In a preferred embodiment, the use is for increasing the nutrient retention of LCFA in cell membranes. The majority of cell membranes fatty acids are bound as phospholipids, which form membrane bilayers. In another preferred embodiment, the use is for increasing the nutrient retention of LCFA in adipose tissue. Adipose tissue stores fatty acids, typically bound as apolar triglycerides.

[0034] Use according to any of the preceding claims, wherein the use is for increasing the nutrient retention of LCFA in the brain. The brain is a particular target area where an LCFA of interest has a health effect.

[0035] In an embodiment, the use is for increasing the nutrient retention of LCFA in the hippocampus. The hippocampus is a part of the brain of particular interest.

[0036] In another preferred embodiment, the use is for increasing the nutrient retention of LCFA in the cerebellum. The cerebellum is another part of the brain of particular interest.

[0037] It is advantageous if the nutrient retention of LCFA in the brain is for both the hippocampus and the cerebellum.

[0038] Long-chain fatty acids (LCFAs), particularly LC-PUFAs such as EPA and DHA, are crucial for optimal brain function and development. They play a vital role in maintaining the structure and function of brain cell membranes, supporting synaptic plasticity, and contributing to overall cognitive functioning. Thus, in some embodiments, the invention relates to the non-therapeutical use of phosphatidylcholine and / or lyso- phosphatidylcholine for increasing the nutrient retention of at least one Long-Chain Fatty Acid (LCFA) (e.g. EPA and / or DHA) in the brain of an animal or a human, wherein said use comprises improving cognitive function in the animal or human.

[0039] In preferred embodiments, the nutrient retention is increased in the hippocampus and / or cerebellum regions of the brain, wherein said use comprises improving cognitive function in the animal or human subject. The hippocampus is a brain structure primarily involved in memory formation, especially the transfer of short-term memories to long-term storage, and spatial navigation. While traditionally considered a motor control center, the cerebellum is also considered to contribute to cognitive and emotional functions. As demonstrated in the Examples, the effects of the invention in vivo have been shown to afford pronounced nutrient retention in these areas of the brain that are associated with cognitive functioning. It is preferred if the nutrient retention in a mammal animal of at least one LCFA was increased by at least 2.5%, preferably at least 5%, preferably at least 10%, more preferably at least 15%, compared to a supplement oral composition without phosphatidylcholine and lyso-phosphatidylcholine. Such an increase in nutrient retention is believed to have a significant health impact.

[0040] In a preferred embodiment, the use is for a human.

[0041] In another preferred embodiment, the use is for a mammal animal. Examples include domestic animals, for instance cats, dogs, horses, cow, goat and sheep.

[0042] In yet another preferred embodiment, the animal is a marine animal. Examples include fish, such as salmon, mackerel, sardines, herring, tuna, trout and anchovies; and crustaceans, including mussels, oysters, shrimp, crabs and lobster.

[0043] In a preferred embodiment, the nutrient retention of at least one increased LCFA is a Long-Chain Unsaturated Fatty Acid (LC-UFA).

[0044] Preferably the at least one increased LC-UFA is selected from the group consisting of docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA), eicosaenoic acid, erucic acid, arachidonic acid (ARA), adrenic acid, nervonic acid, vaccenic acid, palmitoleic acid, pentadecanoic acid, alpha-linolenic acid, eicosatrienoic acid, gamma-linolenic acid (GLA), dihomo-gamma-linolenic acid (DGLA), eicosatetraenoic acid, and conjugated linoleic acid.

[0045] In a preferred embodiment, the at least one increased LC-UFA is an omega-3 LC-PUFA. More preferably, the at least one increased LC-UFA is DGLA, DHA or, EPA. Most preferably, the increased LC-UFA is DHA.

[0046] In a preferred embodiment, at least 2 different LC-UFA are increased, preferably at least 3, more preferably at least 4.

[0047] In some embodiments, it is preferred if, at the same time, the nutrient retention of arachidonic acid (ARA) is decreased. In some subjects (e.g. adult subjects), lowering of the nutrient retention of ARA while increasing another LC-UFA (preferably a LC-PUFA, such as DHA) is desirable. It has been found by the inventors that a diet supplemented with algal oil can modify the distribution of fatty acids in the human or animal body and induce a decrease in ARA retention. Since the present invention has been found to be particularly effective in increasing DHA retention, the present invention can therefore also be used for modifying the DHA : ARA ratio in tissues of the human or animal subject (e.g. in an adult human or adult animal subject). Alternatively, the composition of the present invention may be used to ensure the presence of triglyceride bound DHA, in combination with the substantial absence of ARA. This may also represent a means for increasing the proportion of DHA in the tissues, relative to the amount of ARA.

[0048] Thus, in some embodiments, the use further comprises increasing the proportion of one or more LC-UFA (preferably a LC-PUFA, such as DHA) in one or more tissues (e.g. brain or visceral fat tissue) of an animal or a human relative to the proportion of ARA in the same one or more tissues. In preferred embodiments, the tissue of the animal or human where the proportion of one or more LC-UFA (preferably a LC-PUFA, such as DHA) is increased relative to the proportion of ARA is in the brain tissue of the subject (e.g. in the hippocampus and / or the cerebellum regions of the brain).

[0049] It has been found by the inventors that the present invention is particularly effective in increasing LC-UFA (preferably LC-PUFA, such as DHA) levels in the brain, preferably the adult brain. By supplementing with algal oil, or by ensuring an absence of ARA in the supplementing composition, it has been found to be possible to modify the relative ratio of DHA : ARA in the brain tissue, particularly in the hippocampus and cerebellum. Without being bound to a particular theory, DHA and ARA are believed to compete for space within cell membranes and therefore applying the invention for specifically increasing retention of DHA can lead to a decrease in ARA through competition for space. This is believed by the inventors to be of particular benefit as increasing DHA levels in the brain - relative to ARA levels - can have a positive role in balancing pro- and anti-inflammatory effects. While ARA is an essential fatty acid for brain development (e.g. in infancy) and function, excessive amounts, particularly in an adult brain, can disrupt the balance of fatty acids, increase inflammation, and negatively impact cognitive abilities. By ensuring a greater proportion of DHA in the brain tissue of adults relative to levels of ARA, the present invention is believed to exert a positive effect on (maintaining or improving) cognitive functioning in the human or animal subject.

[0050] Thus, the present invention offers multiple means by which the cognitive functioning of an animal or human subject may be improved: i) by increasing retention of one or more LCFAs, particularly LC-PUFAs, in the brain tissue (e.g. in the hippocampus and / or the cerebellum regions of the brain) thereby maintaining the structure and function of brain cell membranes, and supporting synaptic plasticity; and ii) by increasing the proportion of DHA in the brain tissue of a preferably adult subject relative to ARA, thereby balancing pro- and anti-inflammatory effects favourably.

[0051] The invention further provides an ingestible composition for increasing the nutrient retention of at least one Long-Chain Fatty Acid (LCFA) as described herein, wherein the composition comprises phosphatidylcholine and / or lyso-phosphatidylcholine and at least one triglyceride-bound LCFA with increased nutrient retention. Such a composition is suitable for the non-medical use as described herein.

[0052] The term “ingestible composition” refers to a composition suitable for ingestion by an animal or human. The ingestible composition may be provided in a form suitable for use as a supplement (e.g. as a tablet, gummy, or capsule) or for dosing into food or beverage compositions and the like. Alternatively, the ingestible composition may be in a form of a nutritional composition intended for consumption as a functional food or beverage. As the skilled person will appreciate, the term “oral composition” herein refers to a composition that is at least suitable for administration or consumption orally (i.e. the composition need not be specifically optimised for that mode of administration or consumption, for instance, in terms of taste or other organoleptics). Similarly, the term “enteral composition” herein refers to a composition that is at least suitable for administration enterally (e.g. is of a suitable nutritional value and suitable formulation for that mode of administration and purpose).

[0053] In connection with the ingestible composition, reference to “at least one triglyceride- bound LCFA with increased nutrient retention” is intended to refer to the presence of at least one triglyceride bound LCFA having an increased nutrient retention potential as a consequence of the presence of phosphatidylcholine and / or lyso-phosphatidylcholine in the composition.

[0054] The ingestible composition according to the present invention comprises at least one triglyceride-bound LCFA. The at least one triglyceride-bound LCFA may be provided in the composition as part of one or more LCFA-containing fats or oils, including liquid oils, semi-solid fats or solid fats. The triglyceride-bound LCFA may also be diluted with one or more additional oils or fats to form a mixed fat or oil fraction in the composition. Suitable liquid oils which may provide triglyceride bound LCFA, or which may be used to form a mixed fat or oil fraction comprising triglyceride-bound LCFA, include those selected from rapeseed oil, high oleic rapeseed oil, high erucic acid rapeseed oil, soybean oil, sunflower oil, high oleic sunflower oil, linseed oil, olive oil, corn oil, cottonseed oil, carinata oil, groundnut oil, safflower oil, high oleic safflower oil, peanut oil, walnut oil, avocado oil, rice brand oil, camelina oil, shea olein, sesame oil, MCT oil, SCT oil, marine oils (e.g. fish oil or krill oil), and single cell organism oils (e.g. algal oil, microbial oil, or fungal oil), any fraction thereof, any mixture thereof, or any esterified or interesterified product, fraction or mixture thereof. As will be appreciated, those liquid oils recited above which do not themselves comprise LCFA (e.g. MCT oil or SCT oil) may be utilised in a mixture with an oil or fat which does comprise LCFA.

[0055] Suitable semi-solid or solid fats include those selected from palm fat, coconut fat, shea butter, shea stearin, cocoa butter, cocoa stearin, cocoa olein, allanblackia fat, kokum fat, mango kernel fat, sal fat, illipe butter, palm kernel fat, babassu fat, coconut oil stearin, coconut oil olein, palm kernel olein, palm olein, palm kernel stearin, avocado oil, any fraction thereof, any mixture thereof, or any esterified or interesterified product, fraction or mixture thereof.

[0056] In some embodiments, the composition comprises a melted fat selected from shea butter, shea stearin, shea olein, cocoa butter, cocoa stearin, cocoa olein, allanblackia fat, kokum fat, mango kernel fat, sal fat, illipe butter, coconut oil, coconut oil stearin, coconut oil olein, palm kernel oil, palm kernel olein, palm olein, rapeseed oil, palm oil, palm kernel stearin, babassu oil, any fraction thereof, any mixture thereof, or any esterified or interesterified product, fraction or mixture thereof.

[0057] The term “melted fat” as used herein is used to refer to a fat or fat composition that is a solid or semi-solid at 20°C; and that has been heated to above 20°C so as to have a solid fat content of less than 1% as determined by ISO 8292-1. Preferably, the term “melted fat” as used herein is used to refer to a fat or fat composition that is a solid at 20°C, and that has been heated to above 20°C so as to have a solid fat content of less than 1% as determined by ISO 8292-1 . The term “melted fat” is thus not used to refer to a liquid oil that is liquid at room temperature (e.g. 20°C) and that does not require heating to above this temperature in order to be a liquid. Such oils typically include sunflower oil and rapeseed oil. In a preferred embodiment, the triglyceride-bound LCFA (preferably a LC-PUFA, e.g. DHA or EPA) is a component of an oil or fat included in the composition, wherein the oil or fat is derived from a marine source (e.g, fish or krill) or single cell organism (e.g. algae, bacteria or fungus), preferably wherein the oil or fat is krill oil or algal oil.

[0058] The ingestible composition comprises a combination of i) phosphatidylcholine and / or lyso-phosphatidylcholine; and ii) a triglyceride bound LCFA. In some embodiments, the phosphatidylcholine and / or lyso-phosphatidylcholine is incorporated within a phospholipid fraction (i.e. as part of mixture of phospholipids) included within the composition. Phosphatidylcholine and / or lyso-phosphatidylcholine used in the ingestible composition may be provided from any suitable source. For example, phosphatidylcholine may be derived from botanical (e.g. soybean lecithin, rapeseed lecithin, sunflower lecithin), animal (e.g. egg yolk), marine (e.g. fish or krill), or singlecelled organism (algal, bacterial or fungal) sources. Lyso-phosphatidylcholine is typically obtained from enzymatic hydrolysis of phosphatidylcholine (i.e. to remove one of the two fatty acid chains from the glycerol backbone of phosphatidylcholine). Alternatively, lyso- phosphatidylcholine may be extracted directly from a suitable source, such as a botanical source or marine source (e.g. fish or krill).

[0059] The total combined amount of phosphatidylcholine and / or lyso-phosphatidylcholine in a phospholipid fraction of use in the present invention may be from at least 20 wt%, more preferably at least 25 wt%, more preferably at least 30 wt%, more preferably at least 35 wt%. For example, the total combined amount of phosphatidylcholine and / or lyso- phosphatidylcholine in a phospholipid fraction of use in the present invention is from 20 to 70 wt%, preferably from 20 to 50 wt%, more preferably from 20 to 40 wt%, still more preferably from 25 to 35 wt%.

[0060] An example of a phospholipid fraction comprising phosphatidylcholine and / or lyso- phosphatidylcholine includes a natural source of phospholipids, for example lecithin. Thus, in some embodiments, the ingestible composition of the invention includes a phospholipid fraction which derives from lecithin, as a source of phosphatidylcholine and / or lyso-phosphatidylcholine.

[0061] Lecithin is an amphiphilic fat made of mixtures of glycerophospholipids, including those comprising negatively charged organophosphates (such as phosphatidic acid, phosphatidylethanolamine and phosphatidylinositol), and those comprising zwitterionic organophosphates (such as phosphatidylcholine and phosphatidylserine). Lecithin may undergo extraction and / or processing to varying extents. Suitable forms of lecithin for use in the present invention include deoiled enzymatically hydrolyzed lecithin (providing a source of lyso-phosphatidyl choline), deoiled standard lecithin, standard lecithin, hydrolyzed lecithin, fractionated lecithin, or combinations thereof. The lecithin may be derived from a botanical source (such as soya, rapeseed, or sunflower) an animal source (e.g. egg yolk), a marine source (e.g. fish or krill), or single-celled organism source (e.g. algal, bacterial or fungal).

[0062] In some embodiments, it can be useful to include one or more phospholipid solubilizers in the composition which can help to maintain miscibility of a phospholipid fraction in the LC-FA containing fats or oils of the composition. This can help to avoid phospholipid sedimentation, extending the shelf-life of the composition. Sedimentation of phospholipid is not only undesirable from a formulation perspective, but the protective effects of phosphatidylcholine and / or lyso-phosphatidylcholine upon the LCFA component may also be negatively impacted by sedimentation. Consequently, the presence of phospholipid solubilizers can be particularly beneficial in preserving the quality of the composition and retaining oxidative stability of LCFA in the composition. Phospholipid solubilizers may also improve product handling.

[0063] Examples of phospholipid solubilizers include MCT (medium chain triglycerides) oil, oilsoluble monoglycerides (e.g. glycerol mono-oleate), and waxes. Examples of suitable waxes includes rice bran wax, candelilla wax, carnauba wax, and sunflower wax. In preferred embodiments, the composition comprises MCT oil, which has been found by the inventors to be particularly advantageous in preserving desirable properties of the composition, not only in reducing phospholipid sedimentation risk, but also in preserving oxidative- and thermo- stability of the composition. MCT contains all three positions on the glycerol backbone occupied by medium chain-fatty acids, wherein the length of the acyl chains is from 6-12 carbon atoms (C6-Ci2).

[0064] In preferred embodiments, the composition comprises MCT oil in combination with the LCFA-containing fat or oil, and optionally any additional fats or oils. For example, the composition may comprise MCT oil in combination with an LCFA-containing liquid oil such as a marine oil (e.g. fish oil or krill oil) or single-celled organism oil (algal, bacterial or fungal oil). In particularly preferred embodiments, the composition comprises the combination of a phospholipid fraction (which may be derived from lecithin) comprising 25 to 35 wt% combined weight of phosphatidylcholine and / or lyso-phosphatidylcholine, MCT oil as a phospholipid solubilizer, and an LCFA-containing liquid oil (e.g. algal oil, fish oil, or krill oil).

[0065] In a preferred embodiment, the composition comprises a combined amount of at least 0.05 wt% of phosphatidylcholine and lyso-phosphatidylcholine, and at least 0.01 wt% of the at least one triglyceride-bound LCFA with increased nutrient retention.

[0066] Preferably, the composition comprises a combined weight of at least 0.01 wt% of phosphatidylcholine and lyso-phosphatidylcholine, preferably 0.1 wt% of phosphatidylcholine and lyso-phosphatidylcholine, preferably at least 1.0 wt% of phosphatidylcholine and lyso-phosphatidylcholine, more preferably at least 2 wt% of phosphatidylcholine and lyso-phosphatidylcholine.

[0067] More preferably, the composition comprises from 0.01-80 wt% of phosphatidylcholine and lyso-phosphatidylcholine, preferably from 0.1-60 wt% phosphatidylcholine and lyso- phosphatidylcholine, more preferably from 1-50 wt% phosphatidylcholine and lyso- phosphatidylcholine.

[0068] In particularly preferred embodiments, the composition comprises from 0.1-10 wt%, such as from 0.5-5.0 wt%, or from 0.5-3.0 wt% phosphatidylcholine and lyso- phosphatidylcholine.

[0069] It is preferred if the composition comprises at least 0.01 wt% of the at least one triglyceride-bound LCFA with increased nutrient retention, preferably at least 0.05%, more preferably at least 0.1 wt%, more preferably at least 0.5 wt%, more preferably at least 1%.

[0070] Preferably, the composition comprises from 0.01- 20 wt% of the at least one triglyceride- bound LCFA with increased nutrient retention, such as 0.05-15 wt%, 0.05-10 wt%, 1-10 wt% or 0.5-5 wt%.

[0071] Preferably, the composition comprises (i) phosphatidylcholine and lyso- phosphatidylcholine; and ii) the at least one triglyceride LCFA in a weight ratio (i) I (ii) of at least 0.1 , preferably at least 0.2, preferably at least 0.5, more preferably at least 0.8, more preferably at least 1 , more preferably at least 2, even more preferably at least 5, more preferably at least 8.

[0072] Preferably, the composition comprises: (i) phosphatidylcholine and lyso- phosphatidylcholine and (ii) at least one triglyceride-bound LCFA in a weight ratio (i) I (ii) of from 0.01 to 400, preferably from 0.1 to 100, more preferably from 1 to 20, most preferably from 8 to 15. In a preferred embodiment, the composition is a fat blend of algal oil, a phospholipid fraction comprising phosphatidylcholine and / or lyso- phosphatidylcholine (e.g derived from lecithin) and optionally another liquid oil, for example selected from MCT oil, high oleic sunflower oil (HOSO), and combinations thereof. The optional additional liquid oil may be present in the composition, for example, in an amount from 0-80 wt%, such as 0-50 wt%, or 0-20 wt%, depending on the nature of the formulation.

[0073] In one preferred embodiment, the composition is a fat blend of algal oil, a phospholipid fraction comprising phosphatidylcholine and / or lyso-phosphatidylcholine (e.g. derived from lecithin) and MCT oil. In another preferred embodiment, the composition is a fat blend of algal oil, a phospholipid fraction comprising phosphatidylcholine and / or lyso- phosphatidylcholine (e.g derived from lecithin), MCT oil and HOSO. In another preferred embodiment, the composition is a fat blend of algal oil, a phospholipid fraction comprising phosphatidylcholine and / or lyso-phosphatidylcholine (e.g derived from lecithin), and HOSO.

[0074] In the compositions of the invention, the triglyceride-bound LCFA may be selected from any of those for which the use in increasing nutrient retention is described herein. Preferably, the composition comprises at least one triglyceride bound LC-UFA, for example, selected from the group consisting of docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA), eicosaenoic acid, erucic acid, arachidonic acid (ARA), adrenic acid, nervonic acid, vaccenic acid, palmitoleic acid, pentadecanoic acid, alpha-linolenic acid, eicosatrienoic acid, gamma-linolenic acid (GLA), dihomo-gamma-linolenic acid (DGLA), eicosatetraenoic acid, and conjugated linoleic acid. In preferred embodiments, the composition of the invention comprises triglyceride-bound DHA. In a preferred embodiment, the at least one increased LC-UFA is an omega-3 LC-PUFA. More preferably, the at least one increased LC-UFA is DGLA, DHA or, EPA. Most preferably, the increased LC-UFA is DHA. In a particularly preferred embodiment, the composition comprises i) phosphatidylcholine and / or lyso-phosphatidylcholine; and ii) DHA and / or EPA, preferably DHA and EPA.

[0075] As discussed herein, in some embodiments, the use relates to increasing nutrient retention of one or more LC-UFA, whilst also increasing the proportion of one or more LC-UFA relative to ARA in the tissue of a human or animal. In some embodiments, ARA (in triglyceride-bound form or free fatty acid) is present in the composition as a minor fraction (e.g. less than 10wt%, preferably less than 5 wt%, more preferably less than 3 wt%) of the amount of total LC-UFA (in triglyceride-bound form or free fatty acid) present in the composition. In a preferred embodiment, ARA (in triglyceride-bound form or free fatty acid) is present in the composition in an amount less than 10 wt%, preferably less than 5 wt%, more preferably less than 3 wt%) of the amount of DHA (in triglyceride- bound form or free fatty acid) present in the composition.

[0076] It has been found by the inventors that the ability of phosphatidylcholine and lyso- phosphatidylcholine to increase the nutrient retention potential of LCFA, particularly in cell membranes, is superior to alternative choline containing species (i.e. nonphospholipid bound), such as choline salts (e.g. choline chloride). In preferred embodiments, the composition is essentially free, or free, of choline salts. For example, the composition may comprise less than 500 ppm, preferably less than 100 ppm, of total choline salts. This can also confer advantages since choline salts have high hygroscopicity, easily agglomerate, and can drive unwanted oxidation. As shown in the Examples, phosphatidylcholine and lyso-phosphatidylcholine are capable of achieving nutrient retention of LCFA in the tissues of a human or animal subject receiving a supplement of a composition of the invention where, in comparison, this effect is either not observed (e.g. in the case of the certain brain tissues) or only observed minimally in others receiving choline chloride in place of phosphatidylcholine and lyso- phosphatidylcholine. Furthermore, phosphatidylcholine and lyso-phosphatidylcholine also does not present the processability challenges of choline salts in providing an ingestible composition.

[0077] Phosphatidylcholine or lyso-phosphatidylcholine may be used alone or in combination as the species for increasing the nutrient retention potential of the LCFA of the composition. Lyso-phosphatidylcholine is naturally less abundant than phosphatidylcholine in, for instance, lecithin and other natural sources of phosphatidylcholine and lyso-phosphatidylcholine. Therefore, lyso-phoshatidylcholine may typically be present in minor proportion compared to the amount of phosphatidylcholine present in the compositions of the invention. Nevertheless, in some embodiments, the lyso-phosphatidylcholine may be used alone, or in greater proportion than phosphatidylcholine, in the uses and compositions of the invention. In other embodiments, phosphatidylcholine is used alone, or in greater proportion than lyso- phosphatidylcholine, in the uses and compositions of the invention.

[0078] An additional advantage of phosphatidylcholine and lyso-phosphatidylcholine over choline salts found by the inventors is that supplementation with choline salts is associated with the formation of trimethylamine N-oxide (TMAO). Elevated TMAO levels have been linked to an increased risk of cardiovascular disease and other health problems. Surprisingly, it has been found by the inventors that phosphatidylcholine and lyso-phosphatidylcholine provide up to 110% more bioavailable choline to a subject than an equivalent amount (in terms of choline) of choline bitartrate, yet the choline salt induces production of 250% more unwanted TMAO.

[0079] Compositions according to the present invention have been found to have particularly advantageous oxidative- and thermo-stability properties and may retain shelf-life for substantial periods, even at room temperature (15-21 °C). Thus, unlike typical LCFA- containing compositions, the compositions of the present invention may be effectively stored without the need for refrigeration.

[0080] The ingestible composition may be an oral composition in the form of a food or health supplement. The composition may also be an animal feed or animal feed supplement.

[0081] In a preferred embodiment, the composition is a capsule, a gummy, a chewing gum, a gel, a tablet, a powder, a bar (e.g. a nutritional bar), a liquid beverage, a shot or a pouch. In other preferred embodiments, the composition is a dairy-based drink or fermented product (e.g. a yoghurt). In other preferred embodiments, the composition is a plantbased drink or dairy-analogue product (e.g. a plant-based yoghurt).

[0082] The invention further provides a method of preparing an ingestible composition (e.g. an oral or enteral composition) as described herein, comprising the mixing of a phospholipid fraction comprising phosphatidylcholine and / or lyso- phosphatidylcholine, and an oil or fat comprising triglyceride-bound LCFA. Any conventional mixing techniques may be used for mixing the ingredients. Optionally, additional ingredients may be added. Typically, an ingestible composition is prepared first comprising the mixture of phosphatidylcholine and / or lyso-phosphatidylcholine and triglyceride-bound LCFA. Once the ingestible composition has been prepared, this may be diluted or mixed with other ingredients intended for preparing a nutritional composition intended for consumption.

[0083] As part of the method for preparing the ingestible composition, the phospholipid fraction comprising phosphatidylcholine and / or lyso-phosphatidyl choline (e.g. a phosphatidylcholine enriched lecithin) may be heated in order to reduce its viscosity to optimise mixing with triglyceride bound LCFA. For example, the phospholipid fraction (e.g. derived from lecithin) may be heated to 30 to 50 °C, preferably to 35 to 45 °C, prior to mixing with the triglyceride bound LCFA.

[0084] LCFAs, such as DHA, are prone to oxidation when exposed, for example, to oxygen, light, heat, and metals, leading to rancidity and an unpleasant fishy taste or odor. Thus, preparation of the ingestible composition may also benefit from steps to reduce the possibility of oxidation during preparation. Therefore, in some embodiments, the method for preparing the ingestible composition is undertaken under an inert atmosphere, for example, under a nitrogen atmosphere.

[0085] To prevent oxidation, triglyceride bound LCFA are also often stored and transported frozen. Typically, the triglyceride bound LCFA is provided at a temperature of from about 10 to 25 °C, such as 15 to 20 °C for mixing with the phospholipid fraction, provided the triglyceride bound LCFA is fully dissolved. Nevertheless, it has been found by the inventors that triglyceride bound LCFA may be combined with the phospholipid fraction and blended at higher temperatures, for example from 30 to 45 °C, preferably from 35 to 40 °C, without detriment to the triglyceride bound LCFA fraction.

[0086] The composition which is formed has been found to exhibit long shelf-life and high oxidative- and thermo-stability, which is believed to derive from the protective effects exerted by the phosphatidylcholine component. The ingestible composition may be diluted with additional oil or fat, or have additional ingredients incorporated therein, as desired. For instance, additional ingredients may be included when providing a nutritional drink formulation.

[0087] Thus, in a third aspect, the present invention provides a use of phosphatidylcholine and / or lyso-phosphatidylcholine for increasing the thermal stability of at least one Long- Chain Unsaturated Fatty Acid (LC-UFA), preferably at least one Long-Chain Polyunsaturated Fatty Acid (LC-PUFA), in an ingestible composition, such as those described herein.

[0088] As mentioned above, LCFAs are prone to oxidation when exposed, for example, to oxygen, light, heat, and metals, leading to rancidity and an unpleasant fishy taste or odor. Thermal instability is known to be a particular issue for LC-UFA, and especially LC- PUFA, which can impact the shelf life and nutritional value of products, particularly functional food products.

[0089] Hadaruga et al ref

[0014] describe a study of the thermal stability of omega-3 fatty acids, EPA and DHA, in Atlantic salmon oil showing that significant degradation was observed even at a temperature of 50 °C. In one part of the study, their relative concentrations are shown to decrease from 6.1% for EPA and 4.1% for DHA to 1.7% and 1.5%, respectively, after thermal treatment at 150 °C. Similar results are also shown by Holser, R.A., et al ref

[0015] who monitored degradation of DHA methyl esters (as opposed to triglyceride bound DHA) at 120 °C. Following heat treatment up to 120°C, the esters showed several spectral changes associated with oxidative degradation. Longer heating times were also found to produce no additional major changes in the spectra, indicating that temperature alone was critical to the extent of degradation, as opposed to the duration of time at elevated temperature.

[0090] The inventors have surprisingly found that phosphatidylcholine and / or lyso- phosphatidylcholine confer increased thermal stability to LC-UFA, such as DHA, in an ingestible composition, such as the nutritional drink formulations described herein. As shown in the accompanying examples, a heat treatment of nutritional drink formulations containing LC-UFA, including DHA, at 120°C was found not to give rise to any appreciable loss of LC-UFA, particularly DHA, as a result of degradation (e.g. by oxidative mechanisms). This is particularly advantageous for ingestible compositions, such as the nutritional drink formulations described herein, which may undergo UHT pasteurization treatment to extend shelf-life, without compromising the nutritional value of the composition.

[0091] Thus, in some embodiments, the use of phosphatidylcholine and / or lyso- phosphatidylcholine for increasing the thermal stability of at least one Long-Chain Unsaturated Fatty Acid (LC-UFA), preferably at least one Long-Chain Polyunsaturated Fatty Acid (LC-PUFA), is in an ingestible composition which is heat treated to a temperature of at least 100 °C, such as at least 120 °C, or at least 140 °C. For example, the ingestible composition may be a composition that is heat treated as part of a UHT pasteurization treatment, such as at 142 °C for at least 4 seconds.

[0092] As will be appreciated, compatible embodiments described herein in connection with the first and second aspects apply equally to the third aspect of the invention. Thus, the LC- UFA, sources of lyso-phosphatidlycholine and / or phosphatidylcholind, and features of the ingestible composition set out in embodiments herein in connection with the first and second aspects of the invention apply equally to the third aspect of the invention.

[0093] Nutritional drink formulation

[0094] The ingestible composition described herein may be provided in the form of a nutritional drink formulation for the uses described herein. Such a drink formulation may be an enteral drink formulation or an oral drink formulation, comprising phosphatidylcholine and triglyceride-bound LCFA as well as optional additional ingredients intended to optimise nutritional composition, palatability and other organoleptics of the formulation. For example, the nutritional drink formulation may further comprise water, a carbohydrate source, a protein source, a dietary fiber source and / or a further fat source. The nutritional drink formulation may be a dairy-based drink or plant-based drink.

[0095] In some embodiments, the nutritional drink formulation comprises water in an amount of from 10% to 90% by weight of the composition, for example from 50% to 85% by weight.

[0096] The carbohydrate source may be any carbohydrate source suitable for use in nutritional compositions, for example digestible carbohydrates, such as maltodextrin, maltose, galactose, dextrose (e.g. dextrose monohydrate or glucose powder), dextrin, sucrose, lactose, glucose, fructose, com syrup, corn syrup solids, rice syrup solids, starch, such as cereal starch, rice starch, corn starch and mixtures thereof. In some embodiments, the nutritional drink formulation comprises one or more of: i) polysaccharides and / or modified polysaccharides, preferably selected from methylcellulose, hydroxypropyl methylcellulose, carboxymethyl cellulose, maltodextrin, carrageenan and salts thereof, alginic acid and salts thereof, agar, agarose, agaropectin, pectin and alginate; ii) hydrocolloids; and iii) gums, preferably selected from xanthan gum, guar gum, locust bean gum, gellan gum, gum arabic, vegetable gum, tara gum, tragacanth gum, konjac gum, fenugreek gum, and gum karaya. Typically, the carbohydrate source is present in an amount of from 0.1% to 30% by weight of the composition.

[0097] The protein source may be any suitable dietary protein for example animal proteins (such as milk proteins, meat proteins and egg proteins), vegetable proteins (such as soy protein, oat protein, wheat protein, rice protein, chickpea protein, potato protein and pea protein), mixtures of free amino acids or combinations thereof. In some embodiments, the nutritional drink formulation comprises animal-milk derived proteins such as casein, whey protein concentrate, whey protein isolate, or milk protein isolate. Preferably, the animal-milk derived protein comprises casein, such as micellar casein isolate (MCI). MCI has been found to be particularly advantageous in conferring desirable viscosity properties to the drink formulation. MCI has also been found by the inventors to provide a desirable balance of organoleptics - conferring a creamy / milky flavour and rich texture. MCI is also more temperature stable when subjected to UHT treatment in comparison to, for instance, whey protein concentrate (WPC) and whey protein isolate (WPI). Nevertheless, MCI may be used in combination with, preferably UHT-stable, WPC and WPI in some embodiments in order to confer improvements in viscosity of the nutritional drink formulation. Typically, the protein source is present in the composition in an amount of from 0.1% to 20% by weight of the composition.

[0098] The dietary fiber (non-digestible carbohydrates) may also be present in the nutritional formulation, if desired. Numerous types of dietary fibers are available. For example, oligosaccharides, such as fructo-oligosaccharides, galacto- oligosaccharides, xylooligosaccharides, fuco-oligosaccharides, and manno-oligosaccharides.

[0099] The nature of any additional ingredients that may be added to the composition in formulating a nutritional drink are dependent on the particular characteristics that are desired in the composition and include, for example, one or more sweeteners, one or more flavourings, one or more colouring additives, or any combination thereof. Other additives known in the art for inclusion in such compositions may of course also be included. In an example embodiment, the composition may comprise cocoa powder.

[0100] The supplement composition of the present invention can have a bitter taste. The inventors have found that top note flavours can be effective in masking the bitterness and improving the taste profile. Examples of top note flavours include raspberry, caramel, vanilla, banana, and coffee flavours. Raspberry and caramel flavours have been found to be particularly effective in masking bitterness in the composition. The combination of cocoa powder and caramel flavour are believed to act synergistically in masking bitterness and also improving aftertaste of the composition.

[0101] The nutritional drink formulation preferably comprises other constituents, such as macro- and micronutrients, for example functional food ingredients. The nutritional composition is preferably designed to meet the nutritional needs of the consumer or provide further benefits or functionalities. In one embodiment the nutritional composition is a “nutritionally complete formula”, that is it contains adequate nutrients to sustain healthy human life for extended periods (e.g., an enteral composition). Preferably, the nutritional composition comprises vitamins and minerals.

[0102] If necessary, the product may contain emulsifiers and stabilizers. The emulsifier may be selected from the group consisting of for example mono- and di-glycerides, acetic acid esters of mono / di-glycerides, lactic acid esters of mono / di-glycerides, diacetyl tartaric acid esters of mono / di-glycerides, succinic acid esters of mono / di-glycerides, sorbitan esters, sucrose esters, polyglycerol esters, calcium stearoyl lactate and mixtures thereof. The product may also comprise lecithin as an emulsifier and / or stabilizer. Thus, lecithin may fulfil separate roles of providing a source of phosphatidyl choline and lyso- phosphatidylcholine and also providing emulsifying / stabilizing properties.

[0103] In some embodiments, the nutritional drink formulation has a substantially monomodal particle size distribution (e.g. where at least 90 vol.%, preferably at least 95 vol.% of the composition is defined by a single peak in a particle size distribution curve). In order to provide such a formulation, the formulation may undergo high pressure homogenization (e.g. at 200-275 bar, such as 250 bar). Providing a formulation with a substantially monomodal particle size distribution provides advantages in food processing and product quality, particularly in terms of texture, stability, and flow properties. This can lead to improved consistency, better mouthfeel, and enhanced processability.

[0104] In some embodiments, the nutritional drink formulation has an average particle size less than 10 pm, such as less than 5 pm or less than 2 pm. In preferred embodiments, the nutritional drink formulation has an average particle size of less than 0.5 pm, preferably less than 0.2 pm, most preferably less than 0.1 pm. In some embodiments, the viscosity of the nutritional drink formulation is less than or equal to 250 mPa. s at a shear rate of 100 S'1, preferably less than or equal to 200 mPa.s a shear rate of 100 s’1, for example from 150 to 250 mPa.s a shear rate of 100 s’1. Viscosity measurements of the drink formulation may by measured using an Anton Paar rheometer equipped with a concentric cylinder geometry system, following a shear rate sweep rheological protocol. This protocol allows the viscosity of a sample to be assessed as a function of increasing shear rate.

[0105] In an example embodiment, the nutritional drink formulation comprises the following ingredients:

[0106] In another embodiment, the nutritional drink formulation comprises the following ingredients:

[0107] As part of a method for preparing a nutritional drinks formulation, milk or vegetable protein may be dispersed in heated water (e.g. 50-60 °C) and left to hydrate (e.g. for 1 to 2 hours) before sugar, maltodextrin / cocoa powder and any stabilisers and emulsifiers are added to the mixture (e.g. at 50-60 °C) and blended (e.g. 4000-8000 rpm). The supplement composition prepared as described herein may then be added to the mixture and thoroughly mixed (e.g., at 50-60 °C, 4000-8000 rpm), before flavouring and any salts are subsequently added. The resulting drink formulation may then undergo an optional pasteurisation step (at or above 140 °C, for up to 5 seconds) before a high-pressure homogenisation (HPH) step (e.g at 60-70°C and 200-300 bar). HPH has been by the inventors to confer benefits in terms of particle size distribution in the drinks formulation, as well as increased physical product stability. Following HPH, the solution may be cooled and stored.

[0108] The invention will now be further elucidated by the following non-limiting examples.

[0109] Examples

[0110] The invention will be further elucidated by the study described below. In the study, mice were fed with a diet supplemented with a composition comprising phosphatidylcholine and Long-Chain Fatty Acid (LCFA) rich in LC-PUFA, in particular DHA. The modulation of nutrient retention in cell membranes, and a number of specific organs, was determined.

[0111] Study Outline

[0112] Rationale for the animal model, custom diet, and daily oral gavage. The current study was performed in male wild-type C57BL / 6J mice. The mice were placed on a standard synthetic rodent diet (AIN-93M), to avoid any seasonal variation and ensure exact knowledge of the fatty acid composition. Mice were first habituated on AIN-93M diet for at least two weeks, to stabilize the nutritional levels in the animal. Thereafter, on top of this synthetic diet, different formulations of an algae oil - phospholipid blends and control (high oleic sunflower oil) formulation were administered orally on a daily basis for 28 days in total. Subsequently, tissue enrichment with fatty acids was monitored.

[0113] Sourcing and housing of mice

[0114] Thirty-two (32) male C57BL / 6J mice were sourced from Charles River and shipped at an age of 7-8 weeks in a single shipment. The health status was Specific Pathogen Free (SPF) according to FELASA 2014 recommendations. Mice were group housed in the same social pairs until the end of the study. However, certain mice had to be removed due to aggressive behaviour prior to study end. Cages were provided with sawdust as bedding material, gnawing sticks and a house / shelter to hide. Each cage was supplied with domestic mains tap water suitable for human consumption. Water was provided ad libitum from the arrival of the mice until the end of the study.

[0115] Upon arrival, mice were directly placed on the AIN93M synthetic (ad libitum) diet throughout the study. Welfare of animals was monitored daily. For all groups, body weight was monitored once per week. During the whole study any signs of discomfort was registered.

[0116] Experimental design

[0117] Table 1 describes the experimental groups. The pairs of mice in the same cage were assigned to the same treatment group to avoid any possibility of interaction between interventions (i.e., mice are coprophagic). Cages were randomly assigned to the different experimental groups. However, to ensure similar body weights among the groups, the average body weight per cage was stratified among groups to ensure that the distribution of the weights of the mice was approximately equal in all groups.

[0118] One group had the diet supplemented with DHA-free High Oleic Sunflower Oil (HOSO) as a negative control. A second group was supplemented with DHA-rich algal oil, which also contained other LCFA. A third group was supplemented with a combination of the same DHA-rich algal oil, and lecithin rich in phosphatidylcholine (PC) and also containing a minor proportion (approximately 2.5 wt%) of lyso-phosphatidylcholine (LPC). A fourth group was supplemented with a combination of the same DHA-rich algal oil, and choline chloride salt. The supplement oils, lecithin and choline chloride were supplied by AAK, and ingredients were mixed and homogenized prior to use. Groups were treated for 28 days, and dissected. Samples were collected from the brain and visceral fat. Visceral fat was fractionated into free fatty acids, triglycerides (representing stored fat), and phospholipids (representing cell membranes) and each fraction was analyzed for fatty acid composition. Brain regions were analyzed for fatty acid composition without fraction, this since the brain fat is predominantly made up of phospholipids.

[0119] Table 1 : Experimental groups of mice

[0120] Group Model Group Intervention Samples size* Oral gavage collected

[0121] 1 Male 7 Neg control oil Brain,

[0122] C57BL / 6J (HOSO) visceral fat,

[0123] 2 Male 7 Algal oil Brain,

[0124] C57BL / 6J visceral fat,

[0125] 3 Male 8 Algal oil + high Brain,

[0126] C57BL / 6J PC lecithin visceral fat,

[0127] 4 Male 7 Algal oil + ChCI Brain,

[0128] C57BL / 6J visceral fat,

[0129] Total

[0130] * following exclusion of aggressive mice before study end

[0131] For the diet, the basic chow was the commercially available AIN - 93M derived from Altromin (Lage, Germany). Fatty acid levels as present in the chow, without the supplements, are shown in Table 2. According to the product sheet, the AIN - 93 M diet composition comprises 4.1% fat and 12.2% protein. Total amount of fatty acids in the chow is 38.120 mg / kg. Daily intake of fatty acids through the chow is on average, based on literature, between 114 and 152 mg / day (3-4 gram / d chow

[0011] ). Although adequate in essential fatty acids, this diet lacks DGLA, DHA, DPA, EPA, adrenic acid and ARA.

[0132] Table 2: Fatty acid composition of the chow

[0133] Fatty acid Chow (mg / kg)

[0134] C16:0 5.400

[0135] C16:1 200

[0136] C18:0 1.200

[0137] C18:1 9.400

[0138] C18:2 21.000

[0139] C18:3 400

[0140] C20:0 320

[0141] C20:1 400

[0142] C20:2 40

[0143] Mice were dosed with 0.2 g / day of the blends depicted in table 3. The algal oil provided per day 1.3 mg DHA, and the PC / LPC lecithin provided per day 15 mg phosphatidylcholine + lyso-phosphatidycholine equivalent to 2 mg choline. Choline chloride was dosed in the algal oil emulsion to provide an equivalent amount of choline as for the PC / LPC lecithin (2 mg choline). Fatty acid levels, in the test samples, analysed after preparation, are depicted as mg intake of the fatty acid per day.

[0144] Phosphatidylcholine also contains fatty acid residues. Differences in fatty acid levels between the algal oil and algal oil + PC / LPC below 0.25 mg / day are indicated as minimal differences. These differences are considered not to contribute to differences found in the tissues of the animals. Fatty acids not shown were not detectable in the product. ND indicates that the dose of the fatty acid was below detection level. Since choline chloride has no fatty acid residues associated with it, the fatty acid levels of the algal oil and algal oil + ChCI compositions are the same.

[0145] Table 3: Fatty acid profile of the tested interventions

[0146] HOSO Algal oil / Algal Algal oil +

[0147] Oil + ChCI PC / LPC mg FA / day mg FA / day mg FA / day

[0148] Fatty acids with minimal difference, less than 0.25mg / day, between the algal oil and algal oil + PC / LPC

[0149] 10:0 0,005 0,004 0,006

[0150] 12:0 0,089 0,034 0,062 14:0 0,004 0,049 0,038

[0151] 16:0 1 ,496 2,171 2,221

[0152] 17:0 0,020 0,035 0,060

[0153] DMA 18:0 0,016 ND ND

[0154] 18:1(n-7) 0,250 0,297 0,186

[0155] 18:3(n-3) ND 0,025 0,033

[0156] 20:0 ND 0,102 0,000

[0157] 20:5(n-3) 0,002 0,004 ND

[0158] 22:0 0,424 0,261 0,156

[0159] 24:0 0,139 0,089 0,043

[0160] 22:5(n-6) 0,001 0,004 0,273

[0161] 22:6(n-3) 0,001 1 ,310 1 ,075

[0162] Fatty acids with a difference, bigger than 0.25mg / day, between the algal oil and algal oil + PC

[0163] 18:0 1 ,282 1 ,047 0,743

[0164] 18:1(n-9) 32,305 27,042 10,100

[0165] 18:2(n-6) 2,177 1 ,895 8,589

[0166] 22:5(n-3) DPA ND 0,343 ND

[0167] Fatty acid composition of the interventions in % of fatty acids in the triglyceride (TG) and the phospholipid (PL) fraction is depicted in Table 4. Table 4: Fatty acids as wt% of total

[0168] TG PL Total

[0169] HOSO 99,1 0,9 100,0

[0170] Algal oil / Algal oil + ChCI 98,8 1 ,2 100,0

[0171] Algal oil + PC 48,8 51 ,2 100,0

[0172] When analyzing the phospholipid fraction into the different phospholipids (sphingomyelin, phosphatidylcholine, lysophospatidylcholine, phosphatidylethanolamine and a phosphatidylserine / phosphatidylinositol fractions) the levels of these fractions were below the detection limit for the control (HOSO and the algae fractions). For the Algae PC / LPC fraction weight % of fatty acid levels were only significant in the phosphatidylcholine fraction (90,0% of the total fatty acids) and in the phosphatidylethanolamine fraction (10,0% of the total fatty acids). A dose of 200 pl supplement was administered orally per day. Fatty acid composition of the interventions in mg / ml are shown in Table 5. Table 5: Fatty acid composition of the interventions in mg / ml.

[0173] Mg / ml HOSO Algae oil / Algae oil + PC Algal oil +

[0174] ChCI

[0175] 10:0 0.03 0.02 0.03

[0176] 12:0 0.45 0.17 0.31

[0177] 14:0 0.02 0.25 0.19

[0178] 16:0 7.48 10.86 11.11

[0179] 17:0 0.10 0.18 0.30

[0180] DMA 18:0 0.08 ND ND

[0181] 18:1(n-7) 1.25 1.49 0.93

[0182] 18:3(n-3) ND 0.13 0.17

[0183] 20:0 ND 0.51 0.00

[0184] 20:5(n-3) 0.01 0.02 ND

[0185] 22:0 2.12 1.31 0.78

[0186] 24:0 0.70 0.45 0.22

[0187] 22:5(n-6) 0.01 0.02 1.37

[0188] 22:6(n-3) 0.01 6.55 5.38

[0189] 18:0 6.41 5.24 3.72

[0190] 18:1(n-9) 161.53 135.21 50.50

[0191] 18:2(n-6) 10.89 9.48 42.95

[0192] 22:5(n-3) DPA ND 1.72 ND

[0193] Tissue collection and Sample analysis

[0194] On day 29, at 24h (+ / -3) hours after the final oral administration of test product, mice received an overdose anaesthetic. Mice were euthanized via cervical dislocation, after which visceral fat was collected, and the brain was dissected. The hemispheres were separated, and each was immediately processed to isolate the hippocampus, cortex, striatum, and cerebellum. The collected tissues were weighed and stored at -80°C for further analysis. Tissues were prepared for fatty acid analysis according to the method as described in reference Couedelo et al. ref.

[0011] , Brain samples were extracted and analyzed for fatty acid composition as methyl esters (FAME) using gas-chromatography equipped with a flame ionization detector.

[0195] Adipose tissue samples were extracted and fractionated by column chromatography into free fatty acids, triglyceride-bound fatty acids, and phospholipid-bound fatty acids (deemed to represent cell membranes). Each fraction was analyzed for fatty acid composition as methyl esters (FAME) using gas-chromatography equipped with a flame ionization detector.

[0196] For each fatty acid and each compartment analysed, the change in concentration was determined versus the control treatment (HOSO).

[0197] For adipose tissue, three classes of lipids were considered: free fatty acids, triglycerides, and phospholipids. Phospholipids were deemed as representative of membranes (cell, organelle, etc.).

[0198] Percentage increase of the nutrient retention of fatty acids, relative to the control (HOSO) condition are shown in Table 6.

[0199] Results: Brain

[0200] Figures 1a)-1e) describe the % increase of DHA nutrient retention as compared to the control (HOSO), in the brain areas in which a significant effect was detected, 1a. hippocampus and 1 b. cerebellum. The % increase as compared to the control (HOSO) condition for the different adipose tissue fractions are depicted in figure 1c. triglyceride fraction, figure 1d. phospholipid fraction and figure 1e. the free fatty acid fraction.

[0201] For the fatty acid DHA, it was observed that PC / LPC elevated DHA nutrient retention in two brain regions: the hippocampus and the cerebellum. For these brain areas, it was surprisingly found that the combination of algal oil and PC / LPC generates a stronger increase compared to algal oil alone. The DHA levels in the cortex and the striatum did not significantly change in the intervention groups.

[0202] Supplementation of algal oil has been found by the inventors to have an effect of reducing the levels of the proinflammatory fatty acid ARA, such as in the phospholipid fraction, in the brain tissue. This is particularly true for the hippocampus and cerebellum, where the effects of the invention in increasing DHA retention have been found to be most pronounced.

[0203] By integrating algal oil into the composition of the invention as a source of triglyceride bound DHA, the relative ratio of DHA : ARA in the brain tissue, particularly in the hippocampus and cerebellum, can be significantly increased. Since the present invention has been found to be particularly effective in increasing DHA levels in the brain, by supplementing with algal oil, or by ensuring an absence of ARA in the supplementing composition, it is possible to modify the relative ratio of DHA : ARA in the brain tissue, particularly in the hippocampus and cerebellum. This is believed by the inventors to be of particular benefit as increasing DHA levels, relative to ARA levels, in the brain may have a positive role in balancing pro and anti-inflammatory effects. While ARA is an essential fatty acid for brain development in adolescence and function, excessive amounts can disrupt the balance of fatty acids in the adult brain, increase inflammation, and negatively impact cognitive abilities.

[0204] Results: adipose tissue

[0205] The ratio of the change in nutrient retention observed for the combination of A) algal oil + PC / LPC, or B) algal oil + ChCI, over algal oil alone was calculated for all adipose tissue samples. An increase in ratio is considered an amplification of nutrient retention, showing an increase in fatty acid incorporation in the specified target as a result of the addition of PC / LPC to the algal oil. In line with the DHA effect found in the cerebellum and hippocampus, it was found that PC / LPC enhanced the presence of the C20 fatty acids and most of the VL-UFA in the phospholipid fraction of the cells.

[0206] These increases were found for both fatty acids present at comparable levels in the interventions (EPA and DHA) as well as fatty acids which were not present in the interventions nor in the chow (DGLA, ARA, Adrenic acid and Lignoceric acid).

[0207] This effect, a PC / LPC induced increase in C20 and VLCFA’s was in the triglyceride fraction only found for DHA and nervonic acid (table below).

[0208] The table 6 below shows the ratios between algae oil + PC / LPC I algae oil for the different lipid fractions of the adipose tissue (ND=non-detectable levels of the specific fatty acid). Increases in FA levels induced by PC / LPC that exceed 10% (considered a physiologically relevant effect) are highlighted in bold. Table 6: A) Algal oil + PC / LPC / Algae oil nutrient retention ratio for adipose tissue; and B) Algal Oil + ChCI / Algal oil nutrient retention ratio for adipose tissue Figures 2a)-2e) show the effect on nutrient retention of the intervention depicted in % increase or decrease as compared to the control (HOSO) for the group of FA showing an increased algae oil + PC / LPC / algae oil ratio in the PL fraction, (2a. ARA, 2b. DGLA, 2c. EPA, 2d. DHA, and 2e. Adrenic acid.). Supplementation of algae oil reduces the levels of the proinflammatory fatty acid ARA in the phospholipid fraction of the adipose tissue cells. Addition of PC / LPC partially counteracts the reduction of ARA (Figure 2a), but nevertheless still results in a reduction of ARA in the phospholipid fraction in the Algae PC / LPC condition. Conversely, addition of ChCI leads to a greater reduction of ARA.

[0209] The level of the anti-inflammatory fatty acids DGLA, EPA, adrenic acid and DHA in the PL fraction is in all conditions increased by adding the PC / LPC in the algal oil supplement. Notably, the level of increase of DHA in the PL fraction is far superior to that associated with algal oil containing ChCI, instead of PC / LPC (Figure 2d).

[0210] Supplement Compositions and Preparation Thereof

[0211] Table 7

[0212] *comprising triglyceride bound DHA oil as the major component, as well as triglyceride bound ARA (20:4(n- 6) Arachidonic), EPA (20:5(n-3) Eicosapentaenoic), and DPA (22:6(n-6) Docosapentaenoic) as minor components.

[0213] Compositions 1 and 2 as recited in Table 7 above were prepared with different levels of DHA bound triglyceride and lecithin. PC enriched lecithin (containing a minor proportion of LPC) was gently heated to 35-40°C under a nitrogen atmosphere before DHA oil (at a temperature of 15-20°C) was slowly added over the course of 15 minutes with stirring (500 rpm) until a homogenous mixture was achieved which was then stored in aluminium jars under nitrogen blanketing. The resulting compositions are concentrates capable of being used as supplements, or dosed into a functional food or beverage, such as a nutritional drink formulation.

[0214] Application

[0215] The composition comprising the mixture of triglyceride bound LCFA with phosphatidylcholine / lyso-phosphatidylcholine, as described above, can be dosed in common supplement forms such as a liquid oil, capsules or tablets. The composition can be used to gain health benefits in both humans and animals (including marine animals). For animals, compositions could be added to for instance pet food or animal feed.

[0216] A specific application option is to incorporate the composition in the fat fraction of a nutritional drink formulation (e.g. a medical drink or a sports drink for humans). For this, the composition can be diluted with water, and / or solid or liquid fats or oils, as described herein, depending on the desired dosage. Suitable oils for mixing include liquid oils like sunflower oil, high oleic sunflower oil, rapeseed oil, canola oil, coconut oil, olive oil, soybean oil and palm kernel oil.

[0217] Nutritional Drink Formulation 1

[0218] A nutritional drink formulation was prepared having the composition recited in Table 8 below.

[0219] Table 8

[0220] *comprising 68.90% HOSO, 27.51 % sunflower lecithin PC / LPC, 3.59% algal (triglyceride-bound DHA containing) oil

[0221] Table 9: Nutritional Information ( / 100g)

[0222] The drink formulation was prepared by the following process, which includes an optional UHT pasteurization step: i) Water was heated to 60°C and MCI was slowly added; ii) MCI was mixed with the heated water (6000 rpm) until fully dispersed (approximately 30 minutes); iii) MCI was left to hydrate in the water solution (at 60°C) for 1 .5 hour; iv) Maltodextrin and sugar were slowly added to the mixture before being mixed for 10min at 6000 rpm at 60 °C; v) HOSO was melted and slowly blended with sunflower lecithin until fully dispersed (50 rpm at 60°C) before algal oil (containing triglyceride-bound DHA) was added to the sunflower oil I lecithin composition which was then in turn added to the water-based mixture and mixed (6000 rpm) at 50-60°C for 5 min;vi) Flavouring was then added and mixed at 60 °C for a further 5 mins at 6000 rpm; vii) The mixture was then pre-heated to 70°C before undergoing a heat treatment at 142°C for 4 s; viii) The mixture was then cooled and high pressure homogenised at 70°C and 250 bar; ix) The final mixture was then cooled further to below 10°C and clean-fill packed into containers for storage.

[0223] The homogenisation step has been found by the inventors to be effective in providing a monomodal particle size distribution. As shown in the particle size distribution curve of Figure 3, the HOSO / Lecithin / Algal (DHA) oil concentrate composition without homogenization exhibits a multimodal particle size distribution, similar to that seen in lecithin (shown for comparison purposes). Post-homogenisation, both the HOSO / Lecithin / Algal (DHA) oil concentrate composition and lecithin exhibit a monomodal particle size distribution, within a relatively narrow range of particle sizes. Additionally, the UHT pasteurisation step has also been found to contribute to the attainment of a monomodal particle size distribution.

[0224] Viscosity measurements of the nutritional drink formulation were measured using an Anton Paar rheometer equipped with a concentric cylinder geometry system, following a shear rate sweep rheological protocol allowing the viscosity of the sample to be assessed as a function of increasing shear rate. This methodology provided information about the rheological behavior of the drink and its viscosity under different shear rate values. The nutritional drink formulation was found to exhibit a shear thinning rheological behavior, since its viscosity was decreased while the shear rate was increased.

[0225] Nutritional Drink Formulations 1a to 1d

[0226] Nutritional Drink Formulations 1a to 1d were prepared in accordance with the method for preparing Nutritional Drink Formulation 1 above (albeit excluding heat treatment and homogenisation steps), with the exception of using a modified algal oil source having varying concentrations of DHA. The composition of each drink formulation is recited in Table 9a below, and the nutritional information for each formulation is recited in Table 9b. None of Nutritional Drink Formulations 1a to 1d included any flavouring.

[0227] Table 9a

[0228] *contained in a HOSO / sunflower lecithin / algal oil composition

[0229] Table 9b: Nutritional Information ( / 100g) Nutritional Drink Formulation 2

[0230] A plant-based nutritional drink formulation was prepared having the composition recited in Table 10 below.

[0231] Table 10

[0232] *comprising 27.5% high oleic sunflower oil (HOSO), 26% coconut oil, 20% rapeseed oil, 23% sunflower lecithin PC / LPC, 3.5% algal (triglyceride-bound DHA containing) oil Table 11 : Nutritional Information ( / 100g)

[0233] The drink formulation was prepared by the following process, which includes an optional UHT pasteurization step: i) Water was heated to 60°C and the oat powder was slowly added; ii) oat powder was mixed with the heated water (3000 rpm) until fully dispersed (approximately 30 minutes); iii) Oat powder was left to hydrate in the water solution (at 60°C) for 1 .5 hour; iv) Cocoa powder was slowly added to the mixture before being mixed for 54 min at 6000 rpm at 60 °C, followed by addition of sugar, stabilizers and emulsifier, as well as carrageenan; v) HOSO was melted and slowly blended with coconut oil, rapeseed oil and with sunflower lecithin before algal oil (comprising triglyceride bound DHA) was added to the oil I lecithin composition which was then in turn added to the water-based mixture and mixed (6000 rpm) at 50-60°C for 5 min;vi) Flavouring and salts (NaCI, Na2HPO4, CaHPO4, and CaCO3) were then added and mixed at 60 °C for a further 5 mins at 5000 rpm; vii) The mixture was then pre-heated to 70°C before undergoing a heat treatment at 142°C for 4 s; viii) The mixture was then cooled and high pressure homogenised at 70°C and 250 bar; ix) The final mixture was then cooled further to below 10°C and clean-fill packed into containers for storage.

[0234] Viscosity measurements were measured as for Nutritional Drink Formulation 1. Similar shear thinning characteristics were also exhibited in Nutritional Drink Formulation 2.

[0235] Assessing thermostability conferred by lysophosphatidylcholine and / or phosphatidylcholine in Nutritional Drink Formulations 1a to 1d, and 2

[0236] Nutritional Drink Formulations 1 a to 1 d, and 2 were prepared in a Thermomix® apparatus in accordance with the methods described above, albeit without the described heat and homogenisation treatments,. Instead, the mixtures underwent primary homogenisation in the Thermomix® apparatus after which samples were taken for analysis prior to UHT heat treatment. The formulations were subsequently each subjected to a heat treatment of 120°C under ambient pressure (exposed to environment) for 2 minutes to mimic the effects of a typical UHT treatment before being allowed to cool (a longer heating timescale implemented to compensate for higher UHT treatment temperatures achieved when conducted in larger-scale apparatus). Secondary homogenisation of the drink formulations was subsequently conducted at 220 bar using a two-stage homogenizer.

[0237] Further samples were then taken of the formulations after the UHT heat treatment.

[0238] Analysis of the fatty acid (triglyceride bound) content of the drink formulations was conducted on the samples of the formulations taken before and after the heat treatment and analysed using the same measurement method following oil extraction of the samples. Results of the compositional analysis are presented below in Table 11a (for the 4 most prevalent long-chain unsaturated fatty acids present in the drink formulations). The results demonstrate a high level of retention of the fatty acids, particularly DHA, with little loss of unsaturated fatty acids as a result of the heat treatment (e.g. as a result of degradation mechanisms (e.g. oxidation or hydrolysis) that become increasingly prevalent at higher temperatures, particularly for unsaturated fatty acids). Without being bound by any particular theory, the inventors believe that the presence of lyso- phosphatidylcholine and / or phosphatidylcholine in the drink formulations provides a protective effect on long-chain unsaturated fatty acids, particularly DHA, when subjected to elevated temperatures, such as those representative of a UHT pasteurization treatment.

[0239] Table 11a B = before heat treatment

[0240] A = after heat treatment

[0241] Nutritional Drink Formulation 3 An example recipe for a further drink formulation is provided in T able 12. T able 13 shows the constituents of DHA, choline equivalents and phosphatidylcholine. The choline is mostly bound as phosphatidylcholine. The rapeseed oil and coconut oil could be replaced with other liquid oils or liquid oil blends.

[0242] Table 12: Recipe for nutritional liquid supplement fat blend

[0243] Oil Amount (g / 100g product)

[0244] Triglyceride-bound DHA Oil*+ high 35

[0245] PC lecithin mixture

[0246] Coconut oil 25

[0247] Rapeseed oil 40

[0248] * sourced from marine (e.g. fish or krill) or single-celled organism (e.g. algal, bacterial or fungal) oils

[0249] Table 13: DHA / Choline / PC levels: medical nutrition liquid supplement recipe levels for DHA, Choline and PC.

[0250] Compound Amount (g / 100g product)

[0251] DHA 1.35

[0252] Choline 1.97

[0253] Phosphatidylcholine 14.83

[0254] Rati o w / wPC : D HA 11 :1

[0255] Ratio w / w Choline:DHA 1 ,5:1

[0256] The supplement fat blend can be used in a nutritional drinks formulation, for instance for recovering patients or sports practitioners.

[0257] For some nutritional drinks formulations the recommended DHA level is 250 - 500 mg DHA / day.

[0258] For example, the supplement nutritional blend could be used in a drink containing 10% of the fat blend, resulting in 135 mg DHA / 100g product. A drink dose of 100 ml, ingested 2 times a day, would result in 270 mg DHA / day. Alternatively, a drink of 200 ml, ingested 1 time a day, would also result in 270 mg DHA / day.

[0259] In another example, the supplement nutritional blend could be used in a drink containing 20% of the fat blend results in 270 mg DHA / 100g product. Hence, a drink dose of 50 ml, ingested 2 times a day, would result in 270 mg DHA / day. Alternatively, a drink dose of 100 ml, ingested 1 time a day, would also result in 270 mg DHA / day.

[0260] Literature 1. Troesch, B., et al., Expert Opinion on Benefits of Long-Chain Omega-3 Fatty Acids (DHA and EPA) in Aging and Clinical Nutrition. Nutrients, 2020. 12(9).

[0261] 2. Fuentes, N.R., et al., Membrane therapy using DHA suppresses epidermal growth factor receptor signalling by disrupting nanocluster formation. Journal of Lipid Research, 2021 . 62.

[0262] 3. Banaszak, M., et al., Role of Omega-3 fatty acids eicosapentaenoic (EPA) and docosahexaenoic (DHA) as modulatory and anti-inflammatory agents in noncommunicable diet-related diseases - Reports from the last 10 years. Clinical Nutrition ESPEN, 2024. 63: p. 240-258.

[0263] 4. Mustonen, A.M. and P. Nieminen, Dihomo-y-Linolenic Acid (20:3n-6)- Metabolism, Derivatives, and Potential Significance in Chronic Inflammation. Int J Mol Scl, 2023. 24(3).

[0264] 5. Brouwers, H., et al., Adrenic acid as a novel anti-inflammatory player in osteoarthritis. Osteoarthritis and Cartilage, 2018. 26: p. S126.

[0265] 6. Bingham, C.O., 3rd and K.F. Austen, Phospholipase A2 enzymes in eicosanoid generation. Proc Assoc Am Physicians, 1999. 111 (6): p. 516-24.

[0266] 7. Shimizu, T., Lipid mediators in health and disease: enzymes and receptors as therapeutic targets for the regulation of immunity and inflammation. Annu Rev Pharmacol Toxicol, 2009. 49: p. 123-50.

[0267] 8. Kim, A.S. and M.S. Conte, Specialized pro-resolving lipid mediators in cardiovascular disease, diagnosis, and therapy. Adv Drug Deliv Rev, 2020. 159: p. 170- 179.

[0268] 9. So, J., et al., EPA and DHA differentially modulate monocyte inflammatory response in subjects with chronic inflammation in part via plasma specialized proresolving lipid mediators: A randomized, double-blind, crossover study. Atherosclerosis, 2021. 316: p. 90-98.

[0269] 10. Oakes, E.G., et al., Joint effects of one year of marine omega-3 fatty acid supplementation and participant dietary fish intake upon circulating lipid mediators of inflammation resolution in a randomized controlled trial. Nutrition, 2024. 123: p. 112413.

[0270] 11 . Couedelo, L., et al., Effect of increased levels of dietary a-linolenic acid on the n-3 PUFA bioavailability and oxidative stress in rat. Br J Nutr. 2022 May 14; 127(9): 1320-1333

[0271] 12. Lewkowicz, N., et al, Naturally Occurring Nervonic Acid Ester Improves Myelin Synthesis by Human Oligodendrocytes. Cells. 2019 Aug; 8(8): 786. 13. Smolders, L, et al. Natural Choline from Egg Yolk Phospholipids Is More Efficiently Absorbed Compared with Choline Bitartrate; Outcomes of A Randomized Trial in Healthy Adults. Nutrients. 2019, 11 , 2758

[0272] 14. Hadaruga, D. I., et al. Beilstein, J Org Chem, 2016, Feb 2; 12: 179-191 15. Holser, R.A., et al. American Journal of Analytical Chemistry, 2014, 5, 373-

[0273] 377.

Claims

Claims1 . Non-therapeutical use of phosphatidylcholine and / or lyso-phosphatidylcholine for increasing the nutrient retention of at least one Long-Chain Fatty Acid (LCFA) in an animal or a human.

2. Use according to claim 1 , wherein the use is for increasing the nutrient retention of LCFA as phospholipids.

3. Use according to claim 1 or 2, wherein the use is for increasing the nutrient retention of LCFA in membranes (e.g. cell and organelle membranes).

4. Use according to any of the preceding claims, wherein the use is for increasing the nutrient retention of LCFA in adipose tissue.

5. Use according to any of the preceding claims, wherein the use is for increasing the nutrient retention of LCFA in the brain, preferably wherein said use comprises improving cognitive function in the animal or human.

6. Use according to claim 5, wherein the use is for increasing the nutrient retention of LCFA in the hippocampus.

7. Use according to claim 5 or 6, wherein the use is for increasing the nutrient retention of LCFA in the cerebellum.

8. Use according to any of the preceding claims, wherein the nutrient retention in an animal of at least one LCFA was increased by at least 2.5%, preferably at least 5%, preferably at least 10%, more preferably at least 15%, when compared to a supplement oral composition without phosphatidylcholine.

9. Use according to any of the preceding claims, wherein the use is for increasing nutrient retention in a human.

10. Use according to any of the preceding claims 1-8, wherein the use is for increasing nutrient retention in an animal.11 . Use according to claim 10, wherein the animal is a marine animal.

12. Use according to any of the preceding claims, wherein the nutrient retention of at least one increased LCFA is a Long-Chain Unsaturated Fatty Acid (LC-UFA).

13. Use according to claim 12, wherein the at least one increased LC-UFA is selected from the group consisting of docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA), eicosaenoic acid, erucic acid, adrenic acid, nervonic acid, vaccenic acid, palmitoleic acid, pentadecanoic acid, alphalinolenic acid, eicosatrienoic acid, gamma-linolenic acid (GLA), dihomo-gamma- linolenic acid (DGLA), eicosatetraenoic acid, and conjugated linoleic acid.

14. Use according to claim 12 or 13, wherein the at least one increased LC-UFA is an omega-3 Long-Chain Polyunsaturated Fatty Acid (LC-PUFA).

15. Use according to claim 14, wherein the at least one increased LC-PUFA is DGLA, DHA or, EPA, for example DHA and EPA.

16. Use according to claim 15, wherein the increased LC-PUFA is DHA.

17. Use according to claim 16, wherein the proportion of DHA is increased in one or more tissues relative to the amount of arachidonic acid (ARA) in the same one or more tissues, preferably wherein the one or more tissues comprises brain tissue (e.g. in hippocampus and / or the cerebellum regions of the brain).

18. Use according to any of the claims 12-16, wherein at least 2 LC-UFA are increased, preferably at least 3, more preferably at least 4.

19. Ingestible composition for increasing the nutrient retention of at least one Long- Chain Fatty Acid (LCFA) according to any of the preceding claims, wherein the composition comprises phosphatidylcholine and / or lyso-phosphatidycholine, and at least one triglyceride-bound LCFA with increased nutrient retention.

20. Composition according to claim 19, wherein the composition comprises a combined amount of at least 0.05 wt% of phosphatidylcholine and lyso- phosphatidylcholine and at least 0.01 wt% of the at least one triglyceride-bound LCFA with increased nutrient retention.

21. Composition according to claim 20, wherein the composition comprises a combined amount of at least 0.01 wt% of phosphatidylcholine and lyso- phosphatidylcholine, preferably 0.1 wt% of phosphatidylcholine and lyso- phosphatidylcholine, preferably at least 1.0 wt% phosphatidylcholine and lyso- phosphatidylcholine, more preferably at least 2 wt% phosphatidylcholine and lyso-phosphatidylcholine.

22. Composition according to any of the claims 19-21 , wherein the composition comprises a combined amount of from 0.01-80 wt%, preferably from 0.1-60 wt% of phosphatidylcholine and lyso-phosphatidylcholine, preferably from 1-50 wt%.

23. Composition according to any of the claims 19-22, wherein the composition comprises a combined amount of 0.1-10 wt%, such as from 0.5-5.0 wt%, or from 0.5-3.0 wt%, of phosphatidylcholine and lyso-phosphatidylcholine.

24. Composition according to any of the claims 19-23, wherein the composition comprises at least 0.01 wt% of the at least one triglyceride-bound LCFA with increased nutrient retention, preferably at least 0.05%, more preferably at least 0.1 wt%, more preferably at least 0.5 wt%, more preferably at least 1 wt%.

25. Composition according to claim 24, wherein the composition comprises from 0.01- 20 wt% of the at least one triglyceride-bound LCFA with increased nutrient retention, preferably from 0.05-10 wt%, preferably 1-10 wt%, more preferably from 0.5-5 wt%.

26. Composition according to any of the claims 19-25, wherein the composition comprises: (i) phosphatidylcholine and lyso-phosphatidylcholine, and (ii) the at least one triglyceride-bound LCFA in a weight ratio (i) I (ii) of at least 0.1 , preferably at least 0.2, preferably at least 0.5, more preferably at least 0.8, more preferably at least 1 , more preferably at least 2, even more preferably at least 5, more preferably at least 8.

27. Composition according to any of the claims 19-26, wherein the composition comprises: (i) phosphatidylcholine and lyso-phosphatidylcholine, and (ii) the at least one triglyceride-bound LCFA in a weight ratio (i) I (ii) of from 0.01 to 400, preferably from 0.1 to 100, more preferably from 1 to 20, most preferably from 8 to 15.

28. Composition according to any of the claims 19-27, wherein the composition is essentially free, or free, of choline salts (e.g. choline chloride).

29. Composition according to any of claims 19-28, wherein the at least one triglyceride-bound LCFA is as defined in claims 12-15.

30. Composition according to any of the claims 19-28, wherein ARA (in triglyceride- bound form or free fatty acid) is present in an amount of less than 10 wt%, preferably less than 5 wt%, more preferably less than 3 wt%, of the total amount of LC-UFA (in triglyceride-bound form or free fatty acid) present in the composition.

31. Composition according to any of the claims 19-29, wherein ARA (in triglyceride- bound form or free fatty acid) is present in the composition in an amount less than 10 wt%, preferably less than 5 wt%, more preferably less than 3 wt%) of the total amount of DHA (in triglyceride-bound form or free fatty acid) present in the composition.

32. Composition according to any of the claims 19-30, wherein at least one triglyceride-bound LCFA is provided in the composition as part of one or more LCFA-containing fats or oils, including liquid oils, semi-solid fats or solid fats, optionally diluted with one or more additional oils or fats (e.g. additional non- LCFA containing liquid oils) to form a mixed fat or oil composition.

33. Composition according to any of the claims 19-32, wherein the composition comprises a liquid oil selected from rapeseed oil, high oleic rapeseed oil, higherucic acid rapeseed oil, soybean oil, sunflower oil, high oleic sunflower oil, linseed oil, olive oil, corn oil, cottonseed oil, carinata oil, groundnut oil, safflower oil, high oleic safflower oil, peanut oil, walnut oil, avocado oil, rice brand oil, camelina oil, shea olein, sesame oil, MCT oil, SCT oil, marine oils (e.g. fish oil or, krill oil), and single cell organism oils (e.g. algal oil, or microbial oil, or fungal oil), any fraction thereof, any mixture thereof, or any esterified or interesterified product, fraction or mixture thereof.

34. Composition according to any of the claims 19-33, wherein the composition comprises a semi-solid or solid fat selected from palm fat, coconut fat, shea butter, shea stearin, cocoa butter, cocoa stearin, cocoa olein, allanblackia fat, kokum fat, mango kernel fat, sal fat, illipe butter, palm kernel fat, babassu fat, coconut oil stearin, coconut oil olein, palm kernel olein, palm olein, palm kernel stearin, avocado oil, any fraction thereof, any mixture thereof, or any esterified or interesterified product, fraction or mixture thereof.

35. Composition according to any of the claims 19-34, wherein the composition comprises a melted fat selected from shea butter, shea stearin, shea olein, cocoa butter, cocoa stearin, cocoa olein, allanblackia fat, kokum fat, mango kernel fat, sal fat, illipe butter, coconut oil, coconut oil stearin, coconut oil olein, palm kernel oil, palm kernel olein, palm olein, rapeseed oil, palm oil, palm kernel stearin, babassu oil, any fraction thereof, any mixture thereof, or any esterified or interesterified product, fraction or mixture thereof.

36. Composition according to any of the claims 19-35, wherein the composition is a blend of algal oil, a phospholipid fraction comprising phosphatidylcholine and / or lyso-phosphatidylcholine (e.g. derived from lecithin), and optionally another liquid oil, for example as defined in claim 33.

37. Composition according to any of the claims 19-36, wherein phosphatidylcholine and / or lyso-phosphatidylcholine in the composition are derived from lecithin, for example wherein the lecithin is derived from a botanical source (such as soya, rapeseed, or sunflower) an animal source (e.g. egg yolk), a marine source (e.g. fish or krill), or single-celled organism source (e.g. algal, bacterial or fungal). Composition according to any of the claims 19-35, wherein the phosphatidylcholine and / or lyso-phosphatidylcholine is derived from a botanical source (such as soya, rapeseed, or sunflower) an animal source (e.g. egg yolk), a marine source (e.g. fish or krill), or single-celled organism source (e.g. algal, bacterial or fungal).

38. Composition according to any of the preceding claims 19-37, wherein the composition is a functional food or health supplement.

39. Composition according to claim 38, wherein the composition is an animal feed or animal feed supplement.

40. Composition according to any of the claims 19-39, wherein the composition is an oral composition.

41. Composition according to any of the claims 19-40, wherein the composition is a capsule, a gummy, a gel, a chewing gum, a tablet, a powder, a bar (e.g. a nutritional bar), a liquid beverage (e.g. a nutritional drink), a shot or a pouch.

42. Composition according to any of claims 19-41 , wherein the composition is a dairybased drink or fermented product (e.g. a yoghurt) or a plant-based drink or dairyanalogue product (e.g. a plant-based yoghurt).

43. Composition according to any of the claims 19-37, wherein the composition is an enteral composition.

44. Composition according to any of claims 19-42, wherein the composition is a nutritional drink formulation further comprising one or more of, preferably all of, the additional ingredients selected from water, a carbohydrate source, a protein source, a dietary fiber source, and a further fat source.

45. Method of preparing a composition according to any of the claims 19-44, comprising the mixing of a phospholipid fraction comprising phosphatidylcholine and / or lyso-phosphatidylcholine, and an oil or fat comprising triglyceride-bound LCFA.

46. Method according to claim 45, wherein the phospholipid fraction comprising phosphatidylcholine and / or lyso-phosphatidylcholine is heated to from 30 to 45° C prior to mixing with an oil or fat.

47. Method of according to claim 45 and 46, further comprising formulating a nutritional drinks formulation by adding one or more, preferably all, additional ingredients selected from water, a carbohydrate source, a protein source, a dietary fiber source, and a further fat source.

48. Method according to claim 47, wherein formulation of the drink includes a pasteurisation step and / or a high pressure homogenisation step.

49. Use of phosphatidylcholine and / or lyso-phosphatidylcholine for increasing the thermal stability of at least one Long-Chain Unsaturated Fatty Acid (LC-UFA), preferably at least one Long-Chain Polyunsaturated Fatty Acid (LC-PUFA), in an ingestible composition, such as an ingestible composition as defined in any one of claims 19-44.

50. Use according to claim 49, wherein the LC-UFA is as defined in any one of claims 13 to 16.

51. Use according to claim 49 or claim 50, wherein the use is for increasing the thermal stability of at least one Long-Chain Unsaturated Fatty Acid (LC-UFA) in an ingestible composition which is heat treated to a temperature of at least 100°C, such as at least 120 °C, or at least 140 °C, preferably in an ingestible composition that is heat treated as part of a UHT pasteurization treatment, such as at 142 °C for at least 4 seconds.

Images