Microalgal oil emulsions and uses thereof

Abstract

The present invention relates generally to nutritional compositions comprising an emulsion comprising one or more non-mammalian lipids and / or triacylglycerides, including such compositions having a lipid and / or triacylglyceride content comparable to that of mammalian milk, such as human milk. Particularly contemplated are nutritional compositions having a lipid and / or triacylglyceride content comparable to that of human milk, wherein at least some of the lipid and / or triacylglyceride is from microalgae.

Description

[0001] MICROALGAL OIL EMULSIONS AND USES THEREOF

[0002] TECHNICAL FIELD

[0003] The invention relates generally to nutritional compositions comprising one or more nonmammalian lipids and / or triacylglycerides, including such compositions having a lipid and / or triacylglyceride content comparable to that of mammalian milk, such as human milk.

[0004] BACKGROUND CONTEMPLATED HEREIN

[0005] The following includes information that may be useful in understanding the present inventions. It is not an admission that any of the information provided herein is prior art, or relevant, to the presently described or claimed inventions, or that any publication or document that is specifically or implicitly referenced is prior art. Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0006] The composition of mammalian milk is adapted to support normal growth and development of the neonate, infant or child. However, there are many circumstances where a suitable or plentiful supply of milk is not available. Frequently, milk from a mammalian species other than that of the subject can be substituted. However, the composition of mammalian milk differs across species - for example, the nutritional composition of human milk differs in some respects to that of non-human milks. Non-human whole milk such as cow, goat, or sheep milk, contains a very different complement of milk oligosaccharides to that of human milk.

[0007] Significant resource has been applied to the development of milk substitutes, such as maternal formulas, infant formulas, follow-on formulas, growing-up formulas, dietetic products and other milk / dairy-containing compositions, particularly those for human consumption and which are typically produced using non-human milk.

[0008] There remains a need for milk substitutes, for example, human milk substitutes that provide comparable functionality to that of human milk. For example, components of human milk have been associated with cognitive development. Optimal cognitive development and growth is fundamental to healthy development of infants and children. Impaired cognitive development has been reported to have significant immediate and long-term effects on quality of life.

[0009] Healthy nutrition is important not only for growth and development in growing infants and children, but also for juvenile and adult health and well-being.

[0010] Cognitive decline is characterised by reduced learning ability, memory and attention span is widespread among the elderly. Agents and compositions that prevent, ameliorate, slow, or delay cognitive decline will have widespread benefits on the quality of life of the elderly.

[0011] There is a growing demand for non-animal nutritional products, such as non-animal substitutes for dairy and / or milk products that are suitable for vegetarian or vegan consumption.

[0012] There thus remains a need to provide compositions suitable for providing comparable nutrition to that provided by mammalian milk, without relying or relying solely on milk products, such as dairy lipids.

[0013] The invention disclosed herein seeks to overcome at least some of the disadvantages associated with existing nutritional compositions and / or their production, and / or to provide nutritional compositions suitable for use in the preparation of nutritional formulas, and / or for use in methods of providing one or more benefits associated with healthy nutrition, such as methods of supporting cognitive development and / or minimising cognitive impairment and / or cognitive decline, and / or to provide nutritional compositions comprising a non-mammalian lipid and / or triacylglyceride content enriched in palmitate, such as a composition comprising microalgal-derived lipids and / or triacylglycerides having a lipid and / or triacylglyceride content comparable to that of human milk, and / or to overcome at least some of the disadvantages associated with the state of the art as set out above, or to at least provide a useful alternative to existing compositions and methods, or to at least provide the public with a useful choice.

[0014] SUMMARY OF THE INVENTION

[0015] In a first aspect the invention relates to a composition comprising, consisting essentially of, or consisting of an emulsion, the emulsion comprising:

[0016] (a) triglyceride, wherein said triglyceride comprises at least 15% w / w palmitic acid, wherein at least 50% w / w of said palmitate being present at the sn-2 position; and

[0017] (b) one or more phospholipids; and

[0018] (c) one or more sphingolipids; and

[0019] (d) one or more gangliosides; and

[0020] (e) one or more cholesterols.

[0021] In certain examples, the triglyceride comprises, consists essentially of, or consists of microalgal- derived triglyceride.

[0022] In one example, the triglyceride consists of microalgal-derived triglyceride.

[0023] In certain examples, the microalgal-derived triglyceride is from any one or more of the group consisting of: Amphora spp.; Arthospira spp.; Audouinella eugena; Aurantiochytrium limacinum; Balbiania investiens; Chlamydomonas spp., such as Chlamydomonas reinhardtii; Chlorella spp., such as Chlorella protothecoides, Chlorella pyrenoidosa, Chlorella sorokiniana, Chlorella vulgaris: CV-15 and CV-395, or Chlorella zofingiensis; Chlorococcum spp.; Crypthecodinim cohnii; Desmodesmus spp.; Dunaliella spp., such as Dunaliella tertiolecta; Haematococcus spp., such as Haematococcus pluvialis; Isochrysis spp., such as Isochrysis galbana; Myrmecia bisecta; Nannochloropsis spp., such as Nannochloropsis limnetica, Nannochloropsis occulata, Nannochloropsis oceanica, or Nannochloropsis salina; Navicula pelliculosa; Neochloris oleabundans; Nitzchia spp., such as Nitzchia laevis; Palmodictyon varium; Phaeodactylum spp., such as Phaeodactylum tricornutum; Porphyridium spp., such as Porphyridium cruentum; Pseudochantransia spp.; Scenedesmus spp., such as Scenedesmus obliquus; Schizochytrium spp., such as Schizochytrium mangrove! or Schizochytrium limacinum; Skeletonema marinoi; Spirulina spp.; Synechococcus spp.; Thorea ramosissima; Trachydiscus minutus; and Trisochrysis spp..

[0024] In certain examples, the triglyceride comprises, consists essentially of, or consists of triglyceride from Nannochloropsis oceanica, such as Nannochloropsis oceanica CCMP531 or Nannochloropsis oceanica UTEX 2164.

[0025] In certain examples, the one or more phospholipids, the one or more sphingolipids, the one or more gangliosides, and the one or more cholesterols are provided as a composition, such as a dairy composition.

[0026] In certain examples, the emulsion is formed from triglyceride and a composition comprising one or more of the one or more phospholipids, the one or more sphingolipids, the one or more gangliosides, and / or the one or more cholesterols. In certain examples, the composition is a pharmaceutical or nutraceutical composition.

[0027] In certain examples, the emulsion has a mean particle size of from about 1 pm to about 10 pm in diameter.

[0028] In certain examples, the triglyceride comprises at least about 20% w / w palmitic acid.

[0029] In certain examples, the fatty acid present in the microalgal-derived triglyceride comprises at least about 50% w / w palmitic acid.

[0030] In certain examples, at least about 80% w / w of the palmitic acid present is present at the sn-2 position.

[0031] In certain examples, the one or more phospholipids comprises, consists essentially of, or consists of one or more milk phospholipids.

[0032] In certain examples, the one or more phospholipids is selected from the group consisting of:

[0033] (a) phosphatidylcholine;

[0034] (b) phosphatidylethanolamine;

[0035] (c) phosphatidylserine;

[0036] (d) phosphatidylinositol;

[0037] (e) phosphatidic acid;

[0038] (f) a derivative, extract, or fraction of one or more of the phospholipids defined in a) - e); and

[0039] (g) any combination of two or more of any of a) to f) above.

[0040] In certain examples, the one or more sphingolipids is selected from the group consisting of:

[0041] (a) sphingomyelins;

[0042] (b) Ceramides;

[0043] (c) Gangliosides

[0044] (d) Lactosylceramide

[0045] (e) Glucosylceramide

[0046] (f) a derivative, extract, or fraction of one or more of the sphingolipids defined in a) - e); and

[0047] (g) any combination of two or more of any of a) to f) above.

[0048] In certain examples, the one or more gangliosides is selected from the group consisting of:

[0049] (a) monosialodihexosylganglioside (GM3);

[0050] (b) disialoganglioside (GD3);

[0051] (c) a derivative, extract, or fraction of one or more of the gangliosides defined in a) - b); and

[0052] (d) any combination of two or more of any of a) to c) above.

[0053] In certain examples, the one or more cholesterols is and phytosterols selected from the group consisting of:

[0054] (a) Cholesterol

[0055] (b) p-sitosterol;

[0056] (c) Campesterol

[0057] (d) Stigmasterol

[0058] (e) Brassicasterol:

[0059] (f) a derivative, extract, or fraction of one or more of the cholesterols defined in a) - e); and

[0060] (g) any combination of two or more of any of a) to f) above. In certain examples, the composition comprising one or more of the one or more phospholipids, the one or more sphingolipids, the one or more gangliosides, and / or the one or more cholesterols, is a composition comprising, consisting essentially of, or consisting of milk fat globule membrane (MFGM), such as bovine MFGM, including for example a composition comprising, consisting essentially of, or consisting of intact MFGM, such as intact bovine MFGM.

[0061] In certain examples, the sole source of phospholipids, and / or the sole source of sphingolipids, and / or the sole source of gangliosides, and / or the sole source of cholesterols in the composition comprising, consisting essentially of, or consisting of an emulsion is a composition comprising, consisting essentially of, or consisting of milk fat globule membrane (MFGM), such as bovine MFGM, including for example a composition comprising, consisting essentially of, or consisting of intact MFGM, such as intact bovine MFGM.

[0062] In certain examples, the composition comprising one or more of the one or more phospholipids, the one or more sphingolipids, the one or more gangliosides, and / or the one or more cholesterols, is bovine beta-serum.

[0063] In certain examples, the sole source of phospholipids present in the composition comprising, consisting essentially of, or consisting of an emulsion is beta-serum, such as bovine beta-serum. In one example, the sole source of phospholipids present in the composition comprising, consisting essentially of, or consisting of an emulsion is beta-serum prepared as described herein in the Examples.

[0064] In certain examples, the sole source of sphingolipids present in the composition comprising, consisting essentially of, or consisting of an emulsion is beta-serum, such as bovine beta-serum. In one example, the sole source of sphingolipids present in the composition comprising, consisting essentially of, or consisting of an emulsion is beta-serum prepared as described herein in the Examples.

[0065] In certain examples, the sole source of gangliosides present in the composition comprising, consisting essentially of, or consisting of an emulsion is beta-serum, such as bovine beta-serum. In one example, the sole source of gangliosides present in the composition comprising, consisting essentially of, or consisting of an emulsion is beta-serum prepared as described herein in the Examples.

[0066] In certain examples, the sole source of cholesterols present in the composition comprising, consisting essentially of, or consisting of an emulsion is beta-serum, such as bovine beta-serum. In one example, the sole source of cholesterols present in the composition comprising, consisting essentially of, or consisting of an emulsion is beta-serum prepared as described herein in the Examples.

[0067] In certain examples, the sole source of any two or more of the group comprising the phospholipids, the sphingolipids, the gangliosides, and the cholesterols, present in the composition comprising, consisting essentially of, or consisting of an emulsion is beta-serum, such as bovine betaserum. In one example, the sole source of any two or more of the group comprising the phospholipids, the sphingolipids, the gangliosides, and the cholesterols, present in the composition comprising, consisting essentially of, or consisting of an emulsion is beta-serum prepared as described herein in the Examples.

[0068] In certain examples, the sole source of all phospholipids, sphingolipids, gangliosides, and cholesterols present in the composition comprising, consisting essentially of, or consisting of an emulsion is beta-serum, such as bovine beta-serum. In one example, the sole source of all phospholipids, sphingolipids, gangliosides, and cholesterols present in the composition comprising, consisting essentially of, or consisting of an emulsion is beta-serum prepared as described herein in the Examples.

[0069] In certain examples, the composition comprising, consisting essentially of, or consisting of an emulsion is prepared using beta-serum that comprises at least about 800 mg cholesterol per 100 g beta-serum (i.e., beta-serum comprising at least about 800 mg / 100 g cholesterol). In one example, the composition comprising, consisting essentially of, or consisting of an emulsion is prepared using beta-serum that comprises at least about 900 mg / 100 g cholesterol. In one example, the composition comprising, consisting essentially of, or consisting of an emulsion is prepared using beta-serum that comprises at least about 1000 mg / 100 g cholesterol. In one example, the composition comprising, consisting essentially of, or consisting of an emulsion is prepared using beta-serum that comprises from about 800 mg / 100 g cholesterol to about 1200 mg / 100 g cholesterol.

[0070] In certain examples, the composition comprising, consisting essentially of, or consisting of an emulsion is prepared using beta-serum that comprises at least about 20g fat per 100 g beta-serum (i.e., beta-serum comprising at least about 20 g / 100 g fat). In one example, the composition comprising, consisting essentially of, or consisting of an emulsion is prepared using beta-serum that comprises at least about 25 g / 100 g fat. In one example, the composition comprising, consisting essentially of, or consisting of an emulsion is prepared using beta-serum that comprises at least about 30 g / 100 g fat. In one example, the composition comprising, consisting essentially of, or consisting of an emulsion is prepared using beta-serum that comprises from about 20 g / 100 g fat to about 35 g / 100 g fat.

[0071] In certain examples, the composition comprises:

[0072] (a) at least about 1 g triglyceride per 100 g; and / or

[0073] (b) at least about 100 mg phospholipid per 100 g; and / or

[0074] (c) at least about 50 mg sphingolipid per 100 g; and / or

[0075] (d) at least about 50 mg ganglioside per 100 g; and / or

[0076] (e) at least about 500 mg cholesterol per 100 g; and / or

[0077] (f) any combination of two or more of any of a) to e) above.

[0078] In certain examples, the triglyceride comprises from about 10 to about 50% w / w palmitic acid and from about 20 to about 70% w / w palmitoleic acid.

[0079] In certain examples, at least about 15% w / w of the triglyceride consists of a C16: l-C16:0- C16: 1 triglyceride species.

[0080] In certain examples, the triglyceride is substantially free of linoleic acid.

[0081] In another aspect, the invention relates to a method of preparing a composition comprising, consisting essentially of, or consisting of an emulsion, the emulsion comprising triglyceride, one or more phospholipids, one or more sphingolipids, one or more gangliosides, and one or more cholesterols, the method comprising: providing Nannochloropsis cells or a cell extract or lysate therefrom; contacting said cells or cell extract with a solvent comprising methanol and chloroform under conditions and for a time sufficient to dissolve one or more lipids; recovering and combining the one or more lipids with a composition comprising one or more phospholipids, one or more sphingolipids, one or more gangliosides, and one or more cholesterols to provide a mixture; homogenising the mixture by applying shear and / or sonication until the mean emulsion particle size is from about 200 nm to about 10 pm, for example from about 1 pm to 5 pm.

[0082] In one example, the Nannochloropsis cells were grown in conditions in which the concentration or amount of nitrogen was growth-limiting.

[0083] In certain examples, the Nannochloropsis cells were grown in conditions in which the concentration or amount of phosphate was growth-limiting.

[0084] In certain examples, the preparation of the emulsion comprises homogenisation and / or sonication.

[0085] In another aspect, the invention relates to a composition as contemplated herein for use in therapeutically increasing growth and / or development of a neonatal, infant, child, or adult subject in need thereof.

[0086] In another aspect, the invention relates to a composition as contemplated herein for use in increasing the growth and / or development of a neonatal, infant, child, or adult subject in need thereof.

[0087] In another aspect, the invention relates to the use of a composition as contemplated herein in the preparation of a medicament, pharmaceutical composition, or nutritional composition for increasing growth and / or development of a subject in need thereof.

[0088] In another aspect, the invention relates to a method for increasing growth and / or development of a neonatal, infant, child, or adult subject, the method comprising orally administering to the subject a composition as contemplated herein.

[0089] In another aspect, the invention relates to a method for increasing cognitive development of a neonatal, infant, child, or adult subject, the method comprising orally administering to the subject a composition as contemplated herein.

[0090] In another aspect, the invention relates to a method of treating or preventing cognitive impairment or cognitive decline in a subject, the method comprising administering to the subject a composition as contemplated herein.

[0091] In another aspect, the invention relates to a non-therapeutic method for increasing growth of a healthy subject, the method comprising orally administering to the subject a composition as contemplated herein, wherein the method is not a method for the treatment of the human or animal body by therapy.

[0092] In another aspect, the invention relates to a non-therapeutic method for increasing cognitive development of a healthy subject, the method comprising orally administering a composition as contemplated herein to the subject, wherein the method is not a method for the treatment of the human or animal body by therapy.

[0093] In another aspect, the invention relates to the use of a composition as contemplated herein in a non-therapeutic method for increasing growth of a healthy subject, the method comprising orally administering the formulation to the subject, wherein the method is not a method for the treatment of the human or animal body by therapy.

[0094] In another aspect, the invention relates to the use of a composition as contemplated herein in a non-therapeutic method for increasing cognitive development of a healthy subject, the method comprising orally administering the formulation to the subject, wherein the method is not a method for the treatment of the human or animal body by therapy.

[0095] In certain examples, the subject is a neonatal, infant, or child subject.

[0096] In certain examples, the subject is an adult subject.

[0097] In certain examples, the composition is an infant formula, follow-on formula, or growing-up formula.

[0098] In certain examples, the composition is an adult formula, such as a nutritional formula, medical food, sports formula, or geriatric formula.

[0099] In a further aspect the invention relates to a method of providing one or more health benefits to a subject, the method comprising administering a composition or nutritional formulation as described herein to the subject.

[0100] In various examples, the one or more health benefits is selected from the group consisting of improved fat absorption, digestion, and / or metabolism; improved gut health, including for example improved gut-microbiome interactions; improved calcium absorption, digestion, metabolism, and / or improved bone health; improved immune response and / or immune modulation; improved cognition, improved cognitive development, and improved brain health; and improved cardiovascular health.

[0101] Relatedly, the invention in a further aspect relates to a method of improving fat absorption, fat digestion, and / or fat metabolism in a subject, the method comprising administering a composition or nutritional formulation as described herein to the subject.

[0102] The invention in a further aspect relates to a method of improving gut health, including for example improving gut-microbiome interactions in a subject, the method comprising administering a composition or nutritional formulation as described herein to the subject.

[0103] The invention in another aspect relates to a method of improving calcium absorption, calcium digestion, calcium metabolism, and / or bone health in a subject, the method comprising administering a composition or nutritional formulation as described herein to the subject.

[0104] The invention in a further aspect relates to a method of improving immune response and / or immune modulation in a subject, the method comprising administering a composition or nutritional formulation as described herein to the subject.

[0105] The invention in another aspect relates to a method of improving cognition, improved cognitive development, and / or improving brain health in a subject, the method comprising administering a composition or nutritional formulation as described herein to the subject.

[0106] The invention in a further aspect relates to a method of improving cardiovascular health in a subject, the method comprising administering a composition or nutritional formulation as described herein to the subject.

[0107] In a further aspect the invention relates to a method of maintaining or increasing brain function or brain development, the method comprising administering a composition or nutritional formulation as described herein to a normal subject.

[0108] In various examples, the composition or nutritional formulation is administered to:

[0109] (a) prevent delayed brain development, or

[0110] (b) maintain the viability or function of neuronal cells, or

[0111] (c) maintain or increase cognitive development, or

[0112] (d) prevent delayed cognitive development, or

[0113] (e) maintain or increase cognitive performance, or (f) prevent cognitive decline, or

[0114] (g) maintain or increase learning, memory or attention span, or

[0115] (h) prevent decline of learning ability, memory or attention span, or

[0116] (I) maintain or increase ability to cope with stress, or

[0117] (j) prevent decline in brain function, or

[0118] (k) maintain or increase the level of gangliosides in the brain, or

[0119] (l) maintain or increase blood, nutrient or oxygen flow to the brain, or

[0120] (m) maintain or increase synaptogenesis, or

[0121] (n) maintain or increase the activity of the gut / brain axis, or

[0122] (o) maintain or increase neurogenesis in an older brain, or

[0123] (p) maintain or increase white matter volume, or

[0124] (q) a combination of any two or more of a) to p).

[0125] In various examples, the subject is a neonatal, infant or child subject.

[0126] In one example, the subject is an infant, and the composition or formulation is administered to the infant to support optimal growth and energy supply, better calcium absorption and stronger bone development, brain development, supporting cognitive functions and neural growth and supporting a healthy microbiome and strengthening the immune system.

[0127] In one example, the subject is an older adult, and the composition or formulation is administered to the older adult to support better energy supply and nutrient uptake, maintain bone density, reducing the risk of osteoporosis and supporting overall bone health, support brain health by aiding in the maintenance of neuronal membranes and cognitive functions, improve lipid profiles and supporting cardiovascular health, support a healthy gut microbiome, improving digestion and overall gut health in older adults.

[0128] In a further aspect the invention relates to a method of treating mild cognitive impairment associated with any one or more of age-related cognitive decline, dementia, Alzheimer's disease, vascular disease, frontotemporal lobar degeneration (FTLD), dementia with Lewy bodies, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, cognitive impairment associated with schizophrenia, chemotherapy-induced neuropathy, Down's syndrome, Korsakoff's disease, cerebral palsy, epilepsy, neuronal ischemia, neuronal reperfusion injury, neuronal trauma, neuronal haemorrhage, neuronal infection, stroke, neuronal exposure to a toxic substance, age-related mental disorders, anxiety disorders, age-related depression, dementia associated with microvascular disorders (such as diabetes, hypotension, stroke induced vascular dementia, and obesity), dementia associated with a disorder of the immune system, dementia associated with a central nervous system (CNS) disorder, dementia associated with hypotension, and dementia associated with obesity, or vascular dementia.

[0129] In a further aspect the invention relates to the use of a composition or emulsion as described herein in the manufacture of a formulation for treating mild cognitive impairment associated with any one or more of age-related cognitive decline, dementia, Alzheimer's disease, vascular disease, frontotemporal lobar degeneration (FTLD), dementia with Lewy bodies, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, cognitive impairment associated with schizophrenia, chemotherapy-induced neuropathy, Down's syndrome, Korsakoff's disease, cerebral palsy, epilepsy, neuronal ischemia, neuronal reperfusion injury, neuronal trauma, neuronal haemorrhage, neuronal infection, stroke, neuronal exposure to a toxic substance, age-related mental disorders, anxiety disorders, age-related depression, dementia associated with microvascular disorders (such as diabetes, hypotension, stroke induced vascular dementia, and obesity), dementia associated with a disorder of the immune system, dementia associated with a central nervous system (CNS) disorder, dementia associated with hypotension, dementia associated with obesity or vascular dementia.

[0130] Other aims, aspects, features and advantages of the present invention will become apparent from the following description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred examples contemplated herein, are given by way of illustration only, since various changes and modifications within the spirit and scope contemplated herein will become apparent to those skilled in the art from this detailed description.

[0131] The invention is exemplified in the following non limiting examples and with reference to the accompanying figures.

[0132] BRIEF DESCRIPTION OF THE FIGURES

[0133] Figure 1 presents a schematic diagram of the method of extraction, purification, and characterization of microalgal oils described in Example 1 herein.

[0134] Figure 2 presents a graph showing biomass growth of Chlamydomonas reinhardtii, Scenedesmus obliquus, Nannochloropsis oceanica, and N. oculata under standard, nutrient limitation (nonitrogen, and no-phosphorous), and light saturating conditions as described herein in Example 1. All experiments were carried out in triplicate and the results shown represent the mean ± standard error of the three replicates. Two-way permutation ANOVA was carried out using R (version 4.1.1, R Core Team, 2021) and the aovp function of the ImPerm R package, version 2.1.0427 with 1 million permutations to determine significant differences between recoveries recorded under different extraction conditions. Post-hoc tests and the resulting significance groupings were calculated using the PredictMeans R package, version 1.0.8438. Statistical difference was assumed at p < 0.05. Different letters represent statistically different groups.

[0135] Figure 3 presents two graphs showing lipid accumulation under different growth conditions (standard; nutrient-limiting: no-nitrogen, and no-phosphorous; and light saturating conditions) for a period of 7 days. (A) Total lipid content (%) of the dry cell weight of Chlamydomonas reinhardtii, Scenedesmus obliquus, Nannochloropsis oceanica, and N. oculata. (B) Total triacylglyceride (TAG) content (mg / g) for C. reinhardtii, S. obliquus, N. oceanica and N. oculata. Statistical difference was assumed at p < 0.05. Different letters represent statistically different groups.

[0136] DETAILED DESCRIPTION

[0137] The present invention relates to compositions comprising triglycerides, such as microalgal triglycerides enriched in palmitate at the sn-2 position, for example nutritional compositions comprising an emulsion formed from a microalgal-derived lipid extract and one or more phospholipids, one or more sphingolipids, one or more gangliosides, and one or more cholesterols. The present invention further relates to methods of preparing and using such compositions.

[0138] The nutritional compositions find particular application in providing developmentally important nutrition to infant and child subjects, and in providing ongoing nutritional support to older subjects including adult and elderly subjects. Selected definitions

[0139] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7). These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[0140] Those skilled in the art will appreciate the meaning of various terms of degree used herein. For example, as used herein in the context of referring to an amount (e.g., "about 9%"), the term "about" represents an amount close to and including the stated amount that still performs a desired function or achieves a desired result, e.g. "about 9%" can include 9% and amounts close to 9% that still perform a desired function or achieve a desired result. For example, the term "about" can refer to an amount that is within less than 10% of, within less than 5% of, within less than 1% of, within less than 0.1% of, or within less than 0.01% of the stated amount. It is also intended that where the term "about" is used, for example with reference to a figure, concentration, amount, integer or value, the exact figure, concentration, amount, integer or value is also specifically contemplated.

[0141] The term "and / or" can mean "and" or "or".

[0142] Those skilled in the art will appreciate the meaning of various terms of degree used herein. For example, as used herein in the context of referring to an amount (e.g., "about 9%"), the term "about" represents an amount close to and including the stated amount that still performs a desired function or achieves a desired result, e.g., "about 9%" can include 9% and amounts close to 9% that still perform a desired function or achieve a desired result. For example, the term "about" can refer to an amount that is within less than 10% of, within less than 5% of, within less than 1% of, within less than 0.1% of, or within less than 0.01% of the stated amount. It is also intended that where the term "about" is used, for example with reference to a figure, concentration, amount, integer or value, the exact figure, concentration, amount, integer, or value is also specifically contemplated.

[0143] The term "beta-serum" means an aqueous dairy ingredient separated from dairy streams containing greater than 60% fat that have been through phase inversion from an oil-in-water to a water-in-oil emulsion. Cream is a useful and common starting material for the production of betaserum. For example, beta-serum is produced during the production of butter-oil (also known as anhydrous milk fat or AMF) from cream. A representative method of producing beta-serum is provided herein in the Examples. In certain examples, the beta-serum is dried, and in certain examples, dried beta-serum is provided as a powder.

[0144] The term "comprising" as used in this specification means "consisting at least in part of". When interpreting each statement in this specification that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises", and the terms "including", "include" and "includes" are to be interpreted in the same manner.

[0145] The term "consisting essentially of" when used in this specification refers to the features stated and allows for the presence of other features that do not materially alter the basic characteristics of the features specified.

[0146] The term "consisting of" as used herein means the specified materials or steps of the claimed invention, excluding any element, step, or ingredient not specified in the claim. The term "dietetic product" means a product specially processed or formulated to satisfy particular dietary requirements which exist because of a particular physical or physiological condition and / or specific diseases and disorders and which are presented as such.

[0147] An "effective amount" as used herein is the amount required to confer one or more therapeutic or prophylactic effects.

[0148] As used herein, the terms "to increase brain health or development" or "increasing brain health and development" are used interchangeably herein and generally refer to enhancing or improving brain function and advancing brain development. These terms include increasing or improving cognitive development or performance, increasing learning, memory or attention span, increasing the ability of a subject to cope with stress, increasing blood, nutrient or oxygen flow to the brain, increasing synaptogenesis, increasing the activity of the gut / brain axis or increasing neurogenesis.

[0149] The terms "increasing cognitive development", "improving cognitive development", "to increase cognitive development", and "to improve cognitive development" are used interchangeably herein and refer to increasing or improving any one or more of the rate, ability, interest, willingness, or openess to learn, remember and / or apply knowledge. In some examples, "cognitive development" refers to brain weight and / or brain composition.

[0150] The terms "increasing growth" or "to increase growth" are used interchangeably herein and refer to increasing healthy growth which may refer to increasing the absolute growth or rate of growth with reference to weight, length, or height, while not increasing adiposity or decreasing bone density.

[0151] As used herein, the terms "to maintain brain function or development" or "maintaining brain function or development" are used interchangeably and generally refer to preventing the decline of or maintaining brain function, maintaining normal brain development or preventing delay in brain development. These terms include preventing delayed brain development, maintaining the viability or function of neuronal cells, maintaining cognitive development, preventing delayed cognitive development, maintaining cognitive performance, preventing cognitive decline, maintaining or preventing decline of learning ability, memory or attention span, maintaining the ability of a subject to cope with stress, maintaining blood, nutrient or oxygen flow to the brain, maintaining synaptogenesis, maintaining the activity of the gut / brain axis, or maintaining neurogenesis.

[0152] The term "oral administration" includes oral, buccal, enteral and intra-gastric administration.

[0153] The term "pharmaceutically acceptable carrier" as used herein refers to a carrier including but not limited to an excipient, diluent, auxiliary or combination thereof that can be administered to a subject as a component of a composition or medicament as contemplated herein that does not reduce the activity of the composition and is not toxic when administered in typically-administered doses or quantities, such as those sufficient to deliver an effective amount of the active ingredient(s).

[0154] A "subject" is an animal, preferably a mammal, more preferably a mammalian companion animal or human. Preferred companion animals include cats, dogs and horses.

[0155] Nutritional compositions

[0156] The compositions contemplated herein are suitable for use in nutritional formulations including maternal formulas, infant formulas, follow-on formulas, growing-up formulas, paediatric formulas, human milk fortifiers, children's foods or drinks, maternal supplements, maternal nutritional formulations, fermented foods, UHT milks, UHT drinking yoghurts, acidified milk drinks, UHT powders, medical foods, sports nutritional formulations, or formulations for senior or aged populations. In various examples, the nutritional composition is a liquid (concentrate or ready-to-drink) or powdered maternal formula, infant formula, follow-on formula, growing-up formula, or dietetic product.

[0157] The term "maternal formula" as used in this specification means a composition for pregnant woman to take during pregnancy. The term "infant formula" as used in this specification means a composition for infants aged between 0 days and 6 months old. The term "follow-on formula" as used in this specification means a composition for infants aged 6 months to 1 year. The term "growing up formula" as used in this specification means a composition for infants and children aged 1 year upwards. Growing-up formula includes growing-up milk powders (GUMPs). The term "paediatric formula" as used in this specification means a composition for children aged from birth.

[0158] It will be appreciated by those skilled in the art that the age ranges for the different compositions: "infant formula", "follow-on formula, "growing-up formula" and "paediatric formula" can vary from child to child depending on the individual's development. These products may be in liquid form as concentrates or ready-to-drink liquids or provided as powder concentrates.

[0159] In various examples the nutritional formulation comprises at least about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4 or 5% w / w triglyceride, and suitable ranges may be selected from between any of these values, (for example, from about 0.001 to about 1.0, about 0.005 to about 5, about 0.005 to about 3, about 0.005 to about 1, about 0.005 to about 0.5, about 0.005 to about 0.05, about 0.01 to about 5, about 0.01 to about 3, about 0.01 to about 1, about 0.01 to about 0.5, about 0.01 to about 0.1, about 0.05 to about 5, about 0.05 to about 4, about 0.05 to about 1, about 0.05 to about 0.1, about 0.1 to about 5, about 0.1 to about 4, about 0.1 to about 1, about 0.1 to about 0.5, about 0.5 to about 5, about 0.5 to about 3, about 0.5 to about 1, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 2 to about 5, about 2 to about 4, about 3 to about 5% w / w triglyceride, such as the microalgal triglyceride enriched in palmitate at the sn-2 position as contemplated herein.

[0160] In various examples the nutritional formulation comprises at least about 0.005% triglyceride, such as the microalgal triglyceride enriched in palmitate at the sn-2 position as contemplated herein, on a weight / volume basis.

[0161] Formulas for infants and children

[0162] In various examples the infant formula comprises at least about 0.1% triglyceride, such as the microalgal triglyceride enriched in palmitate at the sn-2 position as contemplated herein, on a weight / weight solids basis.

[0163] In various examples the follow-on formula comprises at least about 0.15% triglyceride, such as the microalgal triglyceride enriched in palmitate at the sn-2 position as contemplated herein, on a weight / weight solids basis.

[0164] In various examples the growing-up formula comprises at least about 0.15% triglyceride, such as the microalgal triglyceride enriched in palmitate at the sn-2 position as contemplated herein, on a weight / weight solids basis.

[0165] In various examples the maternal formula comprises at least about 0.1% triglyceride, such as the microalgal triglyceride enriched in palmitate at the sn-2 position as contemplated herein, on a weight / weight solids basis.

[0166] As used herein, infant formula generally refers to a nutritional formula for infants aged 0-6 months, follow-on formula refers to a nutritional formula for infants aged 6 months to 1 year, and growing up formula refers to a nutritional formula for infants aged 12 months onwards. Maternal formulas are for woman trying to conceive, pregnant, or breastfeeding.

[0167] Nutritional formulations comprising the triglyceride compositions described herein may be incorporated into formulations known in the art. Suitable formulations and ingredients will be apparent to those skilled in the art.

[0168] In one example, the composition delivers humanised milk-based nutrition in relation to, for example, triglyceride. "Humanised" means that the extract or composition is compositionally or functionally similar to human breast milk in relation to triglyceride content and composition, such as the amount or ratio of triglycerides having palmitate at the sn-2 position.

[0169] One example of humanisation is in relation to palmitic acid and palmitoleic acid composition, such that the compositions contemplated herein are comparable to human milk or colostrum in relation to their palmitic acid and / or palmitoleic acid composition.

[0170] In one example, the optimal triacylglyceride composition, such as the ratio, position, or amount of palmitic acid and / or palmitoleic acid in said triacylglycerides is administered in compositions specifically tailored for the target subject, such as neonatal, infant or child subjects in particular age groups or developmental stages.

[0171] Microalgal lipids

[0172] Microalgae are able to synthesise triacylglycerides (TAGs), generally with species-specific composition. For example, two microalgal genera suitable for use in accordance with the present invention and this disclosure, Nannochloropsis spp., and Chlorella spp., have been reported to accumulate TAGs containing 5.1% myristic acid, 62.2% palmitic acid, 19.0% palmitoleic acid, 0.4% linoleic acid, 0.9% eicosapentanoic acid, and TAGs containing 25.1% palmitic acid, 23.1% oleic acid, 21.6% linoleic acid, and 8.9% o-linoleic acid (based on total lipid mass), respectively. Other microalgal species similarly differ in their production of TAGs.

[0173] Particularly contemplated microalgae from which one or more TAGs suitable for use as described herein include microalgae species and strains selected from the group comprising: Amphora spp.; Arthospira spp.; Audouinella eugena; Aurantiochytrium limacinurrr, Balbiania investiens; Chlamydomonas spp., such as Chlamydomonas reinhardtii; Chlorella spp., such as Chlorella protothecoides, Chlorella pyrenoidosa, Chlorella sorokiniana, Chlorella vulgaris-. CV-15 and CV-395, or Chlorella zofingiensis; Chlorococcum spp.; Crypthecodinim cohnii; Desmodesmus spp.; Dunaliella spp., such as Dunaliella tertiolecta; Haematococcus spp., such as Haematococcus pluvialis; Isochrysis spp., such as Isochrysis galbana; Myrmecia bisecta; Nannochloropsis spp., such as Nannochloropsis limnetica, Nannochloropsis occulata, Nannochloropsis oceanica, or Nannochloropsis salina; Navicula pelliculosa; Neochloris oleabundans; Nitzchia spp., such as Nitzchia laevis; Palmodictyon varium; Phaeodactylum spp., such as Phaeodactylum tricornutum; Porphyridium spp., such as Porphyridium cruentum; Pseudochantransia spp.; Scenedesmus spp., such as Scenedesmus obliquus;

[0174] Schizochytrium spp., such as Schizochytrium mangrovei or Schizochytrium limacinum; Skeletonema marinoi; Spirulina spp.; Synechococcus spp.; Thorea ramosissima; Trachydiscus minutus; and Trisochrysis spp..

[0175] In one example, the triglyceride comprises, consists essentially of, or consists of triglyceride from Nannochloropsis oceanica, such as Nannochloropsis oceanica CCMP531 or Nannochloropsis oceanica UTEX 2164. Particularly contemplated TAGs, such as those conveniently obtained from microalgae, for example as a lipid extract from a microalgal source such as described and exemplified herein in the Examples, include TAGs comprising, consisting essentially of, or consisting of one or more fatty acids selected from the group consisting of: palmitic acid, palmitoleic acid, myristic acid, oleic acid, eicosapentaenoic acid, linoleic acid, and arachidonic acid.

[0176] In various particularly contemplated examples, the one or more triacylglycerides present in the nutritional compositions described herein comprises, consists essentially of, or consists of one or more triacylglyceride species selected from the group consisting of: 016:0016:0014:0, 016:0016:0016:0, 016:0016:0016: 1, 016:0016:0016:2, 016:0016:0:018:0, 016:0016:0:018: 1, 016:0016:0:018:2, 016:0016:0:018:3, 016:0016:0:020:4, 016:0016:0:020:5, 016: 1016:0014:0, 016: 1016:0016:0, 016: 1016:0016: 1, 016: 1016:0016:2, 016: 1016:0:018:0, 016: 1016:0:018: 1, 016: 1016:0:018:2, 016: 1016:0:018:3, 016: 1016:0:020:4, 016: 1016:0:020:5, 016:2016:0014:0, 016:2016:0016:0, 016:2016:0016:1, 016:2016:0016:2, 016:2016:0:018:0, 016:2016:0:018: 1, 016:2016:0:018:2, 016:2016:0:018:3, 016:2016:0:020:4, 016:2016:0:020:5, 018:0016:0014:0, 018:0016:0016:0, 018:0016:0016:1, 018:0016:0016:2, 018:0016:0:018:0, 018:0016:0:018: 1, 018:0016:0:018:2, 018:0016:0:018:3, 018:0016:0:020:4, 018:0016:0:020:5, 018:1016:0014:0, 018: 1016:0016:0, 018: 1016:0016: 1, 018: 1016:0016:2, 018: 1016:0:018:0, 018: 1016:0:018: 1, 018: 1016:0:018:2, 018: 1016:0:018:3, 018: 1016:0:020:4, 018: 1016:0:020:5, 018:2016:0014:0, 018:2016:0016:0, 018:2016:0016: 1, 018:2016:0016:2, 018:2016:0:018:0, 018:2016:0:018: 1, 018:2016:0:018:2, 018:2016:0:018:3, 018:2016:0:020:4, 018:2016:0:020:5, 018:3016:0014:0, 018:3016:0016:0, 018:3016:0016: 1, 018:3016:0016:2, 018:3016:0:018:0, 018:3016:0:018: 1, 018:3016:0:018:2, 018:3016:0:018:3, 018:3016:0:020:4, and 018:3016:0:020:5.

[0177] In various particularly contemplated examples, the one or more triacylglycerides present in the nutritional compositions described herein comprises one or more triacylglyceride species selected from the group consisting of: 016: 1014:0014:0, 016: 1014:0016: 1, 016: 1016: 1016: 1, 016: 1016:0:020:5, 016: 1014:0016: 1, 018:2016: 1016: 1, 016: 1016:0016: 1, 018: 1016: 1016: 1, 016: 1016:0:020:4, 016: 1016:0016:0, 018: 1016:0016: 1, 018: 1016: 1018: 1, and 018: 1016:0018:1.

[0178] In certain contemplated examples, for example compositions for infant nutrition such as one or more of the infant formula contemplated herein, the one or more triacylglycerides present comprises, consists essentially of, or consists of a triacylglyceride species with palmitic acid (016:0) at sn-2 and oleic acid (018: 1) at sn-1 and sn-3, that is, 018: 1016:0018: 1. In certain examples, a 018: 1016:0018: 1 triacylglyceride species comprises from about 1% to about 30% of the triglycerides present.

[0179] In certain contemplated examples, for example compositions for infant nutrition such as one or more of the infant formula contemplated herein, the one or more triacylglycerides present comprises, consists essentially of, or consists of a triacylglyceride species with palmitic acid (016:0) at sn-2 and a polyunsaturated fatty acid, such as linoleic acid (018:2) or alpha-linolenic acid (018:3) at sn-1 or sn-3 or both sn-1 and sn-3.

[0180] For example, the one or more triacylglycerides present comprises, consists essentially of, or consists of a triacylglyceride species selected from the group consisting of 014:0016:0:018:2, 014:0016:0:018:3, 016:0016:0:018:2, 016:0016:0:018:3, 016: 1016:0:018:2, 016: 1016:0:018:3, 018:0016:0:018:2, 018:0016:0:018:3, 018: 1016:0:018:2, 018: 1016:0:018:3, 020:4016:0018:2, 020:4016:0018:3, 020:5016:0018:2, 020:5016:0018:3, 018:2016:0014:0, 018:2016:0016:0, C 18 : 2C16 : 0C 16 : 1, C18:2C16:0C16:2, C18:2C16:0:C18:0, C 18 : 2C16 : 0 : CIS : 1, C18:2C16:0:C18:2, C 18 : 2C16 : 0 : CIS : 3, C18:2C16:0:C20:4, C18:2C16:0:C20:5, C18:3C16:0C14:0, C18:3C16:0C16:0, C18:3C16:0C16:l, C18:3C16:0C16:2, C18:3C16:0:C18:0, C 18 : 3C16 : 0 : CIS : 1, C 18 : 3C16 : 0 : CIS : 2, C 18 : 3C16 : 0 : CIS : 3, C18:3C16:0:C20:4, and C 18 : 3C16 : 0 : C20 : 5.

[0181] In certain examples, from about 10% to about 20% of the triglycerides present are selected from triglyceride species with a polyunsaturated fatty acid, such as linoleic acid (C18:2) or alphalinolenic acid (C18:3) at sn-1 or sn-3 or both sn-1 and sn-3. Phospholipids

[0182] The compositions and emulsions described herein comprising one or more phospholipids. In particularly contemplated examples, the one or more phospholipids comprise one or more milk phospholipids.

[0183] Sources of phospholipids other than milk are contemplated. Such other sources include lecithins, which are mixtures of glycerophospholipids including phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and phosphatidic acid.

[0184] Gangliosides

[0185] Gangliosides are a group of sialic-acid-containing glycosphingolipids and may be found in animal tissues and fluids, such as blood, brain tissue and milk. Gangliosides may be extracted from milk or from other animal sources, such as beef, chicken, pork, and fish and marine species, such as sea urchin. Gangliosides may also be produced synthetically or semi-synthetically.

[0186] Gangliosides may be measured using known techniques, such as by measuring Lipid Bound Sialic Acid (LBSA) or individual species quantified by Thin Layer Chromatography (TLC), High Performance Liquid Chromatography with UV detection (HPLC-UV), or by liquid chromatography linked to mass spectrometry.

[0187] A number of techniques can be employed to extract gangliosides from a variety of materials according to methods well known to those skilled in the art. Particularly contemplated are gangliosides extracted from milk or other dairy sources, particularly any high fat milk fraction, for example: cream, butter, buttermilk, butter serum, beta-serum and whey protein concentrates. Advances in dairy processing have made possible ganglioside enrichment of dairy streams.

[0188] Sphingolipids

[0189] Sphingolipids are lipids comprising a sphingoid base, such as sphingosine, dihydrosphingosine, or phytosphingosine. Representative classes of sphingolipids include ceramides such as dihydroceramide, ceramide, phytoceramide, lactosylceramide, glucosylceramide, their phosphorylated counterparts, and inositol-containing ceramides, such as inositol phosphorylceramide, mannose inositol phosphorylceramide, and mannose diinositol phosphorylceramide; sphingomyelins; glycosphingolipids; cerebrosides; sulfatides; and gangliosides.

[0190] Sphingomyelins are particularly contemplated sphingolipids for use herein. Sphingomyelins are found in the cell membranes of animal cells and reportedly have important roles in cellular integrity as well as various cell signalling pathways.

[0191] Cholesterols

[0192] Cholesterols are sterols and are essential components of animal cell membranes. Cholesterols reportedly interact with sphingolipids within cell membranes, and in certain examples preparations useful herein will comprise cholesterol-sphingolipid complexes, which may in certain examples comprise other cell membrane components such as one or more phospholipids and / or one or more gangliosides.

[0193] Particularly contemplate cholesterols for use as contemplated herein include cholesterol, P- sitosterol, campesterol, stigmasterol, and brassicasterol.

[0194] In various examples, the one or more phospholipids, one or more sphingolipids, one or more gangliosides, and one or more cholesterols are obtained from mammalian milk. It should be appreciated that any fraction or milk derivative can be utilised in the methods and compositions contemplated herein provided it contains one or more of said one or more phospholipids, one or more sphingolipids, one or more gangliosides, and one or more cholesterols.

[0195] In some examples, the one or more phospholipids, one or more sphingolipids, one or more gangliosides, and / or one or more cholesterols is derived from any mammalian milk including but not limited to bovine, sheep, goat, pig, mouse, water buffalo, camel, yak, horse, donkey, llama or human milk fat, with bovine milk being a particularly contemplated source. In some examples the mammalian milk is processed to remove at least a portion of the water, protein, sugar, and / or fat prior to use as contemplated herein.

[0196] Milk fat globule membrane

[0197] Milk fat globule membrane (MFGM) is a glycosylated, protein-embedded, phospholipid fraction that surrounds triglycerides in milk. MFGM is a tri-phospholipid membrane layer, containing a variety of lipids and proteins (glycosylated and associated with the membrane layer) and carbohydrate moieties. Preparations of MFGM are available commercially, and include MFGM-10 and PL-20 (Aria Foods Ingredients, DN), PLCI and BSP2 (Tatua Limited, NZ), and BPC-50 (Fonterra, NZ).

[0198] In certain examples, the one or more phospholipids, one or more sphingolipids, one or more gangliosides, and / or one or more cholesterols is provided by or derived from MFGM, such as via a composition comprising, consisting essentially of, or consisting of MFGM.

[0199] In certain examples, the sole source of phospholipids present in the composition comprising, consisting essentially of, or consisting of an emulsion is a composition comprising, consisting essentially of, or consisting of milk fat globule membrane, for example intact milk fat globule membrane, such as bovine milk fat globule membrane. In one example, the sole source of phospholipids present in the composition comprising, consisting essentially of, or consisting of an emulsion is milk fat globule membrane, for example intact milk fat globule membrane.

[0200] In certain examples, the sole source of sphingolipids present in the composition comprising, consisting essentially of, or consisting of an emulsion is a composition comprising, consisting essentially of, or consisting of milk fat globule membrane, for example intact milk fat globule membrane, such as bovine milk fat globule membrane. In one example, the sole source of sphingolipids present in the composition comprising, consisting essentially of, or consisting of an emulsion is milk fat globule membrane, for example intact milk fat globule membrane.

[0201] In certain examples, the sole source of gangliosides present in the composition comprising, consisting essentially of, or consisting of an emulsion is a composition comprising, consisting essentially of, or consisting of milk fat globule membrane, for example intact milk fat globule membrane, such as bovine milk fat globule membrane. In one example, the sole source of gangliosides present in the composition comprising, consisting essentially of, or consisting of an emulsion is milk fat globule membrane, for example intact milk fat globule membrane. In certain examples, the sole source of cholesterols present in the composition comprising, consisting essentially of, or consisting of an emulsion is a composition comprising, consisting essentially of, or consisting of milk fat globule membrane, for example intact milk fat globule membrane, such as bovine milk fat globule membrane. In one example, the sole source of cholesterols present in the composition comprising, consisting essentially of, or consisting of an emulsion is milk fat globule membrane, for example intact milk fat globule membrane.

[0202] In certain examples, the sole source of any two or more of the group comprising the phospholipids, the sphingolipids, the gangliosides, and the cholesterols, present in the composition comprising, consisting essentially of, or consisting of an emulsion is a composition comprising, consisting essentially of, or consisting of milk fat globule membrane, for example intact milk fat globule membrane, such as bovine milk fat globule membrane. In one example, the sole source of any two or more of the group comprising the phospholipids, the sphingolipids, the gangliosides, and the cholesterols, present in the composition comprising, consisting essentially of, or consisting of an emulsion is milk fat globule membrane, for example intact milk fat globule membrane.

[0203] In certain examples, the sole source of all phospholipids, sphingolipids, gangliosides, and cholesterols present in the composition comprising, consisting essentially of, or consisting of an emulsion is a composition comprising, consisting essentially of, or consisting of milk fat globule membrane, for example intact milk fat globule membrane, such as bovine milk fat globule membrane. In one example, the sole source of all phospholipids, sphingolipids, gangliosides, and cholesterols present in the composition comprising, consisting essentially of, or consisting of an emulsion is milk fat globule membrane, for example intact milk fat globule membrane.

[0204] In certain examples, the composition comprising, consisting essentially of, or consisting of an emulsion is prepared using a composition comprising, consisting essentially of, or consisting of milk fat globule membrane, for example intact milk fat globule membrane, such as bovine milk fat globule membrane or intact bovine milk fat globule membrane that comprises at least about 800 mg cholesterol per 100 g milk fat globule membrane ( I . e. , milk fat globule membrane comprising at least about 800 mg / 100 g cholesterol). In one example, the composition comprising, consisting essentially of, or consisting of an emulsion is prepared using a composition comprising, consisting essentially of, or consisting of milk fat globule membrane, for example intact milk fat globule membrane, such as bovine milk fat globule membrane or intact bovine milk fat globule membrane that comprises at least about 900 mg / 100 g cholesterol. In one example, the composition comprising, consisting essentially of, or consisting of an emulsion is prepared using a composition comprising, consisting essentially of, or consisting of milk fat globule membrane, for example intact milk fat globule membrane, such as bovine milk fat globule membrane or intact bovine milk fat globule membrane that comprises at least about 1000 mg / 100 g cholesterol. In one example, the composition comprising, consisting essentially of, or consisting of an emulsion is prepared using a composition comprising, consisting essentially of, or consisting of milk fat globule membrane, for example intact milk fat globule membrane, such as bovine milk fat globule membrane or intact bovine milk fat globule membrane that comprises from about 800 mg / 100 g cholesterol to about 1200 mg / 100 g cholesterol.

[0205] In certain examples, the composition comprising, consisting essentially of, or consisting of an emulsion is prepared using a composition comprising, consisting essentially of, or consisting of milk fat globule membrane, for example intact milk fat globule membrane, such as bovine milk fat globule membrane or intact bovine milk fat globule membrane that comprises at least about 20g fat per 100 g milk fat globule membrane (i.e., milk fat globule membrane comprising at least about 20 g / 100 g fat). In one example, the composition comprising, consisting essentially of, or consisting of an emulsion is prepared using a composition comprising, consisting essentially of, or consisting of milk fat globule membrane, for example intact milk fat globule membrane, such as bovine milk fat globule membrane or intact bovine milk fat globule membrane that comprises at least about 25 g / 100 g fat. In one example, the composition comprising, consisting essentially of, or consisting of an emulsion is prepared using a composition comprising, consisting essentially of, or consisting of milk fat globule membrane, for example intact milk fat globule membrane, such as bovine milk fat globule membrane or intact bovine milk fat globule membrane that comprises at least about 30 g / 100 g fat. In one example, the composition comprising, consisting essentially of, or consisting of an emulsion is prepared using a composition comprising, consisting essentially of, or consisting of milk fat globule membrane, for example intact milk fat globule membrane, such as bovine milk fat globule membrane or intact bovine milk fat globule membrane that comprises from about 20 g / 100 g fat to about 35 g / 100 g fat.

[0206] By way of example, the source of the one or more phospholipids, one or more sphingolipids, one or more gangliosides, and / or one or more cholesterols may be generated from established milk processing, such as anhydrous milk manufacture that produce a permeate, such as a MFGM preparation, or such as beta-serum.

[0207] It will be appreciated by those skilled in the art that, as exemplified herein in the Examples, beta-serum comprises one or more phospholipids, one or more sphingolipids, one or more gangliosides, and one or more cholesterols, and is particularly contemplated as an ingredient for use in preparing the nutritional emulsions disclosed herein.

[0208] In some examples the dairy composition comprising one or more phospholipid (such as betaserum) comprises at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1 5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 8, 8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 99.5% phospholipid by weight, and useful ranges may be selected between any of these values (for example, about 5 to about 95%, about 10 to about 95%, about 15 to about 95%, about 20 to about 95%, about 25 to about 95%, about 30 to about 95%, about 35 to about 95%, about 40 to about 95%, about 45 to about 95%, about 50 to about 95%, about 5 to about 99%, about 10 to about 99%, about 15 to about 99%, about 20 to about 99%, about 25 to about 99%, about 30 to about 99%, about 35 to about 99%, about 40 to about 99%, about 45 to about 99%, about 50 to about 99%, about 5 to about 70%, about 10 to about 70%, about 15 to about 70%, about 20 to about 70%, about 25 to about 70%, about 30 to about 70%, about 35 to about 70%, about 40 to about 70%, about 45 to about 70%, and about 50 to about 70% phospholipid by weight).

[0209] In some examples the dairy composition comprising one or more sphingolipids (such as betaserum) comprises at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1 5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 8, 8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 99.5% sphingolipids by weight, and useful ranges may be selected between any of these values (for example, about 5 to about 95%, about 10 to about 95%, about 15 to about 95%, about 20 to about 95%, about 25 to about 95%, about 30 to about 95%, about 35 to about 95%, about 40 to about 95%, about 45 to about 95%, about 50 to about 95%, about 5 to about 99%, about 10 to about 99%, about 15 to about 99%, about 20 to about 99%, about 25 to about 99%, about 30 to about 99%, about 35 to about 99%, about 40 to about 99%, about 45 to about 99%, about 50 to about 99%, about 5 to about 70%, about 10 to about 70%, about 15 to about 70%, about 20 to about 70%, about 25 to about 70%, about 30 to about 70%, about 35 to about 70%, about 40 to about 70%, about 45 to about 70%, and about 50 to about 70% sphingolipids by weight).

[0210] In some examples the dairy composition comprising one or more cholesterols (such as betaserum) comprises at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1 5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 8, 8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 99.5% cholesterols by weight, and useful ranges may be selected between any of these values (for example, about 5 to about 95%, about 10 to about 95%, about 15 to about 95%, about 20 to about 95%, about 25 to about 95%, about 30 to about 95%, about 35 to about 95%, about 40 to about 95%, about 45 to about 95%, about 50 to about 95%, about 5 to about 99%, about 10 to about 99%, about 15 to about 99%, about 20 to about 99%, about 25 to about 99%, about 30 to about 99%, about 35 to about 99%, about 40 to about 99%, about 45 to about 99%, about 50 to about 99%, about 5 to about 70%, about 10 to about 70%, about 15 to about 70%, about 20 to about 70%, about 25 to about 70%, about 30 to about 70%, about 35 to about 70%, about 40 to about 70%, about 45 to about 70%, and about 50 to about 70% cholesterols by weight).

[0211] In some examples the dairy composition comprising one or more gangliosides (such as betaserum) comprises at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1 5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 8, 8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 99.5% gangliosides by weight, and useful ranges may be selected between any of these values (for example, about 5 to about 95%, about 10 to about 95%, about 15 to about 95%, about 20 to about 95%, about 25 to about 95%, about 30 to about 95%, about 35 to about 95%, about 40 to about 95%, about 45 to about 95%, about 50 to about 95%, about 5 to about 99%, about 10 to about 99%, about 15 to about 99%, about 20 to about 99%, about 25 to about 99%, about 30 to about 99%, about 35 to about 99%, about 40 to about 99%, about 45 to about 99%, about 50 to about 99%, about 5 to about 70%, about 10 to about 70%, about 15 to about 70%, about 20 to about 70%, about 25 to about 70%, about 30 to about 70%, about 35 to about 70%, about 40 to about 70%, about 45 to about 70%, and about 50 to about 70% gangliosides by weight).

[0212] In some examples the composition comprising one or more gangliosides (such as beta-serum) comprises at least about 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% by weight of one or more phospholipids selected independently from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid, and useful ranges may be selected between any of these values (for example, about 0.1 to about 30%, about 0.5 to about 30%, about 1 to about 30%, about 2 to about 30%, about 3 to about 30%, about 4 to about 30%, about 5 to about 30%, about 10 to about 30%, about 15 to about 30%, about 20 to about 30%, about 0.1 to about 5%, about 0.5 to about 5%, about 1 to about 5%, about 2 to about 5%, about 3 to about 5%, about 0.1 to about 10%, about 0.5 to about 10%, about 1 to about 10%, about 2 to about 10%, about 3 to about 10%, about 4 to about 10%, about 5 to about 10%, about 6 to about 10%, about 0.1 to about 20%, about 0.5 to about 20%, about 1 to about 20%, about 2 to about 20%, about 3 to about 20%, about 4 to about 20%, about 5 to about 20%, about 10 to about 20%, about 15 to about 20% by weight of one or more phospholipids selected independently from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid). In one example the composition comprising one or more gangliosides comprises monosialodihexosylganglioside (GM3) or d isialogang lioside (GD3) or a combination thereof. In one example, the composition comprising one or more gangliosides comprises both GM3 and GD3.

[0213] In various examples, GM3 comprises from about 50% to about 100% of the gangliosides present, such as from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 95% to about 100%, from about 50% to about 99%, such as from about 60% to about 99%, from about 70% to about 99%, from about 80% to about 99%, from about 90% to about 99%, from about 95 to about 99%, from about 50% to about 95% of the gangliosides present, such as from about 60% to about 95%, from about 70% to about 95%, from about 80% to about 95%, or from about 90% to about 95% of the gangliosides present.

[0214] In various examples, GD3 comprises from about 1% to about 20% of the gangliosides present, such as from about 1% to about 15%, from about 1% to about 10%, from about 5% to about 20%, from about 5% to about 15%, or from about 5% to about 10% of the gangliosides present.

[0215] In some examples the composition comprising one or more gangliosides (such as beta-serum) comprises at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% by weight of one or more gangliosides selected independently from GD3 and GM3, and useful ranges may be selected between any of these values (for example, about 0.1 to about 30%, about 0.5 to about 30%, about 1 to about 30%, about 2 to about 30%, about 3 to about 30%, about 4 to about 30%, about 5 to about 30%, about 10 to about 30%, about 15 to about 30%, about 20 to about 30%, about 0.1 to about 5%, about 0.5 to about 5%, about 1 to about 5%, about 2 to about 5%, about 3 to about 5%, about 0.1 to about 10%, about 0.5 to about 10%, about 1 to about 10%, about 2 to about 10%, about 3 to about 10%, about 4 to about 10%, about 5 to about 10%, about 6 to about 10%, about 0.1 to about 20%, about 0.5 to about 20%, about 1 to about 20%, about 2 to about 20%, about 3 to about 20%, about 4 to about 20%, about 5 to about 20%, about 10 to about 20%, about 15 to about 20% by weight of one or more gangliosides selected independently from GM3 and GD3.

[0216] In one example, the dairy composition (such as beta-serum) comprises any one or more of about 10% to about 99% by weight total lipid, about 1% to about 50% by weight phospholipid, about 0.1% to about 10% by weight phosphatidylcholine, about 0.1% to about 10% by weight phosphatidylinositol, about 0.1% to about 10% by weight phosphatidylserine, about 0.1% to about 30% by weight phosphatidylethanolamine, about 0.1% to about 25% by weight sphingolipid, and about 0.1 to about 10% by weight ganglioside, and about 0.1% to about 10% cholesterol.

[0217] In alternative examples, the dairy composition (such as beta-serum) comprises about 20% to about 40 % by weight total lipid, about 1% to about 25% by weight phospholipid, about 1% to about 25% by weight sphingolipids, about 1% to about 25% by weight gangliosides, and about 1% to about 25% cholesterols.

[0218] In further examples, the dairy composition (such as beta-serum) comprises about 70% to about 99 % by weight total lipid, about 25% to about 80% by weight phospholipid, about 0.1% to about 10% by weight sphingolipid, about 0.1 to about 10% by weight ganglioside, and about 0.1% to about 10% cholesterol.

[0219] In another example, the dairy composition (such as beta-serum) comprises about 5% to about 15% by weight phospholipid, for example about 10% by weight phospholipid, about 25% to about 40% by weight protein, such as about 30% to about 35% by weight protein, and about 30% to about 50% by weight lactose, such as about 40% to about 45% by weight lactose.

[0220] In another example, the dairy composition (such as beta-serum) comprises about 15% to about 25% by weight phospholipid, for example about 20% by weight phospholipid, about 25% to about 45% by weight protein, such as about 30% to about 40% by weight protein, and about 25% to about 40% by weight lactose, such as about 30% to about 35% by weight lactose.

[0221] In another example, the dairy composition (such as beta-serum) comprises about 45% to about 55% by weight phospholipid, for example about 50% by weight phospholipid, about 5% to about 20% by weight protein, such as about 10% to about 15% by weight protein, and about 20% to about 30% by weight lactose, such as about 20% to about 25% by weight lactose.

[0222] In another example, the dairy composition (such as beta-serum) comprises about 25% to about 40% by weight phospholipid, for example about 30% to about 35% by weight phospholipid, about 20% to about 35% by weight protein, such as about 25% to about 30% by weight protein, and about 25% to about 40% by weight lactose, such as about 30% to about 35% by weight lactose.

[0223] In another example, the dairy composition (such as beta-serum) comprises about 45% to about 55% by weight phospholipid, for example about 50% by weight phospholipid, about 15% to about 30% by weight protein, such as about 20% to about 25% by weight protein, and about 10% to about 25% by weight lactose, such as about 15% to about 20% by weight lactose.

[0224] In another example, the dairy composition (such as beta-serum) comprises about 1% to about 5% by weight phospholipid, for example about 1% to about 3% by weight phospholipid, about 25% to about 45% by weight protein, such as about 30% to about 40% by weight protein, and about 40% to about 55% by weight lactose, such as about 45% to about 50% by weight lactose.

[0225] Emulsions

[0226] Those skilled in the art will be aware of a variety of methods of making emulsions such as those contemplated herein. A representative method reliant on homogenisation and sonication is exemplified herein in the Examples. Equipment suitable for use in the preparation of emulsions as contemplated herein, such as shakers, stirrers, homogenisers, and sonicators or ultrasonicators, is readily available.

[0227] In certain examples, emulsions contemplated herein have a particle size of from about 200 nm to about 10 pM. The inventors believe, without wishing to be bound by any theory, that emulsions comprising particles of this size provide desired bioavailability and / or stability.

[0228] In certain examples, emulsions contemplated herein have a polydispersity index (PDI) of below about 0.3. The inventors believe, without wishing to be bound by any theory, that this degree of uniformity in particle size is desirable in emulsions contemplated herein.

[0229] In certain examples, emulsions contemplated herein have a high zeta potential, for example, a zeta potential of > ±10-30 mV. The inventors believe, without wishing to be bound by any theory, that this degree of stability confers on emulsions contemplated herein certain benefits, including no or little phase separation or coalescence, and resistance to oxidation.

[0230] Certain examples of the emulsions contemplated herein have desirable interfacial properties, such as the ability to form strong interfacial films due to the presence of proteins and phospholipids.

[0231] In various examples, emulsions contemplated herein have a viscosity suitable to provide desired stability, for example, stability as contemplated above, and sufficient flowability for use in liquid formulations, such as in infant formula. In one example, the emulsions prepared herein are prepared using MFGM or a composition comprising, consisting essentially of, or consisting of MFGM, as the sole source of the one or more phospholipids, and / or the sole source of the one or more sphingolipids, and / or the sole source of the one or more gangliosides, and / or the sole source of the one or more cholesterols.

[0232] In one example, the emulsions prepared herein are prepared using beta-serum as the sole source of the one or more phospholipids, and / or the sole source of the one or more sphingolipids, and / or the sole source of the one or more gangliosides, and / or the sole source of the one or more cholesterols.

[0233] Other ingredients

[0234] In various examples, the compositions contemplated herein comprise one or more other ingredients, such as one or more other ingredients suitable for use in the preparation of nutritional compositions including formulas.

[0235] Representative examples of ingredients suitable for use in the compositions contemplated herein include one or more sugars, protein such as added protein, non-protein nitrogen, probiotics, prebiotics, vitamins, minerals, antioxidants, stabilizers, and emulsifiers.

[0236] In various examples, the nutritional composition comprises one or more sugars. Sugars include monosaccharides (including glucose and galactose), disaccharides (for example sucrose or lactose), and other oligosaccharides including neutral oligosaccharides and acidic oligosaccharides.

[0237] In various examples the extract or composition comprises less than about 85, 84, 83, 82, 81, 80, 79, 78, 77, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, or less than about 20% by weight sugars, and suitable ranges may be selected from between any of these values, (for example, from about 20 to about 85, about 20 to about 80, about 20 to about 75, about 30 to about 85, about 30 to about 80, about 40 to about 85, about 40 to about 80, about 40 to about 75, about 50 to about 85, about 50 to about 80, about 50 to about 75, about 50 to about 70, about 55 to about 85, about 55 to about 80, about 55 to about 75, about 55 to about 70, about 60 to about 85, about 60 to about 80, about 60 to about 75, about 60 to about 70, about 65 to about 85, about 65 to about 80, about 65 to about 75, about 65 to about 70, about 70 to about 85, about 70 to about 80, about 70 to about 75%, about 20 to about 65, about 20 to about 60, about 20 to about 55, about 20 to about 50, about 20 to about 45, about 20 to about 40, about 20 to about 35, about 20 to about 30, or from about 20 to about 25% by weight sugars).

[0238] In some examples the composition comprises about 1% to about 60% by weight lactose, about 1% to about 15 % by weight lactose or about 50% to about 65% by weight lactose.

[0239] In some examples the composition contains one or more neutral oligosaccharides. Neutral oligosaccharides are oligosaccharides that have no charge. Neutral oligosaccharides present in the compositions of the invention include galactooligosaccharides, fructooligosaccharides, N- acetyllactosamine, N-acetylgalactosaminylglucose, N-acetylgalactosaminyllactose, isoglobotriose, 3'- galactosyllactose, 4'-galactosyllactose, 6'-galactosyllactose, novo-lacto-N-pentaose-I, Lacto-N- neotetraose, and Lacto-N-neohexaose.

[0240] In various examples the extract or composition comprises at least about 0.01, 0.05, 0.1, 0.2, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25 or 30% by weight neutral oligosaccharides, and suitable ranges may be selected from between any of these values, (for example, from about 0.01 to about 30, about 0.01 to about 20, about 0.01 to about 15, about 0.01 to about 10, about 0.01 to about 6, 0.01 to about 2, about 0.01 to about 0.5, about 0.05 to about 30, about 0.05 to about 25, about 0.05 to about 20, about 0.05 to about 15, about 0.05 to about 10, about 0.1 to about 30, about 0.1 to about 25, about 0.1 to about 20, about 0.1 to about 15, about 0.1 to about 10, about 0.2 to about 30, about 0.2 to about 25, about 0.2 to about 20, about 0.2 to about 15, about 0.2 to about 10, about 0.25 to about 30, about 0.25 to about 25, about 0.25 to about 20, about 0.25 to about 15, about 0.25 to about 10, about 0.5 to about 30, about 0.5 to about 25, about 0.5 to about 20, about 0.5 to about 15, about 0.5 to about 10, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 2 to about 30, about 2 to about 25, about 2 to about 20, about 2 to about 15, about 2 to about 10, about 5 to about 30, about 5 to about 25, about 5 to about 20, about 5 to about 15, about 10 to about 30, about 10 to about 25, or about 10 to about 20% by weight neutral oligosaccharides).

[0241] Protein

[0242] The nutritional compositions contemplated herein will typically comprise protein so as to provide useful nutrition to consumers and / or subjects to whom the compositions are administered.

[0243] In principle, protein from any source may be suitable for use in the preparation of the nutritional compositions contemplated herein. However, dairy proteins, such as those provided in milk protein concentrates, milk protein isolates, whey protein concentrates, whey protein isolates, caseinates, and the like, are particularly contemplated.

[0244] In various examples the extract or composition comprises about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less than about 1% by weight protein, and suitable ranges may be selected from between any of these values, (for example, from about 1 to about 20, about 1 to about 15, about 1 to about 10, about 1 to about 8, about 1 to about 6, about 1 to about 5, about 2 to about 20, about 2 to about 16, about 2 to about 11, about 2 to about 9, about 2 to about 8, about 2 to about 7, about 3 to about 20, about 3 to about 15, about 3 to about 13, about 3 to about 9, about 3 to about 8, about 3 to about 6, about 4 to about 20, about 4 to about 15, about 4 to about 10, about 4 to about 9, about 4 to about 8, about 4 to about 7, about 5 to about 2,0 5 to about 16, about 5 to about 10, about 5 to about 8, about 5 to about 7, about 6 to about 20, about 6 to about 17, about 6 to about 11, about 6 to about 10, about 6 to about 9, about 7 to about 20, about 7 to about 15, about 7 to about 10, about 8 to about 20, about 8 to about 17, about 8 to about 12, about 9 to about 20, about 9 to about 15, about 10 to about 20, about 10 to about 16, about 11 to about 20 11 to about 16, about 12 to about 20, about 12 to about 17, about 13 to about 20, about 13 to about 16, about 14 to about 20, about 14 to about 18 or about 15 to about 20% by weight protein).

[0245] In various examples, the nutritional compositions contemplated herein comprise one or more probiotics, such as one or more strains of lactobacteria.

[0246] Particularly contemplated vitamins include, but are not limited to, vitamin A, Thiamin (Bl), Riboflavin (B2), Niacin (B3), Pantothenic Acid (B5), vitamin B6, Biotin (B7), Folate (B9), Cobalamin (B12), vitamin C, vitamin D3, vitamin E, and vitamin K.

[0247] Particularly contemplated minerals include, but are not limited to Calcium, Copper, Iodine, Iron, Magnesium, Manganese, Phosphorus, Potassium, Selenium, Sodium, and Zinc.

[0248] In one example a composition, formula, or medicament useful herein further comprises a pharmaceutically acceptable carrier. In another example the composition, formula, or medicament is or is formulated as a food, drink, food additive, drink additive, dietary supplement, nutritional product, medical food, dietetic product, enteral or parenteral feeding product, meal replacement, nutraceutical, medicament or pharmaceutical. In one example the composition, formula, or medicament is in the form of a tablet, a caplet, a pill, a hard or soft capsule or a lozenge. In one example the composition, formula, or medicament is in the form of a cachet, a dispensable powder, granules, a suspension, an elixir, a liquid, or any other form that can be added to food or drink, including for example water, milk or fruit juice. In one example the composition, formula, or medicament further comprises one or more constituents (such as antioxidants) which prevent or reduce degradation of the composition, formula, or medicament during storage or after administration. These formulations may include any edible consumer product which is able to carry lipid. Examples of suitable edible consumer products include aqueous products, baked goods, confectionary products including chocolate, gels, ice creams, reconstituted fruit products, snack bars, food bars, muesli bars, spreads, sauces, dips, dairy products including yoghurts and cheeses, drinks including dairy and non-dairy based drinks, milk, milk powders, sports supplements including dairy and non-dairy based sports supplements, fruit juice, food additives such as protein sprinkles and dietary supplement products including daily supplement tablets. Suitable nutraceutical compositions useful herein may be provided in similar forms.

[0249] Health benefits

[0250] Without wishing to be bound by any theory, the inventors believe the compositions contemplated herein will be suitable to provide one or more health benefits selected from the group consisting of improved fat absorption, digestion, and / or metabolism; improved gut health, including for example improved gut-microbiome interactions; improved calcium absorption, digestion, and / or metabolism; improved immune response and / or immune modulation; improved cognition, improved cognitive development, and improved brain health; and improved cardiovascular health.

[0251] Again without wishing to be bound by any theory, the inventors also believe the compositions contemplated herein will be suitable to provide one or more health benefits reported to be associated with consuming oil enriched in sn-2 palmitate, including reduced loss of dietary calcium, lower incidence of hard stools or constipation, and / or improved gut bacterial composition.

[0252] Similarly, and again without wishing to be bound by any theory, the inventors believe the compositions contemplated herein will be suitable to provide one or more health benefits reported to be associated with consuming oil enriched in palmitoleic acid, particularly for adult or elderly subjects, including increased insulin sensitivity and / or reduced risk of diabetes, improved cholesterol metabolism, improved skin integrity, one or more anti-thrombotic effects, one or more antiinflammatory effects, reduced risk of stroke, improved cognitive development and / or support, and / or improved cognitive function and / or reduced cognitive decline or impairment.

[0253] Certain particularly contemplated health benefits to be achieved by administering the compositions contemplated herein include:

[0254] Enhanced Fat Digestion and Absorption

[0255] Without wishing to be bound by any theory, the inventors believe that the sn-2 position of triglycerides is critical for digestion, as enzymes in the gut, such as pancreatic lipase, preferentially release fatty acids from the sn-1 and sn-3 positions. Palmitic acid in the sn-2 position is reportedly absorbed directly as a monoglyceride, which mimics the natural structure of human milk fat. The inventors expect that the compositions and emulsions contemplated herein, enriched in TAGs with palmitic acid at sn-2, will provide improved fat absorption and improved bioavailability of fatty acids, particularly in infants and individuals with digestive challenges.

[0256] Gut Health and Microbiome Interaction

[0257] Without wishing to be bound by any theory, the inventors expect that the compositions and emulsions contemplated herein, comprising phospholipids, including sphingomyelin, may provide prebiotic-like effects, including promoting a healthy gut microbiome, contributing to the formation of protective barriers in the intestinal lining, reducing gut permeability, and / or improving overall gut health.

[0258] Immune Modulation

[0259] Without wishing to be bound by any theory, the inventors expect that the compositions and emulsions contemplated herein comprising sphingomyelin may provide improved immune regulation. In particular, the inventors expect compositions and emulsions as contemplated herein may help modulate inflammation and immune responses, particularly in infants, by supporting the development of the immune system and the gut barrier.

[0260] Cognitive Development and Brain Health

[0261] Without wishing to be bound by any theory, the inventors expect that the compositions and emulsions contemplated herein, enriched in TAGs with palmitic acid at sn-2, and particularly those comprising emulsions in which such TAGs are encapsulated with one or more buttermilk phospholipids, may support cognitive development in infants and / or provide neuroprotection in adults.

[0262] Calcium Absorption and Bone Health

[0263] Without wishing to be bound by any theory, the inventors believe that the provision of TAGs having palmitic acid positioned at sn-2 may reduce the formation of calcium soaps in the gut which occur when free palmitic acid binds to calcium. This reduction in calcium soap formation is expected to increase calcium bioavailability, promoting better calcium absorption and contributing to bone health, particularly in infants and growing children. In certain examples, the compositions contemplated herein are suitable for use in the treatment or prevention of osteoporosis.

[0264] Functional Lipids for Cardiovascular Health

[0265] Without wishing to be bound by any theory, the inventors believe that phospholipids present in the compositions and emulsions contemplated herein may positively affect lipid metabolism, reducing cholesterol absorption and improving lipid profiles. This is in turn expected to have potential benefits for cardiovascular health in older children and adults.

[0266] In one example the composition as contemplated herein can be used to treat or improve diseases that include cognitive decline as a symptom. For example, the use of the composition can reduce the rate of cognitive decline, maintain cognition in a patient, or improve cognitive ability and / or function in a patient to whom the composition is administered. Examples of diseases that include cognitive decline as a symptom include Alzheimer's disease, Parkinson's disease, Huntington's disease or dementia.

[0267] Therefore, in one example the invention relates to the use of a composition as described herein in the treatment or prevention of cognitive decline, including cognitive decline associated with any one or more or age-related cognitive decline, dementia, Alzheimer's disease, vascular disease, frontotemporal lobar degeneration (FTLD), dementia with Lewy bodies, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, cognitive impairment associated with schizophrenia, chemotherapy-induced neuropathy, Down's syndrome, Korsakoff's disease, cerebral palsy, epilepsy, neuronal ischemia, neuronal reperfusion injury, neuronal trauma, neuronal haemorrhage, neuronal infection, stroke, neuronal exposure to a toxic substance, age-related mental disorders, anxiety disorders, age-related depression, dementia associated with microvascular disorders (such as diabetes, hypotension, stroke induced vascular dementia, and obesity), dementia associated with a disorder of the immune system, dementia associated with a central nervous system (CNS) disorder, dementia associated with hypotension, dementia associated with obesity or vascular dementia, infantile onset severe development delay, or distal myopathy with rimmed vacuoles (DMRV) comprising administering an effective amount of a triglyceride-containing composition as contemplated herein.

[0268] Accordingly, without wishing to be bound by any theory, the applicant believes that the compositions and methods contemplated herein provide useful nutritional and functional benefit(s), including for example to human subjects.

[0269] The compositions, emulsions, and methods contemplated herein may provide one or more further benefits or embody one or more other desirable characteristics, such as improved stability. Without wishing to be bound by any theory, the inventors expect that the compositions and emulsions contemplated herein, enriched in TAGs with palmitic acid at sn-2, and particularly those comprising emulsions in which such TAGs are encapsulated with one or more buttermilk phospholipids, may improve emulsion stability, reducing or preventing coalescence of fat droplets which in turn leads to more stable and homogenous emulsions. This is particularly beneficial in nutritional compositions as contemplated herein, such as infant formula or other functional foods, by enhancing shelf life and preventing separation.

[0270] The invention is further described with reference to the following examples. It will be appreciated that the invention as claimed is not intended to be limited in any way by these examples.

[0271] EXAMPLES

[0272] Example 1:

[0273] This example presents methods for the preparation of infant formula using microalgae oil from N. oceanica UTEX 2164 as a source of palmitoleic fatty acids and sn-2 palmitate. Here, the purified microalgae oil has been emulsified by bovine phospholipids for use in the infant formula.

[0274] Methods

[0275] Microalqae specie and cultivation conditions

[0276] C. reinhardtii, S. obliquus, N. oceanica and N. oculata were selected as commercially relevant microalgae. C. reinhardtii 6145 was purchased from the Chlamydomonas Resource Center (St. Paul, MN, USA), S. obliquus UTEX 3155 and N. oceanica UTEX 2164 were purchased from the UTEX culture collection of algae (The University of Texas, Austin, USA) and N. oculata CCMP531 was purchased from the Bigelow National Center for Marine Algae and Microbiota (East Boothbay, ME, USA). Cultures were grown under the following conditions:

[0277] Standard conditions

[0278] C. reinhardtii was grown on tris-acetate phosphate (Plouviez et al., 2017a), S. obliquus in BG11 media (Plouviez et al., 2017b), and both N. oceanica and N. oculata in f / 2 salt media (Andersen, 2005). All cultures were grown in 2 L Duran bottles at 22°C under constant illumination at 50 pmol photons rrr2s-1. For all species, a dense culture grown under standard conditions was centrifuged and inoculated at a density to allow for optimal growth (30% v / v for S. obliquus and C. reinhardtii or 40% for N. oceanica and N. oculata). Air enriched to 2% CO2 was filtered (0.22 m filters) and continually sparged into the cultures at 1 L / min. The cultures were harvested after 7 days of incubation. Unless otherwise stated, the temperature, bubbling and light intensities were identical in all experiments.

[0279] Nitrogen-free conditions

[0280] All four microalgae were first grown under standard conditions for 7 days as described above. The cultures were then harvested by centrifugation, resuspended in their respective nitrogen-free media, and photosynthetically grown under nitrogen-depletion conditions for a further seven days.

[0281] Phosphorus-free conditions

[0282] All four microalgae were first grown under standard conditions for 7 days as described above. The cultures were then harvested by centrifugation, resuspended in their respective phosphorus-free media, and photosynthetically grown under nitrogen-depletion conditions for another 7 days. As many microalgae can intracellularly store significant amounts of phosphorus (Cliff et al., 2023), the same step was repeated and the cultures were grown for another 7 days in phosphorus-free media.

[0283] Light saturated conditions

[0284] Only C. reinhardtii and S. obliquus were tested under light-saturating conditions. Cultures were grown under standard conditions in triplicate for six days as described above. On day 6, two cultures were illuminated by ~200 mol photons m-2 s-1 for 24 hours, while the control cultures were kept under standard illumination conditions. This control was employed to distinguish the impact of light intensity.

[0285] Harvesting was done in batches by centrifugation (10,000xg for 3.5 min). The harvested biomass (see Figure 2) was frozen at -80°C before freeze-drying in an FD-10F-TP2 freeze dryer (Lab- Kits, Hong Kong). All glassware and media were autoclaved prior to use. Sample preparation and Lipid Extraction

[0286] A Dionex ASE 350 provided with dionium extraction cell purchased from Thermo Fisher Scientific was used to extract lipids from Nannochloropsis ocenica grown under nitrogen limitation conditions. Based on preliminary analysis, Methanol:Chloroform (2: 1, v / v) was chosen as the extraction solvent for all the samples. Extraction temperature was set at 100 °C and the static time (~5 min) and static cycle (~4 cycles) were selected for each extraction batch. Bulk extractions were then carried out.

[0287] Freeze-dried biomass was used for each extraction. Approximately, 1 g of biomass was ground using a mortar and pestle to form a uniform mixture with an ASE adsorbing powder comprising of diatomaceous earth and moisture adsorbing powder at a ratio of 10: 1. A 22 mL stainless-steel ASE extraction cells was filled with the ground biomass adsorbing powder mixture and rest of the headspace was filled with white sand. ASE specific 0.2 pm cellulose fibre filter (Dionex, Sunnyvale, CA, USA) was used to prevent blockage of the frit in the bottom cap. After loading the extraction cells on the cell holder, ASE extraction program was set as follows: (i) cells were first rinsed with the extraction solvent; (ii) the extraction cells containing biomass samples were loaded into the ASE; (ill) the cells were filled with the solvent and pressurized to 10.3 MPa; (iv) the cells were automatically pre-heated for 5 min; (v) extractions were carried out for 40 min with a flush volume ~100% of cell volume and purge time ~60 sec; (vi) the cells were again rinsed using the extraction solvent. The extracted lipid samples were collected in 30 mL ASE glass tubes for analysis.

[0288] A REACTI-THERM III heating module TS-18824 was used to dry the generated lipid extracts at 36-37 °C under nitrogen evaporation to prevent lipid oxidation. The total lipid content was determined g ravi metrically. The dried extracts were then resuspended in chloroform and divided into separate aliquots for further analysis.

[0289] Solid Phase extraction (SPE) - Microalaal TAG separation

[0290] Microalgal TAGs were separated from the crude lipid extracts by SPE extraction method developed by Teng et al., 2019. Two step separation was carried out using NH2(Strata; 55pm, 70 A; 500 mg / 3mL) cartridge first and then by Si (Strata; 55 55pm, 70 A; 500 mg / 3mL) cartridge purchased from Phenomenax, USA. Before loading the crude lipid extract samples all NH2cartridges used were washed with 6 mL of 100% chloroform under vacuum but without using a vacuum pump and not allowing the cartridge to dry.

[0291] Briefly, the crude lipid extracts were reconstituted in 500 uL of chloroform and loaded onto the cartridge and were eluted with 6 mL of chloroform: isopropanol (2: 1, v / v) under vacuum using a pump. At this stage, after collecting each drop of the sample the cartridge was allowed to dry. Subsequently, the eluted samples were dried under nitrogen and further reconstituted in 500 uL of hexane followed by heating at 50 °C for 1 min. Likewise, before loading the eluted samples again 6 mL of 100% hexane was passed through the Si cartridge under vacuum but without using a vacuum pump and not allowing the cartridge to dry. The samples dissolved in hexane were loaded onto the Si cartridge and were eluted with 6 mL of diethyl ether: hexane (1:9, v / v). Each drop of the eluted sample was collected into a vial and the cartridge was allowed to dry. The eluted samples were dried under nitrogen and were ready for the next enzymatic reaction.

[0292] Enzymatic reaction and separation of monoqlvcerides by thin layer chromatography (TLC)

[0293] The samples collected from SPE were redissolved in a kimax tube with 40 uL of hexane, and to it 400 uL of buffer (IM aqueous solution tris-hydroxymethylaminemethane adjusted to pH 8.0 by hydrochloric acid HCI addition) solution containing 16,000 units of lipase (pancreatic lipase type VI) was added. The mixture was shaken carefully and then 0.5 mL of sodium cholate (1 g / L) solution and 0.2 mL of calcium chloride (220 g / L) solution were added. The lids of the kimax tubes were closed and were shaken cautiously (without wetting the lid) and were immediately placed in a thermostat controlled water bath maintained at 40 °C for exactly 1 min. After incubation, the samples were collected from the water bath and vortexed for 2 min. Subsequently, after 2 min 1 mL of 6 (M) HCI and 1 mL of diethyl ether was added and vortexed vigorously. The samples were then centrifuged at 2000 rpm for 10 min and the organic layers were pooled into a separate vial. The pooled samples were evaporated under nitrogen and redissolved in 50 uL of diethyl ether.

[0294] After the enzymatic reaction, the separation of monoglycerides were performed by TLC using HPTLC silica gel plates 60F254 purchased from Merck, Germany. The samples and standards (TAG, MAG, and DAG) were loaded onto the TLC plate with a pipette from about 1.5 cm from the bottom edge of the plate following a thin uniform line. The TLC plates loaded with samples were then placed in a well-saturated TLC-developing tank with a developing solvent at room temperature. The developing solvent used was a mixture of hexane:ethyl acetate: acetic acid (60:40:0.5) v / v / v. Subsequently, the TLC plates after a run through were air dried and dipped in H2SC>4 - 5% methanol solution and heated for colour or band development. The matched band with the standard retention factor were scraped off and, in this case, only monoglyceride (MAG) band was scraped with a spatula and collected in a glass tube. The collected MAG band was again methylated and the Sn-2 fatty acids were determined using the GC method.

[0295] Steps for Sn-2 fatty acid determination Bulk purification of Microalaal TAGs

[0296] After extracting the Nannochloropsis ocenica oil in bulk, the TAGs were purified from the extracted wax mixture containing other impurities using a column chromatographic separation method. Silica gel-column (0.040-0.063 mm; with mesh size 230-400) was prepared by dissolving the silica gel in hexane and pouring the mixture into the column. Excess solvent was drained off, but the column was kept moist in order to avoid cracks that may result in ineffective separation. The extracted dried lipid mixture was dissolved in hexane and loaded onto the column. Excess solvent (hexane) was added separately to the mixture for allowing the TAGs to separate effectively. Each drop of the sample after passing through the column was collected in separate kimax tube and was tested simultaneously by TLC for TAG identification with a TAG standard (data not shown). After all the extracted lipids were passed through the column (done in four batches), collected TAG fractions only were pooled together and dried under nitrogen to avoid oxidation. This purified microalgal TAG was used further for emulsion preparation.

[0297] Beta-serum preparation

[0298] Beta-serum was prepared from 6 L of fresh pasteurised cream containing 40% fat. Briefly, the cream was heated above 40 °C and less than 55 °C in a water bath. Heated cream was centrifuged with preheated centrifuge at 3000 rpm for 25 minutes at 40 °C to get a light phase containing 80% fat. After centrifugation the light phase was scooped out without disturbing the skim phase and was washed with Milli-Q water followed by centrifugation at 3000 rpm for 25 minutes at 40 °C. The light phase was retained and the heavy phase and the skim phase were discarded. The retained washed high fat cream was subjected to the homogenisation process with Ultra Turrax homogeniser at 24000 rpm for 2 minutes (IKA LABORTECHNIK, T25 basic) for phase inversion to occur. The phase inverted cream was well mixed and centrifuged with preheated centrifuge at 4700nrpm for 15 minutes at 40 °C. The aqueous phase beta-serum was collected by 10 mL syringe and kept in a beaker covered with parafilm and refrigerated. This beta-serum was further freeze dried and used for the emulsion preparation.

[0299] Emulsion formation

[0300] Purified microalgae oil and beta-serum prepared as described above were then used to make emulsions. Briefly, 3% of beta-serum (w / v) was dissolved in Milli-Q water and mixed thoroughly by a stirrer at 50 °C in water bath for 1 hr. Subsequently, after an hour a 10% of purified microalgal TAG (w / v) was added to the beta-serum mixture and a coarse emulsion was formed by homogenising with Ultra Turrax homogeniser at 12000 rpm for 1 minutes (IKA LABORTECHNIK, T25 basic). This coarse emulsion was further subjected to ultrasonication to form a fine emulsion. Sonication conditions were as follows: Amplitude ~ 25%; Pulse ~ 5 sec on and 1 sec off; Time ~ 10 minutes. After 10 minutes the particle size was then checked by using a mastersizer (Malvern Mastersizer 3000). Until the particle size of the emulsions reached between 1-5 pm sonication was continued for another two rounds under the same conditions.

[0301] Results

[0302] Table 1 presents the results of lipid analysis of the microalgal oils produced under different growing conditions. Total lipid content (%) of the dry cell weight, and total triacylglyceride (TAG) content (mg / g) of the dry cell weight, is shown in Figure 3A, and Figure 3B, respectively. As can be seen, the distribution of fatty acids at the sn-2 position varied substantially across the different microalgae when grown under nitrogen limiting conditions. Table 1. Positional distribution of sn-2 palmitate in the extracted triacylglycerides on day 7 under nitrogen and phosphorus limiting conditions.

[0303] Microalgal Growth mg TAG / g sn-2 palmitate sn-2 fatty acids (%) species conditions of biomass mg / g of biomass C16:0 C18:l C18:2

[0304] (average) (average)

[0305] Nannochloropsis No nitrogen 211.96 181.22 85.5 ± 1.7 3.2 ± 1.9 0 oceanica No phosphate 209.30 159.73 76.3 ± 1.8 0.30 ± 0.09

[0306] N. oculata No nitrogen 128.06 80.67 64 ± 8 6.734 ± 2.43 2.664

[0307] No phosphate 95.68 75.41 78.8 ± 1.6 0.90 ± 0.36

[0308] Scenedesmus No nitrogen 295.5 41.68 14.1 ± 0.8 44 ± 6 10.0 ± 0.5 obliquus

[0309] Chlamydomonas No nitrogen 172.9 98.15 56.8 ± 3.3 4.3 ± 2.0 3.9 ± 1.3 reinhardtii

[0310] As can be seen in Table 1, the % palmitate at the sn-2 position was comparable in N. oceanica and N. oculata when grown under phosphate limiting conditions, but the absolute amount of triacylglycerol produced per gram of biomass differed substantially in these two species - as it did when each was grown under nitrogen limiting conditions.

[0311] Nannochloropsis oceanica TAG species

[0312] The analysis of the triglyceride fraction of the N. oceanica oil was conducted following the method of Fei et al., 2019, and the results are shown in Table 2 below. As can be seen, these align with the algae fatty acid analysis reported in Table 1 above, showing C16: 1 as the predominant fatty acid in the oil and 016:0 as the main fatty acid at the SN2 position of the triglyceride. The most abundant triglyceride species identified was 016: 1016:0016: 1, accounting for approximately 23% of the identified TAGs.

[0313] Notably, similarly to human milk microalgae oil contains TAG with palmitic acid (C16:0) at sn-2 and oleic acid (CIS: 1) and linoleic acid (C18:2) at sn-1 and sn-3 as predominant positions.

[0314] Table 2. TAG species in the Nannochloropsis oceanica oil

[0315] As can be seen in Table 3 below, the microalgae oil contains higher proportions of palmitic acid at sn-2 position, 44% (85% sn-2) than comparator oils such as canola and the commercially available sn-2 palmitate enriched oil, OPO-1, 32.1% (50% SN-2 (see Table 3). The microalgae oil was also enriched in palmitoleic acid (21%) compared to canola and OPO-1 oil.

[0316] able 3. Microalgae oil composition compared to human milk produced for term and preterm infants, canola and OPO oil

[0317] Term Infants Preterm Infants

[0318] Colostrum Transitional Mature Colostrum Transitional Mature Algae oil (%) Canola 1 OPO 1

[0319] Saturated Fatty Acids

[0320] Caprylic acid (C8:0) 0.07-0.19 0.2-0.31 0.2-0.3 0.03-0.03 0.09-0.11 0.16-0.16

[0321] Capric acid (C10:0) 0.5-1.04 1.2-1.6 1.5-1.8 0.09-0.09 1.0-1.7 1.2-2.1

[0322] Lauric acid (C12:0) 2.8-3.5 5.4-6.6 5.7-6.5 3.2-4.6 5.7-7.5 5.7-8.1

[0323] Myristic acid (C14:0) 5.4-6.0 6.6-7.5 6.5-7.1 5.8-7.2 8.0-9.2 7.4-9.0 7 (100% sn-2)

[0324] Palmitic acid (C16:0) 24.3-25.5 21.9-23.3 21.7-22.7 22.5-24.1 21.5-23.5 20.9-22.3 44 (85% sn-2) 9.7 32.1 (50% SN-2)

[0325] Stearic acid (C18:0) 6.2-6.6 6.1-6.7 6.3-6.6 5.8-6.5 6.0-6.9 6.2-7.13 4 3 6

[0326] Arachidic acid (C20:0) 0.19-0.25 0.20-0.32 0.20-0.26 0.16-0.18 0.15-0.15 0.20-0.300 3 Q 2

[0327] Monounsaturated Fatty Acids

[0328] Myristoleic acid (C14:ln-5) 0.13-0.23 0.19-0.25 0.18-0.22 0.11-0.13 0.22-0.22 0.21-0.21

[0329] Palmitoleic acid (C16:ln-7) 1.9-2.2 2.0-2.4 2.2-2.4 1.7-1.8 2.1-2.5 2.0-2.521 Q 1 Q 1

[0330] Oleic acid (C18:ln-9) 34.7-35.9 31.2-33.2 32.2-33.6 30.6-33.7 30.5-34.3 31.7-36.7 . .97. -□9

[0331] Vaccenic acid (C18:ln-7) 2.6-2.8 1.9-2.0 1.7-2.1 2.3-2.4 2.5-2.6 2.1-2.2 1 5 02

[0332] Erucic acid (C22:ln-9) 0.20-0.24 0.14-0.28 0.10-0.12 0.16-0.16 0.10-0.14 0.08-0.05 n-3 Polyunsaturated fatty acids (n-3 LCPUFAs) a-Linolenic acid (C18:3n-3) 0.74-0.90 0.84-1.06 0.91-1.03 0.69-1.09 0.70-1.02 0.85-1.13 ,9 n l

[0333] Eicosapentaenoic acid (C20:5n-3) 0.08-0.12 0.11-0.17 0.08-0.10 0.06-0.10 0.10-0.16 0.08-0.16 -

[0334] Clupanodonic acid (C22:5n-3) 0.27-0.33 0.19-0.25 0.14-0.16 0.30-0.34 0.24-0.36 0.16-0.24

[0335] Docosahexaenoic acid (C22:6n-3) 0.47-0.55 0.40-0.52 0.28-0.34 0.43-0.71 0.47-0.67 0.31-0.49 n-6 Polyunsaturated fatty acids (n-6 LCPUFAs)

[0336] Linoleic acid (C18:2n-6) 13.5-15.3 13.4-14.8 14.3-15.7 13.7-16.3 11.4-13.6 12.3-14.4350 1 9 3 y-Linolenic acid (C18:3n-6) 0.07-0.11 0.10-0.18 0.14-0.20 0.07-0.07 0.09-0.13 0.11-0.21

[0337] Eicosadienoic acid (C20:2n-6) 0.82-0.96 0.53-0.63 0.35-0.41 0.89-0.95 0.28-0.30 0.24-0.24

[0338] Dihomo-y-Linolenic acid (C20:3n-6) 0.56-0.64 0.46-0.52 0.39-0.43 0.69-0.81 0.47-0.55 0.40-0.50

[0339] Arachidonic acid (C20:4n-6) 0.73-0.81 0.61-0.69 0.45-0.51 0.68-0.90 0.54-0.68 0.48-0.581 6

[0340] Docosatetraenoic acid (C22:4n-6) 0.29-0.39 0.19-0.25 0.09-0.11 0.44-0.49 0.22-0.22 0.13-0.17

[0341] Adrenic acid (C22:5n-6) 0.13-0.21 0.09-0.13 0.06-0.10 0.15-0.17 0.05-0.05 0.05-0.09

[0342] Data shows the relative proportion in total lipids (%) between mothers who had term and preterm infants. Colostrum = 0-5 days of postnatal life (DPL); Transitional = 6-15 DPL; Mature = 16-60 DPL.

[0343] As can be seen in Table 4 below, the beta-serum prepared herein, extracted from cream, contained less protein contaminants than commercially available beta-serum which is typically extracted from cream or whey (Table 4). Without wishing to be bound by any theory, the inventors believe that high protein content may affect emulsification properties of the phospholipid-containing emulsions contemplated herein. Notably, the beta-serum prepared herein contains higher concentrations of cholesterol than commercially available beta-serum. Again without wishing to be bound by any theory, the inventors expect this to help to stabilise the emulsion and provide a useful source of cholesterol suitable to provide one or more health benefits, such as improved infant nutrition and / or development.

[0344] Table 4. Nutritional composition of commercially available beta-serum and in house betaserum used to produce algae emulsions

[0345] Publications

[0346] Andersen, R. A. (2005). Algal Culturing Techniques. Academic Press. ISBN : 978-0120884261.

[0347] Cliff, B., Park, R., & Adhikari, R. (2023). Innovations in fermentation : sustainable food production through microbial systems. Trends in Food Science & Technology, 142(2), 312-321. https: / / doi.Org / 10.1016 / j.tifs.2023.04.011

[0348] Fei, F., Verstraete, W., & Rabaey, K. (2019). Biotechnological production of nutritional food from microalgae: a review. Biotechnology Advances, 37(1), 1-9. https: / / doi.Org / 10.1016 / j.biotechadv.2018. ll.001

[0349] Plouviez, M., Quillet, L., Lechevalier, V., Legrand, J., Ragonnet, C., Lemoine, Y., & Ropars, M. (2017a). Evaluation of microalgae as protein and lipid sources for food and feed : impact of growing parameters and biomass characteristics. Journal of Applied Phycology, 29(3), 1423-1432. https: / / doi.org / 10.1007 / sl0811-017-1059- y

[0350] Plouviez, M., Quillet, L., & Legrand, J. (2017b). Microalgae biomass as an alternative source of proteins: production and characterization under different environmental conditions. Aquaculture Research, 48(7), 3380-3390. https: / / doi.org / 10. llll / are.13159

[0351] Teng, Z., Zhou, Y., Liang, D., & Wang, C. (2019). Fermentation technology for food applications: a comprehensive review. Food Chemistry, 301, 125221. https: / / doi.Org / 10.1016 / j.foodchem.2019.125221

[0352] The entire disclosures of all applications, patents and publications cited herein, if any, are herein incorporated by reference. Where in the foregoing description reference has been made to integers or components having known equivalents thereof, those integers are herein incorporated as if individually set forth.

[0353] It should be noted that various changes and modifications to the presently preferred examples described herein will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope contemplated herein and without diminishing its attendant advantages. It is therefore intended that such changes and modifications be included within the present invention.

[0354] The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features. Aspects contemplated herein have been described by way of example only, and it should be appreciated that variations, modifications and additions may be made without departing from the scope contemplated herein, for example the invention as defined in the claims. Furthermore, where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred in this specification.

Claims

CLAIMS1. A composition comprising, consisting essentially of, or consisting of an emulsion, the emulsion comprising(a) triglyceride, wherein said triglyceride comprises at least 15% w / w palmitic acid, wherein at least 50% w / w of said palmitate being present at the sn-2 position; and wherein the triglyceride comprises, consists essentially of, or consists of microalgal-derived triglyceride.(b) one or more phospholipids; and(c) one or more sphingolipids; and(d) one or more gangliosides; and(e) one or more cholesterols.

2. The composition according to claim 1 wherein the triglyceride consists of microalgal-derived triglyceride.

3. The composition according to claim 2, wherein the microalgal-derived triglyceride is from any one or more of the group consisting of: Amphora spp.; Arthospira spp.; Audouinella eugena; Aurantiochytrium limacinum; Balbiania investiens; Chlamydomonas spp., such as Chlamydomonas reinhardtii; Chlorella spp., such as Chlorella protothecoides, Chlorella pyrenoidosa, Chlorella sorokiniana, Chlorella vulgaris: CV-15 and CV-395, or Chlorella zofingiensis; Chlorococcum spp.; Crypthecodinim cohnii; Desmodesmus spp.; Dunaliella spp., such as Dunaliella tertiolecta; Haematococcus spp., such as Haematococcus pluvialis; Isochrysis spp., such as Isochrysis galbana; Myrmecia bisecta; Nannochloropsis spp., such as Nannochloropsis limnetica, Nannochloropsis occulata, Nannochloropsis oceanica, or Nannochloropsis salina; Navicula pelliculosa; Neochloris oleabundans; Nitzchia spp., such as Nitzchia laevis; Palmodictyon varium; Phaeodactylum spp., such as Phaeodactylum tricornutum; Porphyridium spp., such as Porphyridium cruentum; Pseudochantransia spp.; Scenedesmus spp., such as Scenedesmus obliquus; Schizochytrium spp., such as Schizochytrium mangrove! or Schizochytrium limacinum; Skeletonema marinoi; Spirulina spp.; Synechococcus spp.; Thorea ramosissima; Trachydiscus minutus; and Trisochrysis spp..

4. The composition according to claim 1, wherein the triglyceride comprises, consists essentially of, or consists of triglyceride from Nannochloropsis oceanica, such as Nannochloropsis oceanica CCMP531 or Nannochloropsis oceanica UTEX 2164.

5. The composition according to any one of claims 1 to 4, wherein the one or more phospholipids, the one or more sphingolipids, the one or more gangliosides, and the one or more cholesterols are provided as a composition, such as a dairy composition.

6. The composition according to any one of claims 1 to 5, wherein the emulsion is formed from triglyceride and a composition comprising one or more of the one or more phospholipids, the one or more sphingolipids, the one or more gangliosides, and / or the one or more cholesterols.

7. The composition according to any one of claims 1 to 6, wherein the composition is a pharmaceutical or nutraceutical composition.

8. The composition according to any one of claims 1 to 7, wherein the emulsion has a mean particle size of from about 1 pm to about 10 pm in diameter.

9. The composition according to any one of claims 1 to 8, wherein the triglyceride comprises at least about 20% w / w palmitic acid.

10. The composition according to claim 9, wherein the fatty acid present in the microalgal-derived triglyceride comprises at least about 50% w / w palmitic acid.

11. The composition according to any one of claims 1 to 10, wherein at least about 80% w / w of the palmitic acid present is present at the sn-2 position.

12. The composition according to any one of claims 1 to 11, wherein the one or more phospholipids comprises, consists essentially of, or consists of one or more milk phospholipids.

13. The composition according to any one of claims 1 to 12 wherein the one or more phospholipids is selected from the group consisting of:(a) phosphatidylcholine;(b) phosphatidylethanolamine;(c) phosphatidylserine;(d) phosphatidylinositol;(e) phosphatidic acid;(f) a derivative, extract, or fraction of one or more of the phospholipids defined in a) - e); and(g) any combination of two or more of any of a) to f) above.

14. The composition according to any one of claims 1 to 13 wherein the one or more sphingolipids is selected from the group consisting of:(a) sphingomyelins;(b) Ceramides;(c) Gangliosides(d) Lactosylceramide(e) Glucosylceramide(f) a derivative, extract, or fraction of one or more of the sphingolipids defined in a) - e); and(g) any combination of two or more of any of a) to f) above.

15. The composition according to any one of claims 1 to 14 wherein the one or more gangliosides is selected from the group consisting of:(a) monosialodihexosylganglioside (GM3);(b) disialoganglioside (GD3);(c) a derivative, extract, or fraction of one or more of the gangliosides defined in a) - b); and(d) any combination of two or more of any of a) to c) above.

16. The composition according to any one of claims 1 to 15 wherein the one or more cholesterols is selected from the group consisting of:(a) Cholesterol(b) p-sitosterol;(c) Campesterol(d) Stigmasterol(e) Brassicasterol:(f) a derivative, extract, or fraction of one or more of the cholesterols defined in a) - e); and(g) any combination of two or more of any of a) to f) above.

17. The composition according to any one of claims 5 to 16, wherein the composition comprising one or more of the one or more phospholipids, the one or more sphingolipids, the one or more gangliosides, and / or the one or more cholesterols, is beta-serum or a composition comprising, consisting essentially of, or consisting of milk fat globule membrane.

18. The composition according to any one of claims 1 to 17, wherein the composition comprises:(a) at least about 1 g triglyceride per 100 g; and / or(b) at least about 100 mg phospholipid per 100 g; and / or(c) at least about 50 mg sphingolipid per 100 g; and / or(d) at least about 50 mg ganglioside per 100 g; and / or(e) at least about 500 mg cholesterol per 100 g; and / or(f) any combination of two or more of any of a) to e) above.

19. The composition according to any one of claims 1 to 18, wherein the triglyceride comprises from about 10 to about 50% w / w palmitic acid and from about 20 to about 70% w / w palmitoleic acid.

20. The composition according to any one of claims 1 to 19, wherein at least about 15% w / w of the triglyceride consists of a C16: l-C16:0-C16: l triglyceride species.

21. The composition according to any one of claims 1 to 20, wherein the triglyceride is substantially free of linoleic acid.

22. A method of preparing a composition comprising, consisting essentially of, or consisting of an emulsion, the emulsion comprising triglyceride, one or more phospholipids, one or more sphingolipids, one or more gangliosides, and one or more cholesterols; the method comprising providing Nannochloropsis cells or a cell extract or lysate therefrom; contacting said cells or cell extract with a solvent comprising methanol and chloroform under conditions and for a time sufficient to dissolve one or more lipids; recovering and combining the one or more lipids with a composition comprising one or more phospholipids, one or more sphingolipids, one or more gangliosides, and one or more cholesterols to provide a mixture;homogenising the mixture by applying shear and / or sonication until the mean emulsion particle size is from about 200 nm to about 10 pm.

23. The method according to claim 22, wherein the Nannochloropsis cells were grown in conditions in which the concentration or amount of nitrogen was growth-limiting.

24. The method according to claim 22 or claim 23, wherein the Nannochloropsis cells were grown in conditions in which the concentration or amount of phosphate was growth-limiting25. The method according to any one of claims 22 to 24 wherein the preparation of the emulsion comprises homogenisation and / or sonication.

26. The composition according to any one of claims 1 to 21 for use in therapeutically increasing growth and / or development of a neonatal, infant, child, or adult subject in need thereof.

27. The composition according to any one of claims 1 to 21 for use in increasing the growth and / or development of a neonatal, infant, child, or adult subject in need thereof.

28. The use of a composition according to any one of claims 1 to 21 in the preparation of a medicament, pharmaceutical composition, or nutritional composition for increasing growth and / or development of a subject in need thereof.

29. A method for increasing growth and / or development of a neonatal, infant, child, or adult subject, the method comprising orally administering to the subject a composition according to any one of claims 1 to 21.

30. A method for increasing cognitive development of a neonatal, infant, child, or adult subject, the method comprising orally administering to the subject a composition according to any one of claims 1 to 21.

31. A method of treating or preventing cognitive impairment or cognitive decline in a subject, the method comprising administering to the subject a composition according to any one of claims 1 to 21.

32. A non-therapeutic method for increasing growth of a healthy subject, the method comprising orally administering to the subject a composition according to any one of claims 1 to 21, wherein the method is not a method for the treatment of the human or animal body by therapy.

33. A non-therapeutic method for increasing cognitive development of a healthy subject, the method comprising orally administering a composition according to any one of claims 1 to 21 to the subject, wherein the method is not a method for the treatment of the human or animal body by therapy.

34. The use of a composition according to any one of claims 1 to 21 in a non-therapeutic method for increasing growth of a healthy subject, the method comprising orally administering theformulation to the subject, wherein the method is not a method for the treatment of the human or animal body by therapy.

35. The use of a composition according to any one of claims 1 to 21 in a non-therapeutic method for increasing cognitive development of a healthy subject, the method comprising orally administering the formulation to the subject, wherein the method is not a method for the treatment of the human or animal body by therapy.

36. The method according to any one of claims 29 to 33, or the use according to claim 34 or 35, wherein the subject is a neonatal, infant, or child subject.

37. The method according to any one of claims 29 to 33, or the use according to claim 34 or 35, wherein the subject is an adult subject.

38. The composition according to any one of claims 1 to 21 wherein the composition is an infant formula, follow-on formula, or growing-up formula.

39. The composition according to any one of claims 1 to 21 wherein the composition is an adult formula, such as a nutritional formula, medical food, sports formula, or geriatric formula.

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