Infant formula for improving cognitive development

Abstract

Disclosed is a nutritional composition comprising a digestible carbohydrate, a protein and a lipid wherein the lipid is in the form of lipid globules at least partially coated with phospholipids on the surface for increasing myelination in the brain of an infant suffering from or at risk of suffering from a delayed brain development.

Description

Technical Field

[0001] The present invention relates to a nutritional composition for infants suffering from or at risk of suffering from brain developmental delay. Background Art

[0002] Human milk is the undisputed gold standard for infant nutrition. However, in some cases, breastfeeding is insufficient or unsuccessful due to medical reasons or the choice not to breastfeed. For such cases, infant formula or follow-on formula has been developed. Nowadays, commercial infant formula is commonly used to provide a supplementary or sole source of nutrition in the early life. These formulas contain a range of nutrients to meet the nutritional needs of growing infants and typically include fats, carbohydrates, proteins, vitamins, minerals and other nutrients that are helpful for optimal infant growth and development. Commercial infant formula is designed to mimic as closely as possible the composition and function of human milk.

[0003] The decision to breastfeed is an early parental decision that can affect the child's later cognitive and behavioral functions. The general consensus from large-scale epidemiological studies is that, on average, breastfed children perform better in IQ and cognitive function tests than children fed exclusively with formula, even when taking into account factors such as birth weight, gestational length, and maternal education and socioeconomic status.

[0004] These neuropsychological findings are complemented by adolescent morphometric brain imaging studies, which show increased volumes of total white matter, subcortical gray matter, and parietal cortex thickness in children breastfed in infancy. Non-imaging assessments of early neural pathway maturation using evoked potentials further support the developmental benefits of breastfeeding, while finding that formula-fed infants have longer wave latencies in their visual and auditory pathways at 1 year of age (Khedr et al., 2004, DOI: 10.1111 / j.1651-2227.2004.tb03011.x), indicating delayed or immature myelination of these pathways compared to breastfed infants.

[0005] The main hypothesized substrates for these developmental advantages are the abundant supplements of long-chain fatty acids found in breast milk, particularly docosahexaenoic acid (DHA) and arachidonic acid (AA) (McCann and Ames, 2005, DOI: 10.1093 / ajcn.82.2.281). Together, DHA and AA constitute approximately 20% of the brain fatty acid content and are involved in early neural development by promoting healthy neuronal growth, repair, and myelination (Guesnet and Alessandri, 2011, DOI: 10.1016 / j.biochi.2010.05.005). Optimal fetal neural development depends particularly on DHA. DHA deficiency results in reduced dendritic branching and impaired expression of genes regulating neurogenesis, neurotransmission, and connectivity.

[0006] Myelin is a lipid-rich material that surrounds the axons of nerve cells to insulate them and increase the rate at which electrical impulses, called action potentials, are transmitted along the axon. Myelin is formed in the central nervous system (CNS; brain, spinal cord, and optic nerve) by glial cells called oligodendrocytes and in the peripheral nervous system (PNS) by glial cells called Schwann cells. In the CNS, axons carry electrical signals from one nerve cell body to another. In the PNS, axons carry signals to and from muscles and glands or from sensory organs such as the skin.

[0007] This "insulating" role of myelin is essential for normal motor function (i.e., movement, such as walking), sensory function (e.g., hearing, vision, or the sensation of feeling pain), and cognition (e.g., acquiring and recalling knowledge), as demonstrated by the consequences of disorders affecting myelin, such as genetically determined leukodystrophies of the brain; acquired inflammatory demyelinating disorders, multiple sclerosis; and inflammatory demyelinating peripheral neuropathies.

[0008] The process of producing myelin is called myelination or myelinogenesis. In humans, myelination begins early in the third trimester, although only small amounts of myelin are present in the CNS or PNS at birth. In infancy, myelination progresses rapidly, with an increasing number of axons acquiring myelin. This corresponds to the development of cognitive and motor skills, including language comprehension, speech acquisition, crawling, and walking. Myelination continues into adolescence and early adulthood, and although it is essentially complete at this time, myelin can be added to gray matter regions (such as the cerebral cortex) throughout life.

[0009] Deoni et al., 2018, doi:10.1016 / j.neuroimage.2017.12.056 described an examination of the longitudinal trajectories of brain and neurocognitive development in children breastfed exclusively for at least 3 months compared to those fed formula. They further examined development among children receiving different formula compositions. The results revealed that overall myelination was significantly improved in breastfed children compared to those fed formula exclusively, along with enhanced general, language, and non-verbal cognitive abilities. These differences were found to persist into childhood, even among groups matched for important socioeconomic and demographic factors. They also found that significant developmental differences depended on the formula composition received, and in particular, long-chain fatty acids, iron, choline, sphingomyelin, and folic acid were significantly associated with early myelination trajectories.

[0010] WO 2017 / 102720 describes a nutritional composition for infants and toddlers that comprises phospholipids, their metabolic precursors, and / or metabolites. The phospholipids promote and / or support optimal myelination trajectories in the brain that are similar to those observed in infants breastfed exclusively with human milk.

[0011] Oshida et al., 2003, DOI:10.1203 / 01.PDR.0000054654.73826.AC described that dietary sphingomyelin (SM) contributes to CNS (central nervous system) myelination in developing rats, where sphingolipid biosynthesis was L chemically blocked by cycloserine.

[0012] Tanaka et al., 2013, http: / / dx.doi.org / 10.1016 / j.braindev.2012.03.004 described a randomized controlled trial to examine the effects of sphingomyelin (SM) on the mental, motor, and behavioral development of preterm infants. They reported a positive correlation between nutritional intervention via administration of SM-enriched milk and the neurobehavioral development of low-birth-weight infants.

[0013] Human milk lipids are known to have a unique physical structure consisting of large lipid globules with a volume-based mode diameter of approximately 4 μm, with a triglyceride core surrounded by a three-layer membrane (milk fat globule membrane (MFGM)). Due to industrial processing procedures applied to achieve a stable product, the volume-based mode diameter of lipid droplets in standard infant formulas is typically approximately 0.3 - 0.5 μm, and these lipid droplets are surrounded mainly by milk proteins rather than MFGM. Infant formulas with lipid globules having a structure more similar to those in human milk have been described.

[0014] WO 2011 / 115490 describes a nutritional composition comprising phospholipids for use in altering the fatty acid composition of the meninges and for improving cognitive and behavioral performance in human subjects.

[0015] WO 2011 / 115491 describes a nutritional composition comprising lipid spheres having a defined particle size distribution for use in altering the fatty acid composition of the meninges and for improving cognitive and behavioral performance in human subjects.

[0016] WO 2018 / 104512 describes a nutritional composition comprising lipid spheres having a determined particle size distribution and further comprising an increased level of sn-2 palmitate for altering the fatty acid composition of cell membranes, particularly in the brain and red blood cell membranes, and for improving cognitive development and behavioral performance in human subjects. SUMMARY OF THE INVENTION

[0017] The inventors of the present invention have unexpectedly found that, in addition to the presence of sphingomyelin or specific fatty acids (such as DHA and ARA) in nutritional compositions for infants and young children, the supramolecular lipid structures in the nutritional compositions also affect the myelin formation rate in infants with delayed brain development or at risk of having delayed brain development.

[0018] Accordingly, the present invention relates to a nutritional composition selected from infant formula, follow-on formula, and toddler formula, the nutritional composition comprising digestible carbohydrates, protein, and lipids, wherein the lipids are in the form of lipid spheres, and wherein

[0019] a. based on volume, the lipid spheres have a mode diameter of at least 1.0 μm; and / or

[0020] b. based on the total lipid volume, at least 45 vol% of the lipid spheres have a diameter of 2 to 12 μm; and

[0021] c. the lipids comprise 0.5 wt.% to 20 wt.% of phospholipids based on the total lipids, and wherein the lipid spheres are at least partially coated with phospholipids on the surface,

[0022] for use in increasing myelin formation in the brains of infants with delayed brain development or at risk of having delayed brain development.

[0023] The inventors conducted an experiment in which mouse dams were exposed to a diet lacking omega-3 fatty acids during pregnancy and lactation, resulting in impaired brain development in the offspring (Basak et al., 2020, doi:10.3390 / nu12123615). The offspring were then fed two different diets. The control diet contained small lipid droplets and phospholipids added in a dry mix form (i.e., not present in a coating). The test diet contained large phospholipid-coated lipid spheres. Other than these differences, the control diet and the test diet were identical. The offspring received the control diet or the test diet from postnatal day 16 to day 42. The fatty acid content in the brains of the offspring was determined. In the hippocampal region, the gene expression (qPCR) of myelin basic protein (MBP) and myelin-associated glycoprotein (MAG), which are markers of myelin, was determined.

[0024] In the offspring group consuming the test diet, the levels of palmitic acid (C16:0) and stearic acid (C18:0) in the brain cell membranes were increased compared to the levels observed in the offspring group consuming the control diet. C16:0 and C18:0 are two of the most abundant saturated fatty acids in the brain during early life [Martínez et al., 1998, doi:10.1046 / j.1471-4159.1998.71062528.x.]. Myelin is particularly rich in these fatty acids [Manzoli et al., 1970, doi:10.1016 / 0014-5793(70)80462-8].

[0025] In the offspring group consuming the test diet, the levels of MBP and MAG in the hippocampal region were increased compared to the levels observed in the offspring group consuming the control diet. The hippocampus is an important region for brain functions that mature relatively late. The myelination pattern of the hippocampus is crucial for brain function. During the dietary intervention, myelination of the hippocampus was ongoing, see Nickel et al., 2018, doi:10.1155 / 2018 / 6436453.

[0026] The effect of dietary treatment with a nutritional composition containing large phospholipid-coated lipid droplets was unexpected. Although it is known that the fatty acid composition of the diet, particularly the addition of sphingomyelin to the diet, can affect brain fatty acid status, myelination, and cognitive function, the fatty acid composition and sphingomyelin levels were identical in both the control diet and the test diet. Thus, the additional increase in brain myelin in the test group may be related to the phospholipids present in the coating and the larger size of the lipid spheres. Detailed Description

[0027] A first aspect of the present invention relates to a nutritional composition selected from infant formula, follow-on formula and toddler formula, the nutritional composition comprising digestible carbohydrates, protein and lipids, wherein the lipids are in the form of lipid droplets, and wherein

[0028] a. based on volume, the lipid droplets have a mode diameter of at least 1.0 μm; and / or

[0029] b. based on the total lipid volume, at least 45% by volume of the lipid droplets have a diameter of 2 to 12 μm; and

[0030] c. the lipids comprise 0.5 wt.% to 20 wt.% of phospholipids based on the total lipids, and wherein the lipid droplets are at least partially coated with phospholipids on the surface,

[0031] for use in increasing myelination in the brain of an infant suffering from or at risk of suffering from delayed brain development.

[0032] For some jurisdictions, the present invention may also be formulated as a method for increasing myelination in the brain of an infant suffering from or at risk of suffering from delayed brain development, the method comprising feeding to the infant a nutritional composition selected from infant formula, follow-on formula and toddler formula, the nutritional composition comprising digestible carbohydrates, protein and lipids, wherein the lipids are in the form of lipid droplets, and wherein

[0033] a. based on volume, the lipid droplets have a mode diameter of at least 1.0 μm; and / or

[0034] b. based on the total lipid volume, at least 45% by volume of the lipid droplets have a diameter of 2 to 12 μm; and

[0035] c. the lipids comprise 0.5 wt.% to 20 wt.% of phospholipids based on the total lipids, and wherein the lipid droplets are at least partially coated with phospholipids on the surface.

[0036] For some jurisdictions, the present invention may also be formulated as the use of digestible carbohydrates, protein and lipids in the manufacture of a nutritional composition for increasing myelination in the brain of an infant suffering from or at risk of suffering from delayed brain development, wherein the nutritional composition is selected from infant formula, follow-on formula and toddler formula, the nutritional composition comprising digestible carbohydrates, protein and lipids, wherein the lipids are in the form of lipid droplets, and wherein

[0037] a. based on volume, the lipid droplets have a mode diameter of at least 1.0 μm; and / or

[0038] b. at least 45% by volume of the lipid globules, based on the total lipid volume, have a diameter of 2 to 12 μm; and

[0039] c. The lipids comprise 0.5 wt.% to 20 wt.% of phospholipids based on total lipids, and wherein the lipid globules are at least partially coated with phospholipids on the surface.

[0040] In some jurisdictions, administration of a nutritional composition to an infant is considered non-therapeutic. In those cases, the present invention may be described as being carried out by a method of administering a nutritional composition as defined above. For the sake of clarity, the method may also be defined as a non-therapeutic method. By definition, the term "non-therapeutic" excludes any therapeutic effect.

[0041] Application

[0042] The "level of myelination in the infant brain" can be appropriately determined in an MRI scanner.

[0043] As used herein, the term "infant" refers to a child of 0-36 months of age, preferably a child of 0-24 months of age, more preferably a child of 0-12 months of age, and most preferably a child of 0-6 months of age. Preferably, the infant is a human infant.

[0044] The brain development of infants can be appropriately assessed with the Bayley III test. The test refers to the Bayley Scales of Infant Development, 3rd edition (BSID-III), which is a standard series measurement mainly used to assess the movement (fine and rough), language (acceptance and expression) and cognitive development of infants and young children from 16 days to 42 months of age. The main purpose of Bayley-III is to identify suspected developmental delays in children by using standard reference scores. (Bayley N. Bayley Scales of Infant and Toddler Development [Bayley Infant Development Scale]. 3rd edition. San Antonio, TX: Harcourt Assessment Inc [San Antonio, Texas: Harcourt Assessment Company], 2006).

[0045] In a preferred embodiment, the brain development delay in an infant suffering from brain development delay is treated or reduced by increased myelination.

[0046] In another preferred embodiment, for infants at risk of suffering from brain development delay, the risk of suffering from brain development delay is reduced by increased myelination. In other words, preferably, brain development delay is prevented by increasing myelination of infants at risk of suffering from brain development delay.

[0047] Preferably, the increased myelination helps to improve the cognitive development of the infant and / or improve motor function.

[0048] Preferably, the increased myelination is around the axons connected to the hippocampus and within the hippocampus.

[0049] Preferably, the increased myelination is by higher levels of myelin-associated glycoprotein (MAG), myelin basic protein (MBP), or a combination thereof in the brain. More preferably, the increased myelination is by higher levels of MAG, MBP, or a combination thereof in the hippocampal region of the brain.

[0050] Preferably, the increased myelination is by increasing the levels of palmitic acid (C16:0), stearic acid (C18:0), or a combination thereof in the brain cell membrane.

[0051] Preferably, the increased myelination in the infant's brain is compared to a similar infant with or at risk of having a brain development delay who consumed a nutritional composition selected from infant formula, follow-on formula, and toddler formula, the nutritional composition comprising digestible carbohydrates, protein, and lipids, and wherein the lipids are in the form of lipid globules, and wherein:

[0052] a. Based on volume, the lipid globules have a mode diameter of about 0.5 μm, and

[0053] b. Based on the total lipid volume, less than 45 volume % of the lipid globules have a diameter greater than 2 μm, and

[0054] c. wherein the lipids comprise less than 0.5 wt.% phospholipids based on the total lipids, and wherein the lipid globules are not coated with phospholipids.

[0055] Preferably, infants with or at risk of having a brain development delay are selected from premature infants, small for gestational age infants, infants with phenylketonuria (PKU), infants with epilepsy, infants with cerebral palsy, infants born with brain injury, infants exposed to perinatal hypoxia, or a combination thereof.

[0056] In a preferred embodiment, the infant is selected from premature infants, small for gestational age infants, or a combination thereof. As used herein, the term "premature infant" refers to a subject born before 37 weeks of gestational age. Premature infants are at a greater risk of cerebral palsy, developmental delay, hearing problems, and vision problems. The earlier the infant is born, the greater these risks are. As used herein, the term "small for gestational age infant" refers to an infant born with a smaller size than the normal size for its gestational age, most commonly defined as having a weight below the 10th percentile for its gestational age.

[0057] In another preferred embodiment, the infant is a full-term born infant who is not small for gestational age, and wherein the infant is additionally one or more of an infant with phenylketonuria (PKU), an infant with epilepsy, an infant with cerebral palsy, an infant born with brain injury, and an infant exposed to perinatal hypoxia.

[0058] Lipid

[0059] The nutritional composition for use according to the invention comprises lipids. The lipids in the present invention comprise one or more selected from the group consisting of: triglycerides, polar lipids (such as phospholipids, cholesterol, glycolipids, sphingomyelins), free fatty acids, monoglycerides, and diglycerides. Preferably, the composition comprises at least 70 wt.%, more preferably at least 80 wt.%, even more preferably at least 85 wt.%, and most preferably at least 90 wt.% of triglycerides based on the total lipids.

[0060] The lipids preferably account for 30% to 60% of the total calories of the nutritional composition. More preferably, the nutritional composition comprises lipids providing 35% to 55% of the total calories, and even more preferably the nutritional composition comprises lipids providing 40% to 50% of the total calories. The lipids are preferably present in an amount of 3 to 7 g / 100 kcal, more preferably in an amount of 4 to 6 g of lipids / 100 kcal, and most preferably in an amount of 4.5 to 5.5 g of lipids / 100 kcal. When in liquid form (such as an instant liquid), the nutritional composition preferably comprises 2.1 to 6.5 g of lipids / 100 ml, more preferably 3.0 to 4.0 g / 100 ml. Based on dry weight, the nutritional composition preferably comprises 10 wt.% to 50 wt.%, more preferably 12.5 wt.% to 40 wt.%, and even more preferably 19 wt.% to 30 wt.% of lipids.

[0061] The lipids preferably comprise plant lipids. The presence of plant lipids advantageously enables a high content of polyunsaturated fatty acids and / or an optimal fatty acid profile more similar to human milk fat. Lipids from only non-human mammalian milk (such as cow's milk) cannot provide an optimal fatty acid profile. The amount of essential fatty acids in non-human mammalian milk is too low.

[0062] Preferably, the nutritional composition comprises at least one, preferably at least two plant lipid sources selected from the group consisting of: linseed oil (linseed oil or flaxseed oil), rapeseed oil (rape seed oil) (such as colza oil, low erucic acid rapeseed oil, and canola oil), sunflower oil, high oleic sunflower oil, safflower oil, high oleic safflower oil, olive oil, coconut oil, palm oil, and palm kernel oil.

[0063] In a preferred embodiment, the nutritional composition comprises from 30 wt.% to 90 wt.%, more preferably from 35 wt.% to 80 wt.%, more preferably from 40 wt.% to 70 wt.%, more preferably from 40 wt.% to 60 wt.% of vegetable lipids, based on the total lipids.

[0064] The lipids in the nutritional composition preferably further comprise mammalian milk fat, preferably ruminant milk fat. More preferably, the mammalian milk fat is derived from cow milk, goat milk, sheep milk, buffalo milk, yak milk, reindeer milk, and / or camel milk. Most preferably, the mammalian milk fat is cow milk fat. Preferably, the mammalian milk fat is not human milk fat. Preferably, the mammalian milk fat comprises at least 70 wt.%, more preferably at least 90 wt.%, more preferably at least 97 wt.% triglycerides, by weight of the mammalian milk fat.

[0065] Preferably, the mammalian milk fat is derived from butter, butter fat, butter oil, and / or anhydrous milk fat. More preferably, the mammalian milk fat is derived from anhydrous milk fat and / or butter oil. Such mammalian milk fat sources have a relatively high triglyceride level. These mammalian milk fat sources can be in the form of a continuous lipid phase or a water-in-oil emulsion. Using these mammalian milk fat sources during the manufacture of the nutritional composition of the present invention enables the formation of lipid globules, each containing a mixture of vegetable fat and mammalian milk fat.

[0066] The mammalian milk fat in the present invention refers to all lipid components of milk, such as produced by a mammal (such as a cow) and present in commercial milk and milk-derived products. Butter in the present invention is a water-in-oil emulsion containing more than 80 wt.% milk fat. Milk fat in the present invention refers to all fat components in milk that can be separated by churning (in other words, present in butter). Anhydrous milk fat (AMF) is a term known in the art and refers to extracted milk fat. Typically, AMF contains more than 99 wt.% lipids, based on the total weight. It can be prepared by extracting milk fat from cream or butter. Anhydrous butter oil in the present invention is synonymous with AMF. Butter oil is also a term known in the art. It typically refers to a milk fat extract having more than 98 wt.% lipids and is typically a precursor in the method for preparing anhydrous milk fat or anhydrous butter oil.

[0067] Preferably, the composition comprises from 10 wt.% to 70 wt.%, more preferably from 20 wt.% to 65 wt.%, more preferably from 30 wt.% to 60 wt.%, more preferably from 40 wt.% to 60 wt.% of mammalian milk fat, based on the total lipids.

[0068] Preferably, the ratio of vegetable fat to mammalian milk fat is in the range of 3 / 7 to 9 / 1. In a preferred embodiment, the lipids in the nutritional composition comprise:

[0069] a) vegetable lipids in an amount of 35 wt.% to 80 wt.%, based on the total lipids, and

[0070] b) mammalian milk fat in an amount of 20 wt.% to 65 wt.%, based on the total lipids, wherein the mammalian milk fat is selected from butter, butter fat, oily butter or anhydrous milk fat.

[0071] More preferably, the lipids in the nutritional composition comprise:

[0072] a) vegetable lipids in an amount of 40 wt.% to 70 wt.%, based on the total lipids, and

[0073] b) mammalian milk fat in an amount of 30 wt.% to 60 wt.%, based on the total lipids, wherein the mammalian milk fat is selected from butter, butter fat, oily butter or anhydrous milk fat.

[0074] Most preferably, the lipids in the nutritional composition comprise:

[0075] a) vegetable lipids in an amount of 40 wt.% to 60 wt.%, based on the total lipids, and

[0076] b) mammalian milk fat in an amount of 40 wt.% to 60 wt.%, based on the total lipids, wherein the mammalian milk fat is selected from butter, butter fat, oily butter or anhydrous milk fat.

[0077] Compared with vegetable fat, mammalian milk fat is known to have a higher content of palmitic acid (PA) at the sn-2 position of triglycerides. In a preferred embodiment, the lipids in the nutritional composition comprise at least 10 wt.% of PA, based on the total fatty acids, and at least 15 wt.% of PA is located at the sn-2 position of triglycerides, based on the total palmitic acid. Preferably, the amount of PA is less than 30 wt.%, based on the total fatty acids. More preferably, the amount of PA is from 12 wt.% to 26 wt.%, even more preferably from 14 wt.% to 24 wt.%, based on the total fatty acids.

[0078] Preferably, at least 15 wt.%, more preferably at least 20 wt.%, even more preferably at least 25 wt.%, most preferably at least 30 wt.% of PA, based on the total PA, is in the sn-2 or β position in the triglycerides. Preferably, the amount of PA in the sn-2 position in the triglycerides does not exceed 45 wt.%, preferably does not exceed 40 wt.%, based on the total PA present in the lipids. Preferably, the amount of PA in the sn-2 position in the triglycerides is from 25 wt.% to 40 wt.%, based on the total PA.

[0079] Compared with plant fats, mammalian milk fat is known to have higher levels of the short-chain fatty acids (SCFAs) butyric acid (BA; C4:0) and caproic acid (CA; C6:0). In a preferred embodiment, the lipids in the nutritional composition comprise from 0.6 wt.% to 5 wt.% SCFAs, being the sum of BA and CA based on total fatty acids. Preferably, the nutritional composition comprises less than 5 wt.%, preferably less than 4 wt.%, of BA based on total fatty acids. Preferably, the nutritional composition comprises at least 0.5 wt.%, preferably at least 0.6 wt.%, preferably at least 0.9 wt.%, more preferably at least 1.2 wt.%, of BA based on total fatty acids.

[0080] In a preferred embodiment, the lipids in the nutritional composition comprise:

[0081] · at least 10 wt.% of PA based on total fatty acids, and based on total PA, at least 15 wt.% of PA is located at the sn-2 position of triglycerides; and

[0082] · from 0.6 wt.% to 5 wt.% SCFAs, being the sum of BA and CA based on total fatty acids.

[0083] The nutritional composition preferably further comprises one or more lipids selected from fish oil, egg lipids and microbial, algal, fungal or single cell oils.

[0084] Fatty acid composition

[0085] SFA refers to saturated fatty acids and / or acyl chains, MUFA refers to monounsaturated fatty acids and / or acyl chains, PUFA refers to polyunsaturated fatty acids and / or acyl chains having 2 or more unsaturated bonds; LC-PUFA refers to long-chain polyunsaturated fatty acids and / or acyl chains having at least 20 carbon atoms in the fatty acyl chain and having 2 or more unsaturated bonds; medium-chain fatty acids (MCFA) refer to fatty acids and / or acyl chains having a chain length of 6, 8 or 10 carbon atoms. n3 or ω-3 PUFA refers to polyunsaturated fatty acids and / or acyl chains having 2 or more unsaturated bonds and the unsaturated bonds are located at the third carbon atom from the methyl end of the fatty acyl chain; n6 or ω-6 PUFA refers to polyunsaturated fatty acids and / or acyl chains having 2 or more unsaturated bonds and the unsaturated bonds are located at the sixth carbon atom from the methyl end of the fatty acyl chain.

[0086] In the context of the present invention, the weight percentages of fatty acids based on total fatty acids are calculated on the basis that all fatty acids are free fatty acids, and thus do not take into account whether the fatty acids are attached to the glycerol backbone.

[0087] DHA refers to docosahexaenoic acid and / or acyl chain (22:6n3); DPA refers to docosapentaenoic acid and / or acyl chain (22:5n3); n6 DPA refers to ω-6 docosapentaenoic acid and / or acyl chain (22:5n6). EPA refers to eicosapentaenoic acid and / or acyl chain (20:5n3); ARA refers to arachidonic acid and / or acyl chain (20:4n6). LA refers to linoleic acid and / or acyl chain (18:2n6); ALA refers to α-linolenic acid and / or acyl chain (18:3n3).

[0088] LA refers to linoleic acid and / or acyl chain, and is a precursor of n6 PUFA (18:2n6) and n6 LC-PUFA, and is an essential fatty acid as it cannot be synthesized by the human body. The nutritional composition preferably contains LA. LA is preferably present in an amount sufficient to promote healthy growth and development, but the amount should be as low as possible to prevent a negative, competitive effect on the formation of n3 PUFA and an excessive n6 / n3 ratio. Thus, the nutritional composition preferably contains less than 20 wt.% of LA, preferably 5 wt.% to 16 wt.%, more preferably 10 wt.% to 14.5 wt.%, based on total fatty acids. Preferably, the nutritional composition contains at least 5 wt.% of LA, preferably at least 6 wt.% of LA, more preferably at least 7 wt.% of LA, based on total fatty acids. Per 100 kcal, the nutritional composition preferably contains 350–1400 mg of LA.

[0089] ALA refers to α-linolenic acid and / or acyl chain, and is a precursor of n3 PUFA (18:3n3) and n3 LC-PUFA, and is an essential fatty acid as it cannot be synthesized by the human body. The nutritional composition preferably contains ALA. Preferably, ALA is present in an amount sufficient to promote healthy growth and development of infants. Based on total fatty acids, the nutritional composition preferably contains at least 1.0 wt.%, more preferably, the nutritional composition contains at least 1.5 wt.%, even more preferably at least 2.0 wt.% of ALA. Preferably, based on total fatty acids, the nutritional composition contains less than 10 wt.% of ALA, more preferably less than 5.0 wt.%.

[0090] Preferably, the nutritional composition contains an LA / ALA weight ratio of 2 to 20, more preferably 3 to 16, even more preferably 4 to 14, most preferably 5 to 12.

[0091] Based on total fatty acids, the lipids in the nutritional composition preferably contain 5 wt.% to 35 wt.% of PUFA, containing LA and ALA with an LA / ALA weight ratio of 2 to 20.

[0092] Preferably, the nutritional composition comprises n3 LC-PUFA, such as EPA, DPA and / or DHA, more preferably DHA. Since the efficiency of conversion of ALA to DHA in infants may be low, it is preferred that both ALA and DHA are present in the nutritional composition. Preferably, the nutritional composition comprises at least 0.05 wt.%, preferably at least 0.1 wt.%, more preferably at least 0.2 wt.% of DHA based on total fatty acids. Preferably, the nutritional composition comprises not more than 2.0 wt.%, preferably not more than 1.0 wt.% of DHA based on total fatty acids.

[0093] The nutritional composition preferably comprises ARA. Preferably, the nutritional composition comprises at least 0.05 wt.%, preferably at least 0.1 wt.%, more preferably at least 0.2 wt.% of ARA based on total fatty acids. The group of n6 fatty acids, in particular arachidonic acid (ARA) counteracts the group of n3 fatty acids, in particular DHA, and thus the nutritional composition preferably comprises a relatively low amount of ARA. Preferably, the nutritional composition comprises not more than 2.0 wt.%, preferably not more than 1.0 wt.% of ARA based on total fatty acids. Preferably, the weight ratio between DHA and ARA is between 1:4 and 4:1, more preferably between 1:2 and 2:1, even more preferably between 0.6 and 1.5.

[0094] Lipid globule size

[0095] The lipids in the nutritional composition are in the form of lipid globules, wherein:

[0096] a. Based on volume, the lipid globules have a modal diameter of at least 1.0 μm; and / or

[0097] b. Based on total lipid volume, at least 45 volume % of the lipid globules have a diameter between 2 and 12 μm.

[0098] Lipids typically exist in the nutritional composition in the form of lipid globules. When the nutritional composition is in liquid form, these lipid globules are emulsified in the aqueous phase. Alternatively, when the nutritional composition is in powder form, the lipid globules are present in the powder and the powder is suitable for reconstitution with water or another food-grade aqueous phase. The lipid globules may comprise a core and a surface.

[0099] Based on volume, the lipid globules in the nutritional composition preferably have a modal diameter of at least 1.0 μm, more preferably at least 2.0 μm, and most preferably at least 3.0 μm. Preferably, based on volume, the lipid globules have a modal diameter between 1.0 and 10 μm, more preferably between 2.0 and 8.0 μm, even more preferably between 3.0 and 7.0 μm, and most preferably between 4.0 μm and 6.0 μm.

[0100] Alternatively or preferably additionally, the size distribution of the lipid globules is preferably effected in such a way that, based on the total lipid volume, at least 45 volume % (vol. %), preferably at least 55 vol. %, even more preferably at least 65 vol. %, and most preferably at least 75 vol. % of the lipid globules have a diameter between 2 and 12 μm. In a more preferred embodiment, based on the total lipid volume, at least 45 vol. %, preferably at least 55 vol. %, more preferably at least 65 vol. %, and most preferably at least 75 vol. % of the lipid globules have a diameter between 2 and 10 μm. In an even more preferred embodiment, based on the total lipid volume, at least 45 vol. %, more preferably at least 55 vol. %, even more preferably at least 65 vol. %, and most preferably at least 75 vol. % of the lipid globules have a diameter between 4 and 10 μm. Preferably, based on the total lipid volume, less than 5 vol. % of the lipid globules have a diameter above 12 μm.

[0101] Standard infant formula, follow-on formula or growing-up milks typically have lipid globules which, based on volume, have a modal diameter of about 0.3 - 0.5 μm and / or, based on the total lipid volume, less than 45 vol. % of the lipid globules have a diameter above 2 μm.

[0102] The volume percentages of the lipid globules are based on the total lipid volume. The modal diameter relates to the diameter which is present in the highest amount based on volume % of the total lipid, or the peak in a graph which represents the diameter on the x-axis and the volume % on the y-axis.

[0103] The volume of the lipid globules and their size distribution can be suitably determined using a particle size analyzer such as a Mastersizer 2000 (Malvern Instruments, Malvern, UK), for example by the method described by Michalski et al., 2001, Lait 81:787 - 796.

[0104] Phospholipid

[0105] The nutritional composition comprises from 0.5 wt.% to 20 wt.% of phospholipids, preferably from 0.5 wt.% to 10 wt.%, even more preferably from 0.75 wt.% to 8 wt.%, even more preferably from 1.2 wt.% to 8 wt.%, and most preferably from 1.5 wt.% to 5 wt.% of phospholipids, based on the total lipids.

[0106] These lipid globules are at least partially coated with phospholipids on the surface. By "coated" it is meant that the outer surface layer of the lipid globules contains phospholipids, while phospholipids are present in negligible amounts in the lipid globule core. A suitable way to determine whether phospholipids are located on the surface of the lipid globules is confocal laser scanning microscopy or transmission electron microscopy; see for example Gallier et al. (A novel infant milk formula concept: Mimicking the human milk fat globule structure, Colloids and Surfaces B: Biointerfaces 136 (2015) 329 - 339).

[0107] The nutritional composition preferably contains glycerophospholipids. Examples of glycerophospholipids are phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylglycerol (PG). Preferably, the nutritional composition contains one or more of PC, PS, PI and PE, and more preferably, the nutritional composition contains at least PC.

[0108] The nutritional composition preferably contains sphingomyelin. Sphingomyelin has a phosphocholine or phosphoethanolamine molecule esterified to a ceramide with a 1 - hydroxy group. They are classified as phospholipids and sphingolipids, but not as glycerophospholipids or glycosphingolipids. Preferably, the nutritional composition contains from 0.05 wt.% to 10 wt.%, more preferably from 0.1 wt.% to 5 wt.%, even more preferably from 0.2 wt.% to 2 wt.% of sphingomyelin, based on total lipids. Preferably, the nutritional composition contains at least 5 wt.%, more preferably from 5 wt.% to 40 wt.%, more preferably from 10 wt.% to 35 wt.%, even more preferably from 15 wt.% to 35 wt.% of sphingomyelin, based on total phospholipids.

[0109] The nutritional composition preferably contains glycosphingolipids. Preferably, the nutritional composition contains from 0.1 wt.% to 10 wt.%, more preferably from 0.5 wt.% to 5 wt.%, even more preferably from 2 wt.% to 4 wt.% of glycosphingolipids, based on total lipids. The term glycosphingolipid herein specifically refers to a glycolipid having the amino alcohol sphingosine. The sphingosine backbone is O - linked to a charged head group (such as an ethanolamine, serine or choline backbone). The backbone is also linked to an amide group of a fatty acyl group. Glycosphingolipids are ceramides having one or more sugar residues, which are linked by a β - glycosidic bond at the 1 - hydroxy position and include gangliosides. Preferably, the nutritional composition contains gangliosides, and more preferably at least one ganglioside selected from the group consisting of GM3 and GD3.

[0110] The nutritional composition preferably contains phospholipids derived from mammalian milk. Preferably, the nutritional composition contains phospholipids and glycosphingolipids derived from mammalian milk. The nutritional composition preferably contains phospholipids and optionally glycosphingolipids from mammalian milk, which mammalian milk is from cows, mares, sheep, goats, water buffalo, horses, and / or camels. More preferably, the nutritional composition contains phospholipids and optionally glycosphingolipids from cow's milk.

[0111] Phospholipids derived from milk preferably include phospholipids isolated from milk fat, milk cream lipids, cream serum lipids, butter serum lipids (β-serum lipids), whey lipids, cheese lipids, and / or buttermilk lipids. Buttermilk lipids are typically obtained during the manufacture of buttermilk. Casein serum lipids or β-serum lipids are typically obtained during the manufacture of anhydrous milk fat from butter. Preferably, the phospholipids and optionally the glycosphingolipids are obtained from milk cream. Examples of suitable commercially available sources of phospholipids from milk are BAEF, SM2, SM3, and SM4 powders from Corman, Salibra from Glanbia, Lipamin M20 from Lecico, Vivinal from FrieslandCampina MFGM and LacProdan MFGM-10 or PL20 from Arla.

[0112] The use of phospholipids from milk fat advantageously includes the use of milk fat globule membrane, which is more similar to the situation in human milk. Thus, the concomitant use of phospholipids derived from milk and triglycerides from a mixture of plant lipids and mammalian milk fat enables the manufacture of coated lipid globules with a coating more similar to human milk while providing an optimal fatty acid profile.

[0113] Preferably, the phospholipids are derived from mammalian milk fat, more preferably from bovine mammalian milk fat. Preferably, the phospholipids are derived from or form part of the milk fat globule membrane (MFGM), more preferably from bovine MFGM or form part of bovine MFGM.

[0114] Preferably, the nutritional composition contains phospholipids and glycosphingolipids. In a preferred embodiment, the weight ratio of phospholipids:glycosphingolipids is from 2:1 to 12:1, more preferably from 2:1 to 10:1, and even more preferably 2:1 to 5:1.

[0115] Methods for obtaining lipid globules with increased size and having a phospholipid coating are disclosed, for example, in WO 2010 / 027258 and WO 2010 / 027259.

[0116] Digestible carbohydrate

[0117] The nutritional composition comprises digestible carbohydrates. The digestible carbohydrates preferably provide from 25% to 75% of the total calories of the nutritional composition. Preferably, the digestible carbohydrates provide from 40% to 60% of the total calories. Based on calories, the nutritional composition preferably comprises from 5 to 20 g of digestible carbohydrates / 100 kcal, more preferably from 6 to 16 g of digestible carbohydrates / 100 kcal. When in liquid form (e.g., as an instant liquid), the nutritional composition preferably comprises from 3 to 30 g of digestible carbohydrates / 100 ml, more preferably from 6 to 20 g, even more preferably from 7 to 10 g / 100 ml. Based on dry weight, the nutritional composition preferably comprises from 20 wt.% to 80 wt.%, more preferably from 40 wt.% to 65 wt.% of digestible carbohydrates.

[0118] Preferred sources of digestible carbohydrates are one or more of lactose, glucose, sucrose, fructose, galactose, maltose, starch, and maltodextrin. Lactose is the main digestible carbohydrate present in human milk. Lactose has a low glycemic index. The nutritional composition preferably comprises lactose. The nutritional composition preferably comprises digestible carbohydrates, wherein at least 35 wt.%, more preferably at least 50 wt.%, more preferably at least 75 wt.%, even more preferably at least 90 wt.%, most preferably at least 95 wt.% of the digestible carbohydrates are lactose. Based on dry weight, the nutritional composition preferably comprises at least 25 wt.% of lactose, preferably at least 40 wt.% of lactose.

[0119] Protein

[0120] The nutritional composition comprises protein. The protein preferably provides from 5% to 20% of the total calories. Preferably, the nutritional composition comprises protein providing from 6% to 12% of the total calories. Preferably, the nutritional composition comprises less than 3.5 g of protein / 100 kcal, more preferably, the nutritional composition comprises from 1.5 to 2.1 g of protein / 100 kcal, even more preferably from 1.6 to 2.0 g of protein / 100 kcal. The low protein concentration is advantageously closer to human milk, since human milk contains a lower amount of protein based on total calories compared to cow's milk. The protein concentration in the nutritional composition is determined by the sum of protein, peptides, and free amino acids. Based on dry weight, the nutritional composition preferably comprises less than 12 wt.% of protein, more preferably between 9.6 wt.% and 12 wt.%, even more preferably between 10 wt.% and 11 wt.%. Based on ready-to-drink liquid products, the nutritional composition preferably comprises less than 1.5 g of protein / 100 ml, more preferably between 1.2 and 1.5 g / 100 ml, even more preferably between 1.25 and 1.35 g / 100 ml.

[0121] The source of the protein is preferably selected in such a way that it meets the minimum requirements for the essential amino acid content and ensures satisfactory growth. Thus, protein sources based on milk proteins such as whey proteins, caseins and mixtures thereof, and proteins based on soy, potato or pea are preferred. In the case of using whey proteins, the protein source is preferably based on acid whey or sweet whey, whey protein isolate or mixtures thereof. Preferably, the nutritional composition comprises at least 3 wt.% casein based on dry weight. Preferably, the casein is intact and / or non-hydrolyzed.

[0122] Indigestible carbohydrate

[0123] The nutritional composition preferably comprises indigestible oligosaccharides. Preferably, the nutritional composition comprises indigestible oligosaccharides having a degree of polymerization (DP) between 2 and 250, more preferably between 3 and 60.

[0124] Preferably, the nutritional composition comprises fructooligosaccharides, galactooligosaccharides and / or galacturonic acid oligosaccharides, more preferably fructooligosaccharides and / or galactooligosaccharides, even more preferably galactooligosaccharides, most preferably trans-galactooligosaccharides. In a preferred embodiment, the nutritional composition comprises a mixture of galactooligosaccharides and fructooligosaccharides, more preferably trans-galactooligosaccharides and fructooligosaccharides. Suitable indigestible oligosaccharides are for example Vivinal GOS (FrieslandCampina DOMO), Raftilin HP or Raftilose (Orafti).

[0125] Preferably, the nutritional composition comprises 80 mg to 2 g of indigestible oligosaccharides per 100 ml, more preferably 150 mg to 1.5 g per 100 ml, even more preferably 300 mg to 1 g per 100 ml. Based on dry weight, the nutritional composition preferably comprises 0.25 wt.% to 20 wt.%, more preferably 0.5 wt.% to 10 wt.%, even more preferably 1.5 wt.% to 7.5 wt.% of indigestible oligosaccharides.

[0126] Formula food

[0127] The use according to the invention requires the administration of an infant formula, a follow-on formula or a growing-up milk. This means that the nutritional composition is not human milk. This also means that the nutritional composition is not natural cow's milk or natural milk from another mammal. In the context of the present invention, a growing-up milk may also be referred to as a growing milk.

[0128] Alternatively, as used herein, the term "infant formula" or "follow-on formula" or "toddler formula" means that it refers to an artificially manufactured composition, or in other words it is synthetic. Thus, in one embodiment, the administered nutritional composition is an artificial infant formula or an artificial follow-on formula or an artificial toddler formula or a synthetic infant formula or a synthetic follow-on formula or a synthetic toddler formula.

[0129] In the present invention, infant formula refers to an artificially manufactured nutritional composition intended for infants from 0 to about 4 to 6 months of age and intended as a substitute for human milk. Typically, infant formula is suitable for use as the sole source of nutrition. Such infant formula is also referred to as first stage formula. Follow-on formula for infants from 4 to 6 months to 12 months of age is intended as a complementary feeding for infants who are starting to be weaned onto other foods. Infant formula and follow-on formula are strictly regulated, such as by EU Regulations No. 609 / 2013 and No. 2016 / 127. In the context of the present invention, toddler formula refers to an artificially manufactured nutritional composition intended for infants from 12 months to 36 months of age, which is intended as a complementary feeding for infants.

[0130] The nutritional composition is preferably an infant formula or a follow-on formula. More preferably, the nutritional composition is an infant formula.

[0131] The nutritional composition comprises digestible carbohydrates, proteins and lipids, wherein the lipids preferably provide 30% to 60% of the total calories, the proteins provide 5% to 20% of the total calories, and the digestible carbohydrates provide 25% to 75% of the total calories.

[0132] The nutritional composition is preferably an infant formula or a follow-on formula and preferably comprises 3 to 7 g of lipids / 100 kcal, preferably 4 to 6 g of lipids / 100 kcal, more preferably 4.5 to 5.5 g of lipids / 100 kcal, preferably comprises 1.7 to 3.5 g of proteins / 100 kcal, more preferably 1.8 to 2.1 g of proteins / 100 kcal, more preferably 1.8 to 2.0 g of proteins / 100 kcal, and preferably comprises 5 to 20 g of digestible carbohydrates / 100 kcal, preferably 6 to 16 g of digestible carbohydrates / 100 kcal, more preferably 10 to 15 g of digestible carbohydrates / 100 kcal.

[0133] Preferably, the nutritional composition is an infant formula or a follow-on formula, and preferably when in ready-to-drink form, it has an energy density of 60 to 75 kcal / 100 ml, more preferably 60 to 70 kcal / 100 ml. This density ensures an optimal balance between hydration and caloric intake.

[0134] In one embodiment, the nutritional composition is a powder. Suitably, the nutritional composition is in powder form, which can be reconstituted with water or other food-grade aqueous liquids to form a ready-to-drink liquid, or in liquid concentrate form, which should be diluted with water to form a ready-to-drink liquid. It has been found that the lipid globules maintain their size and coating upon reconstitution.

[0135] A second aspect of the invention relates to a nutritional composition selected from infant formula, follow-on formula, and toddler formula, the nutritional composition comprising digestible carbohydrates, protein, and lipid, wherein the lipid is in the form of lipid globules, and wherein

[0136] a. based on volume, the lipid globules have a mode diameter of at least 1.0 μm; and / or

[0137] b. based on the total lipid volume, at least 45 vol% of the lipid globules have a diameter of 2 to 12 μm; and

[0138] c. the lipid comprises 0.5 wt.% to 20 wt.% of phospholipids based on the total lipid, and wherein the lipid globules are at least partially coated with phospholipids on the surface,

[0139] For use in increasing the levels of myelin-associated glycoprotein (MAG), myelin basic protein (MBP), or a combination thereof, in the brain of an infant with delayed brain development or at risk of having delayed brain development.

[0140] A third aspect of the invention relates to a nutritional composition selected from infant formula, follow-on formula, and toddler formula, the nutritional composition comprising digestible carbohydrates, protein, and lipid, wherein the lipid is in the form of lipid globules, and wherein

[0141] a. based on volume, the lipid globules have a mode diameter of at least 1.0 μm; and / or

[0142] b. based on the total lipid volume, at least 45 vol% of the lipid globules have a diameter of 2 to 12 μm; and

[0143] c. the lipid comprises 0.5 wt.% to 20 wt.% of phospholipids based on the total lipid, and wherein the lipid globules are at least partially coated with phospholipids on the surface,

[0144] For use in increasing the levels of palmitic acid (C16:0), stearic acid (C18:0), or combinations thereof in the brain cell membranes of infants suffering from or at risk of suffering from brain developmental delay.

[0145] The embodiments previously described herein with respect to the first aspect of the invention are preferably also applicable to the second and third aspects of the invention.

[0146] In this document and its claims, the verb "comprise" and its conjugations are used in their non-limiting sense, meaning including the items following the word, but not excluding items not specifically mentioned. Furthermore, the reference to an element by the indefinite article "a" or "an" does not exclude the possibility of there being more than one element, unless the context clearly requires that there be one and only one element. Thus, the indefinite article "a" or "an" generally means "at least one".

[0147] Examples

[0148] Experimental diets used in Examples 1 and 2

[0149] The progeny diet (control or test) was provided to the animals in the form of freshly prepared soft dough daily. The soft dough was placed on the cage floor to allow easy access for the animals.

[0150] The progeny diet contained macronutrient and micronutrient compositions following AIN93G (Reeves PG et al., AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr 1993;123:1939-51). The progeny diet consisted of 28.3% (w / w) infant milk formula (IMF). The fat fraction was entirely derived from the IMF. Proteins, carbohydrates, and micronutrients were added to match AIN93G. In each experiment, the fat content and fatty acid profile of the control diet and the test diet were kept the same.

[0151] Table 1: Fatty acid composition (g / 100g IMF powder)

[0152]

[0153] Test IMF

[0154] The test IMF was prepared in a manner similar to that described in Example 1B of WO 2010 / 0027259. The lipid spheres of the test IMF are larger and coated with phospholipids. Test 1 IMF contains phospholipids derived from egg yolk. Test 2 IMF contains β-lactoserum powder as a source of milk-derived phospholipids, including a part of the milk fat globule membrane (MFGM). The phospholipid source was added before homogenization, which resulted in the presence of phospholipids in the coating of the lipid spheres.

[0155] Control IMF

[0156] The control IMF was prepared by high-pressure homogenization in a conventional manner, producing small lipid spheres. The control IMF contains the same source and the same amount of phospholipids as the test IMF. The phospholipid source was added after homogenization and thus is not located in the coating of the lipid spheres.

[0157] The size distribution of the lipid droplets was determined by laser scattering analysis (Mastersizer 2000, Hydro 200G, Malvern Instruments Limited, Worcestershire, UK). Table 2 shows the detailed characteristics of the lipid sphere structure in different IMFs.

[0158] Table 2

[0159]

[0160] Example 1

[0161] During pregnancy and lactation, maternal mice were exposed to a ω3-deficient diet, which led to impaired brain development in the offspring (Carrié et al., 2000, DOI: https: / / doi.org / 10.1016 / S0022-2275(20)34486-2). C57BL / 6J mice were used in this experiment. Starting 4 weeks before breeding, female mice were maintained on a semi-synthetic AIN-93G-based rodent diet, in which the total fat was 7% (w / w) by weight of the diet, which included 2.58% (w / w) of linoleic acid C18:2(n6) and 0.01% (w / w) of α-linolenic acid C18:3(n3) by weight of the diet, as the sources of ω6 and ω3 PUFA, respectively (= ω3-deficient diet). The ω3-deficient diet was continued throughout pregnancy and lactation. The day of litter birth was considered postnatal (PN) day 0. At PN2, litters were culled to 6 pups per dam (litters contained at least 2 males and 2 females). Starting at PN16, litters (and dams) were randomly assigned to receive either of two experimental diets. At PN21, male offspring were weaned and continued on their respective diets. At PN42, mouse offspring were sacrificed. Brains were harvested and the hippocampi were dissected.

[0162] One hippocampus from each brain was homogenized (Ultra-Turrax T25 basic, IKA, VWR international) in 50 volumes of ice-cold deionized water (MiliQ). Subsequently, the brain fatty acid (FA) profile was quantified by gas chromatography analysis. 1 ml of brain homogenate was extracted according to the procedure of Bligh & Dyer (dichloromethane / methanol extraction). Lipids were converted to methyl esters with sulfuric acid in methanol. Fatty acid methyl esters (FAME) were extracted from the methanol solution with hexane and analyzed on a gas chromatograph (GC) equipped with a flame ionization detector (FID).

[0163] The other hippocampus from each animal was used to quantify the gene expression (qPCR) of myelin basic protein (MBP) and myelin-associated glycoprotein (MAG), which are markers of myelin. Total RNA was isolated using the RNeasy mini kit ( QIAGEN), and then cDNA synthesis was performed using the iScript tm cDNA synthesis kit (Bio-rad). The obtained cDNA was used for qPCR to quantify the gene expression of MBP and MAG using SYBR TM Select Master Mix (Applied Biosystems TM ).

[0164] All data were analyzed using Student's t - test with SPSS 19.0 software (SPSS Benelux, Gorinchem, The Netherlands). Individual animals were considered the experimental units.

[0165] Results

[0166] Brain fatty acid species are presented in Table 3 as a percentage of total fatty acids (% FA). Values in parentheses are standard deviations.

[0167] Table 3

[0168]

[0169] The fatty acids palmitic acid (C16:0) and stearic acid (C18:0) are the major saturated fatty acids in brain tissue. Brain myelin is particularly rich in these fatty acids. Both C16:0 and C18:0 were increased in the brains of offspring fed the Test 1 diet compared to the brains of offspring fed Control 1.

[0170] The gene expression of hippocampal MAG and MBP (relative to the expression of the housekeeping genes 18s, B2M, Gusb, HRPT1, TBP, Tubb3, and actin) in the brains of offspring fed Test 1 was higher than that in the brains of offspring fed Control 1. See Table 4 below. Values in parentheses are the standard error of the mean.

[0171] Table 4

[0172]

[0173] Example 2

[0174] C57BL / 6J mice were used in this experiment. The day of litter birth was considered postnatal (PN) day 0. At PN2, litters were randomized and culled to a standardized litter size of 3 pups per dam (litters contained at least 1 male and 1 female). Starting at PN16, litters (and dams) were randomly assigned to receive either of two experimental diets. Five days after diet exposure, and thus at PN21, the mouse offspring were sacrificed.

[0175] One hemisphere of each brain was homogenized (Ultra-Turrax T25 basic, IKA, VWR International) in 50 volumes of ice-cold deionized water (MiliQ). Subsequently, the brain fatty acid (FA) profiles were quantified by gas chromatography. One milliliter of brain homogenate was extracted according to the procedure of Bligh & Dyer (dichloromethane / methanol extraction). Lipids were converted to methyl esters with concentrated sulfuric acid in methanol. Fatty acid methyl esters (FAME) were extracted from the methanol solution with hexane and analyzed on a gas chromatograph (GC) equipped with a flame ionization detector (FID).

[0176] Data were analyzed using Student's t-test with SPSS 19.0 software (SPSS Benelux, Gorinchem, the Netherlands). Individual animals were considered the experimental units.

[0177] Results

[0178] Brain fatty acid species are presented in Table 5 as a percentage of total fatty acids (%FA). Values in parentheses are standard deviations.

[0179] Table 5

[0180]

[0181] Both C16:0 and C18:0 were increased in the brains of offspring fed the Test 2 diet compared to the brains of offspring fed Control 2.

[0182] Example 3

[0183] An infant formula, intended for infants from 0 to 6 months of age, after reconstitution of 13.7 g of powder to a final volume of 100 ml, contains per 100 ml:

[0184] - 66 kcal

[0185] - 1.3 g of protein (whey protein / casein weight ratio 1 / 1)

[0186] - 7.3 g of digestible carbohydrates (mainly lactose)

[0187] - 3.4 g of fat (containing 97 wt.% vegetable oil, about 1.5 wt.% milk-derived phospholipids, the remainder being fish oil and microbial oil, by weight of total lipids)

[0188] - 0.8 g of indigestible oligosaccharides, of which 0.08 g is long-chain inulin (source Raftiline HP) and 0.72 g is trans-galactooligosaccharides (source Vivinal GOS)

[0189] - Minerals, vitamins, trace elements and other micronutrients in accordance with the infant formula specifications.

[0190] The formulated food contains lipid globules having a volume mode diameter of about 5.6 μm, and the volume percentage of lipid globules having a mode diameter between 2 and 12 μm is higher than 45.

Claims

1. A nutritional composition selected from infant formula, follow-on formula, and toddler formula, the nutritional composition comprising digestible carbohydrates, protein, and lipids, wherein the lipids are in the form of lipid droplets, and wherein a. based on volume, the lipid droplets have a mode diameter of at least 1.0 μm; and / or b. based on the total lipid volume, at least 45% by volume of the lipid droplets have a diameter of 2 to 12 μm; and c. the lipids comprise 0.5 wt.% to 20 wt.% of phospholipids based on the total lipids, and wherein the lipid droplets are at least partially coated with phospholipids on the surface, for use in increasing myelination in the brain of an infant suffering from or at risk of suffering from brain developmental delay.

2. The nutritional composition for use according to claim 1, wherein the increased myelination contributes to improving the cognitive development and / or motor function of the infant.

3. The nutritional composition for use according to claim 1 or 2, wherein the increased myelination is around axons connected to the hippocampus and within the hippocampus.

4. The nutritional composition for use according to any one of the preceding claims, wherein the infant is a child aged 0 - 12 months.

5. The nutritional composition for use according to any one of the preceding claims, wherein the infant suffering from or at risk of suffering from brain developmental delay is selected from premature infants, small for gestational age infants, infants with phenylketonuria (PKU), infants with epilepsy, infants with cerebral palsy, infants born with brain injury, infants exposed to perinatal hypoxia, or combinations thereof.

6. The nutritional composition for use according to any one of the preceding claims, wherein the phospholipids comprise at least 5 wt.% of sphingomyelin based on the total phospholipids.

7. The nutritional composition for use according to any one of the preceding claims, wherein the phospholipids are derived from mammalian milk fat.

8. The nutritional composition for use according to any one of the preceding claims, wherein the lipids contain at least 10 wt.% of palmitic acid based on the total fatty acids, and based on the total palmitic acid, at least 15 wt.% of the palmitic acid is in the sn-2 position of the triglyceride.

9. The nutritional composition for use according to any one of the preceding claims, wherein the increased myelination is through higher levels of myelin-associated glycoprotein (MAG), myelin basic protein (MBP), or a combination thereof in the brain.

10. The nutritional composition for use according to any one of the preceding claims, wherein the increased myelination is through increasing the levels of palmitic acid (C16:0), stearic acid (C18:0), or a combination thereof in the brain cell membrane.

11. The nutritional composition for use according to any one of the preceding claims, wherein the nutritional composition is infant formula or follow-on formula.

12. A nutritional composition according to any one of the preceding claims, wherein the increased myelination in the brain of the infant is compared to a similar infant suffering from or at risk of suffering from delayed brain development, the similar infant having consumed a nutritional composition selected from infant formula, follow-on formula and growing-up milks, the nutritional composition comprising digestible carbohydrates, protein and lipids, and wherein the lipids are in the form of lipid globules, and wherein: a. based on volume, the lipid globules have a modal diameter of about 0.5 μm, and b. based on total lipid volume, less than 45% by volume of the lipid globules have a diameter greater than 2 μm, and c. wherein the lipids comprise less than 0.5 wt.% phospholipids based on total lipids, and wherein the lipid globules are not coated with phospholipids.

13. A nutritional composition selected from infant formula, follow-on formula and growing-up milks, the nutritional composition comprising digestible carbohydrates, protein and lipids, wherein the lipids are in the form of lipid globules, and wherein a. based on volume, the lipid globules have a modal diameter of at least 1.0 μm; and / or b. based on total lipid volume, at least 45% by volume of the lipid globules have a diameter of 2 to 12 μm; and c. the lipids comprise 0.5 wt.% to 20 wt.% phospholipids based on total lipids, and wherein the lipid globules are at least partially coated with phospholipids on the surface, for use in increasing the levels of myelin-associated glycoprotein (MAG), myelin basic protein (MBP), or a combination thereof, in the brain of an infant suffering from or at risk of suffering from delayed brain development.

14. A nutritional composition selected from infant formula, follow-on formula and growing-up milks, the nutritional composition comprising digestible carbohydrates, protein and lipids, wherein the lipids are in the form of lipid globules, and wherein a. based on volume, the lipid globules have a modal diameter of at least 1.0 μm; and / or b. based on total lipid volume, at least 45% by volume of the lipid globules have a diameter of 2 to 12 μm; and c. the lipids comprise 0.5 wt.% to 20 wt.% phospholipids based on total lipids, and wherein the lipid globules are at least partially coated with phospholipids on the surface, for use in increasing the levels of palmitic acid (C16:0), stearic acid (C18:0), or a combination thereof, in the brain cell membranes of an infant suffering from or at risk of suffering from delayed brain development.

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