Vitamin-containing composition and use thereof

Abstract

The present invention relates to a composition comprising at least one vitamin for use in promoting, supporting or optimizing one or more of the following: (i) de novo myelination; (ii) brain structure; (iii) brain connection; (iv) intellectual potential; (v) cognitive potential; (vi) learning potential; and (vii) cognitive function, wherein the composition is for administration to the female subject.

Description

[0001] This application is a divisional application of Chinese patent application 201780016224.3, with an application date of March 30, 2017, an earliest priority date of March 30, 2016, and a publication number of CN108778279A. Technical Field

[0002] The present invention relates to a composition for promoting, supporting or optimizing de novo myelination, and / or brain structure, and / or brain connectivity, and / or one or more of cognitive potential, learning potential and intellectual potential, and / or cognitive function in the offspring of a female individual. Background Art

[0003] In utero and during the first weeks or months of life, there are a number of external factors that can influence a baby's growth and development. In particular, continuing research is increasingly showing that a mother's diet can influence a baby's neurodevelopment, particularly brain development, and that this influence can have irreversible short-term and long-term consequences.

[0004] In utero and during the first 2 or 3 years of life, neurological development, especially brain development, is rapid and places particularly high demands on the infant for key nutrients. Failure to meet these nutrient requirements during this critical period can lead to suboptimal neurological development, especially suboptimal brain development.

[0005] One process that plays a key role in neural development, particularly brain development, and may be particularly sensitive to nutritional factors is de novo myelination, by which bare axons are incorporated into myelin sheaths. This process, and particularly its subsequent trajectory, plays a key role in determining brain structure, particularly the amount and / or spatial distribution of myelinated material throughout the brain.

[0006] The correlation of brain structure, in particular the amount and / or spatial distribution of myelinated material throughout the brain, is well documented for cognitive function and intelligence. Essentially, the myelin sheath in the brain provides insulating sheets along neurons, thereby allowing nerve impulses to be conducted faster. However, it is the brain structure that affects brain connections, in particular the amount and / or spatial distribution of myelin sheaths throughout the brain, for example, by which pathways and how quickly and effectively to transmit messages in the form of nerve impulses within the brain, particularly between different brain regions. This intercerebral communication can play a role in cognitive function and learning, and may affect and even be used to physiologically limit intelligence, cognition and / or learning potential.

[0007] Therefore, it is necessary to identify the required nutrients to promote, support or optimize de novo myelination, particularly de novo myelination trajectory, thereby brain structure, particularly the amount and / or spatial distribution of myelinated material in the whole brain. It is also necessary to provide a composition that can be taken before pregnancy, during pregnancy and during lactation to ensure that the mother is fully supplied with any of the nutrients to meet the needs of her offspring, and thereby promote, support or optimize de novo myelination, particularly de novo myelination trajectory, and thereby brain structure, more particularly the amount and / or spatial distribution of myelinated material in the whole brain. In addition, it is necessary to find a way to optimize the intellectual potential and / or cognitive potential and / or learning potential and / or cognitive function of the offspring.

[0008] Furthermore, it is important to ensure that the mother is adequately supplied with said nutrients to ensure her health and to avoid any depletion of said nutrients in her body which might result from her offspring's requirements for said nutrients, and any associated health effects.

[0009] Surprisingly, the inventors have now found that certain vitamins may promote, support or optimize de novo myelination in infants, in particular the trajectory of de novo myelination, and / or brain structure, in particular the amount and / or spatial distribution of myelinated material throughout the brain. Summary of the invention

[0010] The present invention is based on a maternal supplement comprising at least one vitamin for administration to a female individual before or during pregnancy and / or when the offspring is receiving breast milk from the female individual. The maternal supplement of the present invention is considered to have a beneficial effect on the de novo myelination of the offspring. Promoting, supporting and / or optimizing de novo myelination by maternal supplements enables mothers to provide health benefits to their offspring, including long-term beneficial effects for the infant's later life when the infant is no longer breastfed.

[0011] A first aspect of the invention therefore relates to a composition comprising at least one vitamin for use in promoting, supporting or optimizing one or more of the following in offspring of a female individual:

[0012] (i) de novo myelination;

[0013] (ii) brain structure;

[0014] (iii) brain connectivity;

[0015] (iv) intellectual potential;

[0016] (v) cognitive potential;

[0017] (vi) learning potential; and

[0018] (vii) cognitive function,

[0019] The composition is for administration to a female individual.

[0020] Surprisingly, the inventors have found that a composition comprising at least one vitamin can promote, support or optimize de novo myelination, in particular the trajectory of de novo myelination, and / or brain structure, in particular the amount and / or spatiotemporal distribution of myelinated material throughout the brain, in an individual.

[0021] The current findings result from a nutritional analysis of the results of a longitudinal cognitive and brain imaging study in which de novo myelination, particularly de novo myelination trajectories, and / or brain structure, including the amount and spatial distribution of myelinated material throughout the brain, were examined and compared. Additional details and results of the study are given in the accompanying examples.

[0022] A second aspect of the invention relates to at least one vitamin for use in promoting, supporting or optimizing one or more of the following in offspring of a female individual:

[0023] (i) de novo myelination;

[0024] (ii) brain structure;

[0025] (iii) brain connectivity;

[0026] (iv) intellectual potential;

[0027] (v) cognitive potential;

[0028] (vi) learning potential; and

[0029] (vii) cognitive function,

[0030] Wherein the at least one vitamin is for administration to a female individual.

[0031] A third aspect of the invention relates to a method of promoting, supporting or optimizing one or more of the following in the offspring of a female individual:

[0032] (i) de novo myelination;

[0033] (ii) brain structure;

[0034] (iii) brain connectivity;

[0035] (iv) intellectual potential;

[0036] (v) cognitive potential;

[0037] (vi) learning potential; and

[0038] (vii) cognitive function,

[0039] The method comprises administering to the female individual a therapeutically effective amount of at least one vitamin.

[0040] A fourth aspect of the invention relates to the use of at least one vitamin for the preparation of a composition for promoting, supporting or optimizing one or more of the following in offspring of a female individual:

[0041] (i) de novo myelination;

[0042] (ii) brain structure;

[0043] (iii) brain connectivity;

[0044] (iv) intellectual potential;

[0045] (v) cognitive potential;

[0046] (vi) learning potential; and

[0047] (vii) cognitive function,

[0048] The composition is for administration to a female individual.

[0049] A fifth aspect of the invention relates to a composition comprising at least one vitamin, wherein the composition is used as a pre-conception supplement for promoting, supporting or optimizing one or more of the following in the offspring:

[0050] (i) de novo myelination;

[0051] (ii) brain structure;

[0052] (iii) brain connectivity;

[0053] (iv) intellectual potential;

[0054] (v) cognitive potential;

[0055] (vi) learning potential; and

[0056] (vii) Cognitive function. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 - Shown are the mean whole brain (whole white matter) myelination trajectories of breastfed infants and toddlers compared to those of infants and toddlers fed two commercial formulas containing different levels of folic acid.

[0058] Figure 1a - Shown are the mean regional myelination trajectories of breastfed infants and toddlers compared with those of infants and toddlers fed two commercial formulas containing different levels of folic acid.

[0059] Figure 1b - is a brain image showing myelinated brain areas associated with folate.

[0060] Figure 2 - is a brain image showing myelinated brain areas associated with vitamin B12.

[0061] Figure 2a - is a brain image showing myelinated brain areas associated with iron.

[0062] Figure 2b - is a brain image showing myelinated brain areas associated with zinc.

[0063] Figure 2c - is a brain image showing myelinated brain areas associated with calcium.

[0064] Figure 2d - is a brain image showing myelinated brain areas associated with phosphorus.

[0065] Figure 2e - is a brain image showing myelinated brain areas associated with magnesium.

[0066] Figure 2f - is a brain image showing myelinated brain areas associated with sphingomyelin.

[0067] Figure 2g - is a brain image showing myelinated brain areas associated with phosphatidylinositol.

[0068] Figure 2h - is a brain image showing myelinated brain areas associated with phosphatidylcholine.

[0069] Figure 2i - is a brain image showing myelinated brain areas associated with choline.

[0070] Figure 2j - is a brain image showing myelinated brain areas associated with docosahexaenoic acid.

[0071] Figure 2k - is a brain image showing myelinated brain areas associated with arachidonic acid.

[0072] Figure 3 : Shown are the effects of neuraminic acid on neuronal cell density and astrocyte density.

[0073] Figure 4 : Shown are the effects of stearic acid on neuronal cell density and astrocyte density.

[0074] Figure 5 : Shown are the effects of octanoic acid on neuronal cell density and astrocyte density.

[0075] Figure 6 : Shown are the effects of sphingomyelin on neurosphere number and neuronal proliferation.

[0076] Figure 7 : Relative abundance of major SM species in ingredients, infant formula, bovine milk and human milk are shown. (Error bars represent standard deviation when n=3).

[0077] Figure 8 : Relative fatty acid abundance of SM fractions from ingredients, infant formula, bovine milk and human milk is shown. (Error bars represent standard deviation when n=3).

[0078] Fig. 9 : Trajectory of myelination over 5 years in different brain regions of children fed Formula 3 (green) or Formula 6 (blue).

[0079] Fig.10 : Trajectory of myelination over 5 years in different brain regions of children fed Formula 6 (blue) or breastfed (breastfed for a minimum of 90 days) (purple).

[0080] Fig.11 : Trajectory of myelination over 5 years in different brain regions of children fed Formula 3 (green) or breastfed (breastfed for minimum 90 days) (purple).

[0081] Fig.12 : The effect of DHA on MBP, NF and / or MBP / NF at day 18 and / or day 30 is shown.

[0082] Fig.13 : The effect of stearic acid on A2B5, MBP, MAG, NF, MBP / NF and / or MAG / NF at day 14, day 18 and / or day 30 is shown.

[0083] Fig.14 : The effect of vitamin B12 on A2B5, NF, MBP / NF and / or MAG at day 12, day 18 and / or day 30 is shown.

[0084] Fig.15 : The effects of folic acid on A2B5, NF, MAG, MAG / NF and / or MBP / NF at day 12, day 18 and / or day 30 are shown.

[0085] Fig.16 : The effects of choline on A2B5, MAG and / or MBP at day 12, day 18 or day 30 are shown.

[0086] Fig.17: The effect of iron on A2B5, MBP, MAG, NF and / or MAG / NF at day 12, day 18 and / or day 30 is shown.

[0087] Fig.18 : The effect of zinc on MBP, NF and / or MBP / NF at day 12, day 18 and / or day 30 is shown.

[0088] Fig.19 : The effect of phosphorus on MAG, NF and / or MAG / NF at day 12, day 18 and / or day 30 is shown.

[0089] Fig. 20 : The effect of magnesium on A2B5, MBP, NF, MAG, MBP / NF and / or MAG / NF at day 22, day 18 and / or day 30 is shown.

[0090] Fig.21 : The effect of copper on A2BF, MAG and / or MAG / NF at day 12 and / or day 18 is shown.

[0091] Fig. 22 : Shown are the effects of phosphatidylcholine on A2B5 at day 12 and on MAG at day 18.

[0092] Fig.23 : The effects of phosphatidylinositol on A2B5, MBP, MAG, NF, MAG / NF at day 12, day 18 and / or day 30 are shown.

[0093] Fig.24 : The effect of phosphatidylserine on A2B5, NF and / or MAG / NF at day 12 and / or day 18 is shown.

[0094] Fig.25 : The effect of sphingomyelin on A2B5, MAG and / or MBP at day 12, day 18 and / or day 30 is shown.

[0095] Fig.26 : Shown are the effects of ceramide on A2B5 at day 12 and on MAG at day 18.

[0096] Fig. 27 : The effects of galactosylceramide on A2B5, MBP, NF and / or MBP / NF at day 12 and / or day 30 are shown.

[0097] Fig.28 : The effects of glucosylceramide on A2B5 at day 12 and on NF at days 12 and 18 are shown.

[0098] Fig.29: Shown are the effects of D-erythroceramide on A2B5 at day 12 and on MAG at day 18.

[0099] Fig.30 : Shown are the effects of ceramide-1-phosphate on A2B5 at day 12 and on NF and MAG at day 18.

[0100] Fig.31 : The effects of monosialoganglioside-3 (GM3) on A2B5, MBP, MAG and / or MBP / NF at day 12, day 18 and / or day 30 are shown.

[0101] Fig.32 : The effects of disialoganglioside 3 (GD3) on A2B5, MBP, NF and / or MAG at day 12, day 18 and / or day 30 are shown.

[0102] Fig.33 : The fatty acid profiles of phosphatidylinositol (PI), phosphatidylcholine, phosphatidyl (PC), phosphatidylserine (PS), and sphingomyelin used in Example 7 are shown.

[0103] Fig.34 : Shown are the effects of vitamin B12 on MAG and MBP mRNA expression and on the co-expression of MBP and BetaIII.

[0104] Fig.35 : Shown are the effects of ARA on MAG and MBP mRNA expression and the effects on the co-expression of MBP and BetaIII.

[0105] Fig.36 : Shown are the effects of stearic acid on MAG and MBP mRNA expression and on the co-expression of MBP and BetaIII.

[0106] Fig.37 : Shown are the effects of zinc on MAG and MBP mRNA expression and on the co-expression of MBP and BetaIII.

[0107] Fig.38 : Shows the effect of phosphatidylinositol on MAG and MBP mRNA expression.

[0108] Fig.39 : Shown are the effects of GD3 on MAG and MBP mRNA expression and the effects on the co-expression of MBP and BetaIII.

[0109] Fig.40 : Shows the effect of DHA on MAG and MBP mRNA expression and on the co-expression of MBP and BetaIII.

[0110] Fig.41 : Shows the effect of neuraminic acid on MAG and MBP mRNA expression and the effect on MBP and BetaIII co-expression.

[0111] Fig.42 : Shown are the effects of iron on MAG and MBP mRNA expression and on the co-expression of MBP and BetaIII.

[0112] Fig.43 : Shows the effect of phosphatidylcholine on MAG and MBP mRNA expression and on the co-expression of MBP and BetaIII.

[0113] Fig.44 : Shows the effect of phosphatidylserine on MAG and MBP mRNA expression and on the co-expression of MBP and BetaIII.

[0114] Fig.45 : Shows the effect of folic acid on MAG and MBP mRNA expression and on the co-expression of MBP and BetaIII.

[0115] Fig.46 : Shown are the effects of choline on MAG and MBP mRNA expression and on the co-expression of MBP and BetaIII.

[0116] Fig.47 : Shown are the effects of ceramide on MAG and MBP mRNA expression and on the co-expression of MBP and BetaIII.

[0117] Fig.48 : Shown are the effects of galactosylceramide on MAG and MBP mRNA expression and on the co-expression of MBP and BetaIII.

[0118] Fig.49 : Shown are the effects of glucosylceramide on MAG and MBP mRNA expression and on the co-expression of MBP and BetaIII.

[0119] Fig.50 : Shown are the effects of ceramide-1-phosphate on MAG and MBP mRNA expression and on the co-expression of MBP and BetaIII.

[0120] Fig.51 : Shows the effect of D-erythroceramide on the mRNA expression of MAG and MBP and the co-expression of MBP and BetaIII.

[0121] Fig.52 : Shows the effect of sphingomyelin on the co-expression of MBP and BetaIII.

[0122] Fig.53 : Shows the effect of GM3 on the co-expression of MBP and BetaIII.

[0123] Fig.54a - is a brain image showing myelinated brain areas associated with maternal DHA intake.

[0124] Fig.54b - is a brain image showing myelinated brain areas associated with maternal choline intake.

[0125] Fig.54c - is a brain image showing myelinated brain areas associated with maternal iron intake.

[0126] Fig.54d - is a brain image showing myelinated brain areas associated with maternal folate intake.

[0127] Fig.54e - is a brain image showing myelinated brain areas associated with maternal vitamin B12 intake.

[0128] Fig.55 The experimental procedure of the maturation experiment in Example 8 is shown.

[0129] Fig.56 The experimental procedure of the myelination experiment in Example 8 is shown. DETAILED DESCRIPTION

[0130] The present invention will now be described in more detail. It should be noted that the various aspects, features, embodiments and implementations described in this application may be compatible and / or may be combined together.

[0131] For the sake of clarity, all embodiments and aspects reported hereinafter shall apply mutatis mutandis to the different embodiments and aspects described for the present invention.

[0132] In one aspect of the invention, a synthetic composition comprising at least one vitamin is provided for promoting, supporting or optimizing de novo myelination, in particular the trajectory of de novo myelination, and / or brain structure, in particular the amount and spatial distribution of myelinated material throughout the brain (in particular as determined by de novo myelination and the trajectory of de novo myelination), and / or brain connectivity, and / or intellectual potential and / or cognitive potential and / or learning potential, and / or cognitive function in the offspring of a female individual, wherein the composition is for administration to a female individual.

[0133] The composition of the invention may prevent, reduce the risk of and / or alleviate suboptimal de novo myelination, in particular de novo myelination trajectory, and / or brain structure, in particular the amount and spatial distribution of myelinated material throughout the brain (in particular as determined by de novo myelination and de novo myelination trajectory), and / or intellectual potential and / or cognitive potential and / or learning potential, and / or cognitive function in the individual by promoting, supporting and / or optimizing de novo myelination, in particular de novo myelination trajectory, and / or brain structure, in particular the amount and spatial distribution of myelinated material throughout the brain (in particular as determined by de novo myelination and de novo myelination trajectory), and / or intellectual potential and / or cognitive potential and / or learning potential. This may be non-therapeutic or therapeutic.

[0134] In the context of the present invention, the term "comprising" or "including" does not exclude other possible elements. The composition of the present invention (including the various embodiments described herein) may comprise, consist of or consist essentially of the following elements: the basic elements and necessary limitations of the present invention described herein, and any other or optional ingredients, components or limitations described herein or as required.

[0135] As used herein, the term "promote" or "promoting" refers to a factor or factors that cause a process to occur.

[0136] As used herein, the term "support" or "supporting" refers to the factor or factors that maintain a process once it begins to occur.

[0137] As used herein, the term "optimize" or "optimizing" means to improve or enhance.

[0138] Unless otherwise indicated, all indicated nutrient amounts are expressed as amounts based on weight of the dry nutritional composition.

[0139] Unless otherwise indicated, all percentages expressed herein are by weight.

[0140] The term "cfu" is to be understood as colony forming units.

[0141] As used herein, the term "therapeutically effective amount" refers to an amount of an "active substance" (here a vitamin or a mixture thereof) that produces a therapeutic effect, for example, with respect to one or more of the following effects on the offspring: (i) de novo myelination; (ii) brain structure; (iii) brain connectivity; (iv) intellectual potential; (v) cognitive potential; (vi) learning potential and (vii) cognitive function.

[0142] As used herein, "metabolite" refers to a substance produced during metabolism.

[0143] As used herein, "metabolic precursor" refers to a substance from which another substance is formed through a metabolic reaction.

[0144] Where the nutrients may be contained in the composition in different forms (as such or in the form of salts, complexes or more complex structures contained in the nutrients), the amounts reported hereinafter are intended to refer to the amounts of the nutrients as such.

[0145] In a preferred embodiment, the composition promotes, supports or optimizes the amount and / or spatial distribution of myelinated material throughout the brain, in particular the amount and / or spatiotemporal distribution of myelinated material throughout the brain.

[0146] In a preferred embodiment, the composition promotes, supports or optimizes the de novo myelination trajectory.

[0147] Since human breast milk is the gold standard in infant nutrition, the de novo myelination trajectory measured or observed in individuals who are breastfed, more particularly exclusively breastfed from well-nourished or well-nourished mothers, may be considered optimal. Thus, a composition of the invention may be considered to optimize the myelination trajectory of an individual if it causes the de novo myelination trajectory of an individual to meet or be closer to the de novo myelination trajectory measured or observed in individuals who are breastfed, more particularly exclusively breastfed from well-nourished or well-nourished mothers.

[0148] If the distance between the de novo myelination trajectory of the offspring and any equivalent / same measurement point on the trajectory of the breastfed individual is at most 50%, particularly at most 25%, more particularly at most 20%, then the de novo myelination trajectory of the offspring can be considered to be consistent with or closer to the de novo myelination trajectory measured or observed in a breastfed, more particularly fully breastfed individual (particularly the breast milk of a well-nourished or nutritious mother). Non-limiting examples within the range of at most 50% include 50%, 40%, 30%, 25%, 20%, 10%, 5%, 1%, 0.5% and 0.01%. In particular, the trajectory will be considered bioequivalent.

[0149] The myelination trajectory can be measured at any combination of time points. In particular, the time points are within the first 5 years of a human individual's life, more particularly the first 2 or 3 years of a human's life, and even more particularly in the first year of a human's life.

[0150] The de novo myelination trajectory can be determined by measuring the myelin-associated water fraction and / or myelin-associated water pool of the individual at different time points, particularly at different time points through the first 5 years of life of the human individual, more particularly the first 2 or 3 years of life of the human individual, and even more particularly the first year of life of the human individual. The myelin-associated water fraction and / or myelin-associated water pool of the individual can be measured using multi-component relaxation (MCR) magnetic resonance imaging (MRI) technology, and in particular using the mcDESPOT technology (Deoni et al., 2008). In particular, the de novo myelination trajectory can be determined by measuring the myelin-associated water pool using the mcDESPOT technology (Magn. Reson. Med. 2008 60: 1372-1387), the subject matter of which is hereby incorporated by reference).

[0151] If the composition of the present invention makes one or more offspring in a standard neurodevelopment test, such as the score on the Mullen Early Learning Scale, meet or are closer to the score measured or observed in a breastfed, more particularly fully breastfed individual (particularly well-nourished or nutritious mother's breast milk), then the composition can be considered to optimize the cognitive function of the offspring. If the difference between one or more of the standardized neurodevelopmental test scores of the offspring (such as Mullen's T score) and one or more of the standardized neurodevelopmental test scores of the breastfeeding individual is less than one standard deviation, more particularly less than half of the standard deviation of the standardized test score, such as less than 10 points, more particularly less than 5 points of Mullen's T score, particularly less than 2 points, the cognitive and neurodevelopmental function of the offspring can be considered to meet or be closer to the cognitive and neurodevelopmental function measured in the breastfeeding individual. The standardized neurodevelopmental test score (such as Mullen's T score) is measured at the same time point in the individual and the breastfed individual.

[0152] The Mullen score may be measured at any suitable time point, and in particular within the first 3 years after birth of a human individual, more particularly within the first 2 years after birth of a human individual, and even more particularly within the first year after birth of a human individual.

[0153] As used herein, the term "female individual" refers to a female, especially before or during pregnancy, or shortly after a child is born. Preferably, the female individual is a mammalian individual, more preferably, a cat, a dog or a human. Even more preferably, the female individual is a human. In all cases, the female individual may be deficient or borderline deficient (subclinically deficient) in one or more vitamins, or the female individual cannot obtain an adequate daily supply of one or more vitamins through her diet.

[0154] As used herein, unless otherwise indicated, the term "offspring" encompasses offspring of any developmental stage of a female individual, including fetuses, newborns, infants, children and adults or other mammalian fetuses, cubs, or adult animals (e.g., cats and dogs). Preferably, in any embodiment of the invention, the term offspring is associated with humans and refers to the neonatal, infant and child stages, more preferably the neonatal and infant stages. Preferably, in any embodiment of the invention, the term offspring is associated with other mammals (e.g., cats and dogs) and refers to the neonatal or infant stage.

[0155] As used herein, the term "neonatal" refers to a newborn individual. In humans, the term neonate generally refers to an infant less than 4 weeks old.

[0156] As used herein, the term "infant" refers to a human infant up to 12 months of age, and includes premature and very premature infants, low birth weight infants (i.e., a newborn weighing less than 2500 g (5.5 pounds) whether due to prematurity or fetal growth restriction), and small for gestational age (SGA) infants (i.e., an infant with a birth weight below the 10th percentile for infants of the same gestational age).

[0157] As used herein, the term "child" refers to a human between 1 and 18 years old, more preferably between 1 and 10 years old, even more preferably between 1 and 5 years old, and even more preferably between 1 and 2 years old.

[0158] The expression "infant" means a child between the ages of one and five years (including toddlers).

[0159] A "preterm baby" or "preterm infant" is a baby or toddler born before the due date. It usually refers to a baby born before 37 weeks of gestation.

[0160] The expression "full-term baby" refers to a baby born after 37 weeks of gestation.

[0161] The expression "postpartum period" or "puerperium" refers to the period of time that begins immediately after the birth of a child and lasts for about six weeks.

[0162] In a preferred embodiment, the offspring is a formula-fed infant or child.As used herein, the term "formula-fed infant or child" refers to an infant or child who is fed infant formula and / or growing-up milk.

[0163] An exclusively breastfed infant or toddler / an infant or toddler fed exclusively with breast milk refers to an infant whose majority of nutrients and / or energy comes from human breast milk ("majority" is preferably at least 90% or at least 95%, or at least 99%).

[0164] An infant / young child who is primarily fed with infant formula generally refers to an infant / young child whose nutritional source of nutrients and / or energy is primarily derived from synthetic infant formula, follow-on milk or growing-up milk. "Mainly" means at least 50% of those nutrients and / or energy, or at least 75%.

[0165] As used herein, the term "de novo myelination" refers to the process by which exposed axons in the brain of an individual undergo myelination during growth and development. The process begins in utero and is most abundant in the first 5 years of life of a human individual, particularly the first 2 or 3 years of life of a human. More preferably, the term refers to the de novo myelination trajectory.

[0166] As used herein, the term "de novo myelination trajectory" refers to the extent of myelination as a function of time (as measured, for example, by the myelin water fraction), and particularly through infancy and childhood, particularly early childhood, and more particularly in the first 5 years of life of a human individual, more particularly in the first 2 or 3 years of life, or the first year of life in a human individual.

[0167] As used herein, the term "brain structure" refers to the structure of gray and white matter in the brain and specific brain regions, and particularly refers to the myelinated white matter in the brain and specific brain regions as determined by de novo myelination and particularly de novo myelination trajectory (i.e., by de novo structural deposition of myelin sheaths). More particularly, the term refers to the amount and / or spatial distribution of myelinated material in the whole brain and / or specific brain regions, and even more particularly the amount and / or spatiotemporal distribution of myelinated material in the whole brain and / or specific brain regions.

[0168] As used herein, the term "intelligence potential" refers to the possible intelligence and intelligence that an individual can obtain as determined by physiological factors. In particular, intelligence potential can refer to fluid intelligence.

[0169] As used herein, the term "fluid intelligence" refers to an individual's neural potential and / or an individual's ability to solve novel or abstract problems as determined by physiological factors. This is different from crystallized intelligence, which is determined at least in part by learned or culturally adapted knowledge.

[0170] As used herein, the term "cognitive potential" refers to the possible cognitive and / or mental abilities or mental capacity that an individual acquires as determined by physiological factors. In particular, the term may refer to one or more of the following: information processing potential, perceptual potential, attention potential, thinking potential, reasoning potential, comprehension and memory potential, psychomotor potential (including gross motor and fine motor potential), visual potential (including visual reception potential), language potential (including expression and reception language potential), memory and recall potential, concentration potential, executive function potential (including problem solving, decision making and inhibition potential).

[0171] As used herein, the term "learning potential" refers to the possible ability or capacity that an individual has to learn (e.g., how easily and / or quickly an individual can acquire knowledge or skills through experience, study, or being taught), as determined by physiological factors, and the possible ability that an individual has to adapt in response to environmental factors, as determined by physiological factors.

[0172] Vitamins (particularly B9 and / or B12) may be particularly effective in supporting, promoting or optimizing de novo myelination in the following brain regions, in particular de novo myelination trajectories, and / or brain structures, in particular the amount and / or spatial distribution of myelinated material throughout the brain: cerebellum, brainstem, temporal lobe, frontal lobe, visual cortex, motor and somatosensory cortex. Thus, vitamins may be particularly effective in promoting, supporting or optimizing visual potential, motor function (including coordination and execution of motor potential) and psychomotor potential.

[0173] In a preferred embodiment of the invention, the cognitive potential is selected from the group consisting of visual potential, motor function (including coordination and execution of movements) and psychomotor potential.

[0174] By optimizing these potentials, the compositions of the invention may optimize motor function (including coordination and execution of movements) and / or psychomotor function and / or vision.

[0175] Vitamins

[0176] The compositions of the present invention comprise one or more vitamins.

[0177] Vitamins are organic compounds and important nutrients that living organisms require in limited quantities. Vitamins are usually not synthesized by the body in sufficient quantities and must be obtained through the diet. Supplementation is important in treating certain health problems.

[0178] Vitamins are classified by their biological and chemical activity rather than by their chemical structure. Thus, each vitamin refers to a number of vitamer compounds that share a common biological activity associated with that particular vitamin. Such chemicals are grouped into general categories, such as "vitamin A," which includes the compounds retinal, retinol, and the four known carotenoids. Vitamers, by definition, can be converted into the active form of the vitamin in the body, and can sometimes be converted into each other.

[0179] There are currently 13 vitamins that are generally recognized, namely A, B1, B2, B3, B5, B6, B7, B9, B12, C, D, E and K.

[0180] Vitamins have different biochemical functions. Some vitamins (such as vitamin D) have hormone-like functions, acting as regulators of mineral metabolism or regulators of cell and tissue growth and differentiation (e.g., some forms of vitamin A). Others are used as antioxidants (e.g., vitamin E and sometimes vitamin C). The largest number of vitamins, the B complex vitamins, are used as enzyme cofactors (coenzymes). In this role, vitamins can be tightly bound to enzymes as part of a prosthetic group; for example, biotin is part of an enzyme involved in the production of fatty acids. They may also not be as tightly bound to enzyme catalysts as coenzymes. For example, folic acid can carry methyl, formyl, and methylene groups in cells.

[0181] Vitamins have traditionally been obtained through food intake alone, and changes in diet may drastically alter the type and amount of vitamins ingested. However, since the mid-20th century, vitamins have been produced as commodity chemicals and are widely available as multivitamin dietary and food supplements and additives of inexpensive semisynthetic and synthetic origin.

[0182] In a preferred embodiment, the composition comprises vitamin B9 (also known as folic acid or folate). Folic acid or folate is also known as vitamin M, vitamin B c (or folic acid analogs), pteroyl-L-glutamic acid and pteroyl-L-glutamate salts.

[0183]

[0184] Folic acid is produced synthetically and used in fortified foods and supplements on the basis that it is converted to folate, a synthetic oxidized form of folic acid that is converted to tetrahydrofolate (TEF) by the enzyme dihydrofolate reductase (DHFR).

[0185] Vitamin B9 is essential for many body functions. Humans are unable to synthesize folate de novo; therefore, folate must be provided through the diet to meet their daily needs. The body requires folate to synthesize, repair, and methylate DNA, as well as to act as a cofactor in certain biological reactions. This is especially important for aiding rapid cell division and growth. Children and adults alike need folate to produce healthy red blood cells and to prevent anemia.

[0186] Folic acid can be incorporated into the nutritional composition of the present invention as such or in the form of its physiologically acceptable salt (folate) or a mixture thereof. In the context of the present invention, the term "folic acid" includes all folic acid present in the composition of the present invention as such or in the form of its physiologically acceptable salt or a mixture thereof.

[0187] Preferably, folic acid is included in the composition in an amount from about 0.001% to about 99.999% of the composition.

[0188] In a preferred embodiment, the composition according to the present invention comprises a level of folic acid such that the total daily intake from the composition of the present invention is from about 50 μg to about 1000 μg, more preferably from about 60 μg to about 1000 μg, even more preferably from about 70 μg to about 700 μg, even more preferably from about 100 μg to about 500 μg, more preferably from about 200 μg to about 400 μg.

[0189] In a preferred embodiment, the composition comprises an amount of folic acid such that the total daily intake from the composition of the present invention will not exceed about 1000 μg.

[0190] In a preferred embodiment, the composition of the present invention comprises vitamin B12. Vitamin B12 (also known as cobalamin) is a water-soluble complex vitamin that plays a key role in the normal functioning of the brain / nervous system and red blood cell formation. Vitamin B12 is involved in cellular metabolism, particularly DNA synthesis / regulation, and also plays a role in fatty acid metabolism and amino acid metabolism. Animals cannot produce vitamin B12, but can only produce it industrially through bacterial fermentation synthesis.

[0191] Vitamin B12 encompasses a class of chemically related compounds (vitaminophen) that all display similar biological activity. It contains cobalt at the center of a planar tetrapyrrole ring called a corrin ring. The basic structure of the vitamin is biosynthesized as hydroxocobalamin, but conversion between different forms of the vitamin can occur in the body after consumption.

[0192]

[0193] The most common synthetic form of the vitamin is cyanocobalamin, produced by chemically modifying bacterial hydroxocobalamin. Given its superior stability and low cost, this form is used in many drugs and supplements and for the fortification of foods. In the body, it is converted into the human physiological forms methylcobalamin and 5'-deoxyadenosylcobalamin.

[0194] Vitamin B12 may be included in the composition in an amount from about 0.001% to about 99.999% of the composition.

[0195] In a preferred embodiment, the composition comprises an amount of vitamin B12 such that the total daily intake from the composition of the present invention is from about 0.2 μg to about 250 μg, more preferably from about 0.26 μg to about 50 μg, more preferably from about 0.5 μg to about 30 μg, even more preferably from about 1 μg to about 10 μg, more preferably from about 2 μg to about 6 μg.

[0196] In a highly preferred embodiment, the composition comprises an amount of vitamin B12 such that the total daily intake from the composition of the present invention is about 250 μg.

[0197] In a preferred embodiment, the composition comprises an amount of vitamin B12 such that the total daily intake from the composition of the present invention does not exceed about 50 μg.

[0198] Vitamin B12 may be incorporated into the composition of the invention as such or in the form of one of its physiologically acceptable salts or mixtures thereof.

[0199] If the composition of the invention comprises B12 and / or folic acid, it is particularly advantageous if it additionally comprises B6. The presence and amounts of specific minerals and other vitamins will vary depending on many factors, for example the age, weight and condition of the person or animal to which the composition is to be administered.

[0200] In a preferred embodiment, the composition of the present invention comprises vitamin B1.

[0201] Vitamin B1 is also known as thiamine or thiamine. Its structure consists of an aminopyrimidine and a thiazole ring connected by a methylene bridge. The thiazole is substituted with methyl and hydroxyethyl side chains.

[0202]

[0203] Thiamine is on the World Health Organization's Essential Medicines List, a list of the most important medicines needed for a basic health system. The phosphate derivative of vitamin B1 is involved in many cellular processes. The best characterized form is thiamine pyrophosphate (TPP), which is a coenzyme in the catabolism of sugars and amino acids. In yeast, TPP is also required for the first step of alcoholic fermentation. Thiamine mononitrate is a stable, non-hygroscopic salt that is often used for food fortification.

[0204] All living organisms use thiamine, but it is synthesized only in bacteria, fungi, and plants. Animals must obtain it from their diet, so, for humans, it is an essential nutrient. In mammals, deficiency can lead to Korsakoff syndrome, optic neuropathy, and a disease called beriberi that affects the peripheral nervous system (polyneuritis) and / or the cardiovascular system. In less severe cases, nonspecific signs include malaise, weight loss, irritability, and confusion. If thiamine deficiency is not treated, it can be fatal.

[0205] Vitamin B1 may be included in the composition in an amount from about 0.001% to about 99.999% of the composition.

[0206] Vitamin B1 may be incorporated into the composition of the invention as such or in the form of one of its physiologically acceptable salts or a mixture thereof.

[0207] In a preferred embodiment, the composition of the present invention comprises vitamin B2. Vitamin B2 is also known as riboflavin. Specifically, the active forms of riboflavin, namely flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), serve as cofactors for a variety of flavoprotein enzyme reactions, including the activation of other vitamins. It was formerly known as vitamin G.

[0208]

[0209] Vitamin B2 may be included in the composition in an amount from about 0.001% to about 99.999% of the composition.

[0210] Vitamin B2 may be incorporated into the composition of the invention as such or in the form of one of its physiologically acceptable salts or mixtures thereof.

[0211] In a preferred embodiment, the composition of the present invention comprises vitamin B3. Vitamin B3 (also known as niacin or nicotinic acid) is a derivative of pyridine with a carboxyl group (COOH) at the 3-position. Other forms of vitamin B3 include the corresponding amides and nicotinamide ("niacinamide"), in which the carboxyl group is replaced by a carboxamide group (CONH2), as well as more complex amides and esters.

[0212]

[0213] Drugs / supplementsNiacin is used primarily to treat hypercholesterolemia and pellagra (niacin deficiency). Inadequate dietary niacin can lead to nausea, skin and mouth lesions, anemia, headaches, and fatigue. Niacin deficiency is also observed in pandemic deficiency syndromes caused by deficiencies of five key vitamins (niacin, vitamin C, thiamine, vitamin D, and vitamin A) and is common in areas of widespread poverty and malnutrition.

[0214] Nicotinic acid and nicotinamide are precursors of the coenzymes nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) in the body. In the presence of the enzyme NAD+ kinase, NAD is converted into NADP by phosphorylation. NADP and NAD are coenzymes of many dehydrogenases and participate in hydrogen transfer processes. NAD is important in the catabolism of fats, carbohydrates, proteins and ethanol, as well as in cell signaling and DNA repair. NADP is important in anabolic reactions such as fatty acid and cholesterol synthesis.

[0215] Vitamin B3 may be included in the composition in an amount from about 0.001% to about 99.999% of the composition.

[0216] Vitamin B3 may be incorporated into the composition of the invention as such or in the form of one of its physiologically acceptable salts or mixtures thereof.

[0217] In a preferred embodiment, the composition of the present invention comprises vitamin B5. Vitamin B5 (also known as pantothenic acid or pantothenate) is a water-soluble vitamin that is an amide formed by pantothenic acid and beta-alanine. Only the dextrorotatory (D) isomer of pantothenic acid is biologically active, and studies have shown that the levorotatory (L) form can antagonize the effects of the dextrorotatory isomer.

[0218]

[0219] Pantothenic acid is essential for all life forms and is involved in a wide variety of biological processes. A deficiency in pantothenic acid can have many widespread effects.

[0220] Animals require pantothenic acid for the synthesis of coenzyme A (CoA), as well as for the synthesis and metabolism of proteins, carbohydrates, and fats. Pantothenic acid is found in small amounts in almost every food, and in large amounts in avocados, whole-grain cereals, beans, eggs, meat, royal jelly, and yogurt. It is commonly found as its alcohol analogs the provitamin panthenol (pantothenic acid) and calcium pantothenate. Pantethine is a dietary supplement whose chemical structure has two pantothenic acid molecules connected by two sulfur atoms. The effective dose of pantethine is more than 100 times higher than the vitamin requirement for pantothenic acid.

[0221] Vitamin B5 may be included in the composition in an amount from about 0.001% to about 99.999% of the composition.

[0222] Vitamin B5 may be incorporated into the composition of the invention as such or in the form of one of its physiologically acceptable salts or mixtures thereof.

[0223] In a preferred embodiment, the composition of the present invention comprises vitamin B6. Vitamin B6 refers to a group of chemically similar compounds that can be interconverted in biological systems. The active form of vitamin B6 (pyridoxal 5'-phosphate, PLP) is used as a coenzyme in many enzyme reactions in amino acid, glucose and lipid metabolism. More specifically, PLP is involved in many aspects of macronutrient metabolism, neurotransmitter synthesis, histamine synthesis, hemoglobin synthesis and function, and gene expression.

[0224]

[0225] Several forms (vitamin isoforms) of vitamin B6 are known: pyridoxine (PN; the most common form found in vitamin B6 supplements), pyridoxine 5'-phosphate (PNP), pyridoxal (PL), pyridoxal 5'-phosphate (PLP; the metabolically active form), pyridoxamine (PM), pyridoxamine 5'-phosphate (PMP), 4-pyridoxic acid (PA), and pyridoxine (a semisynthetic derivative of pyridoxine in which two pyridoxine moieties are joined by a disulfide bridge).

[0226] All forms except pyridoxal and pyritinol are interconvertible. Absorbed pyridoxamine is converted to PMP by pyridoxal kinase, which is further converted to PLP by pyridoxamine-phosphotransaminase or pyridoxine 5'-phosphate oxidase, which also catalyzes the conversion of PNP to PLP. Pyridoxine 5'-phosphate oxidase is dependent on flavin mononucleotide (FMN) as a cofactor produced by riboflavin (vitamin B2).

[0227] Vitamin B6 may be included in the composition in an amount from about 0.001% to about 99.999% of the composition.

[0228] Vitamin B6 may be incorporated into the composition of the invention as such or in the form of one of its physiologically acceptable salts or mixtures thereof.

[0229] In a preferred embodiment, the composition of the present invention comprises vitamin B7. Vitamin B7 is also known as biotin, vitamin H or coenzyme R. Structurally, it comprises a ureido (tetrahydroimidazoline) ring fused to a tetrahydrothiophene ring, wherein the valeric acid substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring.

[0230]

[0231] Biotin is necessary for cell growth, fatty acid production, and fat and amino acid metabolism. More specifically, biotin is a coenzyme for carboxylases involved in the synthesis of fatty acids, isoleucine and valine, and gluconeogenesis.

[0232] Biotin deficiency is rare because, in general, intestinal bacteria produce more biotin than the daily needs of the human body. However, there are many metabolic diseases in which individual metabolism of biotin is abnormal, such as the deficiency of the holocarboxylase synthetase that covalently links biotin to the carboxylase, in which biotin acts as a cofactor. Biotin deficiency may be due to insufficient dietary intake or the inheritance of one or more congenital genetic diseases that affect biotin metabolism. Subclinical deficiency can lead to mild symptoms, while congenital genetic diseases may have serious (even fatal) consequences. Biotin-thiamine sensitive basal ganglia disease is another potential life-threatening condition that requires biotin (and thiamine) for treatment.

[0233] Vitamin B7 may be included in the composition in an amount from about 0.001% to about 99.999% of the composition.

[0234] Vitamin B7 may be incorporated into the composition of the invention as such or in the form of one of its physiologically acceptable salts or mixtures thereof.

[0235] In a preferred embodiment, the composition of the present invention comprises vitamin A. Vitamin A encompasses a class of unsaturated organic compounds that includes retinol, retinal, retinoic acid, and several provitamin A carotenoids and beta-carotene. All forms of vitamin A have a beta-ionone ring attached to an isoprenoid chain, known as a retinyl group. Both structural features are essential for vitamin activity.

[0236]

[0237] Beta-carotene has two linked retinyl groups which serve in the body to contribute to vitamin A levels. Alpha-carotene and gamma-carotene have a single retinyl group which imparts some vitamin activity. The carotenoid beta-cryptoxanthin has an ionone group and has vitamin activity in humans.

[0238] Vitamin A is important for growth and development, for maintaining the immune system and good vision. More specifically, the retina of the eye requires vitamin A in the form of retinal, which binds to the protein opsin to form rhodopsin, a light-absorbing molecule essential for dark and color vision. Vitamin A also acts very differently from retinoic acid (the irreversibly oxidized form of retinol), an important hormone-like growth factor for epithelial and other cells.

[0239] In foods of animal origin, the main form of vitamin A is an ester, mainly retinyl palmitate, which is converted into retinol in the small intestine. Retinol is used as a storage form of the vitamin and can be converted into and from its visually active aldehyde form (retinal). Retinol is commercially produced and administered as an ester (such as retinyl acetate or palmitate). Vitamin A can also be in the form of the carotene alpha-carotene, beta-carotene, gamma-carotene, and lutein beta-cryptoxanthin (all containing a beta-ionone ring).

[0240] Vitamin A may be included in the composition in an amount from about 0.001% to about 99.999% of the composition.

[0241] Vitamin A may be incorporated into the composition of the invention as such or in the form of one of its physiologically acceptable salts or mixtures thereof.

[0242] In a preferred embodiment, the composition of the present invention comprises vitamin C. Vitamin C or L-ascorbic acid or just ascorbate (the anion of ascorbic acid) is an essential nutrient for humans and certain other animal species. Vitamin C encompasses several vitamin equivalents with similar activity in animals, including ascorbic acid and its salts, as well as some oxidized forms of the molecule, such as dehydroascorbic acid. When any of these is introduced into cells, both ascorbate and ascorbic acid are naturally present in the body, as the forms interconvert depending on pH.

[0243]

[0244] Vitamin C is a cofactor in at least eight enzymatic reactions, including several collagen synthesis reactions, which, when dysfunctional, cause the most severe symptoms of scurvy. Ascorbate can also act as an antioxidant to protect against oxidative stress.

[0245] All animals and plants require ascorbate for a range of essential metabolic reactions. It is made internally by nearly all living organisms. The biological role of ascorbate is to act as a reducing agent, donating electrons for a variety of enzymatic reactions and a few non-enzymatic reactions. The one-electron and two-electron oxidized forms of vitamin C, hemi-dehydroascorbic acid, and dehydroascorbic acid, respectively, can be reduced in vivo by glutathione- and NADPH-dependent enzymatic mechanisms. The presence of glutathione in cells and extracellular fluids helps maintain ascorbate in a reduced state.

[0246] Vitamin C may be included in the composition in an amount from about 0.001% to about 99.999% of the composition.

[0247] Vitamin C may be incorporated into the composition of the invention as such or in the form of one of its physiologically acceptable salts or mixtures thereof.

[0248] In a preferred embodiment, the composition of the present invention comprises vitamin D. Vitamin D refers to a group of fat-soluble steroidal compounds responsible for enhancing the intestinal absorption of calcium, iron, magnesium, phosphate and zinc. Vitamin D plays an important role in calcium homeostasis and metabolism. In humans, the most important compounds in this group are vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol). Cholecalciferol and ergocalciferol can be taken from the diet and supplements. Few foods contain vitamin D, and the main natural source of the vitamin is synthesized in the skin from cholesterol, which depends on sunlight (especially UVB radiation).

[0249] Vitamin D synthesized from the diet or by the sun's skin is biologically inactive; activation requires enzymatic conversion (hydroxylation) in the liver and kidneys. In the liver, cholecalciferol (vitamin D3) is converted into calciferol (also known as calcidiol (INN)), 25-hydroxycholecalciferol (also known as 25-hydroxyvitamin D3, abbreviated as 25 (OH) D3). Ergocalciferol (vitamin D2) is converted into 25-hydroxyergocalciferol (also known as 25-hydroxyvitamin D2, abbreviated as 25 (OH) D2) in the liver. A portion of calciferol is converted into calcitriol by the kidneys, which is the biologically active form of vitamin D. Calcitriol circulates as a hormone in the blood, thereby regulating the concentration of calcium and phosphate in the bloodstream and promoting healthy growth and remodeling of bones. Calcitriol also affects neuromuscular and immune function.

[0250] Vitamin D may be included in the composition in an amount from about 0.001% to about 99.999% of the composition.

[0251] Vitamin D may be incorporated into the composition of the invention as such or in the form of one of its physiologically acceptable salts or mixtures thereof.

[0252] In a preferred embodiment, the composition of the present invention comprises vitamin E. Vitamin E refers to a group of compounds including tocopherols and tocotrienols. Gamma-tocopherol is the most common form in the Western diet and is commonly found in corn oil, soybean oil, margarine and dressings.

[0253] Alpha-tocopherol is the most biologically active form of vitamin E and the second most common form of vitamin E in the diet. The nutritional content of vitamin E is defined by alpha-tocopherol activity. This variant is found in wheat germ oil, sunflower oil, and safflower oil. As a fat-soluble antioxidant, it stops the production of reactive oxygen species that are formed when fats undergo oxidation.

[0254]

[0255] The molecules that contribute to the activity of alpha-tocopherol are the four tocopherols and four tocotrienols identified by the prefixes alpha-, beta-, gamma-, and delta-. Natural tocopherols occur only in the RRR configuration. Synthetic forms contain eight different stereoisomers and are referred to as "all-racemic"-alpha-tocopherol. Water-soluble forms (such as d-alpha-tocopherol succinate) are used as food additives.

[0256] Vitamin E has many biological functions, and the antioxidant function is the most well-known. α-Tocopherol is a fat-soluble antioxidant, which is used as an antioxidant in the glutathione peroxidase pathway, thereby protecting cell membranes from oxidation by reacting with the lipid free radicals produced in the lipid peroxidation chain reaction. This will remove free radical intermediates and prevent the oxidation reaction from continuing. The oxidized α-tocopherol free radicals produced in the process can be recycled back to the active reduced form by the reduction of other antioxidants (such as ascorbate, retinol or panthenol). Other forms of vitamin E have their own unique characteristics; for example, γ-tocopherol is a nucleophilic substance that can react with electrophilic mutagens. Tocotrienols are not well understood, but are believed to have a special role in protecting neurons from damage and cholesterol reduction by inhibiting the activity of HMG-CoA reductase. δ-Tocotrienols prevent the processing of sterol regulatory element binding protein (SREBP).

[0257] Vitamin E may be included in the composition in an amount from about 0.001% to about 99.999% of the composition.

[0258] Vitamin E may be incorporated into the composition of the invention as such or in the form of one of its physiologically acceptable salts or mixtures thereof.

[0259] In a preferred embodiment, the composition of the invention comprises vitamin K. Vitamin K encompasses a group of structurally similar fat-soluble vitamins that the human body requires to fully synthesize certain proteins that are prerequisites for blood clotting. Vitamin K-related modifications of proteins allow them to bind calcium ions, which they otherwise would not be able to do. In the absence of vitamin K, blood clotting is severely impaired and uncontrolled bleeding occurs. Low levels of vitamin K can also weaken bones and promote calcification of arteries and other soft tissues.

[0260] Chemically, the vitamin K family includes 2-methyl-1,4-naphthoquinone (3-) derivatives. Vitamin K includes two naturally occurring isovitaminosis: vitamin K1 and vitamin K2. Vitamin K2 in turn consists of a number of related chemical subtypes, in which carbon side chains of varying lengths are composed of isoprenoid groups of atoms. In addition, three synthetic types of vitamin K are known: vitamin K3, K4, and K5.

[0261] Vitamin K1 (also called phylloquinone, phytomenaquinone or phytomenaquinone) is synthesized by plants, especially green leafy vegetables. It acts as a vitamin in animals and performs the traditional functions of vitamin K, including its activity in the production of blood clotting proteins. Animals can also convert it to vitamin K2. Both forms of the vitamin contain a functional naphthoquinone ring and an aliphatic side chain. Phylloquinone has a phytyl side chain.

[0262]

[0263] Bacteria in the colon can convert K1 to vitamin K2. In addition, bacteria often lengthen the isoprenoid side chains of vitamin K2 to produce a series of vitamin K2 forms, most notably the MK-7 to MK-11 homologs of vitamin K2. All forms of K2 except MK-4 can only be produced by bacteria, which utilize these forms in anaerobic respiration. Vitamin K can be included in the composition in an amount of about 0.001% to about 99.999% of the composition.

[0264] Vitamin K may be incorporated into the composition of the invention as such or in the form of one of its physiologically acceptable salts or mixtures thereof.

[0265] In a highly preferred embodiment, the composition comprises one or more of the following: 1000 to 3000 [mu] g beta carotene, 10 to 80 mg vitamin C, 1 to 2 mg vitamin Bl, 0.5 to 1.5 mg vitamin B6, 0.5 to 2 mg vitamin B2, 5 to 18 mg niacin, 0.5 to 2.0 [mu] g vitamin B12, 100 to 800 [mu] g folic acid, 30 to 70 [mu] g biotin, 1 to 5 [mu] g vitamin D, 3 to 10 IU vitamin E.

[0266] Additional components

[0267] In a highly preferred embodiment, the composition of the present invention further comprises one or more of the following ingredients: minerals, fatty acid derivatives, choline and phospholipids or metabolites or metabolic precursors thereof.

[0268] The amount of each of these additional ingredients is selected depending on whether the composition is intended to be administered / taken once or more daily.

[0269] When the composition of the present invention comprises one or more of vitamins and these additional ingredients, it can have an improved effect in promoting, supporting and / or optimizing de novo myelination, particularly de novo myelination track, and / or brain structure, particularly the amount and spatial distribution of myelinated material of the whole brain, and / or brain connections, and / or cognitive potential, and / or intelligence potential, and / or learning potential, and / or cognitive function. For example, this may be because the composition realizes de novo myelination in identical and / or separate complementary brain regions. The improved effect can be synergistic.

[0270] In a preferred embodiment, the composition comprises a phospholipid, a metabolite thereof, or a metabolic precursor thereof. As used herein, the term "phospholipid" refers to any phospholipid. As used herein, the term "phospholipid" refers to a molecule consisting of two fatty acids attached to a glycerol "head." The glycerol molecule is also attached to a phosphate group.

[0271] Preferably, the phospholipid is a compound of formula (I)

[0272]

[0273] in:

[0274] R 1 is O;

[0275] X is NH or O;

[0276] R 2 is a C2-C44 saturated or unsaturated, linear or branched acyl group;

[0277] R 3 is a substituent of formula (II) or formula (III):

[0278]

[0279] R 4 is selected from C5 or C6 substituted or unsubstituted cyclic alkyl or cyclic alkenyl groups, and -(CH2) n —R 7 ;

[0280] R 5 is a C2-C44 saturated or unsaturated straight chain or branched acyl group;

[0281] R 6 is a C2-C44 saturated alkyl or alkenyl group;

[0282] R 7 -N(CH3)3 + NH3 + or a substituent of formula (IV):

[0283]

[0284] And n is an integer of 1 to 4, preferably 1 or 2.

[0285] As used herein, the term "alkyl" includes saturated straight and branched chain alkyl groups, which may be substituted (mono- or poly-) or unsubstituted. The term cyclic alkyl will be understood accordingly. Preferably, the cyclic alkyl group is C3-8, more preferably a C3-6 cyclic alkyl group.

[0286] As used herein, the term "alkenyl" refers to a carbon chain containing one or more double bonds, which carbon chain may be branched or unbranched, and may be substituted (mono- or poly-) or unsubstituted.

[0287] As used herein, the term "acyl" refers to the group R'(C=O)-, where R' is a saturated (alkyl) or unsaturated (alkenyl) carbon chain.

[0288] As used herein, the term "acyclic" refers to a non-cyclic group.

[0289] In a preferred embodiment, R 4 is a C6 cyclic alkyl or cyclic alkenyl group substituted by one or more hydroxyl groups. In a preferred embodiment, R 4 Derived from inositol (C6H 12 O6), even more preferably derived from inositol, i.e. R 4 for:

[0290]

[0291] Non-limiting examples of phospholipids include phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, and phosphatidylcholine.

[0292] In a preferred embodiment, the phospholipid is selected from phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, sphingomyelin and mixtures thereof, and metabolic precursors and metabolites of any of the foregoing substances, and mixtures thereof.

[0293] Phosphatidylinositol is a compound of formula (V):

[0294]

[0295] Where R 8 and R 9 Each is independently a C2 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group.

[0296] More preferably, R 8 and R 9 Each is independently a C13 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group, which together with the adjacent carbonyl group corresponds to a C14 to C44 saturated or unsaturated fatty acid residue, and even more preferably a C14 to C24 saturated or unsaturated fatty acid residue.

[0297] More specifically, R 8 and R 9is a C13 to C23 branched or unbranched acyclic alkyl or acyclic alkenyl group, which together with their adjacent carbonyl groups is a C14 to C24 saturated or unsaturated fatty acid residue, wherein the fatty acid from which the fatty acid residue is derived is selected from: C14:0, C15:0, C16:0, C18:0, C20:0, C20:3, C20:4, C21:0, C22:0, C23:0, C24:0, C18:1n-9, C18:2n-6 and C24:1n-9. Even more particularly C18:0, C18:1n-9, C18:2, C20:3 and C20:4.

[0298] As will be appreciated by the skilled artisan. As used herein, the term phosphatidylserine refers to phosphatidyl-L-serine.

[0299] Phosphatidylserine is a compound of formula (VI):

[0300]

[0301] Where R 10 and R 11 Each is independently a C2 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group.

[0302] More preferably, R 10 and R 11 Each is independently a C13 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group, which together with the adjacent carbonyl group corresponds to a C14 to C44 saturated or unsaturated fatty acid residue, and even more preferably a C14 to C24 saturated or unsaturated fatty acid residue.

[0303] More specifically, R 10 and R 11 is a C13 to C23 branched or unbranched acyclic alkyl or acyclic alkenyl group, which together with its adjacent carbonyl group is a C14 to C24 saturated or unsaturated fatty acid residue, wherein the fatty acid from which the fatty acid residue is derived is selected from: C14:0, C15:0, C16:0, C18:0, C20:0, C20:3, C20:4, C21:0, C22:0, C23:0, C24:0, C18:1n-9, C18:2n-6 and C24:1n-9. Even more particularly C18:0, C18:1n-9, C20:4 and C22:6.

[0304] Phosphatidylethanolamine is a compound of formula (VII):

[0305]

[0306] Where R 12 and R 13Each is independently a C2 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group.

[0307] More preferably, R 12 and R 13 Each is independently a C13 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group, which together with the adjacent carbonyl group corresponds to a C14 to C44 saturated or unsaturated fatty acid residue, and even more preferably a C14 to C24 saturated or unsaturated fatty acid residue.

[0308] As used herein, the term "sphingomyelin" refers to a lipid molecule, or a mixture of lipid molecules, in which the sphingosine backbone is acylated with a fatty acid residue at the amino group (-NH2), and in which the hydroxyl group at position 1 of the sphingosine backbone is linked to a phosphocholine or phosphoethanolamine group.

[0309] Preferably, the sphingomyelin is a compound of formula (VIII) or a mixture of compounds of formula (VIII):

[0310]

[0311] Where R 14 and R 15 Each is independently a C2 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group.

[0312] More preferably, R 14 is a C13 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group, which together with the adjacent carbonyl group corresponds to a C14 to C44 saturated or unsaturated fatty acid residue.

[0313] Non-limiting examples of C14 to C44 saturated or unsaturated fatty acids from which fatty acid residues can be derived include: C14:0, C15:0, C16:0, C18:0, C20:0, C21:0, C22:0, C23:0, C24:1, C25:0, C28:1, C30:2, C30:1, C30:0, C32:3, C32:2, C32:1, C32:0, C33:1, C34:3, C34:2, C34:1, C34:0. :0, C35:2, C35:0, C36:4, C36:3, C36:2, C36:1, C36:0, C37:1, C37:0, C38:4, C38:3, C38:1, C38:0, C39:1 , C39:0, C40:2, C40:1, C40:0, C41:2, C41:1, C41:0, C42:47, C42:3, C42:2, C42:1, C42:0, C44:3, C44:1.

[0314] Even more preferably, R14 It is a C13 to C23 branched or unbranched acyclic alkyl or acyclic alkenyl group, which together with the adjacent carbonyl group corresponds to a C14 to C24 saturated or unsaturated fatty acid residue, wherein the fatty acid from which the fatty acid residue is derived is selected from: C14:0, C15:0, C16:0, C18:0, C20:0, C21:0, C22:0, C23:0, C24:0, C18:1n-9, C18:2n-6 and C24:1n-9.

[0315] Even more preferably, the sphingomyelin is a mixture of compounds of formula (VIII), wherein the mixture is such that the fatty acid residues (R 14 The total amount of fatty acids (together with the adjacent carbonyl groups) is mainly saturated fatty acids, and the least dominant is unsaturated fatty acids. More preferably, the mixture will be such that 80% to 96% of the fatty acid residues in the mixture are saturated fatty acids, particularly C14, C15, C16, C18, C20, C22, C23, C24 saturated fatty acids, more particularly C16, C18, C20, C22 and C24.

[0316] Phosphatidylcholine is a compound of formula (IX):

[0317]

[0318] Where R 16 and R 17 Each is independently a C2 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group.

[0319] More preferably, R 16 and R 17 Each is independently a C13 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group, which together with the adjacent carbonyl group corresponds to a C14 to C44 saturated or unsaturated fatty acid residue, and even more preferably a C14 to C24 saturated or unsaturated fatty acid residue.

[0320] More specifically, R 16 and R 17is a C13 to C23 branched or unbranched acyclic alkyl or acyclic alkenyl group, which together with its adjacent carbonyl group is a C14 to C24 saturated or unsaturated fatty acid residue, wherein the fatty acid from which the fatty acid residue is derived is selected from: C14:0, C15:0, C16:0, C16:1, C18:0, C20:0, C20:1, C20:3, C20:4, C21:0, C22:0, C22:6, C23:0, C24:0, C18:1n-9, C18:2n-6 and C24:1n-9. Even more particularly C14:0, C16:0, C18:0, C18:1n-9, C18:2n-6, C20:1, C20:3, C20:4 and C22:6.

[0321] Particularly preferred phospholipids include phosphatidylcholine, phosphatidylinositol, phosphatidylserine and sphingomyelin, more particularly phosphatidylcholine and / or sphingomyelin.

[0322] In a preferred embodiment of the present invention, the phospholipid is phosphatidylcholine, phosphatidylinositol, phosphatidylserine or sphingomyelin, or a metabolic precursor or metabolite of any of the foregoing substances. More preferably, the phospholipid is phosphatidylcholine or sphingomyelin, or a metabolic precursor or metabolite of any of the foregoing substances.

[0323] Preferably, the phospholipids, metabolic precursors and / or metabolites thereof are contained in the composition in an amount of at most 99.999% of the composition.

[0324] Preferably, sphingomyelin, its metabolic precursors and / or metabolites are contained in the composition in an amount of at most 99.999% of the composition.

[0325] More preferably, the composition comprises sphingomyelin in an amount of 200 mg to 1000 mg, 400 mg to 700 mg, 650 mg.

[0326] Preferably, phosphatidylcholine, its metabolic precursors and / or metabolites are contained in the composition in an amount of at most 99.999% of the composition.

[0327] More preferably, the composition comprises phosphatidylcholine in an amount of 300 mg to 5000 mg, 1000 mg to 5000 mg, 3000 mg to 5000 mg, 4000 mg to 4500 mg.

[0328] Preferably, phosphatidylinositol, its metabolic precursors and / or metabolites are contained in the composition in an amount of at most 99.999% of the composition.

[0329] More preferably, the composition comprises phosphatidylinositol in an amount of 50 mg to 400 mg, 100 mg to 250 mg, 200 mg to 210 mg.

[0330] Preferably, phosphatidylserine, its metabolic precursors and / or metabolites are contained in the composition in an amount of at most 99.999% of the composition.

[0331] More preferably, the composition comprises phosphatidylserine in an amount of 50 mg to 500 mg, 200 mg to 500 mg, 400 mg.

[0332] Preferably, phosphatidylethanolamine, its metabolic precursors and / or metabolites are contained in the composition in an amount of at most 99.999% of the composition.

[0333] More preferably, the composition comprises phosphatidylethanolamine in an amount of 50 mg to 500 mg, 200 mg to 500 mg, 400 mg.

[0334] If one or more metabolic precursors and / or metabolites of phospholipids are used in the composition instead of or in combination with phospholipids, the amount of the compound is such that the level of phospholipids physiologically delivered by the composition complies with those listed above. Determining the appropriate amount is well within the capabilities of those skilled in the art.

[0335] As used herein, the term metabolic precursor and / or metabolite of one or more phospholipids does not include choline.

[0336] Non-limiting examples of metabolic precursors and / or metabolites of phospholipids, in particular sphingomyelin, phosphatidylcholine, phosphatidylinositol, phosphatidylserine and / or phosphatidylethanolamine, are: galactosylceramide, glucosylceramide, sphingosine, sphingosine-1-phosphate, ceramide, D-erythro-dihydroceramide and ceramide-1-phosphate, and gangliosides.

[0337] Particularly useful phospholipids include phosphatidylcholine, phosphatidylserine, phosphatidylinositol and / or sphingomyelin, in particular sphingomyelin.

[0338] In a preferred embodiment of the present invention, the phospholipid is a metabolic precursor and / or metabolite of phosphatidylcholine, phosphatidylserine, phosphatidylinositol, sphingomyelin and / or any one of the above and / or any one of the above combinations. Preferably, the phospholipid is sphingomyelin, its metabolic precursor and / or metabolite.

[0339] Particularly effective metabolic precursors and / or metabolites of phospholipids, in particular sphingomyelin, include ceramides and gangliosides and gangliosides as well as ceramide-1-phosphate and d-erythro-dihydroceramide.

[0340] The term "ceramide" indicates a lipid molecule in which the sphingosine backbone is acylated with a fatty acid residue. When the term ceramide is used in the present specification, it may identify a single ceramide species as well as a mixture of single ceramide species.

[0341] Preferably, the ceramide is a compound of formula (IXa) or a mixture of compounds of formula (IXa):

[0342]

[0343] Where R 16a and R 17a Each is independently a C2 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group.

[0344] More preferably, R 16a is a C13 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group, which together with the adjacent carbonyl group corresponds to a C14 to C44 saturated or unsaturated fatty acid residue.

[0345] Non-limiting examples of C14 to C44 saturated or unsaturated fatty acids from which fatty acid residues can be derived include: C14:0, C15:0, C16:0, C18:0, C20:0, C21:0, C22:0, C23:0, C24:1, C25:0, C28:1, C30:2, C30:1, C30:0, C32:3, C32:2, C32:1, C32:0, C33:1, C34:3, C34:2, C34:1, C34:0. :0, C35:2, C35:0, C36:4, C36:3, C36:2, C36:1, C36:0, C37:1, C37:0, C38:4, C38:3, C38:1, C38:0, C39:1 , C39:0, C40:2, C40:1, C40:0, C41:2, C41:1, C41:0, C42:47, C42:3, C42:2, C42:1, C42:0, C44:3, C44:1.

[0346] Even more preferably, R 16a It is a C13 to C23 branched or unbranched acyclic alkyl or acyclic alkenyl group, which together with the adjacent carbonyl group corresponds to a C14 to C24 saturated or unsaturated fatty acid residue, wherein the fatty acid from which the fatty acid residue is derived is selected from: C14:0, C15:0, C16:0, C18:0, C20:0, C21:0, C22:0, C23:0, C24:0, C18:1n-9, C18:2n-6 and C24:1n-9, and more particularly selected from C16:0, C18:0, C20:0, C22:0 and C24:0.

[0347] Even more preferably, the ceramide is a mixture of compounds of formula (IXa) wherein the mixture is such that the fatty acid residues (R 16aThe total amount of fatty acids (together with the adjacent carbonyl groups) is mainly saturated fatty acids, and the least dominant is unsaturated fatty acids. More preferably, the mixture will be such that 80% to 96% of the fatty acid residues in the mixture are saturated fatty acids, particularly C14, C15, C16, C18, C20, C22, C23, C24 saturated fatty acids, more particularly C16, C18, C20, C22 and C24.

[0348] As used herein, the term "ganglioside" refers to an oligosaccharide ceramide lipid molecule comprising a residue of ceramide of formula IXa as defined herein. When the term ganglioside is used in this specification, it may identify a single ganglioside species as well as a mixture of single ganglioside species comprising a residue of ceramide of formula IXa as defined herein.

[0349] Particularly effective gangliosides may be monosialoganglioside-3 (GM3) ganglioside and / or disialoganglioside 3 (GD3) ganglioside.

[0350] Ceramide-1-phosphate and d-erythro-dihydroceramide comprise the residue of ceramide of formula IXa as defined herein.

[0351] Sphingomyelin can be synthesized from ceramide and phosphatidylcholine. Therefore, it may be particularly beneficial if ceramide and / or one or more gangliosides are used in combination with phosphatidylcholine, its metabolic precursors or metabolites.

[0352] The phospholipids, metabolic precursors and / or metabolites included in the compositions of the present invention may be natural, synthetic or mixtures thereof. The metabolic precursors and / or metabolites may be used in the compositions of the present invention in their pure form or substantially pure form. Alternatively, they may be added in the form of a source comprising them.

[0353] Any source of phospholipids, metabolic precursors and / or metabolites thereof suitable for ingestion by an individual for whom the composition is intended to be consumed may be used in the present invention.

[0354] In particular, phospholipids, metabolic precursors or metabolites thereof will be from natural sources, non-limiting examples of which include eggs, soy, bovine brain and / or mammalian milk or extracts thereof. Non-limiting examples of soy sources include soy lecithin food additives, and non-limiting examples of mammalian milk include cow's milk, camel's milk, sheep's milk, goat's milk, including skim milk. Non-limiting extracts of milk include protein extracts (e.g., whey proteins and caseins), milk fat globule membranes (MFGM) and extracts containing them.

[0355] A particularly useful source of phospholipids, metabolic precursors or metabolites thereof (particularly sphingomyelin) useful in the present invention is a bovine milk whey protein concentrate enriched in α-lactalbumin and / or non-pure α-lactalbumin, which has been extracted from milk whey protein (particularly bovine milk whey protein).

[0356] α-lactalbumin is a high quality, easily digestible whey protein and is the major protein found in HM. Since α-lactalbumin and / or milk fractions enriched in α-lactalbumin have a high content of essential amino acids (particularly tryptophan), they are ideal for use in low protein infant formulas. Although α-lactalbumin itself is a protein, non-purified sources may contain sphingomyelin.

[0357] In a preferred embodiment, the phospholipids, their metabolic precursors or metabolites (in particular sphingomyelin) are used in the form of a whey protein concentrate enriched in α-lactalbumin or as α-lactalbumin.

[0358] In a more preferred embodiment, use is made of a bovine whey protein concentrate enriched in alpha-lactalbumin or alpha-lactalbumin having a phospholipid content, in particular a sphingomyelin content, which is higher than 500 mg / 100 g dry weight of the composition.

[0359] Another particularly useful source of phospholipids, their metabolic precursors or metabolites may be MFGM or an extract containing them, in particular MFGM or an extract containing them from cow's milk. It may be particularly beneficial if MFGM or an extract containing them contains at least 1%, 2%, 5%, 10%, 20%, 30%, 40% phospholipids and / or at least 0.1%, 0.2%, 0.5% to 5%, 0.8% to 3%, 1% to 2%, 1.6%, 1.9%, 1.8% of phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and / or sphingomyelin. MFGM may further contain magnesium, phosphorus and / or calcium, preferably in a concentration range of 0.05% to 2%, 0.1% to 0.4%.

[0360] Compositions of the invention comprising phospholipids and / or their metabolic precursors and / or metabolites (particularly sphingomyelin, phosphatidylcholine and / or phosphatidylinositol) are particularly effective in supporting, promoting or optimizing de novo myelination, particularly de novo myelination trajectories, and / or brain structures in one or more of the following brain regions: cerebellum, visual cortex, corpus callosum, internal capsule, frontal lobe, parietal lobe, temporal lobe, motor cortex, frontal cortex. These brain regions are associated with one or more of the following: vision, motor function (including coordination and execution of movement), hemispheric interaction, language function, auditory function (including listening and attention), working memory, executive function (including problem solving, social processing, and behavioral interaction, spatial reasoning, and language).

[0361] In a preferred embodiment, the composition further comprises mineral nutrients. As used herein, the term mineral may refer to any mineral. Non-limiting examples of minerals include: iron, zinc, calcium, phosphorus, copper, magnesium, iodine, manganese, chloride, potassium, sodium, selenium, chromium and combinations thereof. Minerals are typically added in the form of salts.

[0362] Particularly effective minerals include iron and / or copper and / or zinc and / or calcium and / or phosphorus and / or magnesium, in particular iron.

[0363] In a preferred embodiment, the composition of the invention comprises iron.

[0364] Iron is preferably contained in the composition of the invention in an amount of up to 99.999% of the composition.

[0365] Iron may be incorporated into the compositions of the invention in the form of a physiologically acceptable salt, such as, for example, ferric citrate, ferric phosphate, ferric pyrophosphate, ferrous ascorbate, ferrous carbonate, ferrous citrate, ferrous fumarate, ferrous gluconate, ferrous lactate, ferrous sulfate, or mixtures thereof.

[0366] Iron may also be incorporated into the compositions of the invention in the form of a physiologically acceptable iron complex (such as, for example, sodium iron EDTA salt) or a mixture thereof.

[0367] If the iron is added to the composition in the form of a ferrous salt or complex (e.g., the ferrous salts listed above), the Fe2+ is more bioavailable and therefore may be more beneficial.

[0368] In a preferred embodiment, the composition according to the present invention comprises a level of iron such that the total daily intake from the composition of the present invention is from about 2 mg to about 50 mg, more preferably from about 2.5 mg to about 45 mg, even more preferably from about 2.7 mg to about 45 mg, more preferably from about 3 mg to about 30 mg, or from about 5 mg to about 20 mg.

[0369] In a preferred embodiment, the composition according to the present invention comprises iron at a level such that the total daily intake from the nutritional composition of the present invention will not exceed about 45 mg.

[0370] In another preferred embodiment, the composition according to the invention comprises iron at a level such that the total daily intake from the nutritional composition of the invention will not exceed 40 mg.

[0371] In the context of the present invention, the term "iron" includes all iron present in the composition of the present invention, either in free form or in the form of a physiologically acceptable salt or a complex thereof.

[0372] In one embodiment, the composition according to the invention comprises zinc.

[0373] Preferably, zinc is comprised in the composition of the invention in an amount of up to 99.999% of the composition.

[0374] Preferably, zinc is included in the composition in an amount such that the total daily intake from the composition of the present invention is from about 1 mg to about 50 mg, more preferably from about 1 mg to about 40 mg, more preferably from about 1.1 mg to about 40 mg, even more preferably from about 2 mg to about 20 mg, even more preferably from about 5 mg to about 15 mg, even more preferably from about 8 mg to about 12 mg.

[0375] In a highly preferred embodiment, zinc is included in the composition in an amount such that the total daily intake from the composition is about 10 mg.

[0376] In a preferred embodiment, the composition according to the present invention comprises zinc at a level such that the total daily intake from the nutritional composition of the present invention will not exceed about 40 mg.

[0377] Zinc may be incorporated into the composition of the invention in the form of a physiologically acceptable salt and / or by comprising any source of zinc, more particularly Zn2+, such as, for example: zinc nitrate, zinc sulfate, zinc gluconate, zinc acetate or mixtures thereof, or in the form of a physiologically acceptable zinc complex, such as zinc picolinate, or mixtures thereof.

[0378] In a preferred embodiment, the composition of the present invention comprises copper.

[0379] Preferably, copper is contained in the composition of the invention in an amount of up to 99.999% of the composition.

[0380] Preferably, copper is included in the composition in an amount such that the total daily intake from the composition of the present invention is from about 0.1 mg to about 10 mg, more preferably from about 1 mg to about 8 mg, more preferably from about 2 mg to about 6 mg, even more preferably from about 2 mg to about 5 mg.

[0381] In a preferred embodiment, the composition according to the present invention comprises copper at a level such that the total daily intake from the nutritional composition of the present invention will not exceed about 10 mg.

[0382] Copper (more specifically Cu2+) can be incorporated into the composition of the present invention as such or in the form of a physiologically acceptable salt, and / or via any source comprising copper. For example, copper can be incorporated into the composition as: copper sulfate and / or copper gluconate and / or copper carbonate, and / or copper citrate, and / or copper-lysine complex.

[0383] In a preferred embodiment, the composition of the invention comprises magnesium.

[0384] Preferably, magnesium is contained in the composition of the invention in an amount of up to 99.999% of the composition.

[0385] Preferably, magnesium is included in the composition in an amount such that the total daily intake from the composition of the present invention is from about 35 mg to about 350 mg, more preferably from about 50 mg to about 250 mg, more preferably from about 100 mg to about 200 mg.

[0386] In a preferred embodiment, the composition according to the invention comprises magnesium at a level such that the total daily intake from the nutritional composition of the invention will not exceed 350 mg.

[0387] Magnesium (more specifically Mg2) can be incorporated into the composition of the present invention as such or in the form of a physiologically acceptable salt, and / or through any source containing magnesium. For example, magnesium carbonate, magnesium chloride, magnesium oxide, magnesium sulfate, magnesium gluconate, magnesium hydroxide, magnesium citrate, magnesium orthophosphate.

[0388] In a preferred embodiment, the composition of the invention comprises calcium.

[0389] Preferably, calcium is comprised in the composition of the invention in an amount of up to 99.999% of the composition.

[0390] Preferably, calcium is included in the composition in an amount such that the total daily intake from the composition of the present invention is from about 100 mg to about 2500 mg, more preferably from about 200 mg to about 2000 mg, more preferably from about 250 mg to about 1500 mg, even more preferably from about 500 mg to about 1000 mg.

[0391] In a preferred embodiment, the composition according to the invention comprises calcium at a level such that the total daily intake from the nutritional composition of the invention will not exceed 2500 mg.

[0392] Calcium (more specifically Ca2+) can be incorporated into the composition of the present invention as is or in the form of a physiologically acceptable salt, and / or from any source containing calcium. For example, calcium carbonate, calcium chloride, calcium citrate, calcium gluconate, calcium glycerophosphate, calcium lactate, calcium hydroxide, calcium orthophosphate.

[0393] In a preferred embodiment, the composition of the invention comprises phosphorus.

[0394] Preferably, phosphorus is contained in the composition of the invention in an amount of at most 99.999% of the composition.

[0395] Preferably, phosphorus is included in the composition in an amount such that the total daily intake from the composition of the present invention is from about 70 mg to about 3500 mg, more preferably from about 100 mg to about 2500 mg, more preferably from about 200 mg to about 2000 mg, even more preferably from about 300 mg to about 1500 mg, even more preferably from about 500 mg to about 1000 mg.

[0396] In a preferred embodiment, the composition according to the invention comprises phosphorus at a level such that the total daily intake from the nutritional composition of the invention will not exceed 3500 mg.

[0397] Phosphorus may be incorporated into the composition of the invention as such or in the form of a physiologically acceptable salt and / or via any source comprising phosphorus, such as dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, trisodium phosphate, calcium phosphate, calcium hydrogen phosphate and mixtures thereof.

[0398] Compositions comprising minerals, particularly one or more of iron, zinc, copper, calcium, magnesium and phosphorus, may be particularly effective in supporting, promoting or optimizing de novo myelination, particularly de novo myelination trajectories, and / or brain structures in one or more of the following brain regions: cerebellum, brainstem, visual cortex, motor and somatosensory cortex, corpus callosum, frontal cortex, temporal white matter, internal capsule, prefrontal cortex, motor cortex. These brain regions are associated with motor function (including coordination and execution of movement), visual function, hemispheric interaction, executive function, working memory, problem solving, social-emotional function, language, auditory function, problem solving and / or working memory.

[0399] In a preferred embodiment, the composition according to the invention comprises choline.

[0400] Choline is preferably comprised in the composition of the invention in an amount of up to 99.999% of the composition.

[0401] The term "choline" identifies a quaternary ammonium salt containing the N,N,N-trimethylethanolammonium cation and having the structure shown below:

[0402]

[0403] In a preferred embodiment, the counterion X- is selected from chloride, hydroxide, citrate, bitartrate and mixtures thereof.

[0404] Unless otherwise indicated, in the context of the present invention the term "choline" shall be intended to identify all choline present in the nutritional composition of the present invention in free form (or as a salt thereof), such as for example choline hydroxide.

[0405] Preferably, choline is included in the composition in an amount such that the total daily intake from the composition of the present invention is from about 100 mg to about 1000 mg, more preferably from about 200 mg to about 600 mg, more preferably from about 250 mg to about 550 mg, even more preferably from about 300 mg to about 500 mg.

[0406] Preferably, choline is included in the composition in an amount such that the total daily intake of choline derived from the composition of the present invention is about 450 mg.

[0407] Choline may be incorporated into the composition of the invention as such or in the form of a physiologically acceptable salt such as, for example, choline chloride, choline citrate, choline bitartrate or mixtures thereof.

[0408] Choline can be incorporated into the composition of the present invention as a single substance, as a component of a mixture of different choline substances, or by adding natural or synthetic ingredients containing one or more choline substances.

[0409] If a metabolic precursor and / or metabolite of choline is used instead of or in combination with choline, the amount of the compound is such that the level of choline physiologically delivered by the composition complies with those listed above. Determining the appropriate amount is well within the capabilities of one skilled in the art.

[0410] Compositions of the invention comprising choline may be particularly effective in supporting, promoting or optimizing de novo myelination, in particular de novo myelination trajectories, and / or brain structures in the following brain regions: cerebellum, internal capsule, motor cortex, visual cortex, thalamus, parietal cortex and frontal lobe. These brain regions are associated with motor function (including coordination and execution of movement), vision, working memory and / or executive function and / or social emotional reasoning and / or spatial reasoning.

[0411] In a preferred embodiment, the composition of the invention comprises fatty acid derivatives.

[0412] Preferably, fatty acid derivatives may be included in the composition of the invention in an amount of up to 99.999% of the composition.

[0413] As used herein, the term "fatty acid derivatives" includes fatty acids (particularly free fatty acids), and / or monoacylglycerols (hereinafter referred to as MAG), and / or diacylglycerols (hereinafter referred to as DAG), and / or triacylglycerols (hereinafter referred to as TAG) and / or cholesterol esters. In a preferred embodiment, the fatty acid derivatives are free fatty acids.

[0414] In another preferred embodiment, the fatty acid derivative is MAG, DAG, TAG and / or cholesterol ester. Even more preferably, the fatty acid derivative is TAG.

[0415] As used herein, the term "MAG" refers to a glycerol molecule in which one of the OH groups is modified to form an ester bond with a fatty acid.

[0416] Preferably, MAG is a compound of formula (X)

[0417]

[0418] Where R 18 , R 19 and R 20 Two of them are H, and R 18 , R 19 and R 20 One of the groups is a C4 to C44 saturated or unsaturated acyl group.

[0419] More preferably, R 18 , R 19 and R 20 Two of them are H, and R 18 , R 19 and R 20 One of the groups is a C10 to C24 (more preferably a C14 to C24) saturated or unsaturated acyl group.

[0420] As used herein, the term "DAG" refers to a glycerol molecule in which two of the OH groups are modified to form ester bonds with fatty acids.

[0421] Preferably, DAG is a compound of formula (X), wherein R 18 , R 19 and R 20 One of them is H, and R 18 , R 19 and R 20 Two of them are each independently a C4 to C44 saturated or unsaturated acyl group.

[0422] More preferably, R 18 , R 19 and R 20 Two of R are each independently a C10 to C24 (more preferably a C14 to C24) saturated or unsaturated acyl group. 18 , R 19 and R 20 The two C4 to C44 saturated or unsaturated acyl groups may be the same or different.

[0423] As used herein, the term "TAG" refers to a glycerol molecule that forms ester bonds with three fatty acids.

[0424] Preferably, as used herein, TAG is a compound of formula (X) wherein R18 , R 19 and R 20 Each is independently a C4 to C44 saturated or unsaturated acyl group, more preferably a C10 to C24 saturated or unsaturated acyl group, more preferably a C14 to C24. 18 , R 19 and R 20 The three C4 to C44 saturated or unsaturated acyl groups in may all be the same, all different, or two may be the same and one different.

[0425] As used herein, the term "cholesterol ester" refers to a compound of formula (XI):

[0426]

[0427] Where R 21 is a C2 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group.

[0428] More preferably, R 21 It is a C9 to C43 branched or unbranched acyclic alkyl group, or an acyclic alkenyl group, which together with the adjacent carbonyl group corresponds to a C10 to C44 saturated or unsaturated fatty acid residue, more preferably a C14 to C24 saturated or unsaturated fatty acid residue.

[0429] As used herein, the term "fatty acid" refers to a compound of formula (XII)

[0430]

[0431] Where R 22 is a C2 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group.

[0432] More preferably, R 22 is a C9 to C43 branched or unbranched acyclic alkyl or acyclic alkenyl group.

[0433] Non-limiting examples of C10 to C44 saturated or unsaturated fatty acids that may be included in the fatty acid derivative (i.e., the fatty acid derivative may be a free fatty acid or a MAG, DAG, TAG and / or cholesterol ester whose fatty acid residue is derived from a fatty acid) include: C10:0, C12:0, C14:0, C15:0, C16:0, C16:1n-7, C18:0, C18:1n-7, C18:1n-9, C18:2n-6, C20:3n-3, C20:0, C20:1n-9, C20:2n-6, C20:3n-6, C20:4n-6, 20:5n-3, C21:0, C22:0, C22:1n-9, C22:6n-3, C23: :0, C24:1, especially 24:1n-9, C25:0, C28:1, C30:2, C30:1, C30:0, C32:3, C32:2, C32:1, C32:0, C33:1, C34:3, C34:2, C34:1, C34:0, C35:2, C35:0, C36:4, C36:3, C36:2, C36:0 6:1, C36:0, C37:1, C37:0, C38:4, C38:3, C38:1, C38:0, C39:1, C39:0, C40:2, C40: 1. C40:0, C41:2, C41:1, C41:0, C42:47, C42:3, C42:2, C42:1, C42:0, C44:3, C44:1. In particular, the fatty acid will be selected from: C10:0, C12:0, C14:0, C16:0, C16:1n-7, C18:0, C18:1n-7, C18:1n-9, C18:2n-6, 18:3n-3, C20:0, C20:1n-9, C20:2n-6, C20:3n-6, C20:4n-6, 20:5n-3, C22:0, C22:1n-9, C22:6n-3, C24:1, 24:1n-9.

[0434] Any fatty acid derivative suitable for ingestion by the individual for whom the composition is intended to be consumed may be used in the present invention.

[0435] In particular, fatty acid derivatives will come from natural sources, non-limiting examples of which include eggs, algae, fish oil, molds, yeasts, seeds, plants such as soybeans, and animal sources such as bovine brains, and / or mammalian milk or extracts thereof. Non-limiting examples of soybean sources include soy lecithin food additives, and non-limiting examples of mammalian milk include cow's milk, camel's milk, sheep's milk, goat's milk, including skim milk. Non-limiting extracts of milk include protein extracts, milk fat globule membranes (MFGM) and extracts containing them. Fatty acid derivatives can also come from palm oil, tallow, lard, cottonseed oil, peanut oil.

[0436] It may be particularly advantageous if the fatty acid derivative comprises a saturated or unsaturated fatty acid selected from C20:4n-6, C22:6n-3, C24:1n-9, C16:0, C18:1n-9 and C18.0, in particular C20:4n-6 and / or C22:6n-3 and / or C18:0, more in particular 22:6n-3 and / or C18:0.

[0437] Compositions comprising phospholipids, particularly sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, and more particularly sphingomyelin, may be particularly effective when used in combination with one or more of these fatty acids.

[0438] C20:4n-6 is arachidonic acid (hereinafter referred to as ARA). C22:6n-3 is docosahexaenoic acid (hereinafter referred to as DHA). 24:1n-9 is nervonic acid. C18.0 is stearic acid. C16:0 is palmitic acid. C18:1n-9 is oleic acid.

[0439] Preferably, the fatty acid derivative is DAA and / or ARA and / or stearic acid. Most particularly, it is a fatty acid derivative containing DHA and / or stearic acid.

[0440] In a preferred embodiment, the composition according to the invention contains fatty acid derivatives comprising DHA and / or ARA and / or nervonic acid and / or stearic acid, in particular fatty acid derivatives containing DHA and / or ARA.

[0441] Preferably, fatty acid derivatives comprising DHA and / or ARA and / or nervonic acid and / or stearic acid may be comprised in the composition of the present invention in an amount of up to 99.999% of the composition.

[0442] In a preferred embodiment, the composition comprises DHA in an amount such that the total daily intake from the composition of the present invention is from about 100 mg to about 1500 mg, more preferably from about 250 mg to about 1200 mg, even more preferably from about 800 mg to about 1200 mg.

[0443] In a preferred embodiment, the composition comprises ARA in an amount such that the total daily intake from the composition of the present invention is from about 100 mg to about 500 mg, more preferably from about 150 mg to about 450 mg, even more preferably from about 200 mg to about 400 mg, more preferably from about 250 mg to about 350 mg, or about 300 mg.

[0444] In a preferred embodiment, the composition comprises nervonic acid in an amount such that the total daily intake from the composition of the present invention is from about 5 mg to about 80 mg, more preferably from about 5 mg to about 50 mg, even more preferably from about 8 mg to about 32 mg.

[0445] In a preferred embodiment, the composition comprises stearic acid in an amount such that the total daily intake from the composition of the present invention is from about 5 mg to about 80 mg, more preferably from about 5 mg to about 50 mg, even more preferably from about 4 mg to about 20 mg.

[0446] Fatty acid derivatives including stearic acid occur in natural sources such as palm oil, tallow, lard, cottonseed oil, peanut oil.

[0447] Fatty acid derivatives containing nervonic acid are present in natural sources, such as seed oils of Cardaminegracea, Heliphila longifola, Thlaspi perfoliatum, Tropaeolum speciosum, Lunaria biennis, Lunaria annua, and Malania oleifera; the fungi Neocallismastix frontalis, Erysiphe graminis, and Sphaerotheca humuli; the bacterium Pseudomonas atlantica; the yeast Saccharomyces cerevisiae, and the marine diatom Nitzschia cylindrus.

[0448] Fatty acid derivatives containing DHA and / or ARA are found in natural sources, such as eggs, algae, fungi or fish oils, and plants.

[0449] The oil comprising fatty acid derivative can have various sources, and this fatty acid derivative comprises DHA and / or ARA, and comprises other polyunsaturated fatty acids (PUFA) conventionally, particularly EPA (eicosapentaenoic acid).Preferably, the fatty acid derivative comprising DHA provides in the form of fish oil containing the fatty acid derivative comprising DHA and / or ARA.Fish oil comprises 5 % by weight or more conventionally, preferably 10 % by weight or more fatty acid derivative comprising DHA and / or ARA.Conventionally also can obtain the oil containing the fatty acid derivative comprising DHA and / or ARA obtained from algae or microorganism in a large number.For example, can use from comprising 10 % by weight or more, for example 20 % by weight or more oil of the algae gathering of fatty acid derivative.

[0450] If the nutritional composition according to the invention contains fatty acid derivatives comprising ARA and DHA, the ingredients may, for example, be contained in the composition of the invention in an amount such that the weight ratio of DHA:ARA is in the range of 4:1 to 1:4, such as 3:1 to 1:3, such as 2:1 to 1:2, such as 1.5:1 to 1:1.5, in particular 1.1:1 to 1:1.1.

[0451] It may also be beneficial if the composition of the present invention comprises a mixture of fatty acid derivatives, wherein the mixture is such that the weight ratio of unsaturated fatty acids and / or fatty acid residues to saturated fatty acids and / or fatty acid residues in the composition of the present invention is in the range of 1:1 to 1:2, 1:1.2 to 1:1.9, 1:1.25 to 1:1.5, 1:3 to 1:4.

[0452] In addition, when a large amount of fatty acid ester derivatives comprising DHA and / or ARA are included in the composition of the present invention, it may be particularly beneficial if the total amount of fatty acid derivatives comprising saturated long-chain fatty acids (particularly C20 / 24) increases. These saturated long-chain fatty acids can be important components of myelin sheaths, enabling them to surround and coat axons. Preferably, in the composition of the present invention, the weight ratio of DHA and / or AA to these unsaturated long-chain fatty acids is, for example, in the range of 1:1 to 1:10, 1:2 to 1:9, 1:3 to 1:4.5, 1:3.5 to 1:4.5.

[0453] Compositions comprising fatty acid derivatives (e.g., fatty acid derivatives comprising DHA and / or AA) of the present invention may be particularly effective in supporting, promoting or optimizing de novo myelination, particularly de novo myelination trajectories, and / or brain structures in one or more of the following brain regions: cerebellum, internal capsule, temporal lobe, frontal lobe, parietal lobe, motor and sensory cortex (including coordination and execution of movement), visual cortex, frontal cortex. This brain region is associated with visual function, motor function and psychomotor function (including coordination and execution of motor function), and / or executive function, and social-emotional function.

[0454] Those skilled in the art can identify appropriate amounts of the above nutrients, their metabolic precursors or metabolites according to the nature and purpose of the composition.

[0455] In a preferred embodiment, the composition according to the invention comprises phospholipids, preferably phosphatidylcholine and / or sphingomyelin, at least one vitamin, preferably B9 and / or B12, iron, choline and DHA.

[0456] In a preferred embodiment, the composition according to the invention comprises phospholipids, preferably phosphatidylcholine and / or sphingomyelin, at least one vitamin, preferably B9 and / or B12, iron, choline, DHA, AA and zinc.

[0457] In a preferred embodiment, the composition according to the invention comprises at least one vitamin, preferably B9 and / or B12, iron and choline.

[0458] In another preferred embodiment, the composition according to the invention comprises at least one vitamin, preferably B9 and / or B12, iron and DHA.

[0459] In another preferred embodiment, the composition according to the invention comprises at least one vitamin, preferably B9 and / or B12, iron and folic acid.

[0460] In another preferred embodiment, the composition according to the invention comprises sphingomyelin, DHA and choline.

[0461] In another preferred embodiment, the composition according to the invention comprises at least one vitamin, preferably B9 and / or B12, DHA and iron.

[0462] In another preferred embodiment, the composition according to the invention comprises sphingomyelin, DHA and at least one vitamin (preferably B9 and / or B12).

[0463] In another preferred embodiment, the composition according to the invention comprises sphingomyelin, choline and folic acid.

[0464] In another preferred embodiment, the composition according to the invention comprises sphingomyelin, at least one vitamin (preferably B9 and / or B12), iron and DHA.

[0465] In another preferred embodiment, the composition according to the invention comprises sphingomyelin, at least one vitamin (preferably B9 and / or B12), choline and iron.

[0466] In another preferred embodiment, the composition according to the invention comprises sphingomyelin, at least one vitamin (preferably B9 and / or B12), DHA and choline.

[0467] In another preferred embodiment, the composition according to the invention comprises at least one vitamin, preferably B9 and / or B12, iron, DHA and choline.

[0468] In another preferred embodiment, the composition according to the invention comprises sphingomyelin, at least one vitamin (preferably B9 and / or B12), iron, choline and DHA.

[0469] In one embodiment, the composition according to the invention contains fatty acid derivatives comprising DHA and / or ARA, vitamin B12 and / or folic acid, sphingomyelin and iron.

[0470] Those skilled in the art can identify appropriate amounts of the above nutrients, their metabolic precursors or metabolites according to the nature, purpose, target individual and dosage of the composition (e.g., how many times a day the composition will be taken by an individual). Generally, the effective dose will depend on the age, body shape and health status of the individual, on the lifestyle of the individual, on the amount of the nutrients in the composition, and may depend on the sex of the individual.

[0471] One skilled in the art will be able to identify appropriate amounts of the above nutrients, metabolic precursors or metabolites in a composition to achieve their maximum permitted level following administration.

[0472] Preferred composition types

[0473] The composition of the present invention may be any type of composition suitable for direct administration to a female subject.

[0474] Compositions according to the present invention are preferably in solid form. Compositions can be in the form of, for example, chewable tablets, dispersible tablets, capsules, lozenges, lozenges, chewing gums, powders (e.g., pouches), strip pouches, or bottles with lids containing powder. Preferably, compositions are in the form of tablets, capsules, or powders. Tablets or capsules can be provided in unit dosage form, for example, once or twice daily, preferably once daily. Powder compositions can be contained in pouches. Powder compositions according to the present invention can be used for sprinkling on food or beverages. Particularly preferred embodiments provide compositions according to the present invention in the form of pouches comprising powder, wherein the powder can be dispersed in beverages (e.g., water, fruit juice, milk, etc.), to provide a delicious nutrient liquid for oral administration.

[0475] In a preferred embodiment, the composition is a nutritional composition.The nutritional composition may be, for example, a nutritionally complete formula, a nutritional supplement, a food product (such as a dairy product, a refrigerated or shelf-stable beverage or soup), a dietary supplement, a meal replacement or a nutritional bar.

[0476] In a preferred embodiment, the composition is a foodstuff intended for consumption by adults, particularly pregnant women.

[0477] As used herein, the term "nutritional composition" refers to a composition that nourishes an individual. The composition can be a nutritionally complete formula, for example, comprising a source of protein, carbohydrates, and fat. The nutritional composition can be administered enterally, parenterally, or intravenously. In a preferred embodiment, the composition is ingested enterally and more preferably orally.

[0478] In a preferred embodiment of the present invention, the composition comprises one or more of a protein source, a lipid source and a carbohydrate source.

[0479] For example, such compositions may comprise protein in the range of about 2 g / 100 kcal to 6 g / 100 kcal, lipid in the range of about 1.5 g / 100 kcal to 3 g / 100 kcal and / or carbohydrate in the range of about 1.7 g / 100 kcal to 12 g / 100 kcal.

[0480] If the composition is a liquid, its energy density may be between 60 kcal / 100 mL and 75 kcal / 100 mL.

[0481] If the composition is solid, its energy density may be between 60 kcal / 100 g and 75 kcal / 100 g.

[0482] In a preferred embodiment, the composition is a synthetic nutritional composition.

[0483] The expression "synthetic composition" refers to a mixture obtained by chemical and / or biological methods.

[0484] Preferably, the composition is a hypoallergenic nutritional composition.As used herein, the term "hypoallergenic nutritional composition" refers to a nutritional composition that is unlikely to cause an allergic reaction.

[0485] In a preferred embodiment of the present invention, the composition is selected from the group consisting of pharmaceutical compositions, food products, food extracts, beverages, food additives, pet care products, nutritional products and nutritional supplements.

[0486] In a highly preferred embodiment, the composition is a maternal supplement. The supplement is preferably taken throughout pregnancy to build up maternal stores of the various constituent components, but supplementation in mid-pregnancy and more particularly in late pregnancy is believed to be particularly advantageous. Likewise supplementation can continue postpartum, either by consumption of the composition by the mother if the infant will be breastfed, or by direct administration of a similar composition to the infant, such as through an infant formula for feeding the infant.

[0487] In a highly preferred embodiment, the composition is used in combination with infant formula and / or stage 1 infant formula and / or growing-up milk, which is administered to infants after birth. The composition may also be used in combination with baby food and / or a fortifier. Preferably, the infant formula and / or stage 1 infant formula and / or growing-up milk and / or baby food and / or a fortifier also comprises a phospholipid or a metabolite or metabolic precursor thereof, which further promotes, supports or optimizes one or more of the following in the infant: (i) de novo myelination; (ii) brain structure; (iii) brain connectivity; (iv) intellectual potential; (v) cognitive potential; and (vi) learning potential; (vii) cognitive function.

[0488] The expression "infant formula" means a food intended specifically for the nutrition of infants during the first four to six months of life and which meets the multiple nutritional requirements of such infants (as defined in Article 1.2 of Council Directive 91 / 321 / EEC of 14 May 1991 on infant formula and stage 2 infant formula).

[0489] The expression "stage 1 infant formula" means a food intended specifically for the nutrition of infants under 4 months of age.

[0490] The term "stage 2 infant formula" refers to a food that is designed to provide nutrition to infants over 4 months of age and is the main liquid component of the gradually diversified diet of such infants.

[0491] In the context of the present invention, the term "growing-up milk (GUM)" refers to a nutritional formula that can be given to children after infant formula has been stopped. "Growing-up milk" (or GUM) is used from the age of one year. It is usually a milk-containing beverage adapted to the special nutritional needs of young children.

[0492] The term "baby food" refers to food intended specifically for the nutritional supply of infants under one year of age.

[0493] The expression "fortifier" refers to a liquid or solid nutritional composition suitable for mixing with breast milk or infant formula.

[0494] The term "weaning" refers to the period during which other foods gradually replace the mother's milk in the infant's diet.

[0495] "Mother's milk" refers to mother's breast milk or colostrum (=human breast milk=HBM).

[0496] The compositions of the present invention may also contain any other additional ingredients or excipients known for the type of composition involved. Non-limiting examples of such additional ingredients include: proteins, amino acids, carbohydrates, oligosaccharides, lipids, prebiotics or probiotics, essential fatty acids, nucleotides, nucleosides, other vitamins, minerals and other micronutrients.

[0497] The type of protein is considered to be insignificant to the present invention. Therefore, protein sources such as whey, casein and mixtures thereof can be used. As for whey protein, acid whey or sweet whey or mixtures thereof can be used, as well as alpha-lactalbumin and beta-lactoglobulin mixed in any desired proportion. The whey protein can be modified sweet whey. Sweet whey is a byproduct of the cheese making process that is more easily available and is commonly used to prepare infant formula based on cow's milk. However, sweet whey contains a component called casein glycomacropeptide (CGMP), which is undesirably rich in threonine and has very little tryptophan. Removing CGMP from sweet whey can make the content of threonine in the protein closer to the content of threonine in human milk. Then, those low-content amino acids (mainly histidine and tryptophan) can be supplemented in this modified sweet whey. A method for removing CGMP from sweet whey is described in EP880902, and an infant formula based on such modified sweet whey is described in WO 01 / 11990. The protein may be complete or hydrolyzed, or a mixture of complete protein and hydrolyzed protein. For example, it may be desirable to provide partially hydrolyzed protein (hydrolysis degree between 2% and 20%) for individuals who are considered to be at risk of milk allergy. If hydrolyzed protein is required, a hydrolysis process may be performed as required and as known in the art. For example, the whey fraction may be enzymatically hydrolyzed by two steps as described in EP 322589 to prepare a whey protein hydrolyzate. For fully hydrolyzed protein, Alcalase 2.4L (EC 940459) may be used first at 55°C, then Neutrase 0.5L (available from Novo Nordisk Ferment AG), and then pancreatin may be used to triple hydrolyze whey protein. If the whey fraction used as a raw material is substantially lactose-free, it is found that the protein undergoes much less lysine blockage during the hydrolysis process. This enables the extent of lysine blocking to be reduced from about 15 wt % total lysine to less than about 10 wt % lysine; for example about 7 wt % lysine, which greatly improves the nutritional quality of the protein source.

[0498] Any suitable dietary protein may be used, for example, animal protein (such as milk protein, meat protein, and egg protein); plant protein (such as soy protein, wheat protein, rice protein, and pea protein); a mixture of free amino acids; or a combination thereof. Non-limiting examples of proteins include casein, α-lactalbumin, whey, soy protein, rice protein, corn protein, oat protein, barley protein, wheat protein, rye protein, pea protein, egg protein, sunflower protein, potato protein, fish protein, meat protein, lactoferrin, serum albumin, immunoglobulin, and combinations thereof. Milk protein (such as casein and whey) and soy protein are particularly preferred.

[0499] The compositions of the present invention may comprise one or more amino acids. Non-limiting examples of amino acids include leucine, threonine, tyrosine, isoleucine, arginine, alanine, histidine, isoleucine, proline, valine, cysteine, glutamine, glutamic acid, glycine, serine, arginine, lysine, methionine, phenylalanine, tryptophan, asparagine, aspartic acid, and combinations thereof.

[0500] The composition of the present invention may include a carbohydrate source. Any carbohydrate source may be used, such as lactose, saccharose, maltodextrin, fructose, glucose, honey, sucrose, corn syrup solids, starch, and combinations thereof.

[0501] The composition of the present invention may include a lipid source. The lipid source may be any lipid. The lipid source preferably provides 5% to 40% of the energy of the composition, for example 20% to 30% of the energy. A blend of rapeseed oil, corn oil, and high oleic sunflower oil may be used to obtain a suitable fat profile. Preferred fat sources include milk fat and vegetable oils. Essential fatty acids, i.e., linoleic acid and α-linolenic acid, may also be added. In one embodiment, a small amount of oil containing rich primary processed arachidonic acid (AA) and docosahexaenoic acid (DHA), such as fish oil or microbial oil, may be added. The ratio of n-6 and n-3 fatty acids of the lipid source is preferably from about 5:1 to about 15:1; for example, from about 8:1 to about 10:1. Non-limiting examples of lipids include: palm oil, palm olein, high oleic sunflower oil, high oleic safflower oil, rapeseed oil, fish oil, coconut oil, cow fat, and combinations thereof.

[0502] The composition of the present invention may include one or more essential fatty acids. Non-limiting examples of essential fatty acids include linoleic acid (LA), alpha-linolenic acid (ALA) and polyunsaturated fatty acids (PUFA). The composition of the present invention may also include gangliosides (monosialyl ganglioside 3 (GM3) and disialyl ganglioside 3 (GD3)), other phospholipids (such as, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine) and combinations thereof.

[0503] The composition may also include at least one prebiotic, preferably in an amount of about 0.3% to about 10%. Prebiotics are non-digestible food ingredients that have a beneficial effect on the host by selectively stimulating the growth and / or activity of one or a limited number of bacteria in the colon, thereby improving the host's health. These ingredients are non-digestible, meaning that they cannot be broken down and absorbed in the stomach or small intestine, so they can pass through the stomach and small intestine intact to the colon, where they are selectively fermented by beneficial bacteria. Examples of prebiotics include specific oligosaccharides. Non-limiting examples of prebiotics include: oligosaccharides optionally containing fructose, galactose, mannose; dietary fiber, particularly soluble fiber, soy fiber; inulin; and combinations thereof. Preferred prebiotics are fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), isomalto-oligosaccharides (IMO), xylo-oligosaccharides (XOS), arabino-oligosaccharides (AXOS), manno-oligosaccharides (MOS), soybean oligosaccharides, saccharose (GS), lactosucrose (LS), lactulose (LA), palatinose oligosaccharides (PAO), maltooligosaccharides, gums and / or their hydrolysates, pectin and / or their hydrolysates, and combinations of the foregoing.

[0504] Additional examples of oligosaccharides are described in Wrodnigg, TM; Stutz, AE (1999) Angew. Chem. Int. Ed. 38:827-828 and WO 2012 / 069416 (incorporated herein by reference).

[0505] The composition may also comprise at least one probiotic strain.Probiotics are preparations or fractions of microbial cells that have a beneficial effect on the health or well-being of the host. Non-limiting examples of probiotics include: Bifidobacterium, Lactobacillus, Lactococcus, Enterococcus, Streptococcus, Kluyveromyces, Saccharoymces, Candida, in particular selected from Bifidobacterium longum, Bifidobacterium lactis, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium adolescentis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, paracasei, Lactobacillus salivarius, Lactobacillus lactis, Lactobacillus rhamnosus, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus salivarius, Lactococcus lactis, Enterococcus faecium, Saccharomyces cerevisiae, Saccharomyces boulardii or a mixture thereof,Preferably selected from Bifidobacterium longum NCC3001 (ATCC BAA-999), Bifidobacterium longum NCC2705 (CNCM I-2618), Bifidobacterium longum NCC490 (CNCM I-2170), Bifidobacterium lactis NCC2818 (CNCM I-3446), Bifidobacterium breve strain A, Lactobacillus paracasei NCC2461 (CNCM I-2116), Lactobacillus johnsonii NCC533 (CNCM I-1225), Lactobacillus rhamnosus GG (ATCC53103), Lactobacillus rhamnosus NCC4007 (CGMCC1.3724), Enterococcus faecium SF 68 (NCC2768; NCIMB10415), and combinations thereof. If probiotics are present, their amount preferably varies according to the age of the human or animal. For example, the probiotics may be present in an amount of about 5 million to about 2,500 million, about 10 million to about 2,500 million, about 30 million to about 2,500 million, about 50 million to about 2,500 million, about 50 million to about 1,000 million, about 75 million to about 2,500 million, about 75 million to about 1,000 million, about 100 million to about 2,500 million, about 100 million to about 1,000 million, about 250 million to about 2,500 million, about 250 million to about 1,000 million, about 500 million to about 2,500 million, about 500 million to about 1,000 million, about 750 million to about 2,500 million, or about 750 million to about 1,000 million, about 1 billion to about 2.5 billion, about 1.5 billion to about 2.5 billion.

[0506] Compositions of the present invention may also include dietary fiber.Dietary fiber is not digested by enzymes through the small intestine, and functions as a natural bulking agent and laxative.Dietary fiber may be soluble or insoluble, and generally two types of blends are preferred. Suitable sources of dietary fiber include soy, peas, oats, pectin, guar gum, gum arabic, oligofructose, galacto-oligosaccharides, sialyllactose and oligosaccharides derived from animal milk.Preferred fiber mixtures are mixtures of inulin and shorter chain oligofructose.Preferably, if there is fiber, the fiber content is between 10g / l and 40g / l of the formula food consumed.

[0507] According to government agency recommendations (such as USRDA), the composition may also include minerals and micronutrients, such as trace elements and vitamins. For example, the composition of the present invention may also include one or more minerals that are known to be essential and nutritionally significant in the daily diet. The minimum requirements for certain minerals have been determined. Non-limiting examples of minerals include: calcium, phosphorus, iodine, magnesium, copper, manganese, chloride, potassium, sodium, selenium, chromium, molybdenum, and combinations thereof. Minerals are usually added in the form of salts. The composition may also include other components, such as inositol, taurine and / or L-carnitine.

[0508] For example, the composition may contain one or more of the following micronutrients in daily doses within the ranges given below: 300 mg to 500 mg of calcium, 50 mg to 100 mg of magnesium, 150 mg to 250 mg of phosphorus, 5 mg to 20 mg of iron, 1 mg to 7 mg of zinc, 0.1 mg to 0.3 mg of copper, 50 [mu] g to 200 [mu] g of iodine, 5 [mu] g to 15 [mu] g of selenium, 1000 [mu] g to 3000 [mu] g of beta-carotene, 10mg to 80mg of vitamin C, 1mg to 2mg of vitamin B1, 0.5mg to 1.5mg of vitamin B6, 0.5mg to 2mg of vitamin B2, 5mg to 18mg of niacin, 0.5[mu]g to 2.0[mu]g of vitamin B12, 100[mu]g to 800[mu]g of folic acid, 30[mu]g to 70[mu]g of biotin, 1[mu]g to 5[mu]g of vitamin D, and 3IU to 10IU of vitamin E.

[0509] If desired, one or more food grade emulsifiers may be incorporated into the formula; for example, diacetyl tartaric acid mono- and di-glycerides, lecithin, and mono- and di-glycerides. Similarly, suitable salts and stabilizers may be included.

[0510] The formula is preferably administered enterally; for example in the form of a powder or liquid concentrate for reconstitution with milk or water, a solid product or a ready-to-drink beverage.

[0511] The composition may optionally contain other substances that may have a beneficial effect, such as nucleotides, nucleosides, etc. Non-limiting examples of nucleotides include cytidine monophosphate (CMP), uridine monophosphate (UMP), adenosine monophosphate (AMP), guanosine monophosphate (GMP), and combinations thereof.

[0512] Other suitable and desired ingredients of compositions that can be used in the compositions of the present invention are described in the guidelines issued by the Codex Alimentarius for the type of nutritional composition involved.

[0513] The composition used in the present invention can be prepared by any suitable means. For example, the composition can be prepared by blending a protein source, a carbohydrate source and a fat source together in a suitable proportion. If an emulsifier is used, it can be included in the blend. Vitamins and minerals can be added at this time, but in order to avoid thermal degradation, they are usually added a little later. Before blending, any lipophilic vitamins, emulsifiers and the like can be dissolved in the fat source. Then water (preferably water subjected to reverse osmosis) can be mixed to form a liquid mixture. The liquid mixture can then be heat treated to reduce the bacterial load. For example, the liquid mixture can be rapidly heated to a temperature in the range of about 80°C to about 110°C for about 5 seconds to about 5 minutes. This can be done by steam injection or a heat exchanger (e.g., a plate heat exchanger). Then, the liquid mixture can be cooled to about 60°C to about 85°C, for example, by rapid cooling. The liquid mixture can then be homogenized; for example, in two stages: the first stage is carried out at about 7MPa to about 40MPa, and the second stage is carried out at about 2MPa to about 14MPa. The homogenized mixture can then be further cooled to add any heat-sensitive components; such as vitamins and minerals. The pH and solid content of the homogenized mixture are conveniently standardized at this moment. The homogenized mixture is transferred to a suitable drying device (such as a spray dryer or a freeze dryer) and converted into a powder. The water content of the powder should be less than about 5 weight %. If it is necessary to increase one or more probiotics, the probiotics can be cultivated according to any suitable method, then prepared, for example, by freeze drying or spray drying, for addition to formula food. Alternatively, a bacterial preparation that has been made into a suitable form can be purchased from professional suppliers such as Danish Hansen Company (Christian Hansen) and Japan Morinaga Milk Industry Co., Ltd. (Morinaga) for addition to food products. Such bacterial preparations can be added to formula food by dry mixing.

[0514] Phospholipids, such as sphingomyelin, may be added at any stage during the process, but are preferably added after the heating step.

[0515] In a preferred embodiment, the composition comprises triglycerides with high sn-2 palmitate, preferably triglycerides with greater than 33% palmitic acid at the sn-2 position.

[0516] In some preferred embodiments, palmitic acid comprises from about 15% to about 25%, such as from about 15% to about 20%, by weight, of the total fatty acid content of the formula, and at least about 30%, e.g., from about 35% to about 43%, of the total palmitic acid content is located at the sn-2 position.

[0517] The commercially available composition sold by Lipid Nutrition is Betapol™ B-55, a triglyceride mixture derived from vegetable oils, wherein at least 54% of the palmitic acid is in the sn-2 position of the glycerol molecule. In a preferred embodiment, the fat content of the composition of the present invention is about 40% to 50% by weight (e.g., about 43% to about 45% by weight) of Betapol™ B-55. Those skilled in the art will appreciate that the percentage of high sn-2 fat used in the formula and the total amount of sn-2 palmitate can vary, and different high sn-2 palmitate oils can be used, without departing from the spirit and scope of the present invention.

[0518] In another embodiment, a conventional food product, such as yogurt or breakfast cereal, may be enriched with at least one vitamin.

[0519] In another embodiment, the composition comprising at least one vitamin in an amount sufficient to realize the desired effect in an individual can be prepared. The composition can be in the form of, for example, tablets, capsules, lozenges or liquids. The supplement can also include protective aqueous colloids (such as gums, proteins, modified starches), binding agents, film formers, encapsulating agents / materials, wall / shell materials, matrix compounds, coatings, emulsifiers, surface active agents, solubilizers (oils, fats, waxes, lecithin, etc.), sweeteners, texturizing agents, adsorbents, carriers, chelating agents, fillers, co-compounds, dispersants, wetting agents, processing aids (solvents), flow agents, taste masking agents, weighting agents, gelling agents, gel forming agents, antioxidants and antimicrobial agents. The composition may also contain conventional pharmaceutical additives and adjuvants, excipients and diluents, including but not limited to: water, gelatin from any source, vegetable gums, lignin sulfonates, talc, sugars, starches, gum arabic, vegetable oils, polyalkylene glycols, flavors, thickeners, preservatives, stabilizers, emulsifiers, buffers, lubricants, colorants, wetting agents, fillers, etc.

[0520] Health Benefits

[0521] The compositions of the present invention have a positive effect on de novo myelination of the brain, particularly the trajectory of de novo myelination, of infants or young children whose mothers are administered such compositions before or during pregnancy, or during postpartum lactation.

[0522] Such positive effects may include promoting and / or supporting optimal de novo myelination in infants or young children, particularly de novo myelination trajectories, which may determine appropriate cognitive, intellectual and learning potential, development of cognitive skills and abilities, and learning. Optimal de novo myelination trajectories may also prevent the development of cognitive impairment or cognitive delay.

[0523] The health effects may be observed after days, weeks or months or years of using the composition comprising at least one vitamin.

[0524] The effects of the invention may be preventive (e.g. avoiding suboptimal de novo myelination, in particular de novo myelination trajectory in the brain, brain structure, brain connectivity, cognition, intelligence and / or learning potential or cognitive function) or therapeutic (e.g. restoring optimal de novo myelination, in particular de novo myelination trajectory in the brain, brain structure, brain connectivity, cognition, intelligence and / or learning potential or cognitive function).

[0525] Infant-related health effects can be measured by various methods as shown in the examples below.

[0526] As is apparent from the above disclosure, the composition of the present invention can be used to promote, support or optimize de novo myelination, particularly the trajectory of de novo myelination, and / or brain structure, particularly the amount and / or spatial distribution of myelinated material in the brain, and / or brain connectivity and / or intellectual potential, and / or cognitive potential and / or learning potential and / or cognitive function, of an individual whose mother has taken the composition before or during pregnancy or during postpartum lactation.

[0527] In another aspect of the invention, a method is provided for promoting, supporting or optimizing de novo myelination, in particular the trajectory of de novo myelination, and / or brain structure, in particular the amount and / or spatial distribution of myelinated material in the brain, and / or brain connectivity, and / or intellectual potential and / or cognitive potential and / or learning potential, and / or cognitive function in an individual (in particular a formula-fed individual), the method comprising feeding a composition comprising at least one vitamin according to the invention to the mother of the individual before, during or after pregnancy, or during lactation.

[0528] The effects of the compositions of the invention described herein may have long-term health benefits. Dementia (e.g., Alzheimer's disease) results in a decline in an individual's ability to think and remember, as well as emotional and language problems. The risk of an individual suffering from dementia, particularly Alzheimer's disease, is often associated with a person's intellectual ability or intelligence. Therefore, by optimizing an individual's intelligence, cognition, and / or learning potential, the risk of an individual suffering from dementia, particularly Alzheimer's disease, may be reduced.

[0529] Various other mental disorders and / or neurological disorders (e.g., autism and schizophrenia) are also relevant to brain structure, and are particularly relevant to the amount and / or spatial distribution of white matter of the whole brain. By promoting, supporting or optimizing individual de novo myelination, particularly de novo myelination track, and / or brain structure, mental disorders and / or neurological disorders (e.g., autism) may be prevented, or the risk of suffering from these disorders may be reduced, or the severity of the illness may be reduced.

[0530] As will be apparent to those skilled in the art, mothers can obtain the same benefits as disclosed herein by taking the vitamins directly rather than in the form of a composition. Thus, the vitamins can be used directly in any of the methods or uses described herein in place of the compositions of the present invention.

[0531] As will further be apparent to a person skilled in the art, it may be particularly beneficial if the vitamins, in particular B12 and / or B9, are administered separately, sequentially and / or simultaneously with one or more of the following ingredients: minerals, fatty acid derivatives, choline and phospholipids or their metabolites or metabolic precursors.

[0532] As used herein, "concurrently" means that the additional ingredients are administered simultaneously with at least one vitamin. Preferably, the additional ingredients are administered simultaneously as part of the composition according to the invention.

[0533] As used herein, "sequentially" means that the additional ingredient is administered within a certain period of time before or after at least one vitamin. The time delay will vary depending on the nature of the additional ingredient and / or the phospholipid, or its metabolite or metabolic precursor.

[0534] As used herein, "separately" means, for example, that there is a substantial interval between administration of the additional agent and at least one vitamin, such that the vitamin may no longer be present in the bloodstream of the female individual in therapeutically effective amounts when the additional ingredient is administered.

[0535] In a preferred embodiment, the additional ingredient is administered at least 2 hours, or at least 4 hours, or at least 8 hours, or at least 12 hours, or at least 24 hours, or at least 48 hours before the at least one vitamin.

[0536] In another preferred embodiment, the additional ingredient is administered at least 2 hours, or at least 4 hours, or at least 8 hours, or at least 12 hours, or at least 24 hours, or at least 48 hours after the at least one vitamin.

[0537] All details of the invention apply equally to compositions comprising at least one vitamin, in particular B12 and / or B9, and to the direct use of at least one vitamin, in particular B12 and / or B9 and / or any other ingredients.

[0538] In one embodiment, the invention provides means for promoting, supporting or optimizing de novo myelination in infants or young children who display a suboptimal de novo myelination trajectory in the brain, which can lead to cognitive deficits, impaired cognitive abilities and / or suboptimal cognitive development. Such infants may be premature or low birth weight infants or infants that are small for gestational age.

[0539] The present invention is also applicable to promoting, supporting or optimizing myelination in infants or young children born at term.All infants can benefit from the present invention, as all infants are or may be susceptible to developing a suboptimal myelination trajectory in the brain.

[0540] In such infants or young children, it is particularly advantageous to obtain a myelination trajectory in the brain that approximates that of infants who are breastfed from well-nourished or adequately nourished mothers, preferably infants who are exclusively breastfed in the first few months of life. It does provide them with a healthy state in terms of cognitive abilities that are consistent with those observed in infants who are breastfed from well-nourished or adequately nourished mothers.

[0541] In one embodiment, infants and toddlers are 0 to 3 months old, 0 to 6 months old, or 0 to 12 months old, or 0 to 36 months old, or 0 to 60 months old.

[0542] Application

[0543] The compositions of the present invention may be administered to a female individual before pregnancy, during pregnancy, or during lactation, or a combination thereof.

[0544] In one embodiment, the composition of the invention is administered to a female individual prenatally and is thus indirectly transmitted to a developing embryo or fetus, for example via the placenta or amniotic fluid. In other words, when the composition is administered to the mother during pregnancy, the offspring is exposed to the composition of the invention in utero. Prenatal administration of the composition can prevent the onset or reduce the risk of suboptimal myelination of the offspring and the effects associated therewith.

[0545] In yet other alternative embodiments, the compositions of the invention are administered to lactating females postpartum, and are thus indirectly transferred to the neonate or infant via ingestion of breast milk, ie, exposure of the offspring to the compound is achieved solely through breast milk.

[0546] In a highly preferred embodiment, the composition is for administration prior to conception to a female individual, such as a female individual desiring pregnancy.Pre-conception administration of a composition according to the invention may affect the intrauterine environment.

[0547] Preferably, in any aspect or embodiment of the invention, pre-conception supplementation or administration refers to administration about 1-24 months, 1-18 months, 1-12 months, 1-6 months or 1-3 months before pregnancy.

[0548] In a preferred embodiment, the composition is administered at least 1 week, or at least 2 weeks, or at least 3 weeks, or at least 1 month, or at least 2 months, or at least 3 months, or at least 4 months, or at least 5 months, or at least 6 months, or at least 12 months, or at least 18 months, or 24 months before pregnancy, e.g., to a woman intending or trying to become pregnant.

[0549] In another preferred embodiment, the composition is for administration to a female individual during pregnancy (eg, prenatally).As used herein, "prenatally" refers to the period before birth, during pregnancy, or associated with pregnancy.

[0550] As used herein, unless otherwise noted, reference to compositions according to the present invention for administration during pregnancy (i.e., antenatal administration) refers in particular to administration during any part of the gestational period or throughout the gestational period. In a preferred embodiment of the present invention, administration of the compositions begins as soon as possible after pregnancy, at least until the end of the embryogenic period. For example, in humans, the embryogenic period encompasses the first 8 weeks of development (10 weeks of the gestational period).

[0551] In a preferred embodiment, the composition is administered throughout pregnancy.

[0552] In another preferred embodiment, the composition of the present invention is applied during most of the gestational period. In any embodiment of the present invention, the application time can be within the following range: the first week of pregnancy, the first two weeks, the first month, the first three months of pregnancy, the second three months of pregnancy or the third month of pregnancy. Preferably, the application can continue at least until the birth of offspring. In a preferred embodiment, the composition is applied as soon as possible after pregnancy until birth (i.e., the entire gestational period). In humans, the application preferably continues for the following cycle: about 1 week to birth, about 2 weeks to birth, about 4 weeks to birth, about 8 weeks to birth, about 12 weeks to birth, about 18 weeks to birth, about 24 weeks to birth.

[0553] In any embodiment of the present invention, the composition of the present invention may additionally be administered to a female individual prior to pregnancy and to a female individual during pregnancy and / or during lactation.

[0554] In a highly preferred embodiment, the composition is for administration to a female individual after birth (e.g., during the postpartum lactation period). During this period, the infant can receive the beneficial effects of the composition via breast milk from the mother. After birth, the composition can be administered during part or all of the lactation period.

[0555] Thus, in one embodiment, the present invention relates to a composition for a lactating postpartum female individual, the composition comprising at least one vitamin for promoting, supporting or optimizing one or more of the following in an infant breastfed by said female individual:

[0556] (i) de novo myelination;

[0557] (ii) brain structure;

[0558] (iii) brain connectivity;

[0559] (iv) intellectual potential;

[0560] (v) cognitive potential;

[0561] (vi) learning potential; and

[0562] (vii) Cognitive function.

[0563] References to lactating females are to females that feed their offspring fully or partially with breast milk.

[0564] As used herein, unless otherwise indicated, references to administering a composition according to the invention during lactation include administering the compound at any time postpartum during which the offspring ingests breast milk (the individual's breast milk) in whole or in part. For example, the administration period during lactation may be from the onset of lactation until the end of the weaning process, i.e. until the offspring stops ingesting breast milk. During this period, the offspring may ingest breast milk in whole or in part.

[0565] Preferably, administration of the composition during lactation includes administration for a period of 1-24 months, 2-20 months, 3-18 months, 6-12 months, 4-12 months or 4-8 months after lactation occurs, during which the offspring fully or partially ingests breast milk. It is foreseeable that administration of the composition of the present invention to infants immediately after birth (0-4 weeks, 0-8 weeks, 0-12 weeks, 0-24 weeks) is particularly advantageous because the myelination process has already begun and has significantly developed at this time.

[0566] In a particularly preferred embodiment, if the offspring is fully or partially ingesting breast milk, the composition of the invention is administered for two weeks during lactation after lactation has occurred.

[0567] In a preferred embodiment, administration of the composition to the female individual begins postpartum, ie, not during pregnancy, but from the onset of lactation.

[0568] The composition may advantageously be used to promote, support or optimize one or more of the above aspects in an infant, wherein the composition is administered to the infant's mother during the postpartum lactation period, and the beneficial effects may be observed in the infant later in life, for example during childhood and / or adolescence.

[0569] In another embodiment, the composition of the present invention is administered both prenatally (i.e., any time period from pregnancy to birth) and postpartum during lactation (i.e., any time period from birth until the end of the weaning process (i.e., the offspring has stopped consuming breast milk)). In addition, in any embodiment of the present invention, the composition may be administered at any time period prenatally as defined above for prenatal administration, and at any time period postpartum as defined above for during lactation, as well as in any combination of these times as described above.

[0570] In another embodiment, the composition of the present invention is used as a dietary supplement for female individuals before pregnancy, during pregnancy or during lactation to promote de novo myelination. In addition, when used alone as a pre-pregnancy supplement, or in combination as a supplement during pregnancy and / or lactation, the composition of the present invention can provide health benefits to future offspring.

[0571] In a preferred embodiment, the composition of the present invention is administered to a female individual for a period of 2 weeks to 52 weeks. In a preferred embodiment, it is administered to a female individual for a period of 2 weeks to 24 weeks, or a period of 2 weeks to 12 weeks.

[0572] In a more preferred embodiment, the composition of the present invention is administered to a female individual for 2 weeks to 52 weeks, and begins shortly after the birth of an infant or young child or the interruption of breastfeeding. In one embodiment, the composition of the present invention is fed to the mother for 2 weeks to 24 weeks, or 2 weeks to 12 weeks, and begins shortly after the birth of an infant or young child or the interruption of breastfeeding.

[0573] In one embodiment, the maternal supplement is used in conjunction with infant formula and / or 1st stage infant formula and / or growing-up milk, which is applied to infants after birth. The composition can also be used in conjunction with baby food and / or a fortifier. Preferably, the infant formula and / or 1st stage infant formula and / or growing-up milk and / or baby food and / or a fortifier also contain at least one vitamin, which promotes, supports or optimizes one or more of the following items of the infant: (i) de novo myelination; (ii) brain structure; (iii) brain connections; (iv) intellectual potential; (v) cognitive potential; and (vi) learning potential; (vii) cognitive function.

[0574] It should be understood that all features of the present invention disclosed herein can be freely combined, and that variations and modifications may be made to these features without departing from the scope of the present invention as defined in the claims. In addition, if there are known equivalents for specific features, these equivalents should be incorporated into this specification as if these equivalents were explicitly mentioned in this specification.

[0575] The following is a series of non-limiting examples used to illustrate the present invention.

[0576] Experimental Section :

[0577] Methods, definitions, and materials

[0578] MRI (Magnetic Resonance Imaging): MRI brain scans of infants and children between 0 and 5 years of age were obtained using white matter imaging techniques. This technique provides a quantitative measure, myelin water fraction (MWF), which is a surrogate marker of myelin content in the brain. When mapped as a function of time in early childhood, a myelination trajectory can be generated.

[0579] Infant formula food composition : Six infant formulas were fed to participating infants and their composition / levels of myelin-related nutrients were analyzed.

[0580] The nutritional compositions were tested in standard commercial infant formulas from different brands / suppliers and revealed different levels of nutrients contained therein.

[0581] Cognitive ability : Age-standardized (T) scores for gross motor, visual reception, and language (expressive and receptive) were derived from the Mullen Scales of Early Learning, which are standardized and validated measures of early cognitive development for infants and children 6 years of age or younger.

[0582] Maternal nutritional intake : Maternal nutrient intake during pregnancy was assessed at 34 weeks' gestation using the Automated Self-Administered 24-Hour (ASA24) Dietary Assessment Tool, a web-based tool that enables multiple, automatically coded, self-administered 24-hour recalls.

[0583] Nutrient analysis

[0584] The nutrients in each of the 6 infant formula compositions are shown in Table 1.

[0585] Table 1

[0586]

[0587]

[0588] Clinical study 1

[0589] Infant Participants

[0590] The infants included in the study were drawn from a larger longitudinal study of normal brain and behavioral development: the Brown University Assessment of Myelination and Behavior during Maturation (BAMBAM). In order to focus on neurotypical development, children with known potential risk factors for learning, neurological, or psychiatric disorders were specifically excluded during recruitment and enrollment. Thus, children with in utero alcohol or illicit substance exposure, preterm birth (<37 weeks of gestation) or multiple births, fetal ultrasound abnormalities, complicated pregnancies (e.g., preeclampsia), APGAR scores <8, NICU admission, neurological disorders (e.g., head injury, epilepsy), psychiatric disorders or developmental disorders in the infant, parents, or siblings (including maternal depression requiring medication) were excluded. Ongoing screening (such as the Autism MCAT, or the Behavior Problems CBCL) was further used to remove enrolled children with clinically concerning behaviors or obvious medical conditions (such as autism spectrum disorders).

[0591] A combination of retrospective and prospective data was obtained from parents via detailed medical records and parent interviews to understand the type of infant formula used, the percentage of breastfeeding versus formula feeding, and the length of time of exclusive breastfeeding. This information was updated at each study visit, which was conducted approximately every 6 months for children under 2 years of age and annually for older children. Using this information, children were classified into 2 groups: No. 1, completely formula-fed; and No. 2, completely breastfed for at least 90 days (3 months). Children who were fed a combination of breast milk and formula within 3 months were excluded from our analysis. Infants in the complete formula feeding group were further subdivided according to parental reports of the main infant formula used in the first 3 months. The main formula was defined as given 90% of the time or more (e.g., in the case where parents use alternative brands during vacation).

[0592] Using these criteria, 94 exclusively formula-fed infants and toddlers were selected to enter Group 1. These children included 13 children receiving Formula 2; 28 children receiving Formula 5; 8 children receiving Formula 3; 39 children receiving Formula 4; 5 children receiving Formula 1; and 1 child receiving Formula 6. A sample of 52 exclusively breastfed infants was also selected and matched to the formula-fed groups with respect to mean age, duration of pregnancy, birth weight, sex ratio, ethnicity ratio, maternal education, family size, and number of languages ​​(other than English) spoken at home. The groupings for each formula are provided in Table 1a.

[0593] Table 1a Details of data used for longitudinal and nutritional analyses

[0594] Formula 1 Formula 2 Formula 5 Formula 3 Formula 4 Formula 6 Breastfeeding <![CDATA[N 儿童 ]]> 5 13 28 8 39 1 52 <![CDATA[N 测量 ]]> 11 27 56 14 64 3 106

[0595] Imaging methods and analysis

[0596] Each infant was scanned using mcDESPOT (Multicomponent Drive Balanced Single Pulse Observation of T1 and T2) white matter imaging, Deoni et al. (Magn. Reson. Med. 2008, 60: 1372-1387), which provides quantitative measurements of myelin water fraction (MWF) at every point throughout the brain - a measure of myelin content. All infants were scanned using an acoustically anechoic mcDESPOT imaging protocol during natural (i.e., non-sedated) sleep. Total imaging time ranged from 19 minutes for the youngest toddlers to 24 minutes for older 4-year-olds.

[0597] All data are acquired on the Siemens 3T Tim Trio scanner equipped with 12 channel head RF arrays. In order to minimize the internal motion of the scan, children are wrapped with pediatric MedVac vacuum fixation bags (CFI Medical Solutions, USA) and foam pads. By reducing the peak gradient amplitude and conversion rate, and using noise isolation scanner hole inserts (quiet barrier HD composite materials, UltraBarrier, USA) to reduce scanner noise. MiniMuff pediatric earmuffs and electric headphones (MRConfon, Germany) have also been used. Children are continuously monitored with pediatric pulse oximetry system and infrared camera. All children keep sleeping during the duration of MRI scanning, and there is no motion artifact in the analysis data.

[0598] After image alignment, non-brain signal removal, and correction for main and transmission magnetic field (B0 and B1) inhomogeneities, a three-pool signal model (including myelin-associated water; axonal and extra-axonal water; and a non-exchange free water pool) was fit to the mcDESPOT data to derive a voxel-based MWF map.

[0599] Each child's map was then nonlinearly aligned to a study-specific template. White matter masks corresponding to five bilateral regions (frontal, temporal, occipital, parietal, and cerebellar WM) and the body, genu, and splenium of the corpus callosum were created from a common database, registered to a common template, and superimposed on each child's MWF map. The mean value for each region was then determined for each child and used for subsequent developmental analyses and trajectory modeling.

[0600] Developmental differences :

[0601] To examine developmental differences between breast-fed and formula-fed infants, and between infants fed different formulas, a nonlinear mixed-effects modeling approach was used. Modified Gompertz growth models were fit independently to Groups 1 and 2, and to each formula subgroup. Each of the four Gompertz model parameters was then compared between the breast-fed and formula-fed groups using unpaired t tests, and between the 4 formula subgroups using analysis of variance, followed by post hoc Tukey tests to determine which differed within the formula groups.

[0602] Cognitive Assessment and Analysis

[0603] In addition to MR imaging, general cognitive abilities and skills were assessed for each child within 7 days of scanning using the Mullen Scales of Early Learning (MSEL) (Mullen EM, 1995). The MSEL provides a broad assessment of behavioral development in the areas of fine and gross motor control, receptive and expressive language, and visual reception. Age-standardized T scores from these domains can be combined into three composite scores: the Early Learning Composite (ELC, which includes fine motor, visual reception, expressive, and receptive language); the Nonverbal Developmental Quotient (NVDQ, which includes fine motor and visual reception scores); and the Verbal Developmental Quotient (VDQ, which includes expressive and receptive language scores).

[0604] As with the MWF MRI data, potential group mean differences in ELC, VDQ, and NVDQ between breast-fed and formula-fed infants and between the different formula subgroups were examined. In addition to mean comparisons, mixed-effects modeling assuming linear trends was used to investigate longitudinal changes in these three composite values.

[0605] Example 1

[0606] Identification of nutritional drivers from cross-sectional analyses

[0607] From the above cohort, children under 5 years of age who were fed different infant formulas during infancy were included in a large correlation analysis to examine the relationship between the nutritional composition of the formulas and brain myelination. The 6 most commonly used formulas in this cohort were analyzed for their nutritional composition. A single general linear model (GLM) was constructed to simulate all quantitative nutrients and the age of the children.

[0608] The Spearman rank correlation between nutrient content and myelin content value (adjusted for children's age) is then calculated at each image voxel or point in the brain. Using a cluster-based correction method, significance is defined as p<0.05 for the first type of error correction. Association or trend is defined as p<0.15. In the initial analysis, all 22 kinds of nutrients shown in Table 1 lead to an underpowered model. In order to reduce the number of nutritional components in the model, we checked the correlation between nutrition. Using a conservative threshold of 0.9, we excluded the nutritional components that are highly correlated to each other in various formula foods. This produces a final model, which includes iron, sphingomyelin, folic acid, choline, DHA, zinc and phosphatidylcholine.

[0609] P<0.05: iron, sphingomyelin, folate, choline, and DHA.

[0610] P < 0.15: zinc and phosphatidylcholine.

[0611] The nutritional components found to be highly correlated with each other were:

[0612] Folic acid and vitamin B12.

[0613] DHA and AA.

[0614] Zinc, calcium, magnesium, copper and phosphorus.

[0615] Phosphatidylcholine, phosphatidylserine and phosphatidylinositol.

[0616] For folic acid, an association with myelination (myelin water fraction) was observed over time in the brain, particularly in the cerebellum, motor cortex, and visual cortex. Results are reported in Figure 1b middle.

[0617] For vitamin B12, an association with myelination (myelin water fraction) was observed over time in the brain, particularly in the cerebellum, visual cortex, motor and somatosensory cortex. Figure 2 Report in.

[0618] For iron, an association with myelination (myelin water fraction) was observed over time in the brain, particularly in the cerebellum, visual cortex, internal capsule, motor and somatosensory cortex, corpus callosum, frontal cortex, temporal lobe white matter. Results are reported in Figure 2a middle.

[0619] For zinc, an association with myelination (myelin water fraction) was observed over time in the brain, particularly in the cerebellum, visual cortex, internal capsule, motor and somatosensory cortex, corpus callosum, frontal cortex, and temporal lobe white matter. Figure 2b Report in.

[0620] For calcium, an association with myelination (myelin water fraction) was observed over time in the brain, particularly in the cerebellum, visual cortex, motor and somatosensory cortex, corpus callosum, frontal cortex, and temporal lobe white matter. Figure 2c Report in.

[0621] For phosphorus, an association with myelination (myelin water fraction) was observed over time in the brain, particularly in the cerebellum, visual cortex, motor and somatosensory cortex, and prefrontal cortex. Results are reported in Figure 2d middle.

[0622] For magnesium, an association with myelination (myelin water fraction) was observed over time in the brain, particularly in the cerebellum, visual cortex, internal capsule, corpus callosum, frontal cortex, and motor cortex. Figure 2e Report in.

[0623] For sphingomyelin, an association with myelination (myelin water fraction) was observed over time in the brain, particularly in the cerebellum, visual cortex, internal capsule, frontal lobe, parietal lobe, and temporal lobe. Figure 2f Report in.

[0624] For phosphatidylinositol, an association with myelination (myelin water fraction) was observed over time in the brain, particularly in the cerebellum, visual cortex, motor cortex, and frontal cortex. Results are reported in Figure 2g middle.

[0625] For phosphatidylcholine, an association with myelination (myelin water fraction) was observed over time in the brain, particularly in the cerebellum, visual cortex, internal capsule, frontal lobe, parietal lobe, and temporal lobe. Figure 2h Report in.

[0626] For choline, an association with myelination (myelin water fraction) was observed over time in the brain, particularly in the cerebellum, visual cortex, thalamus, parietal cortex, and frontal lobe. Figure 2i Report in.

[0627] For DHA, an association with myelination (myelin water fraction) was observed over time in the brain, particularly in the cerebellum, primary and secondary motor cortices, internal capsule, visual cortex, frontal cortex. Figure 2j Report in.

[0628] For AA, an association with myelination (myelin water fraction) was observed over time in the brain, particularly in the cerebellum, internal capsule, parietal lobe, motor and sensory cortices, visual cortex, and frontal cortex. Figure 2k Report in.

[0629] Example 2

[0630] a) Whole-brain myelination trajectories from longitudinal studies

[0631] From the available data, the trajectories of longitudinal myelin development (de novo myelination) were calculated using repeated MWF data from children whose infant formulas (the compositions of such formulas are reported in Table 3 below) contained varying amounts of folic acid and vitamin B12. The trajectories were calculated using a longitudinal nonlinear mixed effects approach. A modified Gompertz growth model was fitted to the data for the children in each formula group. The results are reported in Figure 1 middle.

[0632] Table 3 :

[0633] (Low in folate / B12) (High in folate / B12) Folic acid 107mcg / 100g 306mcg / 100g Vitamin B12 4.93mcg / 100g 8.34mcg / 100g

[0634] b) Average regional myelination trajectories from a longitudinal study

[0635] From the available data, mean regional trajectories of longitudinal myelin development (de novo myelination) were calculated using repeated MWF data from children whose infant formulas (the compositions of such formulas are reported in Table 2a below) contained varying amounts of folic acid and vitamin B12. Trajectories were calculated using a longitudinal nonlinear mixed effects approach, and a modified Gompertz growth model was fit to the data for children in each formula group. The results are presented in Figure 1a Report in.

[0636] Table 3a

[0637] (Low in folate / B12) (High in folate / B12) Folic acid 98.8mcg / 100g 306mcg / 100g Vitamin B12 4.66mcg / 100g 8.34mcg / 100g

[0638] Clinical Study 2

[0639] Participants: Mother-infant pairs

[0640] A usable dataset of 21 mother-child pairs was collected in a mixed cross-sectional / longitudinal cohort at Children's Hospital Colorado, USA.

[0641] Pregnant women were selected from the local community with the following inclusion criteria: at least 18 years of age, English-speaking (first language), singleton pregnancy, no smoking, alcohol, or illicit drug use during pregnancy, no abnormal fetal ultrasound, no history of hypertension or diabetes before or during pregnancy, no personal history of major psychiatric or learning disabilities (including major depression) or use of psychiatric medications, no reports of major psychiatric or learning disabilities in other children, suspected metals or other MRI contraindications. These criteria were intended to provide a generally healthy group without major risk factors for abnormalities or alterations in neurodevelopment. Infants were included as follows: parents spoke English (first language), singleton pregnancy, born at term (after 37 weeks of gestation), no exposure to tobacco, alcohol, or illicit drugs during pregnancy, no abnormal fetal ultrasound, no admission to the neonatal intensive care unit, APGAR score >= 8, no history of hypertension or diabetes in the mother before or during pregnancy, no history of major psychiatric or learning disabilities in the parents or siblings, no history of neurological trauma in the infant, no other neurological conditions (e.g., epilepsy), suspected metals or other MRI contraindications. Obtain informed consent from all parents, including a description of the US, MRI, and other laboratory facilities, the overall study protocol, and specific details about each measurement that will be obtained.

[0642] Imaging methods and analysis

[0643] Each infant was scanned using mcDESPOT (multicomponent driven balanced single pulse observation of T1 and T2) white matter imaging technology, Deoni et al. (Magn. Reson. Med. 2008, 60: 1372-1387), which provides quantitative measurements of myelin water fraction (MWF) at each point throughout the brain - a measurement of myelin content. All infants were scanned using an acoustically anechoic mcDESPOT imaging protocol during natural (i.e., non-sedated) sleep. Whole-brain (1.7×1.7×1.7) mm3 data was acquired using an optimized protocol that included additional steps for correction of main and emission magnetic field inhomogeneities (Deoni et al., 2008; Deoni, 2011; Deoni et al., 2013). After acquisition and processing (including motion correction, skull stripping, B0 and B1 map calibration, and voxel-based parameter estimation), the computed T1, T2, and MWF “maps” were nonlinearly aligned to MNI space using a multistep registration approach (Deoni et al., 2012; Dean et al., 2014c; 2014d).

[0644] Children included in the analysis had one MRI scan between 90 and 180 days of age, with a mean of 117 days + / - 71 days of age.

[0645] Correlation analysis :

[0646] Correlation analyses (Pearson) were performed to investigate the association between maternal nutritional intake data at 34 weeks of gestation and myelin content in infant brains at approximately 3 to 6 months of age.

[0647] Example 3

[0648] From the above groups, it can be seen that maternal nutrient intake data are associated with infant brain myelin content. The following nutrients are significant at the p<0.001 level: DHA, choline, iron, B12 and folic acid.

[0649] Using the identified regions of mask significance, and then looking at the correlations between myelin water fraction in those masks and maternal nutritional values ​​(actually partial correlations since we included the effect of age), we find the following:

[0650] B12: r 2 =0.39

[0651] Choline: r 2 =0.33

[0652] DHA:r 2 =0.44

[0653] Folic acid: r 2 =0.173

[0654] Iron: r 2 =0.18

[0655] For DHA, choline, iron, B12, and folate, associations with maternal intake and myelination (myelin water fraction) were observed in the following brain regions:

[0656] B12: Cerebellum, temporal lobe, frontal lobe

[0657] Folic acid: cerebellum, brainstem

[0658] Iron: cerebellum, brainstem

[0659] Choline: cerebellum, internal capsule, motor cortex

[0660] DHA: cerebellum, internal capsule, temporal lobe, frontal cortex, motor cortex

[0661] The results are reported in Figures 54a to 54e middle.

[0662] Example 4

[0663] Suppliers and Stock Solutions

[0664] Compound company Cat No. Cas Original solution bitter Sigma O3907 12407-2 50mM Neural acid Fluka 87117 50637-6 10mM Stearic acid Fluka 85679 57-114 10mM Sphingomyelin Sigma S0756 8518710-6 10mM

[0665] Vehicle and dosage

[0666]

[0667]

[0668] Culture medium composition and culture method

[0669] 1) Neural Basal Complete Medium

[0670] Neurobasal medium (LIFE TECHNOLOGIES CORP, No. 21103-049)

[0671] 50X B27 Supplement (LIFE TECHNOLOGIES CORP, No. 12587-010)

[0672] 2mM L-glutamine (LIFE TECHNOLOGIES CORP, No. 25030-149)

[0673] 1X Penicillin-Streptomycin (LIFE TECHNOLOGIES CORP, No. 15140-122)

[0674] 2) Neural Basal Complete Medium with Growth Factor (GF)

[0675] Above recipe with GF blend

[0676] 1M Tris (MW 121.14, Fisher SCI BP152)

[0677] Heparin (sigma H3149)

[0678] BSA (Sigma A7030)

[0679] DNA enzyme, RNA enzyme, protease free water (Fisher SCI AC327390010)

[0680] EGF(GIBCO PHG0311)

[0681] bFGF vial (GIBCO PHG0021)

[0682] 3) Choline-free Neurobasal Medium (Lifetechnologies, custom formulated, no L-glutamine, no phenol red)

[0683] Note: Complete medium and complete medium with GF are the same as above.

[0684] Generation of Neural Progenitor Cell (NPC) Banks:

[0685] Dissociation of E14 mouse neocortex

[0686] Required Reagents:

[0687] DPBS (1X) + 10% Penicillin / Streptomycin

[0688] Neurobasal Medium / 10% Penicillin / Streptomycin / 10X Hepes

[0689] Process :

[0690] E14 pup brains were harvested and placed in ice-cold DPBS (1X) + 10% penicillin / streptomycin and then dissected using a dissecting microscope. From each pup, one brain hemisphere was placed in 2 mL Neurobasal medium / 10% P / S / 10X Hepes and the other brain hemisphere was placed in another tube.

[0691] The tissue from each tube was aseptically and manually dissociated into single cells, added to Neurobasal complete medium and centrifuged at 130G for 5 minutes. The tissue was then resuspended in Neurobasal complete medium with GF and placed in a 100mm×20mm Corning suspension dish (No. 430591). The cells were passaged twice using a 1:3 ratio, after which they were centrifuged (130g, 5 minutes), resuspended in freezing medium (10% DMSO and Neurobasal complete medium, no GF) and frozen in liquid nitrogen (LN2).

[0692] Thawing cells for compound screening

[0693] The vial was removed from LN2, thawed quickly, and the cells were transferred dropwise to a 15 mL conical flask. 10 mL of complete neurobasal medium was added. The cells were transferred to a suspension culture dish and placed in an incubator for 2 hours. At 1.5 hours, the cells were checked. According to health and number, the required number of plates was estimated, and the appropriate amount of complete neurobasal medium was heated. After 2 hours, the cells were placed in a 15 mL conical tube and spun at 130G for 5 minutes. The cells were then resuspended in neurobasal medium with GF (3uL of GF per 10 mL culture medium). The cells were then grown overnight and subsequently used in the experiment.

[0694] Plate cells in 96-well plates for neurosphere count and diameter only

[0695] Corning Costar 3474, 96-well plate, ultra-thin attachment

[0696] Dissociation and plating of cells

[0697] Remove 3-4 mL of cells from the tilted plate and add to the 15 mL conical. Use some of the remaining medium to rinse the plate. Pull all remaining medium out and place into a 15 mL conical tube and spin at 130G for 5 minutes. Remove all medium. Gently resuspend the cells in 5 mL of warm PBS and spin again. Then remove the PBS and then gently resuspend the cells in 500 μL Accutase (Corning TM Accutase TM Cells were then gently pipetted with a 1000 μL tip to break up clumps and then placed in a shaking water bath for 5-10 minutes, after which they were frequently vortexed by hand.

[0698] Culture media were prepared as indicated below, all with GF:

[0699] Compound bitter Control / vehicle Low Choline / Carrier 1% BSA Low medium 1:5 Caprylic Acid:Capric Acid Neural acid Control / vehicle Low Choline / Carrier Moderate Choline / Carrier Low medium high Stearic acid Control / vehicle Low Choline / Carrier Moderate Choline / Carrier Low medium high Sphingomyelin Control / vehicle Low Choline / Carrier Moderate Choline / Carrier Low medium high

[0700] Control and compound media were prepared with Medium 2 and contained 29uM choline,

[0701] Low (5uM) and medium (70uM) choline media were prepared using Medium 3.

[0702] Compound company Cat cas number Original solution Carrier / Dissolution Dose 1 Dose 2 Choline chloride Sigma 26978 67-48-L 7mM PBS 5μm 70μm

[0703] Gently pipette the medium using a 1000 μL tip, and then use a 200 μL tip to further disperse the cells.

[0704] Clumps should be no larger than about 3-5 cells. Add 5-10 ml of warm medium (GF) to dilute enzyme. Add 2 mL of medium. Pipette this with a 1000 uL pipette, then add 3 mL with a disposable pipette. Filter cells through a cell culture approved 40 μM filter before plating.

[0705] Remove 1 mL to count the cells. Spin the cells again. Remove the medium from the cell clumps and add 1 mL of the preparation medium (no GF). Pipette the cells with a 1000uL pipette. Prepare a cell dilution (24,000 cells / well) in 250μl of the appropriate medium. Vortex the cells daily and grow for 2 days.

[0706] Fixation and staining

[0707] 1. Fix the cells in the hood. For fixation and subsequent immunohistochemical analysis, remove 100 μL of culture medium and add 100ul 4% PFA in 1×PBS to fix the cells, count the neurospheres manually, then wash the cells twice with 1×PBS for 5 minutes, and leave in 1×PBS, wrap in foil and place overnight at 4°C, or perform Dapi staining. Remove 100uL PBS and add 100uL antibody (AB) staining solution (1% goat serum, 1×PBS, and 0.1% triton X) block at room temperature for 1 hour. Remove AB staining solution. Then the cells are stained with Dapi 1:5000 in AB staining solution, 100 μL per well, and then the cells are incubated in the dark at room temperature for 15 minutes. Then the cells are washed 2 times in AB staining solution for five minutes. Use GE Cytell imager or LSM 710, Zeiss confocal microscope for imaging, and analyze the diameter of neurospheres with ImageJ software (National Institutes of Health).

[0708] Plate cells in 24-well plates for monolayer differentiation or EdU incorporation assays

[0709] Compound bitter Control / vehicle Low Choline / Carrier Moderate Choline / Carrier Low medium high Neural acid Control / vehicle Low Choline / Carrier Moderate Choline / Carrier Low medium high Stearic acid Control / vehicle Low Choline / Carrier Moderate Choline / Carrier Low medium high Sphingomyelin Control / vehicle Low Choline / Carrier Moderate Choline / Carrier Low medium high

[0710] Prior to use in the following assays, 24-well glass bottom plates (Mat Tek P24G-1.0-13-F Case, Glass Bottom 24 Well Plates) were coated with poly-L-ornithine (Sigma P4957) and fibronectin (Sigma F1141).

[0711] See above for dissociation and plating of cells.

[0712] Cells were plated (10,000 cells per well) in complete media with GF for 24 hours (500 uL per well). Once cells were attached, they were switched to choline-deficient media, other compound media, or appropriate media.

[0713] Differentiation assays: quantification of neuronal, glial, and NPC marker expression

[0714] After 24 h, ensure that the cells are attached to the plate and then carefully remove the culture medium.

[0715] Add 500 μL of 2% Nu serum (serum replacement) (Corning TM Nu-Serum Growth Medium Supplement, No. CB55004), control, low choline, or medium choline medium. Note: The medium does not contain GF.

[0716] Control and compound media were prepared with Medium 2 and contained 29uM choline, low (5uM) and medium (70uM) choline media were prepared with Medium 3.

[0717] The cells were cultured in medium supplemented with 2% Nu serum for 9 days, with the medium changed every 2 days. For fixation and subsequent immunohistochemical analysis, the medium was removed, the cells were rinsed once with 1× PBS for 5 minutes, and fixed with 4% PFA in 1× PBS for 15 minutes at 4°C. The cells were then washed twice with 1× PBS for 5 minutes, left in 1× PBS, wrapped in foil and placed at 4°C overnight, or they were immediately stained with primary antibodies.

[0718] Staining for differentiation

[0719] Remove PBS and add enough AB staining solution (1% goat serum, 1× PBS, and 0.1% triton X) to cover the bottom, and keep the block at room temperature for 1 hour.

[0720] Prepare primary antibody dilutions in an appropriate amount of AB staining solution, 250uL per well (antibodies are only kept on ice for a short time, mouse anti-MAP2 or TUJ1 1:500 (neuronal markers), rabbit anti-GFAP (glial marker) 1:1000, chicken anti-nestin CFP (EGFP antibody (progenitor cell marker)) 1:1000. Remove AB staining solution and add a solution of primary antibodies to each chamber. Wrap cells in foil and keep at 4°C overnight. Then wash cells once with 400uL of AB staining solution for 5 minutes to remove primary antibodies. Prepare secondary antibody solution (250uL is sufficient for each chamber) (goat anti-mouse alexa488 1:2000, anti-rabbit Cy3 (1:500), anti-chicken alexa 647 1:500, and 1:5000 Dapi).

[0721] The cells were incubated at room temperature for 1 hour in the dark and washed twice for five minutes in AB staining solution. They were then kept at 4°C or imaged using a GE Cytell imager or LSM 710, Zeiss confocal microscope and analyzed with ImageJ software (National Institutes of Health, USA).

[0722] Microtubule-associated protein 2 (MAP2), neuronal β-tubulin III (TuJ1), glial fibrillary acidic protein (GFAP) and Nestin CFP (EGFP antibody) .

[0723] Each marker expression was measured on collected images (integrated density measurement in ImageJ) and normalized to DAPI fluorescence, labeling all nuclei (integrated density measurement).

[0724] Octanoic acid was labeled with neuronal β-tubulin III (TuJ1), and subsequent compounds were labeled with microtubule-associated protein 2 (MAP2).

[0725] NPC proliferation assay in monolayer culture (incorporation of EdU-S phase marker)

[0726] After 24 hours, ensure that cells have attached to the plate and then carefully remove the medium. Add 500 μL of compound medium plus GF. Incubate cells in appropriate medium for 3 days.

[0727] Control and compound media were prepared with Medium 2 and contained 29uM choline, low (5uM) and medium (70uM) choline media were prepared with Medium 3.

[0728] Use Click- EdU Alexa EdU incorporation was measured using the 555 Imaging Kit (Life technologies, No. c10338).

[0729] At the end of day 3, EdU was added to each well at 10 μM for 30 min prior to fixation.

[0730] For fixation and subsequent immunohistochemical analysis, the medium was removed, cells were rinsed once with 1× PBS for 5 min, and fixed with 4% PFA in 1× PBS for 15 min at 4°C, then cells were washed twice with 1× PBS for 5 min, left in 1× PBS, wrapped in foil and left overnight at 4°C, or stained.

[0731] Remove PBS and add enough AB staining solution (1% goat serum, 1× PBS, and 0.1% triton X) to cover the bottom, and keep the block at room temperature for 1 hour. Stain the cells for EDU. Incubate the cells at room temperature in the dark for 30 minutes. Wash the cells with 1× PBS and stain with Dapi 1:5000uL for 15 minutes. Wash the cells once with 1× PBS and then place in PBS at 4°C or image immediately using a GE Cytell imager (cell viability application) or LSM 710, Zeiss confocal microscope (analyzed using ImageJ software (National Institutes of Health)).

[0732] The results are shown in Tables 4 to 8 and Figures 3 to 6 middle.

[0733] Table 4 - Effects of neuraminic acid on neuronal cell density and astrocyte density

[0734] Neurons Astrocytes Control 0.436189 0.642448 Low NA 0.467588 0.621784 Moderate NA 0.56563 0.721512 High NA 0.539448 0.70279

[0735] Table 5 Effects of stearic acid on neuronal cell density and astrocyte density

[0736] Neurons Astrocytes Control 0.44 0.64 Low SA 0.54 0.68 Medium SA 0.66 0.81 High SA 0.64 0.85

[0737] Table 6 Effects of octanoic acid on neuronal cell density and astrocyte density

[0738] Neurons Astrocytes Control 1.1 4.1 Low OA 1.2 3.7 Moderate OA 1.8 6.1 High OA 1.7 7.9

[0739] Table 7 Effect of sphingomyelin on the number of neurospheres

[0740] Sphingomyelin Control Low medium high Average (mean) 103 94 121 219

[0741] Table 8 Effects of sphingomyelin on neuronal proliferation

[0742] DAPI Control 262 Low SM 280 Moderate SM 314 High SM 305

[0743] Example 5

[0744] Experimental Section

[0745] sample

[0746] Component C2: whey protein concentrate enriched with alpha lactalbumin (Sample Manager ID: K2Q-00030); first infant milk containing whey protein concentrate enriched with alpha lactalbumin (Sample Manager ID: K2Q-00032); bovine milk (whole milk); human breast milk (quality control pool of 6 individual samples collected after the 4th week after delivery; Lee Biosolutions, St Louis, MI, USA).

[0747] Extraction of phospholipids from dairy products

[0748] Milk powder: weigh 1 g of the homogenized powder into a 50 mL glass flask and dilute into 20 mL of pure distilled water. Heat the solution in a water bath at 40 °C for 30 minutes. Place a volume of 500 μL of this solution in a 10 mL glass tube.

[0749] Cow's and human milk: Divide 500 μL of the homogenized liquid into 10 mL glass tubes.

[0750] Post-extraction analytes of MP in human breast milk were quantified in triplicate by UPLC-MS / MS (RDLS-MP-80138-Rev01) using a mixture of 9.5 mL of chloroform / methanol (2+1). Briefly, the tubes were shaken and placed in an ultrasonic bath at 40°C for 15 minutes, then centrifuged at 2500 rpm for 10 minutes. A volume of 2 mL of potassium chloride solution (0.88%, m / m) was added to the liquid phase, followed by shaking and centrifugation at 2500 rpm for 10 minutes. The lower organic phase was transferred to a glass vial, evaporated to dryness under a gentle N2 flow, and reconstituted in 500 μL of chloroform / methanol (9+1) before injection into the LC-MS.

[0751] Analysis of phospholipids by liquid chromatography coupled to mass spectrometry (LC-MS)

[0752] The analysis was performed on a QExactive plus orbital trap equipped with a Thermo Scientific Dionex UltiMate 3000 rapid separation LC system (Thermo Fisher Scientific, Bremen, Germany). Separation was performed on a HILIC column (100×2.1 (id) mm; 1.7 μm) with a mobile phase composition of (A) ammonium acetate 10 mM and (B) acetonitrile. The injection volume was set to 10 μL, and the gradient started from 95% B to 70% B in 15 minutes, held at 70% B for 1 minute, returned to the initial conditions in 3 minutes and equilibrated for 6 minutes.

[0753] The Q Exactive plus orbital trap was equipped with an atmospheric pressure chemical ionization (APCI) probe operating in positive ion mode. The APCI and MS parameters were as follows: corona discharge current 4.0 μA, sheath gas and auxiliary gas 24 and 5 arbitrary units, respectively; capillary and vaporizer temperatures 320 °C and 390 °C, respectively, sweep gas flow 0 arbitrary units, and s-lens RF level 80. The automatic gain control (AGC) target value was set to 1 × 10 6 The AGC was set to 1 × 10 6 The ion density was 400 nm and the ion density was 400 nm. The ion density was 400 nm and the ion density was 500 nm. The ion density was 500 nm and the ion density was 500 nm. The ion density was 4 ...

[0754] SM species were extracted from the total ion chromatogram using accurate mass. The parent ion corresponds to the in-source loss of phosphatidylcholine to ceramide. Based on the LipidView database and literature, an inclusion list was used to perform specific fragmentation of 57 SM regioisomers built on the parent ion corresponding to the loss of ceramide to water [Cer-H2O+H + ] m / z (Trenerry VC, Akbaridoust G., Plozza T., Rochfort S., Wales WJ, Auldist M., Ajilouni S. Ultra-high-performance liquid chromatography-ion trap mass spectrometry characterization of milk polar lipids from dairy cows feddifferent diets. Food Chemistry 2013, 141, 1451-1460); Godzien J., Ciborowski M., Martinez-Alcazar MP, Samczuk P., Kretowski Godzien J., Ciborowski M., Martinez-Alcazar MP, Samczuk P., Kretowski A., Barbas C. Rapid and reliable identification of phospholipids for untargeted metabolomics with LC-ESI-QTOF-MS / MS. Journal of Proteome Research 2015, 14, 3204-3216.

[0755] Analysis of fatty acid methyl esters (FAME) by gas chromatography with flame ionization detector (GC-FID)

[0756] SM fractions were collected between 8.5 and 10 minutes into glass tubes, 5 times per sample. After evaporation of the solvent under a stream of N2, quantification of fatty acids in human milk by gas chromatography after MP was performed in triplicate by FAME analysis (RDLS-MP-8980-00030-Rev01-FAME_Human Milk Fat, 2012, Version 1.0).

[0757] Results and discussion

[0758] Hydrophilic interaction liquid chromatography (HILIC) was used to separate PL classes (i.e., phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), and SM). The carbon number and degree of unsaturation within individual SM species were assigned based on the accurate masses of pseudomolecular ions detected in an orbitrap mass spectrometer. The relative abundance of SM species was determined for comparison between ingredients, infant formula, bovine milk, and human milk.

[0759] SM substances in different dairy products

[0760] 45 SM species were detected in the analyzed samples (Table 9).

[0761] Table 9: SM substances detected in ingredients, infant formula, bovine milk and human milk samples (indicated by x). SM substances detected only in human milk are indicated in bold.

[0762]

[0763] The substances SM 24:1, SM 38:4, SM 38:3 and SM 42:4 are found only in trace levels in human milk.

[0764] Relative abundance of SM species

[0765] The relative abundance (%) of SM in different dairy products was estimated from the peak area divided by the sum of all peak areas corresponding to SM species in the chromatogram of each sample. Figure 7 The relative abundance of the major SM species in ingredients, infant formula, bovine milk, and human milk is shown.

[0766] The relative abundance of SM substances present in ingredients and infant formula is comparable to that in bovine milk and slightly different from the human milk fraction of some species (e.g., SM 32:1, SM 32:0, SM 33:1, SM 34:1, SM 38:0, SM 39:1, SM 39:0, and SM 41:1), being lower in human milk than in ingredients, infant formula, and bovine milk. Whereas SM 36:2, SM 36:1, SM 36:0, SM 37:1, SM 38:2, SM 38:1, SM 40:1, SM 42:2, and SM 42:1 have higher relative abundance in human milk than in other dairy products.

[0767] Human milk samples included a quality control pool of 6 individual samples collected within or after 4 weeks of life. Knowing that SM abundance in human milk varies with diet and duration of lactation, this may partially explain the observed differences. However, despite differences in the relative abundance of some SM species, >85% of the SM species detected in human milk were also found in infant formula and bovine milk.

[0768] It is noteworthy that for a given m / z extracted from the MS trace, different LCB FA combinations can be suggested (e.g., SM34:1 can correspond to SM d18:1 / 16:0, d18:0 / 16:1, d16:1 / 18:0, etc.). Therefore, we evaluated the GC FA profiles to gather more information about the molecular structure of SM between different dairy products.

[0769] Fatty acid profiles in SM fractions from different milk products

[0770] The regioisomeric structure of SM was investigated by first fractionating SM and then analyzing the FAs present in the fractions by GC-FID. Fractionation of SM for LC-MS analysis was performed as described above, but in this case the effluent was directed into 5 mL glass tubes instead of MS. Each fraction was then subjected to a methylation procedure prior to subsequent GC analysis. The relative abundance of FAs within the SM fractions was quantified in Figure 8 Shown in.

[0771] like Figure 8 As shown in Table 10, the SM fraction contains mainly saturated FA (i.e., myristic acid 14:0, pentadecanoic acid 15:0, palmitic acid 16:0, stearic acid 18:0, arachidic acid 20:0, behenic acid 22:0, tricosanoic acid 23:0 and tetracosanoic acid 24:0). A higher proportion of SFA was observed in the SM fractions from all dairy products (Table 10). This is consistent with the literature, revealing a high distribution of SFA with carbon chains higher than 18 in the SM fraction. This high amount of SFA reflects the structural effect of SM, i.e., reducing the fluidity of the milk fat globule membrane and maintaining rigidity.

[0772] Table 10: Percentages of SFA, MUFA and PUFA detected in SM fractions from different milk products .

[0773]

[0774] Monounsaturated FA (MUFA) accounts for about 4%-11% of FA in the SM fraction. Oleic acid 18:1n-9 and nervonic acid 24:1n-9 are 2 MUFA detected. Interestingly, compared with other dairy products, 24:1n-9 is found to have a relatively high ratio in human milk, and this is consistent with the literature. Compared with other products, the only PUFA linoleic acid 18:2n-6 is found to be relatively high in the infant formula and human milk tested. Finally, ω-3PUFA is not detected in the SM fraction. This is also consistent with the data found in the literature, showing that arachidonic acid (AA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) are mainly present in PE, PI and PS.

[0775] Example 5

[0776] The two main formulas reported for infant feeding in the cohort study were Infant Formula #3 and Infant Formula #6. Infant Formula #3 was associated with increased global myelin development during the first 5 years of life compared with #6. Fig. 9 , Fig.10 , Fig.11 ). Trajectories calculated via our longitudinal mixed effects approach show that Gompertz growth curve parameters differ in each brain region investigated. Specific nutrient analysis of myelin-related nutrients for infant formula #5 and #6 is provided in Table 12.

[0777] Example 6

[0778] The two main formulas reported for infant feeding in the cohort study were Infant Formula #3 and Infant Formula #6. Infant Formula #3 was associated with increased global myelin development during the first 5 years of life compared with #6. Fig. 9 , Fig.10 , Fig.11 ). Trajectories calculated via our longitudinal mixed effects approach show that Gompertz growth curve parameters differ in each brain region investigated. Specific nutrient analysis of myelin-related nutrients for infant formula #5 and #6 is provided in Table 12.

[0779] Table 12-Nutrient analysis of myelin-related nutrients in 6 infant formula foods .

[0780]

[0781]

[0782] Example 7

[0783] Co-culture of neurons and OLs

[0784] Neurons / oligodendrocytes were cultured as previously described by Charles et al. (2000).

[0785] Pregnant female rats at 17 days of gestation were killed by cervical dislocation (Wistar rats) and the fetuses were removed from the uterus. The forebrain was removed and placed in Leibovitz ice-cold medium (L15) containing 2% penicillin-streptomycin (PS) and 1% bovine serum albumin (BSA). The forebrain was dissociated by trypsinization at 37°C for 20 minutes (trypsin EDTA 1X). The reaction was terminated by adding Dulbecco's modified Eagle's medium (DMEM) containing grade II DNase I (0.1 mg / mL) and 10% fetal calf serum (FCS). The cells were then mechanically dissociated by aspiration and extrusion 3 times with a 10 mL pipette. The cells were then centrifuged at 180×g for 10 minutes at 4°C on a layer of BSA (3.5%) in L15 medium. The supernatant was discarded and the pelleted cells were resuspended in DMEM containing 10% FCS. The cells were then centrifuged at 515×g for 10 minutes at 4°C. The supernatant was discarded, and the cells of the pellet were resuspended in a culture medium comprising a neurobasal composition supplemented with 2% B27, 2mM L-glutamine (L Glu), 2% PS solution, 1% FCS and 10ng / mL platelet-derived growth factor (PDGF-AA). Live cells were counted in a Neubauer hemocytometer using a trypan blue exclusion test. Cells were seeded in a 96-well plate pre-coated with poly-L-lysine and laminin at a density of 20,000 cells / well.

[0786] One day after seeding (culture day 1), cells were incubated with test compounds ( Selected from those listed in Table 13 ) or estradiol. Control cells were not incubated with test compounds or estradiol. Estradiol was used as a positive control. It is known that estradiol induces OPC proliferation. The positive effect of estradiol on OL differentiation has also been confirmed because it has an effect on the early myelination process. The positive effect of estradiol on neurite outgrowth has also been published (see Alevaro et al., 2010 for review).

[0787] In a humidified incubator, the plate was maintained at 37°C in an atmosphere of air (95%)-CO2 (5%). Half of the culture medium was replaced with fresh culture medium and test compound or control compound every other day. The test compound or control compound was maintained at a defined concentration for the duration of the experiment. Compounds were tested on 1 culture (6 wells per condition). Cells were then used on the 12th, 18th or 30th day of culture to measure OPC proliferation, differentiation of OPC to OL and early myelination process (myelin sheath wrapping), or one of the maturation (myelin maturation) and mature myelination process (myelin sheath wrapping) of OL.

[0788] Proliferation of OPCs-Measurement of A2B5-positive cells and total neurite length (NF)

[0789] On day 12 of culture, cells were fixed with a cold mixture of absolute ethanol (95%) and pure acetic acid (5%) for 5 minutes. Cells were then permeabilized and nonspecific sites were blocked with a solution containing 0.1% saponin and 1% FCS in phosphate-buffered saline (PBS) for 15 minutes at room temperature.

[0790] The cells were then incubated with monoclonal anti-A2B5 generated in mice conjugated to Alexa 488 at 1 / 200 dilution in PBS with 1% FCS, 0.1% saponin for 2 hours at room temperature, and with anti-NF (phosphorylated and non-phosphorylated neurofilament 200) raised in rabbit at 1 / 500 dilution in PBS with 1% FCS, 0.1% saponin for 2 hours at room temperature. The antibody was revealed with Alexa Fluor 568 goat anti-rabbit at 1 / 400 dilution in PBS with 1% FCS, 0.1% saponin for 1 hour at room temperature.

[0791] The total number of OPCs (number of A2B5 positive cells) was quantified (to assess proliferation) and the axonal network (total neurite length (NF)) was measured to assess the effect of the compounds on the neuronal network (the quality of myelination is directly related to the quality of the axonal network).

[0792] Differentiation from OPC to OL and myelination process (myelin sheathing) - number, area and overlap of MAG-positive cells Measurement of MAG / NF packaging and total neurite length (NF)

[0793] On day 18 of culture, cells were fixed with a cold mixture of absolute ethanol (95%) and pure acetic acid (5%) for 5 minutes. Cells were then permeabilized and nonspecific sites were blocked with a solution containing 0.1% saponin and 1% FCS in phosphate-buffered saline (PBS) for 15 minutes at room temperature.

[0794] The cells were then incubated with monoclonal anti-MAG produced in mouse at a 1 / 400 dilution in PBS containing 1% FCS, 0.1% saponin, and with anti-NF (phosphorylated and non-phosphorylated neurofilament 200) produced in rabbit at a 1 / 500 dilution in PBS containing 1% FCS, 0.1% saponin for 2 hours at room temperature. These antibodies were revealed with CF 488A goat anti-mouse at a 1 / 800 dilution in PBS with 1% FCS, 0.1% saponin, and with Alexa Fluor 568 goat anti-rabbit at a 1 / 800 dilution in PBS with 1% FCS, 0.1% saponin for 1 hour at room temperature.

[0795] The total number of OLs (number and area of ​​MAG positive cells) (to assess differentiation process) and the wrapping of OPCs around axons (overlapping MAG / NF wrapping) (myelination process) were quantified. The axonal network (total axon length (NF)) was measured to assess the effect of compounds on neuronal networks.

[0796] OL maturation (myelin maturation) - number and area of ​​MBP-positive cells, overlapping MBP / NF wrapping, and total axons Measurement of length (NF)

[0797] On day 30 of culture, cells were fixed with a cold mixture of absolute ethanol (95%) and pure acetic acid (5%) for 5 minutes. Cells were then permeabilized and nonspecific sites were blocked with a solution containing 0.1% saponin and 1% FCS in phosphate-buffered saline (PBS) for 15 minutes at room temperature.

[0798] The cells were then incubated with monoclonal anti-MBP produced in mouse at a 1 / 1000 dilution in PBS containing 1% FCS, 0.1% saponin, and with anti-NF (phosphorylated and non-phosphorylated neurofilament 200) produced in rabbit at a 1 / 500 dilution in PBS containing 1% FCS, 0.1% saponin for 2 hours at room temperature. These antibodies were revealed with CF 488A goat anti-mouse at a 1 / 800 dilution in PBS with 1% FCS, 0.1% saponin, and with Alexa Fluor 568 goat anti-rabbit at a 1 / 400 dilution in PBS with 1% FCS, 0.1% saponin for 1 hour at room temperature.

[0799] The total number of OLs (number and area of ​​MBP-positive cells) (to assess OL maturation) and the wrapping of myelin around axons (overlap MBP / NF(wrapping)) were assessed. The axonal network (total neurite length (NF)) was measured to assess the effect of compounds on neuronal networks.

[0800] For all measurements, one culture (6 wells per condition) was completed. For each test condition, 30 pictures were collected per well (each picture represents one field) and analyzed with 20 times magnification using ImageXpress (Molecular Devices) equipped with LED lights (excitation 360 / 480 / 565 and emission 460 / 535 / 620). The 30 pictures were automatically taken and accounted for 80% of the total surface of the culture well.

[0801] Results are expressed as the cumulative mean length (in μm) of the neurite network or myelin per field for a given marker (MAG or MBP). The overlap area between NFs and MAG or MBP was measured to assess encapsulation.

[0802] To assess the OPC population, MAG-positive cell population, MBP-positive cell population, an automatic count of the number of positive cells per picture (=field) was performed. The results are expressed as the average number of positive cells per field.

[0803] All images were taken under the same conditions.

[0804] Table 13

[0805]

[0806]

[0807] The results are shown in Figures 12 to 32 middle

[0808] Example 8

[0809] Materials and methods

[0810] 1. Feeding Layer Preparation: Dissociation of Neonatal Cortices and Maintenance of Mixed Glial Cultures

[0811] Freshly dissected brains were added to a 37°C water bath for 3 minutes, and then the cortex was cut by a P1000 pipette tip to produce smaller fragments. 75 μL of OPC papain solution was added to each brain, and the tissue was then incubated in a 37°C water bath for 20 minutes. Mixed glial culture was then added to the tissue suspension to inactivate the OPC papain solution.

[0812] The tissue was then triturated using a sterile fire-polished glass Pasteur pipette and 4 mL of mixed glial culture medium was added per brain. The cells were centrifuged at 1200 rpm (approximately 300 g) for 5 minutes and then resuspended in warm mixed glial culture medium and plated into PLL-coated flasks.

[0813] 4 hours after inoculation, a complete medium change was performed to remove most of the debris generated by grinding and to promote culture viability. After 3 days of culture, a 2 / 3 medium change was performed and no subsequent medium changes were performed. The cells were then maintained in culture until confluence.

[0814] 2. Hippocampal Neuron Preparation

[0815] Hippocampal neurons were isolated from embryonic (E18) pups of Sprague Dawley rats. Briefly, after animal sacrifice, the brain was isolated, the meninges were removed from the medial side of the cerebral hemispheres, and the hippocampus was dissected out and kept at 4°C until the process was completed.

[0816] The tissue was then incubated with 2.5% trypsin in a 37°C water bath for 15 min and then gently washed and kept in culture medium. Hippocampal dissociation was performed by repeated pipetting of the hippocampus with a functionalized sterile Pasteur pipette. After mechanical dissociation, cells were plated at the desired density in neuronal plating medium, allowed to recover for 4 h, and then placed in competitive neuronal medium.

[0817] 3. Purification of OPCs from Mixed Glial Cultures to Establish OL / Hippocampal Neuron Co-cultures

[0818] On day 9 of the mixed glial culture, the flasks were shaken at 50 rpm for 45 min on an orbital shaker in a 5% CO2 tissue culture incubator. The purpose of this shaking was to remove any loosely adherent contaminating cells from the monolayer.

[0819] The medium was then changed and replaced with 4 mL of fresh mixed glial medium supplemented with 5 μg / mL insulin. The flask was then placed back on the shaker and equilibrated for approximately 3 hours, followed by shaking at 220 rpm for approximately 16 hours (overnight).

[0820] The next morning, the mixed glial culture medium containing microglia and OPC cells was collected and pre-plated in P100 dishes (untreated) for 30 minutes to purify OPC cells; microglia immediately began to adhere to the dish, while OPC cells remained in the supernatant culture medium.

[0821] 30 minutes after pre-plating, the culture medium was collected, and OLs were counted and plated on hippocampal neurons in a final volume of 1 mL OL culture medium.

[0822] A complete OL medium (minus CNTF) exchange was performed and cells were then maintained in culture until the appropriate experimental time.

[0823] For the mature experiment, the experimental procedure is as follows: (see Fig.55 )

[0824] a. OPCs grown on astrocyte feeder layer at 10 DIV

[0825] b. Isolation of OPCs (Day 0)

[0826] c. Administration of Compounds (Day 3)

[0827] d. Quantitative evaluation of maturity at days 4, 7 and 10.

[0828] For the myelination experiment, the experimental procedure was as follows: (see Fig.56 )

[0829] a. Growth of hippocampal neurons until complete neuronal network maturity (14 DIV)

[0830] b. OPCs growing on astrocyte feeder layer at 10 DIV

[0831] c. Isolation of OPCs and co-culture with neurons (Day 14)

[0832] d. Administration of Compounds (Day 15)

[0833] e. On the 15th, 18th, 21st / 23rd and 28th / 29th day of co-culture (after inoculation of co-culture plates),

[0834] Quantitative evaluation of myelination (day 1, before compound treatment)

[0835] 4. Image acquisition

[0836] All cultures at different experimental time points were fixed in 4% paraformaldehyde and 4% sucrose for 10 minutes at room temperature (RT). Primary and secondary antibodies were applied to GDB buffer (30mM phosphate buffer, pH 7.4, containing 0.2% gelatin, 0.5% Triton X-100 and 0.8M NaCl) at room temperature for 2 hours. Cells were stained with appropriate markers (primary antibodies used: anti-A2B5 antibody (ABCAM cat.ab53521), rat anti-MBP (BIO-RAD cat.aa82-87), oligodendrocyte marker O4 antibody (R&D Systems cat.MAB1326), anti-βIII tubulin mAb (Promega cat.G7121); secondary antibodies used: Alexa anti-rat 555 (Life Tech A-21434), Alexa anti-mouse 488 (Life Tech A-11009)). After immunocytochemical staining, all images were acquired with Array Scan XTI (ThermoScientific); the objective was magnified 20 times in 2 × 2 bins. For each condition and replicate (triplicate), a minimum of 15 images were taken.

[0837] To analyze all acquired images, use HCS Studio Cell Analysis Software, specifically the "Scan" application.

[0838] OPC papain solution (made in MEM)

[0839] Papain solution 1.54 mg / mL

[0840] L-cysteine ​​360 ​​μg / mL

[0841] DNase I 60 μg / mL

[0842] Mixed glial culture medium (made in DMEM)

[0843] FBS10%

[0844] Penicillin / Streptomycin (0.33%, from stock solution) 33 units / mL penicillin and 33 μg / mL streptomycin GlutaMAX 1%

[0845] OL medium

[0846] DMEM

[0847] 100X OL-Supplement

[0848] Bovine insulin (from 1 mg / mL stock solution)

[0849] GlutaMAX

[0850] Human holo-transferrin (from 33 mg / mL stock solution)

[0851] B27 Supplements

[0852] FBS CNTF (from 50ng / μL stock solution)

[0853] The results are shown in Figure 34 to Figure 53 middle.

Claims

1. A composition comprising at least one vitamin for promoting, supporting or optimizing one or more of the following in offspring of a female individual: (i) de novo myelination; (ii) brain structure; (iii) brain connectivity; (iv) intellectual potential; (v) cognitive potential; (vi) learning potential; and (vii) cognitive function, wherein the composition is for administration to the female individual, and wherein the offspring is a fetus, a newborn, an infant or a child, and wherein the composition is for administration to the female individual before conception, during pregnancy or during lactation, or a combination thereof.

2. The composition of claim 1, wherein the composition promotes, supports or optimizes the amount and / or spatial distribution of myelinated material throughout the brain.

3. A composition according to any preceding claim, wherein the composition promotes, supports or optimizes the de novo myelination trajectory.

4. A composition according to any preceding claim, wherein the female subject is a human.

5. A composition according to any preceding claim, wherein the vitamin is B12, folic acid or a combination thereof.

6. A composition according to any preceding claim, wherein the composition further comprises one or more of the following ingredients: minerals, fatty acid derivatives, choline and phospholipids or metabolites or metabolic precursors thereof.

7. The composition according to claim 7, wherein the phospholipid is selected from phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, sphingomyelin and mixtures thereof, and metabolic precursors and metabolites of any of the foregoing, and mixtures thereof.

8. The composition of claim 7, wherein the composition further comprises choline.

9. A composition according to any preceding claim, wherein the composition is in the form of a antenatal (before pregnancy) supplement and / or a maternal supplement (pregnancy and / or lactation supplement).

10. At least one vitamin for promoting, supporting or optimizing one or more of the following in the offspring of a female individual: (i) de novo myelination; (ii) brain structure; (iii) brain connectivity; (iv) intellectual potential; (v) cognitive potential; (vi) learning potential; and (vii) cognitive function, The vitamins are for administration to the female individual before conception, during pregnancy or during lactation, or a combination thereof.

11. A method of promoting, supporting or optimizing one or more of the following in offspring of a female individual: (i) de novo myelination; (ii) brain structure; (iii) brain connectivity; (iv) intellectual potential; (v) cognitive potential; (vi) learning potential; and (vii) cognitive function, The method comprises administering to the female individual a therapeutically effective amount of at least one vitamin, wherein the vitamin is administered to the female individual before conception, during pregnancy, or during lactation, or a combination thereof.

12. The method of claim 12, wherein the at least one vitamin is administered to the female individual separately, sequentially or simultaneously with one or more of the following ingredients: minerals, fatty acid derivatives, choline and phospholipids or metabolites or metabolic precursors thereof.

13. Use of at least one vitamin for the preparation of a composition for promoting, supporting or optimizing one or more of the following in offspring of a female individual: (i) de novo myelination; (ii) brain structure; (iii) brain connectivity; (iv) intellectual potential; (v) cognitive potential; (vi) learning potential; and (vii) cognitive function, The composition is for administration to the female individual before pregnancy, during pregnancy or during lactation, or a combination thereof.

14. A composition comprising vitamin B12 and / or folic acid, and choline and / or sphingomyelin and / or fatty acids comprising DHA and / or iron, wherein the composition is in the form of a pre-conception (before pregnancy) supplement and / or a maternal supplement (pregnancy and / or lactation supplement).

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