RU2795441C2 - Nutritional composition and infant formula for stimulation of brain myelinization - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: use of a synthetic nutritional composition containing free form or salt form of choline to prevent, reduce the risk of, or mitigate a suboptimal de novo myelination trajectory in a subject fed with infant formula, wherein the subject is a human infant or a child, such as a human infant or child being fed with an infant formula, and where the specified composition contains choline in an amount of from 50 to 1,000 mg/100 g by dry weight of the composition, and where the human infant is an infant under the age of 12 months, and the child is a person aged from 1 to 18 years, preferably from 1 to 5 years; and where the de novo myelination trajectory is sub-optimal when the distance between any equivalent and/or identical infant trajectory measurement points and the trajectory achieved in exclusively breastfed infants during their first 3 months of life is greater than 50%. Synthetic nutritional composition containing free form or salt form of choline to prevent, reduce the risk of, or mitigate a suboptimal de novo myelination trajectory in a subject fed with an infant formula that also contains folic acid and vitamin B12, and a fatty acid compound other than phospholipid, including docosahexaenoic acid and/or arachidonic acid, and sphingomyelin, in which choline is present in the specified composition in an amount from 50 to 1,000 mg/100 g, while sphingomyelin is present in the specified composition in an amount from 300 mg/kg to 2.5 g/kg, while folic acid is present in the specified composition in an amount from 110 to 500 μg/100 g, and vitamin B12 is present in the specified composition in an amount from 5 to 10 μg/100 g, and iron is present in the specified composition in an amount from 5 to 40 mg/100 g, and the fatty acid compound containing DHA is present in the composition in an amount from 60 to 350 mg/100 g, and the fatty acid compound containing arachidonic acid (ARA) is present in the composition in an amount from 60 to 350 mg/100 g of the dry weight of the composition, in which the entire weight is indicated based on the dry weight of the composition. Wherein the said fatty acid compound is monoacylglycerol and/or diacylglycerol and/or triacylglycerol and is a composition selected from the group consisting of the following: infant formula, complementary milk, infant formula, which is intended to be added or diluted with breast milk and a food product intended for consumption by an infant and/or child, either alone or in combination with human breast milk, where the infant is an infant under 12 months of age and the child is a human between 1 and 18 years of age and preferably of 1 up to 5 years.

EFFECT: invention allows promoting stimulation, support or optimization of primary myelination.

10 cl, 49 dwg, 18 tbl, 7 ex

Description

The field of technology to which the invention belongs

The present invention relates to a composition designed to promote, maintain or optimize primary myelination and/or brain structure and/or neuronal density, and/or one or more of the following: cognitive potential, learning ability and intellectual potential, and/ or cognitive function in the subject.

State of the art

Breastfeeding is recommended for all babies. However, in some cases, breastfeeding is insufficient or impossible for medical reasons. In such situations, infant formula can be used as an alternative to breast milk. However, studies have shown that infant formula does not always have the same effects on the body as mother's milk.

A recent study showed that the brain structure and/or spatiotemporal distribution of myelinated matter in the brains of exclusively breastfed infants may differ from those observed in formula fed infants, and that such differences may be correlated with increased levels of intelligence. , more effective learning and/or cognitive function in infants, particularly later in life, even when concomitant factors are taken into account ("Breastfeeding and early white matter development: a cross sectional study", Deoni et al, NeuroImage 82, ( 2013), 77-86). The said study also clearly demonstrates the existence of a connection, in particular a temporal relationship, between primary myelination and, in particular, between the primary myelination trajectory with brain structure.

In the past, long-term structural studies of the brain of breastfed and formula-fed infants have not been conducted, and differences in cognitive function and ability found were often simply attributed to factors such as socioeconomic status and educational status of the mother. The possible differences in brain structure between breastfed and formula-fed infants have not been considered or measured on the scale that is available today since infant brain MRI became common practice, see Deoni et al.

The importance of brain structure, in particular the amount and/or spatial distribution of myelinated matter within the brain for cognitive function and intelligence, is well established. Actually, myelin in the brain creates an insulating sheath along the length of the neuron, providing a much faster transmission of nerve impulses. However, it is the structure of the brain, in particular the amount and/or spatial distribution of myelin within the brain, that affects the density of neural connections, such as how and how quickly and efficiently messages in the form of nerve impulses are transmitted within the brain and in particular between different areas of the brain. This exchange of information within the brain may play a role in cognition and learning and may influence or even serve to physiologically limit intellectual, cognitive and/or learning potential and regulate cognitive function.

Accordingly, there is a need to find methods to stimulate, support or optimize primary myelination, in particular the primary myelination trajectory, and/or brain structure, in particular the amount and/or spatial distribution of myelinated matter within the brain, and/or the density of neuronal connections in a subject. , in particular in a subject receiving infant formula.

In addition, there is a need to find ways to optimize the intellectual potential and/or cognitive potential and/or learning ability and/or cognitive function in a subject, in particular in a subject receiving infant formula.

The present inventors have surprisingly found that a choline-containing composition can promote, support or optimize primary myelination, in particular the primary myelination trajectory, and/or brain structure, in particular the amount and/or spatial distribution of myelinated matter within the brain, and /or the density of neural connections in the subject, in particular in the subject receiving infant formula.

More specifically, the authors of the present invention have found that a composition containing choline can help stimulate, support or optimize primary myelination, in particular the trajectory of primary myelination, and/or the structure of the brain, in particular the amount and/or spatial distribution of myelinated substance within the brain in a subject receiving infant formula, and may bring one or more of the above characteristics in full or more close correspondence with those observed in a subject who is breastfed, in particular who is exclusively breastfed.

This conclusion is based on a nutritional analysis of a long-term cognitive and brain imaging study that examined and compared primary myelination, in particular the trajectory of primary myelination, and/or brain structure, in particular the amount and/or spatial distribution of myelinated matter. within the brain, specifically as determined by primary myelination and the trajectory of primary myelination in breastfed and infant formula subjects. Additional details of this study and the results are given in the examples included in the text of this document.

Disclosure of invention

The main provisions of the invention are set forth in the claims and are presented in more detail in the detailed description included in the text of this document.

The present invention relates to an artificial nutritional composition containing choline, which can be used to promote, support or optimize one or more of the following:

• primary myelination, in particular the trajectory of primary myelination,

• structures of the brain, in particular the amount and spatial distribution of myelinated matter within the brain and/or certain areas of the brain,

• density of neural connections,

• intellectual potential,

• cognitive potential,

• ability to learn,

• cognitive function

in a subject, in particular in a subject receiving infant formula. More specifically, said subject may be a human infant or child, and more specifically, a human infant or child receiving infant formula.

Said artificial nutritional composition may be a composition selected from the group consisting of; infant formula, milk formula for children from 1 to 3 years old, a composition for infants that is intended to be added or diluted with human breast milk, and a food product intended for consumption by an infant and / or child in pure form or in combination with human breast milk.

Said composition can optimize the primary myelination trajectory in a subject, in particular a subject receiving infant formula, and bring them any amount closer to the average primary myelination trajectory observed in breastfed subjects, in particular exclusively breastfed subjects, in the brain in general or in one or more areas of the brain when compared to a composition in which all other components are identical except for the content of choline.

Said composition may further comprise any one or more of the following ingredients:

• a phospholipid, in particular a phospholipid of formula (I) or a mixture of compounds of formula (I) as described herein, and in particular may be phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, sphingomyelin or a mixture thereof,

• a vitamin, in particular vitamin B12 and/or folic acid,

• mineral matter, in particular iron, zinc, copper, calcium, phosphorus, magnesium and any combination thereof,

• a fatty acid derivative, wherein said fatty acid derivative is a fatty acid-containing compound other than a phospholipid, wherein said fatty acid derivative may be selected from the group consisting of free fatty acid, monoacylglycerol, diacylglycerol, triacylglycerol, cholesterol ester, and combinations thereof and wherein said fatty acid may be docosahexaenoic acid and/or arachidonic acid and/or stearic acid and/or nervonic acid.

If the composition additionally contains one or more of these ingredients, it may be more effective.

Values for particularly effective concentrations of the above ingredients are given in the claims and in the following detailed description, included in the text of this document.

Brief description of the figures

In FIG. Figure 1 shows mean whole brain (whole white matter) myelination trajectories in breastfed infants and young children compared to those receiving two commercial infant formulas with different choline levels.

In FIG. 1a shows the mean trajectories of myelination across brain regions in breastfed infants and young children compared to those receiving two commercial infant formulas with different levels of choline.

In FIG. 1b is a brain tomogram showing myelinated areas of the brain associated with choline.

In FIG. 1c is a brain tomogram showing myelinated areas of the brain associated with sphingomyelin.

In FIG. 1d is a brain image showing myelinated areas of the brain associated with phosphatidylinositol.

In FIG. 1e is a brain tomogram showing myelinated areas of the brain associated with phosphatidylcholine.

In FIG. 1f is a brain tomogram showing myelinated areas of the brain associated with folic acid.

In FIG. 1g is a brain image showing myelinated areas of the brain associated with vitamin B12.

In FIG. 1h is a brain tomogram showing myelinated areas of the brain associated with iron.

In FIG. 1i is a brain tomogram showing myelinated areas of the brain associated with zinc.

In FIG. 1j is a brain image showing myelinated areas of the brain associated with calcium.

In FIG. 1k is a brain tomogram showing myelinated areas of the brain associated with phosphorus.

In FIG. 1l is a brain image showing myelinated areas of the brain associated with magnesium.

In FIG. 1m is a brain image showing myelinated areas of the brain associated with docosahexanoic acid.

In FIG. 1n is a brain tomogram showing myelinated areas of the brain associated with arachidonic acid.

In FIG. 2 shows the effect of nervonic acid on neuronal density and astrocyte density.

In FIG. 3 shows the effect of stearic acid on neuronal density and astrocyte density.

In FIG. 4 shows the effect of octanoic acid on neuronal density and astrocyte density.

In FIG. 5 shows the effect of sphingomyelin on the number of neurospheres and

proliferation of neurons.

In FIG. 6 shows the relative abundance of the main types of sphingomyelin (SM) in the ingredient, infant formula, cow's milk and breast milk. (Error bars reflect the standard deviation at n = 3).

In FIG. 7 shows the relative content of fatty acid (FA) in the SM fraction in the ingredient, infant formula, cow's milk and breast milk. (Error bars reflect the standard deviation at n = 3).

In FIG. 8 shows the effects of docosahexaenoic acid (DHA) on myelin basic protein (MBP), neurofilaments (NF) and/or MBP/NF on day 18 and/or day 30.

In FIG. 9 shows the effect of stearic acid (SA) on A2B5, MBP, monoacylglycerol (MAG), NF, MBP/NF and/or MAG/NF on day 12, day 18 and/or day 30.

In FIG. 10 shows the effect of vitamin B12 on A2B5, NF, MBP/NF and/or MAG on day 12, day 18 and/or day 30.

In FIG. 11 shows the effect of folic acid (FA) on A2B5, NF, MAG, MAG/NF and/or MBP/NF on day 12, day 18 and/or day 30.

In FIG. 12 shows the effect of choline (Cho) on A2B5, MAG and/or MBP on day 12, day 18 or day 30.

In FIG. 13 shows the effect of iron on A2B5, MBP, MAG, NF and/or MAG/NF on day 12, day 18 and/or day 30.

In FIG. 14 shows the effect of zinc on MBP, NF and/or MBP/NF on day 12, day 18 and/or day 30.

In FIG. 15 shows the effect of phosphorus on MAG, NF and/or MAG/NF on day 12, day 18 and/or day 30.

In FIG. 16 shows the effects of magnesium on A2B5, MBP, NF, MAG, MBP/NF and/or MAG/NF on day 12, day 18 and/or day 30.

In FIG. 17 shows the effect of copper on A2BF, MAG, and/or MAG/NF on day 12 and/or day 18.

In FIG. 18: Phosphatidylcholine shows the effect of phosphatidylcholine on A2B5 at day 12 and on MAH at day 18.

In FIG. 19 shows the effect of phosphatidylinositol on A2B5, MBP, MAG, NF, MAG/NF on day 12, day 18 and/or day 30.

In FIG. 20 shows the effect of phosphatidylserine on A2B5, NF and/or MAG/NF on day 12 and/or day 18.

In FIG. 21 shows the effect of sphingomyelin on A2B5, MAG and/or MBP on day 12, day 18 and/or day 30.

In FIG. 22 shows the effect of ceramide (Cer) on A2B5 on day 12 and on MAG on day 18.

In FIG. 23 shows the effect of galactoceramide (GalCer) on A2B5, MBP, NF and/or MBP/NF on day 12 and/or day 30.

In FIG. 24 shows the effect of glucoceramide (GluCer) on A2B5 on day 12 and on NF on day 12 and day 18.

In FIG. 25 shows the effect of D-erythroceramide (D-ery) on A2B5 on day 12 and on MAG on day 18.

In FIG. 26 shows the effect of ceramide-1-phosphate (C1P) on A2B5 on day 12 and on NF and MAG on day 18.

In FIG. 27 shows the effect of monosialoganglioside-3 (GM3) on A2B5, MBP, MAG and/or MBP/NF on day 12, day 18 and/or day 30.

In FIG. 28 shows the effect of disialoganglioside-3 (GD3) on A2B5, MBP, NF and/or MAG on day 12, day 18 and/or day 30.

In FIG. 29 shows the fatty acid profile for phosphatidylinositol (PI), phosphatidylcholine, phosphatidyl (PC), phosphatidylserine (PS), and sphingomyelin used in Example 6.

In FIG. 30 shows the effect of vitamin B12 on MAG and MBP mRNA expression and on MBP and BetaIII co-expression.

In FIG. 31 shows the effect of arachidonic acid (ARA) on MAG and MBP mRNA expression and on MBP and BetaIII co-expression.

In FIG. 32 shows the effect of stearic acid on MAG and MBP mRNA expression and on MBP and BetaIII co-expression.

In FIG. 33 shows the effect of zinc on MAG and MBP mRNA expression and on MBP and BetaIII co-expression.

In FIG. 34 shows the effect of phosphatidylinositol on MAG and MBP mRNA expression.

In FIG. 35 shows the effect of GD3 on MAG and MBP mRNA expression and on MBP and BetaIII co-expression.

In FIG. 36 shows the effect of DHA on MAG and MBP mRNA expression and on MBP and BetaIII co-expression.

In FIG. 37 shows the effect of nervonic acid on MAG and MBP mRNA expression and on MBP and BetaIII co-expression.

In FIG. 38 shows the effect of iron on MAG and MBP mRNA expression and on MBP and BetaIII co-expression.

In FIG. 39 shows the effect of phosphatidylcholine on MAG and MBP mRNA expression and on MBP and BetaIII co-expression.

In FIG. 40 shows the effect of phosphatidylserine on MAG and MBP mRNA expression and on MBP and BetaIII co-expression.

In FIG. 41 shows the effect of folic acid on MAG and MBP mRNA expression and on MBP and BetaIII co-expression.

In FIG. 42 shows the effect of choline on MAG and MBP mRNA expression and on MBP and BetaIII co-expression.

In FIG. 43 shows the effect of ceramide on MAG and MBP mRNA expression and on MBP and BetaIII co-expression.

In FIG. 44 shows the effect of galactoceramide on MAG and MBP mRNA expression and on MBP and BetaIII co-expression.

In FIG. 45 shows the effect of glucoceramide on MAG and MBP mRNA expression and on MBP and BetaIII co-expression.

In FIG. 46 shows the effect of ceramide-1-phosphate on MAG and MBP mRNA expression and on MBP and BetaIII co-expression.

In FIG. 47 shows the effect of D-erythroceramide on MAG and MBP mRNA expression and on MBP and BetaIII co-expression.

In FIG. 48 shows the effect of sphingomyelin on MBP and BetaIII co-expression.

In FIG. 49 shows the effect of GM3 on the co-expression of MBP and BetaIII.

Implementation of the invention

In one aspect of the present invention, an artificial composition containing choline is provided, and such a composition can be used to stimulate support or optimize primary myelination, in particular the primary myelination trajectory, and/or brain structure, and/or neuronal density, and/or intellectual potential and/or cognitive potential and/or learning ability and/or cognitive function in a subject, in particular in a subject receiving infant formula.

By stimulating, supporting or optimizing primary myelination, in particular the primary myelination trajectory, and/or brain structure, in particular the amount and/or spatial distribution of myelinated matter within the brain, in particular as determined by primary myelination and the primary myelination trajectory, and/ or intellectual potential, and/or cognitive potential, and/or learning ability, and/or cognitive function in a subject, in particular in a subject receiving infant formula, the composition of the present invention can prevent, reduce the risk and/or mitigate the consequences of insufficient primary myelination , in particular the trajectory of insufficient primary myelination, and / or insufficient brain structure, and / or insufficient density of neuronal connections, and / or insufficient intellectual potential, and / or insufficient cognitive potential, and / or insufficient learning ability, and / or insufficient cognitive function in said subject. It can be non-therapeutic or therapeutic.

The term "encourage" as used herein refers to one or more factors leading to the development of a particular process.

The term "support" as used herein refers to one or more factors that support a particular process once that process has begun to develop.

The term "subject" as used herein refers to a mammal, in particular a cat, dog or human, more specifically, the term refers to a human, even more specifically to a human infant or child, and even more specifically to a human infant or child, receiving infant formula and/or milk formula for children from 1 to 3 years.

The term "infant" as used herein refers to a human infant under 12 months of age and includes preterm and very preterm infants, low birth weight infants, i. newborns weighing less than 2500 g (5.5 lbs), both due to preterm birth and limited fetal growth, and infants born small for their gestational age (SGA), i.e. babies with birth weight below the 10th percentile for babies of the same gestational age.

The term "child" as used herein refers to a person between 1 and 18 years of age, more specifically a person between 1 and 10 years of age, even more specifically a person between 1 and 5 years of age, and even more specifically - to a person aged 1 to 2 years.

The term "infant or child receiving infant formula" as used herein refers to an infant or child receiving both infant formula and/or infant formula for children from 1 to 3 years of age.

The term "breastfed subject" as used herein refers to a mammal, in particular a cat, dog or human, more specifically, the term refers to a human, even more specifically to a human infant or child, and even more specifically to an infant or the child of a person receiving breast milk, in particular from a well-nourished mother.

The term "primary myelination" as used herein refers to the development of myelination and, in particular, to the process by which unsheathed axons in the brain of a subject are myelinated during growth and development. It is a process that begins, in particular in specific areas of the brain, in the womb and continues after childbirth and which is most active in the human subject, in particular in the first 2 or 3 years of human life, more specifically in the first year of life. person.

The term "primary myelination trajectory" as used herein refers to the degree of primary myelination or accumulation of new myelin (as measured by myelin water fraction, for example) over time, and in particular during and during infancy and childhood, in particular early childhood, and more specifically in the first 5 years of a human subject's life, more specifically in the first 2 or 3 years of a person's life, even more specifically in the first year or in the first 6 months of a person's life, when infant formula can be the only form of nutrition for some babies.

The term "brain structure" as used herein refers to the structure of gray and white matter in the brain and specific areas of the brain, in particular the myelinated white matter in the brain of specific areas of the brain, as determined by primary myelination, in particular the trajectory of primary myelination. , i.e. primary structural deposition of myelin. More specifically, the term refers to the amount and/or spatial distribution of myelinated matter within the brain and/or in specific regions of the brain, and more specifically, to the amount and/or spatial and temporal distribution of myelinated matter within the brain and/or in specific areas. areas of the brain.

The term "intellectual potential" as used herein refers to the possible intellectual abilities or capabilities achievable for a subject, as determined by physiological factors. In particular, intellectual potential may refer to fluid intelligence.

The term "fluid intelligence" as used herein refers to a subject's neural potential and/or the subject's ability to solve novel or abstract problems as determined by physiological factors. It is distinct from "crystallized intelligence", which is at least partly determined by learned or progressive knowledge.

The term "cognitive potential" as used herein refers to the possible cognitive and/or mental abilities or capabilities attainable by a subject as determined by physiological factors. In particular, the term may refer to one or more of the following; information processing potential, perception potential, attention potential, thinking potential, reasoning potential, comprehension and memorization potential, psychomotor potential, including gross motor and fine motor skills, visual potential, including visual perception potential, auditory perception potential, language potential , including the potential of language skills for transmitting and perceiving information, the potential for memorization and recall, the potential for concentration, the potential for executive function, including the potential for problem solving, decision making and inhibition.

The term "learning ability" as used herein refers to a subject's possible ability or ability to learn, such as how easily and/or quickly a subject may be able to acquire knowledge or skills through experience, study, or education, as determined by physiological factors. And also to the possible ability of the subject to adapt in response to external factors, which is determined by physiological factors.

The term "learning" as used herein refers to the acquisition of knowledge or skills through experience, study, or education.

The term "cognition" as used herein refers to the intellectual processes by which an individual learns, perceives or understands ideas; those. to the ability to think and understand. Cognition includes all aspects of information processing, perception, attention, thinking, reasoning, understanding and memory, as well as psychomotor function, language function, memory function, concentration, executive function and problem solving abilities.

The term "optimize" as used herein refers to improvement or enhancement.

Since human breast milk is the ideal standard when it comes to infant nutrition, the primary myelination trajectory measured or observed in breastfed subjects, in particular in exclusively breastfed subjects, in particular fully nourished mothers, may be considered optimal. Therefore, a composition of the present invention can be considered as optimizing the myelination trajectory in a subject if it brings the primary myelination trajectory in a subject, in particular in a subject receiving infant formula, into full or closer agreement with that measured or observed in a breastfed subject, more specifically - in an exclusively breastfed subject, in particular a fully nourished mother.

The primary myelination trajectory in a subject can be considered to match or more closely match that measured or observed in a breastfed subject, more specifically in an exclusively breastfed subject, in particular a fully fed mother, if the distance between any equivalent/identical measurement points is the trajectory of the subject and the trajectory of said breastfed subject is up to 50%, in particular up to 25%, more specifically up to 20%. Non-limiting examples in the range up to 50% include 50%, 40%, 30%, 25%, 20%, 10%, 5%, 1%, 0.5% and 0.01%. In particular, trajectories will be considered bioequivalent.

The myelination trajectory can be measured over any combination of time points. In particular, the time points are within the first 5 years of a person's life, more specifically the first 2 or 3 years of a person's life, even more specifically the first year or the first 6 months of a person's life.

The trajectory of primary myelination can be determined by repeated measurements of any myelination marker in a subject over time. In particular, the primary myelination trajectory can be assessed by measuring the myelin-associated water fraction (myelin water fraction) and/or the myelin-associated water depot (myelin water depot) in a subject at various time points, in particular at various time points during the first 5 years of the life of the human subject, more specifically the first 2 or 3 years of the human life, even more specifically the 1st year or the first 6 months of the human life. The myelin-associated water fraction and/or myelin-associated water depot in a subject can be assessed by magnetic resonance imaging (MRI) multi-component relaxation (MCR) measurements and in particular using the mcDESPOT technique (Deoni et al 2008). In particular, the trajectory of primary myelination can be determined by repeatedly measuring myelin-associated water depot using the mcDESPOT technique over time (Magn.Reson.Med.2008 60:1372-1387, the contents of which are incorporated herein by reference).

A composition of the present invention may be considered to optimize a subject's cognitive function and/or intelligence if it scores in one or more subjects, in particular a subject receiving infant formula, on a standardized cognitive test, including an intelligence test, a school achievement test, and/or a neurodevelopmental test, such as the Mullen Early Learning Scales, in full or closer agreement with those measured or observed in a breastfed subject, more specifically in an exclusively breastfed subject, in particular a fully nourished mother. Cognitive and neuropsychiatric function in a subject may be considered to be in full or nearer agreement with those assessed in said breastfed subject if the difference between the scores administered to said subject on one or more standardized neurodevelopmental tests, such as Mullen T-scores, and such indicators in said breastfed subject is less than one standard deviation, in particular less than half the standard deviation of standardized test scores, for example, less than 10 points, more specifically, less than 5 points for Mullen T-scores, in particular, less than 2 points. Said scores of the standardized neurodevelopmental test, eg Mullen T-scores, are measured at the same time point in said subject and said breastfed subject.

Said Mullen score can be measured at any suitable point in time, in particular during the first 5 years of a person's life, 3 years of a person's life, more specifically the first 2 years of a person's life, even more specifically the first year or first 6 months of a person's life.

By stimulating the maintenance or optimization of cognitive potential, learning ability and/or intellectual potential, the compositions of the invention can have a short or long-term effect on cognitive function, including the development of cognitive functions and/or learning, and on the prevention or minimization of any neurocognitive abnormalities, impairments or delays in development.

This short-term effect may only appear for days, weeks or months.

Said long-term effect may occur over years, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 years.

Compositions containing choline may be particularly effective in supporting, stimulating or optimizing primary myelination, in particular the primary myelination trajectory, and/or brain structure in the following areas of the brain; cerebellum, visual cortex, thalamus, parietal cortex and frontal lobe. These areas of the brain are associated with motor function (including coordination and execution of movements), vision, short-term memory and/or executive function, and/or social-emotional thinking, and/or spatial thinking. Accordingly, a choline-containing composition may be particularly effective in stimulating, supporting, or optimizing motor function potential (including motor coordination and exercise) and/or psychomotor potential, visual potential, short-term memory potential, and/or executive function potential, and/or social-emotional thinking potential, and/or spatial thinking potential.

In one embodiment of the invention, the cognitive potential is selected from the group consisting of motor function potential (including coordination and exercise of motor potential), and/or psychomotor potential, visual potential, short-term memory potential, executive function potential, socio-emotional thinking potential, and spatial potential. thinking.

When stimulating, maintaining or optimizing the potential of motor function (including coordination and exercise of motor potential), visual potential, short-term memory potential, and / or executive function potential, and / or social-emotional thinking potential, and / or spatial thinking potential of the composition according to this inventions may have a short-term or long-term effect, such as an improving effect on motor and psychomotor function (including coordination and movement performance), and/or vision, and/or short-term memory, and/or executive function, and/or socio-emotional thinking, and /or spatial thinking. This short-term effect may only appear for days, weeks or months.

Said long-term effect may occur over years, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 years.

The term "choline" refers to quaternary ammonium salts containing the N,N,N-trimethylethanolammonium cation and having the following structure:

Choline

The counterion X ^- may in particular be chloride, hydroxide, citrate, bitartrate and mixtures thereof.

Unless otherwise indicated, in the context of the present invention, the term "choline" is intended to refer to all cholines present in the nutritional composition of the present invention in free form (or in the form of their salts).

Choline may be present in the composition of the present invention in an amount up to 99.999% of the composition.

In particular, choline may be contained in the composition in an amount of more than 30 mg/100 g dry composition.

In one embodiment, the composition in accordance with the present invention contains choline in an amount selected from the group consisting of the following; more than 30 mg, more than 50 mg, more than 100 mg, more than 111 mg, more than 170 mg, in the range of 30 to 1000 mg, in the range of 30 to 700 mg, in the range of 50 to 1000 mg, in the range of 50 to 700 mg, in the range of 50 to 500 mg, in the range of 100 to 400 mg, in the range of 111 to 180 mg, in the range of 170 mg to 300 mg, all weights per 100 g dry weight of the composition.

In one embodiment, a composition according to the present invention contains such levels of choline in free form (or in the form of its salts) or in the form of derivatives from structures containing it, that the total daily intake provided by the nutritional composition of the invention will not exceed 1 g.

Choline can be included in the composition of the present invention in pure form or in the form of one of the physiologically acceptable salts, such as, for example: choline chloride, choline citrate, choline bitartrate, or mixtures thereof.

It may be particularly useful if the composition according to the present invention additionally contains one or more of the following ingredients: vitamins and/or minerals and/or phospholipids and/or fatty acid derivatives.

If the composition of the invention contains choline and one or more of the ingredients mentioned above, it can be improved to stimulate the support and/or optimization of primary myelination, in particular the trajectory of primary myelination, and/or the structure of the brain, and/or the density of neuronal connections, and/or cognitive potential, and/or intellectual potential, and/or learning ability, and/or cognitive function in a subject, in particular in a subject receiving infant formula. This may be due, for example, to the fact that said ingredients act on primary myelination in the same and/or different complementary areas of the brain. This enhanced effect may be synergistic.

In one embodiment, the composition of the invention further comprises a vitamin and/or mineral and/or a phospholipid and/or a fatty acid derivative.

The term "vitamin" as used herein refers to any vitamin. Non-limiting examples of vitamins include: vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin K, vitamin C, vitamin D, niacin, biotin, pantothenic acid, folic acid, vitamin B12, and combinations thereof.

Particularly effective vitamins can be folic acid, vitamin B12 and vitamin B6, in particular folic acid and vitamin B12, in particular folic acid.

In one embodiment of the invention, the vitamin is vitamin B12, folic acid, and/or a combination thereof.

The vitamin may be present in the composition of the present invention in an amount up to 99.999% of the composition.

Folic acid may be present in the composition of the present invention in an amount up to 99.999% of the composition.

In particular, folic acid may be contained in an amount of more than 50 μg/100 g dry composition, more specifically from 50 μg to 500 μg/100 g dry composition.

In one embodiment, the composition of the invention contains folic acid in an amount selected from the group consisting of the following; more than 50 mcg, more than 65 mcg, more than 70 mcg, more than 100 mcg, more than 110 mcg, more than 160 mcg, in the range of 50 to 500 mcg, in the range of 50 to 400 mcg, in the range of 70 to 170 mcg, in the range 110 to 500 µg, in the range of 110 to 400 µg, in the range of 110 to 400 µg, in the range of 110 to 350 µg, all weights being given per 100 g dry weight of the composition.

In one embodiment, the composition in accordance with the present invention contains such an amount of folic acid that the total daily intake provided by the nutritional composition of the invention will not exceed 400 micrograms.

Folic acid may be included in the nutritional compositions of the present invention as such or in the form of its physiologically acceptable salt (folate) or mixtures thereof.

Vitamin B12 may be present in the composition of the present invention in an amount up to 99.999% of the composition.

In particular, vitamin B12 may be present in the composition in an amount selected from the group consisting of the following; more than 0.01 µg, in particular more than 0.04 µg, in particular more than 0.05 µg, all weights being given per 100 g dry weight of the composition.

In one embodiment, the composition of the invention contains vitamin B12 in an amount selected from the group consisting of the following; more than 0.01 µg, more than 0.5 µg, more than 0.7, more than 5 µg, in the range from 0.1 to 10 µg, from 0.4 to 5 µg, from 0.5 to 2 µg, from 1 to 1.5 µg, 4 to 8.5 µg, in the range of 5 to 8 µg, all weights being per 100 g dry weight of the composition.

In one embodiment, a composition according to the present invention contains such an amount of vitamin B12 that the total daily intake provided by the nutritional composition of the invention will not exceed 7.6 µg/100 g dry composition (77.6 µg/kg dry composition).

Vitamin B12 may be included in the nutritional compositions of the invention as such or in the form of its physiologically acceptable salt, or mixtures thereof, or using any source containing vitamin B12. In particular, vitamin B12 may be included in the composition in its pure form, as cyanocobalamin, hydroxocobalamin, and any combination thereof.

Compositions containing a vitamin, in particular folic acid and/or vitamin B12, may be particularly effective in supporting, stimulating or optimizing primary myelination, in particular the primary myelination trajectory, and/or brain structure in one or more of the following brain regions; cerebellum, visual cortex, motor and somatosensory cortex. These areas of the brain are associated with motor function (including coordination and execution of movements), visual function and psychomotor function.

The term "mineral matter" as used herein refers to any mineral matter. Non-limiting examples of minerals include: iron, zinc, calcium, phosphorus, copper, magnesium, iodine, manganese, chloride, potassium, sodium, selenium, chromium, and combinations thereof. Minerals are usually added in the form of a salt.

Particularly effective minerals can be iron, zinc, calcium, phosphorus, copper and magnesium, in particular iron.

In one embodiment, the composition according to the present invention contains iron and/or zinc and/or calcium and/or phosphorus and/or copper and/or magnesium, in particular iron and zinc, more specifically iron.

In one embodiment, the nutritional composition according to the present invention contains iron. Iron may be present in the composition of the present invention in an amount up to 99.999% of the composition.

In particular, iron may be contained in the composition in an amount of more than 5 mg/100 g dry composition.

In one embodiment, the composition in accordance with the present invention contains iron in an amount selected from the group consisting of the following; more than 4 mg, more than 9 mg, in the range of 5 to 40 mg, in the range of 9 to 40 mg, in the range of 5 to 20 mg, in the range of 9 to 20 mg, in the range of 5 to 15 mg, in the range from 9 to 15 mg, in the range from 3.5 to 7 mg, with all weights based on 100 g dry weight of the composition.

Iron can be included in the compositions of the invention in the form of one of the physiologically acceptable salts, such as, for example, iron III citrate, iron III phosphate, iron III pyrophosphate, iron II ascorbate, iron II carbonate, iron II citrate, iron II fumarate, gluconate iron II, iron II lactate, iron II sulfate, or mixtures thereof.

Iron may be included in the composition of the present invention in the form of a physiologically acceptable iron complex (such as, for example, EDTA sodium iron) or mixtures thereof.

Fe2+ is more bioavailable and therefore it may be more appropriate to add iron to the composition in the form of an iron II salt or complex, for example in the form of the iron II salts listed above.

In one embodiment, the composition in accordance with the present invention contains such levels of iron that the total daily intake provided by the nutritional composition of the invention will not exceed 40 mg.

In one embodiment, the nutritional composition according to the present invention contains zinc. Zinc may be present in the composition of the present invention in an amount up to 99.999% of the composition.

In particular, zinc may be present in the composition in an amount greater than 0.08 mg, greater than 0.3 mg, greater than 0.5 mg, all weights being given per 100 g dry weight of the composition.

In one embodiment, the composition in accordance with the present invention contains zinc in an amount selected from the group consisting of the following; in the range of 0.5 to 8 mg, 2 to 5.5 mg, 2.5 to 4.5 mg, 3 to 4 mg, 4 to 7.5 mg, 6 to 7.5 mg, and all weights are based on 100 g of dry composition.

In one embodiment, the composition in accordance with the present invention contains such levels of zinc that the total daily intake provided by the nutritional composition of the invention will not exceed 302.4 mg/day, or will not exceed 245 mg/day, or will not exceed 166 mg/day. day, or will not exceed 98.9 mg/day, or will not exceed 95.6 mg/day.

Zinc may be present in the composition of the invention in the form of a physiologically acceptable salt, 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, for example, zinc picolinate), or their mixtures.

In one embodiment, the composition in accordance with the present invention contains copper. Copper may be present in the composition of the present invention in an amount up to 99.999% of the composition.

In particular, copper may be present in the composition in amounts greater than 10 μg, greater than 40 μg, greater than 60 μg, all weights being given per 100 g dry weight of the composition.

In one embodiment, the composition in accordance with the present invention contains copper in an amount selected from the group consisting of the following; more than 100 mcg, in the range from 100 mcg to 850 mcg, from 180 to 650 mcg, from 200 to 400 mcg, from 210 to 300 mcg, from 210 to 240 mcg, from 450 to 850 mcg, from 800 to 840 mcg, and all weights are based on 100 g dry weight of the composition.

In one embodiment, the composition in accordance with the present invention contains such levels of copper that the total daily intake provided by the nutritional composition of the invention will not exceed 1426 micrograms per day or will not exceed 488 micrograms per day.

Copper can be included in the composition of the present invention as such or in the form of a physiologically acceptable salt and/or through any source containing copper, in particular Cu2+. For example, copper may be included in the composition in the form of: copper sulfate and/or copper gluconate and/or copper carbonate and/or copper citrate and/or copper-lysine complex.

In one embodiment, the composition in accordance with the present invention contains magnesium. Magnesium may be contained in the composition of the present invention in an amount up to 99.999% of the composition.

In particular, magnesium may be present in the composition in an amount greater than 0.2 mg, greater than 0.35 mg, greater than 0.5 mg, all weights being given per 100 g dry weight of the composition.

In one embodiment, the composition in accordance with the present invention contains magnesium in an amount selected from the group consisting of the following; 0.35 to 90 mg, 25 to 70 mg, 30 to 65 mg, 35 to 60 mg, 40 to 50 mg, 35 to 55 mg, all weights per 100 g dry composition.

In one embodiment, the composition in accordance with the present invention contains such levels of magnesium that the total daily intake provided by the nutritional composition of the invention will not exceed 110 mg per day or will not exceed 65 mg per day.

Magnesium can be included in the composition of the present invention as such or in the form of a physiologically acceptable salt and/or through any source containing magnesium, in particular Mg2+. For example, magnesium carbonate, magnesium chloride, magnesium oxide, magnesium sulfate, magnesium gluconate, magnesium hydroxide, magnesium salts of citric acid, magnesium salts of phosphoric acid.

In one embodiment, the composition in accordance with the present invention contains calcium. Calcium can be contained in the composition of the present invention in an amount up to 99.999% of the composition.

In particular, calcium may be present in the composition in an amount greater than 0.84 mg, greater than 2.52 mg, greater than 4.62 mg, all weights being given per 100 g dry weight of the composition.

In one embodiment, the composition in accordance with the present invention contains calcium in an amount selected from the group consisting of the following; in the range of 84 to 760 mg, in the range of 200 to 550 mg, in the range of 250 to 450 mg, in the range of 280 to 520 mg, from 350 to 650 mg, all weights per 100 g of dry composition.

In one embodiment, the composition in accordance with the present invention contains levels of calcium such that the total daily intake provided by the nutritional composition of the invention will not exceed 482 mg per day or will not exceed 477 mg per day.

Calcium can be included in the composition of the present invention as such or in the form of a physiologically acceptable salt and/or through any source containing calcium, in particular Ca2+. For example, calcium carbonate, calcium chloride, calcium salts of citric acid, calcium gluconate, calcium glycerophosphate, calcium lactate, calcium hydroxide, calcium salts of phosphoric acid.

In one embodiment, the composition in accordance with the present invention contains phosphorus. Phosphorus may be present in the composition of the present invention in an amount up to 99.999% of the composition.

In particular, phosphorus may be contained in the composition in an amount of more than 1.7 mg, more than 14.3 mg, more than 27.3 mg per 100 g dry weight of the composition.

In one embodiment, the composition in accordance with the present invention contains phosphorus in an amount selected from the group consisting of the following; 17 to 516 mg, 129 to 400 mg, 140 to 390 mg, 150 to 370 mg, 160 to 365 mg, 270 to 350 mg, 200 to 360 mg, all weights being given per 100 g dry weight of the composition.

In one embodiment, the composition in accordance with the present invention contains such levels of phosphorus that the total daily intake provided by the nutritional composition of the invention will not exceed 863 mg per day or will not exceed 787 mg per day.

Phosphorus can be included in the composition of the present invention as such or in the form of a physiologically acceptable salt and/or through any source containing phosphorus, such as calcium phosphate, calcium hydrogen phosphate.

A composition containing a mineral, for example one or more of iron, zinc, copper, calcium, magnesium and phosphorus, may be particularly effective in supporting, stimulating or optimizing primary myelination, in particular the primary myelination trajectory, and/or brain structure in one or more of the following brain regions; cerebellum, visual cortex, motor and somatosensory cortex, corpus callosum, frontal cortex, temporal white matter, internal capsule, prefrontal cortex, motor cortex. These areas of the brain are associated with motor function (including coordination and execution of movements), visual function, hemispheric interaction, executive function, short-term memory, problem solving, socio-emotional functioning, language, auditory function, problem solving, and/or short-term memory.

In one embodiment, the composition of the invention contains a phospholipid, its metabolic precursor or metabolite.

The term "phospholipid" as used herein refers to any phospholipid and in particular to a compound of formula (I)

(I)

Where

R ¹ is O,

X is NH or O

R ² is a saturated or unsaturated linear or branched C2-C44 acyl group;

R ³ is a substituent with formula (II) or formula (III)

(II)

(III)

where R ⁵ is a saturated or unsaturated linear or branched C2-C44 acyl group, and

R ⁶ is a saturated C2-C44 alkyl or alkenyl group; And

R ⁴ is selected from; C5 or C6 substituted or unsubstituted cyclic alkyl or alkenyl group, or

-(CH2)nR ⁷ where n is an integer ranging from 1 to 4, in particular from 1 to 2, and R ⁷ is -N(CH ₃ ), NH ₃ or a substituent of formula (IV) and,

(IV)

in particular, R ⁴ is a C6 cyclic alkyl or alkenyl group substituted with one or more hydroxy groups, more specifically R ⁴ is derived from inositol (C6H12O6), and more specifically myoinositol, ie. ^R4 is:

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

In one embodiment of the present invention, the phospholipid is selected from the group consisting of; phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, and/or combinations thereof.

Phosphatidylinositol is a compound of formula (V)

(V)

where R ⁸ represents a branched or straight chain acyclic alkyl or acyclic alkenyl group from C2 to C43, and

R ⁹ is a C2 to C43 branched or straight acyclic alkyl or acyclic alkenyl group.

More specifically, R ⁸ and R ⁹ are independently branched or straight chain acyclic alkyl or acyclic alkenyl groups C13 to C43 which, together with their adjacent carbonyl groups, correspond to saturated or unsaturated fatty acid residues C14 to C44, and yet more specifically, R ⁸ and R ⁹ independently of each other represent C13 to C23 branched or straight acyclic alkyl or acyclic alkenyl groups which, together with their adjacent carbonyl group, correspond to C14 to C24 saturated or unsaturated fatty acid residues.

More specifically, R ⁸ and R ⁹ are C13 to C23 branched or straight acyclic alkyl or acyclic alkenyl groups which, together with their adjacent carbonyl group, correspond to C14 to C24 saturated or unsaturated fatty acid residues, the fatty acids defining such residues fatty acids are selected from the group consisting of; 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. More specifically, C18:0, C18:1n-9, C18:2, C20:3 and C20:4.

As will be obvious to a person skilled in the art. The term "phosphatidylserine" as used herein refers to phosphatidyl-L-serine.

Phosphatidylserine is a compound of formula (VI)

(VI)

(VI)

where R ¹⁰ represents a branched or straight chain acyclic alkyl or acyclic alkenyl group from C2 to C43, and

R ¹¹ is a C2 to C43 branched or straight acyclic alkyl or acyclic alkenyl group.

More specifically, R ¹⁰ and R ¹¹ are independently branched or straight chain C13 to C43 acyclic alkyl or acyclic alkenyl groups which, together with their adjacent carbonyl groups, correspond to C14 to C44 saturated or unsaturated fatty acid residues, and yet more specifically, R ¹⁰ and R ¹¹ independently of each other represent C13 to C23 branched or straight acyclic alkyl or acyclic alkenyl groups which, together with their adjacent carbonyl group, correspond to C14 to C24 saturated or unsaturated fatty acid residues.

More specifically, R ¹⁰ and R ¹¹ are C13 to C23 branched or straight acyclic alkyl or acyclic alkenyl groups which, together with their adjacent carbonyl group, correspond to C14 to C24 saturated or unsaturated fatty acid residues, the fatty acids defining such residues fatty acids are selected from the group consisting of; 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. More specifically, C18:0, C18:1n-9, C20:4 and C22:6.

Phosphatidylethanolamine is a compound of formula (VII)

(VII)

where R ¹² represents a branched or straight chain acyclic alkyl or acyclic alkenyl group from C2 to C43, and

R ¹³ is a C2 to C43 branched or straight acyclic alkyl or acyclic alkenyl group.

More specifically, R ¹² and R ¹³ are independently branched or straight chain C13 to C43 acyclic alkyl or acyclic alkenyl groups which, together with their adjacent carbonyl groups, correspond to C14 to C44 saturated or unsaturated fatty acid residues, and yet more specifically, R ¹² and R ¹³ independently of each other are C13 to C23 branched or straight acyclic alkyl or acyclic alkenyl groups which together with their adjacent carbonyl group correspond to C14 to C24 saturated or unsaturated fatty acid residues.

The term "sphingomyelin" as used herein refers to a lipid molecule or mixture of lipid molecules in which the sphingosine or sphinganine backbone is esterified with a fatty acid residue at the amino group (-NH2) via amide bond formation and in which the hydroxyl group at position 1 of the sphingosine backbone is linked to a phosphorylcholine functional group.

In particular, sphingomyelin is a compound of formula (VIII) or a mixture of compounds of formula (VIII)

(VIII)

Where

R ¹⁴ is a C2 to C43 branched or straight acyclic alkyl or acyclic alkenyl group,

R ¹⁵ is a C2 to C43 branched or straight acyclic alkyl or acyclic alkenyl group.

More specifically, R ¹⁴ is a C13 to C43 branched or straight chain acyclic alkyl or acyclic alkenyl group which, together with its adjacent carbonyl group, corresponds to C14 to C44 saturated or unsaturated fatty acid residues.

Non-limiting examples of C14 to C44 saturated or unsaturated fatty acids that may define such fatty acid residues 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, 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.

More specifically, R ¹⁴ is a C13 to C23 branched or straight chain acyclic alkyl or acyclic alkenyl group which, together with its adjacent carbonyl group, corresponds to C14 to C24 saturated or unsaturated fatty acid residues, the fatty acids defining such fatty acid residues selected from the group consisting of; 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.

Even more specifically, sphingomyelin is a mixture of compounds of formula (VIII), the mixture being formulated in such a way that the total number of fatty acid residues (R ¹⁴ together with the adjacent carbonyl group) contained in the mixture is predominantly saturated fatty acids and least unsaturated fatty acids predominate. More specifically, the mixture is formulated in such a way that 80% to 96% of said fatty acid residues in the mixture are saturated fatty acids, in particular saturated fatty acids C14, C15, C16, C18, C20, C22, C23, C24, more specifically C16 , C18, C20, C22 and C24.

Phosphatidylcholine is a compound of formula (IX)

(IX)

where R ¹⁶ represents a branched or straight chain acyclic alkyl or acyclic alkenyl group from C2 to C43, and

R ¹⁷ is a C2 to C43 branched or straight acyclic alkyl or acyclic alkenyl group.

More specifically, R ¹⁶ and R ¹⁷ are independently branched or straight chain C13 to C43 acyclic alkyl or acyclic alkenyl groups which, together with their adjacent carbonyl groups, correspond to C14 to C44 saturated or unsaturated fatty acid residues, and yet more specifically, R ¹⁶ and R ¹⁷ independently of each other represent C13 to C23 branched or straight acyclic alkyl or acyclic alkenyl groups which together with their adjacent carbonyl group correspond to C14 to C24 saturated or unsaturated fatty acid residues.

More specifically, R ¹⁶ and R ¹⁷ are C13 to C23 branched or straight acyclic alkyl or acyclic alkenyl groups which, together with their adjacent carbonyl group, correspond to C14 to C24 saturated or unsaturated fatty acid residues, the fatty acids defining such residues fatty acids are selected from the group consisting of; 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. More specifically, C14:0, C16:0, C18:0, C18:1n-9, C18:2n-6, C20:1, C20:3, C20:4 and C22:6.

The phospholipid, its metabolic precursor and/or metabolite may be present in the composition of the present invention in an amount up to 99.999% of the composition.

In particular, sphingomyelin, its metabolic precursor and/or metabolite may be present in the composition of the present invention in an amount of up to 99.999% of the composition.

More specifically, the composition will contain sphingomyelin in an amount greater than 200 mg/kg dry weight of the composition, more specifically in the range of 200 mg to 2.5 g/kg dry weight of the composition.

In one embodiment, the composition contains sphingomyelin in an amount selected from the group consisting of; more than 200 mg/kg, more than 300 mg/kg, in the range from 200 mg to 2.5 g/kg, in the range from 200 mg to 2 g/kg, in the amount in the range from 300 mg to 1.5 g/kg or from 400 mg to 1 g/kg, in the range from 200 to 850 mg/kg, or from 300 to 820 mg/kg. All weights are based on the dry weight of the composition.

In particular, phosphatidylcholine, its metabolic precursor and/or metabolite may be present in the composition of the present invention in an amount of up to 99.999% of the composition.

More specifically, the composition will contain phosphatidylcholine in an amount greater than 200 mg/kg dry weight of the composition, more specifically in the range of 200 mg to 2.5 g/kg dry weight of the composition.

In one embodiment, the composition contains phosphatidylcholine in an amount selected from the group consisting of; more than 200 mg/kg, more than 300 mg/kg, more than 400 mg/kg, in the range from 200 mg to 2.5 g/kg, in the range from 200 mg to 2 g/kg, in an amount in the range from 300 mg to 1.5 g/kg or 400 mg to 1 g/kg, 500 to 1.3 g/kg. All weights are based on the dry weight of the composition.

In particular, phosphatidylinositol, its metabolic precursor and/or metabolite may be present in the composition of the present invention in an amount of up to 99.999% of the composition.

More specifically, the composition will contain phosphatidylinositol in an amount greater than 50 mg/kg dry weight of the composition, more specifically in the range of 200 mg to 1.5 g/kg dry weight of the composition.

In one embodiment, the device contains phosphatidylinositol in an amount selected from the group consisting of; more than 200 mg/kg, more than 300 mg/kg, in the range from 200 mg to 2.5 g/kg, in the range from 200 mg to 2 g/kg, in an amount in the range from 250 mg to 800 mg/kg, or from 400 mg to 1.5 g/kg, or 400 mg to 800 mg/kg. All weights are based on the dry weight of the composition.

In particular, phosphatidylserine, its metabolic precursor and/or metabolite may be contained in the composition of the present invention in an amount up to 99.999% of the composition.

More specifically, the composition will include phosphatidylserine in an amount of more than 50 mg/kg dry weight of the composition, more than 200 mg/kg dry weight of the composition, more specifically in the range from 150 mg to 1.5 g/kg dry weight of the composition, from 200 mg to 1 g/kg dry weight of the composition.

In one embodiment, the device contains phosphatidylserine in an amount selected from the group consisting of; more than 150, more than 200 mg/kg, more than 300 mg/kg, in the range from 200 mg to 2.5 g/kg, in the range from 200 mg to 2 g/kg, in an amount in the range from 250 mg to 1000 mg/kg kg or from 400 mg to 1 g/kg. All weights are based on the dry weight of the composition.

In particular, phosphatidylethanolamine, its metabolic precursor and/or metabolite may be present in the composition of the present invention in an amount of up to 99.999% of the composition.

More specifically, the composition will contain phosphatidylethanolamine in an amount of more than 150 mg/kg dry weight of the composition, more than 200 mg/kg dry weight of the composition, more specifically in the range from 150 mg to 1.5 g/kg dry weight of the composition.

In one embodiment, the device contains phosphatidylethanolamine in an amount selected from the group consisting of; more than 170 mg/kg, more than 180 mg/kg, more than 200 mg/kg, in the range from 200 mg to 2.5 g/kg, in the range from 200 mg to 2 g/kg, in an amount in the range from 250 mg to 800 mg/kg or 200 mg to 1 g/kg. All weights are based on the dry weight of the composition.

In one embodiment, the composition of the invention contains phospholipids, including phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, and phosphatidylcholine, such that the total concentration does not exceed 15.4 g/kg.

If a metabolic precursor and/or metabolite of one or more phospholipids is used in a composition in place of, or in combination with, a phospholipid, said compounds may be used in such amounts that the level of phospholipids physiologically delivered by said composition is as defined above. The ability to determine the appropriate amounts is within the purview of a person skilled in the art.

The term "metabolic precursor" and/or "metabolite" of one or more phospholipids as used herein does not include choline.

Non-limiting examples of metabolic precursors and/or metabolite of phospholipids, in particular sphingomyelin, phosphatidylcholine, phosphatidylinositol, phosphatidylserine and/or phosphatidylethanolamine, are: galactoceramides, sphingosine, sphingosine-1-phosphate, ceramide, D-erythrodihydroceramide and ceramide-1-phosphate and gangliosides.

Particularly effective phospholipids may be phosphatidylcholine, phosphatidylserine, phosphatidylinositol and/or sphingomyelin, in particular sphingomyelin.

In one embodiment of the present invention, the phospholipid is phosphatidylcholine, phosphatidylserine, phosphatidylinositol, sphingomyelin, and/or a metabolic precursor and/or metabolite of any of the above, and/or a combination of any of the above. In particular, the phospholipid is sphingomyelin, its metabolic precursor and/or metabolite.

Particularly effective metabolic precursors and/or metabolite of phospholipids, in particular sphingomyelin, can be ceramide and gangliosides, and ceramide-1-phosphate, and d-erythrodihydroceramide.

The term "ceramide" refers to a lipid molecule in which the backbone of sphingosine or sphinganine is esterified with a fatty acid residue to form an amide bond. When using the term "ceramide" in the present description, it can mean an individual ceramide compound, as well as a mixture of individual ceramide compounds.

In particular, the ceramide is a compound of formula (IXa) or a mixture of compounds of formula (IXa)

(IXa),

Where

R ^16a is a C2 to C43 branched or straight acyclic alkyl or acyclic alkenyl group,

R ^17a is a C2 to C43 branched or straight acyclic alkyl or acyclic alkenyl group.

More specifically, R ^16a is a C13 to C43 branched or straight chain acyclic alkyl or acyclic alkenyl group which, together with its adjacent carbonyl group, corresponds to C14 to C44 saturated or unsaturated fatty acid residues.

Non-limiting examples of C14 to C44 saturated or unsaturated fatty acids that may define such fatty acid residues 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, 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.

More specifically, R ^16a is a C13 to C23 branched or straight chain acyclic alkyl or acyclic alkenyl group which, together with its adjacent carbonyl group, corresponds to C14 to C24 saturated or unsaturated fatty acid residues, the fatty acids defining such fatty acid residues selected from the group consisting of; 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 in particular the group consisting of: C16:0, C18:0, C20:0, C22:0 and C24:0.

Even more specifically, ceramide is a mixture of compounds of formula (IXa), the mixture being formulated in such a way that the total number of fatty acid residues (R ^16a together with the adjacent carbonyl group) contained in the mixture is predominantly represented by saturated fatty acids, and to a lesser extent unsaturated fatty acids predominate. More specifically, the mixture is formulated in such a way that 80% to 96% of said fatty acid residues in the mixture are saturated fatty acids, in particular saturated fatty acids C14, C15, C16, C18, C20, C22, C23, C24, more specifically C16 , C18, C20, C22 and C24.

The term "ganglioside" as used herein refers to an oligoglycosylceramide lipid molecule containing a ceramide residue of formula IXa as defined herein. When using the term "ganglioside" in the present description, it can mean an individual ganglioside compound, as well as a mixture of individual ganglioside compounds containing a ceramide residue of formula IXa, as defined herein.

Particularly effective gangliosides may be gangliosides from monosialoganglioside-3 (GM3) and/or gangliosides from disialoganglioside-3 (GD3).

Ceramide-1-phosphate and d-erythrodihydroceramide will contain a ceramide residue of formula IXa as defined herein.

Gangliosides and/or ceramides and/or ceramide-1-phosphate and/or d-erythrodihydroceramide can be included in the composition in any amount.

Concentrations in the range of 2-11.5 mg/100 g for GD3 and/or GM3 may be particularly effective.

Sphingomyelin can be synthesized from ceramide and phosphatidylcholine, respectively, and it may be particularly advantageous to use ceramide and/or one or more gangliosides in combination with phosphatidylcholine, its metabolic precursor or metabolite.

The phospholipid, its metabolic precursors and/or metabolite contained in the composition of the present invention may be natural, artificial or a mixture of the above. Said metabolic precursors and/or metabolite may be used in their pure form or in substantially pure form in the composition of the invention. In an alternative embodiment, they may be added in the form of a source containing them.

Any source of its metabolic precursor phospholipid and/or metabolite suitable for ingestion by the subject for consumption of which the composition is intended may be used in the present invention.

In particular, the phospholipid, its metabolic precursor or metabolite will be derived from natural sources, non-limiting examples of which include eggs, soy, cow brains and/or mammalian milk or extracts thereof. Non-limiting examples of soy sources include the dietary supplement lecithin, non-limiting examples of mammalian milk include cow, camel, sheep, goat milk, including skim milk. Non-limiting examples of milk extracts include protein extracts such as whey protein and casein, milk fat globule membranes (MFGM) and extracts containing them.

A particularly useful source of phospholipids, their metabolic precursor or metabolite, in particular sphingomyelin, which can be used in the present invention may be bovine whey protein concentrate enriched with alpha-lactalbumin and/or crude alpha-lactalbumin that has been extracted from whey milk protein. , in particular whey protein cow's milk.

Alpha-lactalbumin is a high quality whey protein that is easily digested by, for example, human infants and is the main protein present in GM. Alpha-lactalbumin and/or alpha-lactalbumin-enriched milk fraction is ideal for low protein infant formula due to its high content of essential amino acids, in particular tryptophan. Although alpha-lactalbumin itself is a protein, unpurified sources may contain sphingomyelin.

In one embodiment, the phospholipid, its metabolic precursor or metabolite, in particular sphingomyelin, is used in the form of an alpha-lactalbumin enriched whey protein concentrate or as alpha-lactalbumin.

In a more specific embodiment, a cow's milk whey protein concentrate enriched with alpha-lactalbumin, or alpha-lactalbumin with a phospholipid content, in particular with a sphingomyelin content of more than 500 mg/100 g, 900 mg/100 g, 1000 mg/100 g dry weight is used. compositions.

Another particularly useful source of phospholipids, their metabolic precursor or metabolite may be milk fat globule membranes (hereinafter MFGM) or extracts containing them, in particular MFGM or cow's milk extracts containing them. It may be particularly useful if MFGM or extracts containing them contain 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% phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and / or sphingomyelin. MFGM may also additionally contain magnesium, phosphorus and or calcium, in particular in concentrations in the range from 0.05% to 2%, from 0.1% to 0.4%.

A composition containing a phospholipid and/or its metabolic precursor and/or metabolite, in particular sphingomyelin, phosphatidylcholine and/or phosphatidylinositol, may be particularly effective in supporting, stimulating or optimizing primary myelination, in particular the primary myelination trajectory, and/or brain structure in one or more of the following areas of the brain; cerebellum, visual cortex, corpus callosum, internal capsule, frontal lobe, parietal lobe, temporal lobe, motor cortex, frontal cortex. These areas of the brain are associated with one or more of the following functions: vision, motor function (including coordination and movement), hemispheric interaction, language function, auditory function (including listening and attention), short-term memory, executive function, including problem solving , social information processing and behavioral interaction, spatial reasoning and language.

In one embodiment, the composition of the invention contains a fatty acid derivative.

The fatty acid derivative may be present in the composition of the present invention in an amount up to 99.999% of the composition.

The term "fatty acid derivative" as used herein refers to a fatty acid-containing compound other than a phospholipid, and in particular a free fatty acid and/or monoacylglycerol (hereinafter MAG) and/or diacylglycerol (hereinafter DAG) , and/or triacylglycerol (hereinafter TAG), and/or cholesterol ester. More specifically, this term refers to MAG, DAG, TAG or cholesterol ester. Even more specifically, the term refers to TAG.

The term "MAG" as used herein refers to a glycerol molecule in which one of the OH groups has formed an ester bond with a fatty acid. In particular, the term "MAG" as used herein refers to a compound of formula (X)

(X)

Where

two of R ¹⁸ , R ¹⁹ or R ²⁰ are H and where one of R ¹⁸ , R ¹⁹ or R ²⁰ is a C4 to C44 saturated or unsaturated acyl group.

More specifically, two of R ¹⁸ , R ¹⁹ or R ²⁰ are H and one of R ¹⁸ , R ¹⁹ or R ²⁰ is a saturated or unsaturated acyl group from C10 to C24, and even more specifically a saturated or unsaturated acyl group from C14 to C24.

The term "DAG" as used herein refers to a glycerol molecule in which two of the OH groups have formed an ester bond with two fatty acids. In particular, the term "DAG" as used herein refers to a compound of formula (X),

Where

one of R ¹⁸ , R ¹⁹ or R ²⁰ is H, and where two of R ¹⁸ , R ¹⁹ or R ²⁰ are a saturated or unsaturated C4 to C44 acyl group. More specifically, a saturated or unsaturated acyl group from C10 to C24, and even more specifically, a saturated or unsaturated acyl group from C14 to C24. The two saturated or unsaturated acyl groups C4 to C44 of R ¹⁸ R ¹⁹ or R ²⁰ may be the same or different.

The term "TAG" as used herein refers to a glycerol molecule in which three OH groups have formed an ester bond with three fatty acids. In particular, the term "TAG" as used herein refers to a compound of formula (X),

Where

where all of R ¹⁸ , R ¹⁹ or R ²⁰ are saturated or unsaturated acyl groups C4 to C44, more specifically saturated or unsaturated acyl groups C10 to C24, and even more specifically saturated or unsaturated acyl groups C14 to C24 . The three saturated or unsaturated acyl groups C4 to C44 may all be the same, all different, or two groups may be the same and one different.

The term "cholesterol ester" as used herein refers to a compound of formula (XI)

(XI),

Where

R ²¹ is a C2 to C43 branched or straight acyclic alkyl or acyclic alkenyl group.

More specifically, R ²¹ represents C9 to C43 branched or straight acyclic alkyl or acyclic alkenyl groups which, together with their adjacent carbonyl groups, correspond to C10 to C44 saturated or unsaturated fatty acid residues, and even more specifically to saturated or unsaturated fatty acid residues. acids from C14 to C24.

The term "fatty acid" as used herein refers to a compound of formula (XII)

(XII)

Where

R ²² is a C2 to C43 branched or straight acyclic alkyl or acyclic alkenyl group.

More specifically, R ²² is a C9 to C43 branched or straight chain acyclic alkyl or acyclic alkenyl group, even more specifically a C13 to C23 branched or straight chain acyclic alkyl or acyclic alkenyl group.

Non-limiting examples of C10 to C44 saturated or unsaturated fatty acids that may be present in the fatty acid derivative, i. may be a free fatty acid or a fatty acid which defines the residue(s) of such fatty acid in MAG, DAG, TAG and/or cholesterol ester, 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, 18: 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, in particular 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 :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, said fatty acids will be selected from the group consisting of: 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-.

Any fatty acid derivative suitable for ingestion by the subject for consumption of which the composition is intended may be used in the present invention.

In particular, such a fatty acid derivative may come from natural sources, non-limiting examples of which are eggs, algae, fish oil, molds, yeast, seeds, plants, such as soybeans, and animal sources, such as cow brains and/or milk. mammals or its extracts. Non-limiting examples of soy sources include the dietary supplement lecithin, non-limiting examples of mammalian milk include cow, camel, sheep, goat milk, including skim milk. Non-limiting examples of milk extracts include protein extracts, milk fat globule membranes (MFGM) and extracts containing them. The source of fatty acid derivatives can also be palm oil, beef tallow, lard, cottonseed oil, peanut oil.

It may be particularly useful if the fatty acid derivative contains a saturated or unsaturated fatty acid selected from the group consisting of: 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 specifically, 22:6n-3 and/or C18:0.

A composition containing a phospholipid, in particular sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, more particularly sphingomyelin, can be particularly effective when used in combination with one or more of the listed fatty acids.

C20:4n-6 denotes arachidonic acid (hereinafter ARA or AA). C22:6n-3 stands for docosahexaenoic acid (hereinafter DHA). 24:1n-9 stands for nervonic acid. C18.0 is stearic acid. C16:0 is palmitic acid. C18:1n-9 is oleic acid.

In one embodiment, the composition according to the present invention comprises a fatty acid derivative containing DHA and/or ARA and/or nervonic acid and/or stearic acid, in particular a fatty acid derivative containing DHA and/or ARA and/or stearic acid. More specifically, a fatty acid derivative containing DHA and/or stearic acid.

A fatty acid derivative containing DHA and/or ARA and/or nervonic acid and/or stearic acid may be present in the composition of the present invention in an amount up to 99.999% of the composition.

In particular, a fatty acid derivative containing DHA and/or ARA and/or nervonic acid and/or stearic acid may be contained in the composition of the present invention in an amount of from 15 to 350 mg/100 g dry weight of the composition, more specifically from 30 mg to 300 mg/100 g dry weight of the composition.

In one embodiment, the composition according to the present invention comprises a fatty acid derivative containing DHA and/or ARA and/or nervonic acid and/or stearic acid in an amount selected from the group consisting of the following; more than 15 mg/100 g, more than 30 mg/100 g, more than 50 mg/100 g, more than 55 mg/100 g, in the range of 30 to 300 mg/100 g, in the range of 30 to 200 mg/100 g, or 30 to 150 mg/100 g, in the range of 50 to 300 mg/100 g, in the range of 50 to 200 mg/100 g, in the range of 50 to 150 mg/100 g, in the range of 150 to 350, in range from 60 to 350 mg/100 g, in the range from 60 to 120 mg/100 g, in the range from 100 to 110 mg/100 g. All concentrations are based on the dry weight of the composition.

Fatty acid derivatives containing stearic acid are present in natural sources such as palm oil, beef tallow, lard, cottonseed oil, peanut oil.

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

Fatty acid derivatives containing DHA and/or ARA are present in natural sources such as, for example, eggs, algae, mushrooms, fish oils, and plants.

Oils containing fatty acid derivatives containing DHA and/or ARA and, in general, other polyunsaturated fatty acids (PUFAs), in particular EPA (eicosapentaenoic acid), may have different origins. Preferably, the fatty acid derivatives containing DHA are supplied in the form of fish oil containing fatty acid derivatives containing DHA and/or ARA. Fish oil usually contains 5 wt. % or more, preferably 10 wt. % or more fatty acid derivatives containing DHA and/or ARA. Also commonly available are oils containing fatty acid derivatives containing DHA and/or ARA derived from algae or microorganisms. For example, oils derived from algae containing 10 wt. % or more, for example 20 wt. % or more fatty acid derivatives.

If the nutritional composition according to the present invention contains fatty acid derivatives containing ARA and DHA. Said ingredients may, for example, be present in the composition of the present invention in amounts that result in a DHA:ARA weight ratio in the range of 4:1 to 1:4, such as 3:1 to 1:3, such as 2:1 to 1 : 2, for example from 1.5:1 to 1:1.5, in particular from 1.1:1 to 1:1.1.

It may also be useful if the composition of the present invention contains a mixture of derivatives of fatty acids, and this mixture is formed in such a way that the weight ratio of unsaturated to saturated fatty acids and/or fatty acid residues in the composition of the invention is in the range from 1 : 1 to 1 : 2 ; from 1:1.2 to 1:1.9, from 1:1.25 to 1:1.5; from 1:3 to 1:4.

Moreover, if the composition of the present invention contains large amounts of fatty acid derivatives containing DHA and/or ARA, it may be especially useful to increase the total amount of fatty acid derivatives containing saturated long chain fatty acids, in particular C20/24. Such saturated long chain fatty acids may be an important component of myelin, aiding in its wrapping and sheathing around axons. The weight ratio of DHA and/or AA to such saturated long chain fatty acids in the composition of the invention may, for example, range from 1:1 to 1:10; from 1:2 to 1:9, from 1:3 to 1:4.5, from 1:3.5 to 1:4.5.

A composition of the invention containing a fatty acid derivative, for example a fatty acid derivative containing DHA and/or AA, may be particularly effective in supporting, stimulating or optimizing primary myelination, in particular the primary myelination trajectory, and/or brain structure in one or more of the following areas of the brain: cerebellum, internal capsule, parietal lobe, motor and sensory cortex (including coordination and execution of movements), visual cortex, frontal cortex. These areas of the brain are associated with visual function, motor function and psychomotor function (including coordination and execution of movements) and/or executive function and socio-emotional function.

One skilled in the art will be able to determine the appropriate amounts of the above nutrients, their metabolic precursors or metabolites, based on their nature, intended use, target subject, and dose of the composition, such as how many times per day the subject will ingest the composition. In general, an effective dose will depend on the age, size and health of the subject, the lifestyle of the subject, the amount of nutrients in the composition, and possibly the sex of the subject.

An effective dose can be any dose that promotes support, stimulation or optimization of primary myelination, in particular the trajectory of primary myelination, and / or brain structure, and / or neuronal density, and / or intellectual potential, and / or cognitive potential, and /or learning ability, and/or cognitive function in the subject.

For infant formula or formula for infants 1 to 3 years of age, one skilled in the art can determine the starting amounts or ratios of the compounds or nutrients described herein, such as choline, relative to those present in human breast milk produced for an infant or a child of the same age, in particular a fully nourished mother.

The ability to determine an effective dose based on the information provided herein and knowledge in the art is within the purview of a person skilled in the art.

In one embodiment, the composition according to the present invention contains choline, a vitamin, in particular vitamin B12 and/or folic acid, a phospholipid, in particular phosphatidylcholine and/or phosphatidylserine and/or phosphatidylinositol and/or sphingomyelin and/or a metabolic precursor or a metabolite of any of the above, a mineral substance, in particular iron and/or zinc and/or calcium and/or magnesium and/or phosphorus and/or copper and a fatty acid derivative, in particular containing DHA and/or AA , and/or nervonic acid and/or stearic acid.

Particularly useful concentrations/amounts of said ingredients in said composition may be sphingomyelin in an amount of at least 300 or 420 mg/kg, in particular more than 800 m/kg and more specifically more than 900 mg/kg, phosphatidylcholine in an amount of at least 1000 mg /kg, phosphatidylserine in an amount of at least 900 mg/kg, phosphatidylinositol in an amount of at least 700 mg/kg, folic acid in an amount of at least 140 mg/kg, in particular at least 160 mg/kg, vitamin B12 in an amount of at least 2.34 µg/100 g, in particular more than 5 µg/100 g, more specifically 7 µg/100 g, iron in an amount of at least 6 mg/100 g or at least 8.6 mg/ 100 g, more specifically 11.5 mg/100 g, even more specifically more than 16 mg/kg, choline in an amount of at least 124 mg/kg, more specifically 140 mg/kg, a fatty acid derivative containing DHA, in an amount of at least 89 mg/100 g, a fatty acid derivative containing AA in an amount of at least 89 mg/100 g or at least 175 mg/100 g, zinc in an amount of at least 4 or at least 4 .7 mg/100 g, more specifically 7 mg/100 g, calcium in an amount of at least 200 mg or at least 500 mg/100 g, phosphorus in an amount of at least 140 mg/100 g or at least 350 mg/100 g, copper in an amount of at least 250 µg/100 g or at least 600 µg/100 g, magnesium in an amount of at least 30 mg/100 g or at least 50 mg/100 g. weights are based on the dry weight of the composition.

A concentration falling within the specified error for any analytical procedure used to measure the concentration of one or more of the ingredients listed above should be considered to fall within the concentration range given in this document.

In one embodiment, the composition according to the present invention contains choline, vitamin B12 and/or folic acid, sphingomyelin and/or its metabolic precursor or metabolite, iron, zinc, and a fatty acid derivative containing DHA and/or AA.

In one embodiment, the composition in accordance with the present invention includes a fatty acid derivative containing DHA and/or ARA, vitamin B12 and/or folic acid, sphingomyelin and iron.

In a more specific embodiment, the composition according to the present invention comprises a fatty acid derivative containing DHA at a concentration of 102 mg/100 g, ARA at a concentration of 102 mg/100 g, vitamin B12 at a concentration of 5.4 μg/100 g, folic acid at a concentration of 169 mcg/100 g, sphingomyelin at a concentration of 81.4 mg/100 g and iron at a concentration of 6.7 mg/100 g.

The composition of the present invention may be any kind of composition suitable for direct administration to a subject.

In particular, the composition will be an artificial nutritional composition.

As used herein, the term "nutritional composition" refers to a synthetic composition that provides nutrition to a subject. Such a nutritional composition may be administered enterally, parenterally or intravenously. In particular, the composition will be taken enterally and more particularly orally.

In one embodiment of the present invention, said composition will be an artificial nutritional composition selected from the group consisting of the following; formula for infants 1 to 3 years of age, an infant formula or composition for infants that is intended to be added to or diluted with human breast milk (hereinafter "GM"), such as a GM fortifier, or a food intended for consumption by an infant and/or child both in pure form and in combination with GM, for example complementary foods.

The compositions of the present invention may also contain any other ingredients or excipients known to be useful in this type of composition, such as infant formula.

Non-limiting examples of such ingredients include: proteins, amino acids, carbohydrates, oligosaccharides, lipids, prebiotics or probiotics, nucleotides, nucleosides, other vitamins, minerals, and other trace elements.

In one exemplary embodiment of the present invention, the composition comprises a protein source, a lipid source, and a carbohydrate source.

For example, such a composition may contain protein in the range of about 2 to 6 g/100 kcal, lipids in the range of about 1.5 to 3 g/100 kcal, and/or carbohydrates in the range of about 1.7 to 12 g/100 kcal. .

If the composition is a liquid, its energy value may be from 60 to 75 kcal/100 ml.

If the composition is a solid, its energy value may be from 60 to 75 kcal/100 ml.

It is assumed that the type of protein is not of great importance for the present invention. However, in the case of artificial compositions for infants and/or young children, such as infant formula or milk formulas for children from 1 to 3 years, said proteins must support the growth of the infant and/or child, so that said infant and/or child can match growth curves specific to their genetic background, birth weight and health status.

Non-limiting examples of proteins include: casein, alpha-lactalbumin, whey, beta-lactoglobulin, soy protein, rice protein, corn protein, oat protein, barley protein, wheat protein, rye protein, pea protein, egg protein, seed protein sunflower, potato protein, fish protein, meat protein, lactoferrin, serum albumin, immunoglobins, and combinations thereof.

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.

Non-limiting examples of carbohydrates include lactose, sucrose, maltodextrin, starch, and combinations thereof.

Non-limiting examples of lipids include: palm olein, high oleic sunflower oil, high oleic safflower oil, canola oil, fish oil, coconut oil, cow's milk fat, and combinations thereof.

It may be particularly useful if the composition comprises fat in an amount of 25 to 30 g/100 g dry weight of the composition.

Non-limiting examples of essential fatty acids include: linoleic acid (LA), α-linolenic acid (ALA). Compositions of the invention may further comprise monosialoganglioside-3 (GM3) and disialoganglioside-3 (GD3) gangliosides, and combinations thereof.

Non-limiting examples of prebiotics include: oligosaccharides optionally containing fructose, galactose, mannose; dietary fiber, in particular soluble fiber, soy fiber; inulin; and their combinations. Preferred prebiotics are fructooligosaccharides (FOS), galactooligosaccharides (GOS), isomaltooligosaccharides (IMO), xylooligosaccharides (XOS), arabinoxylooligosaccharides (AXOS), mannanoligosaccharides (MOS), soy oligosaccharides, glycosyl sucrose (GS), lactosucrose (LS), lactulose (LA). ), palatinozaoligosaccharides (PAO), maltooligosaccharides, gums and/or their hydrolysates, pectins and/or their hydrolysates, and combinations of the above.

Additional examples of oligosaccharides are described in Wrodnigg, T. M.; Stutz, A.E. (1999) Angew. Chem. Int. Ed. 38:827-828, as well as in WO 2012/069416, which are incorporated herein by reference.

Non-limiting examples of probiotics include: Bifidobacterium, Lactobacillus, Lactococcus, Enterococcus, Streptococcus, Kluyveromyces, Saccharoymces, Candida, specifically selected from the group consisting of Bifidobacterium longum, Bifidobacterium lactis, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium adolescentis, Lactobacillus acidophilus Lactobacillus casei Lactobacillus paracasei Lactobacillus salivarius Lactobacillus lactis Lactobacillus rhamnosus Lactobacillus johnsonii Lactobacillus plantarum Lactobacillus salivarius Lactococcus lactis Enterococcus faecium Saccharomyces cerevisiae ces boulardii or mixtures thereof, preferably selected from the group consisting of Bifidobacterium longum NCC3001 (ATCC BAA-999) Bifidobacterium longum NCC2705 (CNCM I-2618) Bifidobacterium longum NCC490 (CNCM I-2170) Bifidobacterium lactis NCC2818 (CNCM I-3446) I -2116), Lactobacillus johnsonii NCC533 (CNCM I-1225), Lactobacillus rhamnosus GG (ATCC53103), Lactobacillus rhamnosus NCC4007 (CGMCC 1.3724), Enterococcus faecium SF 68 (NCC2768; NCIMB10415) and combinations thereof.

Non-limiting examples of nucleotides include: cytidine monophosphate (CMP), uridine monophosphate (UMP), adenosine monophosphate (AMP), guanosine monophosphate (GMP), and combinations thereof.

Other suitable and desirable nutritional composition ingredients that may be used in the nutritional composition of the invention may be described in the guidelines of the Codex Alimentarius for the type of nutritional composition in question, e.g. intended for infant consumption, such as complementary foods.

In an even more specific embodiment, the composition of the invention is an infant formula, the composition of which is defined in example 5 and, in particular, in table 11a.

Artificial nutritional composition, such as infant formula used in the invention, can be obtained in any suitable way. For example, an infant formula can be made by mixing together a protein source, a carbohydrate source, and a fat source in appropriate proportions. If used, emulsifiers may be included in the mixture. Choline, any vitamins and any minerals can be added at this stage, but these are usually added later to avoid thermal decomposition. Any lipophilic vitamins, emulsifiers, and the like may be dissolved in the fat source prior to mixing. Water, preferably reverse osmosis water, can then be mixed in to form a liquid mixture. The liquid mixture can then be heat treated to reduce bacterial loads. For example, the liquid mixture can be rapidly heated to a temperature in the range from about 80°C to about 110°C for about 5 seconds to about 5 minutes. This can be done by steam injection or by means of a heat exchanger; such as a plate heat exchanger. Then the liquid mixture can be cooled to a temperature of from about 60°C to about 85°C; such as quenching. The liquid mixture can then be homogenized; for example, in two stages: under pressure from about 7 MPa to about 40 MPa in the first stage and from about 2 MPa to about 14 MPa in the second stage. The homogenized mixture can then be further cooled to add any heat sensitive components; such as vitamins and minerals. At this point, the pH and solids content of the homogenized mixture is standardized for convenience. The homogenized mixture is transferred to a suitable drying apparatus such as a spray dryer or freeze dryer and powdered. The moisture content of the powder should be less than about 5 wt. %. If it is necessary to add the probiotic(s), then it(them) can be cultured in accordance with any suitable method and prepared for addition to infant formula by, for example, freeze drying or spray drying. Alternatively, bacterial preparations can be purchased from specialized suppliers such as Christian Hansen and Morinaga already prepared in a suitable form for addition to foods such as infant formula. Such bacterial preparations can be added to powdered infant formula by dry mixing.

As is clear from the above description, the composition of the present invention can be used to stimulate, support or optimize primary myelination, in particular the trajectory of primary myelination, and/or the structure of the brain, and/or the density of neuronal connections, and/or intellectual potential, and/or cognitive potential and/or learning ability and/or cognitive function in a subject, in particular in a subject receiving infant formula.

In another aspect of the present invention, a method is provided for stimulating, supporting or optimizing primary myelination, in particular the primary myelination trajectory and/or brain structure and/or neuronal density and/or intellectual potential and/or cognitive potential and/or learning ability, and/or cognitive function in a subject, in particular in a subject receiving infant formula, and said method includes feeding said subject with a composition containing choline, as defined herein.

In another aspect of the present invention, there is provided a choline-containing composition as defined herein for use in preparing a composition for stimulating, supporting or optimizing primary myelination, in particular primary myelination trajectory and/or brain structure and/or neuronal density. , and/or intellectual potential, and/or cognitive potential, and/or learning ability, and/or cognitive function in a subject, in particular in a subject receiving infant formula.

It may be particularly advantageous to administer the composition of the present invention to an infant 9 months of age or less, in particular 6 months of age or less, more particularly 3 months of age or less.

Non-limiting examples of an age of 3 months or less are under 2 weeks of age, under 1 month of age, under 2 months of age, under 3 months of age, 1 to 3 months of age.

Exposure to the composition of the invention described herein may have long term health benefits. Dementia, such as Alzheimer's disease, causes a decrease in the subject's ability to think and remember, and also leads to emotional and language problems. The risk that a subject will suffer from dementia, in particular Alzheimer's disease, has been associated with the intellectual capacity or intelligence of the individual. Accordingly, by optimizing the intellectual, cognitive potential and or learning ability, it is possible to reduce the risk of a subject developing dementia, in particular Alzheimer's disease. Said long-term effect may take several years, for example 40, 50, 60, 70, 80, 90 years.

In addition, a variety of psychiatric and/or neurological disorders, such as anxiety, depression, autism, and schizophrenia, are associated with brain structure. By stimulating, supporting or optimizing primary myelination, in particular the primary myelination trajectory, and/or the structure of the brain, in particular defined by primary myelination and the primary myelination trajectory, it is possible for the subject to prevent psychiatric and/or neurological disorders, such as anxiety, depression. , autism and schizophrenia, or reduce the risk of their development, or alleviate the severity of the mentioned disorder(s). Said effect may manifest itself over several years, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 years.

Unless otherwise indicated, all percentages given herein are wt. %.

In the context of the present invention, the terms "comprising" or "comprises" do not exclude other possible elements. The composition of the present invention, including many of the embodiments described herein, may contain the essential elements and limitations of the invention described herein, as well as any additional or optional ingredients, components or limitations described herein, or otherwise, depending on the needs. , consist or essentially consist of them.

As will be apparent to one of skill in the art, the same benefits as disclosed herein can be obtained if choline is taken directly rather than in the form of a composition. Accordingly, choline can be used directly instead of the composition of the present invention in any method or application described herein.

As will also be apparent to one of skill in the art, it may be particularly useful if choline is administered, i. are consumed separately, sequentially and/or simultaneously with one or more of the following ingredients: a vitamin, in particular vitamin B12 and/or folic acid, and/or a mineral, in particular iron and/or zinc and/or calcium, and/or phosphorus and/or magnesium and/or copper and/or a fatty acid derivative, in particular a fatty acid derivative containing docosahexaenoic acid and/or arachidonic acid and/or nervonic acid and/or stearic acid and/or a phospholipid , in particular phosphatidylcholine, phosphatidylserine, phosphatidylinositol, and/or sphingomyelin.

All details of the present invention are equally applicable to the composition containing choline, as well as to the direct use of choline.

It should be understood that all features of the present invention described herein may be freely combined and that changes and modifications may be made without departing from the scope of the invention as defined in the claims. In addition, if there are known equivalents of specific features, such equivalents are included as if they were specifically mentioned in this description.

The terms "in particular" or "more specifically" when used in the present invention should not be considered as limiting, but they should be interpreted as synonymous with the expressions "for example" or "namely".

Below are a number of non-limiting examples that illustrate the invention.

Experimental section

Methods, definitions and materials

MRI (magnetic resonance imaging). Brain MRI scans of infants and children aged 0 to 5 years were performed using a white matter imaging technique. This technique provides a quantitative indicator "myelin water fraction" (MWF), which is a surrogate marker of myelin content in the brain. When plotted as a function of time over early childhood, myelination trajectories can be obtained.

Composition of infant formula. The six infant formulas consumed by the infants participating in the study were analyzed in terms of their composition/level of myelin-associated nutrients.

The nutritional compositions were tested in standard commercially available infant formulas from various brands/suppliers and at various nutrient levels.

Cognitive abilities. Age-standardized (T)-scores of gross motor skills, visual perception, and language (transmission and comprehension) are derived from the Mullen Early Learning Scales, a standardized and validated measure of early cognitive development in infants and children 6 years of age or younger.

Nutrient Analysis

Table 1 lists the nutrients for each of the 6 infant formula formulations.

Table 1

Nutrient Unit Concentration range for the 6 infant formulas included in the analysis Alpha lactalbumin g/100 g protein n.d.-1.01 Fat g/100 g 26.3-29.5 AA mg/100 g 94.2-180 DHA mg/100 g 42.3-89.8 Iron mg/100 g 8.42-11.7 Calcium mg/100 g 397-566 Phosphorus mg/100 g 234-358 Sodium mg/100 g 135-189 Potassium mg/100 g 593-834 Copper mcg/100 g 471-834 Zinc mg/100 g 4.23-7.25 Magnesium mg/100 g 38.4-53.9 Manganese mcg/100 g 60.6-140.3 Vitamin B12 mcg/100 g 4.93-8.34 Folic acid mcg/100 g 98.8-306 Choline mg/100 g 35.7-170 Beta lactoglobulin g/100 g n.d.-4.21 Phosphatidylcholine mg/kg 397-1287 Phosphatidylinositol mg/kg 266-788 Phosphatidylserine mg/kg < LQ(144)-977 Phosphatidylethanolamine mg/kg < LQ (174) Sphingomyelin mg/kg <LQ(100)-480

Clinical Study

Infant members

The infants included in this study were selected from a larger longitudinal study of normal brain development and behavioral development: the Brown University Assessment of Myelination and Behavior Across 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 selection and enrollment in the study. Therefore, children with intrauterine exposure to alcohol or illicit substances, preterm (< 37 weeks gestation) or multiple birth abnormalities, fetal abnormalities on ultrasound complicated by pregnancy (eg, preeclampsia), APGAR score < 8, NICU stay, neurological impairment ( eg head trauma, epilepsy), psychiatric or developmental disorders in the infant, parents or siblings (including maternal depression requiring medication). Current screening, such as the MCAT for autism or the CBCL for behavioral problems, was additionally used to exclude already selected children with clinically disturbing behaviors or obvious medical conditions (eg, autism spectrum disorders).

A combination of retrospective and prospective data was collected from parents as part of a detailed history and parental survey of infant formula used, percentage of breastfeeding versus formula feeding, and duration of exclusive breastfeeding. This information was updated at each study visit, which occurred approximately every 6 months for children under 2 years of age and annually for older children. Based on this information, children were assigned to one of 2 groups: No. 1. Received exclusively infant formula; and #2. Exclusively breastfed for at least 90 days (3 months). Children who received a combination of breast milk and infant formula for 3 months were excluded from our analysis. Infants in the exclusive infant formula group were further subdivided based on information from parents regarding the primary infant formula used during the first 3 months. Primary infant formula was defined as one that was used 90% of the time or more (eg, if parents used an alternative brand while on vacation).

Based on these criteria, 94 infants and young children were selected for Group 1, who received exclusively infant formula. This included 13 children who received Formula No. 2; 28 treated with mixture No. 5; 8 receiving mixture No. 3; 39 who received mixture No. 4; 5 who received formula 1 and 1 who received formula 6. A sample of 52 exclusively breastfed infants were also selected and matched with the infant formula group above for the following: mean age, length of pregnancy, birth weight, ratio boys: girls, ethnicity, mother's education, family size, and number of languages spoken at home (in addition to English). Table 1a shows the grouping data for each mixture.

Table 1a. Breakdown of data for longitudinal analysis and nutritional analysis

Blend 1 Blend 2 Blend 5 Blend 3 Blend 4 Blend 6 Breastfeeding N _children 5 13 28 8 39 1 52 N _measurements eleven 27 56 14 64 3 106

Visualization and analysis methods

Each infant was scanned using the mcDESPOT (Multi-Equilibrium Single Pulse T ₁ and T ₂ ) white matter imaging technique according to Deoni et al. (Magn. Reson. Med. 2008, 60:1372-1387), which provides a quantitative measure of the myelin water fraction (MWF) - an estimate of the content of myelin - at each point in the brain. All infants were scanned during natural (ie, unsedated) sleep using mcDESPOT imaging protocols with acoustic suppression. The total scan time ranged from 19 minutes for the youngest toddlers to 24 minutes for children over 4 years of age.

All data were obtained on a Siemens 3T Tim Trio scanner with a 12-channel RF head coil. To minimize movement during the scan, children were swaddled using a MedVac pediatric vacuum immobilization bag (CFI Medical Solutions, USA) and foam pads. The scanner noise was reduced by reducing the amplitudes of the peak gradient and the rate of change of the gradient, as well as by using noise-insulating liners for the scanner opening (Quiet Barrier HD Composite, UltraBarrier, USA). We also used MiniMuff pediatric headphones and electrodynamic headphones (MR Confon, Germany). The condition of the children was constantly monitored using a pediatric pulse oximetry system and an infrared camera. All children continued to sleep during the MRI scan and no motion-related artifacts were found in the analyzed data.

After image alignment, removal of the non-brain signal, and correction for inhomogeneity of the main and applied magnetic field (B ₀ and B ₁ ), a three-component signal model (including myelin-bound water; water inside and outside axons; and free water, not involved in the exchange processes) to process the mcDESPOT data to obtain MWF voxel maps.

The map for each child was then non-linearly matched to a custom study template. From public databases, white matter masks were formed corresponding to 5 bilateral areas (frontal, temporal, occipital, parietal and cerebellar white matter), as well as for the trunk, knee and ridge of the corpus callosum, they were registered in a common template and superimposed on the MWF map of each child . Then, mean values for each area were determined for each child and used for subsequent developmental analysis and trajectory modeling.

Differences in development

Non-linear mixed effects modeling was used to analyze developmental differences between breastfed and formula-fed infants, and between infants receiving different formulas. Data for groups #1 and #2 and each subgroup for different mixtures were independently processed using modified Gompertz growth models. Each of the four parameters of the Gompertz model was then compared between breastfeeding and infant formula groups using a two-sample t-test, and between subgroups of 4 formulas using analysis of variance followed by Tukey's post hoc test to identify different groups of infant formula.

Assessment and analysis of cognitive function

Along with MRI, each child was assessed for general cognitive abilities and skills within 7 days after the scan using the Mullen Early Learning Scale, MSEL (Mullen EM, 1995). The MSEL provides a general assessment of behavioral development in the areas of fine and gross motor control, perceptual language and communication skills, and visual perception. Age-normalized T-scores for these areas can be combined into three composite scores: Early Learning Composite (ELC, including fine motor skills, visual comprehension, comprehension and communication language skills); non-verbal development index (NVDQ, including indicators of fine motor skills and visual perception); and a verbal development indicator (VDQ, which includes indicators of language skills for transmitting and perceiving information).

As with the MWF MRI data, potential differences in group means for ELC, VDQ, and NVDQ between breastfed infants and infant formula, and between subgroups of different infant formula were analyzed. In addition to mean comparisons, longitudinal changes across these three composites were examined using mixed-effects modeling assuming a linear trend.

Example 1

Determination of nutritional factors based on cross-sectional analysis

From the cohort described above, infants under 5 years of age who received various infant formulas during infancy were included in an extended correlation analysis to identify the relationship between infant formula nutrient composition and brain myelination. From this cohort, the 6 most commonly used infant formulas were analyzed in terms of their nutritional composition. A single general linear model (GLM) was built that modeled all quantified nutrients and the age of the child.

Spearman's rank correlation between nutrient content and myelin content score (adjusted for the child's age) was then calculated for each image voxel or point within the brain. Significance was defined as p < 0.05 adjusted for type 1 errors using the cluster correction method. Association or trend was defined as p < 0.15. In the initial analysis, accounting for all 22 nutrients listed in Table 1 resulted in an invalid model. To reduce the number of nutrient components in the model, we analyzed the internal correlation between nutrients. Based on a conservative cut-off value of 0.9, we excluded nutrients that were highly correlated with each other in different infant formula formulations. The result was a final model that included iron, sphingomyelin, folic acid, choline, DHA, zinc, and phosphatidylcholine.

P < 0.05: iron, sphingomyelin, folic acid, choline, DHA.

P<0.15: zinc and phosphatidylcholine.

Nutrients that showed high correlation with each other included:

folic acid and vitamin B12.

DHA and AA.

Zinc, calcium, magnesium, copper and phosphorus.

Phosphatidylcholine, phosphatidylserine and phosphatidylinositol.

In the case of choline, association with myelination (myelin water fraction) was observed in the brain over time, in particular in the cerebellum, visual cortex, thalamus, parietal cortex and frontal lobe. The results are shown in FIG. 1b.

In the case of sphingomyelin, association with myelination (myelin water fraction) was observed in the brain over time, in particular in the cerebellum, visual cortex, internal capsule, frontal lobe, parietal lobe, temporal lobe. The results are shown in FIG. 1s.

In the case of phosphatidylinositol, the association with myelination (myelin water fraction) was observed in the brain over time, in particular in the cerebellum, visual cortex, motor cortex, frontal cortex. The results are shown in FIG. 1d.

In the case of phosphatidylcholine, the association with myelination (myelin water fraction) was observed in the brain over time, in particular in the cerebellum, visual cortex, internal capsule, frontal lobe, parietal lobe, temporal lobe. The results are shown in FIG. 1e.

In the case of folic acid, the association with myelination (myelin water fraction) was observed in the brain over time, in particular in the cerebellum, motor cortex, visual cortex. The results are shown in FIG. 1f.

In the case of vitamin B12, association with myelination (myelin water fraction) was observed in the brain over time, in particular in the cerebellum, visual cortex, motor and somatosensory cortex. The results are shown in FIG. 1g.

In the case of iron, the relationship with myelination (myelinated water fraction) was observed in the brain over time, in particular in the cerebellum, visual cortex, internal capsule, motor and somatosensory cortex, corpus callosum, frontal cortex, temporal white matter. The results are shown in FIG. 1h.

In the case of zinc, an association with myelination (myelinated water fraction) was observed in the brain over time, in particular in the cerebellum, visual cortex, internal capsule, motor and somatosensory cortex, corpus callosum, frontal cortex, temporal white matter. The results are shown in FIG. 1i.

In the case of calcium, the relationship with myelination (myelinated water fraction) was observed in the brain over time, in particular in the cerebellum, visual cortex, motor and somatosensory cortex, corpus callosum, frontal cortex, temporal white matter. The results are shown in FIG. 1j.

In the case of phosphorus, the relationship with myelination (myelin water fraction) was observed in the brain over time, in particular in the cerebellum, visual cortex, motor and somatosensory cortex, prefrontal cortex. The results are shown in FIG. 1k.

In the case of magnesium, association with myelination (myelin water fraction) was observed in the brain over time, in particular in the cerebellum, visual cortex, internal capsule, corpus callosum, frontal cortex, motor cortex. The results are shown in FIG. 1l.

In the case of DHA, association with myelination (myelin water fraction) was observed in the brain over time, in particular in the cerebellum, primary and secondary motor cortex, internal capsule, visual cortex, frontal cortex. The results are shown in FIG. 1m.

In the case of AA, the association with myelination (myelin water fraction) was observed in the brain over time, in particular in the cerebellum, internal capsule, parietal lobe, motor and sensory cortex, visual cortex, frontal cortex. The results are shown in FIG. 1n.

Example 2

a) Whole brain myelination trajectory from a longitudinal study

Based on the available data, trajectories of longitudinal myelin development (primary myelination) were calculated using repeatedly measured MWF data in children whose infant formulas contained various amounts of choline (the composition of such mixtures is given below in table 2). The trajectories were calculated using the method of longitudinal nonlinear mixed effects. Data for children from each formula group were processed using Gompertz's modified growth models. The results are shown in FIG. 1.

table 2

(low choline) (high in choline) Choline 35.7 mg/100 g 170 mg/100 g

b) Mean trajectory of myelination by region of the brain from a longitudinal study

Based on the available data, average longitudinal myelin development trajectories were calculated by brain regions (primary myelination) using multiple measured MWF data in children whose infant formulas contained various amounts of choline (the composition of such mixtures is given below in table 2a). Trajectories were calculated using the method of longitudinal non-linear mixed effects, and data for children from the group of each of the mixtures were processed using modified Gompertz growth models. The results are shown in FIG. 1a.

Table 2a

(low choline) (high in choline) Choline 111 mg/100 g 170 mg/100 g

The results are shown in FIG. 1a.

Example 3

Suppliers and mother liquors

Compound Company Catalog number Cas No. Uterine Octanoic acid Sigma O3907 12407-2 Nervonic acid Fluca 87117 50637-6 Stearic acid Fluca 85679 57-114 Sphingomyelin Sigma S0756 8518710-6

Carrier media and doses

Compound Carrier medium/solution in Dose 1 Dose 2 Dose 3 Octanoic acid DMSO 10 µM 50 µM 250 µM Nervonic acid DMSO 10 nM 100 nM 1 μM Stearic acid DMSO 100 nM 1 μM 10 µM Sphingomyelin Solution in ETOH/diluted in DMSO 10 nM 100 nM 1 μM

Media compositions and cultivation methods

1) Neurobasal complete environment

Neurobasal Medium (LIFE TECHNOLOGIES CORP, No. 21103-049)

Additive 50X B27 (LIFE TECHNOLOGIES CORP # 12587-010)

2 mM L-glutamine (LIFE TECHNOLOGIES CORP, no. 25030-149)

1X Penicillin-Streptomycin Solution (Pen-Strep) (LIFE TECHNOLOGIES CORP #15140-122)

2) Neurobasal complete medium with growth factors (GF)

The above formulation with the addition of GF

1M Tris (MW 121.14, Fisher SCI BP152)

Heparin (sigma H3149)

BSA (Sigma A7030)

Water free of DNase, RNase, protease (Fisher SCI AC327390010)

EGF (GIBCO PHG0311)

bFGF vial (GIBCO PHG0021)

3) Neurobasal medium without choline (Life technologies, special formula, without L-glutamine, without phenol red)

Note: Complete medium and complete medium with GF were prepared in the same way as above.

Formation of neural progenitor cell (NPC) libraries:

mouse neocortex dissociation E14

Required reagents:

Dulbecco's Phosphate Buffered Saline (DPBS) (1X) + 10% Pen/Strep

Neurobasal Medium / 10% Pen/Strep / 10X HEPES

Procedure

The brains of E14 pups were harvested and placed in ice-cold DPBS(1X) + 10% Pen/Strep, then dissected using a dissecting loupe. For each cub, 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.

Tissue from each tube was aseptically and manually divided into single cells, neurobasal complete medium was added, and centrifuged at 130 G for 5 min. The tissue was then resuspended in neurobasal complete medium with GF and placed in a Corning 100 mm X 20 mm suspension culture dish (#430591). Cells were subcultured twice at a ratio of 1 : 3, after which they were centrifuged (130 g for 5 min), resuspended in freezing medium (10% DMSO and neurobasal complete medium, without GF), and frozen in liquid nitrogen (LN2).

Thawing cells for compound screening

The flasks were removed from LN2, quickly thawed, and the cells were transferred dropwise into a 15 ml conical flask. 10 ml neurobasal complete medium was added. Cells were transferred to a suspension culture dish and placed in an incubator for 2 hours. After 1.5 hours, the condition of the cells was checked. Based on the condition and quantity, the number of plates needed was estimated and the appropriate amount of complete neurobasal medium was warmed up. After 2 hours the cells were placed in a 15 ml conical tube and rotated at 130 g for 5 minutes. The cells were then resuspended in neurobasal complete GF medium (3 μl of GF for every 10 ml of medium). Cells were then grown overnight, after which they were used in experiments.

Seeding cells in 96-well plates only for counting neurospheres and diameters

Corning Costar 3474 96-well plate with Ultra low attachment surface to prevent cell adhesion

Dissociation and seeding of cells

3-4 ml of cells were taken from the inclined plate and added to a 15 ml conical cone. Part of the remaining medium was used to rinse the tablet. The rest of the medium was taken and placed in a 15 ml conical tube and centrifuged at 130 G for 5 min. Deleted the whole environment. The cells were carefully resuspended in 5 ml of warm phosphate-buffered saline (PBS) and centrifuged again. The PBS was then removed and the cells were carefully resuspended in 500 μl of Accutase Cell Detachment Solution (Corning™ Accutase™ Cell Detachment Solution #25058CI). The cells were then gently agitated with a 1000 µl spouted pipette to resuspend the pellet, and then placed in a shaking water bath for 5-10 minutes, followed by occasional manual vortexing.

Media was prepared as follows, all media contained GF:

Compound Octanoic acid Control/carrier medium Low content
choline/carrier 1% BSA bottom. Avg. 15
Oct : Dec Nervonic acid Control/carrier medium Low content
choline/carrier Average choline/carrier medium bottom. Avg. High Stearic acid Control/carrier medium Low content
choline/carrier Average choline/carrier medium bottom. Avg. high Sphingomyelin Control/carrier medium Low content
choline/carrier Average choline/carrier medium bottom. Avg. high

Control medium and compound medium were prepared with medium no. 2 and contained 29 μM choline,

media with low (5 μM) and medium (70 μM) choline content were prepared with medium no. 3.

Compound Company Catalog number cas no. Uterine Carrier/dissolved Dose 1 Dose 2 Choline chloride Sigma 26978 67-48-L PBS 5 µM 70 µM

The medium was CAREFULLY agitated with a pipette with a 1000 µl nose and then with a 200 µl nose for greater cell dispersion.

The clots contained no more than ~3-5 cells. To dilute the enzyme, 5-10 ml of warm medium (GF) was added. Added 2 ml of medium. It was taken with a pipette of 1000 μl, and then 3 ml was added with a serological pipette. Prior to seeding, cells were sieved through a 40 µm approved cell culture sieve.

1 ml was taken for cell counting. Cells were centrifuged again. The medium above the cell sediment was removed. 1 ml of prepared medium (without GF) was added. Cells were collected with a 1000 µl pipette. The cells were diluted (24,000 cells/well) in 250 µl of the appropriate medium. Cells were shaken daily and grown for 2 days.

Fixing and staining

1. Cells were fixed under traction. For fixation and subsequent immunohistochemical analysis, 100 µl of the medium was removed and 100 µl of 4% paraformaldehyde (PFA) in 1X PBS was added to fix the cells, simultaneously manually counting the neurospheres, then the cells were washed twice with 1X PBS for 5 min and left in 1X PBS, wrapped in foil and left overnight at 4°C, or stained with Dapi. 100 μl of PBS was withdrawn and 100 μl of antibody staining solution (AB) (1% goat serum, 1xPBS and 0.1% Triton X) was added at room temperature over 1 hour. The antibody staining solution was removed. The cells were then stained with Dapi 1:5000 in antibody staining solution, 100 µl per well, then the cells were incubated at room temperature for 15 min in the dark. Cells were then washed 2 times in antibody staining solution for five minutes. Images were acquired using a GE Cytell imager or LSM 710 Zeiss confocal microscope and neurosphere diameters were analyzed using ImageJ software (National Institutes of Health).

Seeding cells in 24-well plates for monolayer differentiation or EdU incorporation assay

Compound Octanoic acid Control/carrier Low content
choline/carrier Average choline/carrier medium bottom. Avg. High Nervonic acid Control/carrier Low content
choline/carrier Average choline/carrier medium bottom. Avg. High Stearic acid Control/carrier Low content
choline/carrier Average choline/carrier medium bottom. Avg. high Sphingomyelin Control/carrier medium Low content
choline/carrier Average choline/carrier medium bottom. Avg. high

24-well glass bottom plates (Mat Tek P24G-1.0-13-F Case, 24-well glass bottom plates) were coated with poly-L-ornithine (Sigma P4957) and fibronection (Sigma F1141) before being used in the following analysis.

See above for dissociation and cell seeding.

Cells were seeded (10,000 cells/well) in complete GF medium for 24 hours (500 μl/well). After fixation of the cells, they were transferred to a medium without choline, a medium with another compound, or an appropriate medium.

Differentiation Assay: Quantification of Neuronal, Glial and NPC Marker Expression

After 24 hours, the fixation of the cells on the plate was checked, then the medium was carefully removed.

500 µl Compound Medium containing 2% Nu Serum (serum replacement) (Corning™ Nu-Serum Growth Medium Supplement, # CB55004), control, low or medium choline medium was added. Note: The medium did not contain GF.

Control and compound media were prepared with medium #2 and contained 29 μM choline, low (5 μM) and medium (70 μM) choline media were prepared with medium #3.

Cells were cultured for 9 days in 2% Nu Serum medium with medium changed every 2nd day. For fixation and subsequent immunohistochemical analysis, the medium was removed, the cells were washed once with 1X PBS for 5 min and fixed with 4% PFA in 1X PBS for 15 min at 4°C. Cells were then washed twice with 1X PBS for 5 min, left in 1X PBS, wrapped in foil and left overnight at 4°C, or they were immediately stained with primary antibody.

Staining for differentiation

PBS was removed and enough antibody staining solution (1% goat serum, 1x PBS and 0.1% Triton X) was added to cover the bottom, the block was kept at room temperature for 1 hour.

Dilution of the primary antibody was performed in the appropriate amount of antibody staining solution, 250 µl per well (antibodies kept only briefly on ice, mouse anti-MAP2 or TUJ1 1 : 500 (neuronal marker), rabbit anti-GFAP (glial marker) 1 : 1000 , chicken anti-Nestin CFP (EGFP antibody (progenitor cell marker)) 1 : 1000. Antibody staining solution was removed and primary antibody solution was added to each well. Cells were wrapped in foil and kept overnight at 4°C. Cells were then washed with 400 µl of antibody staining solution for 5 min once to remove primary antibodies Secondary antibody solutions (enough for 250 µl per well) were prepared (goat anti-mouse Alexa 488 1:2000, anti-rabbit Cy3 ( 1:500), anti-chicken Alexa 647 1:500 and 1:5000 Dapi).

Cells were incubated at room temperature for 1 hour in the dark and washed 2 times in antibody staining solution for five minutes. They were kept at 4°C or imaged using a GE Cytell visualizer or LSM 710 Zeiss confocal microscope and analyzed using ImageJ software (National Institutes of Health).

Microtubule-associated protein 2 (MAP2), neuronal beta-tubulin III (TuJ1), gliofibrillary acidic protein (GFAP), and Nestin CFP (EGFP antibody).

The expression of each marker was measured on the obtained images (imageJ cumulative density index) and normalized to DAPI fluorescence with all nuclei labeled (integral density index).

Octanoic acid was labeled with neuronal beta-tubulin III (TuJ1), subsequently used compounds were labeled with microtubule associated protein 2 (MAP2).

NPC Monolayer Culture Proliferation Assay (Inclusion of EdU - S-Phase Marker)

After 24 hours, the fixation of the cells on the plate was checked, then the medium was carefully removed. Added. 500 µl of medium with compound and GF. Cells were cultured for 3 days in the appropriate medium.

Control and compound media were prepared with medium #2 and contained 29 μM choline, low (5 μM) and medium (70 μM) choline media were prepared with medium #3.

EDU incorporation was measured using the Alexa Fluor® 555 Click-iT® EdU Imaging Kit (Life technologies, no. c10338).

At the end of day 3, EdU was added to each well at a concentration of 10 μM for 30 minutes before fixation.

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

PBS was removed and enough antibody staining solution (1% goat serum, 1x PBS and 0.1% Triton X) was added to cover the bottom, the block was kept at room temperature for 1 hour. Cells were stained for EDU. Cells were incubated for 30 min at room temperature in the dark. Cells were washed with 1X PBS and stained with Dapi 1:5000 µl for 15 min. Cells were washed once with 1X PBS, then left in PBS at 4°C or imaged immediately using a GE Cytell Imager (cell viability application) or LSM 710 Zeiss confocal microscope (with analysis using ImageJ software (National Institutes of Health)) .

The results are shown in tables 3-7 and in FIG. 2-5.

Table 3 Effects of Nervonic Acid (NA) on Neuronal Cell Density and Astrocyte Density

neural cells astrocytes control 0.436189 0.642448 bottom. NA 0.467588 0.621784 avg. NA 0.56563 0.721512 high NA 0.539448 0.70279

Table 4 Effects of Stearic Acid (SA) on Neuronal Cell Density and Astrocyte Density

neural cells astrocytes control 0.44 0.64 bottom. SA 0.54 0.68 avg. SA 0.66 0.81 high SA 0.64 0.85

Table 5. Effects of Octanoic Acid (OA) on Neuronal Cell Density and Astrocyte Density

neural cells astrocytes Control 1.1 4.1 bottom. OA 1.2 3.7 avg. OA 1.8 6.1 high OA 1.7 7.9

Table 6. Effect of sphingomyelin on the number of neurospheres

Sphingomyelin Control Bottom. Avg. High Average) 103 94 121 219

Table 7. Effect of sphingomyelin (SM) on neuronal proliferation

DAPI control 262 low SM content 280 average SM content 314 high SM content 305

Example 4

experimental part

Samples

Ingredient C2, whey protein concentrate enriched with alpha-lactalbumin (Sample Control System ID: K2Q-00030); infant first milk containing whey protein concentrate fortified with alpha-lactalbumin (Sample Control System ID: K2Q-00032); cow's milk (whole milk); human breast milk (set of 6 separate quality control samples collected 4 weeks after birth; Lee Biosolutions, St. Louis, MO, USA).

Extraction of phospholipids from dairy products

Powdered milk. A portion of 1 g of the homogenized powder was weighed into a 50 ml glass flask and diluted in 20 ml of pure distilled water. The solution was heated at 40°C for 30 min in a water bath. A 500 µl amount of this solution was transferred into a 10 ml glass tube.

Cow's milk and breast milk: An aliquot of 500 μl of the homogenized liquid was transferred into a 10 ml glass tube.

Test compounds were extracted according to MP for quantification of human milk composition using UPLC-MS/MS (RDLS-MP-80138-Rev01) from three measurements using 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, followed by centrifugation for 10 minutes at 2500 rpm. 2 ml of potassium chloride solution (0.88%, mol/mol) was added to the liquid phase, then shaken and centrifuged for 10 min at 2500 rpm. The lower organic phase was transferred to a glass vessel, evaporated to dryness under a gentle stream of N ₂ nitrogen, and reconstituted in 500 μl chloroform/methanol (9+1) prior to entry into LC-MS.

Analysis of phospholipids by liquid chromatography combined with mass spectrometry (LC-MS)

Analyzes were performed on a Q Exactive Plus Orbitrap (Thermo Fisher Scientific, Bremen, Germany) equipped with a Thermo Scientific Dionex UltiMate 3000 Rapid Separation LC system. Separation was performed on a HILIC column (100 x 2.1 (id. dia.) mm; 1.7 μm) with a mobile phase consisting 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 over 15 min, maintained at 70% B for 1 min, returned to baseline within 3 min, and equilibrated over 6 min.

The Q Exactive Plus Orbitrap was equipped with an atmospheric pressure chemical ionization (APCI) sensor that operated in positive ionization mode. The APCI and MS parameters were as follows: corona discharge current 4.0 μA, shielding gas and auxiliary gas 24 and 5 conventional units, respectively; the capillary and evaporator temperatures were 320 and 390°C, respectively, the purge gas flow rate was 0 r.u., and the s-lens RF level was 80. The automatic gain control (AGC) target was set at 1 × 10 ⁶ charges, and the maximum time input - 100 ms with a resolution of 35,000 and 1 microscan for the entire MS. The AGC was set to 1×10 ⁶ charges and the maximum injection time was 250 ms with a resolution of 17,500 s 1 microscan in data-independent fragmentation mode. The inclusion list of selective precursor ions was used at 30% normalized impact energy. Data accumulation was carried out in the mass range 133–2000 Da in the profile mode. An external mass calibration was used. The system was controlled using Xcalibur 3.0 (Thermo Fisher Scientific).

SM species were extracted from the full ion chromatogram using accurate mass. Precursor ions corresponded to the breakdown of phosphatidylcholine at the source to ceramide. An inclusion list was used for specific fragmentation of the 57 SM regioisomers based on precursor ions corresponding to the m/z of ceramide with the loss of a water molecule [Cer-H ₂ O + H ⁺ ], based on the LipidView database and scientific literature sources (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 fed different diets Food Chemistry 2013, 141 , 1451-1460 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).

Analysis of fatty acid methyl ester (FAME) by gas chromatography with a flame ionization detector (GC-FID)

SM fractions were collected over 8.5 to 10 minutes in glass tubes 5 times for each sample. After evaporation of the solvent in a stream of N ₂ , FAME analyzes were performed three times for each sample in accordance with the MP for the quantitative determination of fatty acid in breast milk by gas chromatography (RDLS-MP-8980-00030-Rev01-FAME_Human milk fat 2012, version 1.0 ).

Results and discussion

Hydrophilic interaction liquid chromatography (HILIC) was used to separate the PL classes (ie, phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC) and SM). The assignment of the number of carbons and unsaturated fragments for individual types of SM was performed based on the exact mass of the pseudomolecular ion recorded by the Orbitrap mass spectrometer. The relative abundance of SM species was determined to compare ingredient, infant formula, cow's milk and breast milk.

SM species in various dairy products

In the analyzed samples, 45 SM species were recorded (Table 8).

Table 8. SM species (indicated by x) were recorded in ingredient, infant formula, cow's milk and breast milk samples. SM species that have only been reported in breast milk are shown in bold.

SM Ingredient Food fusion for kids Cow's milk Breast milk SM Ingredient Food fusion for kids Cow's milk Breast milk SM 24:1 X SM 37:1 X X X X SM 25:0 X X X X SM 37:0 X X X X SM 28:1 X X X X SM 38:4 X SM 28:0 X X X X SM 38:3 X SM 30:2 X X X X SM 38:2 X X X X SM 30:1 X X X X SM 38:1 X X X X SM 30:0 X X X X SM 38:0 X X X X SM 32:3 X X X X SM 39:1 X X X X SM 32:2 X X X X SM 39:0 X X X X SM 32:1 X X X X SM 40:2 X X X X SM32:0 X X X X SM 40:1 X X X X SM 33:1 X X X X SM 40:0 X X X X SM 34:3 X X X X SM 41:2 X X SM 34:2 X X X X SM 41:1 X X X X SM 34:1 X X X X SM 41:0 X X X X SM 34:0 X X X X SM 42:4 X SM 35:2 X X X X SM 42:3 X X SM 35:0 X X X X SM 42:2 X X X X SM 36:4 X X X X SM 42:1 X X X X SM 36:3 X X X X SM 42:0 X X X X SM 36:2 X X X X SM 44:3 X X SM 36:1 X X X X SM 44:1 X X X X SM 36:0 X X X X

Species SM 24:1, SM 38:4, SM 38:3 and SM 42:4 have been found in breast milk only in trace amounts.

Relative abundance of SM species

The relative abundance (%) of SM in various dairy products was determined by the peak area divided by the sum of the areas of all peaks corresponding to the SM species in the chromatogram for each sample. In FIG. 6 shows the relative abundance of the main types of SM in the ingredient, infant formula, cow's milk and breast milk.

The relative abundance of SM species present in ingredient and infant formula was comparable to cow's milk and differed slightly from the proportion in breast milk for 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) were lower in breast milk than in ingredient, infant formula and cow's milk. At the same time, for 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, a higher relative abundance in breast milk compared to other dairy products.

The breast milk sample consisted of a set of 6 separate quality control samples collected at 4 weeks or later after birth. Given that SM content in breast milk varies with diet and lactation time, this may partly explain the observed differences. However, despite fluctuations in the relative abundance of some SM species, >85% of the SM species that have been reported in breast milk have also been found in infant formula and cow's milk.

It should be noted that for a given m/z determined from the MS spectrum, various combinations of LCB-LCDs may be offered (e.g. SM 34:1 may correspond to SM d18:1/16:0, d18:0/16:1, d16 :1/18:0 etc.). Therefore, the GC GC profile of FAs was evaluated to obtain more detailed information on the molecular structures of SM in various dairy products.

Profile of fatty acids (FA) in the SM fraction for various dairy products

The regioisomeric structure of the SM was examined first by fractionating the SM and then by analyzing the FAs present in the fractions using GC-FID. SM fractionation was performed as described above for LC-MS analysis, but in this case the eluate was directed to a 5 ml glass tube rather than to the MS. Each fraction was then methylated prior to subsequent GC analysis. The relative abundance of FAs in the SM fraction is shown in Fig. 7.

As shown in FIG. 7, the SM fraction contained mainly saturated fatty acids (SFAs) (i.e. myristic acid 14:0, pentadecanoic acid 15:0, palmitic acid 16:0, stearic acid 18:0, arachidonic acid 20:0, behenic acid 22 :0, tricosanoic acid 23:0 and lignoceric acid 24:0). A higher proportion of SFA was noted in the SM fraction for all dairy products (table 9). This result is consistent with the scientific literature, indicating a higher distribution of SFAs with a carbon chain greater than 18 in the SM fraction. Such a high amount of SFA reflects the structural role of SM, in particular, they reduce fluidity and maintain the rigidity of the membrane of milk fat globules.

Table 9. Percentage of SFAs, monounsaturated fatty acids (MUFAs) and PUFAs recorded in the SM fraction from various dairy products.

LCD Ingredient Food fusion for kids Cow's milk Breast milk NFA % 14:0-16:0; 18:0; 20:0-24:0 95.1±2.6 92.8 ± 3.9 93.1 ± 8.4 87.9 ± 2.7 MUFA % 18:1n-9 2.5±0.7 3.6±0.9 5.3±1.5 4.4 ± 1.9 24:1n-9 1.7±0.3 1.9 ± 1.7 1.2±0.1 5.9±0.6 PUFA % 18:2n-6 0.8±0.4 1.7±0.2 0.3±0.4 1.7±0.1

Monounsaturated fatty acids (MUFAs) make up approximately 4-11% of the fatty acids in the SM fraction. Oleic acid 18:1n-9 and nervonic acid 24:1n-9 were reported 2 MUFAs. Interestingly, 24:1n-9 was found in a relatively high proportion in breast milk compared to other dairy products, and this observation is consistent with data in the scientific literature. The only PUFA, linoleic acid 18:2n-6, was found in relatively high concentrations in analyzed infant formulas and breast milk compared to other products. Finally, omega-3 PUFAs were not detected in the SM fraction. This is also in line with data reported in the scientific literature, which show that arachidonic acid (AA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n- 3) Mainly present in PE, PI and PS.

Example 5

Examples of artificial nutritional compositions (infant formulas) in accordance with the present invention are shown in table 10 and table 10a

Table 10

Nutrition Information Nutrients Unit
per 100 g Energy value kJ 2216 Protein G eleven whey protein G 7.0 α-lactalbumin G 1.8 Casein G 3.8 fat G 29 linoleic acid mg 4160 α-linolenic acid mg 336 AA mg 203 DHA mg 143 Carbohydrates G 52.5 Alimentary fiber G 2.4 Soluble dietary fiber (as oligofructose) G 2.4 Taurine mg 38 L-carnitine mg 8.0 lutein mcg 64 nucleotides mg 21 L-Tyr mg ≥ 407.4 L-Trp mg ≥ 176.9 Vitamin A mcg retinol equivalent 581 β-carotene mcg 120 Vitamin D mcg 9.6 Vitamin E mg α-tocopherol equivalent 5.9 Vitamin K1 mcg 54 Vitamin B1 mcg 800 Vitamin B2 mcg 880 Vitamin B6 mcg 440 Vitamin B12 mcg 9.5 Niacin mcg 4000 folic acid mcg 346 pantothenic acid mcg 2800 biotin mcg 16 Vitamin C mg 72 choline mg 230 inositol mg 36 Calcium mg 288-588 Phosphorus mg 160-336 Magnesium mg 36 Iron mg 13.1 Zinc mg 7.1 Manganese mcg 40 Copper mcg 266 Iodine mcg 80 Sodium mg 128 Potassium mg 520 Chloride mg 346 Selenium mcg eleven Sphingomyelin mg 93.8

The composition may also contain any additional ingredients commonly found in infant formula formulations.

Table 10a

Nutrient Units per liter Test mixture Energy value kcal 662.0 Energy value kJ 2768.5 water/moisture G 902.6 Ash G 3.4 Protein Protein G 13.4 65% whey G 8.7 as alpha lactalbumin G 2.3 35% casein G 4.7 Carbohydrates Available carbohydrates G 68.6 Carbohydrate G 73.6 including lactose G 68.6 including sugars G 68.6 Soluble Dietary Fiber (as oligofructose) G 5.0 Lipids Total Fat G 36.0 Saturated fatty acids G 14.0 Trans fatty acids G 0.3 Sphingomyelin mg 105.0 Linoleic acid mg 5200.0 Linolenic acid mg 420.5 linoleic : alpha-linolenic acid ratio ratio 12.4 ARA mg 132.0 DHA mg 132.0 ARA/DHA ratio 1.0 vitamins Vitamin A (retinol) μg RE 660.1 beta carotene mcg 150.0 Vitamin D (cholecalciferol) µg D 12.0 Vitamin E (TE) mg 5.8 Vitamin K mcg 53.6 Vitamin B1 (thiamine) mg 0.8 Vitamin B2 (riboflavin) mg 0.9 Vitamin B6 (pyridoxine) mg 0.6 Vitamin B12 (cyanocobalamin) mcg 7.0 Niacin mg 5.0 Folic acid mcg 219.0 pantothenic acid mg 3.5 Biotin mcg 18.0 Vitamin C (ascorbic acid) mg 80.0 Minerals and trace elements Calcium mg 336.7 Phosphorus mg 190.0 Ca :P ratio 1.8 Magnesium mg 45.0 Iron mg 8.6 Zinc mg 5.5 Manganese mcg 50.0 Copper mg 0.3 Iodine mcg 90.0 Sodium mg 175.0 Potassium mg 500.0 Chloride mg 345.0 Selenium mcg 13.5 Other substances Choline mg 160.0 Inositol mg 45.0 Taurine mg 37.6 L-carnitine mg 8.8 Lutein mg 0.1 Nucleotides (total) mg 20.8 CMF mg 10.4 UMF mg 4.0 AMF mg 3.2 HMF mg 1.6 IMF mg 1.6

The composition may also contain any additional ingredients commonly found in infant formula formulations.

Example 6

Co-cultivation of neurons and oligodendrocytes

Neurons/oligodendrocytes (OL) were cultured as previously described in Charles et al., 2000.

Pregnant female rats on day 17 of pregnancy were sacrificed by cervical dislocation (Wistar rats) and the fetuses were removed from the uterus. The forebrain was removed and placed in ice-cold Leibovitz medium (L15) containing 2% penicillin-streptomycin (PS) and 1% bovine serum albumin (BSA). The forebrain was separated by trypsinization for 20 min at 37°C (trypsin EDTA 1X). The reaction was stopped by adding Dulbecco's modified Eagle's medium (DMEM) containing DNase I grade II (0.1 mg/ml) and 10% fetal calf serum (FCS). The cells were then mechanically separated by 3 passages through a 10 ml pipette. After that, the cells were centrifuged at 180 x g for 10 min at 4°C on a layer of BSA (3.5%) in L15 medium. The supernatant was discarded and the cells in the pellet were resuspended in DMEM containing 10% FCS. The cells were then centrifuged at 515 x g for 10 min at 4°C. The supernatant was discarded and the cells in the pellet were resuspended in culture medium containing neurobasal medium supplemented with 2% B27, 2 mM L-glutamine (L Glu), 2% PS solution, 1% FCS, and 10 ng/ml platelet-derived growth factor (PDGF-AA ). Viable cells were counted with a Neubauer cytometer using a trypan blue displacement test. Cells were seeded at a density of 20,000 cells/well in 96-well plates pre-coated with poly-L-lysine and laminin.

One day after seeding (day 1 culture), cells were incubated with a test compound (selected from those listed in Table 11) or with estradiol. Control cells were not incubated with test compound or estradiol. Estradiol was used as a positive control. Estradiol is known to induce the proliferation of oligodendrocyte progenitor cells (OPCs). The positive effect of estradiol on OL differentiation was also demonstrated, as well as its effect on the process of early myelination. Conclusions about the positive effect of estradiol on neurite outgrowth have also been published (see review by Alevaro et al., 2010).

The plates were kept at 37° C. in a humidified incubator under air (95%) - CO2 (5%). Half of the medium was replaced every other day with fresh medium with test compound or control compound. Test or control compounds were kept at the specified concentration throughout all experiments. Compounds were tested in 1 culture (6 wells for each set of conditions). The cells were then used on culture day 12, 18, or 30 to evaluate one of the following: OPC proliferation, OPC differentiation to OL and early myelination process (myelin sheath), or OL maturation (myelin maturation) and mature myelination process (myelin sheath).

OPC proliferation: measurement of A2B5 positive cells and total axon length (NF)

On day 12 of cultivation, the cells were fixed with a cold mixture of absolute ethanol (95%) and pure acetic acid (5%) for 5 min. Then the cell permeability was increased and non-specific sites were blocked with phosphate buffered saline (PBS) containing 0.1% saponin and 1% FCS for 15 min at room temperature.

Cells were then incubated with monoclonal anti-A2B5 conjugated to alexa fluor® 488, produced in mice, at a 1/200 dilution in PBS containing 1% FCS, 0.1% saponin, for 2 hours at room temperature and with anti -NF (phosphorylated and non-phosphorylated neurofilament 200) produced in the body of a rabbit, with a dilution of 1/500 in PBS containing 1% FCS, 0.1% saponin for 2 hours at room temperature. The presence of this antibody was detected using goat anti-rabbit with Alexa Fluor 568 at a 1/400 dilution in PBS with 1% FCS, 0.1% saponin for 1 hour at room temperature.

The total number of OPCs (number of A2B5 positive cells) was quantified (to assess proliferation), the axonal network (total axon length (NF)) was measured to assess the effect of the connection on the neural network (the quality of myelination is directly related to the quality of the axon network).

OPC differentiation into OL and myelination process (myelin sheath): measurement of number and area of MAG-positive cells, MAG/NF sheath overlap, and total axon length (NF)

On day 18 of cultivation, the cells were fixed with a cold mixture of absolute ethanol (95%) and pure acetic acid (5%) for 5 min. Then the cell permeability was increased and non-specific sites were blocked with phosphate buffered saline (PBS) containing 0.1% saponin and 1% FCS for 15 min at room temperature.

The cells were then incubated with mouse-produced monoclonal anti-MAGs diluted 1/400 in PBS containing 1% FCS, 0.1% saponin, and rabbit-produced anti-NF (phosphorylated and non-phosphorylated neurofilament 200). , diluted 1/500 in PBS containing 1% FCS, 0.1% saponin for 2 hours at room temperature. The presence of this antibody was detected using goat anti-mouse antibody CF 488 A diluted 1/800 in PBS with 1% FCS, 0.1% saponin and goat anti-rabbit antibody with Alexa Fluor 568 diluted 1/800 in PBS with 1% FCS, 0 .1% saponin for 1 h at room temperature.

The total number of OLs (number and area of MAG-positive cells) was quantified (to evaluate the process of differentiation) as well as the OPC sheath around axons (overlapping MAG/NF sheath) (myelination process). The axon network (total axon length (NF)) was measured to evaluate the effect of connections on the neural network.

OL maturation (myelin maturation): measurement of the number and area of MBP-positive cells, MBP/NF sheath overlap, and total axon length (NF)

On day 30 of cultivation, the cells were fixed with a cold mixture of absolute ethanol (95%) and pure acetic acid (5%) for 5 min. Then the cell permeability was increased and non-specific sites were blocked with phosphate buffered saline (PBS) containing 0.1% saponin and 1% FCS for 15 min at room temperature.

The cells were then incubated with mouse-produced monoclonal anti-MBP diluted 1/1000 in PBS containing 1% FCS, 0.1% saponin, and rabbit-produced anti-NF (phosphorylated and non-phosphorylated neurofilament 200). , diluted 1/500 in PBS containing 1% FCS, 0.1% saponin for 2 hours at room temperature. The presence of this antibody was detected using goat anti-mouse antibody CF 488 A diluted 1/800 in PBS with 1% FCS, 0.1% saponin and goat anti-rabbit antibody with Alexa Fluor 568 diluted 1/400 in PBS with 1% FCS, 0 .1% saponin for 1 h at room temperature.

The total number of OLs (number and area of MBP-positive cells) was quantified (to assess the maturation process of the OL) as well as the sheath of myelin around axons (overlapping MBP/NF sheath). The axon network (total axon length (NF)) was measured to evaluate the effect of connections on the neural network.

For all measurements, one culture was tested (6 wells for each set of conditions). For each set of conditions tested, 30 images (each image corresponding to one field or another) were obtained per well and analyzed using ImageXpress (Molecular devices) at 20x magnification with LED lamp (excitation 360/480/565 and emission 460/535/620). 30 images were obtained automatically, and they displayed 80% of the total surface of the culture well.

The results were expressed as the cumulative mean length in µm of the neuritic network or myelin sheath labeled for a given marker (MAG or MBP) per field. To evaluate shell formation, the area of overlap between NF and MAG or MBP was measured.

To evaluate the OPC population, MAG-positive cell population, MBP-positive cell population, an automatic count of the number of positive cells in each image (= field) was performed. The results were expressed as the mean values of positive cells per field.

All images were taken under the same conditions.

Table 11

Tablet 1 (A2B5/NF) Control Estradiol (150 nM) DHA (0.15 µM) DHA (1.5 µM) Stearic acid (50 µM) Stearic acid (5 µM) Stearic acid (0.5 µM) B12 (100 nM) B12 (10 nM) B12 (1 nM) Folic acid (250 nM) Folic acid (50 nM) Folic acid (6 nM) Choline (20 µM) Iron (1 µM) Iron (0.1 µM) Zinc (5 µM) Zinc (0.5 µM) Phosphorus (5 mM) Phosphorus (1 mM) Magnesium (25 mM) Copper (0.5 µM) Phosphatidylcholine (100 µM) Phosphatidylinositol (5 µM) Phosphatidylinositol (50 µM) Phosphatidylserine (5 µM) Phosphatidylserine (10 µM) Phosphatidylserine (100 µM) Sphingomyelin (5 µM) Sphingomyelin (25 µM) Ceramide (brain extract): dipalmitoylphosphatidylcholine (DPPC) (1:4) galactoceramides (C18:1/24:1)/(C18:1/18:0) : DPPC (1 : 4) glucoceramides (C18:1/24:1)/(C18:1/18:0) : DPPC (1:4) D-erythrodihydroceramide (C24:1/18:0)/(C18:0/18:1) : DPPC (1:4) Ceramide-1-phosphate (C18:1/24:0) : DPPC (1:4) GM3 : DPPC (1 : 4) GD3 : DPPC (1 : 4)

The results are shown in FIG. 8-28

Example 7

Materials and methods

1. Preparation of the Nutrient Layer: Dissociation of the Neonatal Cortex and Preservation of Mixed Glial Cultures

Freshly prepared brains were placed in a water bath at 37°C for 3 min, then the cortex was crushed by forcing through the nose of a P1000 pipette to obtain smaller fragments. 75 µl papain ORS solution was added to each brain sample, then the tissues were incubated in a water bath at 37°C for 20 min. The mixed glial culture was then added to the tissue suspension to ensure that the papain OPC solution was inactivated.

The tissues were then triturated using a sterile fire-melted Pasteur pipette, after which 4 ml of mixed glial culture medium was added to each brain sample. Cells were centrifuged at 1200 rpm (~300 g) for 5 min, then the cells were resuspended in warm mixed glial culture medium and plated in a PLL-coated flask.

4 hours after inoculation, a complete change of the medium was made in order to remove most of the tissue residues formed during grinding and stimulate the viability of the culture. After 3 days of cultivation, 2/3 of the medium was replaced, after which the medium was no longer changed. The cells were then kept in culture until confluent.

2. Preparation of hippocampal neurons

Hippocampal neurons were isolated from the embryos of pups (E18) of Sprague-Dawley rats. Briefly, after killing the animals, the brains were isolated, the covering membranes were removed from the medial aspect of the cerebral hemispheres, then the hippocampi were dissected and stored at 4°C until the process was completed.

The tissue was then incubated with 2.5% trypsin for 15 min in a water bath at 37°C, after which it was gently washed and stored in culture medium. Separation of the hippocampus was performed by repeated pipetting through a functionalized sterile Pasteur pipette. After mechanical dissociation, cells were seeded at the desired density in neuronal seed medium, allowed to recover for 4 hours, and then transferred to complete neuronal culture medium.

3. Purification of OPCs from mixed glial cultures to form o-cultures of OL/hippocampal neurons

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

Then the medium was changed and replaced with 4 ml of fresh culture mixed glial medium supplemented with 5 μg/ml of insulin. The flasks were placed back on the shaker, equilibrated for about 3 hours, then shaken for about 16 hours at 220 rpm (overnight).

The next morning, mixed glial culture medium containing microglial and OPC cells was harvested and pre-plated in P100 Petri dishes (untreated for cultivation) for 30 min to purify OPC cells; at the same time, microglial cells immediately began to fix on the surface of the Petri dish, while ORF cells remained in the supernatant medium.

30 minutes after the pre-seeding, the medium was collected and the OL count was made, after which they were sown on the hippocampal neurons in a final volume of 1 ml of the OL medium.

Spent a complete replacement of the medium OL (without CNTF), then the cells were kept in culture until the appropriate time of the experiments.

The following procedure was used for maturation experiments:

a. Growth of OPC on astrocyte nutrient layer for 10 DIV (in vitro days)

b. OPC spin-off (Day 0)

c. Adding Connections (Day 3)

d. Quantification of maturation at day 4, 7 and 10.

The following procedure was used for myelination experiments:

a. Growth of hippocampal neurons until full maturation of the neuronal network (14 DIV)

b. Simultaneous growth of OPC on astrocyte nutrient layer for 10 DIV

c. OPC isolation and co-culture with neurons (Day 14)

d. Adding connections (Day 15)

e. Quantification of myelination at day 15 (1 day after seeding of co-cultivation before compound application), 18, 21/23 and 28/29 co-cultivation

4. Acquisition of images

All cultures at different times of the experiment were fixed in 4% paraformaldehyde and 4% sucrose at room temperature (RT) for 10 min. Primary and secondary antibodies were added to GDB buffer (30 mM phosphate buffer, pH 7.4, containing 0.2% gelatin, 0.5% Triton X-100, and 0.8 M NaCl) for 2 h at room temperature. Cells were stained with the appropriate marker (primary antibody used: anti-A2B5 antibody (ABCAM cat. no. ab53521), rat anti-MBP (BIO-RAD cat. no. aa82-87), O4 oligodendrocyte marker antibody (R&D Systems cat. no. MAB1326), monoclonal antibody anti-βIII tubulin (Promega cat. no. G7121) Secondary antibody used: Alexa anti-555 rat (Life Tech A-21434), Alexa anti-488 mouse (Life Tech A-11009) After immunocytochemical staining, all images were obtained using Array Scan XTI (ThermoScientific), objective was 20x with 2 x 2 grouping. A minimum of 15 images were obtained for each condition and reproducing well (three times).

All accumulated images were analyzed using the HCS Studio Cell Analysis software, in particular its "Scan" application.

Papaic OPC solution (formulated in MEM)

Papain solution 1.54 mg/ml

L-cysteine 360 mcg/ml

DNase I 60 µg/ml

Mixed Glial Cell Culture Medium (Formulated in DMEM)

FBS 10%

Penicillin/streptomycin: (0.33% from stock solution) 33 units/ml penicillin and 33 mcg/ml streptomycin

Glutamax 1%

OL environment

DMEM

100X OL additive

Bovine insulin (from 1 mg/ml stock solution)

GlutaMAX

Holotransferrin (from 33 mg/ml stock solution)

Additive B27

FBS

CNTF (from 50 ng/µl stock solution)

The results are shown in FIG. 30-49

Claims (26)

1. Use of a synthetic nutritional composition containing free form or salt form of choline to prevent, reduce the risk of, or mitigate a suboptimal de novo myelination trajectory in a formula-fed subject, where the subject is a human infant or child, such as a human infant or child , fed with a mixture, and where the specified composition contains choline in an amount of from 50 to 1000 mg/100 g per dry weight of the composition, and where the human infant is an infant under the age of 12 months, and the child is a person aged from 1 to 18 years, preferably 1 to 5 years; and where the de novo myelination trajectory is sub-optimal when the distance between any equivalent and/or identical infant trajectory measurement points and the trajectory achieved in exclusively breastfed infants during their first (3) months of life is greater than 50%. 2. Use according to claim 1, wherein said composition further comprises one or more of the following ingredients: a mineral substance and/or a vitamin and/or a phospholipid and/or a fatty acid compound, wherein said fatty acid compound is a fatty acid-containing a compound other than a phospholipid. 3. Use according to claim 2, wherein said vitamin is folic acid and/or vitamin B12, where said mineral is iron, and where said fatty acid compound contains docosahexaenoic acid (DHA) and/or arachidonic acid and is monoacylglycerol, and /or diacylglycerol and/or triacylglycerol. 4. Use according to claim 2 or 3, wherein said phospholipid is a compound of formula (I) or a mixture of compounds of formula (I)

where R ¹ is O, X is NH or O, R ² is a saturated or unsaturated linear or branched C2-C44 acyl group; R ³ is a substituent with formula (II) or formula (III)

where R ⁵ is a saturated or unsaturated linear or branched C2-C44 acyl group, and R ⁶ is a saturated C2-C44 alkyl or alkenyl group; And R ⁴ is selected from a C5 or C6 substituted or unsubstituted cyclic alkyl or alkenyl group, or -(CH2)nR ⁷ where n is an integer ranging from 1 to 4, in particular from 1 to 2, and R ⁷ is -N(CH3), NH3 or a substituent of formula (IV)

in particular R ⁴ is a C6 cyclic alkyl or alkenyl group substituted with one or more hydroxyl groups. 5. Use according to claim 2 or 4 wherein the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and sphingomyelin and any mixture of the above. 6. Application according to any one of paragraphs. 2, 3 or 5, where in the presence of iron in the composition, its content is more than 5 mg/100 g of dry weight of the composition, where in the presence of folic acid in the composition, its content is more than 100 μg/100 g of dry weight of the composition, where in the presence of folic acid in the composition of vitamin B12, its content is more than 5 μg/100 g of the dry weight of the composition, where in the presence of sphingomyelin in the composition its content is more than 300 mg/kg of the dry weight of the composition, where in the presence of a fatty acid compound containing DHA in the composition, its content in the composition is from 60 to 350 mg/100 g of the dry weight of the composition and where, if the fatty acid compound containing arachidonic acid is present in the composition, its content is from 60 to 350 mg/100 g of the dry weight of the composition. 7. Use according to any one of the preceding claims, wherein said composition is a composition selected from the group consisting of an infant formula, a complementary milk formula, an infant composition that is intended to be added to or diluted with human breast milk, and a food product intended for consumption by an infant and/or child in pure form or in combination with human breast milk, where the infant is up to 12 months old and the child is from 1 to 18 years old, preferably from 1 to 5 years old. 8. Use according to any of the preceding claims, wherein the human infant is under 12 months of age. 9. Use according to any preceding claim, where the de novo myelination trajectory is sub-optimal when the distance between any equivalent and/or identical infant trajectory measurement points and the trajectory achieved in infants who are exclusively breastfed during their first (3) months life exceeds 20%. 10. Synthetic nutritional composition containing choline in free form or as a salt thereof to prevent, reduce the risk or mitigate a suboptimal de novo myelination trajectory in a subject fed a formula that also contains folic acid and vitamin B12 and a fatty acid compound, other than a phospholipid, including docosahexaenoic acid and/or arachidonic acid, and sphingomyelin, in which choline is present in the specified composition in an amount of from 50 to 1000 mg/100 g, while sphingomyelin is present in the specified composition in an amount of from 300 mg/kg to 2 .5 g / kg, while folic acid is present in the specified composition in an amount of 110 to 500 μg / 100 g, and vitamin B12 is present in the specified composition in an amount of 5 to 10 μg / 100 g, and iron is present in the specified composition in an amount of 5 to 40 mg/100 g, and a fatty acid compound containing DHA is present in the composition in an amount of 60 to 350 mg/100 g, and a fatty acid compound containing arachidonic acid (ARA) is present in the composition in an amount of 60 to 350 mg/100 g dry weight of the composition, in which the entire weight is indicated based on the dry weight of the composition, wherein said fatty acid compound is monoacylglycerol and/or diacylglycerol and/or triacylglycerol, and wherein said composition is a composition selected from the group consisting of infant formula, complementary milk, an infant composition that is intended to be added or diluted with human breast milk, and a food product intended for consumption by an infant and/or child, or alone , or in combination with human breast milk, where the infant is an infant under 12 months of age and the child is a human between 1 and 18 years of age and preferably between 1 and 5 years of age.

Images