RU2810236C2 - Composition containing breast milk oligosaccharides for use in improving, strengthening, stimulating or modulating gambergic function in central nervous system - Google Patents

Abstract

FIELD: nutritional compositions.

SUBSTANCE: invention relates to a nutritional composition containing breast milk oligosaccharide (HMO). A nutritional composition containing human milk oligosaccharide (HMO) is used to improve, enhance, stimulate or modulate GABAergic function in the central nervous system (CNS) of a mammal, preferably a human infant or young child from birth to 7 years of age.

EFFECT: nutritional composition containing human milk oligosaccharide (HMO) is also used to prevent or compensate for attention deficit hyperactivity disorder, autism spectrum disorder, as well as schizophrenia in subjects in need thereof.

14 cl, 2 dwg, 2 tbl, 1 ex

Description

Prerequisites for creating the invention

The present invention generally relates to the field of nervous system health, nervous system protection and nervous system development. In particular, the invention relates to a composition for use in supporting neuronal development, in particular improving, enhancing, stimulating or modulating function related to or affecting the neurotransmitter gamma-aminobutyric acid (GABA) in the central nervous system (CNS) and to other associated cognitive benefits, particularly in infants and young children, preferably in the target group of (preterm or term) infants or young children from birth to 7 years.

More particularly, the invention relates to the administration of human milk oligosaccharides (HMO), optionally in combination with additional prebiotic oligosaccharides, in particular fructooligosaccharides (FOS), or oligofructose (OF) and/or bovine milk oligosaccharides (BMO) to improve, enhance, stimulate or modulation of GABAergic function in the central nervous system, particularly the filtering of information or the ability to focus or concentrate on mental or physical activity.

The central nervous system, and in particular the brain, controls cognitive functions. The cerebral cortex, which is the outermost layer of neural tissue in the mammalian brain, plays a key role in attention, perceptual perception, higher cognitive function (executive function), and integration of information from afferent inputs.

The development and maturation of the CNS is an extremely complex biological phenomenon that involves many physiological processes, including, for example, the growth and differentiation of neurons and glial cells, neuronal guidance and branching, and the formation of interneuronal interactions (nerve signals) through axonal growth and synaptogenesis.

Neuronal plasticity, defined as the brain's ability to continually adapt function and structural organization to changing demands, is important for neural maturation and mature function. It is required for the proper functioning of the brain and is necessary for the processes of cognition, learning and memory. Some of the neuronal markers, including proteins and neurotrophic factors such as brain-derived neurotrophic factor (BDNF), that are necessary for or at least involved in these physiological processes have been identified and studied in the literature [Huang, E. J., Reichardt, L. F. (2001) ; Neurotrophins: Roles in Neuronal Development and Function, Annu. Rev. Neurosci., 24: 677-736]; [Musumeci, G., Minichiello, L. (2011); BDNF-TrkB signaling in fear learning: from genetics to neural networks, Rev. Neurosci., 22(3):303-15]; [Xiao, J. et al. (2009); The role of neurotrophins in the regulation of myelin development, Neurosignals, 17: 265-276] and [Von Bohlen, Halbach, O. (2011); Immunohistological markers for proliferative events, gliogenesis, and neurogenesis within the adult hippocampus, Cell Tissue Res., 345(1):1-19].

The CNS begins to develop immediately after conception, then during pregnancy, and continues to develop until early adulthood. Specifically, structural maturation predominantly occurs before birth, whereas functional network maturation predominantly occurs after birth. In human embryos, the cerebral cortex develops quite late and over a long period of time.

During the prenatal period, humans experience a peak in neuronal/brain maturation and growth from the 30th week of pregnancy.

The development of GABAergic functions, including the ability to filter information, particularly sensory information such as visual cues, is a very important step in the development of cognitive functions in mammals, particularly in infants and young children. Although this development and improvement of GABAergic functions is of particular importance during the first months/years of life (when neuronal plasticity is greatest), it can also affect older subjects, adolescents and adults, as well as elderly or diseased subjects.

Premature babies, by definition, are born with immature brains, and they actually show only basic electrical activity in the primary sensory areas of the cerebral cortex, that is, those areas responsible for touch, vision and hearing, as well as in the primary motor areas cerebral cortex. In these children, postnatal gradual brain maturation is necessary to compensate for the state of immaturity of the brain at birth, and such compensatory maturation is especially important for the more complex part of the brain that mediates much of the emotional, social and cognitive maturation in the first few years of life [Lubsen, J et al. (2011); Microstructural and functional connectivity in the developing preterm brain, Seminars in Perinatology, 35, 34-43].

Premature babies are born at a time that is very important for the structural and functional development and maturation of the brain, and thus they miss the stage of intrauterine brain development. After birth, they are at risk of pathological conditions, including hemorrhagic and hypoxic-ischemic brain lesions, and at risk of developmental problems in later life, including cognitive impairment. This risk appears to be higher the more prematurely babies are born and the lower their birth weight. Cognitive impairments associated with lower IQ, decreased attention and short-term memory, and problems with executive functions may persist into school and adolescence [Talge, N. et al. (2010). Late-Preterm Birth and its Association with Cognitive and Socioemotional Outcomes at 6 Years of Age. Pediatrics, 126, 1124-1131; van Baar, A., et al. (2009). Functioning at school age of moderately preterm children born at 32 to 36 weeks' gestational age. Pediatrics, 124, 251-257; Farooqi, A et al. (2011). Impact at age 11 years of major neonatal morbidities in children born extremely preterm. Pediatrics, 127, e1247-1257; Nosarti, C. et al. (2010). Neurodevelopmental outcomes of preterm birth. Cambridge: Cambridge University Press].

In general, CNS immaturity or delayed CNS maturation can be observed in infants such as:

preterm infants, low birth weight infants (<2500 g), very low and extremely low birth weight infants (<1500 g), extremely low birth weight infants (<1000 g), and small for gestational age infants [ Allen, M.C. (2008); Neurodevelopmental outcomes of preterm infants, Curr. Opin Neurol., 21(2): 123-8].

Premature or full-term infants with intrauterine growth restriction (IUGR) that occurred as a result of any adverse events during pregnancy (maternal smoking, maternal medication, poor placental quality, placental abnormality, poor maternal and fetal nutrition, excessive stress/ maternal anxiety, etc.); [Gregory, A. et al. (2008); Intrauterine Growth Restriction Affects the Preterm Infant's Hippocampus, Pediatric Research, 63(4): 438–443].

Any newborn child and infant with neurodevelopmental delay as a result of, for example, hypoxemia (ischemia) at birth, postnatal complications, postnatal steroid treatment or any other adverse event (see, for example, Barrett, R.D. et al. (2007); Destruction and reconstruction: hypoxia and the developing brain, Birth Defects Res. C. Embryo Today, 81: 163-76).

These infants exhibit cognitive dysfunction, as well as dysfunction in growth and development, indicating insufficient “compensation” for the process of neurodevelopment. Immaturity or delayed maturation of the cerebral cortex can lead to slow and/or impaired ability to learn, synthesize information, process afferent impulses, lose or underdevelop higher functions of thinking, executive function, concentration, attention, motor skills, and language. This can lead to behavioral problems, pathological decline in intelligence and therefore pathological decline in mental performance.

It has generally been noted that breastfeeding preterm infants may result in improved neuronal development compared with formula feeding. (See, for example: Rozé et al. The apparent breastfeeding paradox in very preterm infants: relationship between breast feeding, early weight gain and neurodevelopment based on results from two cohorts, EPIPAGE and LIFT. BMJ Open 2012;2:e000834.)

It has been noted that in healthy populations, infant breastfeeding may lead to improved cognitive function and educational ability in adulthood compared with formula feeding. (See, for example: Victoria et al. Association between breastfeeding and intelligence, educational attainment, and income at 30 years of age: a prospective birth cohort study from Brazil. Lancet 2015.)

According to the inventors, this generally indicates that certain nutrients present in human breast milk may be missing from traditional/standard formula or being delivered in suboptimal amounts. There is a need to identify the key differences between conventional formula and human breast milk and adapt formula accordingly.

Behavioral and neuronal developmental disorders associated with delayed cortical maturation, particularly abnormal GABAergic function, include disorders such as attention deficit hyperactivity disorder, autism spectrum disorder, and schizophrenia.

An individual's cognitive function can be measured using clinical tests that are age-specific; Pediatricians and child development specialists are familiar with many such tests. For infants and infants, there are screening and neurodevelopmental tests, such as the Bayley Scale of Infant Development (BSID), the Braselton Infant Behavior Scale, the Neuropsychological Development Assessment (NEPSY), and the Griffiths Mental Development Inventory. For preschool and/or school-age children, cognitive ability tests include the Peabody Picture Vocabulary Test (PPVT), Test of Nonverbal Intelligence 2 (TONI-2), Wechsler Preschool and Junior Scale of Intelligence (WPPSI), and Raven's Colored Progressive Matrices ( CPM).

Nutrition is known to play an important role in the maturation of neurons in the brain (reviewed by Huppi, P. S. (2008); Nutrition for the Brain, Pediatric Research, 63(3): 229-231 and Cusik and Georgieff (2016); The Role of Nutrition in Brain Development: The Golden Opportunity of the “First 1000 Days,” Journal of Pediatrics, 175:16-21).

The consequences of poor nutrition may be irreversible and may include reduced cognitive development, memory and learning abilities and hence future economic productivity (see, for example, Horton, R; (2008) The Lancet, Vol. 371, Issue 9608, page 179; Laus, M. F. et al. (2011); Early postnatal protein-calorie malnutrition and cognition: a review of human and animal studies, Int. J. Environ. Res. Public Health., 8(2): 590 -612).

Although mother's breast milk is known to provide the best nutritional support to the developing brain, when breastfeeding is not possible, there is a need to provide artificial nutritional compositions (such as infant formula or formula) that induce improvement or promote the development of optimal cognitive functions.

Thus, oral administration is a suitable route to positively influence neurodevelopment to promote optimal development of cognitive function, memory and mental abilities in preterm or full-term newborns, infants, toddlers, older or adolescent children or young animals.

However, there is little known and reliable data on the ability of nutritious diets or nutritional compositions to influence the development or stimulation of GABAergic functions, and in particular in infants and young children.

Thus, there is a need to stimulate and support the healthy development of cognitive function in general and to improve, enhance, stimulate or modulate GABAergic function in the CNS, particularly the filtering of information or the ability to focus or concentrate on mental or physical activity.

There is a need to prevent, prevent or compensate for attention deficit hyperactivity disorder, autism spectrum disorders, as well as schizophrenia, especially in subjects in need thereof, and especially by activating or modulating GABAergic functions in the CNS.

There is a need to stimulate the development or improve such functions in young subjects, in particular in infants and young children, such as infants or young children, premature or not, from birth to 7 years.

There is a need to provide such nutritional intervention and/or preventative nutritional intervention in a form that is acceptable to a population of subjects, particularly to those individuals in that population who are most vulnerable or most in need. In addition, there is a need to avoid inducing adverse events, side effects or negative effects in such a population.

There is a need to provide a population of subjects in need with such solutions in the simplest and most cost-effective manner, preferably not through the use of specific ingredients considered drugs or medications, but preferably as part of the diet.

The present invention applies to all mammals, including animals and humans, and in particular to infants and young children or young pets, in whom brain plasticity is greatest. The preferred target population is human (preterm or term) infants or young children/pets aged birth to 7 years.

Summary of the invention

The present inventors have unexpectedly discovered that administration of a specific oligosaccharide or a mixture of specific oligosaccharides containing OGM, alone or in combination with OF and/or BMO, is particularly effective in improving, enhancing, stimulating or modulating GABAergic function in the CNS or information filtering or focusing ability or concentrate on mental or physical activity. The administration of these oligosaccharides can be carried out as part of a nutritional intervention or as part of a therapeutic intervention in subjects in need thereof.

Brief description of graphic materials

Fig. 1. The effect of different diets (OHM, BMO, OGM + BMO, OF, OF + OGM) in piglets on the expression of the GABAergic receptor GABBR1 in the hippocampus was shown. OGM, OF and BMO are described in the examples.

Fig. 2. The effect of different diets (OHM, BMO, OGM + BMO, OF, OF + OGM) in piglets on the expression of the GABAergic receptor GABRB2 in the hippocampus was shown. OGM, OF and BMO are described in the examples.

Detailed description of implementation examples

Definitions

As used herein, the following terms have the meanings given below.

The term "infant" means a child under 12 months of age.

The term "young child" means a child between the ages of one and seven years.

The use of singular forms when describing an element or component of the invention should not be limiting in relation to the number of instances (i.e. occurrences) of the element or component. Therefore, an element or component in the singular includes “one or at least one,” and the use of an element or component in the singular also includes the plural unless the number is expressly designated as singular.

The term "human milk oligosaccharide" is used as the abbreviation HMO, collectively refers to the oligosaccharides present in breast milk, and falls within the standard definition accepted by one skilled in the art. HGMs include, but are not limited to, fucosylated oligosaccharides (such as 2'-fucosyllactose, 3'fucosyllactose, difucosyllactose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucohexaose, lacto-N-difucohexaose I, fucosyllacto-N-hexaose, fucosyllacto-N-neohexaose I, fucosyllacto-N-neohexaose II, difucosyllacto-N-hexaose I, difucosyllacto-N-neohexaose I, difucosyllacto-N -neohexaose II, fucosyl-p-lacto-N-hexaose and any combination thereof), N-5 acetylated oligosaccharides (such as lacto-N-tetraose (LNT), N-neotetraose (LNnT) and any combination thereof) and sialylated oligosaccharides .

The term "bovine milk oligosaccharide" (abbreviated BMO) refers to the oligosaccharides present in cow's milk and falls within the standard definition accepted by one skilled in the art. The BMO mixture used in the context of the present invention can, for example, be obtained from bovine whey. Briefly, bovine whey ultrafiltrate, including oligosaccharides such as 3′- and 6′-sialyllactose and GOS, can be demineralized by a combination of electrodialysis and ion exchange as described in the art. BMOs may include, without limitation, fucosylated oligosaccharides (such as 2'-fucosyllactose, 3'fucosyllactose, difucosyllactose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V , lacto-N-fucohexaose, lacto-N-difucohexaose I, fucosyllacto-N-hexaose, fucosyllacto-N-neohexaose I, fucosyllacto-N-neohexaose II, difucosyllacto-N-hexaose I, difucosyllacto-N-neohexaose I, difucosyllacto- N-neohexaose II, fucosyl-p-lacto-N-hexaose and any combination thereof), N-5 acetylated oligosaccharides (such as lacto-N-tetraose (LNT), N-neotetraose (LNnT) and any combination thereof) and sialylated oligosaccharides.

As used herein, the term "oligofructose" (abbreviated OF) refers to a fructose oligomer (i.e., fructose oligosaccharide) having a degree of polymerization of 2 to 10, for example a degree of polymerization of 2 to 8. OF may also be referred to as fructose oligosaccharides, or fructooligosaccharides (abbreviated FOS), or short-chain fructooligosaccharides (abbreviated scFOS). As used herein, the terms OF, FOS and scFOS have the same meaning and may be used interchangeably.

Inulin containing long chain polymers is specifically excluded from this definition of OF. OF is distinguished from inulin by its degree of polymerization (inulin has much longer chains).

FOS/scFOS/OF are generally available commercially, for example under the trade name oligofructose ORAFTI from Beneo GmbH (Mannheim, Germany) (eg ingredient Orafti® P95).

The term "sialylated oligosaccharide" means an oligosaccharide containing one or more sialic acid residues.

The term "fucosylated oligosaccharide" means an oligosaccharide containing one or more fucose residues.

As used herein, the term “sn-2-palmitate” refers to palmitic acid at the sn-2 position of the triglyceride to which it is linked.

Gamma-aminobutyric acid, or “GABA,” is a well-known inhibitory neurotransmitter in the central nervous system and, through its function of inhibiting neuronal activity, is involved, for example, in the synchronization of a network of neurons in the hippocampus. The term "GABAergic" means "relating to or affecting the neurotransmitter GABA." For example, a synapse is GABAergic if it uses GABA as a neurotransmitter. In another example, a GABAergic neuron may produce GABA. In another example, a substance is GABAergic if it is capable of producing effects through interactions with the GABA system, such as by stimulating or blocking the transmission of nerve impulses. In another example, a GABAergic substance or GABAergic agent is any chemical substance capable of modifying the effects of GABA in the body or brain. Classes of GABAergic drugs include, but are not limited to, GABA receptor agonists, GABA receptor antagonists, and GABA reuptake inhibitors. A specific example of a GABA receptor is the GABA receptor type B (GABBR).

The term "GABAergic function in the central nervous system (CNS)" as used herein refers to any GABAergic function exerted by or in relation to any cell, part of a cell, cellular receptor, system or chain of cells contained in the CNS, including, without limitation, neurons and neuroglia such as astrocytes, oligodendrocytes, microglia, and ependymal cells, such as those using GABAergic neurotransmission, GABA transport, or GABA uptake. GABAergic neurotransmission is the basis for filtering incoming information to the CNS as it limits, reduces, or removes unwanted or unnecessary information. Thus, the specific GABAergic function in the CNS involves the functions of filtering information or the ability to focus or concentrate on mental or physical activity. The field has demonstrated many examples of GABAergic modulation of information filtering. For example, GABA content in the hippocampus was correlated with the ability to suppress reminder-triggered unwanted memory retrieval in humans (Schmitz et al. Nat Commun. 2017, 8(1):1311). In the optic roof of the zebrafish, GABAergic interneurons are essential for filtering spatial information, allowing the fish to properly hunt small prey (Del Bene et al. Science 2010, 330(6004):669-673). In addition, changes in GABA levels in the striatum through astrocyte uptake have been shown to modulate the information filtering properties of neurons (Goubard et al. J Physiol 2011, 589(9):2301-2319). Improvement, enhancement, stimulation or modulation of GABAergic functions in the CNS can be assessed, for example, by measuring the expression levels of known genes that are part of the GABAergic system, such as GABAergic receptor or transporter genes. For example, gene expression in the hippocampus may indicate improved, enhanced, stimulated, or modulated GABAergic function.

The term “nutritional composition” means a composition that is fed to a subject. The nutritional composition is typically for oral or intravenous administration and typically includes a lipid or fat source and a protein source. The nutritional composition is preferably a complete nutritional formula that meets all or most of the nutritional needs of a subject (eg, infant formula). Nutritional compositions contain food products.

The term “infant formula” means a food product specifically intended for use by infants during the first four to six months of life and which itself satisfies the nutritional needs of this category of persons (Article 1.2 of European Commission Directive 91/321/EEC of 14 May 1991 about infant formulas and formulas for complementary infants).

The term “infant formula” means a food product specifically intended for consumption by infants over four months of age and forming the main liquid component in the diet of this category of persons, which includes gradually increasing variety.

The term "infant formula" means a food product specifically intended for consumption by infants during the first four months of life.

Infant formula, infant formula and starter formula can be either in the form of a liquid, ready-to-use or concentrated, or in the form of a dry powder that can be reconstituted to form a formula after adding water. Such mixtures are well known in the art.

The term "baby food" means a food product specifically intended for consumption by infants during the first years of life.

The term "infant grain-based composition" means a food product specifically intended for consumption by infants during the first years of life.

The term “formula for children aged 1 to 3 years” refers to a milk-based drink adapted to the specific nutritional needs of young children.

The term "weaning period" refers to the period during which mother's milk is replaced by other foods in the infant's diet.

The term "artificial formula" means a mixture produced by chemical and/or biological means that may be chemically identical to the naturally occurring mixture found in mammalian milk.

The term “prebiotic” refers to a non-digestible carbohydrate that has a beneficial effect on the host by selectively stimulating the growth and/or activity of health-promoting bacteria, such as bifidobacteria, in the human colon (Gibson GR, Roberfroid MB. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics J Nutr 1995;125:1401-12).

The term “probiotic” means preparations of microbial cells or components of microbial cells that have a beneficial effect on the health or well-being of the host (Salminen S, Ouwehand A. Benno Y. et al. Probiotics: how should they be defined. Trends Food Sci. Technol 1999:10 107-10).

Unless otherwise stated, all percentages are by weight.

If quantities of ingredients are stated as weight of ingredient/weight of powdered nutritional composition, it is intended that the present invention also applies to the corresponding amount per liter, taking into account the dilution factor of the dry powdered nutritional composition of 130 g/L (or as otherwise specified in the dilution instructions).

Breast milk oligosaccharides

HMOs are collectively the third largest component of breast milk solids after lactose and fat. OGM typically consists of lactose at the reducing end with a carbohydrate core that often contains fucose or sialic acid at the non-reducing end. More than a hundred milk oligosaccharides have been isolated and characterized, but these represent only a very small fraction of the total number that remain to be characterized.

Infant formulas can be formulated for various purposes using HMO ingredients such as fucosylated oligosaccharides, particularly 2FL, lacto-N-tetraose, lacto-N-neotetraose or sialylated oligosaccharides.

Abbott Laboratories publication EP0975235B1 describes an artificial nutritional composition containing one or more human milk oligosaccharides, wherein the HMOs in the composition are selected from the group consisting of eight HMOs (3-fucosyllactose, lacto-N-fucopentaose III, lacto-N-fucopentaose II, difucosyllactose , 2'-fucosyllactose, lacto-N-fucopentaose I, lacto-N-neotetraose and lacto-N-fucopentaose V), wherein said composition is intended for normal, healthy infants, children, adults or individuals with special needs, for example related to certain pathological conditions. Publication EP0975235B1 states that, in general, oligosaccharides protect infants from viral and bacterial infections of the respiratory, gastrointestinal and genitourinary tracts.

In one embodiment, the composition of the present invention contains an HMO selected from the group consisting of fucosylated oligosaccharides such as 2'-fucosyllactose (2FL), sialylated oligosaccharides and/or N-acetyllactosamine such as lacto-N-neotetraose (LNnT) or lacto -N-tetraose (LNT), or combinations thereof. Experimental examples of compositions containing 2FL are presented herein.

N-acetyllactosamine

In some embodiments, the composition of the invention contains at least one N-acetyllactosamine. In other words, the composition according to the invention contains N-acetyllactosamine and/or an oligosaccharide containing N-acetyllactosamine. Suitable N-acetyllactosamine-containing oligosaccharides include lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT).

Thus, in a preferred embodiment, N-acetyllactosamine is preferably selected from the group consisting of lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT).

LNT and LNnT can be synthesized chemically by enzymatic transfer of saccharide units from donor moieties to acceptor moieties using glycosyltransferases, as described, for example, in US patent No. 5,288,637 and WO 96/10086. In an alternative embodiment, LNT and LNnT can be prepared by chemically converting ketohexoses (eg, fructose), either free or bound to an oligosaccharide (eg, lactulose), into N-acetylhexosamine or N-acetylhexosamine-containing oligosaccharide, as described in Wrodnigg, T.M. ; Stutz, A.E. (1999) Angew. Chem. Int. Ed. 38:827-828. The N-acetyllactosamine thus obtained can then be transferred to lactose as an acceptor moiety.

The composition according to the invention preferably contains from 0.1 to 3 g of N-acetyllactosamine per 100 g of composition based on dry matter weight. It preferably contains from 0.1 to 3 g LNnT per 100 g of composition based on dry matter weight.

In one embodiment, the nutritional composition according to the invention contains N-acetyllactosamine, preferably selected from the group consisting of lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT).

Sialylated oligosaccharides

The composition in accordance with the invention in some embodiments may contain one or more sialylated oligosaccharides.

Sialylated oligosaccharides include all sialylated OGMs, including 3'-sialyllactose and 6'-sialyllactose. Preferably, both 3'-sialyllactose and 6'-sialyllactose are present in said composition. In this embodiment, the ratio of 3'-sialyllactose to 6'-sialyllactose is preferably in the range of 100:1 to 1:100, more preferably 10:1 to 1:10, even more preferably 5:1 to 1:2.

The 3' and 6' forms of sialyllactose can be isolated from a natural source such as animal milk by chromatography or filtration. In an alternative embodiment, they can be produced by biotechnological means using specific sialyltransferases or sialidases, neuraminidases, either by enzyme-based fermentation (recombinant or natural enzymes), by chemical synthesis, or by microbiological fermentation. In the latter case, microorganisms can express their natural enzymes and substrates, or it is possible to create microorganisms that produce the corresponding substrates and enzymes. You can use cultures from microorganisms of the same species or mixed cultures. The formation of sialyloligosaccharides can be initiated by acceptor substrates, starting from any degree of polymerization (DP) from DP = 1 onwards. In an alternative embodiment, sialyllactoses can be produced by chemical synthesis from lactose and free N'-acetylneuraminic acid (sialic acid). Sialyl lactoses are also commercially available, for example from Kyowa Hakko Kogyo, Japan.

The composition according to the invention preferably contains from 0.05 to 10 g, more preferably from 0.1 to 5 g, even more preferably from 0.1 to 2 g sialylated oligosaccharide(s) per 100 g of composition in based on dry matter weight.

In one embodiment, the nutritional composition according to the invention contains a sialylated oligosaccharide, preferably selected from the group consisting of 3'-sialyllactose and 6'-sialyllactose. More preferably, said composition contains both 3'-sialyllactose and 6'-sialyllactose, the ratio of 3'-sialyllactose to 6'-sialyllactose preferably being in the range of 100:1 to 1:100, more preferably 10:1 to 1 : 10, even more preferably from 5: 1 to 1: 2.

Fucosylated oligosaccharide

The composition according to the present invention may contain one or more fucosylated oligosaccharides. Fucosylated oligosaccharides preferably consist of or contain 2'-fucosyllactose (2-FL).

The fucosylated oligosaccharide may be selected from the group consisting of 2'-fucosyllactose, 3-fucosyllactose, difucosyllactose (DiFL), lacto-N-fucopentaose (i.e., lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N -fucopentaose III and lacto-N-fucopentaose V), lacto-N-difucohexaose I, fucosyllacto-N-hexaose, difucosyllacto-N-hexaose I and difucosyllacto-N-neohexaose II. A particularly preferred fucosylated oligosaccharide is 2'-fucosyllactose (2-FL) or DiFL.

The fucosylated oligosaccharide can be isolated from a natural source, such as animal milk, by chromatography or filtration methods. In an alternative embodiment, it can be produced by biotechnological means using specific fucosyltransferases and/or fucosidase, either by using an enzyme-based fermentation method (recombinant or natural enzymes) or a microbiological fermentation method. In the latter case, either microorganisms can express their natural enzymes and substrates, or it is possible to create microorganisms that produce the corresponding substrates and enzymes. Cultures from microorganisms of the same species and/or mixed cultures can be used. The formation of fucosylated oligosaccharide can be initiated by acceptor substrates, starting from any degree of polymerization (DP) from DP = 1 onwards. In an alternative embodiment, fucosylated oligosaccharides can be prepared from lactose and free fucose using chemical synthesis. Fucosylated oligosaccharides are also commercially available, for example from Kyowa Hakko Kogyo, Japan.

The composition according to the invention preferably contains from 0.1 to 3 g of fucosylated oligosaccharide(s) per 100 g of composition on a dry matter basis, most preferably 2FL.

In one embodiment, the nutritional composition of the invention contains a fucosylated oligosaccharide, preferably selected from the group consisting of 2'-fucosyllactose, 3-fucosyllactose, difucosyllactose, lacto-N-fucopentaose (i.e., lacto-N-fucopentaose I, lacto-N- fucopentaose II, lacto-N-fucopentaose III and lacto-N-fucopentaose V), lacto-N-difucohexaose I, fucosyllacto-N-hexaose, difucosyllacto-N-hexaose I and difucosyllacto-N-neohexaose II, and preferably the fucosylated oligosaccharide is is 2'-fucosyllactose (2-FL).

Additional prebiotics

In addition to the basic oligosaccharides that form the subject of the present patent, the composition of the invention may further contain at least one or one additional prebiotic, usually in an amount of from 0.3 to 10% by weight of the composition.

Prebiotics are generally indigestible in the sense that they are not broken down or absorbed in the stomach or small intestine and thus remain in their native form as they pass into the large intestine, where they are selectively fermented by beneficial bacteria.

The composition in accordance with the invention may contain, in some embodiments, oligofructose (OF). An example of such an OF is the commercial ingredient ORAFTI® produced by Beneo GmbH (Mannheim, Germany).

In some embodiments, the prebiotics of the composition of the invention contain other fructooligosaccharides (FOS) and/or galactooligosaccharides (GOS). A combination of prebiotics, such as 90% GOS and 10% short-chain fructo-oligosaccharides, can be used, for example in the form of a product sold by BENEO-Orafti under the brand name Orafti® oligofructose (see http://www.beneo-orafti.com/Our-Products/ Oligofructose) (formerly Raftilose®), or 10% inulin, for example in the form of a product sold by BENEO-Orafti under the brand name Orafti® inulin (see http://www.beneo-orafti.com/Our-Products/Inulin) ( formerly Raftiline®). Another prebiotic combination is 70% short-chain fructo-oligosaccharides and 30% inulin, which is a product sold by BENEO-Orafti® under the brand name Prebio 1.

In one embodiment, the nutritional composition in accordance with the invention contains a prebiotic selected from the list of cow's milk oligosaccharides, inulin, xylooligosaccharides, polydextrose or any combination thereof.

In one embodiment, the nutritional composition of the invention contains bovine milk oligosaccharide (BMO). BMO may contain oligosaccharides that are N-acetylated oligosaccharide, galactooligosaccharide, sialylated oligosaccharide, fucosylated oligosaccharide, or a combination thereof.

Probiotics

The composition of the invention may further contain at least one probiotic. Non-limiting examples of probiotics include: Bifidobacterium, Lactobacillus, Lactococcus, Enterococcus, Streptococcus, Kluyveromyces, Saccharoymces, Candida, particularly those 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, Lactococcus lactis, Enterococcus faecium, Saccharomyces cerevisiae, Saccharomyces boulardii or their mixtures 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), Bifidobacterium breve strain A, Lactobacillus paracasei NCC2461 (CNCM 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 their combinations.

In one embodiment, said probiotic is a probiotic bacterial strain, preferably bifidobacteria and/or lactobacilli. Suitable probiotic bacterial strains include Lactobacillus rhamnosus ATCC 53103, manufactured by Valio Oy, Finland, under the trade name LGG; Lactobacillus rhamnosus CGMCC 1.3724, Lactobacillus paracasei CNCM I-2116, Lactobacillus johnsonii CNCM I-1225, Streptococcus salivarius DSM 13084, sold by BLIS Technologies Limited, New Zealand, under the name KI2; Bifidobacterium lactis CNCM 1-3446, sold among others by Christian Hansen, Denmark, under the trade name Bb 12; Bifidobacterium longum ATCC BAA-999 sold by Morinaga Milk Industry Co. Ltd., Japan, under the trademark BB536; Bifidobacterium breve, sold by Danisco under the trade name Bb-03; Bifidobacterium breve, sold by Morinaga under the trade name M-16V; Bifidobacterium infantis, sold by Procter & Gambie Co. under the trademark Bifantis; and Bifidobacterium breve, sold by Institut Rosell (Lallemand) under the trademark R0070.

The composition according to the invention preferably contains from 10e3 to 10e12 CFU of the probiotic bacterial strain, more preferably from 10e7 to 10e12 CFU per gram of composition on a dry matter basis.

In one embodiment, the nutritional composition contains a probiotic bacterial strain selected from the list consisting of Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus lactis, Lact obacillus delbrueckii, Lactobacillus helveticus, Lactobacillus bulgari, Lactococcus lactis, Lactococcus diacetylactis, Lactococcus cremoris, Streptococcus salivarius, Streptococcus thermophilus, Bifidobacterium lactis, Bifidobacterium animalis, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis or Bifidobacterium adolescentis or any mixture thereof.

Target population

In one embodiment, the present invention is directed to/is intended for mammals. The mammals are preferably human or domestic animals such as a dog or cat.

In a more preferred embodiment, the present invention is directed to young mammals, such as young adults, such as infants (eg, 0 to 6 months or 0 to 12 months) or young children (eg, 1 to 3 years or 1 to 7 years) or for young dogs (eg puppies) or young cats. In an even more preferred embodiment, the mammal is an infant or young child. Without wishing to be bound by any theory, young mammals have high brain plasticity and brain development (or brain connectivity) and benefit most from the present invention. It is further contemplated that the present invention may target specific nutritional intervention windows (e.g., children aged 6 months to 3 years, 3 months to 18 months), subpopulations with specific needs (vulnerable young mammals; senile and semisenile mammals) or in the recovery phase. In one embodiment, the present invention is substantially directed to subjects in need of prevention, prophylaxis, or compensation for attention deficit hyperactivity disorder, autism spectrum disorder, as well as schizophrenia (eg, mammals born prematurely or with suboptimal growth or development). In one embodiment, the present invention is substantially directed to subjects suffering from a condition or disease associated with GABAergic functions, in particular mental or neurological disorders associated with GABAergic functions, such as filtering information or the ability to focus or concentrate on mental or physical activity. Such a disease may include mental or neurological disorders such as attention deficit hyperactivity disorder, autism spectrum disorder, and schizophrenia.

In accordance with a preferred embodiment, the composition in accordance with the invention is intended for use in healthy infants and/or healthy young children. In one embodiment, the invention is of particular relevance to frail infants, premature infants, and/or infants born with abnormally low birth weight, and/or infants who have suffered from intrauterine growth retardation. The preferred period of use is/are during the period(s) of most rapid memory development and/or development of brain connections.

The composition of the invention is intended for infants and/or young children aged 7 years or less, preferably 3 years of age or less, most preferably 1 year of age or less. In one embodiment, the composition is intended for infants 6 months of age or less. In embodiments of the invention, the composition is used during the first 6 months of life, the first 1 year of life, the first 3 years of life, the first 7 years of life, and/or during the recovery period from an illness or developmental disability.

Nutritional composition

The nutritional composition according to the invention is preferably an artificial nutritional composition. The composition of the invention may be, for example, a starter formula, an infant formula, an infant food, a cereal-based infant composition, a baby formula, or a milk formula for children aged 1 to 3 years, and said composition is preferably a starter formula. The composition according to the invention may also be intended for use before and/or during the weaning period. In one embodiment, the nutritional composition may be a complete nutritional composition or supplement for aged, elderly or vulnerable individuals.

The composition in accordance with the invention may be a complete composition providing 100% or most of the nutritional requirements of the target population (for example, in terms of caloric requirements; or in terms of vitamin or mineral requirements, or in terms of protein requirements , lipids or carbohydrates). In an alternative embodiment, the composition of the invention may be a supplement that is in addition to the consumer's normal diet. However, in this case, the dosage and total consumption of the composition are adjusted to provide the claimed information filtering benefit (eg, proportional to the caloric load and caloric needs of the subject).

Use of the composition of the invention may include cases where the composition is a supplement, preferably offered in unit dose form. In one embodiment, the composition is a human breastfeeding supplement.

The composition may be in the form of a powder composition, for example, intended to be diluted with water, or mixed with milk (eg, human breast milk), or consumed as a powder. In one embodiment, the composition of the invention is presented in liquid form; either ready for use or intended to be diluted in water or mixed with milk (such as human breast milk).

The composition of the present invention may also contain a protein source, preferably in an amount of less than 2.5 g per 100 kcal or less than 2.0 g per 100 kcal, even more preferably in an amount of less than 1.8 g per 100 kcal. In one embodiment, the protein content is less than 1.6 g/100 kcal. The type of protein is not considered critical to the present invention, provided that the minimum requirements for essential amino acid content are met and satisfactory growth is achieved. Thus, protein sources based on whey, casein and mixtures thereof, as well as soy-based protein sources can be used. With respect to whey proteins, the protein source may be based on acid whey or sweet whey or mixtures thereof and may include alpha-lactalbumin and beta-lactoglobulin in any desired proportions.

The composition in accordance with the present invention essentially contains a source of carbohydrates. This is particularly preferred when the nutritional composition of the invention is an infant formula. In this case, any carbohydrate source commonly found in infant formulas may be used, such as lactose, sucrose, maltodextrin, starch, and mixtures thereof, although the preferred carbohydrate source is lactose.

The composition in accordance with the present invention essentially contains a source of lipids. This is especially important if the nutritional composition of the present invention is an infant formula. In this case, the source of lipids may be any lipid or fat that is suitable for use in infant formulas. Preferred fat sources include palm olein, high oleic acid sunflower oil and high oleic acid safflower oil. In addition, the essential fatty acids linolenic acid and α-linolenic acid, as well as small amounts of oils containing large amounts of arachidonic acid and docosahexaenoic acid in a formulated form, such as fish oils or microbial oils, can be added. The fat source preferably has a ratio of n-6 to n-3 fatty acids from about 5:1 to about 15:1; for example, from about 8:1 to about 10:1.

The composition of the invention also preferably contains all the vitamins and minerals known to be essential in the daily diet in nutritionally significant amounts. Some vitamins and minerals have minimum requirements. Examples of minerals, vitamins and other nutrients that are optionally present in the composition of the invention include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin E, vitamin K, vitamin C, vitamin D, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chlorine, potassium, sodium, selenium, chromium, molybdenum, taurine and L-carnitine. Minerals are usually added in the form of salts. The presence and amounts of specific minerals and other vitamins may vary depending on the intended population.

If necessary, the composition of the invention may contain emulsifiers and stabilizers such as soy, lecithin, citric acid mono- and diglyceride esters and the like.

The composition of the invention may also contain other substances that may provide beneficial effects, such as lactoferrin, nucleotides, nucleosides and the like.

In one embodiment, the composition of the invention, especially in the form of an infant formula, contains from about 1.8 to about 2.2 g total protein per 100 kcal, for example from about 1.8 to about 2.1 g or from about 1. 9 to about 2.1 g of protein per 100 kcal, with optionally about 0.3 to about 0.4 g/100 kcal of protein being alpha-lactalbumin. The infant formula and weaning formula of the present invention may be in the form of a ready-to-drink liquid, or may be a liquid concentrate or powder formula that can be reconstituted into a ready-to-drink liquid by adding some water to obtain the formula. children, which is the subject of the present invention, includes all ingredients required by US or EU law, including, without limitation, certain vitamins, minerals and essential amino acids. It may also include nucleotides such as CMP, UMP, AMP, GMP and IMP, lutein, zeaxanthin and other ingredients known in the art.

In one embodiment of the invention, the nutritional composition is a food for pets (eg, dogs or cats, or young dogs or young cats).

Effect(s) and use of the composition of the invention

The present invention relates to improving, enhancing, stimulating or modulating GABAergic function in the CNS of a subject. This may include improving, enhancing, stimulating or modulating the filtering of information or the ability to focus or concentrate on mental or physical activity.

Without being bound by any theory, it is believed that in one aspect of the invention, such improvement, improvement and/or activation is related to the effect of the oligosaccharide of the invention on the intestinal microbiota. Oligosaccharides such as OGM (eg, 2FL or LNnT), BMO or OF are only broken down to a small extent in the small intestine, thus leaving most of them available for fermentation by the microbiota in the colon. Indeed, OGM (e.g., 2FL or LNnT), BMO, or OF have been shown to activate the growth of intestinal bifidobacteria, as reflected by increased levels of bifidobacteria in the stool of infants fed formulas supplemented with OGM-containing oligosaccharides (e.g., 2FL or LNnT ), BMO or OF. Both oligosaccharides and probiotics appear to be associated with beneficial changes in GABA receptor expression in the brain. Without being limited by any theory, due to the high presence of GABA in some bacterial species (e.g., Lactobacillus and Bifidobacterium), such probiotic species may act as a carrier medium for the delivery of GABA, and their consumption may have local effects on the gastrointestinal system. which ultimately modulates host neurophysiology (Lyte, BioEssays 2011, 33, 574–581). This connection is even stronger when we consider that both prokaryotes and eukaryotes synthesize GABA through decarboxylation of glutamate by glutamate decarboxylase (GAD), and analyzes from the Human Microbiome Project suggest that genes encoding GAD are present in the human microbiome (Mazzoli, R., Pessione, E. (2016). Front. Microbiol. 7, 1934; Pokusaeva et al., Neurogastroenterol. Motil. 2017, 29(1) doi:10.1111/nmo.12904).

Briefly, the present nutritional composition can be used to improve, enhance, stimulate or modulate GABAergic function through effects on the gut microbiota and the gut-brain axis, which has been shown to influence many aspects of brain function. It may further be envisaged that improvement, enhancement, stimulation or modulation of GABAergic function may be associated with an increase in the concentration of sialic acid (Neu5Ac) in the brain of the subject, mediated by various metabolic pathways influenced by the gut microbiota. Without being limited by theory, in one embodiment, HGM acts synergistically with the probiotic and endogenous microbiota to best influence such metabolic pathways.

In another aspect, the effect underlying the present invention may be associated with altered expression of GABAergic genes in the subject's CNS, in particular GABA receptors or GABA transporters. In one embodiment, the change refers to measured increased or decreased gene expression levels at the mRNA level or at the protein level compared to a control. In one embodiment, gene expression refers to the expression of a gene in the hippocampus of a subject.

In another aspect, the effect underlying the invention may be associated with improving neuronal plasticity in the brain of the subject and/or improving neuronal development, neurogenesis, axonal sprouting, myelination and/or maturation of the brain of the subject (without limitation imposed by any or theory).

GABAergic functions, and in particular the ability to filter information or focus on mental or physical activity, represent important aspects of brain function and development and are critical to a subject's overall cognitive development. They influence, in particular, the processing, classification and control of external signals, the ability to learn, as well as spatial orientation.

In one embodiment of the present invention, GABAergic function is the ability to filter information, such as incoming sensory information. Information filtering is extremely important for social and cognitive development, especially in young subjects. In one embodiment of the present invention, GABAergic function is the ability to focus or concentrate on mental or physical activity.

Thus, in one aspect, the present invention relates to the use (or is determined by the use) of a nutritional composition containing OGM to improve, enhance, stimulate or modulate GABAergic function in the CNS, in particular the ability to filter information or focus or concentrate on mental or physical activity in mammal.

In one aspect, the present invention relates to the use of (or is defined by the use of) a nutritional composition described herein containing HGM for the prevention, prophylaxis or compensation of attention deficit hyperactivity disorder, autism spectrum disorder, as well as schizophrenia, especially in subjects in need thereof. and especially by stimulating or modulating GABAergic functions in the CNS.

In one aspect, the present invention relates to the use (or is determined by the use) of a nutritional composition described herein containing OGM for the improvement, enhancement or modulation of attention deficit hyperactivity disorder, autism spectrum disorder, and schizophrenia, and in particular by stimulating or modulating GABAergic functions in the central nervous system.

In one aspect, the present invention relates to the use (or is determined by the use) of a nutritional composition described herein containing OGM to improve, enhance or modulate GABAergic function in the CNS, particularly information filtering or the ability to focus or concentrate on mental or physical activity.

In one embodiment, the present invention relates to the use of (or is determined by the use of) a nutritional composition described herein, wherein said composition is an infant formula, infant formula, breast milk fortifier, infant formula, or infant food. pets.

In another aspect, the invention relates to OGM (or is defined as OGM) for use in improving, enhancing, stimulating or modulating GABAergic function in the CNS, particularly the ability to filter information or focus or concentrate on mental or physical activity, in a subject in need thereof. In a specific embodiment, the subject is a mammal, wherein said mammal is preferably a human, cat or dog, and wherein said human is preferably an infant or young child.

In another aspect, the invention relates to (or is defined as) a nutritional composition containing HGM for use in improving, enhancing, stimulating or modulating GABAergic function in the CNS, particularly the ability to filter information or focus or concentrate on mental or physical activity in a subject in need thereof. . In a specific embodiment, the subject is a mammal, wherein said mammal is preferably a young mammal, a human, a dog or a cat, more preferably a young human, a dog or a cat, most preferably an infant or young child. The nutritional composition may also contain one or more optional ingredients described herein.

In another aspect, the present invention relates to the use (or is defined by the use) of OGM in the manufacture of a nutritional composition for improving, enhancing, stimulating or modulating GABAergic function in the CNS, in particular the ability to filter information or focus or concentrate on mental or physical activity in a subject in need thereof. . In a specific embodiment, the subject is a mammal, wherein said mammal is preferably a young mammal, a human, a dog or a cat, more preferably a young human, a dog or a cat, most preferably an infant or young child.

Dosage

OGM. In one embodiment, the total amount of OGM is from 0.01 to 50 g/L, from 0.1 to 10 g/L, from 0.3 to 5 g/L, or from 0.5 to 1 g/L, or 0.25, or 0.5, or 1, or 1.5, or 2 g/l.

BMO. In one embodiment, the total amount of BMO is 0.01 to 50 g/L, 0.1 to 10 g/L, 0.3 to 5 g/L, or 0.5 to 1 g/L, or 0.25, or 0.5, or 1, or 1.5, or 2 g/l.

Fructooligosaccharide/FOS/OF. When available, the nutritional composition of the present invention may contain from 0.1 to 20 g of oligofructose (OF) per 100 g of composition on a dry matter basis, for example from 1 to 6 g or from 3 to 5 g of oligofructose (OF) per 100 g of composition based on dry matter weight.

One embodiment of the invention of the nutritional composition contains an amount of fructose oligosaccharide in the following ranges or amounts:

from 0.1 to 20 g/l, or from 0.5 to 10 g/l, or from 1 to 8 g/l, or from 2 to 6 g/l, or 1.5 g/l, or 3 g /l, or 5 g/l of the nutritional composition when the composition is in ready-to-use liquid form, or

from 0.1 to 20 g/l, or from 0.5 to 10 g/l, or from 1 to 8 g/l, or from 2 to 6 g/l, or 1.5 g/l, or 3 g /L, or 5 g/L (liquid reconstituted form) when the composition is in powder form and is intended to be reconstituted to form a reconstituted liquid form, or

the same as above multiplied by 2, 5, 10, 20, 50 or 100 when the nutritional composition is in the form of a concentrated composition intended to be diluted (2, 5, 10, 20, 50 or 100 times, respectively) by water or human breast milk or intended for direct use in concentrated form, or

from 0.4 g to 15 g/100 g of powder nutritional composition, or from 0.8 to 10 g/100 g, or from 1 to 6 g/100 g, or from 2 to 5 g/100 g, or from 2 ,1 to 4 g/100 g or 1.2 g/100 g or 2.3 g/100 g or 3.8 g/100 g or 4 g/100 g or 6 g/100 g powdered nutrition composition if the nutritional composition is in the form of a dry powder.

In one embodiment, the OF content may be from 0.07 g to 3 g/100 kcal of the powdered nutritional composition, or from 0.1 to 2 g/100 kcal, or from 0.4 to 1.5 g/100 kcal, or from 0.45 to 1 g/100 kcal, or from 0.45 to 0.75 g/100 kcal, or 0.3 g/100 kcal, or 0.4 g/100 kcal, or 0.5 g/100 kcal, or 0.75 g/100 kcal, or 1 g/100 kcal of the powdered nutritional composition if the nutritional composition is in the form of a dry powder.

2FL. The nutritional composition of the present invention may contain from 0.02 to 10 g of 2FL per 100 g of composition based on dry matter weight, for example from 0.2 to 0.5 g or from 0.3 to 5 g of 2FL per 100 g of composition based on per dry matter weight.

In one embodiment, the nutritional composition contains 2FL in the following ranges or amounts:

from 0.05 to 20 g/l, or from 0.1 to 5 g/l, or from 0.2 to 3 g/l, or from 0.1 to 2 g/l, or from 0.25 g/ l to 1 g/l, or 0.25 g/l, or 1 g/l of the nutritional composition when the composition is in ready-to-use liquid form, or

from 0.05 to 20 g/l, or from 0.1 to 5 g/l, or from 0.2 to 3 g/l, or from 0.1 to 2 g/l, or from 0.25 g/ l to 1 g/l, or 0.25 g/l, or 1 g/l (liquid reconstituted form), when the composition is in powder form and is intended to be reconstituted to form a reconstituted liquid form, or

the same as above multiplied by 2, 5, 10, 20, 50 or 100 when the nutritional composition is in the form of a concentrated composition intended to be diluted (2, 5, 10, 20, 50 or 100 times, respectively) by water or human breast milk or intended for direct use in concentrated form, or

from 0.04 g to 1.5 g/100 g of powdered nutritional composition, or from 0.08 to 1.2 g/100 g, or from 0.1 to 1 g/100 g, or from 0.2 to 0 .8 g/100 g, or 0.2 g/100 g, or 0.4 g/100 g, or 0.8 g/100 g, or 1 g/100 g, or 1 g/100 g powdered nutritional composition , if the nutritional composition is in the form of a dry powder.

In one embodiment, the 2FL content may be from 0.01 g to 0.3 g/100 kcal of the powdered nutritional composition, or from 0.02 to 0.2 g/100 kcal, or from 0.04 to 0.15 g/ 100 kcal, or 0.02 g/100 kcal, or 0.04 g/100 kcal, or 0.07 g/100 kcal, or 0.15 g/100 kcal, or 0.3 g/100 kcal powder nutritional composition , if the nutritional composition is in the form of a dry powder.

LNnT. In one embodiment, the nutritional composition may contain from 0.01 to 1 g LNnT per 100 g of composition on a dry matter basis, such as 0.1 to 0.25 g or 0.15 to 0.5 g LNnT per 100 g composition based on dry matter weight.

In one embodiment of the invention, the nutritional composition contains an amount of LNnT in the following ranges or values:

from 0.02 to 10 g/l, or from 0.05 to 2.5 g/l, or from 0.1 to 1.5 g/l, or from 0.05 to 1 g/l, or from 0 .12 g/L to 0.5 g/L, or 0.12 g/L, or 0.5 g/L, or 1 g/L of the nutritional composition when the composition is in ready-to-use liquid form, or

from 0.02 to 10 g/l, or from 0.05 to 2.5 g/l, or from 0.1 to 1.5 g/l, or from 0.05 to 1 g/l, or from 0 .12 g/l to 0.5 g/l, or 0.12 g/l, or 0.5 g/l, or 1 g/l (liquid diluted form) when the composition is in powder form and is intended for dilution to obtain a diluted liquid form, or

the same as above multiplied by 2, 5, 10, 20, 50 or 100 when the nutritional composition is in the form of a concentrated composition intended to be diluted (2, 5, 10, 20, 50 or 100 times, respectively) by water or human breast milk or intended for direct use in concentrated form, or

from 0.02 g to 0.75 g/100 g of powdered nutritional composition, or from 0.04 to 0.6 g/100 g, or from 0.05 to 0.5 g/100 g, or from 0.1 up to 0.4 g/100 g, or 0.1 g/100 g, or 0.2 g/100 g, or 0.25 g/100 g, or 0.5 g/100 g, or 1 g/100 g, or 3 g/100 g of powdered nutritional composition if the nutritional composition is in the form of a dry powder.

In one embodiment, the LNnT content may be from 0.01 g to 0.3 g/100 kcal of the powdered nutritional composition, or from 0.02 to 0.2 g/100 kcal, or from 0.04 to 0.15 g/ 100 kcal, or 0.02 g/100 kcal, or 0.04 g/100 kcal, or 0.07 g/100 kcal, or 0.15 g/100 kcal, or 0.3 g/100 kcal powder nutritional composition , if the nutritional composition is in the form of a dry powder.

The composition of the invention, particularly in the form of an infant formula, may contain at least about 0.4 g of oligofructose per 100 kcal. In some embodiments, it contains from about 0.4 to about 0.9 g, from about 0.4 to about 0.7 g, from about 0.4 to about 0.5 g, from about 0.7 to about 0 .8 g or about 0.7 to about 0.9 g oligofructose per 100 kcal. Oligofructose has a degree of polymerization from 2 to 10. In one embodiment, at least 90% of the oligofructose has a degree of polymerization from 2 to 8.

In one embodiment, the nutritional composition contains 2 FL and LNnT, preferably in an amount of 1 g/L 2FL and 0.5 g/L LNnT, or 0.5 g/L 2FL and 0.25 g/L LNnT, or 0. 25 g/l to 2 g/l 2FL and 0.1 to 1 g/l LNnT.

Method for preparing a nutritional composition

The nutritional composition can be prepared by any suitable method known in the art. For example, a commercial infant formula or formula may serve as the base composition to which the required amount of oligosaccharides (eg, OGMs such as 2FL, LNnT or others, OF, BMO) are added, preferably in dry form. In an alternative embodiment, the oligosaccharide can be added as a dry ingredient or a liquid ingredient to a liquid premix that will serve as the basis for the production of the nutritional composition of the invention. The liquid mixture can then be dried using any traditional means.

For example, it can be prepared by mixing together a protein source, a carbohydrate source (other than the combination of oligosaccharides of the present invention) and a fat source in appropriate proportions. At this stage, you can include emulsifiers if used. Vitamins and minerals can be added at this stage, but they are usually added later to avoid thermal decomposition. Any lipophilic vitamins, emulsifiers, etc. may be dissolved in the fat source before mixing. Water, preferably purified by reverse osmosis, can then be added to form a liquid mixture. The water temperature conveniently ranges from about 50°C to about 80°C to facilitate dispersion of the ingredients. Commercially available thinners can be used to form a liquid mixture. 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) are added at this stage if the finished product is to be in liquid form. If the final product is to be a powder, 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) can also be added at this stage if necessary. The liquid mixture is then homogenized, for example, in two stages.

The liquid mixture can then be thermally treated to reduce the bacterial load by rapidly heating the liquid mixture, for example to a temperature in the range of about 80°C to about 150°C, for a period of time from about 5 seconds to about 5 minutes. Heating can be accomplished by injection of steam using an autoclave or a heat exchanger such as a plate heat exchanger. The liquid mixture can then be cooled to a temperature of from about 60°C to about 85°C, for example by flash cooling. The liquid mixture can then be homogenized again, for example in two stages: under a pressure ranging from about 10 MPa to about 30 MPa in the first stage and from about 2 MPa to about 10 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 stage it is convenient to adjust the pH and solids content of the homogenized mixture. The homogenized mixture is transferred to a suitable drying apparatus, such as a spray dryer or freeze dryer, and converted into a powder. The moisture content of the powder should be less than about 5 wt.%.

If a liquid composition is preferred, the homogenized mixture can be sterilized and then aseptically filled into suitable containers, or can be filled into containers first and then autoclaved.

Specific lipids

The composition of the invention may contain selected lipids that have specific effects.

Such lipids may include, but are not limited to, DHA, ARA, linoleic acid or sphingomyelin, preferably in amounts appropriate to provide actual brain health benefits and within the general regulatory requirements for the product type (e.g. WHO infant formula guidelines; CODEX or European Infant Formula Directives).

In some embodiments, the composition of the invention contains a relatively high level of sn-2-palmitate or sphingomyelin. They have been associated with optimal performance and development of the brain and may produce a synergistic effect with the essential compounds of the composition of the invention.

Although feeding an infant a formula containing a high percentage of sn-2-palmitate (in the absence of OF) helps to promote the growth of bifidobacteria in the colon, the combination of high sn-2-palmitate with oligofructose is thought to produce significantly higher growth of bifidobacteria in the colon in formula-fed infants. In addition, a significant reduction in the number of potentially pathogenic bacteria can be achieved. Feeding an infant an infant formula having a high level of sn-2-palmitate and about 3 to about 5 g/L, or about 0.4 to about 0.7 g/100 kcal oligofructose, has been found to be more beneficial than with feeding the baby the same formula without oligofructose. Without being limited by theory, such a synergistic effect between OF and sn-2-palmitate may also activate short-term memory, particularly (but likely not exclusively) through effects on the subject's microbiome and bifidobacteria population. The term “high sn-2 palmitate” is to be understood as ingredients having a high percentage of fatty acids (preferably greater than 33% fatty acids) in the form of palmitate at the sn-2 position of the triglycerides. Such ingredients are commercially available under the trade name BETAPOL® (Loders Croklaan, Wormerveer, the Netherlands) or INFAT® (Advanced Lipids AB, Karlshamn, Sweden, a joint venture of AAK B.V. (Zaandijk, the Netherlands) and Enzymotec Inc, Morristown, USA).

Recent clinical studies in infants have shown that formulas containing at least one omega-6 fatty acid and at least one omega-3 fatty acid in a ratio of about 6 to about 1 increase DHA accumulation in red blood cells and plasma. A balanced ratio of about 6:1 omega-6 fatty acids to omega-3 fatty acids may also provide long-term health benefits, including optimal brain and nervous system development. This balance will be achieved by formulating the compositions of the present invention with fat sources from vegetable oils that contain omega-6 fatty acids, such as, for example, soybean oil and sunflower oil, and that contain omega-3 fatty acids, such as canola, canola, flaxseed, chia, pearla or walnuts. To provide a modified fat mixture, a unique fat mixture with 5 different oils will be used.

The following examples are presented to illustrate certain embodiments and features of the present invention, but should not be construed as limiting the scope of the present invention.

Examples

Example 1. Nutritional composition containing OGM (2FL and/or LNnT)

The nutritional composition of the invention containing OGM 2FL and/or LNnT and OF is presented in Table 1 below. This composition is provided for illustrative purposes only. The composition in Table 1 may be an infant formula. In an alternative embodiment, it may be formulated for use as a formula for breastfed infants.

Table 1

Nutrients per 100 kcal per liter Energy value (kcal) 100 670 Proteins (g) 1.83 12.3 Fat (g) 5.3 35.7 Linoleic acid (g) 0.79 5.3 α-Linolenic acid (mg) 101 675 Lactose (g) 11.2 74.7 Minerals (g) 0.37 2.5 Na (mg) 23 150 K (mg) 89 590 Cl (mg) 64 430 Ca (mg) 62 410 P (mg) 31 210 Mg (mg) 7 50 Mn (µg) 8 50 Se (µg) 2 13 Vitamin A (µg RE) 105 700 Vitamin D (mcg) 1.5 10 Vitamin E (mg TEQ) 0.8 5.4 Vitamin K1 (mcg) 8 54 Vitamin C (mg) 10 67 Vitamin B1 (mg) 0.07 0.47 Vitamin B2 (mg) 0.15 1.0 Niacin (mg) 1 6.7 Vitamin B6 (mg) 0.075 0.50 Folic acid (mcg) 9 60 Pantothenic acid (mg) 0.45 3 Vitamin B12 (mcg) 0.3 2 Biotin (mcg) 2.2 15 Choline (mg) 10 67 Fe (mg) 1.2 8 I (µg) 15 100 Cu (mg) 0.06 0.4 Zn (mg) 0.75 5 2 FL (g) and/or LNnT (g) 0.04 or 0.15
0.01 or 0.07 0.25 or 1
0.1 or 0.5 Oligofructose (OF) (g) (optional) 0.45 or 0.75 3 or 5 3'-SL (g) (optional) 0.22 1.46 6’-SL (g) (optional) 0.05 0.33 Ratio 3'-SL : 6'-SL (optional) 4.4:1 4.4:1 BMO(g) (optional) 0.4 or 2 3 or 15

Example 2: Infant formula containing OGM (2 FL and/or LNnT), optional BMO and/or oligofructose (OF)

The nutritional composition of the invention containing OGM (2FL and/or LNnT) and optionally OF and/or BMO is presented in Table 2 below. This composition is provided for illustrative purposes only. The composition shown in Table 2 is an infant formula. In an alternative embodiment, it may be formulated for use as a formula for breastfed infants.

Another example is based on commercial NAN infant formulas and/or lactogenic infant formulas (manufactured by Nestlé, Switzerland), to which the specific oligosaccharides of the invention are added in the amounts specified below.

table 2

Nutrient per 100 kcal per liter Energy value (kcal) 100 670 Protein (g) 1.83 12.3 Fat (g) 5.3 35.7 Linoleic acid (g) 0.79 5.3 α-Linolenic acid (mg) 101 675 Lactose (g) 11.2 74.7 Prebiotic (70% OF, 30% inulin) (g) (e.g.
Beneo-Orafti® P95 and Inulin Beneo-Orafti®) (optional) 0.64 4.3 2 FL (g) and/or LNnT (mg) 0.3
thirty 2
200 Minerals (g) 0.37 2.5 Na (mg) 23 150 K (mg) 89 590 Cl (mg) 64 430 Ca (mg) 62 410 P (mg) 31 210 Mg (mg) 7 50 Mn (µg) 8 50 Se (µg) 2 13 Vitamin A (µg RE) 105 700 Vitamin D (mcg) 1.5 10 Vitamin E (mg TEQ) 0.8 5.4 Vitamin K1 (mcg) 8 54 Vitamin C (mg) 10 67 Vitamin B1 (mg) 0.07 0.47 Vitamin B2 (mg) 0.15 1.0 Niacin (mg) 1 6.7 Vitamin B6 (mg) 0.075 0.50 Folic acid (mcg) 9 60 Pantothenic acid (mg) 0.45 3 Vitamin B12 (mcg) 0.3 2 Biotin (mcg) 2.2 15 Choline (mg) 10 67 Fe (mg) 1.2 8 I (µg) 15 100 Cu (mg) 0.06 0.4 Zn (mg) 0.75 5 3'-sialyllactose (mg) (optional) thirty 200 6'-sialyllactose (mg) (optional) 6 40 BMO(g) (optional) 0.9 6

Experimental data

A study in piglets examined the effects of various OGM-containing oligosaccharides (e.g., 2FL) with or without the addition of OF and/or BMO on gene expression in the hippocampus. Thirty-six male piglets (12 per treatment group) received control (CON, Purina ProNurse livestock milk replacer), OGM (CON + 1.5 g/l OGM) from 48 hours postfarrow to 33 days of age. consisted of 1.0 g/l 2'FL + 0.5 g/l LNnT), OF [CON + 5 g/l OF], OF + OGM [CON + 5 g/l OF + 1.0 g/l OGM (2'FL), BMO (6.5 g/L BMO) or OGM + BMO (1.0 g/L 2'FL + 0.5 g/L LNnT + 6.5 g/L BMO) All diets contained a reserve for reformulation of 8.0 g/l, which was reserved for the addition of test components of the diet.Piglets were fed 285 ml and 325 ml of diluted diet per kg body weight on days 2–6 after birth (PND 2 –6) and postnatal days 7–33 (PND 7–33), respectively.Piglets were killed on days 32 or 33, and hippocampal tissue was collected for mRNA expression analysis.

The BMO mixture used in infant formulas was derived from cow's milk whey. Briefly, bovine whey ultrafiltrate, including oligosaccharides such as 3′- and 6′-sialyllactose and GOS, was demineralized by a combination of electrodialysis and ion exchange as described in the art.

Gene expression in the hippocampus

Approximately 20 mg of hippocampal tissue was injected into a Lysing Matrix D tube (MP Biomedicals, Santa Ana, CA, USA), placed on ice and added with 650 μl of lysis buffer (Agencourt RNAdvance Tissue Kit, Beckman Coulter, Indianapolis, CA). Indiana, USA). Tubes were vortexed 2 x 1 minute at speed 6 on a FastPrep®-24 (MP Biomedicals, Santa Ana, CA, USA) and then 400 μl of lysate was extracted using the Agencourt RNAdvance Tissue Kit (Beckman Coulter, Indianapolis, USA). Indiana, USA) in accordance with the manufacturer's recommendations. RNA was quantified using the Quant-iT™ RiboGreen™ RNA Assay Kit (Invitrogen, Carlsbad, CA, USA) on a Spectramax M2 (Molecular Devices, Sunnyvale, CA, USA). RNA quality assessment was performed using Fragment Analyzer 96 using Standard Sensitivity RNA Analysis Kit (15 nt) (Advanced Analytical Technologies, Inc., Ankeny, Iowa, USA). Relative mRNA copy numbers for 93 genes were quantified using the NanoString nCounter™ system (NanoString Technologies Inc., Seattle, WA, USA) according to the manufacturer's instructions, using 100 ng of RNA as a starting amount.

results

The OGM, BMO, BMO + OGM, and OF + OGM diets showed differential effects on gene expression compared to the CON control group. Hippocampal mRNA expression of several key genes involved in the GABAergic system is shown in Fig. 1 for GABA receptor type B subunit 1 (GABBR1) and FIG. 2 for GABA receptor type A subunit Beta2 (GABRB2). Administration of OGM, preferably in combination with a prebiotic such as OF and/or BMO, has the remarkable ability to modulate the GABAergic system at the expression level.

In fig. Figure 1 shows that GABBR1 receptor gene mRNA expression is either maintained or even significantly increased compared to the control diet (CON) when OGM, alone (2'FL + LNnt), or in combination with OF or BMO, is also present in the test diet.

In fig. Figure 2 shows that the expression of GABRB2 receptor gene mRNA is particularly increased compared to the control diet when OGM is administered in combination with BMO.

It should be understood that one or more examples in the description are not limiting and that the present description is intended to cover variations and equivalents of these examples.

Claims (14)

1. The use of a nutritional composition containing human milk oligosaccharide (HMO) in a mammal, preferably a human infant or young child from birth to 7 years of age, to improve, enhance, stimulate or modulate GABAergic function in the central nervous system (CNS). 2. The use of a nutritional composition containing human milk oligosaccharide (HMO) in a mammal, preferably a human infant or young child from birth to 7 years of age, for the prevention, prophylaxis or compensation of attention deficit hyperactivity disorder, autism spectrum disorders, as well as schizophrenia in subjects in need, preferably by stimulating or modulating GABAergic functions in the CNS. 3. Use of a nutritional composition according to claim 1 or 2, wherein said GABAergic function in the central nervous system involves filtering information or the ability to focus or concentrate on mental or physical activity. 4. Use of a nutritional composition according to any one of paragraphs. 1-3, wherein said OGM is selected from the group consisting of N-acetyllactosamine, sialylated oligosaccharides, fucosylated oligosaccharides, or a combination thereof. 5. Use of a nutritional composition according to claim 4, wherein OGM is a fucosylated oligosaccharide selected from the group consisting of fucosylated oligosaccharides, 2'-fucosyllactose, 3'fucosyllactose, difucosyllactose, lacto-N-fucopentaose I, lacto-N-fucopentaose II , lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucohexaose, lacto-N-difucohexaose I, fucosyllacto-N-hexaose, fucosyllacto-N-neohexaose I, fucosyllacto-N-neohexaose II, difucosyllacto- N-hexaose I, difucosyllacto-N-neohexaose I, difucosyllacto-N-neohexaose II, fucosyl-p-lacto-N-hexaose and any combination thereof, N-5 acetylated oligosaccharides such as lacto-N-tetraose (LNT), N-neotetraose (LNnT) and any combination thereof, and preferably the fucosylated oligosaccharide is selected from 2'-fucosyllactose (2-FL), diFL, LNnT, LNT and any combination thereof. 6. Use of a nutritional composition according to claim 4, wherein OGM is N-acetyllactosamine selected from the group consisting of lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT). 7. Use of a nutritional composition according to claim 4, wherein the OGM is a sialylated oligosaccharide, preferably selected from the group consisting of 3'-sialyllactose and 6'-sialyllactose, and more preferably said composition contains both 3'-sialyllactose and 6' -sialyllactose, wherein the ratio of 3'-sialyllactose to 6'-sialyllactose is preferably in the range from 100:1 to 1:100, more preferably from 10:1 to 1:10, even more preferably from 5:1 to 1:2. 8. Use of a nutritional composition according to any of the preceding paragraphs, wherein the amount of OGM is from 0.01 to 50 g/l, from 0.1 to 10 g/l, from 0.3 to 5 g/l, or from 0.5 up to 1 g/l, or 0.25, or 0.5, or 1, or 1.5, or 2 g/l. 9. The use of a nutritional composition according to any of the preceding claims, which further contains a prebiotic, and the specified prebiotic is preferably selected from the list consisting of bovine milk oligosaccharide (BMO), inulin, xylooligosaccharides, polydextrose, fructose oligosaccharide (FOS) or oligofructose (OF) or any combination of them. 10. Use of a nutritional composition according to claim 9, wherein said BMO comprises an N-acetylated oligosaccharide, a galactooligosaccharide, a sialylated oligosaccharide, a fucosylated oligosaccharide, or a combination thereof. 11. Use of a nutritional composition according to any of the preceding claims, which further contains a probiotic, wherein the probiotic is preferably selected from the list consisting of Bifidobacterium, Lactobacillus, Lactococcus, Enterococcus, Streptococcus, Kluyveromyces, Saccharomyces, Candida, in particular 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, Lactococcus lactis, Enterococcus faecium, Saccharomyces cerevisiae, Saccharomyces boulardii or mixtures thereof, is 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), Bifidobacterium breve strain A, Lactobacillus paracasei NCC2461 (CNCM I-2116), Lactobacillus johnsonii NCC533 (CNCM I-1225), Lactobacillus rhamnosus GG (ATCC53103), Lactobacillus rhamnosus NCC4007 (CGMCC 1.3724), Enterococcus faec ium SF 68 (NCC2768; NCIMB10415) and their combinations. 12. Use of a nutritional composition according to any one of the preceding claims, wherein the mammal is a human, domestic animal, cat or dog, and preferably the mammal is a young mammal, more preferably an infant or young child. 13. Use of a nutritional composition as claimed in any one of the preceding claims, wherein said composition is an infant formula, infant formula, breast milk fortifier, infant formula for children 1 to 3 years of age, or a pet food or supplement. 14. The use of human milk oligosaccharide (HMO) for the preparation of a nutritional composition for improving, enhancing, stimulating or modulating GABAergic function in the CNS in a subject in need thereof, preferably in a human infant or young child from birth to 7 years of age.