Non-digestible oligosaccharide mixture for infants at risk of brain injury

Abstract

The invention pertains to nutritional composition comprising non-digestible oligosaccharides [NDOs] selected from i) a mixture of galactooligosaccharides and long-chain fructooligosaccharides and / or ii) a mixture of human milk oligosaccharides comprising at least sialylated, fucosylated and N-acetylated oligosaccharides, for brain injury and / or impaired brain development in infants exposed to perinatal hypoxia and / or preterm infants. The invention further pertains to nutritional compositions comprising a mixture of human milk oligosaccharides comprising at least sialylated, fucosylated and N-acetylated oligosaccharides.

Description

[0001] NON-DIGESTIBLE OLIGOSACCHARIDE MIXTURE FOR INFANTS AT RISK OF BRAIN INJURY

[0002] Field of the invention

[0003] The invention relates to nutritional interventions for preterm and / or infants exposed to perinatal hypoxia at risk of brain injury.

[0004] BACKGROUND OF THE INVENTION

[0005] With advances in neonatal intensive care, the survival of infants exposed to perinatal hypoxia, (very) preterm, (very) low birth weight and small for gestational age children has improved considerably.

[0006] Human milk is recognized as the ideal feeding for infants due to its overall nutritional composition. For a preterm infant, very low birth weight [VLBW] and / or infant small for gestational age (SGA infant), however, human breast milk does not always meet their complete nutritional needs, even though the milk of mothers of preterm infants appears to be adapted to the specific needs of preterms. Therefore, for these infants special nutritional formulae and breast milk fortifiers have been designed and marketed, which differ in composition from standard infant formula. Typically, such preterm formulae have a higher energy and protein content, to enable an increased growth rate. A review on the ESPGHAN nutritional guidelines for such formulae is given in Agostoni et al, JPGN 2010, 50 :85-91 .

[0007] A variety of risk factors associated with preterm and low birth weight birth, including perinatal hypoxia, neonatal infections, and inflammatory responses, adversely affect brain maturation processes in these children. Diffuse white matter injury [WMI] often occurs in preterm infants born between 24 and 32 weeks of gestational age, which may be accompanied by widespread damage to oligodendrocytes and disrupted myelination, reduced cortical volume, thalamus, and basal ganglia volume. Diffuse WMI is associated with impaired cognitive, sensory, and psychological functions, and is increasingly recognized as a risk factor for autism spectrum disorders, attention deficit hyperactivity disorder, social difficulties, anxiety, behavioural disorders, and other psychological disorders.

[0008] Small-for-gestational-age infants, although not always preterm, likewise also exhibit susceptibility to WMI due to intrauterine growth restriction and compromised placental function and / or perinatal hypoxia. The consequences of WMI are far-reaching, impacting long-term neurodevelopment, motor function, and cognitive abilities. Consequently, widespread differences in brain development compared to term peers are found. Unfortunately, these unfavourable differences in brain development appear to pertain into childhood and adolescence, and increase the risks for poor motor, cognitive, and behavioural development in very preterm, VLBW and SGA children.

[0009] WO 2014 / 043368 and WO 2013 / 057049 propose methods and compositions for improving brain growth and cognitive development and for enhancing memory functions in individuals by administering human milk oligosaccharides. However, these methods and compositions are not specifically suited for use infants or preterm infants in the very early postnatal period.

[0010] Thus, there is still a need for improved nutritional compositions for use in preventing brain damage and impaired brain development and promoting brain growth and development in preterm infants and infants exposed to perinatal hypoxia, infants with a low or very-low birth weight and infants who experienced intrauterine growth retardation (IUGR) or who suffered from growth stunting because of malnutrition, such as suboptimal intra-uterine nutrition, and / or disease.

[0011] SUMMARY OF THE INVENTION

[0012] In an animal model, rats were exposed to a combination of fetal inflammation and postnatal hypoxia resulting in neonatal brain injury. The consequences of fetal inflammation and postnatal hypoxia resemble the features of perinatal diffuse white matter injury. It was found that supplementation of the rats with a mixture of galactooligosaccharides [GOS] and long chain fructooligosaccharides (IcFOS) or a mixture of specific human milk oligosaccharides [HMOs] comprising at least sialylated, fucosylated and N-acetylated oligosaccharides mitigates the brain injury and impaired brain development. It was observed that supplementation of the rats with a mixture of galactooligosaccharides [GOS] and long chain fructooligosaccharides (IcFOS) or a mixture of specific human milk oligosaccharides [HMOs] restored interneuron development. The mixture of human milk oligosaccharides was further found to improve brain myelination and reduced neuroinflammation. The mixture of specific HMOs consists of 2’-fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3’-sialyllactose (3’-SL) and 6’- sialyllactose (6’-SL).

[0013] Therefore, a nutritional composition comprising a mixture of HMOs comprising at least sialylated, fucosylated and N-acetylated oligosaccharides and / or a mixture of GOS and IcFOS will provide beneficial effects on brain injury and / or impaired brain development in infants exposed to perinatal hypoxia and / or preterm infants. Such nutritional composition can be used to prevent and / or treat brain injury and / or impaired brain development in these infants including infants including preventing and / or treating neuroinflammation, improving interneuron development and / or improving myelination in these infants. The nutritional compositions unexpectedly can further be used in the treatment and prevention of diffuse white matter injury in these infants including preventing and / or treating neuroinflammation, improving interneuron development and / or improving myelination. The invention also pertains to nutritional compositions for preterms.

[0014] LIST OF PREFERRED EMBODIMENTS

[0015] 1 . Nutritional composition comprising non-digestible oligosaccharides [NDOs] selected from i) a mixture of galactooligosaccharides and long-chain fructooligosaccharides and / or ii) a mixture of human milk oligosaccharides comprising at least sialylated, fucosylated and N-acetylated oligosaccharides, for use in preventing and / or treating brain injury and / or preventing and / or treating impaired brain development in infants exposed to perinatal hypoxia and / or preterm infants. 2. Nutritional composition for use according to embodiment 1 , wherein preventing and / or treating brain injury and / or preventing and / or treating impaired brain development comprises improving interneuron development.

[0016] 3. Nutritional composition for use according to the preceding embodiments wherein the infants exposed to perinatal hypoxia and / or preterm infants are at risk of or suffering from diffuse white matter injury.

[0017] 4. Nutritional composition for use according to the preceding embodiments wherein the nutritional composition comprises the NDO ii) a mixture human milk oligosaccharides comprising at least sialylated, fucosylated and N-acetylated oligosaccharides and wherein preventing and / or treating brain injury and / or preventing and / or treating impaired brain development is selected from treating and / or preventing

[0018] - diffuse white matter injury; and / or

[0019] - neuroinflammation; and / or

[0020] - impaired central myelination.

[0021] 5. Nutritional composition for use according to the preceding embodiments wherein the nutritional composition is a preterm formula or a paediatric formula for faltering growth.

[0022] 6. Nutritional composition for use according to the preceding embodiments, wherein the NDOs comprise ii) a mixture of HMOs, wherein the mixture of HMOs consists of 2’-fucosyllactose (2’- FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3’-sialyllactose (3’-SL) and 6’-sialyllactose (6’-SL).

[0023] 7. Nutritional composition for use according to embodiment 6, wherein the NDOs comprise ii) a mixture of HMOs and wherein the HMOs consist of 42 to 62 wt% 2’-FL, 10 to 16 wt% 3-FL, 21 to 31 wt% LNT, 3 to 5 wt% 3’-SL and 4 to 6 wt% 6’-SL, the sum of 2’-FL, 3-FL, LNT, 3’-SL and 6’-SL being 100 % of the total weight of component ii).

[0024] 8. Nutritional composition for use according to the preceding embodiments, wherein the NDOs comprise i) a mixture of GOS and IcFOS, said mixture consisting of 70 to 95 wt% GOS, preferably 85 to 95 wt% GOS, based on total weight of component i), and 5 to 30 wt% IcFOS, preferably 5 to 15 wt% IcFOS, based on total weight of component i).

[0025] 9. Nutritional composition for use according to the preceding embodiments, wherein the nutritional composition is administered in at least the first three months of life of the infant, preferably in at least the first month of the life of the infant.

[0026] 10. Nutritional composition for use according to the preceding embodiments, wherein the nutritional composition comprises 70 to 120 kcal per 100 ml, 4.0 to 6.0 g lipids per 100 kcal; 2.5 to 4.0 g protein perl OO kcal, 9 to 12 g digestible carbohydrates perl OO kcal, and 20 mg to 2.40 g per 100 ml non-digestible oligosaccharides selected from i) and / or ii).

[0027] 11. Nutritional composition comprising 70 to 120, preferably 75 to 85 kcal / 100 ml kcal per 100 ml, 4.0 to 6.0 g lipids per 100 kcal;

[0028] 2.5 to 4.0 g protein per 100 kcal,

[0029] 9 to 12 g digestible carbohydrates per 100 kcal and non-digestible oligosaccharides [NDOs] comprising ii) 25 mg to 500 mg per 100 kcal nutritional composition of a mixture of human milk oligosaccharides [HMOs] consisting of 42 to 62 wt% 2’-FL, 10 to 16 wt% 3-FL, 21 to 31 wt% LNT, 3 to 5 wt% 3’-SL and 4 to 6 wt% 6’- SL, the sum of 2’-FL, 3-FL, LNT, 3’-SL and 6’-SL being 100 % of the total weight of the HMOs and wherein the nutritional composition is a preterm formula or a faltering growth formula.

[0030] 12. Nutritional composition according to embodiment 11 wherein the composition comprises said NDOs, the NDOs further comprising i) 125 mg to 2.5g of galactooligosaccharides and long- chain fructooligosaccharides.

[0031] 13. Nutritional composition according to embodiments 11 and 12 wherein the composition comprises a total of amount of NDOs consisting of i) and ii) of 150 mg to 3.00 g per 100 kcal, of the nutritional composition

[0032] 14. Nutritional composition according to embodiments 11 to 13 wherein the nutritional composition comprises 0.3 to 0.7 g Medium Chain Fatty Acids (MCFAs) per 100 kcal of the nutritional composition.

[0033] 15. Human milk fortifier composition non-digestible oligosaccharides [NDOs], said NDOs comprising ii) 25 mg to 500 mg per 100 kcal of a mixture of human milk oligosaccharides [HMOs] consisting of 42 to 62 wt% 2’-FL, 10 to 16 wt% 3-FL, 21 to 31 wt% LNT, 3 to 5 wt% 3’- SL and 4 to 6 wt% 6’-SL, the sum of 2’-FL, 3-FL, LNT, 3’-SL and 6’-SL being 100 % of the total weight of the HMOs, and preferably comprising 0 to 5.0 g fat per 100 kcal, 7 to 10 g protein per 100 kcal and 6 to 10 g digestible carbohydrates per 100 kcal of the nutritional composition.

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention concerns a nutritional composition for infants comprising a mixture of non- digestible oligosaccharides selected from i) galacto-oligosaccharides (GOS) and long chain fructooligosaccharides (IcFOS), and / or ii) a mixture of human milk oligosaccharides [HMOs] comprising at least sialylated, fucosylated, and N-acetylated oligosaccharides, preferably a mixture of HMOs consisting of 2’-fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3’-sialyllactose (3’-SL) and 6’-sialyllactose (6’-SL). The nutritional composition comprising non-digestible oligosaccharides selected from i) and / or ii) is for use in infants, preferably selected from infants exposed to perinatal hypoxia, (very) low birth weight, small for gestational age and / or preterm infants, preferably infants exposed to perinatal hypoxia and / or preterms, preferably preterms.

[0036] The invention concerns the present nutritional composition for use in therapy. The invention also concerns the use of the present nutritional composition for preventing and / or treating brain injury and / or impaired brain development. The use further comprises preventing and / or treating brain injury and / or impaired brain development in infants at risk of or suffering from diffuse white matter injury. The use further pertains to preventing, reducing and / or treating neuroinflammation, improving interneuron development and / or improving myelination in said infants.

[0037] The invention can also be worded as a nutritional composition comprising a mixture of non-digestible oligosaccharides selected from i) GOS and IcFOS, and / or ii) a mixture of HMOs comprising at least sialylated, fucosylated, and N-acetylated oligosaccharides, preferably a mixture consisting of 2’-FL, 3- FL, LNT, 3’-SL and 6’-SL for use in preventing and / or treating brain injury and / or preventing and / or treating impaired brain development in infants exposed to perinatal hypoxia and / or preterm infants. Preventing and / or treating brain injury and preventing and / or treating impaired brain development further pertains to preventing, reducing and / or treating neuroinflammation, improving interneuron development and / or improving central myelination in said infants. In a preferred aspect the infants are at risk of or suffering from diffuse white matter injury and the use further pertains to treating and / or preventing diffuse white matter injury.

[0038] The invention can also be worded as the use of a mixture of non-digestible oligosaccharides selected from i) GOS and IcFOS, and / or ii) a mixture of HMOs comprising at least sialylated, fucosylated and N- acetylated oligosaccharides, preferably a mixture consisting of 2’-FL, 3-FL, LNT, 3’-SL and 6’-SL in the manufacture of a nutritional composition for preventing and / or treating brain injury and / or preventing and / or treating impaired brain development in infants exposed to perinatal hypoxia and / or preterm infants. The use of a mixture of no-digestible oligosaccharides in the manufacture of nutritional composition for use in preventing and / or treating brain injury and preventing and / or treating impaired brain development further pertains to preventing, reducing and / or treating neuroinflammation, improving interneuron development and / or improving myelination in said infants. The use in an embodiment further comprises preventing and / or treating diffuse white matter injury. The use further comprises preventing and / or treating brain injury and / or impaired brain development in infants at risk of or suffering from diffuse white matter injury. The use further pertains to preventing, reducing and / or treating neuroinflammation, improving interneuron development and / or improving myelination in said infants. In a preferred aspect the infants exposed to perinatal hypoxia and / or preterm infants are at risk of or suffering from diffuse white matter injury and the use further pertains to treating and / or preventing diffuse white matter injury.

[0039] Also, the invention concerns a method of preventing and / or treating brain injury and / or preventing and / or treating impaired brain development in infants exposed to perinatal hypoxia and / or preterm infants, preferably further comprising preventing, reducing and / or treating neuroinflammation, improving interneuron development and / or improving central myelination in said infants. The method further pertains to preventing and / or treating brain injury and / or impaired brain development and / or diffuse white matter injury in infants at risk of or suffering from diffuse white matter injury. The method comprises administering to the infant a nutritional composition comprising a mixture of non-digestible oligosaccharides selected from i) GOS and IcFOS, and / or ii) a mixture of HMOs comprising at least sialylated, fucosylated and N-acetylated oligosaccharides, preferably a mixture consisting of 2’-FL, 3- FL, LNT, 3’-SL and 6’-SL.

[0040] The invention further pertains to nutritional compositions comprising a mixture of non-digestible oligosaccharides selected from i) GOS and IcFOS, and / or ii) a mixture of HMOs comprising at least sialylated, fucosylated and N-acetylated oligosaccharides, preferably a mixture consisting of 2’-FL, 3- FL, LNT, 3’-SL and 6’-SL for infants, preferably for preterms, (V)LBW and / or SGA infants, most preferably preterms.

[0041] DEFINITIONS

[0042] In the context of the present invention the term “prevention” means “reducing the risk of (occurrence)” or “reducing the severity of’. The term “prevention of a certain condition” also includes “treatment of a person at (increased) risk of said condition”.

[0043] An infant is a child under the age of 12 months.

[0044] Diffuse white matter injury (dWMI) as used herein pertains to non-focal, widespread damage to the white matter, affecting multiple regions of the brain rather than being localized to a specific area. dWMI is associated cognitive deficits and developmental delays, motor impairments, and other neurological symptoms.

[0045] Neuroinflammation as used herein is a response of the innate immune system of the central nervous system (CNS) associated with many disorders, including perinatal infections. Microglia cells are thought to be the main cell type involved in neuroinflammation. Microglia are the innate immune cells of the central nervous system, which act quickly on neuroinflammation. However, prolonged activation of microglia, as in chronic neuroinflammation, causes damage to brain tissue and to the blood-brain- barrier.

[0046] Hypoxia is reduced oxygen availability in tissue. As used herein, "hypoxia" means a reduction in oxygen supply to tissues below physiological levels. Hypoxia is a risk for brain injury.

[0047] The term perinatal as used herein means refers the period occurring "around the time of birth".

[0048] Low birthweight infants have a weight of less than 2500 grams at birth. Very low birthweight infants have a weight below 1500 grams at birth. Extremely low birthweight infants have a birthweight below 1000 grams at birth.

[0049] Preterm (or premature) infants are born after 24 weeks and before the end of the 37th week of pregnancy. Extreme preterms are preterms that are born before the end of 28 weeks of pregnancy, very preterm infants are born between 28 to less than 32 weeks of pregnancy. SGA infants are those whose birth weight lies below the 10th percentile for that gestational age. Reasons for SGA can be several; for example, term or preterm infants can be born SGA because they have been the subject of intrauterine growth restriction (IUGR).

[0050] The term “preterm formulation” as used herein is intended to encompass all formulations (e.g. formulas and fortifiers) intended for a preterm infant. Exemplary preterm formulations include preterm formulas and human milk fortifiers as herein defined (e.g., those which are intended to be admixed with human milk before administration to a preterm infant), and a composition comprising human milk and a human milk fortifier.

[0051] The term HMO or HMOs refer to human milk oligosaccharide(s). HMOs are complex carbohydrates found in human breast milk ((Urashima et al.: Milk Oligosaccharides. Nova Science Publisher (2011); Chen Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)). These carbohydrates are resistant to enzymatic hydrolysis by digestive enzymes. Each human milk oligosaccharide is based on a combination of lactose and one or more of four monosaccharides (N-acetyl-D-glucosamine, D- galactose, sialic acid and / or L-fucose) to for an oligosaccharide. HMOs can be divided in neutral or non- acidic HMOs which can either be fucosylated or non-fucosylated, and acidic HMOs that have at least one sialyl residue in their structure. In the context of the present invention lactose is not regarded as an HMO species. HMOs can be manufactured by means known in the art.

[0052] As used herein, the term "degree of polymerization" (DP) means the number of monomer units joined together in a poly- or oligomer.

[0053] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0054] Non-digestible oligosaccharides: / ') galacto-oligosaccharides and long chain fructo-oligosaccharides The nutritional composition comprises non-digestible oligosaccharides selected from i) galactooligosaccharides and long chain fructo-oligosaccharides and / or ii) a mixture of human milk oligosaccharides [HMOs] comprising at least sialylated, fucosylated and N-acetylated oligosaccharides, preferably a mixture of HMOs consisting of 2’-fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), lacto-N- tetraose (LNT), 3’-sialyllactose (3’-SL) and 6’-sialyllactose (6’-SL).

[0055] Non-digestible oligosaccharides are oligosaccharides with an average degree of polymerization ranging from 2 to 100. Non-digestible oligosaccharides are oligosaccharides that are nondigested in the stomach or small intestine and reach the colon intact. Maltodextrin, lactose, and monomers such as galactose, fucose, and sialic acid are not considered non-digestible oligosaccharides, i.e. they are considered digestible carbohydrates. In an embodiment the nutritional composition comprises non-digestible oligosaccharides selected from i) galacto-oligosaccharides and long chain fructo-oligosaccharides. In such embodiments the present nutritional composition comprises galacto-oligosaccharides (GOS). GOS are non-digestible oligosaccharides preferably having the formula ([galactose]n-glucose; wherein n is an integer ranging from 2 to 10, i.e. 2, 3, 4, 5, 6, ....,10;), wherein the galactose units are preferably in majority linked together via a beta linkage. Beta-linked GOS, (bGOS) are for example sold under the trademark Vivinal™ GOS (Borculo Domo Ingredients, Netherlands). Other suitable sources are Oligomate™ (Yakult, Japan). Preferably the present GOS have an average degree of polymerization (DP) ranging from 2 to 7, more preferably ranging from 3 to 5. Preferably the GOS comprise mainly beta-1 ,4 linkages and / or beta-1 ,6 linkages between the galactose units, more preferably predominantly beta-1 ,4 linkages. In a preferred embodiment, the GOS comprise at least 80 % beta-1 ,4 and beta-1 ,6 linkages based on total linkages.

[0056] The present nutritional composition comprises long chain fructo-oligosaccharides (IcFOS). IcFOS are non-digestible oligosaccharides comprising a chain of beta-linked fructose units with an average degree of polymerization (DP) ranging from 10 to 1000, more preferably 10 to 100, even more preferably 20 to 40. IcFOS include inulin, levan and / or a mixed type of polyfructan. An especially preferred IcFOS is inulin. Inulin has a structure of chain-terminating glucosyl moieties and a repetitive fructosyl moiety, which are linked by beta-2,1 linkages. IcFOS suitable for use in the present nutritional composition is also commercially available, e.g. RaftilineOHP (Orafti).

[0057] For the purpose of this invention, when amounts or ranges are expressed per volume this refers to the nutritional composition in a ready-to-use form, unless expressed otherwise. When amounts are expressed as wt%, this refers to the wt% based on dry weight, unless expressed otherwise.

[0058] Preferably, the present nutritional composition comprises 100 mg to 2 g GOS plus IcFOS per 100 ml, more preferably 250 mg to 1 .5 g, even more preferably 500 mg to 1 g per 100 ml. Based on dry weight, the present nutritional composition preferably comprises 0.6 wt% to 11.8 wt%, more preferably 1.5 to 8.8 wt%, even more preferably 2.9 to 5.9 wt% GOS plus IcFOS. Based on 100 kcal the present nutritional composition preferably comprises 125 mg to 2.5 g GOS plus IcFOS, more preferably 312.5 mg to 1 .875g, even more preferably 625 mg to 1 .25 g.

[0059] Based on total weight of GOS plus IcFOS, preferably the amount of GOS ranges from 70 to 95 % and the amount of IcFOS ranges from 5 to 30 %, the sum of GOS and IcFOS being 100 %. More preferably based on total weight of GOS plus IcFOS, the amount of GOS ranges from 85 to 95 % and the amount of IcFOS ranges from 5 to 15 %, the sum of GOS and IcFOS being 100 %.

[0060] Preferably, GOS and IcFOS are provides in a weight ratio of GOS to IcFOS of 99: 1 to 1 : 99, more preferably 20 : 1 to 1 : 1 , most preferably 12 : 1 to 7 : 1. In a particularly preferred embodiment the weight ratio GOS to IcFOs is 9:1 .

[0061] It is preferred to combine GOS with IcFOS instead of other longer oligosaccharides such as polydextrose. A mixture of GOS and IcFOS was found to have beneficial effects on neuroinflammation, and on interneuron formation in the hippocampus in diffuse white matter injury. Also, mixtures of GOS with IcFOS were shown to provide beneficial effects on the gut microbiota.

[0062] Non-digestible oligosaccharides: ii) mixture of HMOs

[0063] In an embodiment the nutritional composition comprises non-digestible oligosaccharides selected from ii) a mixture of human milk oligosaccharides (HMOs) comprising at least sialylated, fucosylated and N- acetylated oligosaccharides.

[0064] In a preferred embodiment the nutritional composition according to the invention comprises a specific combination of human milk oligosaccharides consisting of 2’-fucosyllactose (2-’FL), 3-fucosyllactose (3- FL), lacto-N-tetraose (LNT), 3’-sialyllactose (3’-SL) and 6'-sialyllactose (6’-SL).

[0065] The above HMOs may be isolated by chromatography or filtration technology from a natural source such as animal milks. Alternatively, they may be produced by biotechnological means using specific enzymes such as fucosyltransferases and / or fucosidases to produce 2’-FL and 3-FL, sialidases and glycosyltransferases for SLs and LNT, either through the use of enzyme-based fermentation technology (recombinant or natural enzymes) or microbial fermentation technology known in the art. In the latter case, microbes may either express their natural enzymes and substrates or may be engineered to produce respective substrates and enzymes. Single microbial cultures and / or mixed cultures may be used. Alternatively, these HMOs may be produced by chemical synthesis, for example from lactose and free monomers such as fucose, sialic acid, N acetyl glucosamine, galactose. These HMOs are commercially available, for example from Kyowa Hakko, Japan, FrieslandCampina, The Netherlands, DSM / Firmenich, Denmark, Novonesis, Denmark, and Sigma- Aldrich.

[0066] The amount of HMOs is preferably 10 to 400 mg per 100 ml, more preferably 30 to 300 mg per 100 ml, even more preferably 40 to 250 mg per 100 ml. Based on dry weight of the composition the amount of human milk oligosaccharides is preferably 0.11 to 2.35 wt%, more preferably 0.18 to 1.76 wt%, even more preferably 0.24 to 1 .49 wt%. Preferably the amount of human milk oligosaccharides per 100 kcal is 12.5 to 500 mg, more preferably 37.5 to 375 mg, even more preferably 50 to 312.5 mg.

[0067] The amount of each specific HMO is preferably, based on total weight of HMOs, 42 to 62 wt% 2’-FL, 10 to 16 wt% 3-FL, 21 to 31 wt% LNT, 3 to 5 wt% 3’-SL, 4 to 6 wt% 6’-SL, the sum of 2’-FL, 3-FL, LNT, 3’-SL and 6’-SL being 100 %, more preferably 47 to 57 wt% 2’-FL, 11 to 15 wt% 3-FL, 23 to 28 wt% LNT, 3.5 to 4.5 wt% 3’-SL and 4.5 to 5.5 wt% 6’-SL. Such ratios of a mixture of 5 HMOs were found to provide beneficial effects on neuroinflammation, white matter injury, cortical myelination and on interneuron formation in the hippocampus in diffuse white matter injury. Also, mixtures of 5 HMOs were shown to provide beneficial effects on the gut microbiota.

[0068] Combination of component i) GOS and IcFOS and component ii) mixture of 5 HMOs

[0069] In some embodiments the nutritional composition of the present invention comprises a combination of non-digestible oligosaccharides consisting of GOS, IcFOS, 2’-FL, 3-FL, LNT, 3’-SL and 6’-SL. Such a combination may beneficially further improve on neuroinflammation, white matter injury, cortical myelination and on interneuron formation in the hippocampus in diffuse white matter injury.

[0070] The total amount of non-digestible oligosaccharides (consisting of NDOs (i) and ii)) is preferably 120 mg to 2.40 g per 100 ml, more preferably 280 mg to 1 .80 g per 100 ml, even more preferably 540 mg to 1.25 g per 100 ml. Based on dry weight of the composition the amount non-digestible oligosaccharides is preferably 0.71 to 14.12 wt%, more preferably 1.65 to 10.59 wt%, even more preferably 3.18 to 7.35 wt%. Preferably the amount of non-digestible oligosaccharides per 100 kcal is 150 mg to 3.00 g, more preferably 350 mg to 2.25 g, even more preferably 675 mg to 1 .56 g.

[0071] Preferably the nutritional composition according to the present invention comprises 50 to 97.5 wt% of component i) based on total non-digestible oligosaccharides and 2.5 to 50 wt% of component ii) based on total non-digestible oligosaccharides, the sum of component i) and ii) being 100 % of the total non- digestible oligosaccharides. More preferably the present nutritional composition comprises 60 to 95 wt% of component i) based on total non-digestible oligosaccharides and 5 to 40 wt% of component ii) based on total non-digestible oligosaccharides, the sum of component i) and ii) being 100 %. Most preferably the present nutritional composition comprises 75 to 90 wt% of component a) based on total non-digestible oligosaccharides and 10 to 25 wt% of component ii) based on total non-digestible oligosaccharides, the sum of component i and ii being 100 %.

[0072] Preferably the weight ratio of components i to ii ranges from 39 to 1 , more preferably ranges from 19 to 1 .5, even more preferably ranges from 9 to 3. Such ratios will result in further improved effects on neuroinflammation, white matter injury, cortical myelination and on interneuron formation in the hippocampus in diffuse white matter injury.

[0073] Preferably, based on total weight, the mixture of non-digestible oligosaccharides consists of 60 to 90 wt% GOS, 7.5 to 10 wt% IcFOS, 1 .5 to 15 wt% 2’-FL, 0.4 to 4 wt% 3-FL, 0.75 to 7.5 wt% LNT, 0.10 to 1 .2 wt% 3’-SL, and 0.10 to 1 .5 wt% 6’-SL, the sum being 100 %, more preferably, based on total weight, the mixture of non-digestible oligosaccharides consists of 65 to 85 wt% GOS, 7.5 to 9.5 wt% IcFOS, 5 to 12 wt% 2’-FL, 1 to 3 wt% 3-FL, 2 to 6 wt% LNT, 0.2 to 1 .0 wt% 3’-SL, and 0.20 to 1 .2 wt% 6’-SL.

[0074] In a preferred aspect, the total weight ratio of both the mixture of HMOs and the GOS having a DP between 2 and 8 (DP 2-8) to IcFOS have an average DP of 10 to 100 is from 1 / 99 to 99 / 1 , more preferably from 1 / 19 to 19 / 1 , more preferably from 1 / 1 to 19 / 1 , more preferably from 2 / 1 to 15 / 1 , more preferably from 5 / 1 to 12 / 1 , even more preferably from 8 / 1 to 10 / 1 , even more preferably in a ratio of about 9 / 1 . In a preferred aspect a nutritional composition is provided that comprises GOS, IcFOS and the mixture of HMOs and wherein the total ratio of HMO and GOS to IcFOS is about 9:1 .

[0075] In a further preferred aspect, the weight ratio of GOS and IcFOS to HMO ranges from 20 to 1 , more preferably ranges from 10 to 1 , more preferably ranges from 5 to 1 , even more preferably ranges from 2 to 1.

[0076] Nutritional composition

[0077] The present nutritional composition comprises non-digestible oligosaccharides selected from i) galactooligosaccharides and long chain fructo-oligosaccharides and / or ii) a mixture of 5 human milk oligosaccharides [5 HMOs], preferably 2’-fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), lacto-N- tetraose (LNT), 3’-sialyllactose (3’-SL) and 6’-sialyllactose (6’-SL) and preferably further comprises lipids, protein, and carbohydrates. Preferably, the nutritional composition is a preterm or paediatric formula for faltering growth, which is intended for providing nutrition to an infant selected from the group consisting of preterm infants, small for gestational age or infants that fail to thrive or faltering growth meaning infants exhibit significant below expected weight for age, weight for length or body mass index for their age.

[0078] The present nutritional compositions preferably has an increased caloric density, compared to regular infant formulae, which supports growth and development of preterm infants, small for gestational age infants. Preterm infants and small for gestational age infants usually have a little stomach and cannot consume a large amount of nutrition. Preferably, the nutritional composition contains 50 to 200 kcal / 100 ml liquid, more preferably 70 to 120 kcal per 100 ml, more preferably 72 to 100 kcal per 100 ml, even more preferably 75 to 85 kcal per 100 ml. These caloric densities are especially preferred for preterm formulae and low birthweight formulae. For a paediatric formula for faltering growth, the caloric density may be even higher, such as 75 to 150 kcal per 100 ml, preferably 85 to 150 kcal per 100 ml, more preferably 93 to 125 kcal per 100 ml, most preferably 95 to 115 kcal per 100 ml. The osmolarity of the nutritional composition is preferably between 150 and 420 mOsmol / l, more preferably 260 to 380 mOsm / l, even more preferably 280 to 350 mOsm / l. Such an osmolarity is beneficial in the prevention of gastrointestinal stress and ensures proper hydration, which is of importance especially for preterm and SGA infants.

[0079] The nutritional composition according to the invention preferably comprises protein, carbohydrate, and lipid.

[0080] Protein

[0081] Preferably, the protein provides 5 to 20 % of the total calories of the nutritional composition, preferably 8 to 16 %, more preferably 9 to 14 %, more preferably 10 to 13.5 % The nutritional composition preferably comprises 2.0 to 5.0 g protein per '100 kcal, more preferably 2.5 to 4.0, even more preferably 3.0 to 3.5 g / 100 kcal. In ready to drink form the nutritional composition preferably comprises 1 .6 to 4.0 g / 100 ml, more preferably 2.0 to 3.2 g, even more preferably 2.4 to 2.8 g. Based on dry weight the nutritional composition preferably comprises 9.4 to 23.5 g protein / 100 g dry weight, more preferably 11 .8 to 18.8 g, even more preferably 14.1 to 18.8 g.

[0082] In case the nutritional composition is especially designed for infants with a body weight below 1000 g, the protein content is preferably 12.5 to 14 % based on total calories. It is preferred that the nutritional composition comprises 2.1 to 4.1 g protein based on 100 kcal, more preferably 2.4 to 3.4 g per 100 kcal. In case the present nutritional composition is a preterm formula or a low birthweight formula, it is preferred that the composition comprises 2.6 to 3.4 g protein based on 100 kcal. In case the nutritional composition is especially designed for infants with a body weight below 1000 g, the protein content is preferably 3.0 to 3.4 g per 100 kcal. In case the nutritional composition is especially designed for infants with a body weight above 1000 g, the protein content is preferably 2.6 to 3.0 g per 100 kcal.

[0083] In case the present nutritional composition is a pediatric formula for faltering growth, for infants in need of catch-up growth, it is preferred that the composition comprises 2.4 to 2.8 g protein based on 100 kcal. Based on dry weight of the nutritional composition, the amount of protein is preferably 8 to 27 wt.%, more preferably 10 to 25 wt.%, even more preferably 13 - 22 wt.%. In case the present nutritional composition is a preterm formula or a low birthweight formula, it is preferred that the composition comprises 12.5 to 20 wt.% protein, more preferably 13 to 17 wt.%. In case the present nutritional composition is a pediatric formula for faltering growth, it is preferred that the composition comprises 12.5 to 18 wt.% protein, more preferably 13 to 15 wt.%.

[0084] The source of the protein is preferably selected in such a way that the minimum requirements for essential amino acid content are met, and satisfactory growth is ensured. Hence, the source of protein is preferably from bovine milk protein. Preferably, the protein component comprises whey protein and / or casein, more preferably consists of whey protein and / or casein, most preferably is a mixture of whey protein and casein. Preferably, the weight ratio of whey protein: casein is 70 : 30 to 40 : 60, more preferably 65 : 35 to 50 : 50, most preferably about 60 : 40. As such, an optimal amino acid profile is obtained, closely resembling that of human milk, which is beneficial for preterms.

[0085] The protein component may contain intact protein, partially hydrolyzed protein, or free amino acids (i.e. fully hydrolyzed).

[0086] Carbohydrates

[0087] It is preferred that the nutritional composition comprises 8 to 15 g digestible carbohydrates based on 100 kcal, preferably 9 to 12 g, even more preferably 10 to 11 g. Based on dry weight of the nutritional composition, the amount of digestible carbohydrates is preferably 38 to 71 wt.%, more preferably 42 to 56 wt.%, even more preferably 47 to 52 wt%. Based on 100 ml composition, the amount of digestible carbohydrates is preferably 6.4 to 12 g, more preferably 7.2 to 9.6 g, even more preferably 8.0 to 8.8 g per 100 ml.

[0088] Preferred digestible carbohydrate sources are lactose, glucose, sucrose, fructose, galactose, maltose, starch, and maltodextrin, more preferably at least lactose is present, most preferably at least lactose and maltodextrin are present. Lactose is the main digestible carbohydrate present in human milk; thus, the nutritional composition preferably comprises lactose. The nutritional composition preferably comprises digestible carbohydrate, wherein at least 35 wt.%, more preferably at least 50 wt.% of the digestible carbohydrate is lactose. Based on dry weight the present composition preferably comprises at least 25 wt.% lactose.

[0089] Lipid

[0090] The present nutritional composition comprises lipid. The lipid that is present in the nutritional composition provides preferably 30 to 60 % of the total calories of the composition. More preferably the present nutritional composition comprises lipid providing 35 to 55 % of the total calories, even more preferably the present composition comprises lipid providing 40 to 50 % of the total calories. Per 100 kcal, the nutritional composition preferably comprises 3.5 to 7.0 g lipid, more preferably 4.0 to 6.0 g lipid, even more preferably 4.5 to 5.0 g. When in liquid form, e.g. as a ready-to-feed liquid, the nutritional composition preferably comprises 2.8 to 5.6 g lipid per 100 ml, more preferably 3.2 to 4.8 g per 100 ml, even more preferably 3.6 to 4.0 g / 100 ml. Based on dry weight the nutritional composition preferably comprises 16.5 to 32.9 wt.%, more preferably 18.8 to 28.2.9 wt.% lipid, even more preferably 21.2 to 23.5 wt.% lipid.

[0091] The lipid that is present in nutritional composition according to the invention preferably comprises vegetable lipids. The presence of vegetable lipids advantageously enables an optimal fatty acid profile, high in (poly)unsaturated fatty acids and / or more reminiscent to human milk fat. Using lipids from cow’s milk alone, or other domestic mammals, does not provide an optimal fatty acid profile. Preferably the present composition comprises at least one, preferably at least two lipid sources selected from the group consisting of linseed oil, canola oil, coconut oil, sunflower oil, palm kernel oil, and high oleic sunflower oil. In a preferred aspect the present composition comprises coconut oil and / or palm kernel oil.

[0092] The lipid that is present in nutritional composition according to the invention preferably comprises milk fat. The palmitic acid present in milk fat is, compared to vegetable oil, to a higher degree in the sn2 position of the triglyceride molecule. This will result in a better digestion of the palmitic acid and in softer stools as there is less formation of calcium palmitic acid soaps. Softer stools and improved fat digestion is important in preterm infants. Furthermore, cow’s milk fat is a source of dietary butyrate. Butyrate supports the intestinal health. Preferably 10 to 50 wt%, more preferably 20 to 40 wt% of the lipid is derived from milk fat, preferably cow’s milk fat.

[0093] Preferably, the nutritional composition comprises at least one source of vegetable lipid selected from sunflower oil, rapeseed oil, coconut oil and palm oil.

[0094] Furthermore, it is preferred that at least one lipid source selected from fish oil (preferably tuna fish oil), single cell oil (such as algal, microbial oil and fungal oil), MCT oil and egg lipid is present. These sources of oil are suitable as LC-PUFA and MCFA sources. Preferably as a source of n-3 LC-PUFA single cell oil, including algal oil and microbial oil, is used. Faty acid composition

[0095] Herein LA refers to linoleic acid and / or acyl chain (18:2 n6); ALA refers to alpha-linolenic acid and / or acyl chain (18:3 n3); PUFA refers to polyunsaturated fatty acids and / or acyl chains; MUFA refers to monounsaturated fatty acids and / or acyl chains; LC-PUFA refers to long chain polyunsaturated fatty acids and / or acyl chains comprising at least 20 carbon atoms in the fatty acyl chain and with 2 or more unsaturated bonds; DHA refers to docosahexaenoic acid and / or acyl chain (22:6, n3); EPA refers to eicosapentaenoic acid and / or acyl chain (20:5 n3); ARA refers to arachidonic acid and / or acyl chain (20:4 n6); DPA refers to docosapentaenoic acid and / or acyl chain (22:5 n3). Medium chain fatty acids (MCFAs) refer to fatty acids and / or acyl chains with a chain length of 6, 8 or 10 or 12 carbon atoms. Medium chain triglycerides (MCT) in turn refer to medium-chain fatty acid esters of glycerol. A mediumchain triglyceride (MCT) is a type of fat composed of two or three fatty acids with a chain length of 6, 8 or 10 or 12 carbon atoms. These fats are found in foods like coconut oil and palm kernel oil. MCTs are metabolized differently than the long-chain triglycerides (LCTs) found in most other foods.

[0096] The present composition preferably comprises at least 3 wt.% MCFA based on total fatty acids, more preferably at least 9 wt.%. In an embodiment the present composition preferably comprises between 3wt% and 15 wt% MCFA, more preferably between 9wt% and 11wt% based on total fatty acids.

[0097] The present composition preferably comprises at least 0.5 wt.% butyrate based on total fatty acids, more preferably at least 0.9 wt.%. In an embodiment the composition comprises between 1 .5 and 1 .5 wt% butyrate, more preferably between 0.8 and 1 .2 wt% based on total fatty acids.

[0098] The present composition preferably comprises MCTs, since MCTs are well adsorbed by the infants and provide beneficial effects to the developing brain. In a preferred embodiment the source of MCT is coconut oil and / or palm kernel oil. The present composition preferably comprises at least 1 ,8g per 100 g, more preferably 2g per 100g even more preferably 2.2g per 100g powdered composition. It is preferred that the nutritional composition comprises more than 0.2g per 100 kcal, more preferably 0.3 to 0.7 g per 100 kcal, even more preferably 0.4 to 0.6g MCTs per 100 kcal. Alternatively worded it is preferred that the nutrition composition comprises at least 0.18 g MCT, more preferably 0.3g to 0.5g of MCT, even more preferably 0.35 to 0.45 g of MCT per 100 ml.

[0099] Preferably the present nutritional composition comprises n-3 LC-PUFA, since n-3 LC-PUFA provide beneficial effect to the developing brain. More preferably, the present nutritional composition comprises EPA, DPA and / or DHA, even more preferably at least DHA. Since a low concentration of DHA, DPA and / or EPA is already effective and normal growth and development are important, the content of n-3 LC-PUFA in the nutritional composition, preferably does not exceed 15 wt.% based on total fatty acid, preferably does not exceed 10 wt.%, even more preferably does not exceed 5 wt.%. Preferably, the nutritional composition comprises at least 0.2 wt.%, preferably at least 0.5 wt.%, more preferably at least 0.75 wt.%, n-3 LC-PUFA based on total fatty acid. The nutritional composition preferably comprises 0.05 - 0.8 wt.%, more preferably 0.15 - 0.7 wt.%, even more preferably 0.25 - 0.6 wt.%, even more preferably 0.4 - 0.55 wt.%, DHA based on total fatty acid. The n-6 LC-PUFA content preferably does not exceed 5 wt.%, more preferably does not exceed 2.0 wt.%, more preferably does not exceed 0.75 wt.%, based on total fatty acid. Since ARA is important in infants for optimal functional membranes, especially membranes of neurological tissues, the amount of n-6 LC-PUFA is preferably at least 0.02 wt.% more preferably at least 0.05 wt.%, more preferably at least 0.1 wt.% based on total lipid, more preferably at least 0.2 wt.%. The presence of ARA is beneficial in nutrition to be administered to infants below the age of 6 months, since for these infants the infant formulae is generally the only source of nutrition. The nutritional composition preferably comprises 0.05 - 0.8 wt.%, more preferably 0.15 - 0.7 wt.%, even more preferably 0.25 - 0.6 wt.%, most preferably 0.4 - 0.55 wt.% ARA based on total fatty acid. The weight ratio of DHA : ARA is preferably from 1 :0.5 to 1 :2.5, more preferably 1 :0.9 to 1 :1.1 ,

[0100] The present nutritional composition is not human breast milk. The nutritional composition according to the invention preferably comprises other ingredients, such as minerals, trace elements, vitamins and other micronutrients as recommended and known in the art.

[0101] The nutritional composition is preferably in the form of a powder or a liquid. In one embodiment, the nutritional composition is in the form of a powder suitable for making a liquid composition after reconstitution with an aqueous solution, preferably with water. Preferably, the composition is a powder to be reconstituted with water.

[0102] In case the nutritional composition is administered to an infant, it is highly preferred that the composition is in the liquid form. The preferred mode of administration is orally, e.g. bottle feeding, but other modes of administration such as tube feeding are also possible.

[0103] Preferably, the nutritional composition is in a liquid form, with a viscosity below 35 mPa.s, more preferably below 6 mPa.s, most preferably 1 to 6 mPa.s, as measured in a Brookfield viscometer at 20 °C at a shear rate of 100 s-1. Suitably, the composition is in a powdered from, which can be reconstituted with water to form a liquid, or in a liquid concentrate form, which could be diluted with water.

[0104] In one aspect the invention relates to a nutritional composition, comprising protein, carbohydrates, and lipid, wherein:

[0105] (i) the caloric density is 70 to 120 kcal per 100 ml,;

[0106] (ii) lipid is present in 4.0 to 6.0 g per 100 kcal;

[0107] (iii) protein is present in 2.5 to 4.0 g per 100 kcal;

[0108] (iv) carbohydrates are present in 9 to 12 g per 100 kcal;

[0109] (v) non-digestible carbohydrates ii) are present in 12.5 mg to 500 mg per 100 kcal.

[0110] In one aspect the invention relates to a preterm formula, a low birthweight formula, comprising protein, carbohydrates and lipid, wherein:

[0111] (i) the caloric density is 75 to 85 kcal per 100 ml; (ii) lipid is present in 4.0 to 6.0 g per 100 kcal;

[0112] (iii) protein is present in 2.5 to 4.0 g per100 kcal;

[0113] (iv) carbohydrates are present in 9 to 12 g per100 kcal;

[0114] (v) non-digestible oligosaccharides i) are present in 100 mg to 2 g per 100 kcal, and non-digestible carbohydrates ii) are present in 12.5 mg to 500 mg per 100 kcal.

[0115] In one aspect the invention relates to a paediatric formula for faltering growth, comprising protein, carbohydrates, and lipid, wherein:

[0116] (i) the caloric density is 93 to 125 kcal per 100 ml;

[0117] (ii) lipid is present in 4.4 to 6.0 g per 100 kcal;

[0118] (iii) protein is present in 2.4 to 2.8 g perl OO kcal;

[0119] (iv) carbohydrates are present in 10 to 12 g perl OO kcal’

[0120] (v) non-digestible oligosaccharides i) and ii) are present in 120 mg to 3 g per 100 kcal.

[0121] Breast milk fortifier.

[0122] In one aspect the nutritional composition is a human milk fortifier (HMF) comprising NDO ii), the mixture of HMOs being as described above. HMF is a powder or concentrated liquid, preferably a powder, added as a supplement to expressed human milk to enhance the nutritional value. It is intended for a preterm or LBW infant. Particular proteins, minerals and vitamins and trace elements are supplemented. Although breastfed preterm infants already receive HMOs through human milk, the types and amount of HMOs in the milk are subject to genetic and environmental factors, and may be variable after preterm delivery. Therefore, additionally the mixture of HMOS could be supplemented to the vulnerable infant via a HMF. When the nutritional composition is a HMF it comprises preferably NDOs comprising ii) 25 mg to 500 mg per 100 kcal of a mixture of human milk oligosaccharides [HMOs], more preferably consisting of 2’-FL, 3-FL, LNT, 3’-SL and 6’-SL, even more preferably of 42 to 62 wt% 2’-FL, 10 to 16 wt% 3-FL, 21 to 31 wt% LNT, 3 to 5 wt% 3’-SL and 4 to 6 wt% 6’-SL, the sum of 2’-FL, 3-FL, LNT, 3’- SL and 6’-SL being 100 % of the total weight of the HMOs. Preferably in addition to the NDO ii) the HMF additionally comprises the NDO i) a mixture of GOS and IcFOS.

[0123] When the nutritional composition is a HMF, said composition preferably comprises per l OO kcal 0 to 5.0 g lipid, 7 to 10 g protein and 6 to 10 g digestible carbohydrates. Per 100 g dry weight the HMF preferably comprises 0 to 21 .5 g lipid, 30.2 to 43.1 g protein, and 25.9 to 43.1 g digestible carbohydrates.

[0124] Target group

[0125] The present invention relates to the use of non-digestible oligosaccharides selected from i) and / or ii) and / or a method for feeding preterm infants and / or infants exposed to perinatal hypoxia, preferably for feeding premature infants. Said infants are at increased risk of, or suffering from, diffuse white matter injury. In some aspects the infants may also be selected from small for gestational age (SGA) infants, (very) low birth weight infants and / or t infants with faltering growth.

[0126] SGA infants are those whose birthweight lies below the 10th percentile for that gestational age. Reasons for SGA can be several; for example, term or preterm infants can be born SGA because they have been the subject of intrauterine growth restriction (IUGR). Many preterm infants are also small for gestational age. Premature and / or SGA infants include low birth weight infants (LBW infants), very low birth weight infants (VLBW infants), and extremely low birth weight infants (ELBW infants). LBW infants are infants with a birth weight below 2500 g; this group includes term infants born SGA. VLBW and ELBW infants are almost always born preterm and are defined as infants with a birth weight below 1500 g or 1000 g, respectively.

[0127] Preferably the infants are human infants. The nutritional composition according to the present invention is especially beneficial for infants that have, or that may be at risk for impaired brain development. The nutritional composition according to the invention is particularly beneficial for preterm infants, small for gestational age infants and infants exposed to perinatal hypoxia. Thus, the target group for the present invention is selected from preterm infants, small for gestational age infants (SGA infants, including IUGR infants), and infants exposed to perinatal hypoxia. Said infants are at risk of brain injury and / or impaired brain development. In a preferred aspect said infant are suffering from or at risk of diffuse white matter injury.

[0128] Preferably, the target group is selected from preterm infants and SGA infants, more preferably preterm infants. A preferred group of SGA infants are the IUGR (intrauterine growth restricted) infants. These infants are due to their delay in development at increased risk of suffering from diffuse white matter injury. Preterm infants have immature lungs therefore are perinatally exposed to hypoxia as amongst others discussed in Di Fiore JM et al Respir Physiol Neurobiol 2019 Aug; 266: 121 - 129 and Di Fiore JM et al. Exp Neurology 2021 Aug; 342: 113753.

[0129] Preferably, the nutritional composition is an infant formula, more preferably a preterm formula, low birthweight formula, post-discharge formula or paediatric formula for infants having faltering growth, in need of catch-up growth, which is intended for providing nutrition to an infant selected from the group consisting of infants exposed to perinatal hypoxia, preterm infants, small for gestational age infants, and infants with retarded growth. Preferably the nutritional composition is a preterm formula.

[0130] Application

[0131] The present composition is preferably enterally administered, more preferably orally to the infant. The present composition can advantageously be applied as a complete nutrition for infants or as a nutritional supplement.

[0132] The present invention aims to promote neuronal development in infant selected from infants exposed to perinatal hypoxia, preterm infants, small for gestational age infants (SGA infants, including IUGR infants) and infants with retarded growth that are at risk of impaired brain development. These infants are suffering from or at risk of brain injury and / or impaired brain development and / or diffuse white matter injury.

[0133] Diffuse white matter injury is associated with impaired interneuron development, impaired white matter development and neuroinflammation, which in turn affects brain development as neuron functioning and circuit formation are impaired. The alterations can all be associated with impaired neurocognitive development / function and present a risk for the development of a variety of neurodevelopmental disorders including cerebral palsy, schizophrenia, and epilepsy.

[0134] The nutritional composition of the present method or use is preferably suitable for providing nutrition to infants exposed to perinatal hypoxia, preterm infants, small for gestational age infants (SGA infants, including IUGR infants) and infants with retarded growth that are at risk of brain injury and / or impaired brain development, preferably preterms. The nutritional composition of the present invention is preferably suitable for use or in a method of providing nutrition to infants suffering from or at risk of diffuse white matter injury. In a preferred embodiment the infant is a preterm suffering from diffuse white matter injury

[0135] The nutritional composition according to the invention is for use in providing nutrition to an infant, preferably infants exposed to perinatal hypoxia, preterm infants, small for gestational age infants (SGA infants, including IUGR infants) and infants with retarded growth, preferably directly after birth and up to 12 months of age. Infants are particularly vulnerable short after birth hence it is preferred that the nutritional composition according to the invention is administered to the infant starting at least in the first two weeks after birth, preferably within the first week after birth, more preferably at least within 5 days after birth, even more preferably at least within 3 days after birth, most preferably at least within 2 days after birth.

[0136] Preferably in the methods or uses according to the invention, the nutritional composition is administered in the first 24 months of life. However, as there are scientifically-based indications that the window of opportunity wherein the infant is more susceptible to cognition- and brain-related effects of nutritional interventions lies closer to the day of birth, the nutritional composition is preferably administered in the first 12 months of life, more preferably in the first 6 months of life, more preferably in the first 3 months of life, most preferably in the first month of life. In a preferred embodiment, the composition is administered to preterm infants starting at least in the first two weeks after birth, preferably within the first week after birth, more preferably at least within 5 days after birth, even more preferably at least within 3 days after birth, most preferably at least within 2 days after birth.

[0137] The invention further relates to a composition comprising non-digestible oligosaccharides [NDOs] selected from i) a mixture of galactooligosaccharides and long-chain fructooligosaccharides and ii) a mixture of human milk oligosaccharides, or the composition according to the invention for use as a medicament. The invention further relates to a composition comprising non-digestible oligosaccharides [NDOs] selected from i) a mixture of galactooligosaccharides and long-chain fructooligosaccharides and / or ii) a mixture of human milk oligosaccharides for use in treating and or improving brain injury and / or impaired brain development. In a preferred embodiment preventing and / or treating brain injury and / or impaired brain development involves improving interneuron development. In some embodiments the use pertains to preventing and / or treating diffuse white matter injury.

[0138] In an embodiment wherein the NDOs comprise ii) a mixture human milk oligosaccharides comprising at least sialylated, fucosylated, and N-actylated oligosaccharide, preventing and / or treating brain injury and / or preventing and / or treating impaired brain development is selected from treating and / or preventing diffuse white matter injury, neuroinflammation; and / or impaired central myelination.

[0139] The nutritional composition is for use in preventing, reducing and / or treating neuroinflammation, improving interneuron development and / or improving cortical myelination in infants suffering from or at risk of diffuse white matter injury. Said composition is further for use in improving brain development and treating impaired brain development in said infants.

[0140] FIGURES

[0141] Figure 1 illustrates cortical myelination was rescued by HMOS treatment in rats exposed to fetal inflammation and postnatal hypoxia (FIPH). NDO treatment significantly increased the percentage of MBP+ signal in layers l-IV (A) and V-VI (B) of the cortex, C) MBP+ microstructure showed less intersections (F) in layers l-IV in water-treated, but not in HMOS-treated, FIPH rats, D) and E) Total oligodendrocyte-lineage cells were not different between groups (D) but 5 HMO treated animals had more mature oligodendrocytes (I) in layers l-IV.

[0142] Figure 2 HMOS and GOS / FOS treatment rescued the number of Parvalbumin+ interneurons in the hippocampus of rats exposed to fetal inflammation and postnatal hypoxia. A) Water-treated FIPH rats had less PVALB+ cells per mm2 than control rats in the cerebral cortex. B) Water-treated FIPH animals had less PVALB+ interneurons in area CA1 of the hippocampus, while this phenotype was reversed by treatment with HMOS or GOS / FOS

[0143] Figure 3 Microglial morphology was significantly altered by treatment with HMOS or GOS / FOS. At postnatal day 20, FIPH animals had less branched microglia in layers l-IV (D) and V-VI (E) ofthe cortex. Treatment with HMOS restored the number of branch endpoints to control levels while GOS-FOS showed a similar trend.

[0144] EXAMPLES

[0145] The invention is further illustrated by the following examples.

[0146] Example 1 Preterm formula comprising 5 HMOs and GOS / lcFOS

[0147] A preterm or low birth weight formula comprising per 100 ml, of which 87.4 g water and 16.9 g dry matter:

[0148] Energy: 81 kcal

[0149] Protein: 3.3 g (bovine whey protein / casein 6 / 4 wt / wt ratio)

[0150] Digestible carbohydrates: 8.40 (2.37 g starch, 5.73 sugars (mainly lactose))

[0151] Lipid: 3.9 g, a mix of vegetable oils and marine oil

[0152] Non digestible oligosaccharides: 0.172 g of a combination of 5 HMOs consisting of 52 wt% 2’-FL,

[0153] 13 wt% 3-FL, 26 wt% LNT, 4 wt% 3’-SL and 5 wt% 6’-SL (Chr Hansen, Danmark). 0.7 g GOS / lcFOS in a 9:1 wt / wt ratio

[0154] Vitamins, minerals, and other micronutrients according to guidelines.

[0155] Example 2

[0156] Post-discharge formula comprising GOS, IcFOS and 5 HMOs

[0157] A post discharge formula for a preterm or low birth weight infant after discharge comprising per 100 ml, of which 87.4 g water and 14.6 g dry matter:

[0158] Energy: 72 kcal

[0159] Protein: 2.704 g (bovine whey protein / casein 6 / 4 wt / wt ratio)

[0160] Digestible carbohydrates: 7.20 (lactose and maltodextrin))

[0161] Lipid: 3.9 g, a mix of vegetable oils and marine oil

[0162] Non digestible oligosaccharides: 0.7 g GOS / lcFOS in a 9:1 wt / wt ratio

[0163] 0.172 g of a combination of 5 HMOs consisting of 52 wt% 2’-FL,

[0164] 13 wt% 3-FL, 26 wt% LNT, 4 wt% 3’-SL and 5 wt% 6’-SL

[0165] Vitamins, minerals, and other micronutrients according to guidelines.

[0166] Example 3

[0167] The effects of a mixture of 5 HMOs and a mixture of GOS and IcFOS was assessed in a "Fetal- Inflammation Postnatal Hypoxia” rat model of diffuse white matter injury.

[0168] Materials and methods

[0169] Wistar rats (Envigo, Horst, The Netherlands) were kept under standard housing conditions. Timed- pregnant animals received 10 pg / kg lipopolysaccharides (LPS) (UltraPure lipopolysaccharide (LPS) from Escherichia Coli O5:B55 (tlrl-pb5lps, batch #9588-42-01 , Invivogen, France) in 1 ml / kg natrium chloride (NaCI)) interperitoneally at embryonic day (E) 20, which has previously been shown to induce cytokine expression in the fetal brain (Cai, Pan, Pang, Evans, & Rhodes, 2000). Healthy control animals received 1 ml / kg NaCI as a vehicle. Pups were born between E22 and E23. At postnatal day (P) 4, offspring from LPS treated dams were placed in a temperature-controlled hypoxic chamber containing 8% O2 in N2 or in a normoxic temperature-controlled environment (i.e., not in the home cage to circumvent potential confounding effects of maternal deprivation) for 140 min. Healthy control pups from healthy control dams remained under normoxic conditions. Pups of both sexes were used. This doublehit model called the “Fetal Inflammation Postnatal Hypoxia” model e.g., FIPH results in brain injury typically observed in infants that are born preterm including impaired interneuron development, impaired myelination and increased neuroinflammation.

[0170] On P1 , FIPH pups were randomly assigned to one of three treatments: Daily intragastric treatment with water as untreated control, 5HMOs or GOS / FOS (0.25 g oligosaccharides per 100g bodyweight, dissolved in 10 pl sterile water per g bodyweight per day). 5HMOs was a mixture of 5 HMOs consisting of consisting of 52 wt% 2’-FL, 13 wt% 3-FL, 26 wt% LNT, 4 wt% 3’-SL and 5 wt% 6’-SL by Chr Hansen. GOS / FOS was GOS and IcFOS in a 9:1 wt / wt ratio were Vivinal GOS (Friesland Campina) and Raftilin HP (Orafti) respectively.

[0171] At P20, pups were euthanized using intraperitoneal injection with 20% pentobarbital and were transcardially perfused with phosphate buffered saline (PBS), followed by 4% paraformaldehyde (PFA) in PBS. Brains were collected and were fixed in 4% PFA for 24 hr, dehydrated and embedded in paraffin. Coronal tissue sections of 8 pm were made of Bregma -3.3mm. Sections were rehydrated and antigen retrieval was performed using citrate buffer (pH 6.0) at 95°C. Samples were washed and incubated with blocking buffer for 1 h at room temperature, and incubated with primary antibodies against Myelin Basic Protein (MBP; 1 :2000, ab218011 , Abeam, UK), Oligodendrocyte Transcription Factor 2 (OLIG2; 1 :500, ab9610, Chemicon [Merck], Germany), Anti-Adenomatous Polyposis Coli (APC) clone CC1 (CC1 ; 1 :300, OP80, Calbiochem [Merck]), Ionized Calcium-Binding Adaptor Molecule 1 (IBA1 ; 1 :2000, 019- 19741 , FUJIFILM Wako Chemicals, Japan), Parvalbumin (PVALB; 1 :1000, P3088, Merck), and COUP- TF interacting protein 2 (CTIP2; 1 :1000, ab18465, Abeam) at 4°C overnight. The next day, samples were washed and incubated with secondary antibodies (Abeam, Invitrogen [ThermoFisher Scientific], MA, USA) for 1 h at room temperature and with 4',6-dia-midino-2-fenylindool (DAPI; 1 :5000, D9542, Merck) for 5m, and embedded in FluorSave reagent (345789, VWR, the Netherlands). Images were acquired using a fluorescence microscope (Axiovision Z1 , Carl Zeiss, Germany) and ZEN Blue software (Carl Zeiss, Germany) at 10x magnification (OLIG2 / CC1) or 20x magnification (MBP, PVALB, IBA1) in layers l-IV and V-VI in the primary somatosensory cortex (trunk area), at a fixed distance from the cingulum, and at 10x magnification for PVALB cells in the hippocampus (CA1).

[0172] Statistics

[0173] Data discussed below was analyzed using IBM SPSS Statistics (v.29.0, IBM, NY, USA). Data was checked for outliers used Rout’s method (1 %) and any outliers were removed. Normality was assessed using Shapiro-Wilk and Kolmogorov-Smirnov tests, followed by Levene’s test for homogeneity of variances. Data was analyzed using a predefined, step-wise analysis plan, using full factorial linear mixed models, or non-parametric equivalent, including experimental group and sex. Firstly, to determine the presence of a therapeutic window, the FIPH model-effect was tested between the healthy control group and the water-treated FIPH group. If a significant FIPH model-effect was detected (indicated by asterisks in all graphs), all groups were compared using a second linear mixed model, followed by multiple comparisons using Sidak’s correction (family of six comparisons, indicated by dollar-signs). Significant effects were reported when p < .05. Graphs were created using Graphpad Prism software (v.9.3, CA, USA) and data is presented as mean ± standard error of the mean (SEM). As used herein the following symbols have the following meaning: * p < 0.05, ** p < .01 , *** p < .001 , $ corrected p < .05, $$ corrected p < .01 , $$$ corrected p < .001 .

[0174] 5HMOs restore cortical myelin after fetal inflammation and postnatal hypoxia

[0175] To examine whether non-digestible oligosaccharide treatment improved white matter injury in the double-hit model of diffuse white matter injury, MBP+ immunofluorescence was quantified in layers l-IV (CTIP negative) and V-VI (CTIP positive) of the cortex at bregma -3.3mm at P20. In both areas, water- treated FIPH animals had significantly less MBP+ area than controls (layer l-IV: p < .001 , Figure 1A, layer V-VI: p = .002, Figure 1 B), while intragastric treatment with HMOS improved cortical MBP+ area compared to the water-treated group (p = .033 and p = .038, respectively). There was no significant difference between control animals and H MO-treated animals (p = .275 and p = .994) and the difference between 5HMOs and GOS / FOS-treated animals was not significant (p = .733 and p = .088).

[0176] To examine the MBP+ microstructure the number of intersections and total fiber length were quantified in cortical layers l-IV and V-VI. The number of intersections was significantly decreased in the water- treated FIPH groups compared to healthy controls (both p < .001), while intersections were not different between the 5HMOs-treated group and controls (p = .481 , Figure 1 C). Total MBP+ fiber length was reduced in the water-treated group (p = .003) but not in the 5HMOs group (p = .656) or GOS / FOS group (p = .090) wherein the total MBP+ fiber length was found to be comparable to healthy controls.

[0177] Since white matter injury is associated with a reduced number of mature, myelinating oligodendrocytes, these cells were examined in layers l-IV of the cortex. Although there was no difference in the total number of OLIG2+ oligodendrocyte-lineage cells between control vs. water-treated FIPH animals (p = .329, Figure 1 D), the number of mature oligodendrocytes was found to be significantly decreased in the water-treated FIPH group (p = .008), but not in the 5HMOs-treated group (p = .326), compared to healthy controls (Figure 1 E). A similar trend was observed in the GOS / FOS-treated group.

[0178] 5HMOs and GOS / FOS rescue Parvalbumin+ interneurons in the hippocampus

[0179] Diffuse white matter injury has been linked to interneuron deficits, specifically of Parvalbumin+ cells, therefore these cells were examined in the hippocampus and cortex. In the cortex, a decrease of PVALB+ cells per mm2 was observed in FIPH rats treated with water (p = .043), but not when treated with HMOS (p = .921) or GOS / FOS (p = .120) compared to healthy controls (Figure 2A).ln the hippocampal region CA1 , a decrease in the number of PVALB+ interneurons was found in water-treated FIPH animals compared to controls (p = .014), while both treatment with 5HMOs and GOS / FOS restored this deficit (p = .016 and p = .020, respectively compared to water-treated FIPH rats, respectively) (Figure 2B).

[0180] HMOs modulate microglial activation

[0181] To examine whether HMOS and GOS / FOS contribute to improved myelin and interneuron development by exerting an effect on microglia the morphology and activation pattern thereof was assessed.

[0182] Water-treated rats exposed to fetal inflammation and postnatal hypoxia showed an altered microglial morphological state, with less microglial branches per cell, persisting until P20 (layer l-IV: p = .036, layer V-VI: p = .045). HMOS-treated animals showed more microglial ramifications compared to water-treated animals (Figure 3A-B), suggesting that NDO treatment was able to restore this phenotype. In sum, treatment with 5 HMOs induced unique microglial activation states that appear to mitigate injury and potentially modulate neuroinflammation. Activated microglia can modulate neuroinflammation by secreting inflammatory mediators and signaling molecules.

[0183] Overall, using an animal model of diffuse white matter injury it is shown that both the mixture of 5 HMOs as well as a mixture of GOS and IcFOS allows for improvement of interneuron development, in particular in the hippocampus and. Additionally, the mixture of 5 HMOs significantly improved or reduced the exte.nt of the white matter injury as reflected by the restoration of cortical myelination to the level observed in the non-diseased control animals. A similar trend was observed for the combination of GOS-FOS. The mixture of 5 HMOs further reduced the amount of branched microglia ends thereby dampening neuroinflammation.

[0184] These results are indicative of improved neurodevelopmental outcome.

Claims

Claims1 . Nutritional composition comprising non-digestible oligosaccharides [NDOs] selected from i) a mixture of galactooligosaccharides and long-chain fructooligosaccharides and / or ii) a mixture of human milk oligosaccharides comprising at least sialylated, fucosylated and N-acetylated oligosaccharides, for use in preventing and / or treating brain injury and / or preventing and / or treating impaired brain development in infants exposed to perinatal hypoxia and / or preterm infants.

2. Nutritional composition for use according to claim 1 , wherein preventing and / or treating brain injury and / or preventing and / or treating impaired brain development comprises improving interneuron development.

3. Nutritional composition for use according to the preceding claims wherein the infants exposed to perinatal hypoxia and / or preterm infants are at risk of or suffering from diffuse white matter injury.

4. Nutritional composition for use according to the preceding claims wherein the nutritional composition comprises the NDO ii) a mixture human milk oligosaccharides comprising at least sialylated, fucosylated and N-acetylated oligosaccharides and wherein preventing and / or treating brain injury and / or preventing and / or treating impaired brain development is selected from treating and / or preventing- diffuse white matter injury; and / or- neuroinflammation; and / or- impaired central myelination.

5. Nutritional composition for use according to the preceding claims wherein the nutritional composition is a preterm formula or a paediatric formula for faltering growth.

6. Nutritional composition for use according to the preceding claims, wherein the NDOs comprise ii) a mixture of HMOs, wherein the mixture of HMOs consists of 2’-fucosyllactose (2’-FL), 3- fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3’-sialyllactose (3’-SL) and 6’-sialyllactose (6 - SL).

7. Nutritional composition for use according to claim 6, wherein the NDOs comprise ii) a mixture of HMOs and wherein the HMOs consist of 42 to 62 wt% 2’-FL, 10 to 16 wt% 3-FL, 21 to 31 wt% LNT, 3 to 5 wt% 3’-SL and 4 to 6 wt% 6’-SL, the sum of 2’-FL, 3-FL, LNT, 3’-SL and 6’-SL being 100 % of the total weight of component ii).

8. Nutritional composition for use according to the preceding claims, wherein the NDOs comprise i) a mixture of GOS and IcFOS, said mixture consisting of 70 to 95 wt% GOS, preferably 85 to 95 wt% GOS, based on total weight of component i), and 5 to 30 wt% IcFOS, preferably 5 to 15 wt% IcFOS, based on total weight of component i),9. Nutritional composition for use according to the preceding claims, wherein the nutritional composition is administered in at least the first three months of life of the infant, preferably in at least the first month of the life of the infant.

10. Nutritional composition for use according to the preceding claims, wherein the nutritional composition comprises 70 to 120 kcal per 100 ml, 4.0 to 6.0 g lipids per 100 kcal; 2.5 to 4.0 g protein per 100 kcal, 9 to 12 g digestible carbohydrates per 100 kcal, and 20 mg to 2.40 g per 100 ml non-digestible oligosaccharides selected from i) and / or ii).

11. Nutritional composition comprising 70 to 120, preferably 75 to 85 kcal / 100 ml of the nutritional composition,4.0 to 6.0 g lipids per 100 kcal of the nutritional composition;2.5 to 4.0 g protein per 100 kcal of the nutritional composition,9 to 12 g digestible carbohydrates per 100 kcal of the nutritional composition, and non-digestible oligosaccharides [NDOs], said NDOs comprising ii) 25 mg to 500 mg per 100 kcal of the nutritional composition of a mixture of human milk oligosaccharides [HMOs] consisting of 42 to 62 wt% 2’-FL, 10 to 16 wt% 3-FL, 21 to 31 wt% LNT, 3 to 5 wt% 3’-SL and 4 to 6 wt% 6’-SL, the sum of 2’-FL, 3-FL, LNT, 3’-SL and 6’-SL being 100 % of the total weight of the HMOs and wherein the nutritional composition is a preterm formula or a faltering growth formula.

12. Nutritional composition according to claim 11 wherein the composition comprises said NDOs, the NDOs further comprising i) 125 mg to 2.5g of galactooligosaccharides and long-chain fructooligosaccharides.

13. Nutritional composition according to claims 1 1 and 12 wherein the composition comprises a total of amount of NDOs consisting of i) and ii) of 150 mg to 3.00 g per 100 kcal of the nutritional composition.

14. Nutritional composition according to claims 11 to 13 wherein the nutritional composition comprises 0.3 to 0.7 g Medium Chain Fatty Acids (MCFAs) per 100 kcal of the nutritional composition.

15. Human milk fortifier composition non-digestible oligosaccharides [NDOs], said NDOs comprising ii) 25 mg to 500 mg per 100 kcal of the nutritional composition of a mixture of human milk oligosaccharides [HMOs] consisting of 42 to 62 wt% 2’-FL, 10 to 16 wt% 3-FL, 21 to 31wt% LNT, 3 to 5 wt% 3’-SL and 4 to 6 wt% 6’-SL, the sum of 2’-FL, 3-FL, LNT, 3’-SL and 6’-SL being 100 % of the total weight of the HMOs, and preferably comprising 0 to 5.0 g fat per 100 kcal, 7 to 10 g protein per 100 kcal and 6 to 10 g digestible carbohydrates per 100 kcal of the nutritional composition.

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