Nutrition with human milk oligosaccharides for improving behaviour later in life

Abstract

The invention pertains to synthetic nutritional compositions comprising a mixture of human milk oligosaccharides, said mixture comprising at least one sialylated oligosaccharide, at least one fucosylated oligosaccharide, and at least one N-acetylated oligosaccharide, for use in preventing and / or reducing the risk of occurrence and / or severity of a behaviour disorder and / or mental disorder later in life in a human subject.

Description

[0001] Nutrition with human milk oligosaccharides for improving behaviour later in life

[0002] FIELD OF THE INVENTION

[0003] The invention relates to nutritional interventions for infants for improving behaviour later in life.

[0004] BACKGROUND OF THE INVENTION

[0005] Human milk is the most nutritionally complete and natural food for babies. It consists of nutrients, such as proteins, lipids, carbohydrates, minerals, vitamins, and trace elements that babies need to grow healthy. Human milk also contains immune-related components such as IgA, leukocytes, human milk oligosaccharides (HMOs), lysozyme, lactoferrin, interferon-y, nucleotides, cytokines, and others. Based on dry mass, HMOs are the third largest milk compound group present in human milk. Mature human milk contains 5 to 15 g / l of HMOs and to date over 130 individual structures have been identified.

[0006] The structural complexity and abundance of these HMOs is unique for human milk; in milk of other mammalian species the level and number of different non-digestible oligosaccharides is much lower. HMOs play a vital role in various aspects of the early development of young children. HMOs improve the intestinal microbiota by stimulating bifidobacteria and other beneficial lactic acid producing bacteria and thereby inhibit the growth of potentially pathogenic bacteria. HMOs furthermore inhibit binding of pathogenic microorganisms to the infant's epithelial cell surface. HMOs have also been shown to improve neurodevelopment and cognition (Martin et al., 2016, Nutrients 8:279). A link between 2-fucosyllactose (2’-FL) consumption, and cognitive development in the first month of life of breastfed infants has been identified (Berger et al., 2020, PlosOne 15: e0228323). Higher levels of HMO 6’-sialyllactose (6’-SL) in breast milk were associated with better social outcomes and increased myelination in areas involved in social behaviour in infants at 12 months of age, while levels of 3-fucosyllactose (3-FL) were associated with improved language skills (Rajhans et al., 2023, Nutritional Neuroscience, 23: 896-910).

[0007] Breast-feeding is the preferred method of feeding infants. It has been suggested that breast feeding early in life might influence the occurrence of disorders later in life. However, in some cases breastfeeding is inadequate or unsuccessful for medical reasons or not available because of a choice not to breastfeed. In those cases, infant formula and follow-on formula provide a good alternative. The composition of modern infant and follow-on formulas is already highly adapted in such a way that it meets many of the special nutritional requirements of the fast growing and developing infant, and typically include fat, carbohydrate, protein, vitamins, minerals, and other nutrients helpful for optimal infant growth and development. Commercial infant formulas are designed to mimic, as closely as possible, the composition and function of human milk. In recent years HMOs have been included into infant formulae.Still improvements can be made towards the constitution of infant milk formulae. Studies have found that, compared to breast-fed infants, formula fed infants may have higher risk of hyper-activity and attention related issues in adulthood. Breast-fed infants on the other hand may have a slightly reduced risk of developing anxiety and better emotional regulation in life (Castro et al, 2021 , BMC Pregnancy and Childbirth, 21: 62).

[0008] Early-life stress during the prenatal and postnatal periods affects the formation of neural networks that influence brain function throughout life. Early life stress(ELS) may have profound effects on the brain and mental development, and constitute risk factors for brain structural changes and adult psychopathology Rodent studies involving maternal separation have demonstrated that early life stress can lead to decreased brain-derived neurotrophic factor (BDNF) levels, DNA damage, poor synaptic plasticity in key areas of the hippocampus, and an increased risk of later in life behaviour in coping with stressors.

[0009] Fan Yuring et al. Nutrients 2023, 15, 3743 describes the effect of milk oligosaccharides, in particular 2’-FL, 3’-SL and 6’-SL on the brain and on neurocognitive development in early life.

[0010] WO 2014 / 100126 discloses a nutritional composition comprising at least one human milk oligosaccharide selected from 6'sialyllactose (6’-SL), lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), disialylated lacto-N-tetraose (dSLNT), 3-FL, and 3'-sialyllactose (3’-SL). The nutritional composition is used in a method of reducing stress in an acute manner in an individual in need thereof.

[0011] WO 2023 / 237305 describes a nutritional composition comprising a combination of galacto-oligosaccharides (GOS) and a mixture of specific HMOs consisting essentially of 2'FL, difucosyllactose (DFL) or LNT and 6'-SLfor use in treating and / or preventing stress and / or a mood disorder in an infant, young child and / or child, immediate yet no later in life effects are disclosed.

[0012] WO 2020 / 001863 discloses a nutritional composition comprising HMOs selected from 2’-FL, DFL and / or LNT and / or LNnT, or combinations thereof, implicated in improving, enhancing, promoting, or modulating GABAergic function in the CNS, preferably in a human infant or a young child, preterm or term, between birth and 7 years during intervention with the composition.

[0013] WO 2024 / 165630 describes the segregation behaviour of powdered milk formula having large lipid globules and 2’-FL powder particles.

[0014] US 2017 / 151203 describes compositions having large lipid globules in the absence of HMOs for the amelioration of the fatty acid composition of brain membranes later in life as well as the use in the development of cognitive or behavioural performance.US 2017 / 273343 A1 describes a composition with lipid globules for improving behaviour including increasing novel object recognition.

[0015] WO2014 / 043368 discloses methods for enhancing learning and / or memory, enhancing memory acquisition, memory retention and recall by inducing a higher long-term potentiation in hippocampal neuronal synapsis in individuals by and during administration of 2’-FLto the individual.

[0016] There are currently no nutritional solutions improving the behaviour later in life when infants were exposed to stress early in life. There is an ongoing need for improved formulas to stimulate brain and behavioural development.

[0017] SUMMARY OF THE INVENTION

[0018] Using a mouse model wherein mice were exposed to early life stress the inventors showed that early life stress induced later in life behaviour impairment. In adulthood behaviour was abnormal compared to mice that had not been stressed early in life. However, it was found that diet intervention early in life with a nutritional composition comprising a mixture of human milk oligosaccharides [HMOs] programs for improved behaviour later in life, in particular normalizing behaviour abnormalities observed as a result of early life stress. The effect was found to be independent from the effect of non-digestible oligosaccharides (NDO) as such, as the total amount of NDO was kept constant. For some behavioural aspects this was even further improved if the lipids in the early life diet were large lipid globules and / or contained milk phospholipids.

[0019] The findings indicate that infants or young children exposed to early life stress and that are at risk of a behaviour disorder and / or mental disorder later in life benefit from the use of a nutritional composition comprising a mixture of human milk oligosaccharides, said mixture comprising at least one sialylated oligosaccharide, at least one fucosylated oligosaccharide, and at least one N-acetylate oligosaccharides.

[0020] LIST OF PREFERRED EMBODIMENTS

[0021] 1. Synthetic nutritional composition comprising a mixture of human milk oligosaccharides, said mixture comprising at least one sialylated oligosaccharide, at least one fucosylated oligosaccharide, and at least one N-acetylated oligosaccharide, for use in preventing and / or reducing the risk of occurrence and / or severity of a behaviour disorder and / or mental disorder later in life in a human subject,

[0022] wherein the nutritional composition is administered to said human subject at an age of 0 to 48 months and wherein the behaviour disorder and / or mental disorder later in life is at an age of 6 years, preferably 12 years or older.2. Synthetic nutritional composition comprising a mixture of human milk oligosaccharides, said mixture comprising at least one sialylated oligosaccharide, at least one fucosylated oligosaccharide, and at least one N-acetylate oligosaccharide, for use in reducing the risk of occurrence of abnormal behaviour later in life in a human subject, wherein the nutritional composition is administered to a human subject with an age of 0 to 36 months and the reduced risk is at an age of 5 years or older.

[0023] 3. Synthetic nutritional composition for use according to embodiments 1 and 2 wherein the human subject is exposed to early life stress, wherein early life stress exposure occurs during the period covering the third trimester of pregnancy until after birth at an age to 48 months of age.

[0024] 4. Synthetic nutritional composition for use according to embodiments 1 - 3 wherein the human subject is at risk of a behaviour disorder and / or mental disorder or at risk of abnormal behaviour is selected from the group consisting of abnormal low anxiety-like behaviour, increased impulsivity, recklessness, carelessness, abnormal reactivity, excessive level of indifference, excessive risk taking, disinhibited behaviour, excessive temerity, excessive foolhardiness, excessive disregard, excessive nonchalance, excessive insouciance, excessive temerity, excessive negligence, excessive risk taking, hyperactivity, depressive-like behaviour, low self-care behaviour, autism, ADHD, PTSD, ODD, CD, bipolarity, schizophrenia, borderline personality disorder.

[0025] 5. Synthetic nutritional composition for use according to any one of embodiments 3 and 4 wherein the human subject exposed to early life stress is a selected from a preterm, small for gestational age infant, (very) low birth weight infant, an infant exposed to hypoxia, an infant or young child that was hospitalized and / or an infant or young child that was malnourished and / or exposed to nutritional imbalances and / or with faltering growth.

[0026] 6. Synthetic nutritional composition for use according to the preceding embodiments, wherein the nutritional composition comprises lipids in the form of lipid globules and wherein

[0027] a. the lipid globules have a mode diameter based on volume of at least 2.0 pm; and / or at least 40 vol.% of the lipid globules based on total lipid volume have a diameter of 2 to 12 pm; and / or

[0028] b. the lipid comprises 0.5 to 20 wt.% milk-derived phospholipids based on total lipids.

[0029] 7. Synthetic nutritional composition for use according to the preceding embodiments, wherein the mixture of HMOS comprises, preferably consists essentially of 2’-fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3’-sialyllactose (3’SL) and 6’-sialyllactose (6’SL).8. Synthetic nutritional composition for use according to the preceding embodiments, wherein the nutritional composition comprises galactooligosaccharides (GOS) and / or long-chain fructooligosaccharides (IcFOS).

[0030] 9. Synthetic nutritional composition for use according to the preceding embodiments, wherein the nutritional composition provides early life nutrition to infants at 0-12 months.

[0031] 10. Synthetic nutritional composition for use according to the preceding embodiments, wherein the nutritional composition is selected from an infant formula, follow-on formula and young child formula.

[0032] 11. Synthetic nutritional composition for use according to any one of the preceding embodiments, wherein the mixture of 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 HMOs in the composition.

[0033] 12. Non-therapeutic method for improving resilience to a stressor later in life in a human subject, comprising administering a synthetic nutritional composition, said composition comprising a mixture of human milk oligosaccharides comprising at least one sialylated oligosaccharide, at least one fucosylated oligosaccharide, and at least one N-acetylated oligosaccharide, wherein the nutritional composition is administered to a human subject with an age of 0 to 48 months and wherein the improved resilience is at an age of 6 years or older, preferably 12 years or older.

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention concerns a nutritional composition for infants and young children comprising a mixture of human milk oligosaccharides (HMOs). The present invention concerns a nutritional composition for infants comprising 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). In a preferred aspect the nutritional composition beneficially comprises lipids in the form of lipid globules wherein a. the lipid globules have a mode diameter based on volume of at least 1.0 pm; and / or at least 40 vol.% of the lipid globules based on total lipid volume have a diameter of 2 to 12 pm; and / or b. the lipid comprises 0.5 to 20 wt.% milk-derived phospholipids based on total lipids.

[0036] Preferably, the use of the present invention is in a human subject, more preferably in a human young child of 1 , 2 ,3or 4 year of age, or an infant of 0 up to 12 months of age, more preferably in a human young child of 1 or 2 year of age, or an infant of 0 up to 12 months of age, even more preferably an infant 0-12 months and most preferably an infant of 0-6 months.Hence, the nutritional composition is advantageously for use in infants or young children, preferably for infants or young children that were exposed to stress early in life.

[0037] 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 reducing the risk of occurrence and / or severity of a behaviour disorder and / or mental disorder later in life in a human subject, wherein the nutritional composition is administered to said human subject at an age of 0 to 48 months and wherein the behaviour disorder and / or mental disorder later in life is at an age of 6 years, preferably 12 years or older. Preferably said use is for infants or young children that were exposed to stress early in life.

[0038] The invention can also be worded as a nutritional composition comprising a mixture of HMOs comprising at least sialylated, fucosylated, and N-acetylated oligosaccharides, preferably a mixture consisting of2’-FL, 3-FL, LNT, 3’-SLand 6’-SLfor use in preventing and / or reducing the risk of occurrence and / or severity of a behaviour disorder and / or mental disorder later in life in a human subject. Preferably the nutritional composition for use in preventing and / or reducing the risk of occurrence and / or severity of a behaviour disorder and / or mental disorder later in life is administered to said human subject at an age of 0 to 48 months and wherein the behaviour disorder and / or mental disorder later in life is at an age of 6 years, preferably 12 years or older. Preferably said use is for infants or young children that were exposed to stress early in life.

[0039] The invention can also be worded as the use of a mixture of 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 in preventing and / or reducing the risk of occurrence and / or severity of a behaviour disorder and / or mental disorder later in life in a human subject.

[0040] Also, in some jurisdictions the invention concerns a method of preventing and / or reducing the risk of occurrence and / or severity of a behaviour disorder and / or mental disorder later in life in a human subject. The method comprises administering to the infant or young child a nutritional composition comprising a mixture of HMOs comprising at least sialylated, fucosylated and N-acetylated oligosaccharides, preferably a mixture consisting of2’-FL, 3-FL, LNT, 3’-SLand 6’-SL.

[0041] In a further aspect the invention further pertains to a n on-therapeutic method for improving resilience to a stressor later in life in a human subject, comprising administering a synthetic nutritional composition, said composition comprising a mixture of human milk oligosaccharides comprising at least one sialylated oligosaccharide, at least one fucosylated oligosaccharide, and at least one N-acetylated oligosaccharide, wherein the nutritional composition is administered to a human subject with an age of 0 to 48 months and wherein the improved resilience is at an age of 6 years or older, preferably 12 years or older.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. The expression "young child" means a child aged between one and less than three years, also called toddler.

[0044] An "infant, young child or child born by C-section" means an infant, young child or child who was delivered by caesarean section. It means that the infant, young child, or child was not vaginally delivered.

[0045] A "preterm" or "premature" means an infant, young child or child who was not born at term. Generally, it refers to an infant, young child or child born prior 37 weeks of gestation.

[0046] An "infant having a low birth weight" means a newborn having a body weight below 2500g (5.5 pounds) either because of preterm birth or restricted fetal growth. It therefore encompasses: infant, young child or child who has / had a body weight from 1500 to 2500 g at birth (usually called "low birth weight" or LBW) infant, young child or child who has / had a body weight from 1000 to 1500 g at birth (called "very low birth weight" or VLBW) infant, young child or child who has / had a body weight under 1000 g at birth (called "extremely low birth weight" or ELBW).

[0047] An "infant born small for gestational age (SGA)" means a baby with birth weights below the 10th percentile for babies of the same gestational age. It therefore encompasses an infant, young child or child who has / had a birth weight below the 10th percentile for babies of the same gestational age.

[0048] The expression "nutritional composition" means a composition which nourishes a subject. This nutritional composition is to be taken orally or parenterally, and it generally includes a lipid or fat source, a protein source and a carbohydrate source, The nutritional composition of the invention is a synthetic nutritional composition, i.e. the nutritional composition is not human or animal’s milk.

[0049] The composition of the present invention is a "synthetic nutritional composition". The expression "synthetic nutritional composition" means a mixture obtained by chemical and / or biological means, which can be chemically identical to the mixture naturally occurring in mammalian milks (i.e. the synthetic combination or synthetic composition is not breast milk).

[0050] The expression "infant formula" as used herein refers to a foodstuff intended for nutritional use by infants during the first months of life and satisfying by itself the nutritional requirements of this category of person(Article 2(c) of the European Commission Directive 91 / 321 / EEC 2006 / 141 / EC of 22 December 2006 on infant formulae and follow-on formulae). It also refers to a nutritional composition intended for infants and as defined in Codex Alimentarius (Codex STAN 72-1981) and Infant Specialities (incl. Food for Special Medical Purpose). The expression "infant formula" encompasses both "starter infant formula" and "followup formula" or "follow-on formula".

[0051] A "follow-up formula" or "follow-on formula" is given from the 6th month onwards. It constitutes the principal liquid element in the progressively diversified diet of this category of person.

[0052] 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.

[0053] A "fucosylated oligosaccharide" is an oligosaccharide having a fucose residue. It has a neutral nature. Some examples are 2'FL (2'-fucosyllactose), 3-FL (3- fucosyllactose), difucosyllactose (DFL), Lacto-difucotetraose (LDFT)), lacto-N- fucopentaose (e.g. 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, difucosyllacto-N-hexaose I, difucosyilacto-N-neohexaose II and any combination thereof.

[0054] A "sialylated oligosaccharide" is a charged sialic acid containing oligosaccharide, i.e. an oligosaccharide having a sialic acid residue. It has an acidic nature. Some examples are 3-SL (3’sialyllactose) and 6'SL (6'sialyllactose).

[0055] The expression "N-acetylated oligosaccharide(s)" encompasses both "N-acetyllactosamine" and "oligosaccharide(s) containing N-acetyl-lactosamine". They are neutral oligosaccharides having an N-acetyl-lactosamine residue. Suitable examples are LNT (lacto-N-tetraose), para-lacto-N-neohexaose (para-LNnH) and LNnT (lacto-N-neotetraose). Further examples are lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, lacto-N-octaose, lacto-N-neooctaose, iso-lacto-N-octaose, para- lacto-N-octaose and lacto-N-decaose.As used herein, the term "degree of polymerization" (DP) means the number of monomer units joined together in a poly- or oligomer.

[0056] The term "stress" means a state of emotional or psychological strain. Stress may be caused by adverse or demanding circumstances, resulting from adverse or demanding circumstances. Psychological stress is an emotional response to an external trigger. It involves feelings of strain and pressure and can be caused by various factors such as work demands, relationship issues, or significant life changes. Chronic psychological stress can lead to serious health problems, including anxiety, depression, and cardiovascular diseases.

[0057] Early-life stress, i.e., stress experienced while under the age of five years from birth and including stress to the foetus during late pregnancy and / or maternal stress during pregnancy and lactation, has been reported to have a significant detrimental effect on performance later in life, including psychological parameters such as increased rates of or susceptibility to depression, anxiety, and abnormal risk-taking behaviour. Increased rates of abnormal low anxiety-like behaviour, hyperactivity, depressive-like behaviour or depression, autism, attention-deficit hyperactivity disorder (ADHD), post-traumatic stress disorder (PTSD), obsessive-compulsive disorder (OCD), conversion disorder (CD), bipolarity, schizophrenia and / or borderline personality disorder have been reported in individuals having experienced early-life stress. Early-life stress exposure may trigger the development of behaviour and / or mental disorders as described in the DSM-5 criteria.

[0058] The term "stress resilience" means the ability to adapt successfully when faced with stress. When stress resilience is impaired, the subject may avoid situations and / or symptoms associated with stress.

[0059] Stress as used herein is a state of mental or emotional strain. An event of psychological stress is any situation or occurrence that challenges an individual’s mental or emotional equilibrium. These events, known as stressors, can vary widely from person to person based on their experiences, coping mechanisms, and personal resilience. Common examples of psychological stressors include relationship conflicts, disagreements or tensions with family, friends, or partners and work-related pressure such as deadlines, high workloads, or job insecurity. Financial difficulties: Struggles with debt, unexpected expenses, or financial instability. Health problems: Personal illness or the illness of a loved one. Major life changes: Moving to a new place, starting a new job, or significant life transitions. Traumatic events are also stress-full events, including experiences such as accidents, natural disasters, or exposure to violence. These stressors can trigger a range of behaviours later in life, including behaving anxious, have less self-care, hyperactive behaviour, recklessness, depression-like behaviours, disturbed sleeping patterns sleep disturbances.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".

[0060] Human milk oligosaccharides

[0061] The nutritional composition comprises a mixture of human milk oligosaccharides (HMOs). The term “human milk oligosaccharides” or “HMO” as used herein refers to non-digestible oligosaccharides which are present in human breast milk.

[0062] The nutritional composition comprises a mixture of at least one fucosylated oligosaccharide and at least one sialylated oligosaccharide. Preferably the nutrition composition comprises a mixture of at least one fucosylated oligosaccharide and at least one sialylated oligosaccharide, wherein the HMO mixture preferably comprises 55 - 75 wt%, more preferably 60 - 70 wt% fucosylated HMOs based on weight of the HMOs. In a further aspect the mixture of HMOs comprises 7.5 - 42.5 wt% sialylated HMOs, more preferably 9- 40 wt% sialylated HMOs based on weight of the HMOs.

[0063] The nutritional composition preferably comprises at least 2 types of HMO, more preferably at least 3 types of HMO, even more preferably at least 4 types of HMO.

[0064] Preferably the fucosylated oligosaccharide is 2’-fucosyllactose (2’-FL) or 3-fucosyllactose (3-FL), and preferably the mixture comprises both 2’-fucosyllactose and 3-fucosyllactose. Preferably the sialylated oligosaccharide is 3'-sialyllactose (3’-SL) or 6'-sialyllactose (6’-SL), and preferably the mixture comprises both 3’-sialyllactose and 6’-sialyllactose.

[0065] In a preferred embodiment, the HMO is selected from 2’FL, 3-FL, DFL, 3’SL, 6’SL, and combinations thereof. More preferably the HMO is selected from 2’FL, 3-FL, 3’SL, 6’SL and combinations thereof. In a particularly preferred embodiment, the nutritional composition comprises at least 4 types of HMO, said 4 types of HMO being 2’FL, 3-FL, 3’SL, and 6’SL. Preferably the HMOs comprises 35 to 55 wt% 2’-FL, 10 to 25 wt% 3-FL, 4 to 10 wt% 3’-SL and 5 to 30 wt% 6’-SL based on total HMO weight. In a preferred aspect the sum of 2’-FL, 3-FL 3’-SL and 6’-SL is at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 90%, even more preferably 100% based on total HMO weight.

[0066] In an alternative preferred embodiment, the nutritional composition comprises 5 types of HMO, said 5 types of HMO being 2’FL, 3-FL, LNT, 3’SL, and 6’SL. More preferably, the HMO comprises 42-62 wt.% 2’FL, 10-16 wt.% 3-FL, 20-30 wt.% LNT, 3-5 wt.% 3’SL, and 4-6 wt.% 6’SL based on total HMO weight.Suitable single HMO for the preparation of the nutritional composition are commercially available, for example from Kyowa Hakko Bio, Japan; Friesland Campina, The Netherlands; DSM / Firmenich, Denmark and Novonesis, Denmark. Otherwise, it is well within the reach of the skilled person to obtain HMO by isolation from suitable sources or by chemical synthesis using methods known in the art.

[0067] When the nutritional composition is a powdered nutritional composition, the composition preferably comprises 300-4000 mg HMO per 100 g dry weight, more preferably 450-2000 mg HMO per 100 g dry weight. When the nutritional composition is a ready-to-drink liquid nutritional composition, the composition preferably comprises 20-400 mg HMO per 100 ml, more preferably 30-300 mg HMO per 100 ml and most preferably 40-250 mg HMO per 100 ml HMO. When expressed in amounts based on calories, preferably the nutritional composition comprises 30-600 mg HMO per 100 kcal, more preferably 45-450 mg HMO per 100 kcal and most preferably 60-375 mg HMO per 100 kcal.

[0068] In terms of doses, the nutritional composition preferably provides 40-600 mg HMO per serving, more preferably 50-500 mg HMO per serving. In terms of doses, the nutritional composition preferably provides a total daily dose of 0.1-10 g HMO, more preferably a total daily dose of 0.2-7 g HMO and most preferably a daily dose of 0.4-4 g HMO.

[0069] GOS and IcFOS

[0070] The nutritional composition comprises beta-galacto-oligosaccharide (bGOS) and long chain fructooligosaccharide (IcFOS). bGOS and IcFOS are both non-digestible oligosaccharides [NDO] which act as a prebiotic. 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.

[0071] 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. 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 1 to 10, more preferably ranging from 2 to 8. In an embodiment the GOS has an average DP of 3 to 7. 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.The bGOS is preferably transgalacto-oligosaccharide. A suitable bGOS is commercially available, for example VivinalOGOS (FrieslandCampina DOMO). Preferably the bGOS is short chain galactooligosaccharide (bGOS) with an average degree of polymerization (DP) in the range of 1 to 10, more preferably in the range of 2 to 8. In an embodiment the GOS has an average DP of 3 to 7.

[0072] The present nutritional composition comprises long chain fructo-oligosaccharides (IcFOS). Asuitable IcFOS is commercially available, for example RaftilinOHP (Orafti). IcFOS is long chain fructo-oligosaccharide (IcFOS) with an average DP in the range of 10-100, more preferably in the range of 20 to 60. A suitable long chain FOS is RaftilinOHP (Orafti).

[0073] Preferably, the weight ratio of bGOS to IcFOS ranges from 100:1 to 1 :10, more preferably from 20:1 to 1 :1 , even more preferably from 7:1 to 10:1 , and most preferably the weight ratio is 9:1. Preferably these weight ratios apply to bGOS and IcFOS.

[0074] Preferably, the weight ratio of bGOS and IcFOS combined to HMO ranges from 20:1 to 1:10, more preferably from 15:1 to 1 :5 and most preferably from 10:1 to 1 :1.

[0075] Preferably, the nutritional composition comprises 80 mg to 2 g of bGOS and IcFOS per 100 ml, more preferably 150 mg to 1.5 g, most preferably 300 mg to 1 g of bGOS and IcFOS per 100 ml. Preferably, the nutritional composition comprises 120 mg to 3 g of bGOS and IcFOS per 100 kcal, more preferably 225 mg to 2.25 g, most preferably 450 mg to 1.5 g of bGOS and IcFOS per 100 kcal. Based on dry weight, the nutritional composition preferably comprises 0.25-20 wt.%, more preferably 0.5-10 wt.%, and most preferably 1.5-7.5 wt.% of bGOS and IcFOS.

[0076] Mixture ofHMOS and GOS / FOS.

[0077] The nutritional composition preferably comprises a mixture of non-digestible oligosaccharides consisting of HMOs as well as bGOS and IcFOS. Such a mixture may beneficially improve behaviour later in life.

[0078] In a preferred aspect the nutritional composition does not comprise other non-digestible oligosaccharides [NDO] than the mixture of HMOs, bGOS and IcFOS according to the invention. Preferably the NDO in the nutritional composition according to the invention consists of at least 90 wt%, more preferably 95 wt% even more preferably at least 98 wt% of the NDO consisting of a mixture of bGOS-lcFOS and HMOs according to the invention. Preferably the NDOs in the nutritional composition comprising the combination of bGOS-IcFOS and HMOs according to the invention consist of at least 90 wt%, more preferably 95 wt% even more preferably at least 98 wt% of the NDS according to the invention. Preferably the NDO in the nutritional composition consists of the mixture of bGOS-lcFOS and HMOs according to the invention.In a preferred aspect, the total weight ratio of both the mixture of HMOs and the bGOS 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.

[0079] In a preferred aspect a nutritional composition is provided that comprises bGOS, IcFOS and the mixture of HMOs and wherein the total ratio of HMO and bGOS to IcFOS is about 9:1.

[0080] In a further preferred aspect, the weight ratio of bGOS 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.

[0081] In yet another preferred aspect the ratio of bGOS to IcFOS to HMOs ranges from 20:1 :0.5 to 2:1 : 1.

[0082] Preferably a nutritional composition according to the present invention comprises 2.5 to 20 wt% total NDO, more preferably 2.5 to 15 wt%, even more preferably 3.0 to 10 wt%, most preferably 5.0 to 7.5 wt%, based on total dry weight of the composition, i.e., the total wt% of both the mixture of HMO, bGOS and IcFOS. When in liquid form, the nutritional composition according to the invention preferably comprises 0.35 to 2.5 wt% total NDO, more preferably 0.35 to 2.0 wt%, even more preferably 0.4 to 1.5 wt%, based on 100 ml of the composition.

[0083] Lipid

[0084] The nutritional composition for use according to the present invention comprises lipid. Lipid in the present invention comprises one or more selected from the group consisting of triglycerides, polar lipids (such as phospholipids, cholesterol, glycolipids, sphingomyelin), free fatty acids, monoglycerides and diglycerides. Preferably the composition comprises at least 70 wt.%, more preferably at least 80 wt.%, even more preferably at least 85 wt.% triglycerides, most preferably at least 90 wt.% triglycerides based on total lipid.

[0085] The lipid provides preferably 30 to 60% of the total calories of the nutritional composition. More preferably the nutritional composition comprises lipid providing 35 to 55% of the total calories, even more preferably the nutritional composition comprises lipid providing 40 to 50% of the total calories. The lipid is preferably present in an amount of 3 to 7g per 100 kcal, more preferably in an amount of 4 to 6g lipid per 100 kcal and most preferably in an amount of 4.5 to 5.5g lipid per 100 kcal. When in liquid form, e.g. as a ready-to-feed liquid, the nutritional composition preferably comprises 2.1 to 6.5g lipid per 100 ml, more preferably 3.0 to 4.0g per 100 ml. Based on dry weight, the nutritional composition preferably comprises 10 to 50 wt.%, more preferably 12.5 to 40 wt.% lipid, even more preferably 19 to 30 wt.% lipid.The lipid preferably comprises vegetable lipid. The presence of vegetable lipid advantageously enables an optimal fatty acid profile high in polyunsaturated fatty acids and / or more reminiscent to human milk fat. Lipid from non-human mammalian milk alone, e.g. cow milk, does not provide an optimal fatty acid profile. The amount of essential fatty acids is too low in non-human mammalian milk.

[0086] Preferably the nutritional composition comprises at least one, preferably at least two vegetable lipid sources selected from the group consisting of linseed oil (flaxseed oil), rape seed oil (such as colza oil, low erucic acid rape seed oil and canola oil), sunflower oil, high oleic sunflower oil, safflower oil, high oleic safflower oil, olive oil, coconut oil, palm oil and palm kernel oil.

[0087] In a preferred embodiment, the nutritional composition comprises 30 to 90 wt.% vegetable lipids based on total lipid, more preferably 35 to 80 wt.%, more preferably 40 to 70 wt.%, more preferably 40 to 60 wt.% vegetable lipid based on total lipid.

[0088] The lipid in the nutritional composition preferably further comprises mammalian milk fat, preferably ruminants milk fat, more preferably the mammalian milk fat is derived from cow milk, goat milk, sheep milk, buffalo milk, yak milk, reindeer milk, and / or camel milk, most preferably the mammalian milk fat is cow milk fat. Preferably the mammalian milk fat is not human milk fat. Preferably the mammalian milk fat comprises at least 70 wt.% triglycerides, more preferably at least 90 wt.%, more preferably at least 97 wt.% triglycerides by weight of the mammalian milk fat.

[0089] Preferably the mammalian milk fat is derived from butter, butter fat, butter oil, and / or anhydrous milk fat, more preferably the mammalian milk fat is derived from anhydrous milk fat and / or butter oil. Such mammalian milk fat sources are high in triglyceride levels. These mammalian milk fat sources may be in the form of a continuous lipid phase ora water-in-oil emulsion. The use ofthese mammalian milkfat sources during the manufacture of the nutritional composition of the present invention enables the formation of lipid globules, wherein each globule comprises a mixture of vegetable fat and mammalian milkfat.

[0090] Mammalian milk fat in the present invention refers to all lipid components of milk, as produced by the mammalians, such as the cow, and is found in commercial milk and milk-derived products. Butter in the present invention is a water-in-oil emulsion comprised of over 80 wt.% milk fat. Butterfat in the present invention relates to all of the fat components in milk that are separable by churning, in other words, present in butter. Anhydrous milk fat (AMF) is a term known in the art and relates to extracted milk fat. Typically, AMF comprises more than 99 wt.% lipid based on total weight. It can be prepared from extracting milk fat from cream or butter. Anhydrous butter oil in the present invention is synonymous with AMF. Butter oil also is a term known in the art. It typically relates to a milk fat extract with more than 98 wt.% lipid and typically is a precursor in the process of preparing anhydrous milk fat or anhydrous butter oil.Preferably the composition comprises 10 to 70 wt.% mammalian milk fat based on total lipid, more preferably 20 to 65 wt.%, more preferably 30 to 60 wt.%, more preferably 40 to 60 wt.% based on total lipid.

[0091] Preferably the ratio of vegetable fat to mammalian milk fat ranges from 3 / 7 to 9 / 1. In a preferred embodiment, the lipid in the nutritional composition comprises:

[0092] a) 35 to 80 wt.% vegetable lipid based on total lipid, and

[0093] b) 20 to 65 wt.% mammalian milk fat based on total lipid, wherein the mammalian milk fat is selected from butter, butter fat, butter oil or anhydrous milk fat.

[0094] More preferably, the lipid in the nutritional composition comprises:

[0095] a) 40 to 70 wt.% vegetable lipid based on total lipid, and

[0096] b) 30 to 60 wt.% mammalian milk fat based on total lipid, wherein the mammalian milk fat is selected from butter, butter fat, butter oil or anhydrous milk fat.

[0097] Most preferably, the lipid in the nutritional composition comprises:

[0098] a) 40 to 60 wt.% vegetable lipid based on total lipid, and

[0099] b) 40 to 60 wt.% mammalian milk fat based on total lipid, wherein the mammalian milk fat is selected from butter, butter fat, butter oil or anhydrous milk fat.

[0100] Compared to vegetable fat, mammalian milk fat is known to have a higher content of palmitic acid (PA) at the sn-2 position of a triglyceride. In a preferred embodiment, the lipid in the nutritional composition comprises at least 10 wt.% PA based on total fatty acids and at least 15 wt.% of PA, based on total palmitic acid, is located at the sn-2 position of a triglyceride. Preferably, the amount of PA is below 30 wt.% based on total fatty acids. More preferably, the amount of PA is from 12 to 26 wt.% based on total fatty acids, even more preferably from 14 to 24 wt.%.

[0101] Preferably, at least 15 wt.% PA, more preferably at least 20 wt.% PA, even more preferably at least 25 wt.% PA, most preferably at least 30 wt.% PA, based on total PA is in the sn-2 or beta position in a triglyceride. Preferably the amount of PA in the sn-2 position in a triglyceride is not more than 45 wt.%, preferably not more than 40 wt.% based on total PA present in the lipid. Preferably the amount of PA in the sn-2 position in a triglyceride is from 25 to 40 wt.% based on total PA.

[0102] Compared to vegetable fat, mammalian milk fat is known to have a higher content of short-chain fatty acids (SCFA) butyric acid (BA; C4:0) and caproic acid (CA; C6:0). In a preferred embodiment, the lipid in the nutritional composition comprises 0.6 to 5 wt.% SCFA being the sum of BA and CA based on total fatty acids. Preferably the nutritional composition comprises less than 5 wt.% BA based on total fatty acids, preferably less than 4 wt.%. Preferably the nutritional composition comprises at least 0.5 wt.% BA based on total fatty acids, preferably at least 0.6 wt.%, preferably at least 0.9 wt.%, more preferably at least 1.2 wt.% BA based on total fatty acids.In a preferred embodiment, the lipid in the nutritional composition comprises:

[0103] • at least 10 wt.% PA based on total fatty acids and at least 15 wt.% of PA, based on total PA, is located at the sn-2 position of a triglyceride; and

[0104] • 0.6 to 5 wt.% SCFA being the sum of BA and CA based on total fatty acids.

[0105] The nutritional composition preferably also comprises one or more lipids selected from fish oil, egg lipid, and microbial, algal, fungal or single cell oils.

[0106] Lipid globule size

[0107] The lipid in the nutritional composition is in the form of lipid globules, wherein:

[0108] a. the lipid globules have a mode diameter, based on volume, of at least 2.0 pm; and / or b. at least 45 volume %, based on total lipid volume, of the lipid globules have a diameter of 2 to 12 pm.

[0109] Lipid is typically present in the nutritional composition in the form of lipid globules. When the nutritional composition is in liquid form, these lipid globules are emulsified in the aqueous phase. Alternatively, when the nutritional composition is in powder form, the lipid globules are present in the powder and the powder is suitable for reconstitution with water or another food grade aqueous phase. The lipid globules may comprise a core and a surface.

[0110] The lipid globules in the nutritional composition preferably have mode diameter, based on volume, of at least 2.0 pm, more preferably at least 2.5 pm, and most preferably at least 3.0 pm. Preferably, the lipid globules have a mode diameter, based on volume, between 2.0 and 12 pm, more preferably between 2.0 and 8.0 pm, even more preferably between 3.0 and 7.0 pm, and most preferably between 4.0 pm and 6.0 pm.

[0111] Alternatively, or preferably in addition, the size distribution of the lipid globules is preferably in such a way that at least 45 volume % (vol.%), preferably at least 55 vol.%, even more preferably at least 65 vol.%, and most preferably at least 75 vol.%, based on total lipid volume of the lipid globules have a diameter between 2 and 12 pm. In a more preferred embodiment, at least 45 vol.%, preferably at least 55 vol.%, more preferably at least 65 vol.%, and most preferably at least 75 vol.%, based on total lipid volume, of the lipid globules have a diameter between 2 and 10 pm. In an even more preferred embodiment, at least 45 vol.%, more preferably at least 55 vol.%, yet even more preferably at least 65 vol.%, and most preferably at least 75 vol.%, based on total lipid volume, of the lipid globules have a diameter between 4 and 10 pm. Preferably less than 5 vol.%, based on total lipid volume, of the lipid globules have a diameter above 12 pm.Standard infant formulas, follow-on formulas or young child formulas typically have lipid globules with a mode diameter, based on volume, of about 0.3-1 pm and / or less than 45 vol.%, based on total lipid volume, of the lipid globules have a diameter above 2 pm.

[0112] The volume percentage of lipid globules is based on volume of total lipid. The mode diameter relates to the diameter which is the most present based on volume % of total lipid, or the peak value in a graphic representation, having on the X-as the diameter and on the Y-as the volume %.

[0113] The volume of the lipid globule and its size distribution can suitably be determined using a particle size analyser such as a Mastersizer 2000 (Malvern Instruments, Malvern, UK), for example by the method described in Michalski et al, 2001 , Lait 81 : 787-796.

[0114] Phospholipid

[0115] The nutritional composition comprises 0.5 to 20 wt.% phospholipid based on total lipid, preferably 0.5 to 10 wt.%, even more preferably 0.75 to 8 wt.%, even more preferably 1.2 to 8 wt.%, and most preferably 1.5 to 5 wt.% phospholipid based on total lipid.

[0116] The lipid globules are at least partly coated on the surface with phospholipids. By ‘coating’ is meant that the outer surface layer of the lipid globules comprises phospholipid, whereas phospholipid is virtually absent in the core of the lipid globule. A suitable way to determine whether phospholipid is located on the surface of lipid globules is confocal laser scanning microscopy or transmission electron microscopy; see for instance Gallier et al. (A novel infant milk formula concept: Mimicking the human milk fat globule structure, Colloids and Surfaces B: Biointerfaces 136 (2015) 329-339).

[0117] The nutritional composition preferably comprises glycerophospholipids. Examples of glycerophospholipids are phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylglycerol (PG). Preferably the nutritional composition comprises one or more of PC, PS, PI and PE, more preferably the nutritional composition comprises at least PC.

[0118] The nutritional composition preferably comprises sphingomyelin. Sphingomyelins have a phosphorylcholine or phosphoryl ethanolamine molecule esterified to the 1 -hydroxy group of a ceramide. They are classified as phospholipid as well as sphingolipid but are not classified as a glycerophospholipid nor as a glycosphingolipid. Preferably the nutritional composition comprises 0.05 to 10 wt.% sphingomyelin based on total lipid, more preferably 0.1 to 5 wt.%, even more preferably 0.2 to 2 wt.% based on total lipid. Preferably the nutritional composition comprises at least 5 wt.%, more preferably 5 to 40 wt.% sphingomyelin based on total phospholipid, more preferably 10 to 35 wt.%, even more preferably 15 to 35 wt.%, based on total phospholipid.The nutritional composition preferably comprises glycosphingolipids. Preferably the nutritional composition comprises 0.1 to 10 wt.% glycosphingolipids based on total lipid, more preferably 0.5 to 5 wt.%, even more preferably 2 to 4 wt.%, based on total lipid. The term glycosphingolipids in the present context particularly refers to glycolipids with an amino alcohol sphingosine. The sphingosine backbone is O-linked to a charged head-group such as ethanolamine, serine or choline backbone. The backbone is also amide linked to a fatty acyl group. Glycosphingolipids are ceramides with one or more sugar residues joined in a beta-glycosidic linkage at the 1 -hydroxyl position and include gangliosides. Preferably the nutritional composition contains gangliosides, more preferably at least one ganglioside selected from the group consisting of GM3 and GD3.

[0119] The nutritional composition preferably comprises phospholipid derived from mammalian milk. Preferably the nutritional composition comprises phospholipid and glycosphingolipid derived from mammalian milk. The nutritional composition preferably comprises phospholipid and optionally glycosphingolipid from mammalian milk from cows, mares, sheep, goats, buffalos, horses and / or camels. More preferably the nutritional composition comprises phospholipid and optionally glycosphingolipid from cow’s milk.

[0120] Phospholipid derived from milk includes preferably phospholipid that is isolated from milk fat, cream lipid, cream serum lipid, butter serum lipid (beta serum lipid), whey lipid, cheese lipid and / or buttermilk lipid. Buttermilk lipid is typically obtained during the manufacture of buttermilk. Butter serum lipid or beta serum lipid is typically obtained during the manufacture of anhydrous milk fat from butter. Preferably the phospholipid and optionally glycosphingolipid is obtained from milk cream. Examples of suitable commercially available sources for phospholipid from milk are BAEF, SM2, SM3 and SM4 powder of Corman, Salibra of Glanbia, Lipamin M20 of Lecico, Vivinal ® MFGM of FrieslandCampina and LacProdan MFGM-10 or PL20 of Aria.

[0121] The use of phospholipid from milk fat advantageously comprises the use of milk fat globule membranes, which are more reminiscent to the situation in human milk. The concomitant use of phospholipid derived from milk and triglycerides derived from a mix of vegetable lipid and mammalian milk fat therefore enables the manufacture of coated lipid globules with a coating more similar to human milk, while at the same time providing an optimal fatty acid profile.

[0122] Preferably the phospholipid is derived from mammalian milk fat, more preferably from cow’s mammalian milk fat. Preferably the phospholipid is derived from or forms part of the milk fat globule membrane (MFGM), more preferably is derived from or forms part of cow’s MFGM.Preferably the nutritional composition comprises phospholipid and glycosphingolipid. In a preferred embodiment the weight ratio of phospholipid : glycosphingolipid is from 2:1 to 12:1 , more preferably from 2:1 to 10:1 and even more preferably 2:1 to 5:1.

[0123] Methods for obtaining lipid globules with an increased size and coating with phospholipid are for example disclosed in WO 2010 / 027258 and WO 2010 / 027259.

[0124] Digestible carbohydrates

[0125] The nutritional composition comprises digestible carbohydrates. The digestible carbohydrates preferably provide 25 to 75% of the total calories of the nutritional composition. Preferably the digestible carbohydrates provide 40 to 60% of the total calories. Based on calories the nutritional composition preferably comprises of 5 to 20g of digestible carbohydrates per 100 kcal, more preferably 6 to 16g per 100 kcal. When in liquid form, e.g. as a ready-to-feed liquid, the nutritional composition preferably comprises 3 to 30g digestible carbohydrate per 100 ml, more preferably 6 to 20g, even more preferably 7 to 10g per 100 ml. Based on dry weight the nutritional composition preferably comprises 20 to 80 wt.%, more preferably 40 to 65 wt.% of digestible carbohydrates.

[0126] Preferred digestible carbohydrate sources are one or more of lactose, glucose, sucrose, fructose, galactose, maltose, starch and maltodextrin. Lactose is the main digestible carbohydrate present in human milk. Lactose has a low glycaemic index. The nutritional composition preferably comprises lactose. The nutritional composition preferably comprises digestible carbohydrate, wherein at least 35 wt.%, more preferably at least 50 wt.%, more preferably at least 75 wt.%, even more preferably at least 90 wt.%, most preferably at least 95 wt.% of the digestible carbohydrate is lactose. Based on dry weight the nutritional composition preferably comprises at least 25 wt.% lactose, preferably at least 40 wt.% lactose.

[0127] Protein

[0128] The nutritional composition comprises protein. The protein preferably provides 5 to 20% of the total calories. Preferably the nutritional composition comprises protein that provides 6 to 12% of the total calories. Preferably the nutritional composition comprises less than 3.5g protein per 100 kcal, more preferably the nutritional composition comprises between 1.5 and 2.1g protein per 100 kcal, even more preferably between 1.6 and 2.0g protein per 100 kcal. A low protein concentration advantageously is closer to human milk as human milk comprises a lower amount of protein based on total calories compared to cow’s milk. The protein concentration in a nutritional composition is determined by the sum of protein, peptides and free amino acids. Based on dry weight the nutritional composition preferably comprises less than 12 wt.% protein, more preferably between 9.6 and 12 wt.%, even more preferably between 10 and 11 wt.%. Based on a ready-to-drink liquid product the nutritional composition preferably comprises less than 1.5g proteinper 100ml, more preferably between 1.2 and 1.5g per 100ml, even more preferably between 1.25 and 1.35g per 100ml.

[0129] In an embodiment the nutritional composition is a preterm of post-discharge composition. In such cases a higher protein content is desired. 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.

[0130] 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 protein sources based on cows' milk proteins such as whey, casein and mixtures thereof and proteins based on soy, potato or pea are preferred. In case whey proteins are used, the protein source is preferably based on acid whey or sweet whey, whey protein isolates or mixtures thereof. Preferably the nutritional composition comprises at least 3 wt.% casein based on dry weight. Preferably the casein is intact and / or non-hydrolysed.

[0131] Formula

[0132] The use according to the present invention requires the administration of an infant formula, a follow-on formula or a young child formula. This means that the nutritional composition is not human milk. It also means that the nutritional composition is not native cow’s milk or native milk from another mammal. In the context of the present invention, young child formula can also be named growing-up milk.

[0133] Alternatively, the terms as used herein, “infant formula” or “follow-on formula” or “young child formula” means that it concerns a composition that is artificially made or in other words that it is synthetic. Hence in one embodiment, the nutritional composition that is administered is an artificial infant formula or an artificial follow-on formula or an artificial young child formula or a synthetic infant formula or a synthetic follow-on formula or a synthetic young child formula.

[0134] In the present invention, infant formula refers to nutritional compositions, artificially made, intended for infants of 0 to about 4 to 6 months of age and are intended as a substitute for human milk. Typically, infant formulae are suitable to be used as sole source of nutrition. Such infant formulae are also known as starter formula. Follow-on formula for infants starting with at 4 to 6 months of life to 12 months of life are intended to be supplementary feedings for infants that start weaning on other foods. Infant formulae and follow-on formulae are subject to strict regulations, for example for the EU regulations no. 609 / 2013 and no.2016 / 127. In the present context, young child formula refers to nutritional compositions, artificially made, intended for infants of 1 to 3 years of age, which are intended to be supplementary feedings for infants.

[0135] The nutritional composition is preferably an infant formula or a follow-on formula. More preferably the nutritional composition is an infant formula.

[0136] The nutritional composition comprises digestible carbohydrates, protein and lipid, wherein the lipid preferably provides 30 to 60% of the total calories, the protein provides 5 to 20% of the total calories and the digestible carbohydrates provide 25 to 75% of the total calories.

[0137] The nutritional composition is preferably an infant formula or follow-on formula and preferably comprises 3 to 7g lipid / 100 kcal, preferably 4 to 6g lipid / 100 kcal, more preferably 4.5 to 5.5g lipid / 100 kcal, preferably comprises 1.7 to 3.5g protein / 100 kcal, more preferably 1.8 to 2.1g protein / 100 kcal, more preferably 1.8 to 2.0g protein / 100 kcal and preferably comprises 5 to 20g digestible carbohydrate / 100 kcal, preferably 6 to 16g digestible carbohydrate / 100 kcal, more preferably 10 to 15g digestible carbohydrate / 100 kcal.

[0138] Preferably the nutritional composition is an infant formula or follow-on formula and preferably has an energy density of 60 to 75 kcal / 100 ml, more preferably 60 to 70 kcal / 100 ml, when in a ready-to-drinkform. This density ensures an optimal balance between hydration and caloric intake.

[0139] In one embodiment, the nutritional composition is a powder. Suitably, the nutritional composition is in a powdered form, which can be reconstituted with water or other food grade aqueous liquid, to form a ready-to drink liquid, or is in a liquid concentrate form that should be diluted with water to a ready-to-drink liquid. It was found that lipid globules maintained their size and coating when reconstituted.

[0140] Target group

[0141] The present invention relates to the use of a mixture of HMOs and synthetic nutritional compositions comprising such a mixture of HMOs comprising at least one sialylated oligosaccharide, at least one fucosylated oligosaccharide, and at least one N-acetylated oligosaccharide for infants and / or young children exposed to early life stress. Such infants and young children are at risk of behaviour disorder and / or mental disorder later in life in a human subject. Preferably the nutritional composition is administered to said infants and / or young children at an age of 0 to 48 months, more preferably 0 to 36 months and the behaviour disorder and / or mental disorder later in life is at an age of 6 years and older, preferably 12 years and older.

[0142] Preferably the infants and children are human infants and young children. The nutritional composition according to the present invention is especially beneficial for infants and young children that have beenexposed to early life stress. Early life stress expose affects brain development and provide a risk of the development and / or occurrence of a behaviour disorder and / or mental disorder later in life.

[0143] Infants and young children exposed to early life stress that especially benefit from the nutritional composition according to the invention may be selected from an infant born to a mother who experienced stress during the third trimester of pregnancy and / or during lactation, a preterm, small for gestational age infant, (very) low birth weight infant, an infant exposed to hypoxia , an infant or young child that was hospitalized and / or an infant or young child that was malnourished and / or exposed to nutritional imbalances and / or with faltering growth.

[0144] Preferably, the nutritional composition is an infant formula, a follow-on formula and / or a young child formula. In some aspects the nutritional composition may further be a preterm formula, low birthweight formula, postdischarge formula or paediatric formula for infants having faltering growth, in need of catch-up growth, which is intended for providing nutrition to an infant or young child selected from those exposed to early life stress and that are selected from the group consisting of infants preterm infants, small for gestational age infants, infants with retarded growth, and infants that are born to mothers who experienced stress during third trimester and / or during lactation, preferably preterm infants or infants with retarded growth.

[0145] Application

[0146] The present composition is preferably enterally administered, more preferably orally to the infant and / or young child. The present composition can advantageously be applied as a complete nutrition.

[0147] The inventors surprisingly found that the nutritional composition according to the invention is effective in preventing the consequences of early life stress. The present invention aims to promote behaviour development in infant selected from infants and young children exposed to early life stress be selected from a preterm, small for gestational age infant, (very) low birth weight infant, an infant exposed to hypoxia , an infant or young child that was hospitalized and / or an infant or young child that was malnourished and / or exposed to nutritional imbalances and / or with faltering growth. Those infants are at risk of the development and / or occurrence of a behaviour disorder and / or mental disorder later in life.

[0148] In the context of the present invention, “early life ” typically lasts up to the age of 6 years, more preferably up to the age of 5 years, even more preferably up to the age of 36 months, most preferably up to the age of 12 months. Early life in the context of early life stress herein also includes the prenatal stage of the human subject, preferably the foetal stage, more preferably the foetal stage in the third trimester of pregnancy. In one embodiment, early life starts at birth.In a preferred embodiment, the infant or child is at risk or even at high risk of experiencing early life stress or is experiencing early life stress. Examples of such at (high) risk infants or children are born in families from a lower socioeconomic status (e.g. children from families with an income level below average or a low level of education, e.g. not exceeding high school), infants or children whose parents are separated or going through a separation, infants or children separated from one of both parents, infants or children whose parent are both working, neglected infants or children, abused infants or children, infants or children whose parent(s) are suffering from stress, depression, depressed children, ill infants or children, malnourished infants or children and infants or children exposed to violence.

[0149] Early life stress may take any known form. Typical stressors include psychological stressors, physiological stressors and physical stressors. The early life stress may the form of mental stress, physical stress, metabolic stress or any combination thereof. Exemplary stressors include disrupted families (e.g. divorce, separation, blending of families), separation from one or both parents (e.g. incarceration of a parent), maternal depression, both parents working, lack of attention by parent(s), exposure to violence, abuse (e.g. physical, mental, sexual), neglect (e.g. emotional, physical), death of a loved one (in particular death of a parent), illness (e.g. mental, physical), malnutrition. Herein, “parent(s)” may also refer to “care-taker(s)” or “guardian(s)”. Most suitably, the early life stress is selected from malnutrition, maternal stress, depression and abuse.

[0150] The nutritional composition according to the invention is for use in providing nutrition to infants and / or young children exposed to early life stress selected from an infant born that has experienced prenatal stress during the third fetal trimester, a preterm, small for gestational age infant, (very or extremely) low birth weight infant, an infant exposed to hypoxia , an infant or young child that was hospitalized and / or an infant or young child that was malnourished and / or exposed to nutritional imbalances and / or with faltering growth, preferably directly after birth and up to 48 months of age or after the occurrence of the early life stress

[0151] 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.

[0152] Preferably in the methods or uses according to the invention, the nutritional composition is administered in the first 48 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 topreterm 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.

[0153] In a preferred aspect the nutritional composition exhibits a later-in-life effect and is preferably administered up to 48 months of age, more preferably up to 36 months of age, more preferably 24 months of age, more preferably 12 months of age. In an aspect it may be preferred to administer the nutritional composition up to 6 years of age.

[0154] In a preferred aspect the occurrence of the behaviour disorder and / or mental disorder later in life is at an age of 6 years, preferably 12 years or older. The beneficial effects of the nutritional composition may last beyond early life, such as up to or even beyond adulthood. The effects may even last the entire lifespan of the subject. According to a preferred embodiment, the prevention of the consequences of early life stress prolongs into adulthood. Preferably, the prevention of the consequences of early life stress is manifest when the subject is 6 years of age or higher, more preferably 12 years or higher, or has reached adulthood or when the subject has reached an age of 18 years or higher, or even an age of 25 years or higher.

[0155] The invention further relates to the composition comprising a mixture of human milk oligosaccharides according to the invention for use as a medicament and / or for use in the manufacture of a medicament.

[0156] The infants exposed to early life stress are at risk of risk of a behaviour disorder and / or mental disorder or at risk of abnormal behaviour later in life. Said behaviour disorder and / or mental disorder and / or abnormal behaviour, preferably the behaviour disorder and / or mental disorder are selected from abnormal low anxiety-like behaviour, increased impulsivity, recklessness, carelessness, abnormal reactivity, excessive level of indifference, excessive risk taking, disinhibited behaviour, excessive temerity, excessive foolhardiness, excessive disregard, excessive nonchalance, excessive insouciance, excessive temerity, excessive negligence, excessive risk taking, hyperactivity, depressive-like behaviour, low self-care behaviour, autism, attention-deficit hyperactivity disorder (ADHD), post-traumatic stress disorder (PTSD), obsessive compulsive disorder (OCD), conversion disorder (CD), bipolarity, schizophrenia and / or borderline personality disorder.

[0157] As used herein a behaviour disorder and / or mental disorder is not an impaired cognitive development and / or impaired cognition.

[0158] In a preferred embodiment the behaviour disorder and / or mental disorder and / or abnormal behaviour, preferably the behaviour disorder and / or mental disorder are selected from abnormal low anxiety-like behaviour, hyperactivity, depressive-like behaviour or depression, autism, ADHD, PTSD, OCD, CD, bipolarity, schizophrenia and / or borderline personality disorder.In an embodiment, the nutritional composition is for infants or young children exposed to early-life stress and at risk of or presenting with neurodevelopmental disorders, particularly behaviour disorder and / or mental disorder and / or abnormal behaviour, preferably behaviour disorder and / or mental disorder. Preferably, the behaviour disorder and / or mental disorders are selected from neurodevelopmental conditions, neurodevelopmental symptom clusters, autism spectrum disorder (ASD), attention-deficit / hyperactivity disorder (ADHD).

[0159] In an embodiment the use for ADHD comprises improving attention-regulation difficulties, hyperactivity / impulsivity symptom clusters, executive-functioning challenges, and / or unspecified attention-deficit presentations.

[0160] In an embodiment the use for ASD comprised improving disinhibited behaviour, social communication difficulties, restricted / repetitive behaviours

[0161] In an alternative preferred embodiment the behaviour disorders and / or mental disorders are selected from externalizing problems, oppositional defiant disorder (ODD), conduct disorder (CD), intermittent explosive disorder, and impulse-control dysregulation.

[0162] In a further preferred embodiment, the nutritional composition is particularly beneficial for abnormal low anxiety-like behaviour. Worded otherwise, the nutritional composition is preferably useful for subject exposed to early-life stress that are at risk of or suffering from impaired anxiety responses later in life. In an aspect an impaired anxiety response in reaction to a stressor may be abnormal low anxiety-like behaviour or abnormal high anxiety-like behaviour, preferably abnormal low anxiety-like behaviour.

[0163] In yet a further embodiment, the nutritional composition is particularly beneficial for depressive-like behaviour or depression. Worded otherwise, the nutritional composition is preferably useful for subject exposed to early-life stress that are at risk of or suffering from depressive-like behaviour or depression.

[0164] In the context of the invention the behaviour disorder and / or mental disorder are conditions and disorders as defined in the "Diagnostic and Statistic Manual of Mental Disorders” 5thedition as issued by the American Psychiatric Association, also generally called the DSM-5, and describing mental health conditions. The DSM-5 criteria allow to distinguish between behavioural conditions that are of pathologic nature and allow for therapeutic intervention.In an aspect the invention also concerns a non-therapeutic method for improving stress resilience. Impaired stress resilience concerns the ability to adapt successfully when faced with stress. Hence, the individual with impaired stress resilience may avoid symptoms associated with stress. In a preferred aspect the subject with impaired stress resilience is at least 6 years of, preferably at least 12 years of age. Preferably the subject with impaired stress resilience was exposed to early life stress.

[0165] FIGURES

[0166] Figure 1 illustrates the percentage of time spent grooming after being splashed with sucrose of groups of mice exposed to early-life stress (ES) followed by exposure to a control diet (CTL ES), a diet with HMOs (HMO ES), a diet with large lipid globules (NUT ES) or a diet with a combination of HMOs and large lipid globules (NUTHMO ES). a = significantly different vs CTR-CTR; b = significantly different vs CTR-ES.

[0167] Figure 2 shows the behaviour of groups of mice exposed to early-life stress (ES) followed by exposure to a control diet (CTL ES), a diet with HMOs (HMO ES), a diet with large lipid globules (NUT ES) or a diet with a combination of HMOs and large lipid globules (NUTHMO ES) in a light dark box (LDB). Figure 2A shows the amount of entries into the light compartment of the LDB. Figure 2B shows the distance travelled in cm within the LDB. Figure 2C shows the velocity of different groups of mice in the LDB. a = significantly different vs CTR-CTR; b# = trending effect vs CTR-ES.

[0168] Figure 3 shows the behaviour of groups of mice exposed to early-life stress (ES) followed by exposure to a control diet (CTL ES), a diet with HMOs (HMO ES), a diet with large lipid globules (NUT ES) or a diet with a combination of HMOs and large lipid globules (NUTHMO ES) when exposed to the elevated plus maze test (EPM) and shows the percentage of time spent in the unprotected area of the EPM of the different groups of mice, a = significantly different vs CTR-CTR; b = significantly different vs CTR-ES

[0169] EXAMPLES

[0170] The invention is further illustrated by the following examples.

[0171] Example 1 : Later in life effect of an early dietary intervention with HMOS on early-life-stress-induced behaviour impairment

[0172] A mouse model was used for studying impaired neurological development and abnormal behaviour. Mice C57BL6J pups were exposed to early life stress (ES) between postnatal day (PN) 2 and PN9 as previously described (Rice et al, 2008, Endocrinology 149:4892-4900). This period is characterized by rapid growth and development of brain and other organs, and in mice is roughly equivalent to the early infancy stage in humans. Healthy control groups were not exposed to early life stress. From PN9 onwards the ES and control mice pups were exposed to semi-synthetic experimental diet containing either of 4 infant milk formula (IMF) compositions varying in presence of HMOS and lipid droplet structure. The experimental dietswere isocaloric, had a macronutrient composition based on AIN-93G rodent chow. The diets were composed of 72.6% AIN-93G (without lipids) and 27.3% IMF (including lipids) as previously described (Oosting et al, 2008, Br J Nutr, 111 :p215-226). The 4 experimental diets were provided until PN42, and animals were switched to a standard (gain based) rodent chow diet (Teklad 2920) that was continued for 12 weeks. PN42 is considered to correspond to human late adolescence or early adulthood. At adult age (16 weeks of age), behaviour profile of animals was tested using standard rodent behaviour tests.

[0173] Table 1. The following groups of mice were assessed in the experiment. - means no, + means yes Early life stress HMOs Large lipid

[0174] globules + MFGM

[0175] CTR CTR CTR ES HMO ES HMO NUT ES NUT ES

[0176]

[0177] In all diets (PN9-PN42) the amount of dietary fiber was between 47.4 and 47.6 g / kg diet, of this amount of fibers between 41.0 and 41.2 was provided by GOS and IcFOS and, if present by HMOs.

[0178] In the HMO and HMO NUT diets the amount of HMOs was 22.2 g / kg, whereas CTR and NUT diets contained only GOS and IcFOS. HMOS was a mixture of 2’-FL, 3’-FL, LNT, ‘-3SL, 6’-SL in a wt / wt ratio of about 52: 13: 26: 4: 5 (Novonesis, Bagsvaaerd, Danmark). The amount of GOS was lowered in the HMOs containing diets to 18.8 g / kg.

[0179] The lipid component in the diet of the two large lipid globule (hereinafter called NUT) groups was as follows: The IMF comprised lipid globules that had a volume-based mode-diameter of about 4 um, and > 45 % volume % of lipid globules with a diameter between 2 and 12 um. Milk phospholipids were present in an amount of about 1.5 wt% based on total lipids. In the other groups the lipid globules had a volume-based mode diameter of about 1 um, < 10 vol% with a diameter between 2-12 um, and no enrichment in milk PL was present. The nutritional composition of the different diets is summarized in table 2.Table 2. Nutritional composition in g / kg of different rodent diets.

[0180] component g / kg diet CTR Nut HMO Nut+HMO fat total 70 70 70 70 milk phospholipids

[0181] (1.5% of total lipids) - 1.05 - 1.05

[0182] fiber total 47.6 47.6 47.4 47.4 Cellulose 6.4 6.4 6.4 6.4 HMOs - - 22.2 22.2

[0183] GOS 37.1 37.1 14.7 14.7

[0184] FOS 4.1 4.1 4.1 4.1 [GOS+HMOs] / [FOS] 9.05 9.05 9 9 protein total 178.3 178.3 178.3 178.3 carbohydrate total 620.3 620.3 618.5 618.5

[0185]

[0186] Mice were bred in house. The early-life stress paradigm was initiated at PN2 and consisted of the limited nesting / bedding-material procedure described previously (Rice et al, 2008) In brief, control cages were equipped with standard amounts of sawdust bedding and nesting material (one square piece of cotton nesting material (5x5cm; Technilab-BMI, Someren, the Netherlands). In the early life stress cages, the bottom was covered with a little amount of sawdust bedding and a fine-gauge stainless steel mesh was placed 1 cm above the cage floor. On top of the mesh, half a square piece of cotton nesting material (2,5 x 5 cm) was placed. Cages were covered with a filtertop. Mice were left completely undisturbed until P9. This paradigm results in chronic stress in the mother and fragmented maternal care, which results in chronic early-life stress in the pups and impaired brain development and adult behavioural abnormalities (Rice 2008, Naninck et al, 2015, Hippocampus, 25:309-328).

[0187] At adulthood the following tests were performed with the mice:

[0188] SPLASH TEST: The splash test is a test to evaluate self-care and / or depressive-like behaviour in rodents. Mice are “splashed” with sucrose solution and their natural response is to clean their fur (grooming). The total time (%) spent grooming in the first minute after “splashing” was monitored. A lower score in this test is indicative of a reduction in motivation to fulfil the acts of self-preservation, which is considered to parallel with some symptoms of depression such as apathetic behaviour (Isingrini et al. 2010, PLoS One.

[0189] 2010 Apr 28;5(4):e10404. doi: 10.1371 / journal.hpone.0010404).

[0190] LIGHT / DARK BOX: Animals were exposed to the light / dark box (LDB), which is an unconditioned behaviour test that can be used to assess anxiety like behaviour but also impulsivity or risk-taking behaviour (Arrant et al, 2013, Behav Brain Res 256:119-127). Parameters for activity were also collected in this test. Animalsare placed in a square box that is divided in 2 compartments: a dark compartment otters protection and a light compartment that is an unsafe space. Healthy mice naturally avoid the light compartment.

[0191] Animals were allowed to explore the LDB for 10 minutes. More time spent in, shorter latency to enter for the first time and / or more entries made in total to the light compartment of the LDB is indicative of reduced anxiety like behaviour or increased impulsivity / higher risk taking. Parameters for activity during this test are distance (cm) travelled in the LDB (cm) and velocity (cm / s).

[0192] ELEVATED PLUS MAZE (EPM): Animals were exposed to the elevated plus maze test (EPM), which is an unconditioned behaviour test that can be used to assess anxiety like behaviour and impulsivity or risktaking behaviour. Animals are placed in an elevated plus shaped maze that that has 2 open and closed arms: the closed arms offer protection, and open arms are regarded as unsafe space. Healthy mice naturally avoid the unsafe space. The mice were allowed to explore the EPM for 10 minutes and their location on the EPM was monitored. Time (% of total time) spent in the unprotected area of the EPM was measured and number of open arm entries were quantified. More time spent in the unprotected area and more entries made in the open arms of the EPM are indicative of reduced anxiety-like behaviour and / or increased impulsivity / higher risk-taking behaviour (Kishikawa et al, 2014, Behav Brain Res 274: 235-242.). Lower anxiety like behaviour and increased impulsivity / higher risk-taking behaviour can be beneficial or harmful depending on the context. In the current experiments the comparison was made with control mice that had undergone no early life stress.

[0193] Statistical analysis of behaviour parameters was done with SPSS 13.0 in stepwise manner. Step 1 : it was assessed whether ES affected later in life behaviour in the groups exposed to control diet (using students t-test). Step 2 (was done only when step 1 was significant): the effects of intervention with HMDS and / or without large lipid globules and milk phospholipids (NUT) was tested in ES mice by comparing the ES-diet intervention groups to ES-control and to the healthy control using ANOVA followed by pairwise comparisons.

[0194] All data are expressed as mean+ / - SEM. Differences between groups were considered significant at P<0.05. Trending effects were also included in the figures (# sign) when P<0.08.

[0195] Results:

[0196] SPLASH TEST

[0197] Mice on control diet that were exposed to ES were found to spent significantly less time grooming compared to control animals after being splashed with sucrose (figure 1) later in life, which is indicative of poor self-care and / or depression like behaviour and / or apathy. However, the behaviour of groups exposed to ES and diets with large lipid globules and milk phospholipids (NUT), HMO or the combination (NUTHMO) thereof, did not differ significantly from that of the control group not exposed to ES (CTR CTR), indicating that the ES-induced abnormal self-care behaviour was mitigated by these diets. The ES group that wasexposed to a diet comprising HMOS or the combination HMO and large lipid globules and milk phospholipids early in life showed significant improvements compared to the ES control group.

[0198] LIGHT DARK BOX (LDB).

[0199] In the light dark box (LDB) study, mice on control diet that were exposed to ES did not spend more time in the light compartment of the LDB compared to healthy controls (data not shown), but they did make more entries to the light compartment (figure 2A). Entering the light compartment of the box more frequently is a type of behaviour associated with risk-taking. Compared to the healthy control mice, making more entries into the light is abnormal and indicative of excessive risk-taking behaviour, higher impulsivity or diminished anxiety-like behaviour. Moreover, animals on control diet exposed to ES travelled a longer distance (figure 2B) and had a higher velocity (figure 2C) compared to healthy control animals, which is indicative of hyperactivity in this context. The ES- induced impulsivity was however not observed in ES animals exposed to an early life diet comprising HMOS and / or a diet with large lipid globules and milk phospholipids. The ES-induced hyperactivity was fully mitigated by all 3 early life diets comprising HMOS and / or Nuturis (figure 2 B and 2C), behaviour of all 3 groups was similar to the healthy control and the behaviour scores were significantly different from those of the control-ES group.

[0200] Elevated plus maze (EPM):

[0201] In this study it was observed that, compared to normally behaving and healthy mice on control diet, the mice on control diet that had been exposed to ES spent more time in the unprotected area of the EPM (figure 3)). Spending excessive time in open spaces is considered abnormal for mice and is indicative of excessive risk taking or being impulsive (impulsive actions without proper risk assessment), and neurological dysfunction. This is in line with the results observed in LDB. However, these ES- induced deficits in adult behaviour in the EPM test were mitigated with early in life dietary intervention with the combination of HMOS and large lipid globules with milk phospholipids leading to full normalization of behaviour, as evidenced by the absence of a difference in scores between the healthy control group and the ES NUTHMO group (figure 3).

Claims

CLAIMS1. Synthetic nutritional composition comprising a mixture of human milk oligosaccharides, said mixture of HMOs comprises, preferably consists essentially of 2’-fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3’-sialyllactose (3’SL) and 6’-sialyllactose (6’SL) , for use in preventing and / or reducing the risk of occurrence and / or severity of a behaviour disorder and / or mental disorder later in life in a human subject,wherein the nutritional composition is administered to said human subject at an age of 0 to 48 months and wherein the behaviour disorder and / or mental disorder later in life is at an age of 6 years, preferably 12 years or older.

2. Synthetic nutritional composition comprising a mixture of human milk oligosaccharides, said mixture of HMOs comprises, preferably consists essentially of 2’-fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3’-sialyllactose (3’SL) and 6’-sialyllactose (6’SL), for use in reducing the risk of occurrence of abnormal behaviour later in life in a human subject, wherein the nutritional composition is administered to a human subject with an age of 0 to 36 months and the reduced risk is at an age of 5 years or older.

3. Synthetic nutritional composition for use according to claims 1 and 2 wherein the human subject is exposed to early life stress, wherein early life stress exposure occurs during the period covering the third trimester of pregnancy until after birth at an age to 48 months of age.

4. Synthetic nutritional composition for use according to claims 1 - 3 wherein the human subject is at risk of a behaviour disorder and / or mental disorder or at risk of abnormal behaviour is selected from the group consisting of abnormal low anxiety-like behaviour, increased impulsivity, recklessness, carelessness, abnormal reactivity, excessive level of indifference, excessive risk taking, disinhibited behaviour, excessive temerity, excessive foolhardiness, excessive disregard, excessive nonchalance, excessive insouciance, excessive temerity, excessive negligence, excessive risk taking, hyperactivity, depressive-like behaviour, low self-care behaviour, autism, ADHD, PTSD, ODD, CD, bipolarity, schizophrenia, borderline personality disorder.

5. Synthetic nutritional composition for use according to any one of claims 3 and 4 wherein the human subject exposed to early life stress is a selected from a preterm, small for gestational age infant, (very) low birth weight infant, an infant exposed to hypoxia, an infant or young child that was hospitalized and / or an infant or young child that was malnourished and / or exposed to nutritional imbalances and / or with faltering growth.

6. Synthetic nutritional composition for use according to the preceding claims, wherein the nutritional composition comprises lipids in the form of lipid globules and whereina. the lipid globules have a mode diameter based on volume of at least 2.0 pm; and / or at least 40 vol.% of the lipid globules based on total lipid volume have a diameter of 2 to 12 pm; and / orb. the lipid comprises 0.5 to 20 wt.% milk-derived phospholipids based on total lipids.

7. Synthetic nutritional composition for use according to the preceding claims wherein the nutritional composition comprises galactooligosaccharides (GOS) and / or long-chain fructooligosaccharides (IcFOS).

8. Synthetic nutritional composition for use according to the preceding claims, wherein the nutritional composition provides early life nutrition to infants at 0-12 months.

9. Synthetic nutritional composition for use according to the preceding claims, wherein the nutritional composition is selected from an infant formula, follow-on formula and young child formula.

10. Synthetic nutritional composition for use according to any one of the preceding claims wherein the mixture of 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 HMOs in the composition.

11. Non-therapeutic method for improving resilience to a stressor later in life in a human subject, comprising administering a synthetic nutritional composition, said composition comprising a mixture of human milk oligosaccharides comprising at least one sialylated oligosaccharide, at least one fucosylated oligosaccharide, and at least one N-acetylated oligosaccharide, wherein the nutritional composition is administered to a human subject with an age of 0 to 48 months and wherein the improved resilience is at an age of 6 years or older, preferably 12 years or older.

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