Oligosaccharide mixture for brain development

Abstract

The invention relates to a nutritional composition comprising non-digestible oligosaccharides for infants and / or young children. The invention further relates to the use of said nutritional composition for improving the brain function, in particular social recognition.

Description

[0001] OLIGOSACCHARIDE MIXTURE FOR BRAIN DEVELOPMENT FIELD OF THE INVENTION

[0002] The invention relates to a nutritional composition for infants and young children for use in improving brain development.

[0003] BACKGROUND OF THE INVENTION

[0004] Human milk is the uncontested gold standard concerning infant nutrition. However, in some cases breastfeeding is inadequate or unsuccessful for medical reasons or not available because of a choice not to breastfeed. For such situations infant or follow-on formulas have been developed. Commercial infant formulas are commonly used today to provide supplemental or sole source of nutrition early in life. These formulas comprise a range of nutrients to meet the nutritional needs of the growing 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.

[0005] Human milk contains substantial amounts of non-digestible carbohydrates, known as human milk oligosaccharides (HMOs). Mature human milk contains 5 to 15 g / l of HMOs. It is presumed that more than 200 structurally distinct oligosaccharides are present. The building blocks of human milk oligosaccharides are the monosaccharides D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GIcNAc), L-fucose (Fuc), and sialic acid (N-acetyl neuraminic acid (Neu5Ac). Lactose (Galp1-4Glc) forms the reducing end and can be elongated with N-acetyllactosamine repeat units (Galp1-3 / 4GlcNAc). Lactose, type 2 polylactosamine or type I Lacto-N-biose backbone structures can be sialylated at the terminal Gal in a2-3 and / or a2-6 linkages and / or fucosylated at the terminal Gal in a1-2, or on internal Glc or GIcNAc residues in a1-3, and / or a1-4 linkages. The structural complexity and abundance of these non-digestible oligosaccharides is unique for human milk; in milk of other mammalian species the level and number of different non-digestible oligosaccharides is much lower. HMOs in human milk 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). In the past, infant formulas did not contain non-digestible oligosaccharides. Subsequently infant formulas were developed, containing prebiotic, non-digestible oligosaccharides to functionally mimic the role of the HMOs. One of the best studied mixtures of such prebiotics is a mixture of galacto-oligosaccharides (GOS) plus long chain fructo-oligosaccharides (IcFOS) in a weight ratio of 9:1. Supplementation with a prebiotic mixture containing GOS during early post-natal development enhanced positive social interactions during late adolescence compared with controls (Waworuntu et al., 2014, Synapse 70:121-124). In a more recent animal study, male mice that received GOS / FOS supplementation since birth displayed improved social behaviour compared with controls in adulthood (Szklany et al., 2020, Nutritional neuroscience, 23: 896- 910).

[0006] More recently milk oligosaccharides with a structure identical to HMOs have become available as produced by fermentation by genetically modified micro-organisms and infant formulas containing a HMO or a mixture of HMOs have become available.

[0007] WO 2021 / 116236 discloses age staged nutritional compositions comprising a HMOs mix. Optionally, at least one of the nutritional compositions comprises a prebiotic, preferably the prebiotic comprises polydextrose, galacto-oligosaccharides, or a combination thereof.

[0008] WO 2018 / 215572 discloses a nutritional composition comprising a Human Milk Oligosaccharide (HMO) having an effect on the promoting, enhancement or improvement of the short-term memory of the subjects in infants and / or young children. The HMO may be 2FL, difucosyllactose (DFL) and / or lacto-N-tetraose LNT and / or lacto-N-neotetraose (LNnT) or combinations thereof.

[0009] WO 2020 / 001862 discloses a nutritional composition comprising human milk oligosaccharide (HMO) having an effect on a mammal to improve, enhance, promote or modulate a serotonergic function in the CNS, preferably in a human infant or a young child, preterm or term, between birth and 7 years. The HMO may be 2FL, DFL and / or LNT and / or LNnT or combinations thereof.

[0010] WO 2020 / 001863 discloses a nutritional composition comprising human milk oligosaccharides (HMO) 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. The HMO may be 2FL, DFL and / or LNT and / or LNnT, or combinations thereof.

[0011] WO 2017 / 021476 discloses a nutritional composition comprising at least one fucosylated oligosaccharide and at least one N-acetylated oligosaccharide for promoting or inducing a global gut microbiota that is closer to the one of infants fed exclusively with human breast milk, in comparison to infants fed with a conventional nutritional composition.

[0012] WO 2019 / 215289 discloses nutritional compositions that are useful for promoting beneficial bacteria in the gastrointestinal tract of a Caesarean-section (C-section)-delivered infant. The nutritional composition can include a prebiotic composition comprising human milk oligosaccharides (HMO), milkfat globule membrane (MFGM), and galacto-oligosaccharides (GOS) and / or polydextrose (PDX). There are currently no nutritional solutions stimulating social recognition and behaviour in early life. There is an ongoing need for improved formulas to stimulate social recognition and behavioural development.

[0013] SUMMARY OF THE INVENTION

[0014] Using a juvenile mouse model wherein the mice were exposed to antibiotics the effects of a mixture of HMOs comprising fucosylated and sialylated oligosaccharides, a mixture comprising betagalactooligosaccharide (bGOS) and long chain fructo-oligosaccharide (IcFOS), or the combination of these two non-digestible oligosaccharides (NDO) mixtures on social behaviour and brain transcriptomics in brain areas linked to social memory (amygdala and prefrontal cortex) were tested. It was surprisingly found that social recognition memory was improved when the mixtures were administered early in life and that the combination of the HMO mixture plus bGOS and IcFOS was the most potent therein. Antibiotic-induced deficits on social behaviour and social recognition were remedied.

[0015] It was found that genes related to pathways for CNS development, learning and memory were significantly enriched in response to prebiotic administration and the enrichment ratios were the highest when the combination of bGOS, IcFOS and HMOs was administered. Pathways relevant to neuron projection development and myelination were significantly enriched in the amygdala and prefrontal cortex (PFC) of the juvenile offspring in response to the mixture of NDOs. The amygdala and prefrontal cortex parts are known to be involved in social recognition, social memory, and social behaviour. Social recognition includes social memory as well as social recognition memory. These changes in gene expression patterns and pathway enrichment ratios in amygdala and PFC are underlying the improvement of social behaviour and social recognition memory. The inventors thus found that that a combination of bGOS, IcFOS and HMOs exerts beneficial effects on social recognition memory and social behaviour. It was also found that the adverse effects on brain development as a result of antibiotic treatment can be prevented and / or treated. The present invention thus concerns compositions comprising non-digestible oligosaccharides (NDO), wherein the NDO comprise i) betagalacto-oligosaccharide (bGOS), ii) long chain fructo-oligosaccharide (IcFOS) and iii) human milk oligosaccharides (HMOs), wherein the HMOS comprise at least one fucosylated oligosaccharide and at least one sialylated oligosaccharide for use in improving and / or ameliorating social recognition improving and / or ameliorating social behaviour and / or social behaviour development. The nutritional composition is particularly of benefit in an infant or young child, in particular in infants or young children at risk of impaired brain development, in particular infant born by C-section and / or treated with antibiotics.

[0016] LIST OF PREFERRED EMBODIMENTS

[0017] 1. A composition comprising digestible carbohydrate, protein, lipid, and non-digestible oligosaccharides (NDO), wherein the NDO comprise i) betagalacto-oligosaccharide (bGOS), ii) long chain fructo- oligosaccharide (IcFOS) and iii) human milk oligosaccharides (HMOs), wherein the HMOS comprise at least one fucosylated oligosaccharide and at least one sialylated oligosaccharide for use in improving and / or ameliorating social recognition improving and / or ameliorating social behaviour and / or social behaviour development in an infant or young child, wherein said composition is not human milk.

[0018] 2. Composition for use according to embodiment 1 wherein the infant or young child is suffering from or at risk of impaired social recognition, social behaviour and / or social behaviour development.

[0019] 3. Composition for use according to the preceding embodiments wherein social recognition comprises social memory and social recognition memory.

[0020] 4. Composition for use according to the preceding embodiments wherein the infant or young child is a C- section born infant and / or antibiotic treated infant, preferably an antibiotic treated infant or an infant that is being treated with an antibiotic.

[0021] 5. Composition for use according to the preceding embodiments wherein improving and / or ameliorating social recognition, social behaviour and / or social behaviour development comprises improving corticolimbic signaling in the prefrontal cortex and / or amygdala; increasing and / or enhancing glutamate re-uptake and / or myelinization in the amygdala; improving blood brain barrier establishment; and / or improving myelination, axonal growth and / or synapse formation in the amygdala and / or prefrontal cortex.

[0022] 6. Composition for use according to the preceding embodiments wherein the infant is an antibiotic treated infant wherein the adverse effects of antibiotic on social recognition and / or social behaviour; learning; and / or memory are prevented and / or treated.

[0023] 7. Composition for use according to the preceding embodiments wherein the fucosylated oligosaccharide is selected from 2’-fucosyllactose or 3-fucosyllactose, preferably both 2’-fucosyllactose and 3- fucosyllactose, and wherein the sialylated oligosaccharide is selected from 3’-sialyllactose or 6’- sialyllactose, preferably both 3’-sialyllactose and 6’-sialyllactose.

[0024] 8. Composition for use according to the preceding embodiments wherein the weight ratio of HMOs and the bGOS to IcFOS 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 .

[0025] 9. Composition for use according to the preceding claims wherein 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 .

[0026] 10. Nutritional composition for use according to the preceding embodiments, wherein the composition comprises 2.5 to 20 wt% of non-digestible oligosaccharides consisting of i) bGOS, ii) IcFOS) and iii) HMOs, 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, or when in liquid form, 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.

[0027] 11. Composition for use according to the preceding embodiments, wherein the HMOs comprise 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.

[0028] 12. Composition for use according to any one of the preceding embodiments, wherein the composition is a nutritional composition selected from an infant formula, a follow-on formula, or a growing up milk, preferably an infant formula.

[0029] DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention concerns a nutritional composition for infants comprising a mixture of human milk oligosaccharides (HMOs), beta-galacto-oligosaccharides (bGOS) and long chain fructo-oligosaccharides (IcFOS). The invention further pertains to a nutritional composition comprising digestible carbohydrate, protein, lipid, and non-digestible oligosaccharides (NDO), wherein the NDO comprise i) betagalactooligosaccharide (bGOS), ii) long chain fructo-oligosaccharide (IcFOS) and iii) human milk oligosaccharides (HMOs), wherein the HMOS comprise at least one fucosylated oligosaccharide and at least one sialylated oligosaccharide for use in improving and / or ameliorating social recognition, wherein social recognition comprises social memory and / or social recognition memory; improving and / or ameliorating social behaviour and / or social behaviour development in an infant or young child. The nutritional composition according to the invention is not human milk.

[0031] Preferably, the use of the present invention is in a human subject, more preferably in a human young child of 1 , 2 or 3 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.

[0032] Hence, the nutritional composition is advantageously for use in infants or young children, preferably for infant or young children that were born via C-section and / or treated with antibiotics, preferably infants treated with antibiotics.

[0033] DEFINITIONS

[0034] 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”.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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).

[0039] 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".

[0040] 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. 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.

[0041] 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.

[0042] 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).

[0043] 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.

[0044] As used herein, the term "degree of polymerization" (DP) means the number of monomer units joined together in a poly- or oligomer.

[0045] Social recognition as used herein comprises social recognition memory or social memory as used herein is an essential and basic component of social behaviour that is used to discriminate familiar and novel animals / humans. The hippocampus, amygdala, and anterior cingulate cortex (ACC) are critical regions for the formation / consolidation of social recognition whereas the medial prefrontal cortex (mPFC) and amygdala are involved in the regulation of social behaviours such as social interaction and approach. Social recognition can be assessed using a task in mice wherein an adult mouse is allowed to recognize a juvenile mouse through investigations of the juvenile mouse (Tanimizu et al. J Neurosci. 2017 Apr 12; 37(15): 4103— 4116.). The difference in social investigation times between the first and second exposures to a juvenile mouse reflects the familiarity of the two mice.

[0046] Social memory as used herein refers to an individual’s ability to remember information related to social interactions, group identities, and past experiences with other individuals within their social context. It encompasses the collective recollection of events, people, and shared experiences within a community or group.

[0047] Social recognition memory as used herein focuses on the ability to recognize and remember familiar individuals. It allows humans and animals to correctly respond to visitors in cooperative or competitive social situations. Examples include remembering a mating partner or recognizing threatening visitors to an animal’s habitat.

[0048] Both social memory and social recognition memory are part of a broader concept that encompasses both social memory and social recognition memory. Social memory in turn encompasses the broader context of shared group experiences, while social recognition memory specifically focuses on remembering familiar individuals within a social group. Social recognition, on the other hand, includes both memory aspects and the ability to recognize unique characteristics in social interactions.

[0049] 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".

[0050] Human milk oligosaccharides

[0051] 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.

[0052] The nutritional composition comprises a mixture of at least one fucosylated oligosaccharide and at least one sialyllacted 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. 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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. bGOS and IcFOS

[0059] 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.

[0060] 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.

[0061] 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.

[0062] The present nutritional composition comprises long chain fructo-oligosaccharides (IcFOS). A suitable 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).

[0063] 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.

[0064] 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 .

[0065] 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.

[0066] Mixture ofHMOS and GOS / FOS.

[0067] 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 development of social recognition, social behaviour, and cognition.

[0068] 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.

[0069] Preferably the NDOs in the nutritional composition comprising the combination of bGOS-lcFOS 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.

[0070] 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 .

[0071] 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 .

[0072] 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.

[0073] In yet another preferred aspect the ratio of bGOS to IcFOS to HMOs ranges from 20:1 :0.5 to 2:1 : 1 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.

[0074] Digestible carbohydrate

[0075] The nutritional composition comprises digestible carbohydrate. The digestible carbohydrate preferably provides 30 to 80% of the total calories of the nutritional composition. Preferably the digestible carbohydrate provides 40 to 60% of the total calories. Based on calories the nutritional composition preferably comprises of 5 to 20 g of digestible carbohydrate per 100 kcal, more preferably 7.5 to 15 g. When in liquid form, e.g. as a ready-to-feed liquid, the nutritional composition preferably comprises 3 to 30 g digestible carbohydrate per 100 ml, more preferably 6 to 20 g, even more preferably 7 to 10 g per 100 ml. Based on dry weight, the nutritional composition preferably comprises 20 to 80 wt.%, more preferably 40 to 65 wt.% digestible carbohydrate.

[0076] Preferred digestible carbohydrate sources are lactose, glucose, sucrose, fructose, galactose, maltose, starch, and maltodextrin. Lactose is the main digestible carbohydrate present in human milk. Lactose advantageously 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.%, and 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.%.

[0077] Protein

[0078] The nutritional composition comprises protein. The protein preferably provides 5 to 15% of the total calories, more preferably 6 to 12% of the total calories. Preferably protein is present in the nutritional composition below 3.5 gram per 100 kcal, more preferably between 1.8 and 2.1 g protein per 100 kcal, and most preferably between 1.85 and 2.0 g protein per 100 kcal. The protein concentration in a nutritional composition is determined by the sum of protein, peptides, and free amino acids. The amount of protein can be calculated according to the amount of nitrogen multiplied by 6.25. Based on dry weight, the nutritional composition preferably comprises less than 12 wt.% protein, more preferably between 9.6 and 12 wt.%, most preferably between 10 and 11 wt.% protein. Based on a ready-to-drink liquid product the nutritional composition preferably comprises less than 1.5 g protein per 100 ml, more preferably between 1 .2 and 1 .5 g, even more preferably between 1 .25 and 1 .35 g protein per 100 ml.

[0079] 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, sweet whey, whey protein isolate, or mixtures thereof. Preferably the nutritional composition comprises at least 3 wt.% casein based on dry weight.

[0080] Lipid

[0081] The nutritional composition comprises lipid. The term “lipid” as used herein refers to one or more selected from the group consisting of triglycerides, polar lipids (such as phospholipids, cholesterol, glycolipids, sphingomyelin), free fatty acids, monoglycerides and diglycerides.

[0082] 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 lipids providing 40 to 50% of the total calories. The lipids are preferably present in an amount of 4 to 6 g per 100 kcal. When in liquid form, e.g., as a ready-to-feed liquid, the nutritional composition preferably comprises 2.1 to 6.5 g lipids per 100 ml, more preferably 3.0 to 4.0 g per 100 ml. Based on dry weight, the nutritional composition preferably comprises 10 to 50 wt.%, more preferably 12.5 to 40 wt.% lipids, even more preferably 19 to 30 wt.% lipids.

[0083] The lipid preferably comprises vegetable lipid. The presence of vegetable lipids advantageously enables an optimal fatty acid profile, high in polyunsaturated fatty acids and / or more reminiscent to human milk fat. Lipid from mammalian milk alone, e.g., cow’s milk, do not provide an optimal fatty acid profile. The amount of essential fatty acids is too low in mammalian milk. Also, LC-PUFA such as docosahexaenoic acid (DHA) and arachidonic acid (ARA), preferably docosahexaenoic acid is present.

[0084] 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. In addition, animal fat such as milkfat may be present. Suitable sources for providing DHA and ARA are fish oil, marine oil and microbial oils known in the art.

[0085] Nutritional composition

[0086] The nutritional composition is preferably selected from infant formula, follow-on formula, and young child formula. More preferably, the nutritional composition is an infant formula or a follow-on formula. Most preferably, the nutritional composition is an infant formula.

[0087] The terms as used herein, “infant formula” or “follow-on formula” or “young child formula” refers to compositions that are artificially made or that are synthetic This means that the composition that is administered is not human milk. It also means that the composition that is administered is not native cow’s milk or native milk from another mammal. In the present context, 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 formulae are also known as starter formula. Formula for infants starting for 4 to 6 months of life to 12 months of life are intended to be supplementary feedings to infants that start weaning on other foods. Such formulae are also known as follow-on formulae. Infant formulae and follow-on formulae are subject to strict regulations, for example the EU regulations no. 609 / 2013 and no. 2016 / 127. In the present context, young child formulae refer to nutritional compositions, artificially made, intended for infants of 12 months to 36 months, which are intended to be supplementary feedings to infants. Such formulae are also known as growing-up milks.

[0088] The nutritional composition is preferably an infant formula or follow-on formula and preferably comprises 3 to 7 g lipid / 100 kcal, preferably 4 to 6 g lipid / 100 kcal, more preferably 4.5 to 5.5 g lipid / 100 kcal, preferably comprises 1 .7 to 5 g protein / 100 kcal, more preferably 1 .8 to 3.5 g protein / 100 kcal, even more preferably 1.8 to 2.1 g protein / 100 kcal, most preferably 1 .8 to 2.0 g protein / 100 kcal and preferably comprises 5 to 20 g digestible carbohydrate / 100 kcal, more preferably 6 to 16 g digestible carbohydrate / 100 kcal, and most preferably 10 to 15 g digestible carbohydrate / 100 kcal. Non-digestible oligosaccharides have a caloric density of 2 kcal / g and preferably make up 0.4 to 7% of total calories of the nutritional compositions according to the invention.

[0089] Preferably the nutritional composition is an infant formula or follow-on formula and when in a ready-to-drink format has an energy density of 60 kcal to 75 kcal / 100 ml, more preferably 60 to 70 kcal / 100 ml. This density ensures an optimal balance between hydration and caloric intake.

[0090] 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.

[0091] Application

[0092] The compositions according to the invention comprising HMO, bGOS and IcFOS are preferably used for providing nutrition to an infant or young child, preferably an infant. The HMO in the composition according to the invention comprises at least one fucosylated oligosaccharide and at least one sialylated oligosaccharide. The present composition can advantageously be applied as a complete nutrition for infants.

[0093] In an aspect the nutritional composition comprising the HMO, bGOS, IcFOS mixture is a first infant formula for the first 6 months of life, wherein the formula comprises the HMO, bGOS, IcFOS mixture according to the invention and wherein the HMO in the nutritional composition according to the invention comprises at least one fucosylated oligosaccharide and at least one sialylated oligosaccharide.

[0094] Preferably the nutritional composition of the present invention is provided to a human subject during the first 3 years of life. Preferably, the nutritional composition is used in a method or for use for providing nutrition to a human subject in the first 12 months of life, optionally the first 3 years of life.

[0095] The nutritional composition according to the invention is for use in providing nutrition to an infant, preferably infants exposed to antibiotics and / or infants born by C-section, preferably directly after birth and up to 12 months of age.

[0096] Preferably in methods or uses according to the invention, the nutritional composition is administered in the first 12 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.

[0097] In a preferred embodiment, the composition is administered to C-section 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.

[0098] In a preferred embodiment, the composition is administered to infants treated with antibiotics starting at least in the first two weeks after treatment, preferably within the first week after treatment, more preferably at least within 5 days after treatment, even more preferably at least within 3 days after treatment, even more preferably at least within 2 days after treatment, most preferably at the same day as antibiotics treatment is started.

[0099] The invention further concerns the nutritional composition for use in therapy. In an embodiment said nutritional composition according to the invention for therapeutic use is administered to an infant or young child, preferably an infant, more preferably to a C-section born infant and / or antibiotic treated infant, even more preferably an antibiotic treated infant.

[0100] The present invention aims to promote social recognition comprising social memory and / or social recognition memory, social behaviour and / or social behaviour development in infants, preferably C-section born infants and / or antibiotic treated infants, even more preferably antibiotic treated infants. These infants are at risk of impaired social recognition, social behaviour and / or impaired development of social recognition and / or social behaviour. The invention further pertains to the nutritional composition for use in restoring and / or alleviating the effects on brain development in infants, preferably C-section born infants and / or antibiotic treated infants, even more preferably antibiotic treated infants.

[0101] Alternatively worded the nutritional composition of the present invention is preferably suitable for use of, or in a method of providing nutrition to infants suffering from or at risk of impaired social recognition, impaired social memory and / or social recognition memory, impaired social behaviour and / or social behaviour development.

[0102] The invention further pertains to nutritional compositions according to the invention comprising HMO, bGOS and IcFOS for therapeutic use in improving and / or ameliorating social recognition improving and / or ameliorating social recognition comprising improving and / or ameliorating social memory or social recognition memory improving and / or ameliorating social behaviour and / or social behaviour development.

[0103] In an aspect the therapeutic use in improving and / or ameliorating social recognition, social behaviour and / or social behaviour development comprises improving corticolimbic signalling in the prefrontal cortex and / or amygdala; increasing and / or enhancing glutamate re-uptake and / or myelinisation in the amygdala; improving blood brain barrier establishment; and / or improving myelination, axonal growth and / or synapse formation in the amygdala and / or prefrontal cortex.

[0104] Connections between amygdala and PFC are known to be crucial for the development of social recognition.

[0105] The use further pertains to improved central nervous system development (CNS) and blood brain barrier (BBB) establishment.

[0106] Preferably the nutritional composition is for use in improving and / or ameliorating social recognition, improving and / or ameliorating social memory or social recognition memory, improving and / or ameliorating social behaviour and / or social behaviour development and / or improving in an infant, more preferably to a C-section born infant and / or antibiotic treated infant, even more preferably an antibiotic treated infant. The nutritional composition is for use in countering and / or improving and / or ameliorating antibiotic- induced changes in brain and behaviour. Y1

[0107] The nutritional composition is for use in restoring and / or alleviating adverse effects on brain development in infants, preferably C-section born infants and / or antibiotic treated infants, even more preferably antibiotic treated infants. Preferably the nutritional composition is for use in restoring and / or alleviating the adverse effects of an antibiotic on social recognition, social behaviour, learning, memory and / or CNS development. Restoring and / or alleviating adverse effects on brain development in said infants comprises improving corticolimbic signalling in the prefrontal cortex and / or amygdala, increasing and / or enhancing glutamate reuptake and / or myelinisation in the amygdala, improving myelination, axonal growth and / or synapse formation in the amygdala and / or prefrontal cortex.

[0108] Social behaviour impairments are common features among a variety of neuropsychiatric conditions including autism spectrum disorder (ASD). In some embodiments the nutritional composition is for use in improving and / or ameliorating social recognition, improving and / or ameliorating social memory or social recognition memory, improving and / or ameliorating social behaviour and / or social behaviour development in an infant, preferably an infant at risk of autism spectrum disorder.

[0109] The invention can also be worded as the use of HMO, bGOS and IcFOS, wherein the HMO comprises at least one fucosylated oligosaccharide and at least one sialylated oligosaccharide in the manufacture of a nutritional composition for improving and / or ameliorating social recognition, for improving and / or ameliorating social memory or social recognition memory, improving and / or ameliorating social behaviour and / or social behaviour development in an infant.

[0110] The use further pertains to improving corticolimbic signalling in the prefrontal cortex and / or amygdala, increasing and / or enhancing glutamate re-uptake and / or myelinisation in the amygdala; improving myelination, axonal growth and / or synapse formation in the amygdala and / or prefrontal cortex in said infant. The use in the manufacture of a nutritional composition further pertains to restoring and / or alleviating the adverse effects of an antibiotic treatment on social recognition, social behaviour, learning, memory and / or CNS development. Restoring and / or alleviating adverse effects on brain development in said infants comprises improving corticolimbic signalling in the prefrontal cortex and / or amygdala, increasing and / or enhancing glutamate re-uptake and / or myelinisation in the amygdala, improving myelination, axonal growth and / or synapse formation in the amygdala and / or prefrontal cortex.

[0111] Also the invention concerns a method for improving and / or ameliorating social recognition, improving and / or ameliorating social memory or social recognition memory, social behaviour and / or social behaviour development in an infant in infants at risk of or suffering from impaired social recognition comprising impaired social memory and / or impaired social recognition memory, improving and / or ameliorating social behaviour and / or social behaviour development comprising administering to the infant a nutritional composition comprising HMO, bGOS and IcFOS and wherein the HMO comprises at least one fucosylated oligosaccharide and at least one sialylated oligosaccharide. The method thus further pertains to restoring and / or alleviating the adverse effects of an antibiotic on social recognition, social behaviour, learning, memory and / or CNS development. Restoring and / or alleviating adverse effects on brain development in said infants comprises improving corticolimbic signalling in the prefrontal cortex and / or amygdala, increasing and / or enhancing glutamate re-uptake and / or myelinisation in the amygdala, improving myelination, axonal growth and / or synapse formation in the amygdala and / or prefrontal cortex.

[0112] LIST OF FIGURES

[0113] The present invention will be discussed in more detail below, with reference to the attached figures.

[0114] Figure 1. HMOs and GOS / FOS alone or in combination improves social behaviour. Interaction time in social novelty as part of the three-chambers social interaction test at P22 (F: familiar conspecific mouse, N: novel conspecific mouse. Data were analysed with paired-sample t-test, presented here with individual values as mean+-SEM).

[0115] Figure 2. The effect of HMOs, GOS / FOS, and HMOs+GOS / FOS on restoration or gene expression in pathways relevant to learning or memory and CNS development in P23 amygdala in response to ABX- treatment is shown. Pathway enrichment analysis of genes whose expression is restored in response to ABX HMOs, ABX GOS / FOS, or ABX HMOs+GOS / FOS compared with ABX Vehicle in amygdala at P23: Significance (p<0.05) is indicated with a grey circle and the magnitude of the enrichment of each pathway is indicated by the enrichment ratio.

[0116] Figure 3. The effect of HMOs, GOS / FOS, and HMOs+GOS / FOS on restoration or gene expression in pathways relevant to learning or memory and CNS development in P23 prefrontal cortex (PFC) in response to ABX-treatment. Pathway enrichment analysis of genes whose expression is restored in response to ABX HMOs, ABX GOS / FOS, or ABX HMOs+GOS / FOS compared with ABX Vehicle in the prefrontal cortex (PFC) at P23: Significance (p<0.05) is indicated with a grey circle and the magnitude of the enrichment of each pathway is indicated by the enrichment ratio.

[0117] EXAMPLES

[0118] Example 1

[0119] Material and Methods

[0120] Animals and diet

[0121] NIH Swiss (HSD:NIHS) female and male breeder mice were purchased at 8 weeks old from Envigo, UK. After they were acclimated in the animal unit for 1 week in 33x15x13cm white cages with environmental enrichment (bedding, nesting material, cylindrical tube) and access to tap water and chow food ad libitum at 26 degrees Celsius in a 12-hour light, 12-hour dark cycle. Animals were bred for the first time (primiparous), with plug defining the beginning of gestation and the moment of separation of females from the male breeders into their own cage. After separation, the males were sacrificed. Approximately 20 days after plug detection offspring was produced. The offspring in each cage was administered either a broadspectrum antibiotics (ABX) mixture (ampicillin: 0.2mg / g, gentamicin: 0.2mg / g, vancomycin: 0.1 mg / g and imipenem: 0.05mg / g) or tap water (Vehicle) via gavage from postnatal day 3 (P3) to P7.

[0122] A mixture of HMOs (39 wt% 2’-FL, 3-FL, 10 wt% 3’-SL and 28 wt% 6’-SL) or bGOS (GOS sirup Vivinal) / IcFOS (inulin HP) at a 9:1 weight ratio or a combination of HMOs+bGOS / lcFOS consisting of 2.02 parts bGOS and IcFOS and 1.28 parts of the mixture of HMOs (such that the the bGOS, IcFOS, HMO mixture had a 9:1 weight ratio of short chain to long chain oiligosaccharides) was provided to the offspring by oral administration from postnatal day 3 (P3) until P20.

[0123] At P21 the pups were weaned to their respective equicaloric diets: 1) control, 2) HMOs, 3) bGOS / lcFOS or 4) HMOs+bGOS / lcFOS (sniff diets, GmbH, Germany) until P23. All procedures were conducted with approval from the Animal Experimentation Ethics Committee (AEEC) at University College Cork and the Health Products Regulatory Authority (HPRA) in accordance with the recommendations of the European Directive 2010 / 63 / EU and, followed the ARRIVE 2.0 guidelines.

[0124] Behavioural testing

[0125] The short and long-term effects of treatment with ABX and either HMOs, bGOS / lcFOS or HMOs+bGOS / lcFOS intervention on behaviour were evaluated in male offspring both in early life (P9 and P10) and in juvenile period (P22). Behavioural tests were analysed by an independent experimenter blinded to experimental groups. All tests were performed during the lights on phase and between the hours of 9am and 2pm.

[0126] Three-chamber social interaction test

[0127] The three-chamber social interaction test is widely used to evaluate social behaviour as previously described by Desbonnet et al. 2014. In brief, animals were placed in a 36x19x30cm rectangular light grey apparatus divided into three chambers (left and right chambers: 13x19x30cm each], and a smaller centre chamber: 9x19x30cm), small circular openings allowed easy access to all compartments. Social novelty recognition was assessed where the experimental animal is allowed to interact with a familiar or novel conspecific animal under a wired mesh (circular, 9cm diameter). All animals were age- and sex-matched; each chamber was cleaned and lined with fresh bedding between trials. Interaction time were recorded by a video camera mounted above the apparatus.

[0128] The three chambers social interaction test was used to assess the social behaviour in juvenile mice.

[0129] Transcriptomics of amygdala and prefrontal cortex

[0130] Whole RNA from dissected P23 amygdala and prefrontal cortex (PFC) was isolated with the mirVana RNA isolation kit (Invitrogen) and maintained at -80°C until further processing. Paired-end sequence reads were generated using the Illumina NovaSeq 6000. The sequences generated with the NovaSeq 6000 were performed under accreditation according to the scope of BaseClear B.V. (L457; NEN-EN-ISO / IEC 17025). FASTQ read sequence files were generated using bcl2fastq version 2.20 (Illumina). Initial quality assessment was based on data passing the Illumina Chastity filtering. Subsequently, reads containing PhiX control signal were removed using an in-house filtering protocol. In addition, reads containing (partial) adapters were clipped (up to a minimum read length of 50 bp). The second quality assessment was based on the remaining reads using the FASTQC quality control tool version 0.11 .8. Transcripts were annotated using kallisto (v 0.48.0) using the C57BL6 GRCm39 mouse reference genome using default parameters.

[0131] Brain transcriptomics biostatistical analysis

[0132] Further analysis of RNAseq data was handled in R (v 4.3.1). Gene transcription tables were filtered based on prevalence (50% threshold) and subsequently CLR-transformed, using the same 2 / 3 pseudocount imputation to deal with zeroes as described in Lubbe et al., 2021 .

[0133] To determine whether a gene was restored to control levels, a generalised linear model with a treatmentwise planned orthogonal contrast approach (Control Vehicle = 1 / 2, ABX Vehicle = -1 , ABX + <Treatment> = 1 / 2) was used. Briefly, genes were considered to be restored if 1) the pooled control vehicle and ABX + treatment groups together were different from the ABX Vehicle group and 2) additionally, the ABX Vehicle group was significantly different from Control Vehicle (post-hoc test). Principal component analysis was carried out in a treatment-wise fashion, using the R prcomp implementation in the ‘stats’ library. Compositional differences were estimated using the adonis2 implementation of PERMANOVA.

[0134] Targeted enrichment analysis was performed by pre-defining GO terms of interest (astrocyte differentiation G0:0048708, CNS development G0:0007417, cytokine production G0:0001816, ensheathment of neurons G0:0007272, establishment of BBB G0:0060856, fatty acid catabolism G0:0009062, fucose metabolism G0:0006004, ganglioside metabolism G0:0001573, gliogenesis G0:0042063, glutamate metabolism G0:0006536, glutamate reuptake GO:0051935, learning and memory G0:0007611 , myelin sheath G0:0043209, myelination G0:0042552, neurogenesis G0:0022008, neuron projection morphogenesis G0:0048812, oligodendrocyte differentiation G0:0048709, response to AVP GO:1904117, SCFAs biosynthesis G0:0051790, structural constituent of myelin sheath G0:0019911). Enrichment was assessed using hypergeometric tests (e.g., phyperQ in the R stats package) on the subset of genes that were found to be restored on the p < 0.05 level. Figures were generated in R with ggplot2 (version 3.4.4). Data analysis of non-omics data

[0135] All statistical analysis (apart from RNA sequencing for amygdala and prefrontal cortex) was performed using IBM SPSS Statistics (IBM, SPSS Statistics V29). Before statistical analysis, data was analysed for normality using the Shapiro-Wilk test and homogeneity of variances using Levene’s test. For statistical analysis of parametric data, we used an independent sample Student t-test, while for non-parametric data we used the Mann-Witney U-test. For the behavioural results from three-chambers social interaction test we used repeated measures t-test. Data is shown as mean ±SEM. Statistical significance was set at p < 0.05.

[0136] Results

[0137] HMOs and / or GOS / FOS ameliorated the social behaviour deficits in juveniles induced by early-life antibiotic treatment.

[0138] The three chambers social interaction test was used to assess the social behaviour in juvenile mice. It was found that ABX administration in early life resulted in significantly decreased social recognition during juvenile period (t10 = -0.723, p = 0.486), that is restored by HMOs (t7 = -2.422, p = 0.046), bGOSIc / FOS (t9 = -2.815, p = 0.02) and HMOs+bGOS / lcFOS (t8 = -4.276, p = 0.003) treatment (Fig. 1). The combination of HMO and bGOS / lcFOS was found to be the most potent in restoring social recognition behaviour in the mice having ABX-induced decreased social recognition.

[0139] HMOs and / or GOS / FOS restored pathways involved in myelination, CNS development and learning and memory in the juvenile amygdala.

[0140] The amygdala is a brain area relevant for social behaviour, as it is the brain centre for emotional processing (Garrido Zinn et al., 2016; Tanimizu et al., 2017). The overall composition of genes expressed in P23 amygdala was altered by early-life ABX administration as visualised in the principle component analysis of the amygdala (Figure 2A) (Control Vehicle vs ABX vs ABX+HMOs: F2,23 = 1 .4983, p = 0.005, Control Vehicle vs ABX vs ABX+bGOS / lcFOS: F2,25 = 1.4693, p = 0.0016, Control Vehicle vs ABX vs ABX+HMOs+bGOS / lcFOS: F2,24 = 1.6693, p = 0.0001). There is a trend observed for bGOS / lcFOS and HMOs+bGOS / lcFOS to result in altered transcriptomic profile in the P23 amygdala (p = 0.053 and p = 0.079 respectively) compared with ABX Vehicle, an observation that could not be made for the amygdala of mice treated with HMOs alone (p = 0.339).

[0141] Pathway enrichment analysis revealed that pathways involved in learning and memory, CNS development, glutamate re-uptake and establishment of blood brain barrier (BBB) were significantly enriched in animals that received any of the three treatments compared to ABX-treated animals (Figure 2). However, the combination of HMOs with bGOS / lcFOS displayed a slightly higher enrichment ratio in some of those pathways compared with the HMOs alone and bGOS / lcFOS alone within the ABX-treated animals (Figure 2). Remarkably, a considerably higher enrichment ratio in specifically the pathways relevant to establishment of the BBB and glutamate reuptake was observed in the combination of HMOs with bGOS / lcFOS group compared to HMOs alone and bGOS / lcFOS alone within the ABX-treated mice (Figure 2). Additionally, pathways relevant for myelination such as oligodendrocyte differentiation, ensheathment of neurons and structural constituent of myelin sheath were significantly altered in response to HMOs, bGOS / lcFOS and HMOs+bGOS / lcFOS compared with ABX Vehicle animals (Figure 2). However, ganglioside metabolism which is also relevant for myelination, was significantly enriched by GOS / FOS and HMOs+bGOS / lcFOS but not HMOs alone. Other pathways of interest that were significantly enriched for any of the prebiotic treatments were neurogenesis, neuron projection morphogenesis and response to vasopressin (Figure 2).

[0142] HMOs and GOS / FOS restored pathways involved in CNS development and learning and memory in the juvenile PFC.

[0143] The PFC is an area implicated in social recognition memory, social motivation, decision making and is crucial brain area for social behaviour. The overall composition of genes in P23 PFC was not altered by ABX or any of the three prebiotic treatments (Control Vehicle vs ABX vs ABX+HMOs: F2,24 = 1 .065, p = 0.1466, Control Vehicle vs ABX vs ABX+GOS / FOS: F2,25 = 0.993, p = 0.4448, Control Vehicle vs ABX vs ABX+HMOs+GOS / FOS: F2,24 = 0.9942, p = 0.3902).

[0144] However, investigating the genes that were restored by each of the treatments we found 982 restored by HMOs, 1657 by GOS / FOS and 1811 by HMOs+bGOS / lcFOS in the P23 PFC. Pathway enrichment analysis of genes whose expression is restored revealed that learning and memory, CNS development and cytokine production pathways were only significantly enriched in the PFC of animals that received HMOs+bGOS / lcFOS (Figure 3B).

[0145] Interestingly, pathway enrichment analysis revealed a common pathway that was significantly enriched in both amygdala and PFC termed ‘Learning and Memory’ in response to HMOs+GOS / FOS treatment compared with ABX in juvenile male mice. Previous studies have unmasked links between multiple brain areas implicated in social behaviour and connections between amygdala and prefrontal cortex (PFC) are crucial for social recognition memory (Felix-Ortiz et al., 2016). This conservation of pathways relevant for brain development and learning and memory in both PFC and amygdala might be linked with the enhanced social recognition memory in the animals that received the combination of HMOs+bGOS / lcFOS.

[0146] Conclusions

[0147] In the present study ABX-treatment in early life results in impaired social behaviour and this effect was ameliorated by HMOs, bGOS / lcFOS and in particular by the combination of HMOs+bGOS / lcFOS supplementation in male mice. Each of the prebiotic treatments ameliorated the social deficits induced by early-life ABX in the social novelty, revealing that social recognition memory was enhanced in those animals that received prebiotics. This conservation of pathways relevant for brain development and learning and memory in both PFC and amygdala might be linked with the enhanced social recognition memory in the animals that received the combination of HMOs+bGOS / lcFOS. Even though this pathway was not significant in the PFC for HMOs or bGOS / lcFOS alone, the significance persisted in amygdala. Moreover, the behaviour of those animals supports that the recognition memory was enhanced in response to HMOs or bGOS / lcFOS compared with control male mice at P22.

[0148] In terms of brain anatomy, the social brain, which includes PFC and amygdala, is going through outstanding transformations from childhood all the way to adolescence. The macrostructural changes in the PFC correspond to neurodevelopmental mechanisms at the microstructural level with myelination, axonal growth and synaptic refinement taking a central role during this period. Pathways relevant to neuron projection development and myelination were significantly enriched in the amygdala of the juvenile offspring in response to HMOs, GOS / FOS or HMOs+bGOS / lcFOS. Therefore, changes in gene expression patterns in amygdala and PFC may underly the improvement of social behaviour in prebiotic-treated juvenile mice compared with ABX-treated controls.

[0149] In conclusion, this example highlights the power of dietary manipulations in early life to counter the effects of antibiotics-induced changes in brain and behaviour. Specifically, it was found that HMOs, bGOS / lcFOS and HMOs+bGOS / lcFOS supplementation in early life has positive effects on the social brain. Overall, the data suggests that the combination of HMOs+bGOS / lcFOS beneficially reverts the adverse effects of antibiotic exposure on the developing brain, particularly improving social recognition memory and social behaviour which is supported by gene expression patterns associated with the establishment of the relevant connections therefore.

Claims

CLAIMS1. A nutritional composition comprising digestible carbohydrate, protein, lipid, and non-digestible oligosaccharides (NDO), wherein the NDO comprise i) betagalacto-oligosaccharide (bGOS), ii) long chain fructo-oligosaccharide (IcFOS) and iii) human milk oligosaccharides (HMOs), wherein the HMOS comprise at least one fucosylated oligosaccharide and at least one sialylated oligosaccharide for use in improving and / or ameliorating social recognition; improving and / or ameliorating social behaviour and / or social behaviour development. social behaviour, in an infant or young child, wherein said nutritional composition is not human milk.

2. Nutritional composition for use according to claim 1 wherein social recognition comprises social memory or social recognition memory.

3. Nutritional composition for use according to the preceding claims wherein the infant or young child is suffering from or at risk of impaired social recognition, social memory, social recognition memory, social behaviour and / or social behaviour development.

4. Nutritional composition for use according to the preceding claims wherein the infant or young child is a C-section born infant and / or antibiotic treated infant, preferably an antibiotic treated infant.

5. Nutritional composition for use according to the preceding claims wherein improving and / or ameliorating social recognition, social behaviour and / or social behaviour development comprises improving corticolimbic signaling in the prefrontal cortex and / or amygdala; increasing and / or enhancing glutamate re-uptake and / or myelinisation in the amygdala; improving blood brain barrier establishment; and / or improving myelination, axonal growth and / or synapse formation in the amygdala and / or prefrontal cortex.

6. Nutritional composition for use according to the preceding claims wherein the infant is an antibiotic treated infant wherein the adverse effects of antibiotic on social recognition and / or social behaviour; learning; and / or memory are prevented and / or treated.

7. Nutritional composition for use according to the preceding claims wherein the fucosylated oligosaccharide is selected from 2’-fucosyllactose or 3-fucosyllactose, preferably both 2’-fucosyllactose and 3-fucosyllactose, and wherein the sialylated oligosaccharide is selected from 3’-sialyllactose or 6’- sialyllactose, preferably both 3’-sialyllactose and 6’-sialyllactose.

8. Nutritional composition for use according to the preceding claims wherein the weight ratio of HMOs and the bGOS to IcFOS 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 .

9. Nutritional composition for use according to the preceding claims wherein 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 .

10. Nutritional composition for use according to the preceding claims, wherein the nutritional composition comprises 2.5 to 20 wt% of non-digestible oligosaccharides consisting of i) bGOS, ii) IcFOS) and iii) HMOs, 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, or when in liquid form, 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.

11. Nutritional composition for use according to the preceding claims, wherein the HMOs comprise 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.

12. The nutritional composition for use according to any one of the preceding claims, wherein the nutritional composition is selected from an infant formula, a follow-on formula, or a growing up milk, preferably an infant formula.

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