Promoting butyrate-producing bacteria in the gut microbiome

Abstract

The present invention relates to a bifidogenic prebiotic for use in promoting butyrate-producing bacteria in the gut microbiome of an infant or young child; wherein the bifidogenic prebiotic is administered to the infant prior to about 12 months of age; and the butyrate-producing bacteria are promoted in the gut microbiome of the infant or young child between about 1 and 5 years of age.

Description

[0001] PROMOTING BUTYRATE-PRODUCING BACTERIA IN THE GUT MICROBIOME

[0002] Field of the Invention

[0003] The present invention is related to probiotics and prebiotics for use in promoting butyrate- producing bacteria in the gut microbiome of an infant or young child. of the Invention

[0004] The early postnatal development of the gut microbiome follows patterns associated with changes in the diet and immune maturation. These patterns include the initial predominance and subsequent decline of bifidobacteria, followed by the expansion of producers of shortchain fatty acids (SCFA), for example butyrate-producing bacteria. Butyrate-producers are known to be associated with beneficial effects in respect of inflammation, allergy and / or tolerance; for example.

[0005] Co-administration of Bifidobacterium and butyrate producers has been described. In particular, Bifidobacterium produce lactate / acetate which can be consumed by co-localised butyrate-producing bacteria.

[0006] However, there remains a need to develop new strategies for promoting levels of butyrate- producing bacteria in the gut microbiome of an infant or young child. Promoting levels of these bacteria is expected to have beneficial effects on outcomes that are known to be influenced by butyrate-producing bacteria; for example, positive immune effects on inflammation, allergy and / or tolerance.

[0007] Summary of the Invention

[0008] The present invention is based, at least in part, on the inventors’ determination that early colonization of the infant gut microbiome with infant-type Bifidobacterium promotes the subsequent abundance and function of butyrate-producing bacteria in the gut microbiome

[0009] The inventors have determined a negative association between bifidobacteria and butyrate- producing bacteria in multiple cohorts and across ages, indicating a replacement of bifidobacteria with butyrate-producing taxa as the infants get older. Further, in a longitudinal analysis, the inventors determined a positive association in multiple cohorts between infanttype bifidobacteria in infants below 10 months old, and later abundance of butyrate-producing bacteria taxa from 15 months old. Finally, the inventors have determined that the administration of infant formula enriched in human milk oligosaccharides (HMOs) to infants of 3 to 6 months of age results in an increase of Bifidobacterium at that age, and a subsequent increase of butyrate-producing bacteria (e.g. Faecalibacterium prausnitzii and Eubacterium rectale) and levels of butyrate molecule at later ages, compared to a control population. To the best of the inventors’ knowledge, this is the first determination of a longitudinal relationship between bifidobacteria and butyrate-producing taxa as infants get older.

[0010] In a first aspect, the present invention provides a bifidogenic prebiotic for use in promoting butyrate-producing bacteria in the gut microbiome of an infant or young child; wherein the bifidogenic prebiotic is administered to the infant prior to about 12 months of age; and the butyrate-producing bacteria are promoted in the gut microbiome of the infant or young child between about 1 and 5 years of age.

[0011] In a further aspect, the invention provides a Bifidobacterium microorganism for use in promoting butyrate-producing bacteria in the gut microbiome of an infant or young child; wherein the Bifidobacterium is administered to the infant prior to about 12 months of age; and the butyrate-producing bacteria are promoted in the gut microbiome of the infant or young child between about 1 and 5 years of age.

[0012] In another aspect, the invention provides a combination of a bifidogenic prebiotic and a Bifidobacterium microorganism for use in promoting butyrate-producing bacteria in the gut microbiome of an infant or young child wherein the combination is administered to the infant prior to about 12 months of age; and the butyrate-producing bacteria are promoted in the gut microbiome of the infant or young child between about 1 and 5 years of age.

[0013] In a further aspect, the invention provides a bifidogenic prebiotic, a Bifidobacterium microorganism or a combination of a bifidogenic prebiotic and a Bifidobacterium microorganism for use in treating and / or preventing an allergy and / or allergic sensitization in an infant or young child by promoting butyrate-producing bacteria in the gut microbiome of the infant or young child; wherein the bifidogenic prebiotic, Bifidobacterium microorganism or a combination is administered to an infant prior to about 12 months of age; and the butyrate- producing bacteria are promoted in the gut microbiome of the infant or young child between about 1 and 5 years of age.

[0014] In another aspect, the invention relates to a use of a bifidogenic prebiotic, a Bifidobacterium microorganism or a combination of a bifidogenic prebiotic and a Bifidobacterium microorganism to promote butyrate-producing bacteria in the gut microbiome of the infant or young child; wherein the bifidogenic prebiotic, Bifidobacterium microorganism or a combination is administered to an infant prior to about 12 months of age; and the butyrate- producing bacteria are promoted in the gut microbiome of the infant or young child between about 1 and 5 years of age. In a further aspect, the invention provides a method of promoting butyrate-producing bacteria in the gut microbiome of an infant or young child comprising administering a bifidogenic prebiotic, a Bifidobacterium microorganism or a combination of a bifidogenic prebiotic and a Bifidobacterium microorganism to the infant or young child; wherein the bifidogenic prebiotic, Bifidobacterium microorganism or a combination is administered to an infant prior to about 12 months of age; and the butyrate-producing bacteria are promoted in the gut microbiome of the infant or young child between about 1 and 5 years of age.

[0015] In another aspect, the invention relates to method of treating and / or preventing an allergy and / or allergic sensitization in an infant or young child by promoting butyrate-producing bacteria in the gut microbiome of the infant or young child; comprising administering a bifidogenic prebiotic, a Bifidobacterium microorganism or a combination of a bifidogenic prebiotic and a Bifidobacterium microorganism to the infant or young child; wherein the bifidogenic prebiotic, Bifidobacterium microorganism or a combination is administered to the infant prior to about 12 months of age; and administration of bifidogenic prebiotic, Bifidobacterium microorganism or a combination promotes butyrate-producing bacteria in the gut microbiome of the infant or young child between about 1 and 5 years of age.

[0016] Brief Description of the Drawings

[0017] Figure 1 - Scatterplot showing the relationship between the relative abundance of infant type Bifidobacterium spp. at ages below 6 months and the relative abundance of butyrate producers at ages of 12 months and after. Each dot represents one participant; blue line with grey shade represents the non-linear regression line. Values on the top-left corner represent the Spearman correlation coefficient and its associated p-value.

[0018] Figure 2 - Heatmap showing the relationship between different Bifidobacterium species and butyrate producers in two different populations (Central Europe and South Asia). X axis (on TOP) displays infant age at which Bifidobacterium spp. (Bif) and Butyrate producers (BP) were measured. Y axis (on the left) shows the Bifidobacterium spp species to be analyzed and the population (MH = South Asia, Bangladesh; ON = Central Europe). Each coloured square represents one correlation test. Colour represents correlation coefficient. Grey squares represent no measurement. Black Border represents significant associations.

[0019] Figure 3 - Heatmap showing the relationship between Bifidobacterium iuvenis and the different butyrate producers in a cohort of South Asia. X axis (on TOP) displays infant age at which Bifidobacterium iuvenis (B. iuvenis) and butyrate producers (BP) were measured. Y axis (on the left) shows the butyrate producer species to be analyzed and the population. Each coloured square represents one correlation test. Colour represents correlation coefficient. Red Border represents significant associations.

[0020] Figure 4 - Schematic of HMOs trial - Healthy full-term infants were randomly assigned to a standard cow’s milk-based starter infant formula (control group, CG); the same formula with 1.5 g / L HMOs (test group 1 , TG1); or with 2.5 g / L HMOs (test group 2, TG2); or a human milk- fed group (reference, HMG). Fecal samples were collected at enrolment, 3, 6, 12, and 15 months of age.

[0021] Figure 5 - Boxplots representing the association differences between control formula and HMO-enriched formula. X-axis represent time, Y-axis represent relative abundance. On plot (A), colors represent groups (control formula = yellow, HMO formula = Purple). Each dot represents one participant. On plot (B), each panel represents one group. Each dot represents one participant in each formula type. Asterisks represent one-way T-tests (Control vs others, 0.005 = '***'; 0.01 = 0.05 = 0.1 = CG: Control formula (only matrix); TG1 = 1.5g / L

[0022] HMO formula; TG2 = 2.5g / L HMO formula.

[0023] Detailed Description of the invention

[0024] All percentages are by weight unless otherwise stated.

[0025] The terms “about” or “approximatively” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specific value, such as the variation of 1 / -10% or less, 1 / -5% or less, 1 / -1 % or less, and + / 0.1 % or less of and from the specific value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0026] The terms “subject”, “individual” and “patient” are used interchangeably to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include but are not limited to murines, simians, humans, farm animals, sport animals and pets.

[0027] The term “infant” means a human subject under the age of 12 months or an age equivalent non-human animal.

[0028] The terms “young child” or “toddler” as used herein may mean a human subject aged between 12 months and 5 years of age. Suitably, a “young child” may refer to an age equivalent non- human animal. The expressions “composition” or “nutritional composition” refer to any kind of composition or formulation that provides a nutritional benefit to an individual and that may be safely consumed by a human or an animal. A nutritional composition may be in solid (e.g. powder), semi-solid or liquid form and may comprise one or more macronutrients, micronutrients, food additives, water, etc. For instance, the nutritional composition may comprise the following macronutrients: a source of proteins, a source of lipids, a source of carbohydrates and any combination thereof. Furthermore, the nutritional composition may comprise the following micronutrients: vitamins, minerals, fiber, phytochemicals, antioxidants, prebiotics, probiotics, bioactives, metabolites (e.g. butyrate, Docosahexaenoic acid (DHA), Eicosapentaenoic acid (EPA), Gamma-Linolenic acid (GLA)) and any combination thereof. The composition may also contain food additives such as stabilizers (when provided in liquid or solid form) or emulsifiers (when provided in liquid form). The amount of the various ingredients (e.g. the oligosaccharides) can be expressed in g / 100 g of composition on a dry weight basis when it is in a solid form, e.g. a powder, or as a concentration in g / L of the composition when it refers to a liquid form (this latter also encompasses liquid composition that may be obtained from a powder after reconstitution in a liquid such as milk, water, e.g. a reconstituted infant formula or follow-on / follow-up formula or infant cereal product or any other formulation designed for infant or young child nutrition). Generally, a nutritional composition can be formulated to be taken enterally, orally, parenterally, or intravenously, and it usually includes one of more nutrients selected from: a lipid or fat source, a protein source, and a carbohydrate source. Preferably, a nutritional composition is for oral use.

[0029] In one embodiment, the nutritional composition is a “synthetic nutritional composition”. The expression “synthetic nutritional composition” means a mixture obtained by chemical and / or biological means.

[0030] The expression "infant formula" as used herein refers to a foodstuff intended for particular 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 “follow-up formula” or “follow-on formula”.

[0031] 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 expression “baby food” means a foodstuff intended for particular nutritional use by infants or young children during the first years of life.

[0032] The terms “fortifier” refers to liquid or solid nutritional compositions suitable for fortifying or mixing with human milk, infant formula, growing-up milk or human breast milk fortified with other nutrients. Accordingly, the fortifier can be administered after dissolution in human breast milk, in infant formula, in growing-up milk or in human breast milk fortified with other nutrients or otherwise it can be administered as a stand-alone composition. When administered as a stand-alone composition, the milk fortifier can be also identified as being a “supplement”.

[0033] The term “HMO” or “HMOs” refers to human milk oligosaccharide(s). These carbohydrates are highly resistant to enzymatic hydrolysis, indicating they may display essential functions not directly related to their caloric value. It has been especially illustrated they play a vital role in the early development of infants and young children, such as the maturation of the immune system. Many different kinds of HMOs are found in the human milk. Each individual oligosaccharide is based on a combination of glucose, galactose, sialic acid (N- acetylneuraminic acid), fucose and / or N-acetylglucosamine with many and varied linkages between them, thus accounting for the enormous number of different oligosaccharides in human milk - over 130 such structures have been identified so far. Almost all of them have a lactose moiety at their reducing end while sialic acid and / or fucose (when present) occupy the terminal position at the non-reducing ends. Depending on the presence of fucose and sialic acid in the oligosaccharide structure, the HMOs can be divided as non-fucosylated (neutral) or fucosylated (neutral) and sialylated (acidic) and non-sialylated molecules, respectively.

[0034] The HMOs used in the present invention may be obtained by any suitable method. Suitable methods for synthesising HMOs will be well known to those of skill in the art. For example, processes have been developed for producing HMOs by microbial fermentations, enzymatic processes, chemical syntheses, or combinations of these technologies (see e.g. Zeuner et al., 2019. Molecules, 24(11), p.2033).

[0035] The expression “fucosylated oligosaccharide” refers to an oligosaccharide having a fucose residue. It has a neutral nature. Some examples are 2’-fucosyllactose (2-FL), 3-fucosyllactose (3-FL), difucosyllactose (DiFL), 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, difucosyllacto-N-neohexaose II and any combination thereof. Fucosylated oligosaccharides represents the largest fraction of human milk with 2’-FL constituting up to 30% of the total HMOs. Fucosylated oligosaccharides are thought to reduce the risk of infections and inflammations and to boost growth and metabolic activity of specific commensal microbes reducing inflammatory response.

[0036] The expression “N-acetylated oligosaccharide(s)” encompasses both “N-acetyl-lactosamine” 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), LNnT (lacto-N-neotetraose), DSLNT (disialyllacto-N- tetraose), and any combinations thereof. Other 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.

[0037] The expressions “at least one fucosylated oligosaccharide” and “at least one N-acetylated oligosaccharide” should be understood as “at least one type of fucosylated oligosaccharide” and “at least one type of N-acetylated oligosaccharide”.

[0038] The term “sialylated oligosaccharide” refers to an oligosaccharide having a charged sialic acid residue. It has an acidic nature. Some examples are 3’-sialyllactose (3-SL), 6’-sialyllactose (6- SL), sialyllacto-N-tetraose (Lst - e.g. Lst-a, Lst-b or Lst-c).

[0039] The term fibers is used herein to refer to carbohydrates that are indigestible by a human or animal. Such fibers are also discussed in relation to carbohydrates herein. Suitably, the fiber can be fermented by one or more Bifidobacterium microorganisms provided in the present use or composition and / or within one or more regions in the gastrointestinal tract within an organism, such as a human or non-human animal. As used herein, the expressions “fiber” or “fibers” or “dietary fiber” or “dietary fibers” within the context of the present invention indicate the indigestible portion, in small intestine, of food derived from plants which comprises two main components: soluble fiber, which dissolves in water and insoluble fiber. Mixtures of fibers are comprised within the scope of the terms above mentioned. Soluble fiber is readily fermented in the colon into gases and physiologically active byproducts and can be prebiotic and viscous. Insoluble fiber does not dissolve in water, is metabolically inert and provides bulking, or it can be prebiotic and metabolically ferment in the large intestine. Chemically, dietary fiber consists of carbohydrate polymers with three or more monomeric units which are not hydrolyzed by endogenous enzymes in the small intestine such as arabinoxylans, cellulose, and many other plant components such as resistant starch, resistant dextrins, inulin, lignin, chitins, pectins, arabinans, arabinogalactans, galactans, xylans, beta-glucans, and oligosaccharides. Non-limiting examples of dietary fibers are: prebiotic fibers such as Fructooligosaccharides (FOS), inulin, galacto-oligosaccharides (GOS), fruit fiber, vegetable fiber, cereal fiber, resistant starch such as high amylose corn starch. GOS may include raffinose, stachyose, or verbascose; for example.

[0040] As used herein, “added fiber” or “added dietary fiber” indicates an ingredient mainly or totally constituted by fiber which is added to the complementary nutritional composition and whose content in fiber contributes to the total fiber content of the composition. The total fiber content of the complementary nutritional composition is provided by the sum of amount of fiber naturally present in ingredients used in the recipe (for example from whole grain cereal flour) plus amount of added fiber.

[0041] The term “prebiotic” means non-digestible carbohydrates that beneficially affect the host by selectively stimulating the growth and / or the activity of healthy bacteria, such as bifidobacteria, in the colon of humans (Gibson GR, Roberfroid MB. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr. 1995;125:1401-12). A “bifidogenic prebiotic” means non-digestible carbohydrates that beneficially affect the host by selectively stimulating the growth and / or the activity of bifidobacteria in the gut microbiota of the infant or young child.

[0042] The term “probiotic” means microbial cell preparation or components of microbial cells with a beneficial effect on the health or well-being of the host (Salminen S, Ouwehand A. Benno Y. et al. “Probiotics: how should they be defined” Trends Food Sci. Technol. 1999:10 107-10). The microbial cells according to the present invention are generally bacteria. A probiotic may comprise between 103to 1012cfu of probiotic strain, more preferably between 107and 1012cfu such as between 108and 101° cfu of probiotic strain per g of composition on a dry weight basis

[0043] The term “cfu” should be understood as colony forming unit.

[0044] The “gut microbiota” is the composition of microorganisms (including bacteria, archaea and fungi) that live in the digestive tract.

[0045] The term “gut microbiome” may encompass both the “gut microbiota” and their “theater of activity”, which may include their structural elements (nucleic acid, proteins, lipids, polysaccharides), metabolites (signaling molecules, toxins, organic and inorganic molecules) and molecules produced by coexisting hosts and structured by the surrounding environmental conditions (Berg, G., et al., 2020. Microbiome, 8(1), pp.1-22).

[0046] The term “promoting” a bacteria as used herein may refer to increasing and / or augmenting the growth and / or survival of the bacteria in the gut microbiota. Growth and / or survival of a bacteria may be determined by measuring bacteria cell number, cell density (e.g. measured by optical density) and / or the abundance of 16S rDNA - for example using PCR methods. Promoting growth and / or survival of the bacteria may increase the number of a given bacterial microorganism in an anaerobic culture by at least 20%, at least 30%, at least 40%, at least 50%, at least 75% or at least 100% compared to the number of that bacterial microorganism in a control anaerobic culture which does not comprise the treatment or composition.

[0047] Bifidogenic prebiotic

[0048] A bifidogenic prebiotic may refer to a prebiotic that is capable of being metabolized by a Bifidobacterium. Suitably, the bifidogenic prebiotic is capable of promoting growth and / or survival of the Bifidobacterium. A bifidogenic prebiotic capable of promoting growth and / or survival of Bifidobacterium may be determined by e.g. anaerobic culture of Bifidobacterium with the prebiotic. Growth and / or survival of the Bifidobacterium may be determined by measuring bacteria cell number, cell density (e.g. measured by optical density) and / or the abundance of 16S rDNA - for example using PCR methods A bifidogenic prebiotic capable of promoting growth and / or survival of the Bifidobacterium may increase the number of Bifidobacterium in an anaerobic culture by at least 20%, at least 30%, at least 40%, at least 50%, at least 75% or at least 100% compared to the number of Bifidobacterium bacteria in a control anaerobic culture which does not comprise the prebiotic. Suitably, a prebiotic capable of promoting growth and / or survival of the Bifidobacterium may increase the number of Bifidobacterium bacteria in an anaerobic culture by a statistically significant amount (e.g. p- value <0.05 as determined by one-way ANOVA) compared to the number of Bifidobacterium bacteria in a control anaerobic culture which does not comprise the prebiotic.

[0049] Suitable bifidogenic prebiotics are known in the art. For example, the bifidogenic prebiotic may comprise a HMO and / or a GOS.

[0050] Preferably, the bifidogenic prebiotic comprises one or more HMOs, most preferably one or more human HMOs. Suitable HMOs, and mixtures thereof, are described in further detail herein.

[0051] Butyrate-producing bacteria

[0052] Butyrate-producing bacteria are known in the art (see Louis & Flint; FEMS Microbiology Letters; 294(1); 1-8; 2009). Butyrate-producing bacteria represent a functional group of bacteria which are capable of synthesising butyrate. Butyrate plays a key role in maintaining human gut health, as the major source of energy to the colonic mucosa, and as an important regulator of gene expression, inflammation, differentiation and apoptosis in host cells. Butyrate-producing bacteria carry out fermentative metabolism, i.e. they gain energy in the form of ATP by substrate-level phosphorylation during oxidative substrate breakdown. The resulting reducing equivalents (in the form of NADH) are transferred onto metabolic intermediates, leading to the formation of large amounts of reduced end products such as butyrate. Fermentative bacteria generally possess alternative pathways leading to the formation of different end products. Many butyrate producers from the gut environment can also produce lactate, formate, hydrogen and carbon dioxide, and the relative proportions of the different products formed depend on the environmental conditions. Thus, the level of ATP production and maintenance of redox balance can be modulated through shifts between alternative pathways in response to carbon source availability and hydrogen partial pressure.

[0053] Butyrate-producing bacteria may be identified using functionally based assays to determine butyrate production. For example, a fermentation may be performed in the presence of the bacteria before chromatography to determine the presence of butyrate. Alternatively, genomic analysis may be performed to identify whether the bacteria comprises genes required for butyrate production (see Louis & Flint; 2009; FEMS Microbiol Lett; 294(1):1-8). Alternatively, amplification of the butyryl-CoA : acetate CoA transferase gene using degenerate primers that recognize multiple phylogenetic groups can be performed (see Louis & Flint, 2007, Appl Environ Microb73: 2009-2012).

[0054] Suitably, the butyrate-producing bacteria may be a butyrate-producing bacteria that is known to colonize the gut microbiota of an infant or young child between about 1 and 5 years of age, preferably between about 1 and 3 years of age.

[0055] The butyrate-producing bacteria may be selected from Faecalibacterium prausnitzii, Roseburia inulinivorans and / or Eubacterium rectale.

[0056] The butyrate-producing bacteria may be Faecalibacterium prausnitzii.

[0057] The butyrate-producing bacteria may be Roseburia inulinivorans.

[0058] The butyrate-producing bacteria may be Eubacterium rectale.

[0059] Suitably, the butyrate-producing bacteria may comprise each of Faecalibacterium prausnitzii, Roseburia inulinivorans and Eubacterium rectale.

[0060] Species of bacteria may be identified based on 16S rRNA-sequencing or Average Nucleotide Identity (ANI), for example. Suitably, an ANI of >95% is indicative that two bacteria belong to the same species.

[0061] Methods for sequencing microbial genomes are well known in the art (see e.g. Segerman; Front. Cell. Infect. Microbiol.; 2020; 10; Article 527102 & Donkor; Genes; 2013; 4(4); 556-572). By way of example, metagenomics methods may be used. Suitable metagenomics methods may be performed using shotgun sequencing data, for example. Suitable metogenomics methods are known in the art and include MetaPhlAn 3.0, for example (see Beghini et al.; eLife 2021 ;10: e65088; https: / / huttenhower.sph.harvard.edu / metaphlan).

[0062] The “Average Nucleotide Identity (ANI)” is a term of art that refers to a distance-based approach to delineate species based on pair-wise comparisons of their genome sequences and is an in silico alternative to the traditional DNA-DNA hybridization (DDH) techniques that have been used for phylogenetic definition of a species (Goris et al., 2007, “DNA-DNA hybridization values and their relationship to whole-genome sequence similarities”, Int. J. Syst. Evol. Microbiol. 57: 81-91). Based on DDH, strains with greater than 70% relatedness would be considered to belong to the same species (see e.g., Wayne et al., 1987, Report of the Ad- Hoc-Committee on Reconciliation of Approaches to Bacterial Systematics. Int J Syst Bacteriol 37: 463-464). ANI is similar to the aforementioned 70% DDH cutoff value and can be used for species delineation. ANI has been evaluated in multiple labs and has become the gold standard for species delineation (see e.g., Kim et al., 2014, “Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes”, Int. J. Syst. Evol. Mier. 64: 346-351 ; Richter et al., 2009, “Shifting the genomic gold standard for the prokaryotic species definition”, P Natl Acad Sci USA 106: 19126-19131 ; and Chan et al., 2012, “Defining bacterial species in the genomic era: insights from the genus Acinetobacter”, Bmc. Microbiol. 12)).

[0063] The ANI of the shared genes between two strains is known to be a robust means to compare genetic relatedness among strains, and that ANI values of about 95% correspond to the 70% DNA-DNA hybridization standard for defining a species. See, e.g., Konstantinidis and Tiedje, Proc Natl Acad Sci USA, 102(7):2567-72 (2005); and Goris et al., Int Syst Evol Microbiol. 57(Pt 1 ):81 -91 (2007). The ANI between two bacterial genomes is calculated from pair-wise comparisons of all sequences shared between any two strains and can be determined, for example, using any of a number of publicly available ANI tools, including but not limited to OrthoANI with usearch (Yoon et al. Antonie van Leeuwenhoek 110:1281-1286 (2017)); ANI Calculator, JSpecies (Richter and Rossello-Mora, Proc Natl Acad Sci USA 106:19126-19131 (2009)); and JSpeciesWS (Richter et al., Bioinformatics 32:929-931 (2016)). Other methods for determining the ANI of two genomes are known in the art. See, e.g., Konstantinidis, K. T. and Tiedje, J. M., Proc. Natl. Acad. Sci. U.S.A., 102: 2567-2572 (2005); and Varghese et al. , Nucleic Acids Research, 43(14):6761-6771 (2015). In a particular embodiment, the ANI between two bacterial genomes can be determined, for example, by averaging the nucleotide identity of orthologous genes identified as bidirectional best hits (BBHs). Protein-coding genes of a first genome (Genome A) and second genome (Genome B) are compared at the nucleotide level using a similarity search tool, for example, NSimScan (Novichkov et al., Bioinformatics 32(15): 2380-23811 (2016)). The results are then filtered to retain only the BBHs that display at least 70% sequence identity over at least 70% of the length of the shorter sequence in each BBH pair. The ANI of Genome A to Genome B is defined as the sum of the percent identity times the alignment length for all BBHs, divided by the sum of the lengths of the BBH genes. These and ANI determination techniques are known in the art.

[0064] Suitably, the genome relatedness may be compared to a type strain for a given species.

[0065] Suitably, the type strain for Faecalibacterium prausnitzii may be Faecalibacterium prausnitzii ATCC27768.

[0066] Suitably, the type strain for Roseburia inulinivoran may be Roseburia inulinivoran DSM16841.

[0067] Suitably, the type strain for Eubacterium rectale may be Eubacterium rectale ATCC33656.

[0068] The present invention is based on the determination that early colonization of the infant gut microbiome with infant-type bifidobacteria promotes the subsequent abundance and function of butyrate-producing bacteria in the gut microbiome.

[0069] Suitably, the butyrate-producing bacteria may be promoted in the gut microbiota of the infant or young child between about 1 and 5 years of age, between about 1 and 4 years of age, between about 1 and 3 years of age, or between about 1 and 2 years of age.

[0070] Suitably, the butyrate-producing bacteria may be promoted in the gut microbiota of the infant or young child between about 1 and 3 years of age.

[0071] Suitably, the butyrate-producing bacteria may be promoted in the gut microbiota of the infant or young child at about 12, 18, 24 and / or 36 months of age.

[0072] Bifidobacterium microorganism

[0073] Taxonomically classified as members of the Bifidobacteriaceae family, Bifidobacterium are Gram-positive, non-motile, non-spore forming, anaerobic, saccharolytic microorganisms with a Y-shaped or ‘bifid’ morphology, and a high G + C DNA content. The presence of Bifidobacterium in the mammalian intestine supports the development of the host immune system by improving gut homeostasis and functionality, promoting the intestinal barrier integrity, limiting the onset of certain gut diseases and providing protection against pathogen proliferation.

[0074] Bifidobacterium may be identified at the genus level using methods which are known in the art, for example 16S rRNA-sequencing. Species of Bifidobacterium may be identified based on 16S rRNA-sequencing or Average Nucleotide Identity (ANI), for example. Suitably, an ANI of >95% is indicative that two Bifidobacterium belong to the same species.

[0075] Suitably, the bifidobacteria may be selected from one or more of B. longum, B. bifidum, B. breve, and B. kashiwanohense.

[0076] Suitably, the bifidobacteria may be selected from one or more of B. longum infantis, B. longum longum, B. bifidum, B. breve, B. kashiwanohense and B. longum iuvenis.

[0077] Suitably, the B. longum may be selected from B. longum infantis, B. longum longum and B. longum iuvenis.

[0078] Preferably, the B. longum may be B. longum infantis.

[0079] Suitably, the type strain for B. longum infantis may be B. longum infantis ATCC15697. Suitably, the type strain for B. longum longum may be B. longum longum ATCC15707. Suitably, the type strain for B. longum iuvenis may be B. longum iuvenis NCC 5000 (see Modesto et at, International Journal of Systematic and Evolutionary Microbiology 73(10)).

[0080] The GenBank (EMBL) accession numbers for the 16S rRNA gene sequence and genome of Bifidobacterium longum subsp. iuvenis NCC 5000T are OP696622 (GenBank) and Ga0527908 (JGI), respectively.

[0081] In some embodiments, a B. longum iuvenis microorganism for use in the present invention has an ANI of at least 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.6 %, 98.7 %, 98.8 %, 98.9 %, 99 %, 99.1 %, 99.2 %, 99.3 %, 99.4 %, 99.5 %, 99.6 %, 99.7 %, 99.8 %, 99.9 %, or 100 % compared to B. longum iuvenis NCC 5000. In some embodiments, a B. longum iuvenis microorganism for use in the present invention has an ANI of at least 98.6%, of at least

[0082] 98.6 %, of at least 98.7 %, of at least 98.8 %, of at least 98.9 %, of at least 99 %, of at least 99.1 %, of at least 99.2 %, of at least 99.3 %, of at least 99.4 %, of at least 99.5 %, of at least

[0083] 99.6 %, of at least 99.7 %, of at least 99.8 %, of at least 99.9 % or of at least 100% compared to B. longum iuvenis NCC 5000.

[0084] Suitably, the B. longum iuvenis may be B. longum iuvenis NCC 5025. B. longum iuvenis NCC 5025 was deposited with the Collection Nationale de Cultures de Micro-organisms (CNCM), Institute Pasteur by SOCIETE DES PRODUITS NESTLE S.A according to Budapest Treaty on the 29thof March 2023 receiving the deposit number CNCM I-5942.

[0085] A B. longum iuvenis microorganism for use in the present invention may have an ANI of at least 99% compared to the B. longum strain deposited with the CNCM under deposit number CNCM I-5942. In some embodiments, the B. longum iuvenis microorganism for use in the present invention may have an AN I of at least at least 99.0%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%, compared to the B. longum strain deposited with the CNCM under deposit number CNCM I- 5942.

[0086] Suitably, the B. longum iuvenis microorganism for use in the present invention may have an ANI of at least 99.9% compared to the B. longum strain deposited with the CNCM under deposit number CNCM I-5942.

[0087] Suitably, the type strain for B. bifidum may be B. bifidum ATCC29521. Suitably, the type strain for B. breve may be B. breve ATCC15700. Suitably, the type strain for B. kashiwanohense may be B. kashiwanohense DSM21854.

[0088] The present invention may comprise administering a Bifidobacterium microorganism to an infant or young child in order to promote butyrate-producing bacteria in the gut microbiome of the infant or young child. The Bifidobacterium microorganism may be provided as a probiotic as described herein.

[0089] The Bifidobacterium microorganism and / or bifidogenic prebiotic for use according to the present invention promote bifidobacteria in the gut microbiome of the infant or young child. Preferably, the Bifidobacterium microorganism and / or bifidogenic prebiotic for use according to the present invention promote bifidobacteria in the gut microbiome of the infant.

[0090] Suitably, the Bifidobacterium microorganism and / or bifidogenic prebiotic may be administered to the infant prior to about 12 months of age.

[0091] Suitably, the Bifidobacterium microorganism and / or bifidogenic prebiotic may be administered to the infant prior to about 6 months of age.

[0092] Suitably, the Bifidobacterium microorganism and / or bifidogenic prebiotic may be administered to the infant at least from about 0 months to about 6 months after birth. Suitably, the Bifidobacterium microorganism and / or bifidogenic prebiotic may be administered starting about 7 to about 21 days after birth. Suitably, Bifidobacterium microorganism and / or bifidogenic prebiotic may be administered until about 6 months to about 12 months after birth. In some embodiments, the Bifidobacterium microorganism and / or bifidogenic prebiotic is administered starting about 7 to about 21 days after birth until about 12 months after birth, starting about 7 to about 21 days after birth until about 9 months after birth, or starting about 7 to about 21 days after birth until about 6 months after birth. Preferably, the Bifidobacterium microorganism and / or bifidogenic prebiotic may be administered to the infant prior to about 6 months of age.

[0093] Preferably, the Bifidobacterium microorganism and / or bifidogenic prebiotic may be administered starting about 7 to about 21 days after birth until about 6 months after birth.

[0094] Preferably, the bifidogenic prebiotic may be administered to the infant prior to about 6 months of age.

[0095] Preferably, the bifidogenic prebiotic may be administered to the infant until at least about 6 months of age.

[0096] Preferably, the bifidogenic prebiotic may be administered starting about 7 to about 21 days after birth until about 6 months after birth.

[0097] Suitably, the Bifidobacterium microorganism and / or bifidogenic prebiotic may be administered to the infant until at least about 6 months of age and the butyrate-producing bacteria are promoted in the gut microbiota of the infant or young child between about 1 and 3 years of age.

[0098] Suitably, the Bifidobacterium microorganism and / or bifidogenic prebiotic may be administered starting about 7 to about 21 days after birth until at least about 6 months after birth and the butyrate-producing bacteria are promoted in the gut microbiota of the infant or young child between about 1 and 3 years of age.

[0099] Suitably, the bifidogenic prebiotic may be administered to the infant prior to about 6 months of age and the butyrate-producing bacteria are promoted in the gut microbiota of the infant or young child between about 1 and 3 years of age.

[0100] Suitably, the bifidogenic prebiotic may be administered starting about 7 to about 21 days after birth until at least about 6 months after birth and the butyrate-producing bacteria are promoted in the gut microbiota of the infant or young child between about 1 and 3 years of age.

[0101] Treating and / or preventing an allergy and / or allergic sensitization

[0102] ‘Allergy’, as used herein, may refer to an allergic disorder or an allergic reaction (including symptoms thereof).

[0103] The Bifidobacterium microorganism and / or bifidogenic prebiotic may be for use in treating and / or preventing an allergy and / or allergic sensitization in an infant or young child by promoting butyrate-producing bacteria in the gut microbiota of the infant or young child; wherein the Bifidobacterium microorganism and / or bifidogenic prebiotic is administered to the infant prior to about 12 months of age; and the butyrate-producing bacteria are promoted in the gut microbiota of the infant or young child between about 1 and 5 years of age.

[0104] “Treating”, as used herein, may refer to administering the Bifidobacterium microorganism and / or bifidogenic prebiotic to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease and / or to slow down, reduce or block the progression of the disease.

[0105] “Preventing”, as used herein, may refer to administering the Bifidobacterium microorganism and / or bifidogenic prebiotic to a subject who has not yet contracted the disease and / or who is not showing any symptoms of the disease to prevent or impair the cause of the disease or to reduce or prevent development of at least one symptom associated with the disease. The subject may have a predisposition for, or be thought to be at risk of developing, the disease.

[0106] Suitably, the Bifidobacterium microorganism and / or bifidogenic prebiotic may be administered to a subject in order to reduce the likelihood of the infant or young child developing an allergy and / or allergic sensitization.

[0107] Butyrate-producing bacteria, and butyrate perse, have been shown to have important roles in allergic asthma (Yip et al:, Front Immunol.; 2021 ; Feb 15:12:628453 and Depner et al:, Nat Med. 2020 Nov;26(11): 1766-1775.); atopic dermatitis (Sasaki et al.; Allergy. 2022 Aug 2;77(12):3629-3640); food (e.g. egg) allergy (Fazlollahi et al:, Allergy. 2018 Jul;73(7):1515- 1524); atopy (Galazzo et al:, Gastroenterology. 2020 May; 158(6): 1584-1596); allergy and eczema (Wopereis et al:, J Allergy Clin Immunol. 2018 Apr;141 (4):1334-1342.e5 and Hoskinson et al.; Nat Commun. 2023 Aug 29; 14(1):4785); and gut immunity (Yang et al:, Nat Commun. 2020 Sep 8;11(1):4457).

[0108] Allergic sensitization in childhood, especially in early childhood and especially to food allergens, is critical and development of an "allergic phenotype" or "atopy" has been shown to facilitate subsequent sensitization to other allergens. Hence allergies in childhood can be the first step of an allergic cascade leading to multiple allergies later in life, a process commonly referred to as the “Atopic March". For example, children with persistent food hypersensitivity early in life have a dramatically increased risk to develop allergic rhinitis (hay fever) or asthma later in childhood (Ostblom, E. et al. (2008); Clinical and Experimental Allergy, 38 (8): 1325- 1332). Children with milder forms of food hypersensitivity also have increased risk for development of respiratory allergies but to a lesser degree than children with persistent food hypersensitivity. Therefore, attenuating the severity of food hypersensitivity may be crucial for slowing down the "Atopic March". In this context the management of allergic episodes and prevention of allergies are, in childhood and infancy, of the highest importance.

[0109] In one embodiment preventing and / or reducing the risk of developing an allergy and / or allergic sensitization is by primary prevention. "Primary prevention" is the effect of preventing or reducing the risk of sensitization of patients to allergens, characterized by absence or reduced levels of allergen-specific IgE antibodies. Preventing or reducing sensitization may result in absence or reduction of allergic symptoms upon exposure to the same allergen. By modulating the way a patient gets sensitized in regard to one allergen or one group of allergens (primary prevention), the subsequent allergic response may also be modulated.

[0110] Food allergens are among the first allergens that infants encounter in their early life: typically, cow's milk proteins may be encountered by infants not receiving exclusive breast-feeding. Milk-proteins are indeed among the most frequently observed causes for food allergy in infancy, followed by eggs and wheat proteins. In general, food allergies can manifest in cutaneous (rash, eczema, others) and gastrointestinal symptoms (abdominal cramps; pain, especially in the abdomen; vomiting) in infants and young children. Food allergies are the most common trigger of severe allergic reactions, which may lead to life-threatening anaphylaxis.

[0111] Further sensitization and episodes of allergies can also appear when the infant / young child is exposed to a novel food such as cereals, vegetables, fruits, nuts or fish, and also to air-borne allergens such as pollen, house dust mites and animal dander. Adults are affected to a large extent by contact and respiratory allergies. Recent data from the WHO (Clark, M. J. and. Million, R. P (2009); Nature Reviews, Drug Discovery, 8, p.271 -272) indicates that up to 30- 40% of the world's population suffer from some form of respiratory allergy.

[0112] Animals, particularly small animals such as pets - and especially companion animals such as dogs and cats, may also suffer from food allergies and food intolerances, as well as environmental allergens. These typically manifest in similar symptoms to humans, e.g. gastrointestinal disturbances such as diarrhoea, vomiting and abdominal discomfort, and also dermatitis or pruritis. In small animals, particularly dogs, the most frequent cause of chronic diarrhoea is food-responsive enteropathy (diet-responsive enteropathy or food-responsive diarrhoea).

[0113] By treating and / or preventing or reducing the risk of sensitization of subjects to allergens the compounds and compositions of the present invention may be used for preventing or treating food allergies, respiratory allergies and dermatological allergies. In some embodiments an allergic response is a specific IgE-associated immune response and / or a T cell-dependent hypersensitive reaction. Thus, in some embodiments treating and / or preventing and / or reducing the risk of developing an allergy and / or allergic sensitization comprises reducing or preventing specific IgE-associated immune responses and / or a T celldependent hypersensitive reaction. In some embodiments allergic inflammation is reduced and / or tolerance (e.g. oral tolerance) is enhanced.

[0114] Suitably, the allergic disorder is selected from one or more of the group consisting of: a food allergy, a respiratory allergy and a dermatological allergy.

[0115] In one embodiment the allergic disorder is selected from one or more of the group consisting of: rhinitis, asthma, dermatitis, atopic dermatitis, contact dermatitis, eczema, atopic eczema, urticaria, psoriasis, eosinophilic oesophagitis and an eosinophilic-associated gastrointestinal disease.

[0116] In one embodiment the allergen in the allergic disorder is selected from one or more of: a food allergen, dust mite, pollen, molds or mold spores, weed pollen, tree pollen, grass pollen, fleas, pet hair, feathers, pollution or pet dander.

[0117] In one embodiment the allergen in the allergic disorder is a food allergen. Suitably, wherein the food allergen is selected from: a nut, tree nut, peanut, fish, shellfish, molluscs, crustaceans, milk, egg, soy, gluten, cereals, wheat, oats, barley, rye, celery, corn, lupin, sulphites, sesame, mustard, rice, poultry and meat.

[0118] In one embodiment the allergen is an aeroallergen. Suitably, wherein the aeroallergen is selected from dust mite, pollen, moulds or mold spores, weed pollen, tree pollen, grass pollen, fleas, pet hair, feathers, pollution or pet dander.

[0119] Suitably, treating, preventing or reducing the risk of an allergy and / or allergic sensitization may refer to reducing or ameliorating one or more symptoms as described herein.

[0120] A “food allergy” as used herein refers to an abnormal immune response to one or more food allergens, typically an IgE reaction caused by the release of histamine but also encompassing non-lgE immune responses. Symptoms of food allergy may include itchiness, swelling of the tongue, vomiting, diarrhea, hives, trouble breathing, or low blood pressure. When the symptoms are severe, it is known as anaphylaxis.

[0121] As used herein, the term “food allergen” refers to proteins or derivatives thereof that cause abnormal immune responses. Purified food allergens may be named using the systematic nomenclature of the Allergen Nomenclature Sub-Committee of the World Health Organization and International Union of Immunological Societies. Allergen names are composed of an abbreviation of the scientific name of its source (genus: 3-4 letters; species: 1-2 letters) and an Arabic numeral, for example Der p 1 for the first allergen to be described from the house dust mite Dermatophagoides pteronyssinus. Food allergens are derived from proteins with a variety of biologic functions, including proteases, ligand-binding proteins, structural proteins, pathogenesis-related proteins, lipid transfer proteins, profilins, and calcium-binding proteins. A list of food allergens is provided on the official website of the WHO / IUIS Allergen Nomenclature Database, http: / / www.allergen.org / index.php. (Radauer, C., et al., 2014. Allergy, 69(4), pp.413-419 and Pomes, A., et al., 2018. Molecular immunology).

[0122] A “respiratory allergy” or “aeroallergen” as used herein refers to an abnormal immune response to one or more airborne allergens. Airborne allergens may include pollen, molds or mold spores, weed pollen, tree pollen, grass pollen, and dander. Respiratory allergies may include for example allergic rhinitis and allergic asthma. Symptoms of allergic rhinitis (hay fever) include a runny or stuffy nose, sneezing, red, itchy, and watery eyes, and swelling around the eyes. Symptoms of allergic asthma include episodes of wheezing, coughing, chest tightness, and shortness of breath.

[0123] A “dermatological allergy” as used herein refers to an abnormal immune response caused by contact with one or more environmental allergens. Environmental allergens may include a food allergen, dust mite, pollen, molds or mold spores, weed pollen, tree pollen, grass pollen, fleas, pet hair, feathers or pet dander. Dermatological allergies may include for example dermatitis, atopic dermatitis, contact dermatitis, eczema, atopic eczema, urticaria, and psoriasis. These are typically a group of diseases that results in inflammation of the skin and symptoms include itchiness, red skin and a rash.

[0124] Allergic disorders may also include other allergic inflammatory conditions, for example eosinophilic oesophagitis and an eosinophilic-associated gastrointestinal disease. Eosinophilic esophagitis is an allergic inflammatory condition of the esophagus that involves eosinophils, a type of white blood cell. Symptoms are swallowing difficulty, food impaction, vomiting, and heartburn.

[0125] Suitably, the Bifidobacterium microorganism and / or bifidogenic prebiotic may modulate the permeability of the gut epithelial barrier of the infant or young child by promoting butyrate- producing bacteria in the gut microbiota of the infant or young child.

[0126] Suitably, the Bifidobacterium microorganism and / or bifidogenic prebiotic may decrease the permeability of the gut epithelial barrier by promoting butyrate-producing bacteria in the gut microbiota of the infant or young child. Increased permeability of the gut epithelial barrier may be associated with an increase crossing of e.g. haptens and antigens across the intestinal epithelium.

[0127] Commensal gastrointestinal microbes constitute the earliest and most substantial stimulus for the development of the gut associated lymphoid tissue and associated immune system.

[0128] There is evidence from epidemiological studies that Western-type living conditions, e.g. reduced consumption of fermented food, substantial use of antibiotics and other drugs, and increased hygiene, are associated with the rise in allergic diseases. This links to the so-called "hygiene hypothesis" that suggests that a lack of exposure to microbial stimulus early in childhood is a major factor effecting the prevalence of allergic diseases. Indeed, epidemiological studies have demonstrated an association between the development of allergic diseases and disturbance of the gastrointestinal microbiota. Epidemiological data show that atopic children have different intestinal flora compared with non-atopic children.

[0129] Such changes in the intestinal flora may also have a negative impact on the integrity of the intestinal barrier. Impaired barrier function, termed “leaky gut” has long been considered a predisposing factor for gastrointestinal diseases (Heyman M., Eur. J. Gastroenterol. Hepatol. 17:1279-1285; Odenwald M., Nature Reviews Gastroenterology & Hepatology, (2017), (14), 9 21). As such, alterations in gut barrier integrity / function have multiple consequences facilitating the onset of numerous diseases depending on other hits and on genetic and epigenetic constellations. Food allergy patients often demonstrate with increased intestinal permeability, which correlates with the severity of their clinical symptoms (Ventura , M. T et al. 2006. Dig. Dis. Sci. 38:732-736). Preclinical animal models further provide corroborative evidence supporting a role for intestinal barrier dysfunction and leaky gut, predisposing to oral sensitization and subsequent development of food allergy. In addition, Western diet-induced alterations in intestinal permeability promote food allergen sensitization and clinical allergy symptoms in mice in response to dietary antigens (Hussain M. et al. J. Allergy Clin. Immunol. (2019). Probiotics represent one nutritional attempt to improve / reinforce intestinal barrier integrity and / or function (Ewaschuk JB et al., Am J Physiol Gastrointest Liver Physiol. 2008 Nov;295(5):G1025-34). Reinforcing intestinal barrier integrity by means of probiotic supplementation may thus prevent sensitization to oral allergens in at risk individuals. (Tulyeu J, Microorganisms. 2019 Oct 16;7(10). Besides an established role of gut barrier function in allergen sensitization, uncontrolled immune responses towards dietary or environmental antigens foster the development of type-2 immune mediated allergic disorders. Probiotic cultures or mixes of probiotics have well known immunomodulatory properties that can prevent or alleviate allergic responses. The epithelial barrier of human newborns is not fully mature at birth. Transfer of macromolecules or antigens across the intestinal epithelium of an infant or young child induces differentiation of regulatory T-cells (Tregs) and is essential for the induction of tolerance and protection from allergic diseases. However, the route of transepithelial passage of antigens is important in determining the immune response / outcome. Transcellular passage with enterocyte processing may be preferable compared to uncontrolled paracellular passage (leakage), which instead might result in inflammation and / or sensitization.

[0130] As demonstrated in the present Examples, levels of Bifidobacteria in infants are associated with subsequent increased levels of butyrate-producing bacteria. Butyrate-producing bacteria, and butyrate per se, are known to modulate the permeability of the gut epithelial barrier, in particular butyrate is important for maintain gut epithelial barrier integrity (Hodgkinson et al:, Clin Nutr. 2023 Feb;42(2):61-75).

[0131] Suitably, the Bifidobacterium microorganism and / or bifidogenic prebiotic may support and / or improve colonocyte function in the infant or young child by promoting butyrate-producing bacteria in the gut microbiota of the infant or young child.

[0132] Butyrate is critical for maintaining colonocyte health, in large part due to its role as an energy source. Butyrate produced by the gut microbiota is the primary energy source for colonocytes, yielding around 70% of the cell’s energy (Hodgkinson et al:, as above).

[0133] Suitably, the Bifidobacterium microorganism and / or bifidogenic prebiotic may reduce inflammation by promoting butyrate-producing bacteria in the gut microbiota of the infant or young child, for example by inducing differentiation and / or expansion of Treg cells and / or reducing pro-inflammatory cytokine secretion my macrophages.

[0134] Butyrate has been shown to reduce inflammation in many model systems. Butyrate promotes differentiation of T-regulatory immune cells through GPR109A and thus suppresses inflammation, it also increases IL10 levels by increased differentiation of IL10-producing CD4+ T-cells. Butyrate also decreases macrophage secretion of pro-inflammatory cytokines, such as IL17, IL6, and IL12. Butyrate also increases plasma levels of the anti-IL17 cytokines IL10 and IL12 and increases levels of IL18, an anti-inflammatory cytokine, in the colonic epithelium (Hodgkinson et al:, as above).

[0135] Human milk oligosaccharide (HMO)

[0136] Suitably, the biofidogenic prebiotic comprises an HMO. Any suitable HMO, or mixture thereof, may be used in the present invention. The HMO may be a fucosylated oligosaccharide (i.e. an oligosaccharide having a fucose residue; e.g. 2’ fucosyllactose (2-FL), 3-fucosyllactose (3-FL), difucosyllactose (DiFL), 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, difucosyllacto-N- neohexaose II and any combination thereof), an N-acetylated oligosaccharide (e.g. LNT (lacto-N-tetraose), para-lacto-N-neohexaose (para-LNnH), LNnT (lacto-N-neotetraose), DSLNT (disialyllacto-N-tetraose), 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 and any combinations thereof) and / or a sialylated oligosaccharide (e.g. 3’-sialyllactose (3-SL), 6’-sialyllactose (6-SL), or Lst (sialyllacto-N-tetraose), Lst-a, Lst-b or Lst-c)).

[0137] The mixture of HMOs may comprise at least one fucosylated oligosaccharide, at least one N- acetylated oligosaccharide, and at least one sialylated oligosaccharide.

[0138] In some embodiments, the mixture of HMOs comprises at least one fucosylated oligosaccharide, at least one N-acetylated oligosaccharide, and / or at least one sialylated oligosaccharide. In some embodiments, the mixture of HMOs comprises at least one fucosylated oligosaccharide, at least one N-acetylated oligosaccharide, and at least one sialylated oligosaccharide.

[0139] In some embodiments, the mixture of HMOs comprises or consists of 2’-fucosyllactose (2’FL), 2’,3-difucosyllactose (diFL), lacto-N-tetraose (LNT), 3’-sialyllactose (3’-SL), and 6’- sialyllactose (6’-SL).

[0140] In some embodiments, the mixture of HMOs consists of 2’-fucosyllactose (2’FL), 2’,3- difucosyllactose (diFL), lacto-N-tetraose (LNT), 3’-sialyllactose (3’-SL), and 6’-sialyllactose (6’- SL).

[0141] Fucosylated oligosaccharides

[0142] In some embodiments, the mixture of HMOs comprises at least one fucosylated oligosaccharide.

[0143] Suitably, the at least one fucosylated oligosaccharide comprises of consists of 2’- fucosyllactose (2’FL), 2’,3-difucosyllactose (diFL), 3-fucosyllactose (3FL), lacto-N- fucopentaose-l (LNFP-I), lacto-N-fucopentaose-ll (LNFP-II), lacto-N-fucopentaose-lll (LNFP- III), lacto-N-fucopentaose-V (LNFP-V), lacto-neofucopentaose V (LNnFP-V), lacto-N- difucosylhexaose-l (LNDFH-1), lacto-N-neodifucosylhexaose (LNnDFH), monofucosyllacto-n- hexaose-lll (MFNLH-III), difucosyllacto-N-hexaose-a (DFLNHa), or any combination thereof.

[0144] In some embodiments, the at least one fucosylated oligosaccharide comprises of consists of 2’-fucosyllactose (2’FL) and / or 2’, 3-difucosyllactose (diFL). In some embodiments, the at least one fucosylated oligosaccharide consists of 2’-fucosyl lactose (2’FL) and 2’, 3-difucosyllactose (diFL).

[0145] The at least one fucosylated oligosaccharide may be obtained by any suitable method. For example, 2’FL may be produced by biotechnological means using specific fucosyltransferases and / or fucosidases either through the use of enzyme-based fermentation technology (recombinant or natural enzymes) or microbial fermentation technology. In the latter case, microbes may either express their natural enzymes and substrates or may be engineered to produce respective substrates and enzymes. Alternatively, 2’FL may be produced by chemical synthesis from lactose and free fucose. diFL may be synthesized by enzymatic, biotechnological and / or chemical processes.

[0146] N-acetylated oligosaccharides

[0147] In some embodiments, the mixture of HMOs comprises at least one N-acetylated oligosaccharide.

[0148] Suitably, the at least one N-acetylated oligosaccharide comprises of consists of lacto-N- tetraose (LNT), lacto-N-neotetraose (LNnT), a N-acetyl-glucosamine, a N-acetyl- galactosamine, or any combination thereof.

[0149] In some embodiments, the at least one N-acetylated oligosaccharide consists of lacto-N- tetraose (LNT).

[0150] The N-acetylated oligosaccharides may be obtained by any suitable method. For example, LNnT may be synthesised chemically by enzymatic transfer of saccharide units from donor moieties to acceptor moieties using glycosyltransferases. Alternatively, LNnT may be prepared by chemical conversion of Keto-hexoses (e.g. fructose) either free or bound to an oligosaccharide (e.g. lactulose) into N-acetylhexosamine or an N-acetylhexosamine- containing oligosaccharide. LNT may be synthesized by enzymatic, biotechnological and / or chemical processes.

[0151] Sialylated oligosaccharide

[0152] In some embodiments, the mixture of HMOs comprises at least one sialylated oligosaccharide. Suitably, the at least one sialylated oligosaccharide comprises of consists of 3’-sialyllactose (3’-SL), 6’-sialyllactose (6’-SL), syalyllacto-N-tetraose b (LSTb), syalyllacto-N-tetraose c (LSTc), disyallacto-N-tetraose (DSLNT), or any combination thereof.

[0153] In some embodiments, the at least one sialylated oligosaccharide comprises or consists of 3’- sialyllactose (3’-SL) and / or 6’-sialyllactose (6’-SL). In some embodiments, the at least one sialylated oligosaccharide consists of 3’-sialyllactose (3’-SL) and 6’-sialyllactose (6’-SL).

[0154] The sialylated oligosaccharides may be obtained by any suitable method. For example, 3’- sialyllactose (3’-SL) and / or 6’-sialyllactose (6’-SL) may be isolated by chromatographic or filtration technology from a natural source such as animal milks. Alternatively, they may be produced by biotechnological means using specific sialyltransferases or sialidases, neuraminidases, either by an enzyme based fermentation technology (recombinant or natural enzymes), by chemical synthesis or by a microbial fermentation technology. In the latter case microbes may either express their natural enzymes and substrates or may be engineered to produce respective substrates and enzymes. Single microbial cultures or mixed cultures may be used. Sialyl-oligosaccharide formation can be initiated by acceptor substrates starting from any degree of polymerisation (DP), from DP=1 onwards. Alternatively, sialyllactoses may be produced by chemical synthesis from lactose and free sialic acid.

[0155] Form of administration

[0156] The mixture of HMOs may be administered in any suitable form. For example, the mixture of HMOs may be administered in the form of a nutritional composition, a medical food product for clinical nutrition, or a supplement.

[0157] In some embodiments, the mixture of HMOs is administered in the form of a nutritional composition.

[0158] The nutritional composition may be a synthetic nutritional composition. As used herein, a “synthetic nutritional composition” may refer to 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 composition is not breast milk).

[0159] The nutritional composition may be any suitable nutritional composition in which the mixture of HMOs can be incorporated, such as a nutritional composition in the form of a food or beverage product, a nutritional supplement, a nutraceutical composition, or a pharmaceutical composition. The nutritional composition may be in solid (e.g. powder), liquid or semi-liquid form. Suitably, the nutritional composition is in a form suitable for feeding infants, such as an infant formula, a milk fortifier, or a supplement. The nutritional composition can also be in a form for young children such as a yoghurt or a medical food.

[0160] In preferred embodiments, the mixture of HMOs is administered in the form of an infant formula. An infant formula can be a starter infant formula, a preterm infant formula, a milk fortifier, or a follow-up formula.

[0161] In some embodiments, the mixture of HMOs is administered in the form of a starter infant formula. Generally, a “starter infant formula” is intended for infants from birth as breast-milk substitute.

[0162] In some embodiments, the mixture of HMOs is administered in the form of follow-up formula. A “follow-up formula” or “follow-on formula” may be given from the sixth month onwards. It may constitute the principal liquid element in the progressively diversified diet of this category of person.

[0163] In some embodiments, the mixture of HMOs is administered in the form of a preterm infant formula. The term "preterm infant formula" as used herein may refer to an infant formula intended for a preterm infant.

[0164] In some embodiments, the mixture of HMOs is administered in the form of a milk fortifier. The term "milk fortifier" as used herein may refer to liquid or solid nutritional compositions suitable for mixing with infant formula.

[0165] In some embodiments, the mixture of HMOs is administered at least in the form of a starter infant formula, and / or a follow-up formula. In some embodiments, the mixture of HMOs is administered at least in the form of a starter infant formula. In some embodiments, the mixture of HMOs is administered at least in the form of a starter infant formula and a follow-up formula.

[0166] In some embodiments, the mixture of HMOs is administered in the form of a starter infant formula. In some embodiments, the mixture of HMOs is administered in the form of a starter infant formula and a follow-up formula.

[0167] In other embodiments, the mixture of HMOs is administered in the form of a fortifier. The fortifier can be a formula fortifier such as an infant formula fortifier. The fortifier may be a particularly advantageous embodiment when the infant or young child is born preterm.

[0168] In other embodiments, the mixture of HMOs is administered in the form of a supplement. As used herein, a "supplement" or “dietary supplement” may be used to complement the nutrition of a subject (it is typically used as such but it might also be added to any kind of compositions intended to be ingested by the subject).

[0169] When the composition is a supplement, it can be provided in the form of unit doses. Supplements are typically present in the form of a liquid, a gel, a powder, a tablet, or a capsule. Powder supplements typically encompass supplements to be dissolved in water or to be sprinkled on food or in a beverage. Such supplements are intended to provide additional nutrients and / or a health benefit to the subject consuming it. A supplement can be used for providing nutrients and / or a health benefit to human beings, as well as to animals, as defined above. Supplements include for example powder supplements to be added to breast milk, for example for premature or low birth weight infants.

[0170] In other embodiments, the mixture of HMOs is administered in the form of a pharmaceutical product. Pharmaceutical products include for example drops, syrups, powder, tablet or capsule products intended to treat or prevent an adverse medical condition in a subject in need thereof. In other embodiments, the mixture of HMOs is administered in the form of a nutraceutical product.

[0171] Dosage of HMOs

[0172] The mixture of HMOs may be administered in any dosage that is effective to promote Bifidobacterium in the gut microbiota of the infant or young child. The effective dosage may vary depending on e.g. the weight and / or age of the infant.

[0173] If the mixture of HMOs is administered in the form of an infant formula, the infant formula may be administered as normal (e.g. based on the weight of the infant or child) and suitable amounts of an individual HMO e.g. of 2’FL, diFL, LNT, 3’-SL, 6’-SL may be based on the amounts found in human breast milk produced for an infant or child of the same age, in particular by a nutritionally replete mother. The amounts in the infant formula may vary depending on for example bioavailability of said HMOs from infant formula in comparison to human breastmilk. The exemplary concentration of HMOs described herein may refer to the concentration after the composition has been reconstituted e.g. with water.

[0174] The amounts of 2’FL, diFL, LNT, 3’-SL, 6’-SL in human breast milk may fall within the following ranges: 2’FL in an amount of about 0.5 to about 3 g / L (e.g. about 1.8 g / L); diFL in an amount of about 0.1 to about 0.5 g / L (e.g. about 0.26 g / L); LNT in an amount of about 0.05 to about 0.3 g / L (e.g. about 0.77 g / L); 3’SL in an amount of about 0.1 to about 0.4 g / L (e.g. about 0.22 g / L); and 6’SL in an amount of about 0.05 to about 0.75 g / L (e.g. about 0.47 g / L). Suitably, the mixture of HMOs (e.g. 2’FL, diFL, LNT, 3’-SL, and / or 6’-SL) is administered in a total amount of about 0.1 g / day to about 10 g / day. Suitably, the mixture of HMOs is administered in a total amount of about 0.5 g / day or more, about 1 .0 g / day or more, or about

[0175] 1.5 g / day or more. Suitably, the mixture of HMOs is administered in a total amount of about 5.0 g / day or less, 4.5 g / day or less, 4.0 g / day or less, 3.5 g / day or less, 3.0 g / day or less, or

[0176] 2.5 g / day or less. Suitably, the mixture of HMOs is administered in a total amount of from about 0.5 g / day to about 5.0 g / day, from about 1 .0 g / day to about 3.0 g / day, or from about 1.4 g / day to about 2.5 g / day.

[0177] In some embodiments, the mixture of HMOs is administered in a total amount of from about 1 .2 g / day to about 1 .8 g / day (e.g. about 1.46 g / day) or from about 2.0 g / day to about 3.0 g / day (e.g. about 2.44 g / day). In some embodiments, the mixture of HMOs is administered in a total amount of from about 1.2 g / day to about 1.8 g / day. In some embodiments, the mixture of HMOs is administered in a total amount of about 1.46 g / day.

[0178] Suitably, when the mixture of HMOs is administered in the form of a starter infant formula the mixture of HMOs is administered in a total amount of from about 0.5 g / day to about 5.0 g / day, from about 1.0 g / day to about 3.0 g / day, or from about 1.5 g / day to about 2.5 g / day (e.g. about

[0179] 1 .5 g / day or about 2.5 g / day). Suitably, when the mixture of HMOs is administered in the form of a follow-up formula the mixture of HMOs is administered in a total amount of from about 0.1 g / day to about 2.0 g / day, from about 0.2 g / day to about 1.0 g / day, from about 0.3 g / day to about 0.7 g / day, or about 0.5 g / day.

[0180] Suitably, when the mixture of HMOs is administered in the form of a growing-up milk the mixture of HMOs is administered in a total amount of from about 0.05 g / day to about 0.5 g / day, from about 0.1 g / day to about 0.3 g / day, or about 0.2 g / day.

[0181] Suitably, the mixture of HMOs is administered in the following proportions: (i) 2’FL in an amount of from about 55 wt% to about 60 wt% (e.g. about 58%); (ii) diFL in an amount of from about 5 wt% to about 7 wt% (e.g. about 6 wt%); (iii) LNT in an amount of from about 18 wt% to about 20 wt% (e.g. about 19 wt%); (iv) 3’-SL in an amount of from about 6 wt% to about 8 wt% (e.g. about 7 wt%); and (v) 6’-SL in an amount of from about 9 wt% to about 11 wt% (e.g. about 10 wt%), based on the total weight of HMOs.

[0182] Concentration of HMOs

[0183] A nutritional composition (e.g. infant formula) comprising the mixture of HMOs may comprise the mixture of HMOs in any suitable concentrations to provide an effective dosage. As a guide, for e.g. an infant formula, the mixture of HMOs (e.g. 2’FL, diFL, LNT, 3’-SL, and / or 6’-SL) may be present in a total amount of about 0.1 g / L to about 10 g / L. Suitably, the composition comprises the mixture of HMOs in a total amount of about 0.5 g / L or more, about 1 .0 g / L or more, or about 1 .5 g / L or more. Suitably, the composition comprises the mixture of HMOs in a total amount of about 5.0 g / L or less, 4.5 g / L or less, 4.0 g / L or less, 3.5 g / L or less, 3.0 g / L or less, or 2.5 g / L or less. Suitably, the composition comprises the mixture of HMOs in a total amount of from about 0.5 g / L to about 5.0 g / L, from about 1.0 g / L to about 3.0 g / L, from about 1.2 g / L to 3.0 g / L, or from about 1.5 g / L to about 2.5 g / L. In some embodiments, the composition comprises the mixture of HMOs in a total amount of from about 1 .2 g / L to about 1 .8 g / L (e.g. about 1 .5 g / L) or from about 2.0 g / L to about 3.0 g / L (e.g. about 2.5 g / L). In some embodiments, the composition comprises the mixture of HMOs in a total amount of from about 1.2 g / L to about 1.8 g / L.

[0184] In some embodiments, the composition comprises the mixture of HMOs in a total amount of about 1.5 g / L.

[0185] Suitably, a starter infant formula comprises a total amount of HMOs of from about 0.5 g / L to about 5.0 g / L, from about 1.0 g / L to about 3.0 g / L, from about 1.2 g / L to 3.0 g / L, or from about 1.5 g / L to about 2.5 g / L. Suitably, a follow-up formula comprises a total amount of HMOs of from about 0.1 g / L to about 2.0 g / L, from about 0.2 g / L to about 1.0 g / L, from about 0.3 g / L to about 0.8 g / L, from about 0.35 g / L to about 0.65 g / L, or about 0.5 g / L. Suitably, a growing-up milk comprises a total amount of HMOs of from about 0.1 g / L to about 1.0 g / L, from about 0.2 g / L to about 0.8 g / L, from about 0.28 g / L to about 0.52 g / L, from about 0.3 g / L to about 0.5 g / L, or about 0.4 g / L.

[0186] Suitably, a starter infant formula comprises a total amount of HMOs of from about 0.5 g / L to about 1.5 g / L. Suitably, a starter infant formula comprises a total amount of HMOs of about 1.5 g / L.

[0187] Suitably, the mixture of HMOs (e.g. in the form of a nutritional composition, such as an infant formula) comprises or consists of: (i) 2’FL in an amount of from about 50 wt% to about 65 wt%; (ii) diFL in an amount of from about 2 wt% to about 10 wt%; (iii) LNT in an amount of from about 15 wt% to about 25 wt%; (iv) 3’-SL in an amount of from about 4 wt% to about 10 wt%; and (v) 6’-SL in an amount of from about 5 wt% to about 15 wt%, based on the total weight of HMOs.

[0188] In some embodiments, the mixture of HMOs (e.g. in the form of a nutritional composition, such as an infant formula) comprises or consists of: (i) 2’FL in an amount of from about 55 wt% to about 60 wt% (e.g. about 58%); (ii) diFL in an amount of from about 5 wt% to about 7 wt% (e.g. about 6 wt%); (iii) LNT in an amount of from about 18 wt% to about 20 wt% (e.g. about 19 wt%); (iv) 3’-SL in an amount of from about 6 wt% to about 8 wt% (e.g. about 7 wt%); and (v) 6’-SL in an amount of from about 9 wt% to about 11 wt% (e.g. about 10 wt%), based on the total weight of HMOs.

[0189] As a guide, for e.g. an infant formula, the one or more fucosylated oligosaccharide (e.g. 2’FL and / or diFL) may be present in a total amount of from about 0.1 g / L to about 4 g / L. Suitably, the one or more fucosylated oligosaccharide is present in amount of about 0.1 g / L to about 3.5 g / L, about 0.15 g / L to about 3 g / L, from about 0.2 g / L to about 2.5 g / L, from about 0.3 g / L to about 2 g / L, from about 0.4 g / L to about 2 g / L, or from about 0.5 g / L to about 2 g / L. In some embodiments, 2’FL is present in an amount of from about 0.5 g / L to about 3.0 g / L (e.g. about 0.87 g / L or about 1.45 g / L). In some embodiments, 2’FL is present in an amount of about 0.87 g / L.

[0190] In some embodiments, diFL is present in an amount of from about 0.05 g / L to about 0.3 g / L (e.g. about 0.10 g / L or about 0.14 g / L). In some embodiments, diFL is present in an amount of about 0.10 g / L.

[0191] As a guide, for e.g. an infant formula, the one or more N-acetylated oligosaccharide (e.g. LNT) may be present in a total amount of about 0.05 g / L to about 1.0 g / L. Suitably, the one or more N-acetylated oligosaccharide is present in amount of about 0.1 g / L to about 0.5 g / L, or about 0.2 g / L to about 0.5 g / L. In some embodiments, LNT is present in an amount of from about 0.1 g / L to about 1.0 g / L (e.g. about 0.29 g / L or about 0.48 g / L). In some embodiments, LNT is present in an amount of about 0.29 g / L.

[0192] As a guide, for e.g. an infant formula, the one or more sialylated oligosaccharide (e.g. 3’SL and / or 6’SL) may be present in a total amount of from about 0.05 g / L to about 1 g / L. Suitably, the one or more sialylated oligosaccharide is present in amount of about 0.05 g / L to about 0.5 g / L, or about 0.1 g / L to about 0.5 g / L. In some embodiments, 3’SL is present in an amount of from about 0.05 g / L to about 0.3 g / L (e.g. about 0.11 g / L or about 0.18 g / L). In some embodiments, 3’SL is present in an amount of about 0.11 g / L. In some embodiments, 6’SL is present in an amount of from about 0.05 g / L to about 0.5 g / L (e.g. about 0.14 g / L or about 0.24 g / L). In some embodiments, 6’SL is present in an amount of about 0.14 g / L.

[0193] Suitably, the mixture of HMOs (e.g. in the form of a nutritional composition, such as an infant formula) may comprise or consist of: (i) 2’FL in an amount of from about 0.5 g / L to about 3.0 g / L (e.g. about 0.87 g / L or about 1.45 g / L); (ii) diFL in an amount of from about 0.05 g / L to about 0.3 g / L (e.g. about 0.10 g / L or about 0.14 g / L); (iii) LNT in an amount of from about 0.1 g / L to about 1.0 g / L (e.g. about 0.29 g / L or about 0.48 g / L); (iv) 3’-SL in an amount of from about 0.05 g / L to about 0.3 g / L (e.g. about 0.11 g / L or about 0.18 g / L); and (v) 6’-SL in an amount of from about 0.05 g / L to about 0.5 g / L (e.g. about 0.14 g / L or about 0.24 g / L).

[0194] In some embodiments, the mixture of HMOs (e.g. in the form of a nutritional composition, such as a starter infant formula) comprises or consists of: (i) 2’FL in an amount of from about 0.70 g / L to about 1 .05 g / L, preferably about 0.87 g / L; (ii) di FL in an amount of from about 0.05 g / L to about 0.11 g / L, preferably about 0.10 g / L; (iii) LNT in an amount of from about 0.23 g / L to about 0.36 g / L, preferably about 0.29 g / L; (iv) 3’-SL in an amount of from about 0.09 g / L to about 0.13 g / L, preferably about 0.11 g / L; and (v) 6’-SL in an amount of from about 0.12 g / L to about 0.17 g / L, preferably about 0.14 g / L.

[0195] In other embodiments, the mixture of HMOs (e.g. in the form of a nutritional composition, such as a starter infant formula) comprises or consists of: (i) 2’FL in an amount of from about 1.16 g / L to about 1.74 g / L, preferably about 1.45 g / L; (ii) diFL in an amount of from about 0.12 g / L to about 0.18 g / L, preferably about 0.14 g / L; (iii) LNT in an amount of from about 0.39 g / L to about 0.58 g / L, preferably about 0.48 g / L; (iv) 3’-SL in an amount of from about 0.14 g / L to about 0.21 g / L, preferably about 0.18 g / L; and (v) 6’-SL in an amount of from about 0.19 g / L to about 0.28 g / L, preferably about 0.24 g / L.

[0196] In other embodiments, the mixture of HMOs (e.g. in the form of a nutritional composition, such as a follow-up formula) comprises or consists of: (i) 2’FL in an amount of from about 0.19 g / L to about 0.34 g / L, preferably about 0.26 g / L; (ii) diFL in an amount of from about 0.03 g / L to about 0.05 g / L, preferably about 0.04 g / L; (iii) LNT in an amount of from about 0.06 g / L to about 0.11 g / L, preferably about 0.09 g / L; (iv) 3’-SL in an amount of from about 0.04 g / L to about 0.09 g / L, preferably about 0.06 g / L; and (v) 6’-SL in an amount of from about 0.03 g / L to about 0.06 g / L, preferably about 0.05 g / L.

[0197] Period of administration

[0198] The mixture of HMOs may be administered for any suitable period. For example, if administered at least in the form of a starter infant formula, the HMOs may be administered until at least about 6 months after birth. For example, if administered at least in the form of a starter infant formula and a follow-up formula, the HMOs may be administered until at least about 12 months after birth. For example, if administered at least in the form of a starter infant formula, a follow-up formula, and a growing-up milk, the HMOs may be administered until at least about 15 months after birth.

[0199] The mixture of HMOs may be administered at least from about 0 months to about 6 months after birth. Suitably, the mixture of HMOs is administered starting about 7 to about 21 days after birth. Suitably, the mixture of HMOs is administered until about 6 months to about 18 months after birth, until about 6 months to about 15 months after birth, or until about 6 months to about 12 months after birth. In some embodiments, the mixture of HMOs is administered starting about 7 to about 21 days after birth until about 15 months after birth, starting about 7 to about 21 days after birth until about 12 months after birth, starting about 7 to about 21 days after birth until about 9 months after birth, or starting about 7 to about 21 days after birth until about 6 months after birth.

[0200] Other components

[0201] In addition to the mixture of HMOs, a nutritional composition of the invention, and especially the infant formula, generally contains a protein source, a carbohydrate source and a lipid source.

[0202] A nutritional composition according to the invention, and especially an infant formula of the invention, may contain a protein source. The protein may be present in an amount of from about 1.5 to about 3.0 g / 100kcal, from about 1.5 to about 2.5 g / 100kcal, from about 1.6 to about 2.5 g / 100kcal, or from about 1.6 to about 2.25 g / 100kcal. In some embodiments, the protein amount is present in an amount of about 2.0 g g / 100kcal or less, e.g. from about 1.8 to about 2.0 g / 100kcal, or about 1.9 g / 100kcal.

[0203] Protein sources based on, for example, whey, casein and mixtures thereof may be used as well as plant based protein sources, for example, based on soy. As far as whey proteins are concerned, the protein source may be based on acid whey or sweet whey or mixtures thereof and may include alpha-lactalbumin and beta-lactoglobulin in any desired proportions. In some embodiments, the protein source is whey predominant (i.e. more than 50% of proteins are coming from whey proteins, such as 60% or more or 70% or more).

[0204] The proteins may be intact or hydrolysed or a mixture of intact and hydrolysed proteins.

[0205] The term "intact" in the context of the present invention may mean that the main part of the proteins are intact, i.e. the molecular structure is not altered, for example at least 80% of the proteins are not altered, such as at least 85% of the proteins are not altered, preferably at least 90% of the proteins are not altered, even more preferably at least 95% of the proteins are not altered, such as at least 98% of the proteins are not altered. In a particular embodiment, 100% of the proteins are not altered.

[0206] The term "hydrolysed" in the context of the present invention may mean proteins which have been hydrolysed or broken down into its component amino acids. The proteins may be either fully or partially hydrolysed. If hydrolysed proteins are required, the hydrolysis process may be carried out as desired and as is known in the art. For example, whey protein hydrolysates may be prepared by enzymatically hydrolysing the whey fraction in one or more steps. If the whey fraction used as the starting material is substantially lactose free, it is found that the protein suffers much less lysine blockage during the hydrolysis process. This enables the extent of lysine blockage to be reduced from about 15% by weight of total lysine to less than about 10%> by weight of lysine; for example about 7% by weight of lysine which greatly improves the nutritional quality of the protein source. In one particular embodiment, the proteins of the composition are hydrolysed, fully hydrolysed or partially hydrolysed. The degree of hydrolysis (DH) of the protein can be between 2 and 20, or between 8 and 40, or between 20 and 60 or between 20 and 80 or more than 10, 20, 40, 60, 80 or 90. At least 70%, 80%, 85%, 90%, 95% or 97% of the proteins may be hydrolysed. In a particular embodiment, 100% of the proteins are hydrolysed.

[0207] A nutritional composition according to the present invention, and especially an infant formula of the invention, may contain a carbohydrate source. In this case, any carbohydrate source conventionally found in infant formulae such as lactose, sucrose, saccharose, maltodextrin, starch and mixtures thereof may be used although one of the preferred sources of carbohydrates for infant formula is lactose. The carbohydrate may be in an amount of from about 8 to about 15 g / 100kcal or from about 9 to about 14 g / 100kcal. In some embodiments, the carbohydrate is present in an amount of from about 10 to about 13 g / 100kcal, or about 11.1 g / 100kcal.

[0208] A nutritional composition according to the present invention, and especially an infant formula of the invention, may contain lipids and essential fatty acids. Non-limiting examples of lipids include palm olein, high oleic sunflower oil, high oleic safflower oil, canola oil, fish oil, coconut oil, bovine milk fat, and combinations thereof. Non-limiting examples of essential fatty acids include linoleic acid (LA), a-linolenic acid (ALA). Compositions of the invention may further contain gangliosides monosialoganglioside-3 (GM3) and disialogangliosides 3 (GD3), and combinations thereof. The lipid may be in an amount of from about 3.0 to about 8.0 g / 100kcal, from about 4.0 to about 6.0 g / 100kcal, or from about 4.5 to about 5.5 g / 100kcal. In some embodiments, the lipid is present in an amount of from about 5.0 to about 5.5 g / 100kcal, or about 5.3 g / 100kcal.

[0209] A nutritional composition of the invention, and especially an infant formula of the invention, may also contain all vitamins and minerals understood to be essential in the daily diet and in nutritionally significant amounts. Minimum requirements have been established for certain vitamins and minerals. Examples of minerals, vitamins and other nutrients optionally present in the composition of the invention include vitamin A, vitamin B1 , vitamin B2, vitamin B3, vitamin B6, vitamin B12, vitamin E, vitamin K1 , vitamin K2, vitamin C, vitamin D, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorous, iodine, iron, magnesium, copper, zinc, manganese, chlorine, potassium, sodium, selenium, chromium, molybdenum, taurine, and L-carnitine. Minerals are usually added in salt form. The presence and amounts of specific minerals and other vitamins will vary depending on the intended population. If necessary, a nutritional composition of the invention may contain emulsifiers and stabilisers such as soy, lecithin, citric acid esters of mono- and diglycerides, and the like.

[0210] Suitably, a nutritional composition of the invention, and especially an infant formula of the invention, may have an energy density of from about 60 to about 72 kcal per 100 mL or about 67 kcal per 100 mL

[0211] Preparation of compositions

[0212] The compositions according to the present invention may be prepared by any known or otherwise suitable manner.

[0213] For example, a nutritional composition, e.g. an infant formula, may be proposed by blending together a source of protein with a carbohydrate source and a lipid source in appropriate proportions. If used, emulsifiers may be included at this stage. Vitamins and minerals may be added at this stage, but may also be added later to avoid thermal degradation. Water, preferably water which has been subjected to reverse osmosis or deionized water, may then be added and mixed in to form a liquid mixture. The temperature of mixing is preferably room temperature, but may also be higher. The liquid mixture may then be thermally treated to reduce bacterial loads. The mixture may then be homogenized.

[0214] If it is desired to produce a powdered composition, the homogenized mixture may be dried in a suitable drying apparatus, such as a spray drier or freeze drier and converted into powder.

[0215] Processes used in the manufacture of formulae for infants and young children are based on the concept that the products must be nutritionally adequate and microbiologically safe to consume. Thus, steps that eliminate or restrict microbiological growth are central to production processes. The processing technology generally involves the preservation of an oil-in-water (o / w) emulsion by dehydration in the case of powder products or, sterilization in the case of ready-to-feed or concentrated liquid products. Powdered infant formula may be produced using various processes, such as dry blending dehydrated ingredients to constitute a uniform formula or hydrating and wet-mixing a mixture of macro-ingredients, such as fat, protein and carbohydrate ingredients and then evaporating and spray drying the resultant mixture. A combination of the two processes described above may be used where a base powder is first produced by wet-mixing and spray drying all or some of the macro-ingredients and then dry blending the remaining ingredients, including carbohydrate, minerals and vitamins and other micronutrients, to create a final formula. Liquid formulae are available in a ready-to-feed format or as a concentrated liquid, which requires dilution, normally 1 :1 , with water. The manufacturing processes used for these products are similar to those used in the manufacture of recombined milk.

[0216] If it is desired to produce a liquid infant formula, the homogenized mixture may be filled into suitable containers, preferably aseptically. However, the liquid composition may also be retorted in the container, suitable apparatus for carrying out the filling and retorting of this nature is commercially available.

[0217] Combination

[0218] The invention further provides a combination of a bifidogenic prebiotic and a Bifidobacterium microorganism for use according to the present invention.

[0219] The bifidogenic prebiotic and a Bifidobacterium microorganism may be administered separately, simultaneously or sequentially.

[0220] Suitably, the bifidogenic prebiotic and a Bifidobacterium microorganism may be administered in a combined composition.

[0221] Suitably, a combination of a bifidogenic prebiotic and a Bifidobacterium microorganism may be referred to as a “synbiotic”.

[0222] In aspects of the invention in which a combination of bifidogenic prebiotic and a Bifidobacterium microorganism are used, each may be selected such that the Bifidobacterium microorganism is capable of metabolising the bifidogenic prebiotic provided in the combination.

[0223] The combinations of the invention are not limited to requiring that the Bifidobacterium microorganism is capable of metabolizing the bifidogenic prebiotic provided in the combination. As such, any combinations Bifidobacterium microorganism(s) and bifidogenic prebiotic disclosed herein are encompassed by the invention.

[0224] Suitably, the composition comprises one or more glycan substrates as described herein. The composition may comprise between 103to 1012cfu of probiotic strain, more preferably between 107and 1012cfu such as between 108and 1O10cfu of probiotic strain per g of composition on a dry weight basis mixed with a mixture of HMOs as defined herein.

[0225] Methods

[0226] In another aspect, the present invention provides a method of promoting butyrate-producing bacteria in the gut microbiota of an infant or young child comprising administering a bifidogenic prebiotic, a Bifidobacterium microorganism or a combination of a bifidogenic prebiotic and a Bifidobacterium microorganism to the infant or young child; wherein the bifidogenic prebiotic, Bifidobacterium microorganism or a combination is administered to an infant prior to about 12 months of age; and the butyrate-producing bacteria are promoted in the gut microbiota of the infant or young child between about 1 and 5 years of age.

[0227] In a further aspect, the present invention provides a method of treating and / or preventing an allergy and / or allergic sensitization in an infant or young child by promoting butyrate-producing bacteria in the gut microbiota of the infant or young child; comprising administering a bifidogenic prebiotic, a Bifidobacterium microorganism or a combination of a bifidogenic prebiotic and a Bifidobacterium microorganism to the infant or young child; wherein the bifidogenic prebiotic, Bifidobacterium microorganism or a combination is administered to the infant prior to about 12 months of age; and administration of bifidogenic prebiotic, Bifidobacterium microorganism or a combination promotes butyrate-producing bacteria in the gut microbiota of the infant or young child between about 1 and 5 years of age.

[0228] In a further aspect, the present invention relates to the use of a bifidogenic prebiotic, a Bifidobacterium microorganism or a combination of a bifidogenic prebiotic and a Bifidobacterium microorganism to promote butyrate-producing bacteria in the gut microbiota of the infant or young child; wherein the bifidogenic prebiotic, Bifidobacterium microorganism or a combination is administered to an infant prior to about 12 months of age; and the butyrate- producing bacteria are promoted in the gut microbiota of the infant or young child between about 1 and 5 years of age.

[0229] The Bifidobacterium microorganism may be a Bifidobacterium microorganism as described herein.

[0230] The prebiotic may be a prebiotic as described herein.

[0231] The combination of a Bifidobacterium microorganism and a prebiotic may be provided in any form as described herein. For example, the combination may be provided in a composition as described herein. Embodiments

[0232] The present invention provides the embodiments according to the following numbered clauses:

[0233] 1. A bifidogenic prebiotic for use in promoting butyrate-producing bacteria in the gut microbiome of an infant or young child; wherein the bifidogenic prebiotic is administered to the infant prior to about 12 months of age; and the butyrate-producing bacteria are promoted in the gut microbiome of the infant or young child between about 1 and 5 years of age.

[0234] 2. A bifidogenic prebiotic for use according to clause 1 wherein the bifidogenic prebiotic is used in combination with a Bifidobacterium microorganism.

[0235] 3. A Bifidobacterium microorganism for use in promoting butyrate-producing bacteria in the gut microbiome of an infant or young child; wherein the Bifidobacterium is administered to the infant prior to about 12 months of age; and the butyrate-producing bacteria are promoted in the gut microbiome of the infant or young child between about 1 and 5 years of age.

[0236] 4. A Bifidobacterium microorganism for use according to clause 3 wherein the Bifidobacterium microorganism is used in combination with a bifidogenic prebiotic.

[0237] 5. A combination of a bifidogenic prebiotic and a Bifidobacterium microorganism for use in promoting butyrate-producing bacteria in the gut microbiome of an infant or young child wherein the combination is administered to the infant prior to about 12 months of age; and the butyrate-producing bacteria are promoted in the gut microbiome of the infant or young child between about 1 and 5 years of age.

[0238] 6. A bifidogenic prebiotic, Bifidobacterium microorganism or a combination for use according to any preceding clause wherein levels of butyrate are increased in the gut microbiome of the infant or young child between about 1 and 5 years of age.

[0239] 7. A bifidogenic prebiotic, Bifidobacterium microorganism or a combination for use according to any preceding clause wherein the bifidogenic prebiotic, Bifidobacterium microorganism or combination is administered to the infant between birth and about 6 months of age. 8. A bifidogenic prebiotic, Bifidobacterium microorganism or a combination for use according to any preceding clause wherein the butyrate-producing bacteria is promoted in the gut microbiome of the infant or young child between about 1 and 3 years of age.

[0240] 9. A bifidogenic prebiotic, Bifidobacterium microorganism or a combination for use according to any preceding clause wherein the butyrate-producing bacteria is promoted in the gut microbiome of the infant or young child at about 12, 18, 24 and / or 36 months of age.

[0241] 10. A bifidogenic prebiotic, Bifidobacterium microorganism or combination for use according to any of clauses 2 to 9 wherein the Bifidobacterium microorganism is selected from one or more of B. longum infantis, B. longum longum, B. bifidum, B. breve, B. kashiwanohense, B. scardovii and B. longum iuvenis.

[0242] 11. A bifidogenic prebiotic, Bifidobacterium microorganism or combination for use according to clause 10 wherein the Bifidobacterium microorganism is B. longum infantis.

[0243] 12. A bifidogenic prebiotic, Bifidobacterium microorganism or combination for use according to any preceding clause wherein the bifidogenic prebiotic, Bifidobacterium microorganism or combination promotes one or more of B. longum infantis, B. longum longum, B. bifidum, B. breve, B. kashiwanohense and B. longum iuvenis in the gut microbiome of the infant or young child.

[0244] 13. A bifidogenic prebiotic, Bifidobacterium microorganism or combination for use according to any preceding clause wherein the bifidogenic prebiotic, Bifidobacterium microorganism or combination is administered to the infant prior to about 6 months of age.

[0245] 14. A bifidogenic prebiotic, Bifidobacterium microorganism or combination for use according to clause 13 wherein B. longum infantis is promoted in the gut microbiome of the infant or young child.

[0246] 15. A bifidogenic prebiotic, Bifidobacterium microorganism or combination for use according to any preceding clause wherein the butyrate-producing bacteria is selected from one or more of Faecalibacterium prausnitzii, Roseburia inulinivorans and Eubacterium rectale.

[0247] 16. A bifidogenic prebiotic, Bifidobacterium microorganism or a combination for use according to any preceding clause wherein the butyrate-producing bacteria: (a) modulate the permeability of the gut epithelial barrier; preferably wherein the butyrate-producing bacteria decrease the permeability of the gut epithelial barrier; and / or

[0248] (b) support and / or improve colonocyte function; and / or

[0249] (c) reduce inflammation, suitably by inducing differentiation and / or expansion of Treg cells.

[0250] 17. A bifidogenic prebiotic or combination for use according to any of clauses 1 , 2 or 4 to 16, wherein the bifidogenic prebiotic is one or more human milk oligosaccharides (HMOs).

[0251] 18. A bifidogenic prebiotic or combination for use according to clause 17 wherein the HMOs comprise or consist of a mixture of 2’-fucosyllactose (2’FL), 2’,3-difucosyllactose (diFL), lacto-N-tetraose (LNT), 3’-sialyllactose (3’-SL), and 6’-sialyllactose (6’-SL).

[0252] 19. A bifidogenic prebiotic or combination for use according to clause 18 wherein the mixture of HMOs comprises or consists of: (i) 2’FL in an amount of from about 55 wt% to about 60 wt%; (ii) diFL in an amount of from about 5 wt% to about 7 wt%; (iii) LNT in an amount of from about 18 wt% to about 20 wt%; (iv) 3’-SL in an amount of from about 6 wt% to about 8 wt%; and (v) 6’-SL in an amount of from about 9 wt% to about 11 wt%, based on the total weight of HMOs.

[0253] 20. A bifidogenic prebiotic or combination for use according to any of clauses 17 to 19 wherein the HMOs are present in a nutritional composition.

[0254] 21. A bifidogenic prebiotic or combination for use according to clause 20 wherein the nutritional composition is an infant formula, optionally wherein the nutritional composition is administered in the form of a starter infant formula, and / or a follow-up formula.

[0255] 22. A bifidogenic prebiotic, a Bifidobacterium microorganism or a combination of a bifidogenic prebiotic and a Bifidobacterium microorganism for use in treating and / or preventing an allergy and / or allergic sensitization in an infant or young child by promoting butyrate- producing bacteria in the gut microbiome of the infant or young child; wherein the bifidogenic prebiotic, Bifidobacterium microorganism or a combination is administered to an infant prior to about 12 months of age; and the butyrate-producing bacteria are promoted in the gut microbiome of the infant or young child between about 1 and 5 years of age. 23. Use of a bifidogenic prebiotic, a Bifidobacterium microorganism or a combination of a bifidogenic prebiotic and a Bifidobacterium microorganism to promote butyrate-producing bacteria in the gut microbiome of the infant or young child; wherein the bifidogenic prebiotic, Bifidobacterium microorganism or a combination is administered to an infant prior to about 12 months of age; and the butyrate-producing bacteria are promoted in the gut microbiota of the infant or young child between about 1 and 5 years of age.

[0256] 24. A method of promoting butyrate-producing bacteria in the gut microbiome of an infant or young child comprising administering a bifidogenic prebiotic, a Bifidobacterium microorganism or a combination of a bifidogenic prebiotic and a Bifidobacterium microorganism to the infant or young child; wherein the bifidogenic prebiotic, Bifidobacterium microorganism or a combination is administered to an infant prior to about 12 months of age; and the butyrate-producing bacteria are promoted in the gut microbiome of the infant or young child between about 1 and 5 years of age.

[0257] 25. A method of treating and / or preventing an allergy and / or allergic sensitization in an infant or young child by promoting butyrate-producing bacteria in the gut microbiome of the infant or young child; comprising administering a bifidogenic prebiotic, a Bifidobacterium microorganism or a combination of a bifidogenic prebiotic and a Bifidobacterium microorganism to the infant or young child; wherein the bifidogenic prebiotic, Bifidobacterium microorganism or a combination is administered to the infant prior to about 12 months of age; and administration of bifidogenic prebiotic, Bifidobacterium microorganism or a combination promotes butyrate-producing bacteria in the gut microbiome of the infant or young child between about 1 and 5 years of age.

[0258] Those skilled in the art will understand that they can freely combine all features of the present invention disclosed herein. In particular, features described for the product of the present invention may be combined with the method of the present invention and vice versa. Further, features described for different embodiments of the present invention may be combined. Where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to in this specification.

[0259] Further advantages and features of the present invention are apparent from the figures and non-limiting examples. Examples

[0260] Example 1

[0261] Three datasets from different locations were analyzed (Central Europe, North America / USA and South Asia / Bangladesh). Levels of infant-type Bifidobacterium (B; listed below in Table 1) and butyrate producers (P; listed below in Table 2) were characterized as the sum of relative abundances of each group for each participant at each time point.

[0262] Table 1 : Infant-type Bifidobacterium spp. (B) was defined according to Laursen et al. (Nature Microbiology volume 6, pagesl 367-1382 (2021)): Table 2: Butyrate producers (P) were defined according to Louis & Flynt (FEMS Microbiol Lett

[0263] 2009 May;294(1):1-8):

[0264] The levels of B and P were compared at each different timepoint to assess the longitudinal relationship between both bacteria. Comparisons between bifidobacteria and butyrate producers was performed using Spearman correlation, General Mixed Linear Models and Wilcoxon rank test between the abundances of both groups. Comparisons were done both synchronically (same time point) and longitudinally (different time points), always assuming that the lowest timepoint belonged to infant-type Bifidobacterium.

[0265] Example 2

[0266] The longitudinal relationship between Bifidobacterium longum ssp. Infantis and butyrate producers was determined as described in Example 1 : Bifidobacterium infantis in babies (<6m) is associated to toddler (>12m) butyrate producer abundance.

[0267] A general Linear Mixed Model (GLMM) was used to assess the relationship between both groups, and showed a significant relationship between B. infantis and butyrate producers (P <0.05) but did not show an impact of time, recruitment age, or differences between subpopulations (i.e., C-section) (Figure 1).

[0268] Example 3

[0269] Bifidobacterium infantis was shown to be the main factor driving the longitudinal association with butyrate producers.

[0270] B. Infantis and B. bifidum primarily show a strong longitudinal association starting at 2 months until 6 months. Other taxa show limited or no association with butyrate producers before 6 months of age. After 6 months, a strong negative association with butyrate producers was determined for all taxa (Figure 2).

[0271] There was also an association between early implantation of B. iuvenis and particular butyrate producers, for example Roseburia inulinivorans, in a South Asia population (see Figure 3). However, B. Iuvenis did not show a significant positive association between early levels and general late butyrate producers.

[0272] Example 4

[0273] To assess the effect of human milk oligosaccharides (HMOs) in formula feeding, a randomized controlled trial (ClinicalTrials.gov Identifier: NCT03722550) was performed. A study overview is provided in Figure 4.

[0274] Healthy full-term infants (7-21 days old) were randomly assigned to a standard cow’s milkbased starter infant formula (control group, CG, n=154); the same formula with 1.5 g / L HMOs (test group 1 , TG1 , n=155); or with 2.5 g / L HMOs (test group 2, TG2, n=153); or a human milk- fed group (reference, HMG, n=61).

[0275] The standard starter infant was a bovine milk-based whey predominant term infant formula with 67 kcal / 100 mL reconstituted formula, consisting of 1.9 g intact protein (70% whey / 30% casein) / 100 kcal, 11.1 g carbohydrates / 100 kcal, and 5.3 g lipids / 100 kcal. The concentration of individual HMOs in TG1 and TG2 starter infant formula is shown in Table 3 below.

[0276] Table 3 - concentration of individual HMOs in TG1 and TG2 starter infant formula

[0277] The standard follow-up formula was a bovine milk-based whey predominant term infant formula with 67 kcal / 100 mL reconstituted formula, consisting of 2 g intact protein (50% whey / 50% casein) / 100 kcal, 12.4 g carbohydrates / 100 kcal, and 4.7 g lipids / 100 kcal. The concentration of total HMOs in TG1 and TG2 follow-up formula was 0.5 g / L of the same blend as the starter infant formula.

[0278] The standard growing-up milk was a bovine milk-based growing-up milk with 67 kcal / 100 mL reconstituted formula, consisting of 2.25 g intact protein (40% whey / 60% casein) / 100 kcal, 12.6 g carbohydrates / 100 kcal, and 4.5 g lipids / 100 kcal. The concentration of total HMOs in TG1 and TG2 growing-up milk was 0.4 g / L of the same blend as the starter infant formula.

[0279] Fecal samples collected at enrolment, 3, 6, 12, and 15 months of age were used for profiling.

[0280] The promotion of early bifidobacteria lead to an enhanced abundance of butyrate producers later in time. Consumption of infant formula with demonstrated bifidogenic effect before 6 months of age promotes larger abundances of butyrate producers after 12 months of age (Figure 5). Thiseffect is statistically significant using the products containing 1.5g / L blend of 5 HMOs, and a similar trend is seen with the products with a blend of 2.5g / L

[0281] Example 5

[0282] Similar to the observation with Bifidobacterium spp., not all butyrate producers are equally impacted by the consumption of bifidogenic formula. F.prausnitzii, R. inulinivorans & E. rectale are significantly impacted by the early consumption of HMOs (Table 4). The observed impact is statistically significant using products containing 1 ,5g / L blend of 5 HMOs, and a similar trend is seen with the products with 2.5g / L of the same blend.

[0283] Example 6

[0284] The infant-type Bifidobacterium were taken as defined in Laursen et al, Nature Microbiology 2021. It included the sum of abundances of B. longum subspp., B. breve, B. bifidum, and B. scardovii. However, B. scardovii was not detected in the gene catalog used to profile the microbiome data from this trial (Bosheva et al. Front Nutr. 2022; 9:920362); the rest of the species were detected and present in the data.

[0285] Butyrate molecule was measured as part of the organic acids estimated from fecal samples collected in this trial (Bosheva et al.; as above). For statistical analysis, data transformation (cube root) was performed to normalize distributions in order to define low and high based on tertiles and then to run the statistical tests (Fischer’s exact). For visits, V3 refers to 3 months (this visit is after start of the intervention), V7 is at 12 months and V8 is at 15 months.

[0286] Association results for early Low / High infant-type Bifidobacterium abundance at V3 / 3 months to later Low / High butyrate concentration at V7 / 12 months (p-value = 0.030):

[0287] Test statistic - p-value = 0.030

[0288] Association results for early Low / High Bifidobacterium genus abundance at V3 / 3 months to later Low / High Butyrate concentration at V8 / 15 months (p-value = 0.045):

[0289] Test statistic - p-value = 0.045

[0290] These results demonstrate that higher abundance of infant-type Bifidobacterium or Bifidobacterium genus at early visit (3 months) is significantly associated with higher abundance of butyrate molecule at later visits (12m and 15m, respectively). All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

[0291] (Original in Electronic Form)

[0292] (This sheet is not part of and does not count as a sheet of the international application)

[0293] FOR RECEIVING OFFICE USE ONLY

[0294] FOR INTERNATIONAL BUREAU USE ONLY

Claims

CLAIMS1. A bifidogenic prebiotic for use in promoting butyrate-producing bacteria in the gut microbiome of an infant or young child; wherein the bifidogenic prebiotic is administered to the infant prior to about 12 months of age; and the butyrate-producing bacteria are promoted in the gut microbiome of the infant or young child between about 1 and 5 years of age.

2. A Bifidobacterium microorganism for use in promoting butyrate-producing bacteria in the gut microbiome of an infant or young child; wherein the Bifidobacterium is administered to the infant prior to about 12 months of age; and the butyrate-producing bacteria are promoted in the gut microbiome of the infant or young child between about 1 and 5 years of age.

3. A combination of a bifidogenic prebiotic and a Bifidobacterium microorganism for use in promoting butyrate-producing bacteria in the gut microbiome of an infant or young child wherein the combination is administered to the infant prior to about 12 months of age; and the butyrate-producing bacteria are promoted in the gut microbiome of the infant or young child between about 1 and 5 years of age.

4. A bifidogenic prebiotic, Bifidobacterium microorganism or a combination for use according to any preceding claim wherein levels of butyrate are increased in the gut microbiome of the infant or young child between about 1 and 5 years of age.

5. A bifidogenic prebiotic, Bifidobacterium microorganism or a combination for use according to any preceding claim wherein the bifidogenic prebiotic, Bifidobacterium microorganism or combination is administered to the infant between birth and about 6 months of age.

6. A bifidogenic prebiotic, Bifidobacterium microorganism or a combination for use according to any preceding claim wherein the butyrate-producing bacteria is promoted in the gut microbiome of the infant or young child between about 1 and 3 years of age.

7. A bifidogenic prebiotic, Bifidobacterium microorganism or combination for use according to any preceding claim wherein the Bifidobacterium microorganism is selected from one or more of B. longum infantis, B. longum longum, B. bifidum, B. breve, B. kashiwanohense, B. scardovii and B. longum iuvenis.

8. A bifidogenic prebiotic, Bifidobacterium microorganism or combination for use according to claim 7 wherein the Bifidobacterium microorganism is B. longum infantis.

9. A bifidogenic prebiotic, Bifidobacterium microorganism or combination for use according to any preceding claim wherein the bifidogenic prebiotic, Bifidobacterium microorganism or combination is administered to the infant prior to about 6 months of age.

10. A bifidogenic prebiotic, Bifidobacterium microorganism or combination for use according to any preceding claim wherein the butyrate-producing bacteria is selected from one or more of Faecalibacterium prausnitzii, Roseburia inulinivorans and Eubacterium rectale.

11. A bifidogenic prebiotic or combination for use according to any of claims 1 or 3 to 10, wherein the bifidogenic prebiotic is one or more human milk oligosaccharides (HMOs), optionally wherein the HMOs comprise or consist of a mixture of 2’-fucosyllactose (2’FL), 2’, 3- difucosyllactose (diFL), lacto-N-tetraose (LNT), 3’-sialyllactose (3’-SL), and 6’-sialyllactose (6’- SL).

12. A bifidogenic prebiotic or combination for use according to claim 11 wherein the HMOs are present in a nutritional composition, optionally wherein the nutritional composition is administered in the form of a starter infant formula, and / or a follow-up formula.

13. A bifidogenic prebiotic, a Bifidobacterium microorganism or a combination of a bifidogenic prebiotic and a Bifidobacterium microorganism for use in treating and / or preventing an allergy and / or allergic sensitization in an infant or young child by promoting butyrate- producing bacteria in the gut microbiome of the infant or young child; wherein the bifidogenic prebiotic, Bifidobacterium microorganism or a combination is administered to an infant prior to about 12 months of age; and the butyrate-producing bacteria are promoted in the gut microbiome of the infant or young child between about 1 and 5 years of age.

14. Use of a bifidogenic prebiotic, a Bifidobacterium microorganism or a combination of a bifidogenic prebiotic and a Bifidobacterium microorganism to promote butyrate-producing bacteria in the gut microbiome of the infant or young child; wherein the bifidogenic prebiotic, Bifidobacterium microorganism or a combination is administered to an infant prior to about 12 months of age; and the butyrate-producing bacteria are promoted in the gut microbiota of the infant or young child between about 1 and 5 years of age.

15. A method of promoting butyrate-producing bacteria in the gut microbiome of an infant or young child comprising administering a bifidogenic prebiotic, a Bifidobacterium microorganism or a combination of a bifidogenic prebiotic and a Bifidobacterium microorganism to the infant or young child; wherein the bifidogenic prebiotic, Bifidobacterium microorganism or a combination is administered to an infant prior to about 12 months of age; and the butyrate-producing bacteria are promoted in the gut microbiome of the infant or young child between about 1 and 5 years of age.