Compositions for promoting sleep in a young individual

Abstract

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Description

TITLECOMPOSITIONS FOR PROMOTING SLEEP IN A YOUNG INDIVIDUALTECHNICAL FIELD

[0001] The present disclosure generally relates to compositions and methods for use in promoting sleep associated with increase of propionate, in a young individual. Some embodiments are directed to a composition comprising a combination of galactooligosaccharide (GOS), Vitamins Bl, B2 and B6, minerals zinc, iron and copper, and amino acids histidine, isoleucine, lysine, and leucine for use in promoting sleep in a young individual via increase of propionate.BACKGROUND

[0002] Reduced sleep has been associated with multiple negative effects, such as decreased cognitive development, mood regulation, and overall health. Furthermore, short sleep duration and poor sleep quality has been associated with obesity and behavioral problems. The most common sleep disturbances in infants and children are those related to wakefulness (i.e., difficulties in settling at bedtime or failure to sleep through the night without interruptions). These disturbances have been estimated to affect 15 to 35% of infants aged less than 24 months.SUMMARY

[0003] The present inventors found that exposing the microbiota of a young individual, such as an infant or toddler, to a combination of galacto-oligosaccharides and a mix of micronutrients and amino acids specifically increase the biosynthesis of propionate, a shortchain fatty acid positively associated with sleep duration. Thus, the combination promotes sleep in a young individual by modulating the activity of the microbiota of the young individual.

[0004] Accordingly, an aspect of the present disclosure is a composition for use in promoting sleep in a young individual comprising a combination of at least one galactooligosaccharide (GOS) and at least three ingredients selected from vitamin Bl, vitamin B2, vitamin B6, zinc, iron, copper, histidine, isoleucine, lysine, leucine.

[0005] Another aspect of the present disclosure is a method of promoting sleep (e.g., sleep quality) in a young individual, for example an infant or toddler in need of better sleep. The method comprises administering to the young individual a composition comprisingcombination of at least one galacto-oligosaccharide (GOS) and at least three ingredients selected from vitamins Bl, B2 and B6, minerals zinc, iron and copper, and amino acids histidine, isoleucine, lysine, and leucine. Preferably the combination is orally administered in an amount effective to increase propionate in the young individual.

[0006] In an embodiment, the amino acids, including histidine, isoleucine, lysine and leucine, are provided by a protein source selected from the list consisting of: free amino acids or salts thereof, oligopeptides, peptides, proteins such as dairy, animal or plant proteins; and any combinations thereof.

[0007] In an embodiment, the composition is for use in preventing disorders, diseases or conditions later in life associated with lack of sleep in infancy. In an embodiment, the disorders, diseases or conditions later in life associated with lack of sleep in infancy include obesity, overweight, anxiety, depression, social-emotional disorders, neurodevelopment disorders, mood disorders, attention disorders.

[0008] In an embodiment, the present disclosure is a unit dosage form of the composition comprising a combination of at least one galacto-oligosaccharide and at least three ingredients selected from vitamins Bl, B2 and B6, minerals zinc, iron and copper, and amino acids histidine, isoleucine, lysine, and leucine. The unit dosage form comprises an amount of the combination effective to increase propionate in a young individual to whom the unit dosage form is orally administered.

[0009] An advantage of one or more embodiments disclosed herein is to deliver health benefits on sleep in the subject in a manner that does not induce side effects and / or in a manner that is easy to deliver, is well accepted by the parents or health care practitioners, and delivers such benefits in a manner that keeps the cost of such delivery reasonable and affordable by most. Indeed, existing solutions for infant sleep involve supplementation with plant extracts or bioactives, and the present disclosure provides a solution that will use the microbiota’s own ability to produce molecules promoting sleep.

[0010] Additional features and advantages are described herein and will be apparent from the following Figures and Detailed Description.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 is a graph showing faecal levels of short-chain fatty acids acetate, propionate and butyrate in infants in the experimental example disclosed herein.DETAILED DESCRIPTION

[0012] Definitions

[0013] Some definitions are provided hereafter. Nevertheless, definitions may be located in the “Embodiments” section below, and the above header “Definitions” does not mean that such disclosures in the “Embodiments” section are not definitions.

[0014] All percentages expressed herein are by weight of the total weight of the composition unless expressed otherwise. As used herein, “about,” “approximately” and “substantially” are understood to refer to numbers in a range of numerals, for example the range of -10% to +10% of the referenced number, preferably -5% to +5% of the referenced number, more preferably -1% to +1% of the referenced number, most preferably -0.1% to +0.1% of the referenced number. All numerical ranges herein should be understood to include all integers, whole or fractions, within the range. Moreover, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 1 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

[0015] As used in this disclosure and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an amino acid” or “the amino acid” includes two or more amino acids.

[0016] The words “comprise,” “comprises” and “comprising” are to be interpreted inclusively rather than exclusively. Likewise, the terms “include,” “including” and “or” should all be construed to be inclusive, unless such a construction is clearly prohibited from the context. Nevertheless, the compositions and methods disclosed herein may lack any element that is not specifically disclosed herein. Thus, a disclosure of an embodiment using the term “comprising” includes a disclosure of embodiments “consisting essentially of’ and “consisting of’ the components or steps identified.

[0017] The terms “at least one of’ and “and / or” used respectively in the context of “at least one of X and Y” and “X and / or Y” should be interpreted as “X without Y,” or “Y without X,” or “both X and Y.” Where used herein, the terms “example” and “such as,” particularly when followed by a listing of terms, are merely exemplary and illustrative and should not be deemed to be exclusive or comprehensive.

[0018] As used herein, “related to,” “associated with” and “linked with” mean occurring concurrently, preferably mean caused by the same underlying condition, more preferably mean that one of the identified conditions is at least indirectly caused by the other identified condition,and most preferably mean that one of the identified conditions is directly caused by the other identified condition.

[0019] “Prevention” includes reduction of risk, incidence and / or severity of a condition or disorder. The terms “treatment” and “treat” include both prophylactic or preventive treatment (that prevent and / or slow the development of a targeted pathologic condition or disorder) and curative, therapeutic or disease-modifying treatment, including therapeutic measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic condition or disorder; and treatment of patients at risk of contracting a disease or suspected to have contracted a disease, as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition. The terms “treatment” and “treat” do not necessarily imply that a subject is treated until total recovery. The terms “treatment” and “treat” also refer to the maintenance and / or promotion of health in an individual not suffering from a disease but who may be susceptible to the development of an unhealthy condition. The terms “treatment” and “treat” are also intended to include the potentiation or otherwise enhancement of one or more primary prophylactic or therapeutic measures. As non-limiting examples, a treatment can be performed by a patient, a caregiver, a doctor, a nurse, or another healthcare professional.

[0020] The expression "later in life" and "in later life" can be used interchangeably. They refer to effects measured in the individual (infant or young child) after the age of some weeks, some months or some years after birth, such as after the age of 6 months after birth, such as after the age of 8 months after birth, such as after the age of 10 months after birth, such as after the age of 1 year after birth, such as after the age of 2 years, preferably after the age of 4 years, more preferably after the age of 5 years, even more preferably after the age of 7 years after birth, or even more, and as a comparison to average observations for subjects of the same age. Preferably it refers to an effect observed after at least 1 year of life, or after at least 2, 5, 7, 10 or 15 years of life. So the expression "later in life" might refer to an observation during infancy, during childhood, during the adolescent period, or during adulthood. Preferably it refers to an observation during childhood, during the adolescent period, or during adulthood. The term "later in life" encompasses the effect after the termination of the intervention.

[0021] In some embodiments, “promoting sleep” or improvement of “sleep quality” is improvement in one or more of i) sleep efficiency (e.g. measured by actigraphy data); ii) change in sleep latency (e.g actigraphy data); iii) change in wake after sleep onset (e.g. by actigraphy); iv) change in total sleep duration (mins, actigraphy); v) time in bed; vi) minutes spent in bed after waking up. In other embodiments, sleep quality may be assessed by self-reporting (e.g.Karolinska Sleepiness Scale (KSS) or Epworth Sleepiness Scale (ESS). “Sleep quality” can be quantified by one or both of (a) a total duration of slow wave sleep (SWS) and / or (b) a total duration of rapid eye movement (REM). For example, an improved sleep quality can be established by one or both of a longer total duration of SWS and / or a total duration of REM.

[0022] As used herein, a prophylactically or therapeutically “effective amount” is an amount that prevents a deficiency, treats a disease or medical condition in an individual, or, more generally, reduces symptoms, manages progression of the disease, or provides a nutritional, physiological, or medical benefit to the individual.

[0023] The term “composition” may mean a food, beverage, dietary supplement, complete nutrition, or oral nutritional supplement (ONS) or medical food composition, or mixture thereof. The terms “food,” “food supplement,” “food product” and “food composition” mean a product or composition that is intended for ingestion by an individual such as a human and provides at least one nutrient to the individual. The compositions of the present disclosure, including the many embodiments described herein, can comprise, consist of, or consist essentially of the elements disclosed herein, as well as any additional or optional ingredients, components, or elements described herein or otherwise useful in a diet supplement.

[0024] The composition can be in solid form (e.g., powder) or in liquid form. The amount of the various ingredients can be expressed in g / 100 g of the composition on a dry weight basis when it is in a solid form, e.g. a powder, or as a concentration in g / 100 mL 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 water.)

[0025] The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the composition disclosed herein in an amount sufficient to produce the desired effect, in association with an acceptable diluent, carrier or vehicle. The specifications for the unit dosage form depend on the particular compounds employed, the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0026] The term "young individual" as used herein refers to an infant, a toddler, a young child or a child.

[0027] The term “infant” means a child under the age of 12 months. The term infant includes both infants born at term or infant born preterm.

[0028] The terms “toddler” or “young child” mean a child aged between one and three years.

[0029] The term “child” means a child aged between one and 6 years, including toddlers and pre-school children.

[0030] The terms “enhance,” “increase,” “improve,” “promote” and the like mean that a composition according to the present disclosure (which comprises a combination of GOS and at least three ingredients selected from vitamins Bl, B2 and B6; minerals zinc, iron and copper; and amino acids histidine, isoleucine, lysine, and leucine) provides a sleep quality greater than the sleep quality from a reference composition lacking at least one of these components but otherwise identically formulated.

[0031] 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.

[0032] A " follow-up formula" or "follow-on formula" is given from the sixth month onwards and includes growing-up milk. It constitutes the principal liquid element in the progressively diversified diet of this category of person.

[0033] The expression "baby food" means a foodstuff intended for particular nutritional use by infants or young children during the first years of life.

[0034] The expression "infant cereal composition" means a foodstuff intended for particular nutritional use by infants or young children during the first years of life.

[0035] The term "fortifier" refers to liquid or solid nutritional compositions suitable for mixing with breast milk or infant formula.

[0036] The expression “mother’s milk” should be understood as the breast milk or the colostrum of the mother.

[0037] The supplement may be in the form of tablets, capsules, pastilles or a liquid for example. The supplement may further contain protective hydrocolloids (such as gums, proteins, modified starches), binders, film forming agents, encapsulating agents / materials, wall / shell materials, matrix compounds, coatings, emulsifiers, surface active agents, solubilizing agents (oils, fats, waxes, lecithins etc.), adsorbents, carriers, fillers, co compounds, dispersing agents, wetting agents, processing aids (solvents), flowing agents, taste masking agents, weighting agents, jellifying agents and gel forming agents. The supplement may also contain conventionalpharmaceutical additives and adjuvants, excipients and diluents, including, but not limited to, water, gelatine of any origin, vegetable gums, lignin-sulfonate, talc, sugars, starch, gum arabic, vegetable oils, polyalkylene glycols, flavouring agents, preservatives, stabilizers, emulsifying agents, buffers, lubricants, colorants, wetting agents, fillers, and the like.Further, the supplement may contain an organic or inorganic carrier material suitable for oral or parenteral administration as well as vitamins, minerals trace elements and other micronutrients in accordance with the recommendations of Government bodies such as the USRDA.

[0038] The terms "prebiotic", "fibre(s)" and "fiber(s)" can be used interchangeably. They refer to 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).

[0039] The term "probiotic" means microbial cell preparations 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 are generally bacteria or yeasts.

[0061] The term "cfu" should be understood as colony-forming unit.

[0062] All percentages are by weight unless otherwise stated.

[0040] The further probiotic microorganisms most commonly used are principally bacteria and yeasts of the following genera: Lactobacillus spp., Lacticaseibacillus spp, Limosilactobacillus spp, Streptococcus spp., Enterococcus spp., Bifidobacterium spp. and Saccharomyces spp.

[0041] In some particular embodiments, the probiotic is a probiotic bacterial strain. In some specific embodiments, it is particularly Bifidobacteria and / or Lactobacilli.

[0042] Suitable probiotic bacterial strains include Lactobacillus rhamnosus ATCC 53103 available from Valio Oy of Finland under the trademark LGG, Lactobacillus rhamnosus CGMCC 1.3724, Lactobacillus paracasei CNCM 1-2116, Lactobacillus johnsonii CNCM I- 1225, Streptococcus salivarius DSM 13084 sold by BLIS Technologies Limited of New Zealand under the designation KI2, Bifidobacterium lactis CNCM 1-3446 sold inter alia by the Christian Hansen company of Denmark under the trademark Bb 12, B. longum CNCM 1-2618 (B. longum NCC2705), Bifidobacterium breve sold by Danisco under the trademark Bb-03, Bifidobacterium breve sold by Morinaga under the trade mark M-16V, Bifidobacterium infantis sold for example by Procter & Gamble Co. under the trademark Bifantis and Bifidobacterium breve sold by Institut Resell (Lallemand) under the trademark R0070, Bifidobacterium longumsubsp. Infantis LMG 11588 (also known as ATCC 17930), B. kashiwanohense (JCM 15439) and / or B. kashiwanohense (DSM 21854).

[0043] The nutritional composition or combination according to the invention may contain from 10e3 to 10el2 cfu of the at least one (further) probiotic strain, more preferably between 10e7 and 10el2 cfu such as between 10e8 and lOelO cfu of probiotic strain per g of composition on a dry weight basis.

[0044] In one embodiment, the probiotics are viable. In another embodiment, the probiotics are non-replicating or inactivated. There may be both viable probiotics and inactivated probiotics in some other embodiments. Probiotic components and metabolites can also be added.

[0045] B. kashiwanohense was isolated from healthy infant faeces. For example, this bacterium has previously been characterized by determining its phenotypic and biochemical features and phylogenetic positions based on partial 16S rRNA gene sequence analysis (Morita et al., International Journal of Systematic and Evolutionary Microbiology, 2011, 61: 2610- 2615). The GenBank / EMBL / DDBJ accession numbers for the 16S rRNA and partial hsp60 gene sequences of two strains of B. kashiwanohense are (i) AB491757 and AB578933 and (ii) are AB425276.2 and AB491759.2, respectively. One strain of B. kashiwanohense is publicly available from two collections with the accession numbers JCM 15439 and DSM 21854 (Morita et al., International Journal of Systematic and Evolutionary Microbiology, 2011, 61: 2610-2615).

[0046] The B. kashiwanohense may be a B. kashiwanohense having at least 99% (suitably, at least 99.9%) Average Nucleotide Identity (ANI) to any B. kashiwanohense known to the skilled person.

[0047] As used herein the term “Average nucleotide identity (ANI)” refers to a distance-based approach to delineate species based on pair-wise comparisons of their genome sequences. ANI is an in silico approach for phylogenetic definition of a species and has become the gold standard for species delineation (Goris et al., 2007, Int. J. Syst. Evol. Microbiol. 57: 81-91; Kim et al., 2014, Int. J. Syst. Evol. Mier. 64: 346-351; Richter et al., 2009, P Natl Acad Sci USA 106: 19126-19131; and Chan et al., 2012, Bmc. Microbiol. 12).

[0048] The ANI of the shared genes between two strains is known to be a robust means to compare genetic relatedness among strains. Strains with ANI values of at least about 96% can be considered to belong to the same species (Konstantinidis and Tiedje, 2005, Proc Natl Acad Sci USA, 102(7):2567-72; and Goris et al., 2007, Int Syst Evol Microbiol. 57(Pt 1): 81-91), while ANI values of at least about 99% indicate that the bacterial genomes belongto the same strain. 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., 2017, Antonie van Leeuwenhoek 110: 1281-1286); ANI Calculator, JSpecies (Richter and Rossello-Mora, 2009, Proc Natl Acad Sci USA 106: 19126- 19131); and JSpeciesWS (Richter et al., 2016, Bioinformatics 32:929-931). Other methods for determining the ANI of two genomes are known in the art (Konstantinidis, K. T. and Tiedje, 2005, J. M., Proc. Natl. Acad. Sci. U.S.A., 102: 2567-2572; and Varghese et al., 2015, Nucleic Acids Research, 43(14):6761-6771).

[0049] Suitably, the B. kashiwanohense has at least 99% (suitably, 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%, at least 99.9%) ANI to 7>. kashiwanohense JCM 15439 and / or B. kashiwanohense DSM 21854. Preferably, the B. kashiwanohense has at least 99.9% ANI to B. kashiwanohense JCM 15439 and / or B. kashiwanohense DSM 21854.

[0050] The term "oligosaccharide" is a saccharide polymer containing a small number (typically three to ten) of simple sugars (monosaccharides). Oligosaccharide as used herein refers to a carbohydrate having a degree of polymerisation (DP) ranging from 2 to 20 inclusive. "Degree of polymerisation" or "DP" refers to the total number of saccharide units in an oligosaccharide chain or polysaccharide chain.

[0051] The term "galacto-oligosaccharide" as used herein refers to a non-digestible oligosaccharide comprising two or more galactose molecules. The galacto-oligosaccharides (GOS) used in embodiments disclosed herein have a DP of 2 to 20, preferably a DP of 2 to 10. Preferably at least 30% of the saccharide units are galactose units, preferably at least 50%, more preferably at least 60%, based on monomeric subunits.

[0052] Suitable galacto-oligosaccharides are commercially available, and include for example Purimune GOS (from ComProducts International), King GOS (from King Prebiotics), Vivinal GOS (from Friesland Campina). Other suppliers of oligosaccharides include Clasado, Ingredion, Leprino, Yakult, Dextra Laboratories, Sigma-Aldrich Chemie GmbH and Kyowa Hakko Kogyo Co., Ltd.

[0053] Galacto-oligosaccharides (GOS) are a prebiotic, and optionally the infant formula further comprises an additional prebiotic.

[0054] Galacto-oligosaccharides (GOS) as used herein typically consist of P-linked galactose moieties with galactose or glucose at the reducing end. Such GOS contains P-(l — 2), P-(l— >3), P-(l— >4), or P-(l— >6) linked galactose moieties and may have a degree ofpolymerization (DP) of 3-8 galactose units. The term GOS is therefore preferably referred to as oligosaccharide(s) comprising at least three galactose units, more preferably as oligosaccharide(s) comprising at least four galactose units, preferably having a degree of polymerization (DP) of 3-8 galactose units.

[0055] Galacto-oligosaccharides (GOS) are defined as polymers of galactose (minimum 2 galactose monomers) with a terminal P-linked galactose or glucose monomer. GOS is a commonly-added prebiotic substrate in infant formula compositions, for example as commercially available in the form of Vivinal-GOS (sold by Friesland Campina of the Netherlands), or in the form of Bovine Milk-derived Oligosaccharides (BMOS) (Estominos et al., Am J Clin Nutr., 2022,115: 142-153). BMOS in particular promote the selective growth Bifidobacterium species such as Bifidobacterium longum subsp. infantis (Roberfroid et al., 2010, British Journal of Nutrition, 104(S2): S1-S63).

[0056] In some embodiments, the galactose source is selected from galactooligosaccharides (GOS), Bovine Milk-derived Oligosaccharides (BMOS) and combinations thereof.

[0057] In a particular embodiment, the nutritional composition according to the invention can comprise BMOS. BMOS may be oligosaccharide preparations derived from bovine milk and / or whey fractions. One route to increasing oligosaccharides in bovine milk derived fractions is to transform part of the lactose in such fractions to GOS. BMOS can typically be obtained from concentrating whey permeate to obtain a concentrated bovine milk oligosaccharide composition and either adding GOS or generating the GOS in situ from hydrolysis of lactose by the action of a P-galactosidase (Duncan et al., Nutrients, 2020, 12: 2007).

[0058] In a particular embodiment, the nutritional composition comprises an oligosaccharide mixture (“BMOS”) that comprises from 0.1 to 4.0 wt% of N-acetylated oligosaccharide(s), from 92.0 to 98.5 wt% of the galacto-oligosaccharide(s) and from 0.1 to 4.0 wt% of the sialylated oligosaccharide(s).

[0059] A combination of prebiotics may be used such as 90% GOS with 10% short chain fructo-oligosaccharides such as the product sold under the trade mark Raftilose® or 10% inulin such as the product sold under the trade mark Raftiline®. Other examples of optional additional prebiotics that can be used in the infant formula include sialo-oligosaccharides (SOS), fructo-oligosaccharides (FOS), human milk oligosaccharides (HMO), isomaltooligosaccharides (IMO), xylo-oligosaccharides (XOS), arabino-xylo oligosaccharides (AXOS), mannan oligosaccharides (MOS), oligosaccharides of soy, glycosyl sucrose (GS),lactosucrose (LS), sialyl-lactose (SL), Fucosyl-lactose (FL), Lacto-N-Neotetraose (LNNT), lactulose (LA), palatinose-oligosaccharides (PAO), malto-oligosaccharides, gums and / or hydrolysates thereof, pectins, starches, and / or hydrolysates thereof.

[0060] Human milk oligosaccharides are carbohydrates resistant to enzymatic hydrolysis by digestive enzymes (e.g. pancreatic and / or brush border), indicating that they may display functions not directly related to their caloric value. It has especially been illustrated that 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 terminal positions at the non-reducing ends. The HMOs can be acidic (e.g. charged sialic acid containing oligosaccharide) or neutral (e.g. fucosylated oligosaccharide). Some examples of HMOs are the fucosylated oligosaccharides, the N-acetylated oligosaccharides and / or the sialylated oligosaccharides.

[0061] A "fucosylated oligosaccharide" is an oligosaccharide having a fucose residue. It has a neutral nature. Some examples are LNFP-I (lacto-N-fucopentaose I), 2’ -FL (2' fucosyllactose), 3-FL (3-fucosyllactose).

[0062] The expressions “fucosylated oligosaccharides comprising an alpha-1, 2- fucosyl-epitope” and “2-fucosylated oligosaccharides” encompass fucosylated oligosaccharides with a certain homology of form since they contain an alpha- 1,2-fucosyl- epitope, therefore a certain homology of function can be expected.

[0063] 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) 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.

[0064] A "sialylated oligosaccharide" is a charged sialic acid containing oligosaccharide, i.e. an oligosaccharide having a sialic acid residue. It has an acidic nature. Some examples are 3’-SL (3’-sialyllactose) and 6’-SL (6’-sialyllactose). The expressions"sialylated oligosaccharide" and "sialyllactose (SL)" can be used interchangeably. The trisaccharide sialyllactose consists of lactose at the reducing terminus and one sialic acid residue at the non-reducing end via an alpha-2,3 binding or alpha-2,6 binding, resulting in 3'- SL and 6'-SL, respectively.

[0065] A "precursor of HMO" is a key compound that intervenes in the manufacture of HMO, such as sialic acid and / or fucose.

[0066] Because of the configuration of their glycosidic bonds, galacto- oligosaccharides (GOS) largely resist hydrolysis by salivary and intestinal digestive enzymes. GOS are classified as prebiotics, non-digestible carbohydrates that beneficially affect the host by stimulating the growth and / or activity of beneficial bacteria in the colon.

[0067] 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. Suitably, the present composition may be a probiotic composition.

[0068] The term “cfu” should be understood as colony forming unit.

[0069] The “gut microbiota” is the composition of microorganisms (including bacteria, archaea and fungi) that live in the digestive tract.

[0070] 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).

[0071] The term "SCFA" means short chain fatty acid(s).

[0072] The expression "increasing propionate production" means that the amount of systemic and / or colonic propionate is higher in an individual fed with the nutritional composition according to the present invention (which comprises a combination of GOS and at least three ingredients selected from vitamins Bl, B2 and B6; minerals zinc, iron and copper; and amino acids histidine, isoleucine, lysine, and leucine) in comparison with a standard composition (i.e. a nutritional composition not comprising a combination of GOS and at least three ingredients selected from vitamins Bl, B2 and B6; minerals zinc, iron and copper; and amino acids histidine, isoleucine, lysine, and leucine) and / or in comparison with a standardcomposition supplemented with common fibers like polydextrose or pectin. The propionate production may be measured by techniques known by the skilled person such as by Gas-Liquid Chromatography .

[0073] Embodiments

[0074] An embodiment disclosed herein is a composition for use in promoting sleep in a young individual comprising a combination of at least one galacto-oligosaccharide (GOS) and at least three ingredients selected from vitamin Bl, vitamin B2, vitamin B6, zinc, iron, copper, histidine, isoleucine, lysine, leucine.

[0075] Preferably the composition for use is orally administered in an amount effective to increase propionate in the infant or toddler.

[0076] In an embodiment, the composition for use is administered to a young individual of about 2 to about 4 months of age.

[0077] In an embodiment, the composition for use comprises at least one galactooligosaccharide (GOS) in a daily amount ranging from about 0.001 to about 12 g and at least three ingredients selected from- vitamin B 1 in a daily amount ranging from about 0.8 to about 2 mg;- vitamin B2 in a daily amount ranging from about 1 to about 3 mg;- vitamin B6 in a daily amount ranging from about 0.9 to about 5 mg; zinc in a daily amount ranging from about 6 to about 13 mg; iron in a daily amount ranging from about 8 to about 29 mg; copper in a daily amount ranging from about 0.6 to about 1.1 mg; histidine in a daily amount ranging from about 0.2 to about 1 g; isoleucine in a daily amount ranging from about 0.3 to about 2 g; lysine in a daily amount ranging from about 0.4 to about 3 g; leucine in a daily amount ranging from about 0.6 to about 4 g;In an embodiment, the composition comprises at least one galacto-oligosaccharide (GOS) in an amount ranging from about 0.001 to about 2.5 g per 100 ml of liquid and at least three ingredients selected from vitamin Bl in an amount ranging from about 0.2 to about 0.4 mg per 100 ml of liquid; vitamin B2 in an amount ranging from about 0.2 to about 0.6 mg per 100 ml of liquid;- vitamin B6 in an amount ranging from about 0.2 to about 1 mg per 100 ml of liquid; zinc in an amount ranging from about 1 to about 2.5 mg per 100 ml of liquid;iron in an amount ranging from about 2 to about 6 mg per 100 ml of liquid; copper in an amount ranging from about 0.1 to about 0.2 mg per 100 ml of liquid; histidine in an amount ranging from about 0.04 to about 0.2 g per 100 ml of liquid; isoleucine in an amount ranging from about 0.06 to about 0.4 g per 100 ml of liquid; lysine in an amount ranging from about 0.08 to about 0.6 g per 100 ml of liquid; leucine in an amount ranging from about 0.1 to about 0.8 g per 100 ml of liquid.In an embodiment, the composition comprises at least one galacto-oligosaccharide (GOS) in an amount ranging from about 0.001 to about 2.5 g per 100 ml of liquid and at least three ingredients selected from vitamin Bl in an amount ranging from about 0.1 to about 0.4 mg per 100 ml of liquid; vitamin B2 in an amount ranging from about 0.1 to about 0.6 mg per 100 ml of liquid;- vitamin B6 in an amount ranging from about 0.1 to about 1 mg per 100 ml of liquid; zinc in an amount ranging from about 0.5 to about 2.5 mg per 100 ml of liquid; iron in an amount ranging from about 1 to about 6 mg per 100 ml of liquid; copper in an amount ranging from about 0.05 to about 0.2 mg per 100 ml of liquid; histidine in an amount ranging from about 0.02 to about 0.2 g per 100 ml of liquid; isoleucine in an amount ranging from about 0.06 to about 0.4 g per 100 ml of liquid; lysine in an amount ranging from about 0.08 to about 0.6 g per 100 ml of liquid; leucine in an amount ranging from about 0.1 to about 0.8 g per 100 ml of liquid.In a preferred embodiment, the composition comprises at least one galacto-oligosaccharide(GOS) in an amount ranging from about 0.001 to about 2.5 g per 100 ml of liquid and at least three ingredients selected from vitamin Bl in an amount ranging from about 0.1 to about 0.2 mg per 100 ml of liquid; vitamin B2 in an amount ranging from about 0.1 to about 0.2 mg per 100 ml of liquid; vitamin B6 in an amount ranging from about 0.1 to about 0.2 mg per 100 ml of liquid; zinc in an amount ranging from about 0.5 to about 1 mg per 100 ml of liquid; iron in an amount ranging from about 1 to about 2 mg per 100 ml of liquid;copper in an amount ranging from about 0.05 to about 0.1 mg per 100 ml of liquid; histidine in an amount ranging from about 0.02 to about 0.04 g per 100 ml of liquid; isoleucine in an amount ranging from about 0.06 to about 0.4 g per 100 ml of liquid; lysine in an amount ranging from about 0.08 to about 0.6 g per 100 ml of liquid; leucine in an amount ranging from about 0.1 to about 0.8 g per 100 ml of liquid.In another preferred embodiment, the composition comprises at least one galactooligosaccharide (GOS) in an amount of about 0.25 g per 100 ml of liquid and at least three ingredients selected from vitamin Bl in an amount of about 0.15 mg per 100 ml of liquid; vitamin B2 in an amount of about 0.18 mg per 100 ml of liquid; vitamin B6 in an amount of about 0.16 mg per 100 ml of liquid; zinc in an amount of about 0.94 mg per 100 ml of liquid; iron in an amount of about 1.46 mg per 100 ml of liquid; copper in an amount of about 0.1 mg per 100 ml of liquid; histidine in an amount of about 0.03 g per 100 ml of liquid; isoleucine in an amount of about 0.088 per 100 ml of liquid; lysine in an amount of about 0.088 g per 100 ml of liquid; leucine in an amount of about 0.16 g per 100 ml of liquid.In an embodiment, the composition for use comprises at least one galactooligosaccharide (GOS) in a daily amount ranging from about 0.001 to about 12 g and four ingredients, or five ingredients, or six ingredients, or seven ingredients, or eight ingredients, or nine ingredients, or ten ingredients, selected from- vitamin B 1 in a daily amount ranging from about 0.8 to about 2 mg;- vitamin B2 in a daily amount ranging from about 1 to about 3 mg;- vitamin B6 in a daily amount ranging from about 0.9 to about 5 mg; zinc in a daily amount ranging from about 6 to about 13 mg; iron in a daily amount ranging from about 8 to about 29 mg; copper in a daily amount ranging from about 0.6 to about 1.1 mg; histidine in a daily amount ranging from about 0.2 to about 1 g; isoleucine in a daily amount ranging from about 0.3 to about 2 g; lysine in a daily amount ranging from about 0.4 to about 3 g; leucine in a daily amount ranging from about 0.6 to about 4 g;

[0078] In an embodiment, the composition for use comprises at least one galactooligosaccharide (GOS) in an amount ranging from about 0.001 to about 2.5 g per 100 ml of liquid to and at least four ingredients, or at least five ingredients, or at least six ingredients, or at or least seven ingredients, at or least eight ingredients, or at least nine ingredients, or ten ingredients selected from vitamin Bl in an amount ranging from about 0.2 to about 0.4 mg per 100 ml of liquid; vitamin B2 in an amount ranging from about 0.2 to about 0.6 mg per 100 ml of liquid;- vitamin B6 in an amount ranging from about 0.2 to about 1 mg per 100 ml of liquid; zinc in an amount ranging from about 1 to about 2.5 mg per 100 ml of liquid; iron in an amount ranging from about 2 to about 6 mg per 100 ml of liquid; copper in an amount ranging from about 0.1 to about 0.2 mg per 100 ml of liquid; histidine in an amount ranging from about 0.04 to about 0.2 g per 100 ml of liquid; isoleucine in an amount ranging from about 0.06 to about 0.4 g per 100 ml of liquid; lysine in an amount ranging from about 0.08 to about 0.6 g per 100 ml of liquid; leucine in an amount ranging from about 0.1 to about 0.8 g per 100 ml of liquid.

[0079] Where a nutrient may be comprised in a composition under different forms (as such or in the form of salts, complexes or more complex structures comprising the nutrient) the amounts reported hereafter are to be intended to refer to the amount of the nutrient as such.

[0080] In an embodiment, the composition may comprise the ingredients in the following amounts:

[0081] Vitamin Bl in a daily amount of at least about 0.8 mg, preferably in a daily amount ranging from about 0.8 to about 2 mg, or in an amount of at least about 0.2 mg per 100 ml of liquid, preferably in an amount ranging from about 0.2 to about 0.4 mg per 100 ml of liquid, as thiamin, thiamin pyrophosphate, TPP, thiamin triphosphate, TTP, thiamin hydrochloride, thiamin mononitrate.

[0082] Vitamin B2 in a daily amount of at least about 1 mg, preferably in a daily amount ranging from about 1 to about 3 mg, or in an amount of at least about 0.2 mg per 100 ml of liquid, preferably in an amount ranging from about 0.2 to about 0.6 mg per 100 ml of liquid, as riboflavin, flavin mononucleotide, FMN, flavin adenine dinucleotide, FAD, lactoflavin, ovoflavin.

[0083] Vitamin B6 in a daily amount of at least about 0.9 mg, preferably in a daily amount ranging from about 0.9 to about 5 mg, or in an amount of at least about 0.2 mg per 100ml of liquid, preferably in an amount ranging from about 0.2 to about 1 mg per 100 ml of liquid, as pyridoxine, pyridoxal, pyridoxamine, pyridoxine hydrochloride.

[0084] Zinc may be incorporated in the composition of the invention in a daily amount of at least 6 mg, preferably in a daily amount ranging from about 6 to about 7.5 mg, or in an amount of at least about 1 mg per 100 ml of liquid, preferably in an amount ranging from about 1 to about 2 mg per 100 ml of liquid, in the form of a physiologically acceptable salt such as, for example: zinc nitrate, zinc sulfate, zinc gluconate, zinc acetate or mixtures thereof, or in the form of a physiologically acceptable zinc complex (such as for example zinc picolinate) or mixtures thereof.

[0085] Iron may be incorporated in the composition of the invention in a daily amount of at least 8 mg, preferably in a daily amount ranging from about 8 to about 29 mg, or in an amount of at least about 2 mg per 100 ml of liquid, preferably in an amount ranging from about 2 to about 6 mg per 100 ml of liquid, either in free form, or in the form of a physiologically acceptable salt such as, for example: ferric citrate, ferric phosphate, ferric pyrophosphate, ferrous ascorbate, ferrous carbonate, ferrous citrate, ferrous fumarate, ferrous gluconate, ferrous lactate, ferrous sulfate or mixtures thereof or in the form of one physiologically acceptable iron complex such as for example EDTA ferric sodium salt, and mixtures thereof.

[0086] Copper may be incorporated in the composition of the invention in a daily amount of at least about 0.6 mg, preferably in a daily amount ranging from about 0.6 to about 1 mg, or in an amount of at least about 0.1 mg per 100 ml of liquid, preferably in an amount ranging from about 0.1 to about 0.2 mg per 100 ml of liquid, as such or in the form of a physiologically acceptable salt and / or via any source comprising copper. For example, copper may be incorporated into the composition as: copper sulfate and / or copper gluconate and / or copper carbonate, and / or copper citrate, and / or copper-lysine complex.

[0087] In an embodiment, one or more of the following amino acids are comprised in the following amounts:- histidine in a daily amount of at least about 0.2 g, preferably in a daily amount ranging from about 0.2 to about 1 g, or in an amount of at least about 0.04 g per 100 ml of liquid, preferably in an amount ranging from about 0.04 to about 0.2 g per 100 ml of liquid;- isoleucine in a daily amount of at least about 0.3 g, preferably in a daily amount ranging from about 0.3 to about 2 g, or in an amount of at least about 0.06 g per 100 ml of liquid, preferably in an amount ranging from about 0.06 to about 0.4 g per 100 ml of liquid;- lysine in a daily amount of at least about 0.4 g, preferably in a daily amount ranging from about 0.4 to about 3 g, or in an amount of at least about 0.08 g per 100 ml of liquid, preferably in an amount ranging from about 0.08 to about 0.6 g per 100 ml of liquid;- leucine in a daily amount of at least 0.6 g, preferably in a daily amount ranging from about 0.6 to about 4 g, or in an amount of at least about 0.1 g per 100 ml of liquid, preferably in an amount ranging from about 0.1 to about 0.8 g per 100 ml of liquid;

[0088] In an embodiment, the composition may comprise GOS in a daily amount ranging from about 0.001 to about 12 g, or in an amount ranging from about 0.001 to about 2.5 g per 100 ml of liquid, up to 3.5 g per 100 ml of liquid.

[0089] In an embodiment, the composition may comprise GOS in a daily amount of at least 1 or 2 g, preferably at least 3 g, likewise preferably between 2 to 12 g, or between 2 to 10 g, between 2 to 8 g, or between 2 to 7 g, more preferably between 3 to 12 g, likewise more preferably between 3 and 10 g, such as between 3 to 8 g, between 3 to 7 g, between 3 to 6 g, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 g, or any range formed thereby, or per serving.

[0090] In a particular embodiment, the GOS is provided in the combination or nutritional composition of the present invention in a daily amount of 0.003-3.9 g, preferably 0.006-3 g, 0.05-2.5 g, 0.1-2 g or 0.2- 1.5g, for example 0.36 to 1.22 g, or per serving. Suitably, the GOS is provided in the combination or nutritional composition of the present invention in a daily amount of 0.001, 0.005, 0.01, 0.005, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5 or 4 g, or any range formed thereby, or per serving.

[0091] Minerals such as but not limited to zinc, iron, copper, and combinations thereof are usually added to the compositions of the invention in salt form.

[0092] In an embodiment, the amino acids, including histidine, isoleucine, lysine and leucine, are provided by a protein source selected from the list consisting of: free amino acids or salts thereof, oligopeptides, peptides, proteins such as dairy, animal or plant proteins; and any combinations thereof.

[0093] The term “plant-protein” refers to an ingredient of plant origin that can serve as a dietary source of protein. Plant proteins belong to the family of legumes. These ingredients are derived from (for example, but not by way of limitation) pulses or oil seeds, which are used as a starting material for the production of (for example, but not by way of limitation) flours, protein concentrates, or isolates.

[0094] The term “pulse” as used herein will be understood to refer to dried edible seeds of certain plants in the legume family. Pulses are high in dietary fiber, high in protein, rich in micronutrients, and low in fat. Examples of pulses include, but are not limited to:chickpeas; lentils; dry beans (such as, but not limited to, carob, kidney, haricot, lima, butter, adzuki, mungo, golden, green gram, black gram, urd, scarlet runner, rice, moth, and tepary beans); faba beans; dry broad beans; dry peas; dry cow peas; pigeon peas; Bambara beans; vetches; lupins; and pulses nes (such as, but not limited to, lablab or hyacinth beans, jack or sword beans, winged beans, guar beans, velvet beans, and yam beans); and any mixtures or combinations thereof. Pulses can be processed into pulse flours, pulse semolinas, and pulse brans and / or separated into pulse proteins, pulse fibers, and pulse starches.

[0095] In a preferred embodiment the protein source has an optimized aminogram, for example enrichment in amino acids, an adapted protein profile, for example by mixing with casein, and / or adapted peptide profile for metabolic accessibility by Bifidobacterium species, for example by partial hydrolysis.

[0096] As used herein, the term “protein source” refers to a source of amino acids. Thus, the protein source comprises amino acids. Suitably, the protein source may comprise amino acids (e.g. free amino acids) or a salt thereof, oligopeptides, peptides, proteins, amino acid precursors or any combination thereof. Preferably, the protein source may comprise oligopeptides, peptides, proteins, or any combination thereof. More preferably, the protein source may comprise partially hydrolysed or hydrolysed oligopeptides, peptides, proteins, or any combination thereof.

[0097] As used herein, the term “free amino acids” may refer to amino acid monomers, which are not part of an oligopeptide, peptide, or protein. Amino acid salts may include any physiologically acceptable salt, such as hydrochloride, sodium, potassium, calcium, and magnesium salts. Preferably, the salt is a sodium or potassium salt. Preferably, the free amino acids are amino acid monomers, such as histidine, isoleucine, lysine, leucine, tryptophan, tyrosine, phenylalanine or any combination thereof. Most preferably, the free amino acid is histidine, isoleucine, lysine, leucine monomer.

[0098] As used herein, the term “oligopeptides” may refer to short chains of amino acid monomers (e.g. 2 to 20 amino acid monomers) linked via peptide bonds and can include dipeptides, tripeptides, tetrapeptides, and pentapeptides. The oligopeptides may be enriched for one or more amino acids or consist solely of a single type of amino acid. Suitably, the oligopeptides may be enriched for one or more amino acids or consist solely of a single type of amino acid. Preferably, the amino acids are amino acid monomers, such as histidine, isoleucine, lysine, leucine, tryptophan, tyrosine, phenylalanine or any combination thereof. Most preferably, the amino acid is histidine, isoleucine, lysine, leucine monomer.

[0099] As used herein, the term “peptides” may refer to short chains of amino acid monomers (e.g. 20 to 50 amino acid monomers) linked via peptide bonds. The peptides may be enriched for one or more amino acids or consist solely of a single type of amino acid. Suitably, the peptides may be enriched for one or more amino acids or consist solely of a single type of amino acid. Preferably, the amino acids are amino acid monomers, such as histidine, isoleucine, lysine, leucine, tryptophan, tyrosine, phenylalanine or any combination thereof. Most preferably, the amino acid is histidine, isoleucine, lysine, leucine monomer.

[0100] Suitably, the protein source has an optimized aminogram, i.e. the amino acid profile of the protein source has been changed to the desired profile. For example, the protein source may be enriched in amino acids. By way of further example, the protein source may consist of amino acids. Preferably, the amino acids are amino acid monomers, such as histidine, isoleucine, lysine, leucine, tryptophan, tyrosine, phenylalanine or any combination thereof. Most preferably, the free amino acid is histidine, isoleucine, lysine, leucine monomer.

[0101] Suitably, the protein source has an adapted protein profile and / or an adapted peptide profile for metabolic accessibility by Bifidobacterium species. Thus, the protein source may have a structure or composition which avoids digestion of the amino acids by the subject and promotes availability of the amino acids for fermentation by Bifidobacterium species in the large intestine and / or colon of the subject.

[0102] In some embodiments, the protein source has an adapted protein profile.

[0103] In some embodiments, the protein source has an adapted peptide profile.

[0104] The term “adapted protein profile” may refer to the adaptation of the components provided in the protein source, i.e. to adapting the particular oligopeptide, peptide and / or protein components provided in the protein source. The term “adapted peptide profile” may refer to the adaptation of the size distribution of the oligopeptides, peptides and proteins. For example, the peptide profile of the protein source may be adapted by partial hydrolysis and / or the protein profile of the protein source may be adapted by mixing with casein. The present inventors have surprisingly found that partial hydrolysis of the protein source increases availability of the amino acids for fermentation by Bifidobacterium species (see Example). It is known that embedding factors, for example transforming growth factor beta (TGF-P), within casein protects TGF-P from digestion by the subject (e.g. by the upper gastrointestinal tract of the subject) such that TGF-P can exert its effects in the large intestine. Hence, embedding amino acids within casein would be expected to avoid digestion of the amino acids by the subject and promote availability of the amino acids for fermentation by Bifidobacterium species in the large intestine and / or colon of the subject.

[0105] In some embodiments, the protein source has an optimised aminogram and an adapted protein profile.

[0106] In some embodiments, the protein source has an optimised aminogram and an adapted peptide profile.

[0107] In some embodiments, the protein source has an optimised aminogram, and an adapted protein profile and an adapted peptide profile.

[0108] The protein can be in an amount of from 1.4 to 3 g per 100 kcal. In some embodiments, especially when the composition is intended for premature infants, the protein amount can be between 2.4 and 4 g / lOOkcal or more than 3.6 g / lOOkcal. In some other embodiments, the protein amount can be below 2.0 g per 100 kcal, e.g. between 1.8 to 2 g / 100 kcal, or in an amount below 1.8 g per 100 kcal, such as between 1.4 to 1.8 g protein / lOOkcal.

[0109] Degree of hydrolysis of the protein source

[0110] The proteins may be intact or hydrolysed or a mixture of intact and hydrolysed proteins. By the term “intact” is meant 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.

[0111] The term “hydrolysed” means in the context of the present invention an oligopeptide, peptide or protein which has been hydrolysed or broken down into its component amino acids. The oligopeptides, peptides or proteins may be either fully or partially hydrolysed. It may be desirable to supply partially hydrolysed oligopeptides, peptides or proteins (degree of hydrolysis between 2 and 20%), for example for infants or young children believed to be at risk of developing cow’s milk allergy. If hydrolysed oligopeptides, peptides or 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.

[0112] In an embodiment of the invention, at least 70% of the oligopeptides, peptides or proteins are hydrolysed, preferably at least 80% of the oligopeptides, peptides or proteins are hydrolysed, such as at least 85% of the oligopeptides, peptides or proteins are hydrolysed, even more preferably at least 90% of the oligopeptides, peptides or proteins are hydrolysed, such as at least 95% of the oligopeptides, peptides or proteins are hydrolysed,particularly at least 98% of the oligopeptides, peptides or proteins are hydrolysed. In a particular embodiment, 100% of the oligopeptides, peptides or proteins are hydrolysed.

[0113] Accordingly, suitable protein sources for use according to the invention include an intact protein source and a partially hydrolysed protein source.

[0114] Protein sources for use according to the invention may be animal milk, prepared from an animal milk or an animal milk fraction comprising free amino acids, oligopeptides, peptides or proteins.

[0115] Protein sources based on whey, casein and mixtures thereof may be used. 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. Protein sources of bovine, buffalo, goat and sheep origin, or mixtures thereof, may be used. For example, milk protein sources originating from different species may be mixed to provide the desired caseimwhey ratio. By way of further example, milk protein sources originating from different species may be mixed to provide the desired aminogram and / or bifidobacteria accessible peptide profile. Suitably, the desired aminogram may be one enriched in amino acids. Preferably, the amino acids are amino acid monomers, such as histidine, isoleucine, lysine, leucine, tryptophan, tyrosine, phenylalanine or any combination thereof. Most preferably, the amino acid is histidine, isoleucine, lysine, leucine monomer.

[0116] The milk can be used as such (i.e. powder or liquid) as an ingredient, or these milk sources can be split into casein and whey according to the required application.

[0117] Caseins in the form of caseinates (Na, K, Ca), or in the form of micellar caseins originating from microfiltration processes may be used. Any suitable techniques which are known in the art for the preparation of such caseinates or micellar caseins may be used (see, for example, Carter et al., J Dairy Sci., 2021, 104 :2465-2479).

[0118] Whey proteins are typically obtained through processing of an animal milk. For example, sweet whey may be obtained by rennet coagulation of milk, acid whey may be obtained by acid precipitation from milk, and native whey may be obtained by microfiltration of milk. Via various unit operations (such as nanofiltration, ultrafiltration, electrodialysis, ion exchange and any combination thereof), a wide range of whey ingredients can be obtained with various demineralisation degrees, protein contents and nutritional qualities in respect to amino acid profiles as desired.

[0119] Whey proteins in the form of sweet whey, acid whey and native whey, or any derivatives thereof obtained after processing as described herein, may be used.

[0120] Suitably, the sweet whey material for use according to the invention can be one of sweet whey obtained after separation of casein coagulated with rennet, a concentrate of sweet whey, a demineralized sweet whey, a demineralized concentrate of sweet whey, a concentrate of proteins of substantially lactose-free sweet whey obtained by ultrafiltration followed by diafiltration (ultrafiltration with washing), mother liquors of the crystallization of lactose from sweet whey, a permeate of ultrafiltration of a sweet whey, the product of hydrolysis - by a protease - of a native casein obtained by acid precipitation of skimmed milk with an inorganic acid or by biological acidification, obtained by microfiltration of a skimmed milk, or the product of hydrolysis of a caseinate by a protease. Preferably, the sweet whey has a solid content of about 6 to 30 wt. %. Suitably, the sweet whey has a solid content of about 6 to 30 wt. % after its decationisation. Suitably, sweet whey or a sweet whey protein concentrate may be further demineralized by electrodialysis, ion exchange, reverse osmosis, electrodeionisation or a combination of these procedures or other demineralization procedures known in the field (e.g. ultra- and / or nanofiltration).

[0121] Suitably, the range of the protein content in the sweet whey for use according to the invention is between 5 and 90 wt. %, such as between 10 and 80 wt.%, or between 20 and 70 wt. %, or between 30 and 60 wt. %, or between 40 and 50 wt. %, such as 11.5 wt.%.

[0122] Suitably, the acid whey material for use according to the invention can be one of acid whey obtained after separation of casein coagulated with acid, a concentrate of acid whey, a demineralized acid whey, a demineralized concentrate of acid whey, a concentrate of proteins of substantially lactose-free acid whey obtained by ultrafiltration followed by diafiltration (ultrafiltration with washing), mother liquors of the crystallization of lactose from acid whey, a permeate of ultrafiltration of an acid whey, the product of hydrolysis - by a protease - of a native casein obtained by acid precipitation of skimmed milk with an inorganic acid or by biological acidification, obtained by microfiltration of a skimmed milk, or the product of hydrolysis of a caseinate by a protease. Preferably, the acid whey has a solid content of about 6 to 30 wt. %. Suitably, the acid whey has a solid content of about 6 to 30 wt. % after its decationisation. Suitably, acid whey or an acid whey protein concentrate may be further demineralized by electrodialysis, ion exchange, reverse osmosis, electrodeionisation or a combination of these procedures or other demineralization procedures known in the field (e.g. ultra- and / or nanofiltration).

[0123] Suitably, the range of the protein content in the acid whey or native whey for use according to the invention is between 5 and 90 wt. %, such as between 10 and 80 wt.%, orbetween 20 and 70 wt. %, or between 30 and 60 wt. %, or between 40 and 50 wt. %, such as 11.5 wt.%.

[0124] Casein glycomacropeptide or caseinoglycomacropeptide (CGMP) can be extracted from a dairy source such as sweet whey. Any suitable method known in the art for the extraction of CGMP from a dairy source such as sweet whey may be used in the practice of the present invention. For example, the method disclosed in WO2016 / 128254 Al may be used. Examples 1-4 of WO2016 / 128254 Al are thereby incorporated by reference.

[0125] CGMP is a phosphorylated and partially sialylated macropeptide which is formed by the action of a protease, for example rennet, on mammalian milk kappa-casein. CGMP represents about 15 to 20% by weight of the proteins in sweet whey obtained after separation of casein during cheese manufacture.

[0126] A whey material (such as sweet whey) containing CGMP may be subjected to the extraction of CGMP. Suitably, a whey material (such as sweet whey) containing CGMP may be subjected to the extraction of CGMP to provide a protein material having a targeted tryptophan / threonine ratio. The resulting protein material following the extraction of CGMP from the starting whey material may be referred to as CGMP -reduced (acid, native or sweet) whey. Advantageously, the treated liquid material that is obtained from the extraction of CGMP from a whey material (such as sweet whey) has an amino acid profile which is enriched in amino acids such as Tryptophan (Trp) and reduced in Threonine (Thr). Suitably, a CGMP- reduced whey comprises at least an 85 % reduction in the amount of CGMP compared to the starting whey material.

[0127] Suitably, the range of the protein content in the modified sweet whey (CGMP -reduced sweet whey) for use according to the invention is between 5 and 95 wt.%, between 10 and 80 wt.%, or between 20 and 70 wt. %, or between 30 and 60 wt. %, or between 40 and 50 wt. %, preferably 10 wt.%.

[0128] Alpha-lactalbumin enriched whey protein concentrate can be obtained, for example, by partial or complete removal of other major proteins like beta-lactoglobulin from milk. This leads to an increased ratio between alpha-lactalbumin and beta-lactoglobulin compared to the starting material, e.g. milk. Any suitable process for the preparation of alphalactalbumin enriched whey protein concentrate which is known in the art may be used.

[0129] Preferred protein sources for use according to the invention which have an optimised aminogram, an adapted protein profile and / or an adapted peptide profile include: a) skimmed milk and demineralized, casein glycomacropeptide (CGMP)-reduced sweet whey;b) skimmed milk and CGMP-free acid or native whey; c) CGMP-free whey protein isolate and sweet whey protein concentrate; d) demineralized CGMP -reduced sweet whey and demineralized sweet whey; and e) skimmed milk and alpha-lactalbumin-enriched whey protein.

[0130] In some embodiments, the protein source is an intact protein source. Suitably, the intact protein source comprises: a) skimmed milk and demineralized, caseino-glyco-macropeptide (CGMP)-reduced- whey; b) skimmed milk and CGMP-free whey, such as acid or native whey; or c) skimmed milk and alpha-lactalbumin-enriched whey protein.

[0131] Suitably, the CGMP-free whey is demineralized CGMP-free whey, such as demineralized CGMP-free acid or native whey.

[0132] In some embodiments, the protein source is a partially hydrolysed protein source. Suitably, the partially hydrolysed protein source comprises: a) CGMP-free whey protein isolate and demineralized sweet whey protein concentrate; or b) demineralized CGMP -reduced sweet whey and demineralized sweet whey.

[0133] In one embodiment, the protein source comprises or consists of skimmed milk and demineralized CGMP -reduced sweet whey. In one embodiment, the protein source comprises or consists of skimmed milk and CGMP-free whey, such as acid or native whey. Suitably, the protein source comprises or consists of skimmed milk and demineralized CGMP- free whey, such as acid or native whey. In one embodiment, the protein source comprises or consists of CGMP-free whey protein isolate and demineralized sweet whey protein concentrate. In one embodiment, the protein source comprises or consists of demineralized CGMP -reduced sweet whey and demineralized sweet whey. In one embodiment, the protein source comprises or consists of skimmed milk and alpha-lactalbumin-enriched whey protein.

[0134] The casein / whey ratio of the protein source may vary from 80 / 20 to 0 / 100. In some advantageous embodiments, the casein / whey ratio in nutritional compositions comprising an intact protein source is 30 / 70 to 40 / 60. For example, in starter and follow-on formulas, the ratio may be from 40 / 60 to 60 / 40; in growing-up milks the ratio may be from 50 / 50 to 80 / 20. The casein / whey ratio in nutritional compositions comprising a partially hydrolysed protein source (such as pre-term formulas) may be from 20 / 80 to 40 / 60. In some advantageous embodiments, the casein / whey ratio in a nutritional composition comprising a partially hydrolysed protein source is 0 / 100.

[0135] Suitably, the mixture of skimmed milk and demineralized CGMP -reduced sweet whey is provided with a casein / whey ratio from about 20 / 80 to about 60 / 40, preferably of about 20 / 80.

[0136] Suitably, the mixture of skimmed milk and demineralized CGMP -reduced sweet whey comprises 10.1 wt.% of skimmed milk and 16.1 wt.% demineralized CGMP- reduced sweet whey (on dry matter basis). Suitably, the remainder of the mixture is comprised mainly of lactose and lipids.

[0137] Suitably, the mixture of skimmed milk and (demineralized) CGMP -free acid whey comprises 10.1 wt.% of skimmed milk and 7.7 wt.% (demineralized) CGMP -free acid whey (dry matter). Suitably, the remainder of the mixture is comprised mainly of lactose and lipids.

[0138] Suitably, the mixture of skimmed milk and demineralized CGMP -free native whey comprises 10.1 wt.% of skimmed milk and 7.7 wt.% demineralized CGMP -free native whey (dry matter). Suitably, the remainder of the mixture is comprised mainly of lactose and lipids

[0139] Suitably, the mixture of whey protein isolate CGMP -free and sweet whey protein concentrate comprises CGMP -free whey protein isolate: sweet whey protein concentrate at a ratio of 63:37 on protein basis (dry matter).

[0140] Suitably, the mixture of demineralized CGMP -reduced sweet whey and demineralized sweet whey comprises demineralized CGMP -reduced sweet whey: demineralized sweet whey at a ratio of 83 : 17 on protein basis (dry matter).

[0141] In one embodiment, the skimmed milk comprises at least 34 wt.% protein (dry matter), preferably at least 38 wt.% protein, more preferably at least 44 wt.% protein, such as 44 wt.% to 52 wt.% protein, for example 48 wt.% protein (dry matter). In one embodiment, the demineralized CGMP -reduced sweet whey comprises at least 76 wt.% protein (dry matter), preferably at least 80 wt.% protein, more preferably at least 86 wt.% protein, such as 86 wt.% to 94 wt.% protein, for example 90 wt.% protein (dry matter). In one embodiment, the (demineralized) CGMP -free native whey comprises at least 27.5 wt.% protein (dry matter), preferably at least 32 wt.% protein, more preferably at least 38 wt.% protein, such as 38 wt.% to 46 wt.% protein, for example 42 wt.% protein (dry matter). In one embodiment, the CGMP- free whey protein isolate comprises at least 95 wt.% protein (dry matter; such as at least 96 wt.%, at least 97 wt.%, at least 98 wt.% protein (dry matter)) and the sweet whey protein concentrate comprises at least 87 wt.% protein (MSWP87) (dry matter), preferably at least 91 wt.% protein, more preferably at least 95 wt.% protein, such as 95 wt.% to 98 wt.% protein,for example 96 wt.% protein (dry matter). In one embodiment, the demineralized CGMP- reduced sweet whey comprises at least 28 wt.% protein (modified sweet whey protein concentrate 28, MSWP28) and the demineralized sweet whey comprises at least 28 wt.% protein (demineralized sweet whey 28, DWP28) (dry matter). In one embodiment, the alphalactalbumin-enriched whey protein comprises at least 68 wt.% protein (dry matter), preferably at least 72 wt.% protein, more preferably at least 78 wt.% protein, such as 78 wt.% to 86 wt.%, for example 82 wt.% (dry matter).

[0142] Suitable acid whey protein concentrates for use in the invention with 28% protein include aDWP28, sold by Paras of India, acid whey protein concentrate 80 (AWPC 80, LAC 7009) sold by Fonterra of New Zealand. Suitable native whey protein concentrates for use in the invention include native whey 28 (nDWP28) sold by Euroserum of France, native whey protein concentrate 80 (nWPC 80) sold by Leprino of the USA, and native whey protein isolate with 95% protein (Pronative 95) sold by Lactalis of France. Suitable demineralized CGMP-free acid whey protein sources for use in the invention include WPC 80 Lac7009 sold by Fonterra of New Zealand. Suitable demineralized CGMP-free native whey protein sources for use in the invention include Native Whey 28 sold by Euroserum of France. Suitable CGMP- free whey protein isolates for use in the invention include BiPro 9500 sold by Agropur of the USA. Suitable sweet whey protein concentrates for use in the invention include whey protein concentrate 80 (WPC80) sold by Leprino of the USA and whey protein concentrate 87 (WPC87) sold by Milei of Germany or Lacprodan DL8590 sold by Aria Foods of Denmark. Suitable demineralized sweet whey protein for use in the invention include demineralized Whey 90 (DM90) or DWP3 sold by Euroserum of France, as well as demineralized whey protein powder / liquid 28 (DWP28) sold by Euroserum of France, Lactalis of France and Friesland Campina of the Netherlands. Suitable alpha-lactalbumin-enriched whey protein for use in the invention include the Hilmar™ 8800 alpha-lactalbumin enriched whey protein concentrate sold by Hilmar of the USA. Suitable CGMP -reduced sweet whey protein for use according to the invention include modified sweet whey liquid and powder (CGMP reduced more than 85%) sold by Nestle of Switzerland, modified sweet whey powder and liquid 16% protein content sold by Nestle of Switzerland, modified sweet whey powder and liquid 28% protein content MSWP28 sold by Euroserum of France and Leprino of the USA, modified sweet whey protein concentrate with 80% proteins (MSWP 80) sold by Leprino of the USA, and modified sweet whey protein isolate like for example Bipro sold by Agropure of the USA.

[0143] In some preferred embodiments, the protein sources for use in the invention are hydrolyzed at a degree of hydrolysis (DH) of from 7 to 11%, preferably of from 8.3 to 10.8%, as determined using Assay 1 (described below).

[0144] In some preferred embodiments, the protein source for use in the invention are hydrolysed to an average molecular weight of from 941 to 1284 Da (with 3 sigma), preferably to an average molecular weight of 1112 Da. Protein Molecular Weights (MW) are determined by size exclusion chromatography with a specific post-column labelling and a fluorescence detection based on a calibration curve established with known MW of commercial proteins as described below.

[0145] Assay 1

[0146] In protein hydrolysates, the DH is defined as the percentage of peptides bonds cleaved through hydrolysis which is assessed on pure individual protein (Nielsen et al.,2001, J. Food Sci., 66: 642-646). The DH is expressed by equation (1), wherein h represents the number of hydrolyzed peptide bonds in the protein hydrolysate and htot represents the total number of peptide bonds in the protein source material available for hydrolysis.DH (° / ) > nu

[0147] 100

[0148] Equation (1) can be sub-divided into the following subsections:

[0149] Total number of peptide bonds (htot)

[0150] The derivation of htot is described in detail in the publication from Nielsen et al. (supra). Based on these published data for htot a value of 8.8 mmole / g protein is reported for whey protein.

[0151] Number of hydrolysed peptide bonds (h)

[0152] To determine the number of hydrolysed peptide bonds (h), the ratio of free amino nitrogen groups (AN) and total nitrogen content (TN) in the protein source material (ANinitial) is substracted from the ratio AN / TN in the protein hydrolysate (ANfinal) leading to the following equation (2):

[0153] h = ANfinal - ANinitial = % AN / TN (protein hydrolysate) - % AN / TN (protein source material) (2)

[0154] The free amino nitrogen groups (AN) [a- and a- amino groups] are determined by reaction with trinitrobenzenesulfonic acid [TNBS] based on the procedure described by Adler-Nissen (Adler-Nissen, 1979, J Agric Food Chem, 27: 1256-62). This method is a spectrophotometric assay of the chromophore specifically formed by the reactionof TNBS with free amino groups. The reaction takes place in slightly alkaline conditions. The reaction products are measured at an absorbance of 340 nm.

[0155] The total nitrogen content (TN) is determined via the Kjeldahl method (Kjeldahl, J., 1883, Zeitschrift fur analytische Chemie, 22: 366-383).

[0156] Determination of protein molecular weight

[0157] For determination of the hydrolysates molecular weight distribution (MWD), size exclusion chromatography (SEC) with UV detection is used. A complete description of the method used for determination of MWD characterization is described by Johns P.W. et al. and Bourdeau et al. (Johns P.W. et al., 2011, Food Chemistry, 125, 1041-1050; and Bourdeau et al., 2021, Nutrients, 13: 3011).

[0158] In order to obtain MW information for the peptide size distribution, the elution time axis is calibrated (cubic fitting) using the following 17 standard proteins, peptides and free amino acids: (1) serum albumin (bovine, MW -66’354), (2) ovalbumin (chicken, MW -42’750), (3) carbonic anhydrase (bovine, MW -28’964), (4) beta-lactoglobulin (bovine, MW -18’264), (5) alpha-lactalbumin (bovine, MW -14’ 168), (6) ubiquitin (bovine, MW -8’564), (7) insulin (bovine, MW -5’733), (8) vasoactive intestinal peptide (VIP, mouse, MW -3’325), (9) dynorphin A (porcine, MW -2’ 147), (10) substance P (horse, MW -1’347), (11) angiotensin II (human, MW -1’046), (12) MRFA tetrapeptide (MW -607), (13) IPP tripeptide (MW -325), (14) VPP tripeptide (MW -311), (15) IL dipeptide (MW -245), (16) GGG tripeptide (MW -189), (17) AA18 mix (mix of 18 amino acids, average MW -110).

[0159] In some embodiments, the protein source comprises at least one of histidine, isoleucine, lysine, leucine or salts thereof.

[0160] When the amino acids are bound as peptides and / or proteins such as dairy, animal or plant proteins, the skilled person can measure specific amino acid values in methods well known in the art.

[0161] In an embodiment, the young individual is an infant, a toddler, a young child or a child.

[0162] In an embodiment, the combination is orally administered in an amount effective to increase propionate in the young individual.

[0163] In an embodiment, the promoting of sleep is via increase of propionate.

[0164] In an embodiment, the young individual is in need of better sleep.

[0165] In an embodiment, the promoting of sleep comprises increasing sleep quality.

[0166] In an embodiment, the composition is for use in preventing disorders, diseases or conditions later in life associated with lack of sleep in infancy.

[0167] In an embodiment, the disorders, diseases or conditions later in life associated with lack of sleep in infancy include obesity, overweight, anxiety, depression, social-emotional disorders, neurodevelopment disorders, mood disorders, attention disorders.

[0168] In an embodiment, the composition is administered to the young individual in a unit dosage form, wherein the unit dosage form comprises an amount of the combination effective for increasing levels of propionate in the young individual.

[0169] In an embodiment, the unit dosage form is a predetermined amount of a powder comprising the combination, wherein the powder is reconstituted in a diluent to form a nutritional composition in which the combination is orally administered to the young individual.

[0170] In an embodiment, the present invention relates to a method of promoting sleep in a young individual, the method comprising administering to the young individual the composition for use according to the present invention (which comprises a combination of GOS and at least three ingredients selected from vitamins Bl, B2 and B6; minerals zinc, iron and copper; and amino acids histidine, isoleucine, lysine, and leucine). Preferably the combination is orally administered in an amount effective to increase propionate in the young individual.

[0171] In an embodiment, the present invention relates to a method of increasing propionate levels in a young individual, the method comprising administering to the young individual the composition according to the present disclosure (which comprises a combination of GOS and at least three ingredients selected from vitamins Bl, B2 and B6; minerals zinc, iron and copper; and amino acids histidine, isoleucine, lysine, and leucine).

[0172] The young individual may be of any age or state of health, although in particular embodiments the young individual may be susceptible to particular medical conditions or physical states that are treated or prevented directly or indirectly with increased propionate levels or has a medical condition or physical state that are treated or prevented directly or indirectly with increased propionate levels. The skilled person can measure specific propionate levels through plasma or fecal samples, for example, in methods well known in the art. In preferred embodiments, the young individual does not have a gastrointestinal disorder, condition or disease; although other embodiments include a young individual who has a gastrointestinal disorder, condition, or disease.

[0173] The young individual may be known to have a medical condition or physical state that would benefit from increased propionate levels, or the young individual may besuspected of having a medical condition or physical state that would benefit from increased propionate levels. The method may comprise identifying the young individual as having a medical condition or physical state that would benefit from increased propionate levels, and / or the method may comprise identifying the young individual as at risk of a medical condition or physical state that would benefit from increased propionate levels.

[0174] Young individuals particularly in need of better sleep

[0175] The importance of sleep for infants, toddlers, kids, and young individuals. Sleep is an essential component of a child's overall health and well-being, particularly for infants, toddlers, kids, and young individuals. Adequate sleep is crucial for physical development and growth in children. During sleep, the body releases growth hormones that are necessary for the development of bones, muscles, and tissues. Research has shown that sleep- deprived children may experience stunted growth and delayed physical development. Therefore, ensuring that children get enough sleep is crucial for their physical growth and development.

[0176] In addition to physical development, sleep is also important for cognitive development and learning. Sleep plays a critical role in consolidating memories and processing information learned during the day (Stickgold R, Walker MP. Sleep-dependent memory consolidation and reconsolidation. Sleep Med. 2007 Jun;8(4):331-43). Children who do not get enough sleep may have difficulty concentrating, learning, and retaining new information. Furthermore, sleep-deprived children may experience behavioral problems, such as hyperactivity and impulsivity, which can negatively impact their academic performance (Gruber R, Cassoff J, Frenette S, Wiebe S, Carrier J. Impact of sleep extension and restriction on children's emotional lability and impulsivity. Pediatrics. 2012 Nov;130(5):el 155-61). Therefore, ensuring that children get enough sleep is crucial for their cognitive development and academic success (Gruber R, Laviolette R, Deluca P, Monson E, Cornish K, Carrier J. Short sleep duration is associated with poor performance on IQ measures in healthy school-age children. Sleep Med. 2010 Mar;l l(3):289-94).

[0177] Sleep also plays a crucial role in emotional regulation and behavioral control in children. Lack of sleep can lead to increased irritability, emotional instability, and difficulty regulating emotions. This can result in behavioral problems, such as tantrums and aggression, which can negatively impact social interactions and relationships with peers (Owens JA, Weiss MR. Insufficient sleep in adolescents: causes and consequences. Minerva Pediatr. 2017 Aug; 69(4): 326-336). Therefore, ensuring that children get enough sleep is crucial for their emotional well-being and behavioral control. In conclusion, adequate sleep is crucial for theoverall health and well-being of infants, toddlers, kids, and young individuals, and parents and caregivers should prioritize healthy sleep habits to promote optimal growth, development, and success.

[0178] In one embodiment, the use according to the present invention may be non-therapeutic when the composition is for use in a healthy infant, toddler and / or young child.

[0179] Preferably, the composition is orally administered to the young individual in a nutritional composition, such as an infant formula, a starter infant formula, a follow-on or follow-up formula, a baby food, an infant cereal composition, a fortifier such as a human milk fortifier, or a supplement.

[0180] The nutritional composition can be administered (or given or fed) at an age and for a period that depends on the possibilities and needs.

[0181] Since the nutritional composition is also used for prevention purposes (prevention of a later in life health disorder), it can be for example given immediately after birth of the infants. The composition of the invention can also be given during the first week of life of the infant, or during the first 2 weeks of life, or during the first 3 weeks of life, or during the first month of life, or during the first 2 months of life, or during the first 3 months of life, or during the first 4 months of life, or during the first 6 months of life, or during the first 8 months of life, or during the first 10 months of life, or during the first year of life, or during the first two years of life or even more. In some particularly advantageous embodiments of the invention, the nutritional composition is given (or administered) to an infant within the first 4 or 6 months of birth of said infant.

[0182] In some other embodiments, the nutritional composition of the invention is given few days (e.g. 1, 2, 3, 5, 10, 15, 20...), or few weeks (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10...), or few months (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10... ) after birth. This may be especially the case when the infant is premature, but not necessarily.

[0183] In one embodiment the composition of the invention is given to the infant or young child as a supplementary composition to the mother’s milk. In some embodiments the infant or young child receives the mother’s milk during at least the first 2 weeks, first 1, 2, 4, or 6 months. In one embodiment the nutritional composition of the invention is given to the infant or young child after such period of mother’s nutrition, or is given together with such period of mother’s milk nutrition. In another embodiment the composition is given to the infant or young child as the sole or primary nutritional composition during at least one period of time, e.g. after the 1st, 2nd or 4th month of life, during at least 1, 2, 4 or 6 months.

[0184] In a preferred embodiment, the composition is orally administered to the infant or toddler in an infant formula. The infant formula may contain a protein source in an amount up to 4.0 g / lOOkcal, preferably up to 3.0 g / lOOkcal, more preferably up to 2.0 g / lOOkcal, most preferably 1.8 to 2.0 g / lOOkcal. In some embodiments, over 50% by weight of the protein source is whey. In one embodiment, the protein content is between 30% and 80% whey proteins. Protein sources based on whey, casein and mixtures thereof may be used, as well as protein sources based on soy. For whey proteins, the protein source may be based on acid whey or sweet whey or mixtures thereof and may include alpha-lactalbumin and betalactoglobulin in desired proportions.

[0185] The proteins may be intact or hydrolysed or a mixture of intact and hydrolysed proteins. Partially hydrolysed proteins (degree of hydrolysis between 2 and 20%) may be particularly beneficial for infants believed to be at risk of developing cows' milk allergy. If hydrolysed proteins are required, the hydrolysis process may be carried out as desired and as is known in the art. For example, a whey protein hydrolysate 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, the protein suffers much less lysine blockage during the hydrolysis process, which 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.

[0186] The infant formula may contain a carbohydrate source. Any carbohydrate source conventionally found in infant formulae, such as lactose, saccharose, maltodextrin, starch and mixtures thereof, may be used; although the preferred source of carbohydrates is lactose. Preferably the carbohydrate sources contribute between 35% and 65% of the total energy of the formula. In a preferred embodiment, the carbohydrates comprise rice carbohydrates, for example rice carbohydrates in an amount of at least 5% at least 10%, at least 25% or at least 50%, at least 70%, at least 90%, or about 100% of the carbohydrates (w / w), which can bring a substantial benefit in the sleep pattern. The higher the content in rice carbohydrates, the higher the possible sleep improvement.

[0187] The infant formula may contain a source of lipids. The lipid source may be any lipid or fat which is suitable for use in infant formulas. Preferred fat sources include palm olein, high oleic sunflower oil and high oleic safflower oil. The essential fatty acids linoleic and a-linolenic acid may also be added as may small amounts of oils containing high quantities of preformed arachidonic acid and docosahexaenoic acid such as fish oils or microbial oils. In total, the fat content preferably contributew between 30 to 55% of the total energy of theformula. The fat source preferably has a ratio of n-6 to n-3 fatty acids of about 5:1 to about 15 : 1 ; for example about 8 : 1 to about 10: 1.

[0188] The infant formula may optionally contain one or more vitamins additional to the Vitamins Bl, B2 and B6 and / or one or more minerals additional to the zinc, iron and copper. For example, the infant formula may 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 additional minerals and vitamins optionally present in the infant formula include vitamin A, vitamin B12, vitamin E, vitamin K, vitamin C, vitamin D, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorous, iodine, magnesium, manganese, chloride, potassium, sodium, selenium, chromium, molybdenum, taurine, and L-carnitine. Minerals are usually added in salt form. The presence and amounts of specific additional minerals and vitamins will vary depending on the intended infant population.

[0189] If necessary, the infant formula may contain emulsifiers and stabilizers such as soy lecithin, citric acid esters of mono- and di-glycerides, and the like. The infant formula may optionally contain other substances which may have a beneficial effect such as fibres, lactoferrin, nucleotides, nucleosides, and the like. For example, the infant formula may contain a probiotic bacterial strain in an amount of 103to 1012cfu / g infant formula, more preferably 106to 109cfu / g formula.

[0190] The infant formula described above may be prepared in any suitable manner. For example, they may be prepared by blending together the protein, the carbohydrate source, and the fat source in appropriate proportions. If used, the emulsifiers may be included at this point. The vitamins and minerals may be added at this point but are usually added later to avoid thermal degradation. Any lipophilic vitamins, emulsifiers and the like may be dissolved into the fat source prior to blending. Water, preferably water which has been subjected to reverse osmosis, may then be mixed in to form a liquid mixture. The temperature of the water is conveniently about 50°C to about 80°C to aid dispersal of the ingredients. Commercially available liquefiers may be used to form the liquid mixture. The liquid mixture is then homogenised; for example in two stages.

[0191] The liquid mixture may then be thermally treated to reduce bacterial loads, by rapidly heating the liquid mixture to a temperature in the range of about 80°C to about 150°C for about 5 seconds to about 5 minutes, for example. This may be carried out by steam injection, autoclave or by heat exchanger; for example a plate heat exchanger.

[0192] Then, the liquid mixture may be cooled to about 60°C to about 85°C; for example by flash cooling. The liquid mixture may then be again homogenised; for example in two stages at about 10 MPa to about 30 MPa in the first stage and about 2 MPa to about 10 MPa in the second stage. The homogenised mixture may then be further cooled to add any heat sensitive components; such as vitamins and minerals. The pH and solids content of the homogenised mixture are conveniently adjusted at this point.

[0193] The homogenised mixture may be transferred to a suitable drying apparatus such as a spray drier or freeze drier and converted to powder. The powder should have a moisture content of less than about 5% by weight. A probiotic bacterial strain may optionally be added at this stage by dry -mixing. The powder may then be packaged in unit dosage forms, each unit dosage form comprising an amount of the combination of at least one galactooligosaccharide (GOS) and at least three ingredients selected from vitamins Bl, B2 and B6, minerals zinc, iron and copper, and amino acids histidine, isoleucine, lysine, and leucine that is effective to increase propionate in an infant or toddler to whom the unit dosage form is orally administered. The powder may be reconstituted in a diluent, such as water or milk, prior to the administration to the infant or toddler.

[0194] In view of the disclosures herein, an embodiment is a method of promoting sleep in an infant or toddler, the method comprising administering to the infant or toddler a combination of at least one galacto-oligosaccharide (GOS) and at least three ingredients selected from vitamins Bl, B2 and B6, minerals zinc, iron and copper, and amino acids histidine, isoleucine, lysine, and leucine.

[0195] The combination may be orally administered in an amount effective to increase propionate in the infant or toddler.

[0196] The method may comprise identifying the infant or toddler as in need of better sleep.

[0197] The promoting sleep may comprise increasing sleep quality.

[0198] The combination may be administered to the infant or toddler in a unit dosage form, wherein the unit dosage form comprises an amount of the combination effective for increasing levels of propionate in the infant or toddler. The unit dosage form may be a predetermined amount of a powder comprising the combination, and the method comprises reconstituting the powder in a diluent to form a nutritional composition in which the combination is orally administered to the infant or toddler.

[0199] Suitably, the nutritional composition may be intended for administration twice daily. Thus, the nutritional composition may be formulated to provide two servings per day.

[0200] Suitably, the nutritional composition may be provided in a serving size of 31 g or 36 g total dry weight.

[0201] In some embodiments, the composition of the invention may be administered once daily. In other embodiments, the composition of the invention is administered in multiple servings, for example in two servings (unit doses) per day. When the nutritional composition is provided in the form of unit doses (or “servings”) it is particularly useful to define the amount of oligosaccharides and probiotics in terms of the daily dose to be administered to the infant, or young child or child.EXAMPLE

[0202] The following non-limiting example generally illustrates the concepts underlying the embodiments disclosed herein.

[0203] The inventors investigated short-chain fatty acid production (FIG. 1) by infant microbiota exposed to galacto-oligosaccharide (GOS) alone or in combination with a blend (BL) containing Vitamins Bl, B2 and B6; minerals zinc, iron and copper; and amino acids histidine, isoleucine, lysine, and leucine. “GB” is GOS + BL.

[0204] In vitro digestion and fermentationIn vitro fermentation was achieved for 24h in reactor containing:90% of fermentation medium containing microbiota derived from fresh fecal samples, recovered from 6 healthy infants10% of the test ingredients, consisting of- Buffer only- Galacto-oligosaccharides at the final concentration of 4 g / L- Blend (BL) of (g / L) Vitamin Bl (0.001154), Vitamin B2 (0.001751), Vitamin B6 (0.001577), Zinc (0.009360), Iron (0.014576), Copper (0.000950), Histidine (0.367281), Isoleucine (0.882975), Lysine (0.882609) and Leucine (1.594845)- A combination of GOS (4 g / L) and BL (3.76 g / L)

[0205] After 24 hours, fermentation medium was collected for short chain fatty acid analysis.

[0206] Short chain fatty acid concentration determination

[0207] Short chain fatty acids acetate, propionate and butyrate were determined via Gas Chromatography with flame ionization detection (FID), upon diethyl ether extraction. Briefly, 0.5 mL samples were diluted in distilled water (1 :3) and acidified with 0.5 mL 48% sulfuric acid, after which an excess of sodium chloride was added along with 0.2 mL internal standard (2-methylhexanoic acid) and 2 mL diethyl ether. Upon homogenization and subsequent separation of the water and diethyl ether layer, diethyl ether extracts were analyzed on the GC-FID using nitrogen gas as carrier and makeup gas.

[0208] Statistics

[0209] For the statistical evaluation of the treatment effects, a repeated measures ANOVA analysis was performed (based on paired t-testing, thus accounting for fact that values are compared between samples of a given donor). The statistical significance of the potential treatment effects was determined via Benjamini -Hochberg post hoc testing.

[0210] RESULTS

[0211] Exposure of infant microbiota to GOS resulted to a strong increase of acetate and limited increase of propionate levels, while BL had no effect. Combination of GOS with BL (GB) enhanced the production of propionate by microbiota, compared to BL alone but also compared to GOS alone as shown in FIG. 1

[0212] It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims.(Original in Electronic Form)(Original in Electronic Form)(Original in Electronic Form)(Original in Electronic Form)FOR RECEIVING OFFICE USE ONLYFOR INTERNATIONAL BUREAU USE ONLY

Claims

CLAIMS1. A composition for use in promoting sleep in a young individual comprising a combination of at least one galacto-oligosaccharide (GOS) and at least three ingredients selected from vitamin Bl, vitamin B2, vitamin B6, zinc, iron, copper, histidine, isoleucine, lysine, leucine.

2. The composition for use according to claim 1, wherein it comprises at least one galacto-oligosaccharide (GOS) in a daily amount ranging from about 0.001 to about 12 g and at least three ingredients selected from- vitamin Bl in a daily amount ranging from about 0.8 to about 2 mg;- vitamin B2 in a daily amount ranging from about 1 to about 3 mg;- vitamin B6 in a daily amount ranging from about 0.9 to about 5 mg;- zinc in a daily amount ranging from about 6 to about 13 mg;- iron in a daily amount ranging from about 8 to about 29 mg;- copper in a daily amount ranging from about 0.6 to about 1.1 mg;- histidine in a daily amount ranging from about 0.2 to about 1 g;- isoleucine in a daily amount ranging from about 0.3 to about 2 g;- lysine in a daily amount ranging from about 0.4 to about 3 g;- leucine in a daily amount ranging from about 0.6 to about 4 g;3. The composition for use according to claim 1, wherein the young individual is an infant, a toddler, a young child or a child.

4. The composition for use according to any one of the preceding claims, wherein the combination is orally administered in an amount effective to increase propionate in the young individual.

5. The composition for use according to any one of the preceding claims, wherein the promoting of sleep is via increase of propionate.

6. The composition for use according to any one of the preceding claims, wherein the young individual is in need of better sleep.

7. The composition for use according to any one of the preceding claims, wherein the promoting of sleep comprises increasing sleep quality.

8. The composition for use according to any one of the preceding claims for use in preventing disorders, diseases or conditions later in life associated with lack of sleep in infancy.

9. The composition for use according to claim 9, wherein the disorders, diseases or conditions later in life associated with lack of sleep in infancy include obesity, overweight,anxiety, depression, social-emotional disorders, neurodevelopment disorders, mood disorders, attention disorders.

10. The composition for use according to any one of the preceding claims, wherein it is administered to the young individual in a unit dosage form, wherein the unit dosage form comprises an amount of the combination effective for increasing levels of propionate in the young individual.

11. The composition for use according to claim 10, wherein the unit dosage form is a predetermined amount of a powder comprising the combination, wherein the powder is reconstituted in a diluent to form a nutritional composition in which the combination is orally administered to the young individual.

12. The composition for use according to any one of the claims 1 to 11, wherein the amino acids, including histidine, isoleucine, lysine and leucine, are provided by a protein source selected from the list consisting of: free amino acids or salts thereof, oligopeptides, peptides, proteins such as dairy, animal or plant proteins; and any combinations thereof.

13. A method of promoting sleep in a young individual comprising administering the composition for use according to any one of the preceding claims.

14. A method of increasing propionate levels in a young individual comprising administering the composition for use according to any one of claims 1 to 12.