Mixture of HMOS, MOS and b. infantis

Abstract

The present invention provides a combination of a human milk oligosaccharide (HMO) mixture, bovine milk-derived oligosaccharides (MOS) and Bifidobacterium longum subsp. infantis for use in preventing and / or treating constipation and / or improving stool consistency and / or stool frequency in an infant or a young child, wherein the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'- sialyllactose (3SL), and optionally 3-fucosyllactose (3FL) and / or lacto-N-neotetraose (LNnT). The present invention also provides a combination of a human milk oligosaccharide (HMO) mixture and Bifidobacterium longum subsp. infantis, wherein the HMO mixture comprises or consists of 3'-Galactosyllactose (3'GL), 6'-Galactosyllactose (6'GL), 4'-Galactosyllactose (4'GL), 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3FL) and / or lacto-N- neotetraose (LNnT). The present invention further provides a combination of a human milk oligosaccharide (HMO) mixture, milk fat globule membrane (MFGM) and Bifidobacterium longum subsp. infantis, wherein the HMO mixture comprises or consists of 3'- Galactosyllactose (3'GL), 6'-Galactosyllactose (6'GL), 4'-Galactosyllactose (4'GL), 2'- fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3FL) and / or lacto-N-neotetraose (LNnT).

Description

[0001] MIXTURE OF HMOS, MOS AND B. INFANTIS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a combination of a human milk oligosaccharide (HMO) mixture, bovine milk-derived oligosaccharides (MOS) and Bifidobacterium longum subsp. infantis for use in preventing and / or treating constipation and / or improving stool consistency and / or stool frequency in an infant or a young child.

[0004] BACKGROUND TO THE INVENTION

[0005] Infants and young children may suffer from constipation or hard stools, for example due to the immaturity of their gut, the lack of activity at this young age, and undiversified content of their feeding. In particular, compared to breast-fed infants, formula-fed infants typically have less frequent bowel movements and firmer stools that may be difficult to pass. This may lead to discomfort with pain, and in some cases, constipation.

[0006] Prebiotics and / or probiotics may be incorporated into infant formula, fortifiers or other synthetic nutritional compositions and nutritional supplements, to promote health benefits in formula-fed infants. For example, infant formulas containing MOS, HMO blends, or B. infantis are commercially available.

[0007] Oligosaccharides (OS) are non-digestible carbohydrates found in human and bovine milks and are classified as prebiotic fibres. Recent technological advancements have made it possible to produce milk-derived oligosaccharides (MOS) from bovine milk whey permeate for use in infant formulas. MOS can be added to infant formulas to obtain the functional benefits of prebiotic fibres on gut health, particularly on the microbiota profile and intestinal immune defence.

[0008] Human milk oligosaccharides (HMOs) represent the third largest solid component of human milk after lactose and lipids and include fucosylated oligosaccharides, such as 2’-fucosyllactose (2FL), 3-fucosyllactose (3FL) and difucosyllactose (DFL); N-acetylated oligosaccharides, such as lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT); and sialylated oligosaccharides, such as 3’-sialyllactose (3SL) and 6’-sialyllactose (6SL). HMOs may play a crucial role in the development of a balanced intestinal microbiota and provide a competitive growth advantage for bifidobacteria species.

[0009] Bifidobacterium longum subsp. infantis (B. infantis) has been demonstrated to predominate in the gut microbiota of breastfed infants and to benefit the host by accelerating maturation of the immune response, balancing the immune system to suppress inflammation, improving intestinal barrier function, and increasing short-chain fatty acid (SCFA) production.However, some prebiotic fibres produce gases that fertilize the healthy bacteria that live in the lower gut, which may lead to gut discomfort. This can be particularly problematic in infants and young children and lead to crying periods, feeding difficulties, irritability, pain, especially abdominal pain, abdominal cramping, and poor sleep.

[0010] Thus, there is a need for nutritional compositions comprising beneficial synbiotics that can provide improved stool consistency and / or stool frequency without increasing gut discomfort. SUMMARY OF THE INVENTION

[0011] The present inventors have surprisingly found that a combination of a HMO mixture, MOS and Bifidobacterium longum subsp. infantis can improve stool consistency and / or stool frequency in an infant or a young child without increasing gut discomfort.

[0012] A randomized, double-blind, controlled trial was performed to evaluate healthy infants fed a new synbiotic-supplemented formula. Infants were randomized to receive control formula (OF; cow’s milk-based formula) or experimental formula (EF; the same formula with added HMOs, MOS, and B. infantis). Stools were generally softer (1.8 vs. 2.0 at 1.5, 2 and 4mo, p<0.0013) and more frequent (-2 vs. ~1.5 stools / d after 2 mo, p<0.0457) in EF vs. CF-fed infants. Mean Infant Gastrointestinal Symptom Questionnaire (IGSQ) scores were low in both groups at all time points, indicating good gastrointestinal tolerance.

[0013] Accordingly, in a first aspect, the invention provides a combination of a human milk oligosaccharide (HMO) mixture, bovine milk-derived oligosaccharides (MOS) and Bifidobacterium longum subsp. infantis for use in preventing and / or treating constipation and / or improving stool consistency and / or stool frequency in an infant or a young child, wherein the HMO mixture consists of 2'-fucosy I lactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3FL) and / or lacto-N-neotetraose (LNnT).

[0014] In a further aspect, the invention provides a combination of a human milk oligosaccharide (HMO) mixture, bovine milk-derived oligosaccharides (MOS) and Bifidobacterium longum subsp. infantis for use in reducing the incidence and / or severity of constipation in an infant or a young child, wherein the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3FL) and / or lacto-N-neotetraose (LNnT).

[0015] In a further aspect, the invention provides a combination of a human milk oligosaccharide (HMO) mixture, bovine milk-derived oligosaccharides (MOS) and Bifidobacterium longum subsp. infantis for use in reducing stool hardness in an infant or a young child, wherein theHMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyl lactose (3FL) and / or lacto-N-neotetraose (LNnT).

[0016] In a further aspect, the invention provides a combination of a human milk oligosaccharide (HMO) mixture, bovine milk-derived oligosaccharides (MOS) and Bifidobacterium longum subsp. infantis for use in increasing stool frequency in an infant or a young child, wherein the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyl lactose (3FL) and / or lacto-N-neotetraose (LNnT).

[0017] The combination of the invention may prevent and / or treat constipation and / or improve stool consistency and / or stool frequency, reduce the incidence and / or severity of constipation, reduce stool hardness, and / or increase stool frequency without any associated gut discomfort or digestive disorders (e.g. flatulence, bloating, and / or colics).

[0018] The combination may be administered in any suitable manner. Suitably, the combination is administered separately, simultaneously or sequentially. In some embodiments, the combination is administered simultaneously. Suitably, the combination is provided in the form of a nutritional composition. In some embodiments, the nutritional composition is selected from an infant formula, a starter infant formula, a follow-on or follow-up formula, a baby food, an infant cereal composition, a growing-up-milk, a fortifier such as a human milk fortifier, or a supplement. In some embodiments, the nutritional composition is an infant formula.

[0019] In a further aspect, the invention provides a nutritional composition comprising human milk oligosaccharide (HMO) mixture, bovine milk-derived oligosaccharides (MOS) and Bifidobacterium longum subsp. infantis, wherein the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyl lactose (3FL) and / or lacto-N-neotetraose (LNnT). In some embodiments, the nutritional composition is selected from an infant formula, a starter infant formula, a follow-on or follow-up formula, a baby food, an infant cereal composition, a growing-up-milk, a fortifier such as a human milk fortifier, or a supplement. In some embodiments, the nutritional composition is an infant formula.

[0020] The combination or composition may comprise any suitable HMO mixture in any suitable amounts. In some embodiments, the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), 3-fucosyllactose (3FL), and lacto-N-neotetraose (LNnT). In some embodiments, the HMO mixture consists or consists essentially of: i. 20 wt% to 60 wt% of 2FL, preferably 22 wt% to55 wt%; ii. 4 wt% to 30 wt% of LNT, preferably 6 wt% to 20 wt%; iii. 1 wt% to 12 wt % of DFL, preferably 2 wt% to 8 wt%; iv. 7 wt% to 23 wt% of 6SL and 3SL combined, preferably 8 wt% to 22 wt%; v. 10 wt% to 50 wt% of 3FL, preferably 13 wt% to 46 wt% and vi. 3 wt% to 25 wt% of LNnT, preferably 5 wt% to 20 wt%. Suitably, the total HMO concentration is from 0.5 g / L to 10 g / L, preferably from 1 g / L to 5 g / L, or more preferably from 2 g / L to 3 g / L.

[0021] The combination or composition may comprise any suitable MOS in any suitable amounts. Suitably, the total MOS concentration is from 0.5 g / L to 10 g / L, preferably from 2 g / L to 8 g / L, or more preferably from 4 g / L to 5 g / L.

[0022] The combination or composition may comprise any suitable Bifidobacterium longum subsp. infantis in any suitable amounts. Suitably, the Bifidobacterium longum subsp. infantis is Bifidobacterium longum subsp. infantis LMG 11588 or a strain having an Average Nucleotide Identity (ANI) of at least 95%, preferably at least 99%, more preferably at least 99.9% to Bifidobacterium longum subsp. infantis LMG 11588. Suitably, the combination or composition comprises from 103to 1012cfu of Bifidobacterium longum subsp. infantis, preferably from 105to 101° cfu of Bifidobacterium longum subsp. infantis, more preferably from 105to 107cfu of Bifidobacterium longum subsp. infantis per g of combination or composition on a dry weight basis.

[0023] The infant or young child may be at risk and / or in need thereof. In some embodiments, the infant or young child has an age from 0 months to 12 months, preferably from 0 months to 6 months. In some embodiments, the infant or young child is exclusively formula-fed.

[0024] DETAILED DESCRIPTION OF THE INVENTION

[0025] Definitions

[0026] As used herein, the following terms have the following meanings.

[0027] The term "subject" may refer to an infant, young child, child, an infant small for gestational age (SGA) or a preterm infant.

[0028] The term "infant" means a child under the age of 12 months.

[0029] The expression "young child" means a child aged between one and three years, also called toddler.

[0030] The term “child” means a child aged between three and twelve years. Preferably, the term “child” means a child aged between three and six years.A "preterm" or "premature" subject means an infant or young child who was not born at term. Generally it refers to an infant or young child born prior 36 weeks of gestation.

[0031] By the expression "small for gestational age" or "SGA" it is referred to an infant or young child who is smaller in size than normal for their gestational age at birth, most commonly defined as a weight below the 10th percentile for the gestational age. In some embodiments, SGA may be associated with intrauterine growth restriction (IUGR), which refers to a condition in which a foetus is unable to achieve its potential size.

[0032] By the expression “low birth weight”, it should be understood as any body weight under 2500g at birth.

[0033] The expression "nutritional composition" means a composition which nourishes a subject. This nutritional composition is usually to be taken orally or intravenously. It may include a lipid or fat source, a carbohydrate source and / or a protein source. In a particular embodiment the nutritional composition is a ready-to-drink composition such as a ready-to-drink formula. In a particular embodiment, the nutritional composition of the present invention is a "synthetic nutritional composition". The expression "synthetic nutritional composition" means a mixture obtained by chemical and / or biological means, which can be chemically identical to the mixture naturally occurring in mammalian milks (i.e. the synthetic nutritional composition is not breast milk).

[0034] 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).

[0035] The expression "infant formula" encompasses both "starter infant formula" and "follow-up formula" or "follow-on formula".

[0036] A "follow-up formula" or "follow-on formula" is given from the 6th month onwards and includes “growing-up milk”. It constitutes the principal liquid element in the progressively diversified diet of this category of person.

[0037] The expression “growing-up milk” (or “GUM”) refers to a milk-based drink generally with added vitamins and minerals, that is intended for young children or children.The expression "baby food" means a foodstuff intended for particular nutritional use by infants or young children during the first years of life.

[0038] The expression "infant cereal composition" means a foodstuff intended for particular nutritional use by infants or young children during the first years of life.

[0039] The term "fortifier" refers to liquid or solid nutritional compositions suitable for mixing with breast milk or infant formula.

[0040] The expression “weaning period” means the period during which the mother's milk is substituted by other food in the diet of an infant or young child.

[0041] The "mother's milk" should be understood as the breast milk or the colostrum of the mother. An “oligosaccharide” is a saccharide polymer containing a small number (typically three to ten) of simple sugars (monosaccharides).

[0042] The term "HMO" or "HMOs" refers to human milk oligosaccharide(s). These carbohydrates are 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.

[0043] 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).

[0044] 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.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.

[0045] 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.

[0046] A "precursor of HMO" is a key compound that intervenes in the manufacture of HMO, such as sialic acid and / or fucose.

[0047] The term “GOS” as used herein means “Galacto-oligosaccharide". 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-(1— >2), p-(1— >3), p-(1— >4), or p-(1— >6) linked galactose moieties and may have a degree of polymerization (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.

[0048] The nutritional composition of the present invention can be in solid form (e.g. powder) or in liquid form. The amount of the various ingredients (e.g. the oligosaccharides) can be expressed in g / 100g of composition on a dry weight basis when it is in a solid form, e.g. a powder, or as a concentration in g / L of the composition when it refers to a liquid form (this latter also encompasses liquid composition that may be obtained from a powder after reconstitution in a liquid such as milk, water, e.g. a reconstituted infant formula or a follow-on / follow-up formula or a growing-up milk or an infant cereal product or any other formulation designed for infant nutrition).

[0049] The expressions “infants / young children fed exclusively with human breast milk”, “infants or young children exclusively breast fed”, “exclusive breast fed infants or young children” and “breast-fed infants / young children” can be used interchangeably. They refer to infants or young children fed with a great majority (i.e. at least 90%, or at least95%, or at least 99%) or all (100%) of nutrients and / or energy originating from human breast milk.

[0050] The expression “conventional nutritional composition” refers to standard synthetic nutritional compositions such as infant formula, follow-up milks or growing-up milks already found in the market.

[0051] The terms “microbial”, “microflora” and “microbiota” can be used interchangeably.

[0052] The expressions “microbiota in the gut”, “microbiota of the gut”, “gut microbiota” and “intestinal microbiota” can be used interchangeably.

[0053] By the expressions “preventing” or “prevention”, it is meant avoiding that a physical state, a condition or their consequences occurs and / or decreasing its incidence (i.e. reduction of the frequency). Prevention also encompasses delay or prevention of the onset of the symptoms of the disease, disorder or condition. Prevention may be absolute (such that no disease occurs) or may be effective only in some individuals or for a limited amount of time.

[0054] By the expressions “treating” or “treatment”, it is meant a decrease of the duration and / or of the severity of a physical state, a condition or their consequences (e.g. a decrease or elimination of symptoms of the condition). Treatment also encompasses to reduce, alleviate or eliminate one or more symptoms associated with the disease, disorder or condition which is being treated and / or to slow down, reduce or block the progression of the disease, disorder or condition which is being treated.

[0055] The improvement of health, prevention and / or the treatment of a physical state, a condition or their consequences can occur during the treatment (i.e. during the administration of the composition of the present invention, either immediately after the start of the administration or some time after, e.g. some days or weeks after the start). But it can also encompass the improvement of health, prevention and / or the treatment later in life. The term “later in life” encompasses the effect after the termination of the intervention or treatment. The effect “later in life” can be from 1 week to several months, or even years, for example from 2 to 4 weeks, from 2 to 6 weeks, from 2 to 8 weeks, from 1 to 6 months or from 2 to 12 months. Suitably, the effect “later in life” can be from 12 months to 12 years, such as from 2 years to 10 years, or from 4 years to 5 years, after the termination of the intervention or treatment. Suitably, the effect “later in life” lasts until the subject is at least 5 years of age, such as at least 10 years of age, at least 20 years of age or at least 30 years of age.

[0056] The term “prebiotic” means non-digestible carbohydrates that beneficially affect the host by selectively stimulating the growth and / or the activity of healthy bacteria such as bifidobacteriain 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).

[0057] 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. etal. “Probiotics: how should they be defined” Trends Food Sci. Technol. 1999:10 107-10). The microbial cells are generally bacteria or yeasts.

[0058] The term “synbiotic” may refer to a component that contains both probiotics and prebiotics, or a live microbe and a substrate that is selectively utilized by the co-administered live microbe (see e.g. Swanson, K.S., et al., 2020. Nature Reviews Gastroenterology & Hepatology, 17(11), pp.687-701).

[0059] The term “cfu” should be understood as colony-forming unit.

[0060] All percentages are by weight unless otherwise stated.

[0061] All weights expressed in g per 100g of composition are dry weight unless otherwise stated. In general, the terms “about” and “approximately” are used herein to modify a numerical value(s) above and below the stated value(s) by 10%.

[0062] The term “SCFA” means short chain fatty acid(s). The expression “increasing SCFA production” means that the amount of systemic and / or colonic SCFA, is higher in an individual fed with the nutritional composition according to the present invention in comparison with a standard. The SCFA production may be measured by techniques known by the skilled person such as by Gas-Liquid Chromatography.

[0063] In the present context, the term “gastrointestinal tract” includes the mouth, pharynx, oesophagus, stomach, small intestine, large intestine, rectum and anus. The term “intestine” includes the small intestine, the large intestine and rectum.

[0064] In addition, in the context of the invention, the terms "comprising" or "comprises" do not exclude other possible elements. The composition of the present invention, including the many embodiments described herein, can comprise, consist of, or consist essentially of the essential elements and limitations of the invention described herein, as well as any additional or optional ingredients, components, or limitations described herein or otherwise depending on the needs. Any reference to prior art documents in this specification is not to be considered an admission that such prior art is widely known or forms part of the common general knowledge in the field.The invention will now be described in further details. It is noted that the various aspects, features, examples and embodiments described in the present application may be compatible and / or combined together any combination thereof.

[0065] Composition and combination

[0066] The invention provides a composition comprising a HMO mixture, MOS and Bifidobacterium longum subsp. infantis, wherein the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosy I lactose (3FL) and / or lacto-N-neotetraose (LNnT).

[0067] The invention also provides a combination of a HMO mixture, MOS and Bifidobacterium longum subsp. infantis, wherein the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosy I lactose (3FL) and / or lacto-N-neotetraose (LNnT).

[0068] The combination may be administered separately, simultaneously or sequentially. In preferred embodiments, the combination is administered simultaneously. In some embodiments, the combination is administered in the form of a composition, for example any composition described herein, for example a nutritional composition.

[0069] Bovine milk-derived oligosaccharides (MOS)

[0070] The composition or combination of the invention comprises bovine milk-derived oligosaccharides (MOS).

[0071] Recent technological advancements have made it possible to produce milk-derived oligosaccharides (MOS) from bovine milk whey permeate for use in infant formulas (see e.g. Meli F, et al. (2014) BMC pediatrics 14, 306). MOS is also referred to as galactooligosaccharides (GOS) from whey permeate. MOS can be added to infant formulas to obtain the functional benefits of prebiotic fibres on gut health, particularly on the microbiota profile and intestinal immune defence. MOS (e.g. GOS from whey permeate, Nestle GOS) is generally recognized as safe (GRAS) in the US and is authorized for use in the EU and in China (according to Appendix C.2 of GB 14880-2012).

[0072] MOS may refer to a mixture of p-linked polymers that include di- to octasaccharides composed of one to seven galactose units linked to a glucose molecule at the reducing end which is produced by an enzymatic process involving p-galactosidase. The enzyme initially breaks lactose down to its constituent monosaccharides (glucose & galactose) and then reassembles galactose into galacto-oligosaccharides, leaving an excess of glucose along with some mono-and di-saccharides.MOS may be synthesised using concentrated demineralized sweet whey permeate as a source of lactose. Treatment with a p-galactosidase preparation purified from a non-pathogenic and non-toxigenic strain of Aspergillus oryzae catalyses the transgalactosylation reaction that hydrolyses lactose and leads to the formation of MOS. The resulting MOS has a degree of polymerization that typically ranges from 2 to 5. The enzyme is inactivated by heat treatment and the solution is filtered to reduce the mineral content and residual lactose, glucose, and galactose. The product is heat-treated, further concentrated by evaporation, and finally spray-dried to produce MOS powder.

[0073] When the composition or combination is in liquid form, the total MOS concentration is typically in the range from 0.5 to 10 g / L. Specific examples of the concentration level of total MOS, when the composition or combination is in liquid form, include 1 to 8 g / L, 2 to 7 g / L, or 3 to 6 g / L. In some embodiments, the total MOS concentration is 7.8 g / L or less.

[0074] In some embodiments, the total MOS concentration is from 2 g / L to 8 g / L.

[0075] In some embodiments, the total MOS concentration is from 4 g / L to 5 g / L.

[0076] In some embodiments, the total MOS concentration is about 4.6 g / L.

[0077] When the composition or combination is in solid form, the total MOS concentration is typically in the range from 0.5 to 10 wt% (g total MOS / 100 g dry composition). Specific examples of the concentration level of total MOS, when the composition or combination is in dry form, include 0.5 to 5 wt% (g total MOS per 100 g dry composition), 1 to 5 wt% or 2 to 4 wt%. In one embodiment, the total MOS concentration, when the composition or combination is in dry form, is about 3.4 wt%.

[0078] In some embodiments, the MOS is obtained from cows, goats or buffalos’ milk. In some embodiments, the MOS is obtained from cow’s milk.

[0079] In some embodiments, the MOS is obtained according to example 1 of EP 2643336 B1. EP 2643336 B1 is thereby entirely incorporated by reference.

[0080] In some embodiments, the MOS is an oligosaccharide mixture comprising at least one N-acetylated oligosaccharide, at least one galacto-oligosaccharide and at least one sialylated oligosaccharide.

[0081] In some embodiments, the MOS is comprising 3'-Galactosyllactose, 6'-Galactosyl lactose and 4'-Galactosyllactose.In some embodiments, the MOS is an oligosaccharide mixture comprising from 0.1 to 4.0 wt% of the N-acetylated oligosaccharide(s), from 92.0 to 99.5 wt% of the galacto-oligosaccharide(s) and from 0.3 to 4.0 wt% of the sialylated oligosaccharide(s).

[0082] Suitable N-acetylated oligosaccharides of the oligosaccharide mixture include GalNAca1,3Gaipi,4Glc and Gaipi, 6GalNAca1,3Gaipi,4Glc, and any mixture thereof. Suitable galacto-oligosaccharides of the oligosaccharide mixture include Gaipi,3Gaipi,4Glc (also called 3'-Galactosyllactose, 3'GL), Gaipi,6Gaipi,4Glc (also called 6'-Galactosyllactose, 6'GL), Gaipi,3Gaipi,3Gaipi,4Glc, Gaipi, 6Gaipi,6Gaipi,4Glc, Gaipi, 3Gaipi,6Gaipi,4Glc, Gaipi ,6Gaipi ,3Gaipi ,4Glc, Gaipi ,6Gaipi ,6Gaipi ,6Glc, Gaipi ,3Gaipi ,3Glc, Gaipi,4Gaipi,4Glc (also called 4'-Galactosyllactose, 4'GL) and Gaipi, 4Gaipi,4Gaipi,4Glc, and any mixture thereof. Suitable sialylated oligosaccharides of the oligosaccharide mixture include NeuAca2,3Gaipi,4Glc and NeuAca2,6Gaipi,4Glc, and any mixture thereof.

[0083] The skilled person understands that some oligosaccharides can be both originating from bovine and human breast milk, especially 3'-Galactosyllactose, 6'-Galactosyllactose, 4'-Galactosyllactose, and sialylated oligosaccharides such as 3SL and 6SL. Accordingly in the present application the above mentioned oligosaccharidescan also be identified as HMOs and they can be present in the HMO mixture according to the present invention, although originating from a bovine milk-based ingredient.

[0084] HMO mixture

[0085] The composition or combination of the invention comprises a HMO mixture consisting of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3FL) and / or lacto-N-neotetraose (LNnT).

[0086] In some embodiments, the HMO mixture comprises or consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), and 3-fucosyllactose (3FL).

[0087] In some embodiments, the HMO mixture consists of 2'-fucosyl lactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), and 3-fucosyllactose (3FL).

[0088] In some embodiments, the HMO mixture comprises or consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), and lacto-N-neotetraose (LNnT).In some embodiments, the HMO mixture consists of 2'-fucosyl lactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), and lacto-N-neotetraose (LNnT).

[0089] In preferred embodiments, the HMO mixture comprises or consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), 3-fucosyl lactose (3FL), and lacto-N-neotetraose (LNnT).

[0090] In more preferred embodiments, the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), 3-fucosyllactose (3FL), and lacto-N-neotetraose (LNnT).

[0091] The composition or combination of the invention comprises a HMO mixture consisting of 3'-Galactosyllactose (3’GL), 6'-Galactosyllactose (6’GL), 4'-Galactosyllactose (4’GL), 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3FL) and / or lacto-N-neotetraose (LNnT).

[0092] In some embodiments, the HMO mixture comprises or consists of 3'-Galactosyllactose (3’GL), 6'-Galactosyllactose (6’GL), 4'-Galactosyllactose (4’GL), 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), and 3-fucosyllactose (3FL).

[0093] In some embodiments, the HMO mixture consists of 3'-Galactosyllactose (3’GL), 6'-Galactosyllactose (6’GL), 4'-Galactosyllactose (4’GL), 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), and 3-fucosyllactose (3FL).

[0094] In some embodiments, the HMO mixture comprises or consists of 3'-Galactosyllactose (3’GL), 6'-Galactosyllactose (6’GL), 4'-Galactosyllactose (4’GL), 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), and lacto-N-neotetraose (LNnT).

[0095] In some embodiments, the HMO mixture consists of 3'-Galactosyllactose (3’GL), 6'-Galactosyllactose (6’GL), 4'-Galactosyllactose (4’GL), 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), and lacto-N-neotetraose (LNnT).

[0096] In preferred embodiments, the HMO mixture comprises or consists of 3'-Galactosyllactose (3’GL), 6'-Galactosyllactose (6’GL), 4'-Galactosyllactose (4’GL), 2'-fucosyllactose (2FL),difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), 3-fucosyl lactose (3FL), and lacto-N-neotetraose (LNnT).

[0097] In more preferred embodiments, the HMO mixture consists of 3'-Galactosyllactose (3’GL), 6'-Galactosyllactose (6’GL), 4'-Galactosyllactose (4’GL), 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), 3-fucosyllactose (3FL), and lacto-N-neotetraose (LNnT).

[0098] In one embodiment, the HMO mixture comprises 2FL in an amount of from 16 wt% to 85 wt%. Suitably, the HMO mixture may comprise 2FL in an amount of from 31 wt% to 82 wt%, preferably from 41wt% to 70 wt%. Suitably, the HMO mixture may comprise 2FL in an amount of from 16 wt% to 69 wt%, preferably from 22 wt% to 59 wt%. Suitably, the HMO mixture may comprise 2FL in an amount of from 34 wt% to 85 wt%, preferably from 40 wt% to 71 wt%. Suitably, the HMO mixture may comprise 2FL in an amount of from 20 wt% to 60 wt%, preferably from 22 wt% to 55 wt%.

[0099] In one embodiment, the HMO mixture comprises LNT in an amount of from 4 wt% to 40 wt%. Suitably, the HMO mixture may comprise LNT in an amount of from 10 wt% to 27 wt%, preferably from 14 wt% to 23 wt%. Suitably, the HMO mixture may comprise LNT in an amount of from 9 wt% to 24 wt%, preferably 12 wt% to 21 wt%. Suitably, the HMO mixture may comprise LNT in an amount of from 10 wt% to 40 wt%, preferably 12 wt% to 26 wt%. Suitably, the HMO mixture may comprise LNT in an amount of from 4 wt% to 30 wt%, preferably 6 wt% to 20 wt%.

[0100] In one embodiment, the HMO mixture comprises DFL in an amount of from 1 wt% to 14 wt%. Suitably, the HMO mixture may comprise DFL in an amount of from 4 wt% to 11 wt%, preferably from 6 wt% to 10 wt%. Suitably, the HMO mixture may comprise DFL in an amount of from 2 wt% to 10 wt%, preferably from 3 wt% to 8 wt%. Suitably, the HMO mixture may comprise DFL in an amount of from 4 wt% to 14 wt %, preferably from 5 wt% to 10 wt%. Suitably, the HMO mixture may comprise DFL in an amount of from 1 wt% to 12 wt %, preferably from 2 wt% to 8 wt%.

[0101] In one embodiment, the HMO mixture comprises 6SLand 3SL combined in an amount of from 7 wt% to 34 wt%. Suitably, the HMO mixture may comprise 6SL and 3SL combined in an amount of from 9 wt% to 34 wt%, preferably from 11 wt% to 29 wt%. Suitably, the HMO mixture may comprise 6SL and 3SL combined in an amount of from 8 wt% to 26 wt%, preferably from 11 wt% to 22 wt%. Suitably, the HMO mixture may comprise 6SL and 3SL combined in an amount of from 9 wt% to 31 wt%, preferably from 10 wt% to 28 wt%. Suitably, the HMO mixturemay comprise 6SL and 3SL combined in an amount of from 7 wt% to 23 wt%, preferably from 8 wt% to 22 wt%.

[0102] In one embodiment, the HMO mixture comprises 3FL in an amount of from 10 wt% to 50 wt%. Suitably, the HMO mixture may comprise 3FL in an amount of from 10 wt% to 50 wt%, preferably from 11 wt% to 43 wt%. Suitably, the HMO mixture may comprise 3FL in an amount of from 10 wt% to 50 wt%, preferably from 13 wt% to 46 wt%.

[0103] In one embodiment, the HMO mixture comprises LNnT in an amount of from 6 wt% to 30 wt%. Suitably, the HMO mixture may comprise LNnT in an amount of from 6 wt% to 30 wt%, preferably from 7 wt% to 22 wt%. Suitably, the HMO mixture may comprise LNnT in an amount of from 3 wt% to 25 wt%, preferably from 5 wt% to 20 wt%.

[0104] In some embodiments, the HMO mixture comprises, consists or consists essentially of:

[0105] i. 16 wt% to 69 wt% of 2FL;

[0106] ii. 9 wt% to 24 wt% of LNT ;

[0107] iii. 2 wt% to 10 wt% of DFL;

[0108] iv. 8 wt% to 26 wt% of 6SL and 3SL combined; and

[0109] v. 10 wt% to 50 wt% of 3FL.

[0110] In some embodiments, the HMO mixture comprises, consists or consists essentially of:

[0111] i. 22 wt% to 59 wt% of 2FL;

[0112] ii. 12 wt% to 21 wt% of LNT ;

[0113] iii. 3 wt% to 8 wt% of DFL;

[0114] iv. 11 wt% to 22 wt% of 6SL and 3SL combined; and

[0115] v. 11 wt% to 43 wt% of 3FL.

[0116] In some embodiments, the HMO mixture comprises, consists or consists essentially of:

[0117] i. 34 wt% to 85 wt% of 2FL;

[0118] ii. 10 wt% to 40 wt% of LNT ;

[0119] iii. 4 wt% to 14 wt% of DFL;

[0120] iv. 9 wt% to 31 wt% of 6SL and 3SL combined; and

[0121] v. 6 wt% to 30 wt% of LNnT.

[0122] In some embodiments, the HMO mixture comprises, consists or consists essentially of:

[0123] i. 40 wt% to 71 wt% of 2FL;

[0124] ii. 12 wt% to 26 wt% of LNT;iii. 5 wt% to 10 wt% of DFL; and

[0125] iv. 10 wt% to 28 wt% of 6SL and 3SL combined; and

[0126] v. 7 wt% to 23 wt% of LNnT.

[0127] In some preferred embodiments, the HMO mixture comprises, consists or consists essentially of:

[0128] i. 20 wt% to 60 wt% of 2FL;

[0129] ii. 4 wt% to 30 wt% of LNT ;

[0130] iii. 1 wt% to 12 wt % of DFL;

[0131] iv. 7 wt% to 23 wt% of 6SL and 3SL combined;

[0132] v. 10 wt% to 50 wt% of 3FL; and

[0133] vi. 3 wt% to 25 wt% of LNnT.

[0134] In some more preferred embodiments, the HMO mixture comprises, consists or consists essentially of:

[0135] i. 22 wt% to 55 wt% of 2FL;

[0136] ii. 6 wt% to 20 wt% of LNT ;

[0137] iii. 2 wt% to 8 wt % of DFL;

[0138] iv. 8 wt% to 22 wt% of 6SL and 3SL combined;

[0139] v. 13 wt% to 46 wt% of 3FL and

[0140] vi. 5 wt% to 20 wt% of LNnT.

[0141] In some more preferred embodiments, the HMO mixture comprises, consists or consists essentially of:

[0142] i. about 38 wt% of 2FL;

[0143] ii. about 13 wt% of LNT;

[0144] iii. about 4.5 wt% of DFL;

[0145] iv. about 11 wt% of 6SL and 3SL combined;

[0146] v. about 14.5 wt% of 3FL and

[0147] vi. about 19 wt% of LNnT.

[0148] In some more preferred embodiments, the HMO mixture comprises, consists or consists essentially of:

[0149] i. about 20 wt% to 40 wt% of 2FL;

[0150] ii. about 5 wt% to 25 wt% of 3FL;

[0151] iii. about 1 wt% to 10 wt% of DFL;iv. about 5 wt% to 25 wt% of LNnT ;

[0152] v. about 5 wt% to 15 wt% of LNT ;

[0153] vi. about 1 wt% to 8 wt% of 3SL;

[0154] vii. about 0.1 wt% to 5 wt% of 6SL;

[0155] viii. about 1 wt% to 10 wt% of 3’GL;

[0156] ix. about 0.5 wt% to 5 wt% of 4’GL and

[0157] x. about 10 wt% to 30 wt% of 6’GL.

[0158] In some more preferred embodiments, the HMO mixture comprises, consists or consists essentially of:

[0159] i. about 30 wt% of 2FL;

[0160] ii. about 11 wt% of 3FL;

[0161] iii. about 4 wt% of DFL;

[0162] iv. about 15 wt% of LNnT ;

[0163] v. about 10 wt% of LNT ;

[0164] vi. about 3 wt% of 3SL;

[0165] vii. about 1 wt% of 6SL;

[0166] viii. about 5 wt% of 3’GL;

[0167] ix. about 2 wt% of 4’GL and

[0168] x. about 19 wt% of 6’GL.

[0169] In some more preferred embodiments, the HMO mixture comprises, consists or consists essentially of:

[0170] i. about 15 wt% to 35 wt% of 2FL;

[0171] ii. about 10 wt% to 30 wt% of 3FL;

[0172] iii. about 1 wt% to 10 wt% of DFL;

[0173] iv. about 0.5 wt% to 8 wt% of LNnT ;

[0174] v. about 2 wt% to 15 wt% of LNT ;

[0175] vi. about 1 wt% to 10 wt% of 3SL;

[0176] vii. about 0.5 wt% to 5 wt% of 6SL;

[0177] viii. about 2 wt% to 15 wt% of 3’GL;

[0178] ix. about 1 wt% to 10 wt% of 4’GL and

[0179] x. about 15 wt% to 45 wt% of 6’GL.

[0180] In some more preferred embodiments, the HMO mixture comprises, consists or consists essentially of:i. about 25 wt% of 2FL;

[0181] ii. about 20 wt% of 3FL;

[0182] iii. about 3 wt% of DFL;

[0183] iv. about 2 wt% of LNnT ;

[0184] v. about 7 wt% of LNT ;

[0185] vi. about 4 wt% of 3SL;

[0186] vii. about 2 wt% of 6SL;

[0187] viii. about 7 wt% of 3’GL;

[0188] ix. about 3 wt% of 4’GL and

[0189] x. about 28 wt% of 6’GL.

[0190] In some more preferred embodiments, the HMO mixture comprises, consists or consists essentially of:

[0191] i. about 15 wt% to 35 wt% of 2FL;

[0192] ii. about 15 wt% to 35 wt% of 3FL;

[0193] iii. about 0.5 wt% to 8 wt% of DFL;

[0194] iv. about 0.1 wt% to 5 wt% of LNnT;

[0195] v. about 1 wt% to 15 wt% of LNT ;

[0196] vi. about 1 wt% to 10 wt% of 3SL;

[0197] vii. about 0.5 wt% to 5 wt% of 6SL;

[0198] viii. about 2 wt% to 15 wt% of 3’GL;

[0199] ix. about 1 wt% to 10 wt% of 4’GL and

[0200] x. about 15 wt% to 45 wt% of 6’GL.

[0201] In some more preferred embodiments, the HMO mixture comprises, consists or consists essentially of:

[0202] i. about 23 wt% of 2FL;

[0203] ii. about 23 wt% of 3FL;

[0204] iii. about 2 wt% of DFL;

[0205] iv. about 1 wt% of LNnT;

[0206] v. about 7 wt% of LNT ;

[0207] vi. about 4 wt% of 3SL;

[0208] vii. about 2 wt% of 6SL;

[0209] viii. about 7 wt% of 3’GL;

[0210] ix. about 3 wt% of 4’GL and

[0211] x. about 28 wt% of 6’GL.In some more preferred embodiments, the HMO mixture comprises, consists or consists essentially of:

[0212] i. about 22 wt% of 2FL;

[0213] ii. about 23 wt% of 3FL;

[0214] iii. about 2 wt% of DFL;

[0215] iv. about 1 wt% of LNnT;

[0216] v. about 6 wt% of LNT ;

[0217] vi. about 5 wt% of 3SL;

[0218] vii. about 2 wt% of 6SL;

[0219] viii. about 7 wt% of 3’GL;

[0220] ix. about 3 wt% of 4’GL and

[0221] x. about 29 wt% of 6’GL.

[0222]

[0223] In some embodiments, the composition or combination of the invention comprises lacto-N- fucopentaose I (LNFP-I).

[0224] In some embodiments, LNFP-I is present in a total amount of from 10 mg / L to 5000 mg / L of the composition or combination according to the invention or of from 0.01 g / 100 g to 4 g / 100 g of the nutritional composition or combination according to the invention.

[0225] In some embodiments, LNFP-I is present in a total amount of from 25 mg / L to 4000 mg / L of the composition or combination according to the invention or of from 0.02 g / 100 g to 3.75 g / 100 g of the nutritional composition or combination according to the invention. Suitably, LNFP-I is present in a total amount of from 50 mg / L to 2500 mg / L, for example from 60 mg / L to 2000 mg / L, for example from 80 mg / L to 1500 mg / L, for example from 100 mg / L to 1000 mg / L, for example from 200 mg / L to 800 mg / L of the composition or combination according to the invention. Suitably, LNFP-I is present in a total amount of from 0.04 g / 100 g to 2 g / 100 g, for example from 0.05 g / 100 g to 1.6 g / 100 g, for example from 0.06 to 1.2 g / 100g, for example from 0.07 g / 100 g to 0.8 g / 100 g, for example from 0.1 g / 100g to 0.7 g / 100g of the composition or combination (dry weight).

[0226] In one embodiment, the composition or combination comprises from 0.015 wt.% to 3.8 wt.%, preferably from 0.08 wt.% to 1.2 wt.%, of lacto-N-fucopentaose I (LNFP-I) of the total wt.% of the composition or combination.LNFP-I may be isolated by chromatography or filtration technology from a natural source such as animal milks. Suitably, the animal milk as used herein may be cow, sheep, goat, camel or buffalo milk. Preferably, the animal milk is cow’s milk. Preferably, the LNFP-I may be produced by biotechnological means using specific fucosyltransferases and / or fucosidases either through the use of enzyme-based fermentation technology (recombinant or natural enzymes) or microbial fermentation technology. In the latter case, microbes may either express their natural enzymes and substrates or may be engineered to produce respective substrates and enzymes. Single microbial cultures and / or mixed cultures may be used. Fucosylated oligosaccharide formation can be initiated by acceptor substrates starting from any degree of polymerization (DP), from DP = 1 onwards. Suitable techniques for producing LNFP-I are known in the art (see, for example, Hu et al., Carbohydr Polym, 2022, 297: 120017 and Derya et al., J Biotechnol., 2020, 318: 31-38). Alternatively, LNFP-I may be produced by chemical synthesis from lactose as initial acceptor substrate building an LNT backbone and free fucose as final donor substrate or by starting from LNT for example, produced by biotechnology or chemical synthesis, and using fucose.

[0227] Fucosylated oligosaccharides are also available for example from DSM of the Netherlands (for example, GlyCare™ 3FL 9001 and GlyCare™ LNFP-I / 2FL 8001) or from Elicityl of France (for example, 2FL, 3FL, DFL, LNFP-I).

[0228] In the context of the present invention, the LNFP-I and the HMO mixture combined may be collectively referred to as a HMO mixture comprising or consisting of LNFP-I, 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosy I lactose (3FL) and / or lacto-N-neotetraose (LNnT). This terminology may be used interchangeably.

[0229] In one embodiment, the invention provides a combination of a human milk oligosaccharide (HMO) mixture, bovine milk-derived oligosaccharides (MOS) and Bifidobacterium longum subsp. infantis wherein the HMO mixture consists of lacto-N-fucopentaose I (LNFP-I), 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3FL) and / or lacto-N-neotetraose (LNnT).

[0230] In one embodiment, the invention provides a combination of a human milk oligosaccharide (HMO) mixture, bovine milk-derived oligosaccharides (MOS) and Bifidobacterium longum subsp. infantis for use in preventing and / or treating constipation and / or improving stool consistency and / or stool frequency in an infant or a young child, wherein the HMO mixture consists of lacto-N-fucopentaose I (LNFP-I), 2'-fucosyllactose (2FL), difucosyllactose (DFL),lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3FL) and / or lacto-N-neotetraose (LNnT).

[0231] In some embodiments, the LNFP-I and HMO mixture combined (i.e. the HMO mixture consisting of LNFP-I, 2FL, DFL, LNT, 6SL, and 3SL, and optionally 3FL and / or LNnT) consists or consists essentially of:

[0232] i. 20 wt% to 46 wt% of 2FL;

[0233] ii. 11 wt% to 17 wt% of LNT ;

[0234] iii. 2 wt% to 7 wt% of DFL;

[0235] iv. 9 wt% to 21 wt% of 6SL and 3SL combined;

[0236] v. 9 wt% to 34 wt% of 3FL; and

[0237] vi. 5 wt% to 32 wt% of LNFP-I.

[0238] In some embodiments, the LNFP-I and HMO mixture combined (i.e. the HMO mixture consisting of LNFP-I, 2FL, DFL, LNT, 6SL, and 3SL, and optionally 3FL and / or LNnT) consists or consists essentially of:

[0239] i. 22 wt% to 42 wt% of 2 FL;

[0240] ii. 12 wt% to 15 wt% of LNT ;

[0241] iii. 3 wt% to 6 wt% of DFL;

[0242] iv. 9 wt% to 19 wt% of 6SL and 3SL combined;

[0243] v. 11 wt% to 32 wt% of 3FL; and

[0244] vi. 10 wt% to 19 wt% of LNFP-I.

[0245] In some embodiments, the LNFP-I and HMO mixture combined (i.e. the HMO mixture consisting of LNFP-I, 2FL, DFL, LNT, 6SL, and 3SL, and optionally 3FL and / or LNnT) consists or consists essentially of:

[0246] i. 27 wt% to 41 wt% of 2FL;

[0247] ii. 8 wt% to 15 wt% of LNT ;

[0248] iii. 4 wt% to 6 wt% of DFL;

[0249] iv. 8 wt% to 18 wt% of 6SL and 3SL combined;

[0250] v. 13 wt% to 21 wt% of LNnT ; and

[0251] vi. 7 wt% to 33 wt% of LNFP-I.

[0252] In some embodiments, the LNFP-I and HMO mixture combined (i.e. the HMO mixture consisting of LNFP-I, 2FL, DFL, LNT, 6SL, and 3SL, and optionally 3FL and / or LNnT) consists or consists essentially of:i. 32 wt% to 39 wt% of 2FL;

[0253] ii. 10 wt% to 14 wt% of LNT ;

[0254] iii. 4 wt% to 6 wt% of DFL;

[0255] iv. 7 wt% to 15 wt% of 6SL and 3SL combined;

[0256] v. 16 wt% to 20 wt% of LNnT ; and

[0257] vi. 11 wt% to 23 wt% of LNFP-I .

[0258] In some embodiments, the LNFP-I and HMO mixture combined (i.e. the HMO mixture consisting of LNFP-I, 2FL, DFL, LNT, 6SL, and 3SL, and optionally 3FL and / or LNnT) consists or consists essentially of:

[0259] i. 29 wt% to 40 wt% of 2FL;

[0260] ii. 8 wt% to 13 wt% of LNT ;

[0261] iii. 3 wt% to 11 wt % of DFL;

[0262] iv. 3 wt% to 15 wt% of 6SL and 3SL combined;

[0263] v. 11 wt% to 35 wt% of 3FL;

[0264] vi. 1 wt% to 18 wt% of LNnT; and

[0265] vii. 2 wt% to 24 wt% of LNFP-I.

[0266] In some embodiments, the LNFP-I and HMO mixture combined (i.e. the HMO mixture consisting of LNFP-I, 2FL, DFL, LNT, 6SL, and 3SL, and optionally 3FL and / or LNnT) consists or consists essentially of:

[0267] i. 32 wt% to 39 wt% of 2 FL;

[0268] ii. 9 wt% to 12 wt% of LNT ;

[0269] iii. 3 wt% to 11 wt % of DFL;

[0270] iv. 4 wt% to 15 wt% of 6SL and 3SL combined;

[0271] v. 12 wt% to 35 wt% of 3FL;

[0272] vi. 1 wt% to 17 wt% of LNnT; and

[0273] vii. 4 wt% to 14 wt% of LNFP-I.

[0274] In some more preferred embodiments, the HMO mixture comprises, consists or consists essentially of:

[0275] i. about 13 wt% to 33 wt% of 2FL;

[0276] ii. about 8 wt% to 28 wt% of 3FL;

[0277] iii. about 1 wt% to 10 wt% of DFL;

[0278] iv. about 0.5 wt% to 8 wt% of LNnT ;

[0279] v. about 2 wt% to 15 wt% of LNT ;vi. about 1 wt% to 10 wt% of 3SL;

[0280] vii. about 0.5 wt% to 5 wt% of 6SL;

[0281] viii. about 2 wt% to 15 wt% of 3’GL;

[0282] ix. about 1 wt% to 10 wt% of 4’GL;

[0283] ix. about 15 wt% to 40 wt% of 6’GL; and

[0284] x. about 5 wt% to 15 wt% of LNFP-I.

[0285] Total HMO concentration

[0286] When the composition or combination is in liquid form, the total HMO concentration is typically in the range from 0.5 to 10 g / L, preferably in the range from 1 to 7.5 g / L. Specific examples of the concentration level of total HMO, when the composition or combination is in liquid form, include 1 to 5 g / L, 1 to 4 g / L, 2 to 5 g / L, 1 to 3 g / L or 2 to 4 g / L.

[0287] In some embodiments, the total HMO concentration is from 1 g / L to 5 g / L.

[0288] In some embodiments, the total HMO concentration is from 2 g / L to 3 g / L.

[0289] In some embodiments, the total HMO concentration is about 2.6 g / L.

[0290] When the composition or combination is in solid form, the total HMO concentration is typically in the range from 0.35 to 7 wt% (g total HMO / 100 g dry composition), preferably in the range from 0.35 to 5 wt%. Specific examples of the concentration level of total HMO, when the composition or combination is in dry form, include 0.5 to 3.5 wt% (g total HMO per 100 g dry composition), 0.5 to 2.5 wt%, 1 to 3.5 wt%, 0.5 to 2 wt% or 1 to 2.5 wt%. In one embodiment, the total HMO concentration, when the composition or combination is in dry form, is about 2 wt%.

[0291] Bifidobacterium longum subsp. infantis

[0292] The composition or combination of the invention comprises Bifidobacterium longum subsp. infantis.

[0293] Bifidobacterium longum is a bacterium of the Bifidobacterium genus which is present in the human gastrointestinal tract. In 2002, three previously distinct species of Bifidobacterium, B. infantis, B. longum, and B. suis, were unified into a single species named B. longum with the biotypes infantis, longum, and suis, respectively (Sakata, S., et al., 2002. International journal of systematic and evolutionary microbiology, 52(6), pp.1945-1951).Any suitable Bifidobacterium longum subsp. infantis strain may be used in the present invention. Such strains will be well-known to the skilled person. Suitable strains include Bifidobacterium longum subsp. infantis LMG 11588 (also known as Bifidobacterium longum subsp. infantis NCC3039 or Bifidobacterium longum subsp. infantis ATCC 17930) and Bifidobacterium longum subsp. infantis ATCC 15697 (also known as Bifidobacterium longum subsp. infantis NCC 3078).

[0294] The Bifidobacterium longum subsp. infantis may be a strain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% (suitably, at least 99.9%) ANI to Bifidobacterium longum subsp. infantis strain known to the skilled person.

[0295] Suitably, the Bifidobacterium longum subsp. infantis has at least 95%, at least 96%, at least 97%, at least 98%, 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 Bifidobacterium longum subsp. infantis LMG 11588 (also known as Bifidobacterium longum subsp. infantis NCC3039 or Bifidobacterium longum subsp. infantis ATCC 17930). Preferably, the Bifidobacterium longum subsp. infantis has at least 99.9% ANI to Bifidobacterium longum subsp. infantis LMG 11588.

[0296] An example of a microorganism genome that has at least 99.9% ANI with B. longum subsp. infantis LMG 11588 can be found in PATRIC (https: / / www.patricbrc.org), genome ID 1678.111. Hence, suitably the B. longum subsp. infantis having the PATRIC genome ID 1678.111 may be used in the present invention.

[0297] Bifidobacterium longum subsp. infantis LMG 11588 is sold by the Belgian Coordinated Collections of Microorganisms (BCCM) under the LMG accession number LMG 11588.

[0298] Bifidobacterium longum subsp. infantis ATCC 15697 is sold by the American Type Culture Collection (ATCC) under the accession number ATCC 15697.

[0299] The composition or combination according to the invention may contain from 103to 1012cfu of Bifidobacterium longum subsp. infantis, preferably from 105to 1O10cfu of Bifidobacterium longum subsp. infantis, more preferably from 105to 107cfu of Bifidobacterium longum subsp. infantis per g of combination or composition on a dry weight basis. In some embodiments, the composition or combination according to the invention comprises about 106cfu of Bifidobacterium longum subsp. infantis per g of combination or composition on a dry weight basis.

[0300] The composition or combination according to the invention may contain from 103to 1012cfu of Bifidobacterium longum subsp. infantis, preferably from 105to 1O10cfu of Bifidobacteriumlongum subsp. infantis, more preferably from 107to 109cfu of Bifidobacterium longum subsp. infantis per L of combination or composition. In some embodiments, the composition or combination according to the invention comprises 108to 109cfu of Bifidobacterium longum subsp. infantis per L of combination or composition.

[0301] Suitably, the Bifidobacterium longum subsp. infantis is administered to the subject in an amount of at least about 106cfu / day, at least about 107cfu / day, or at least about 108cfu / day. Suitably, the Bifidobacterium longum subsp. infantis is administered to the subject in an amount of about 1012cfu / day or less, about 1011cfu / day or less, or about 101° cfu / day or less. Suitably, the Bifidobacterium longum subsp. infantis is administered to the subject in an amount of from 106cfu / day to 1012cfu / day, from 107cfu / day to 1011cfu / day, or from 108cfu / day to 101° cfu / day.

[0302] In one embodiment, the Bifidobacterium longum subsp. infantis is viable.

[0303] Milk fat globule membrane (MFGM) and Phospholipids

[0304] In an embodiment, the combination or composition according to the invention comprises Milk fat globule membrane (MFGM).

[0305] Milk fat globule membrane (MFGM) is a complex and unique structure composed primarily of lipids and proteins that surrounds milk fat globule secreted from the milk producing cells of humans and other mammals. It is a source of multiple bioactive compounds, including phospholipids, glycolipids, glycoproteins, and carbohydrates.

[0306] In one embodiment of the present invention, MFGM comprise a phospholipid a metabolic precursor or metabolite thereof.

[0307] In one embodiment of the present invention, MFGM comprise phospholipids, for example phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and sphingomyelin.

[0308] In one embodiment of the present invention, MFGM and phospholipids are provided to the composition or combination via an alpha lactalbimun enriched whey protein ingredient.

[0309] In another embodiment, the combination or composition according to the invention comprises a phospholipid a metabolic precursor or metabolite thereof.

[0310] In one embodiment, the composition or combination of the invention comprises phospholipids. Non limiting examples of phospholipids include phosphatidylinositole, phosphatidylserine, phosphatidylethanolamine, sphingomyelin and phosphatidylcholine.In an embodiment of the present invention the phospholipid is selected from the group consisting of: phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, sphingomyelin and / or combinations thereof.

[0311] In an embodiment of the present invention, the phospholipid comprises phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and sphingomyelin. In another embodiment of the present invention, the phospholipid consists of phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and sphingomyelin.

[0312] In another embodiment of the present invention, the expression “total phospholipids” refers to the total amount of phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and sphingomyelin in the combination and / or nutritional composition.

[0313] In an embodiment of the present invention, the phospholipid comprises phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and sphingomyelin wherein sphingomyelin is comprised in an amount ranging from 20 to 30% w / w sphingomyelin based on total phospholipids.

[0314] In an embodiment of the present invention, the phospholipid comprises phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and sphingomyelin which are comprised at respective amounts ranging from 25 to 35 % w / w phosphatidylcholine based on total phospholipids, amounts ranging from 5 to 15% w / w phosphatidylinositol on total phospholipids, amount ranging from 5 to 10% w / w phosphatidylserine on total phospholipids, amount ranging from 21 to 30 % w / w phosphatidylethanolamine on total phospholipids and amount ranging from 20 to 30% w / w sphingomyelin on total phospholipids.

[0315] In one embodiment of the present invention, the phospholipid consists of phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and sphingomyelin which are comprised at respective amounts ranging from 25 to 35 % w / w phosphatidylcholine based on total phospholipids, amounts ranging from 5 to 15% w / w phosphatidylinositol on total phospholipids, amount ranging from 5 to 10% w / w phosphatidylserine on total phospholipids, amount ranging from 21 to 30 % w / w phosphatidylethanolamine on total phospholipids and amount ranging from 20 to 30% w / w sphingomyelin on total phospholipids.

[0316] In an embodiment of the present invention, the phospholipid comprises phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and sphingomyelin which are comprised at respective amounts of 32% w / w phosphatidylcholine on total phospholipids,11% w / w phosphatidylinositol on total phospholipids, 8% w / w phosphatidylserine on total phospholipids, 25 % w / w phosphatidylethanolamine on total phospholipids and 24% w / w sphingomyelin on total phospholipids.

[0317] In an embodiment of the present invention, the phospholipid consists of phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and sphingomyelin which are comprised at respective amounts of 32% w / w phosphatidylcholine on total phospholipids, 11% w / w phosphatidylinositol on total phospholipids, 8% w / w phosphatidylserine on total phospholipids, 25 % w / w phosphatidylethanolamine on total phospholipids and 24% w / w sphingomyelin on total phospholipids.

[0318] In one embodiment of the present invention, the synthetic nutritional composition comprises total phospholipids in an amount of at least 150 mg / 100g of the synthetic nutritional composition. In one embodiment of the present invention, the synthetic nutritional composition comprises total phospholipids in an amount of at least 200 mg / 100g of the synthetic nutritional composition.

[0319] In one embodiment of the present invention, the synthetic nutritional composition comprises total phospholipids in an amount ranging from 200 to 700 mg / 100g of the synthetic nutritional composition.

[0320] In one embodiment of the present invention, the synthetic nutritional composition comprises total phospholipids in an amount ranging from 300 to 600 mg / 100g of the synthetic nutritional composition.

[0321] In one embodiment of the present invention, the synthetic nutritional composition comprises total phospholipids in an amount ranging from 200 to 400 mg / 100g of the synthetic nutritional composition. In one embodiment, the synthetic nutritional composition comprises sphingomyelin in an amount higher than 200mg / kg of the dry weight of the composition, more particularly ranging from 200mg to 2.5g / kg of the dry weight of the composition.

[0322] In an embodiment the synthetic nutritional composition comprises sphingomyelin in an amount selected from the group consisting of; higher than 200 mg / kg, higher than 300 mg / kg, ranging from 200 mg to 2.5 g / kg, ranging from 200mg to 2g / kg, in amount ranging from 300 mg to 1.5 g / kg or from 400 mg to 1g / Kg, ranging from 200 to 850mg / kg, or 300 to 820mg / kg. All weights being per dry weight of the composition. In one embodiment, the composition will comprise phosphatidylcholine in an amount higher than 200mg / kg of the dry weight of the composition, more particularly ranging from 200mg to 2.5g / kg of the dry weight of the composition.

[0323] 1In an embodiment the synthetic nutritional composition comprises phosphatidylcholine in an amount selected from the group consisting of; higher than 200 mg / kg, higher than 300 mg / kg, higher than 400mg / kg, ranging from 200 mg to 2.5 g / kg, ranging from 200mg to 2g / kg, in amount ranging from 300 mg to 1.5 g / kg or from 400 mg to 1g / Kg, 500 mg to 1.3g / Kg. All weights being per dry weight of the composition.

[0324] In one embodiment, the synthetic nutritional composition will comprise phosphatidylinositol in an amount higher than 500mg / kg of the dry weight of the composition, more particularly ranging from 200mg to 1.5g / kg of the dry weight of the composition.

[0325] In an embodiment the composition comprises phosphatidylinositol in an amount selected from the group consisting of; higher than 200 mg / kg, higher than 300 mg / kg, ranging from 200 mg to 2.5 g / kg, ranging from 200mg to 2g / kg, in amount ranging from 250mg to 800mg / kg or from 400 mg to 1.5g / Kg, or from 400 to 800mg / kg. All weights being per dry weight of the composition.

[0326] In one embodiment, the synthetic nutritional composition will comprise phosphatidylserine in an amount higher than 50mg / kg of the dry weight of the composition, higher than 200mg / kg of the dry weight of the composition, more particularly ranging from 150mg to 1.5g / kg of the dry weight of the composition, from 200mg to 1g / kg of the dry weight of the compositio In an embodiment the synthetic nutritional composition comprises phosphatidylserine in an amount selected from the group consisting of; higher than 150, higher than 200 mg / kg, higher than 300 mg / kg, ranging from 200 mg to 2.5 g / kg, ranging from 200mg to 2g / kg, in amount ranging from 200mg to 1000mg / kg or from 250 mg to 1g / Kg. All weights being per dry weight of the composition

[0327] In one embodiment, the composition will comprise phosphatidylethanolamine in an amount higher than 150mg / kg of the dry weight of the composition, higher than 200mg / kg of the dry weight of the composition, more particularly ranging from 150mg to 1.5g / kg of the dry weight of the composition

[0328] In an embodiment the composition comprises phosphatidylethanolamine in an amount selected from the group consisting of; higher than 170mg / kg, higher than 180 mg / kg, higher than 200 mg / kg, ranging from 200 mg to 2.5 g / kg, ranging from 200mg to 2g / kg, in amount ranging from 250mg to 800mg / kg or from 200 mg to 1g / Kg. All weights being per dry weight of the composition.

[0329] If a metabolic precursor and / or metabolite of one or more phospholipid is used in a composition in place of or in combination with a phospholipid, said compounds may be usedin amounts such that the level of phospholipids physiologically delivered by said composition is in line with those set out hereinabove. It is well within the purview of the skilled person to determine appropriate amounts.

[0330] The term metabolic precursor and / or metabolite of one or more phospholipid as used herein does not include choline.

[0331] The phospholipid, metabolic precursors and / or metabolite thereof, comprised in the composition of the invention may be natural, synthetic or a mixture thereof. Said metabolic precursors and / or a metabolite, may be used in the composition of the invention in their pure form, or substantially pure form. Alternatively, they may be added in the form of a source comprising them.

[0332] Any source of a phospholipid metabolic precursors and / or metabolite thereof, suitable for ingestion by a subject for which the composition is intended to be consumed may be used in the invention.

[0333] In particular, the phospholipid a metabolic precursor or metabolite thereof, will come from natural sources, non-limiting examples of which include, eggs, soy, bovine brains, and / or mammalian milk or extracts thereof. Non limiting examples of soy sources include soy lecithinfood additive, non-limiting examples of mammalian milk include bovine, camel, sheep, goat milk including skilled milks. Non limiting extracts of milk include protein extracts e.g. whey protein and casein, milk fat globule membranes (MFGM) and extracts comprising them. A particularly useful source of a phospholipids a metabolic precursor or metabolite thereof, in particular sphingomyelin, that may be used in the present invention may be a bovine milk whey protein concentrate enriched in alpha-lactalbumin, and / or non-pure alpha-lactalbumin which has been extracted from milk whey protein, in particular bovine milk whey protein

[0334] Alpha-Lactalbumin is a high-quality, easy-to-digest e.g. by human infants, whey protein and is the primary protein found in HM. Alpha-lactalbumin and / or an alpha-lactalbumin enriched milk fraction is ideal for use in lower protein infant formulas due to its high content of essential amino acids, particularly tryptophan. Although alpha-Lactalbumin is in itself a protein non pure sources may comprise sphingomyelin.

[0335] Exemplary and non-limiting examples of commercial ingredients enriched in alpha lactalbumin and providing MFGM (including phospholipids) are: Lacprodan™ MFGM 10 (Aria) or 8800 Alpha-lac Enriched (Hilmar).In an embodiment a phospholipid a metabolic precursor or metabolite thereof, in particular sphingomyelin, is used in the form of a whey protein concentrate enriched in alpha-lactalbumin or as alpha-lactalbumin.

[0336] In a more particular embodiment, a bovine whey protein concentrate enriched in alphalactalbumin or alpha-lactalbumin having a phospholipid content, in particular sphingomyelin content higher than 500 mg / 100g, 900mg / 100g, 1000mg / 100g dry weight of the composition is used.

[0337] In one embodiment of the present invention, the combination or composition according to the present invention comprises a bovine whey protein concentrate enriched in alpha-lactalbumin or alpha-lactalbumin having a phospholipid content in an amount ranging from 1 to 2.5 g / 100g. In a further embodiment, the combination or composition according to the present invention comprises a bovine whey protein concentrate enriched in alpha-lactalbumin or alphalactalbumin having a phospholipid content in an amount ranging from 1 to 2 g / 100g.

[0338] Another particularly useful source of phospholipids a metabolic precursor, or metabolite thereof, may be milk fat globule membrane (hereinafter MFGM) or extracts comprising them, in particular MFGM, or extracts comprising them from bovine milk. It may be particularly beneficial if the MFGM or extracts comprising them comprises at least 1%, 2%, 5%, 10%, 20%, 30%, 40% phospholipids and / or at least 0.1%, 0.2%, 0.5% to 5%, 0.8% to 3%, 1% to 2%, 1.6%, 1.9%, 1.8% of phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and / or sphingomyelin. The MFGM may also further comprise magnesium, phosphorus and or calcium, in particularly in concentrations ranging from 0.05% to 2%, 0.1% to 0.4%.

[0339] In one embodiment of the present invention, MFGM comprise osteopontin.

[0340] In one embodiment of the present invention, the combination or composition of the present invention comprises osteopontin in an amount ranging from 40 to 100 mg / 100g of the synthetic nutritional composition.

[0341] In one embodiment of the present invention, MFGM comprise gangliosides.

[0342] In one embodiment of the present invention, the combination or composition of the present invention comprises gangliosides, in an amount ranging from 10 to 20 mg / 100g, of the synthetic nutritional composition.

[0343] In one embodiment of the present invention, the combination or composition of the present invention comprises gangliosides, in particular ganglioside GD3, in an amount ranging from 5 to 30 mg / 100g , for example from 10 to 20 mg / 100g, of the synthetic nutritional composition.In one embodiment of the present invention, the combination or composition of the present invention comprises gangliosides, in particular ganglioside GM3, in an amount ranging from 0.01 to 1 mg / 100g of the synthetic nutritional composition.

[0344] Therapeutic uses

[0345] The combination or composition of the present invention may be used to prevent and / or treat constipation and / or improve stool consistency and / or stool frequency.

[0346] In one aspect, the present invention provides a combination or composition for use in preventing and / or treating constipation and / or improving stool consistency / frequency in a subject, wherein the combination or composition is any described herein.

[0347] In a further aspect, the present invention provides use of a combination or composition for the manufacture of a medicament for preventing and / or treating constipation and / or improving stool consistency / frequency in a subject, wherein the combination or composition is any described herein.

[0348] In a further aspect, the present invention provides a method of preventing and / or treating constipation and / or improving stool consistency / frequency in a subject, the method comprising administering to the subject a composition or combination according to the invention.

[0349] The expression “preventing and / or treating constipation and / or improving stool consistency / frequency” encompasses one or several of the following: preventing constipation; treating constipation; improving stool consistency; and improving stool frequency.

[0350] The combination or composition of the present invention may be administered orally. For example, the combination of the present invention may be fed to the subject in the form of a nutritional composition (e.g. an infant formula).

[0351] In some embodiments, the combination or composition of the invention is given for treatment purposes, e.g. when the composition is used for the treatment of constipation. The combination or composition of the invention can be administered for some days (1, 2, 3, 4, 5, 6...), or for some weeks (1, 2, 3, 4, 5, 6, 7, 8 or even more), or for some months (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or even more), depending on the needs. It may be administered once the symptoms appear or once these health diseases / conditions (e.g. constipation) have been diagnosed. It may be administered up to the symptoms of the treated diseases / conditions (e.g. constipation) disappear, or several days / weeks / months after said disappearance. In some embodiments, the combination or composition of the invention is administered for 1 month ormore, 2 months or more, 3 months or more, 4 months or more, or 5 months or more. In some embodiments, the combination or composition of the invention is administered from 2-5 weeks of age to 6 months of age.

[0352] In some embodiments, the health benefits / effects are observed during the first few weeks or months of life and / or during the first few weeks or months after administration of the combination or composition, e.g. during the first week, the first 2 weeks, the first month, the first 2 months, the first 4 months or the first 6 months. Effects at longer term may be less important. In some embodiments, the combination or composition according to the present invention is for use in improving stool consistency / frequency in an infant during the first month, the first 2 months, the first 4 months or the first 6 months of life and / or after administration of the combination or composition. In some embodiments, the combination or composition according to the present invention is for use in preventing and / or treating constipation in infants during the first month, the first 2 months, the first 4 months or the first 6 months of life and / or after administration of the combination or composition.

[0353] Improving stool consistency and / or frequency

[0354] In one aspect, the present invention provides a combination or composition for use in improving stool consistency / frequency in a subject, wherein the combination or composition is any described herein.

[0355] In a further aspect, the present invention provides use of a combination or composition for the manufacture of a medicament for improving stool consistency / frequency in a subject, wherein the combination or composition is any described herein.

[0356] In a further aspect, the present invention provides a method of improving stool consistency / frequency in a subject, the method comprising administering to the subject a composition or combination according to the invention.

[0357] The expressions “in improving stool consistency / frequency”, “in improving stool consistency and / or frequency” and “in improving stool consistency and / or stool frequency” can be used interchangeably. They encompass the improvement of stool consistency, the improvement of stool frequency and / or both.

[0358] Breast-fed infants have frequent, watery or loose soft stools whereas formula-fed infants typically have less frequent bowel movements, firmer stools that may be difficult to pass and therefore, may lead to discomfort with pain, and in some cases, constipation. These abnormalities or particularities in stooling patterns are perceived as abnormal by parents andthus a common source of parental distress and a frequency cause of consultation to health care provider.

[0359] Moreover, as a result of these abnormalities or particularities, infants who are fed conventional infant formula may also suffer from digestive disorders. Digestive disorders, including colic may manifest as diarrhea, excessive gas, and abdominal discomfort and pain that may cause excessive crying and fussiness in the infant or young child. Hard stools may also lead to constipation, fussiness and crying, poor feeding tolerance, digestive discomfort, and abdominal pain. As tendency for harder stools increases with age, young children may be more prone to constipation than infants.

[0360] Improving stool consistency

[0361] In some embodiments, the combination or composition of the present invention improves stool consistency (e.g. reduces stool hardness).

[0362] In one aspect, the present invention provides a combination or composition for use in improving stool consistency (e.g. reducing stool hardness) in a subject, wherein the combination or composition is any described herein.

[0363] In a further aspect, the present invention provides use of a combination or composition for the manufacture of a medicament for improving stool consistency (e.g. reducing stool hardness) in a subject, wherein the combination or composition is any described herein.

[0364] In a further aspect, the present invention provides a method of improving stool consistency (e.g. reducing stool hardness) in a subject, the method comprising administering to the subject a composition or combination according to the invention.

[0365] The expression “improving stool consistency” means that the stool of a subject feeding a combination or composition according to the invention has a softer consistency than the stool of a subject not feeding a combination or composition according to the invention. The expression “improving stool consistency” may especially encompass or mean “reducing stool hardness”, “softening stool” and / or “providing softer stool”, e.g. there will be less hard lumps, like nuts, that are hard to pass. The improved stool consistency may be closer to that observed in breast-fed-infants.

[0366] The stool consistency can be determined by any suitable method. For example, using a validated stool scale. An example validated 5-point stool scale developed for infants < one year of age is: 0=watery, 1=runny, 2=mushy-soft, 3=formed, 4=hard.In some embodiments, the combination or composition according to the present invention is for use in improving stool consistency (e.g. reducing stool hardness) in an infant during the first month, the first 2 months, the first 4 months or the first 6 months of life and / or after administration of the combination or composition.

[0367]

[0368] In some embodiments, the combination or composition of the present invention improves stool frequency (e.g. promotes regular bowel movements).

[0369] In one aspect, the present invention provides a combination or composition for use in improving stool frequency in a subject, wherein the combination or composition is any described herein.

[0370] In a further aspect, the present invention provides use of a combination or composition for the manufacture of a medicament for improving stool frequency in a subject, wherein the combination or composition is any described herein.

[0371] In a further aspect, the present invention provides a method of improving stool frequency in a subject, the method comprising administering to the subject a composition or combination according to the invention.

[0372] The expression “improving stool frequency” means that the stool of a subject will become more frequent, i.e. the transit will be regulated and / or faster. It may therefore also encompass or mean “improving / increasing the transit speed” or “avoiding a slow transit” or “regulating the transit”. The improvement of stool consistency / frequency may especially allow avoiding pain, especially abdominal pain, abdominal cramping and also further complications like anal fissure, rectal bleeding, the need of a surgical intervention, the use of medication, the use of stool softeners etc. The improved stool may be closer to that observed in breast-fed-infants. In some embodiments, the combination or composition according to the present invention is for use in improving stool frequency in an infant during the first month, the first 2 months, the first 4 months or the first 6 months of life and / or after administration of the combination or composition.

[0373] Preventing and / or treating constipation

[0374] In one aspect, the present invention provides a combination or composition for use in preventing and / or treating constipation in a subject, wherein the combination or composition is any described herein.In a further aspect, the present invention provides use of a combination or composition for the manufacture of a medicament for preventing and / or treating constipation in a subject, wherein the combination or composition is any described herein.

[0375] In a further aspect, the present invention provides a method of preventing and / or treating constipation in a subject, the method comprising administering to the subject a composition or combination according to the invention.

[0376] In some embodiments, the composition or combination of the invention prevents constipation. The expressions “in preventing constipation”, “to prevent constipation”, “in the prevention of constipation” and “for the prevention of constipation” can be used interchangeably and may refer to avoiding constipation occurring and / or decreasing the incidence of constipation (e.g. reduction of the frequency). In some embodiments, the prevention of constipation occurs during the treatment (i.e. during the administration of the composition of the present invention, either immediately after the start of the administration or some time after, e.g. some days or weeks after the start).

[0377] In some embodiments, the composition or combination of the invention treats constipation. The expressions “in treating constipation”, “to treat constipation”, “in the treatment of constipation” and “for the treatment of constipation” can be used interchangeably and may refer the decrease of the duration of constipation (e.g. number of days / weeks / years the infants or young children will suffer from constipation) or the decrease of the severity (or level) of constipation. These expressions also encompass the relieve of the symptoms or consequences of constipation, such as blocking stool, and / or the decrease of complications caused by constipation on the subject, such as anal fissure, rectal bleeding, the need of a surgical intervention, the use of medication, the use of stool softeners, and / or the decrease of pain, and / or the decrease of tiredness, and / or the ease of the sleep and / or the stabilization of the activity of the subject suffering from constipation.

[0378] In some embodiments, the combination or composition of the present invention reduces the incidence and / or severity of constipation in the subject. Said reduction in the incidence and / or severity of constipation may comprise reducing the stool hardness, preferably to render the stool consistency close to that of breast-fed infants or young children with a normal stool pattern.

[0379] In some embodiments, the combination or composition according to the present invention is for use in reducing the incidence and / or severity of constipation in an infant during the first month, the first 2 months, the first 4 months or the first 6 months of life and / or after administration of the combination or composition.Flatulence, bloating, colics and / or gut discomfort

[0380] The combination or composition of the present invention can be used to prevent and / or treat constipation and / or improve stool consistency and / or stool frequency without associated flatulence, bloating, colics and / or gut discomfort.

[0381] In some embodiments, the subject has no ora limited level of flatulence, bloating, colics and / or gut discomfort. The expressions “no or a limited level” and “no particular issue” of flatulence, bloating, colics and / or gut discomfort can be used interchangeably. For example, a subject administered the combination or composition of the present invention may have no flatulence, bloating, colics and / or gut discomfort episodes or they may have a low level of flatulence, bloating, colics and / or gut discomfort. In some embodiments, a subject administered the combination or composition of the present invention may have less (e.g. at least 1% less, or at least 2% less, or at least 5% less, or at least 10% less, or at least 20% less or at least 50% less) flatulence, bloating, colics and / or gut discomfort episodes in comparison with subject fed with a standard composition. The expressions “a limited level” and “a lower level” can be used interchangeably. They may refer to the frequency and / or the occurrence and / or the severity. In one aspect, the present invention provides a combination or composition for use in preventing and / or treating flatulence, bloating, colics and / or gut discomfort in a subject, wherein the combination or composition is any described herein.

[0382] In a further aspect, the present invention provides use of a combination or composition for the manufacture of a medicament for preventing and / or treating flatulence, bloating, colics and / or gut discomfort in a subject, wherein the combination or composition is any described herein. In a further aspect, the present invention provides a method of preventing and / or treating flatulence, bloating, colics and / or gut discomfort in a subject, the method comprising administering to the subject a composition or combination according to the invention.

[0383] In some embodiments, the combination or composition of the present invention reduces the incidence and / or severity of flatulence, bloating, colics and / or gut discomfort in the subject. Said reduction in the incidence and / or severity of flatulence, bloating, colics and / or gut discomfort may comprise reducing the stool hardness, preferably to render the stool consistency close to that of breast fed infants or young children with a normal stool pattern.

[0384] Gut discomfort and Gastrointestinal Tolerance

[0385] In particular embodiments, the combination or composition of the present invention can be used to prevent and / or treat constipation and / or improve stool consistency and / or stool frequency without associated gut discomfort.In some embodiments, the subject has no or a limited level of gut discomfort. In some embodiments, the combination or composition of the present invention reduces the incidence and / or severity of gut discomfort in the subject. Said reduction in the incidence and / or severity of gut discomfort may comprise reducing the stool hardness, preferably to render the stool consistency close to that of breast fed infants or young children with a normal stool pattern. In one aspect, the present invention provides a combination or composition for use in preventing and / or treating gut discomfort in a subject, wherein the combination or composition is any described herein.

[0386] In a further aspect, the present invention provides use of a combination or composition for the manufacture of a medicament for preventing and / or treating gut discomfort in a subject, wherein the combination or composition is any described herein.

[0387] In a further aspect, the present invention provides a method of preventing and / or treating gut discomfort in a subject, the method comprising administering to the subject a composition or combination according to the invention.

[0388] The expression “gut discomfort” includes abdominal pain and / or cramping. Gut discomfort may be determined by any suitable method, for example using an Infant Gastrointestinal Symptom Questionnaire (IGSQ), which is a validated method for assessment of infant Gl-related behaviours (see e.g. Riley AW et al. (2015) Clin Pediatr (Phila) 54, 1167-1174).

[0389] In one embodiment, the combination or composition of the present invention is useful for non -therapeutic promotion of gastro intestinal (Gl) tolerance in a subject, in particular an infant or young child.

[0390] Modulation of Intestinal microbiome

[0391] In one aspect the present invention relates to a combination or composition as defined above for use in: i) preventing and / or treating bacterial infections in an infant or a young child; ii) modulating the microbiota of an infant or a young child; and / or iii) preventing and / or treating allergy of an infant or a young child.

[0392] In one aspect the present invention relates to a combination or a composition as defined above for use for modulating the microbiota of an infant or a young child and administration of said composition results in abundance of Bifidobacteriaceae and / or Bifidobacterium longum subsp. infantis being increased.In one aspect the present invention relates to a combination or composition as defined above for modulating the microbiota of an infant or a young child; and / or preventing and / or treating allergy of an infant or a young child by modulating intestinal short-chain fatty acids (SCFA) production in such infant or young child.

[0393] In one aspect the present invention relates to a method of modulating the microbiota of an infant or a young child to increase the abundance of Bifidobacteriacea and / or Bifidobacterium longum subsp. infantis, the method comprising: administering, to the infant or young child, a a combination or a composition as defined.

[0394] In another aspect of the present invention, it is provided a combination or a composition as above described to promote immunity development and / or immune function in a subject, in particular an infant or young child.

[0395] In one aspect of the present invention, the combination or composition of the present invention is useful for non -therapeutic promote to immunity development and / or immune function in a subject, in particular an infant or young child.

[0396] Subject

[0397] In some embodiments, the subject is a mammal, such as a human.

[0398] In some embodiments, the composition according to the invention is for use in infants, young children or children. In preferred embodiments, the subject is an infant. In other embodiments, the subject is a young child. In other embodiments, the subject is a child.

[0399] All infants and young children can benefit from the invention as all of them are or can be, at a certain age, susceptible to acquiring an unbalanced intestinal / gut microbiota. The composition or combination according to the invention is typically for use in infants or young children. The composition or combination is particularly suitable for infants under 6 months of age. The composition or combination can be administered (or given or fed) at an age and for a period that depends on the needs.

[0400] The composition or combination can be administered (or given or fed) at an age and for a period that depends on the needs. In some embodiments, the subject is 0-36 months of age, such as 0-12 months or 0-6 months of age. In preferred embodiments, the subject is 0-6 months of age. It is foreseen that the composition or combination of the invention may be evenmore beneficial to infants just after birth (0-4 weeks or 0-8 weeks) as their intestinal tract may be more fragile.

[0401] The mammal to be treated is preferably a human being, but the mammal may also be nonhuman mammal, such as a non-human mammal selected from the group consisting of pig, cow, horse, dog, cat, goat, sheep and rabbit. In some embodiments, the subject is a juvenile animal, preferably wherein the animal is a pet. A pet may be a mammal such as dogs or cats, or rodents such as mice, rats, and guinea pigs, rabbits, etc. In some embodiments, the pet is a small dog breed. The term “juvenile” may refer to an individual that has not yet reached adulthood.

[0402] In some embodiments, the composition or combination according to the invention can be for use before and / or during the weaning period. In some embodiments, the composition or combination according to the invention is for use before the weaning period.

[0403] In some embodiments, the subject is bottle-fed and / or formula-fed. In preferred embodiments, the subject is exclusively formula-fed. In preferred embodiments, the composition or combination is given to the subject as the sole or primary nutritional composition during at least one period of time, e.g. after the 1st, 2ndor 4thmonth of life, during at least 1 , 2, 4 or 6 months. In some embodiments, the composition or combination is given to the subject as the sole or primary nutritional composition from 0-6 months of age.

[0404] In preferred embodiments, the composition or combination of the invention is a complete nutritional composition (fulfilling all or most of the nutritional needs of the subject). In another embodiment the composition or combination of the invention is a supplement or a fortifier intended for example to supplement human milk or to supplement an infant formula ora follow-on formula.

[0405] In other embodiments, the composition or combination of the invention is given to the subject as a supplementary composition to the mother's milk. In some embodiments the subject receives the mother's milk during at least the first 2 weeks, first 1, 2, 4, or 6 months. In one embodiment, the composition or combination of the invention is given to the subject after such period of mother's nutrition, or is given together with such period of mother's milk nutrition. In some embodiments, the infant or young child was born by C-section and / or preterm, and / or small for gestational age (SGA) and / or with a low, very low, or extremely low weight at birth (LWB, VLBW, or ELBW) and / or experienced intra-uterine growth retardation (IUGR), and / or was hospitalized the first days following birth and / or took antibiotic the first days following birth and / or was sick at birth, and / or has impaired microbiota and / or dysbiosis of microbiota.The composition or combination may be particularly useful for preterm infants and / or low or very low birth weight infants. In another particularly interesting embodiment, the composition or combination is used in infants delivered via Caesarean section. Antibiotic administration is a recommended medical practice for C-section birth in order to prevent infection. Such interventions are potent disruptors of microbial communities (the mother’s or the child’s) and C-section born infants may have a delayed and less optimal colonization of the large intestinal tract. The composition or combination of the present invention may also be used in an infant or a young child that was born by C-section or that was vaginally delivered.

[0406] The infants, young children or children may be born term or preterm. In a particular embodiment, the composition or combination of the invention is for use in infants, young children or children that were born preterm. Preterm infants may be at increased risk of poor nutrient utilization, impaired lean body mass growth, fat accumulation in the visceral area and metabolic disease later in life. Thus, in one embodiment the composition or combination of the invention is for use in preterm infants. In one embodiment, the subject is an infant or a young child that was born small for gestational age or low birth weight. Infants or young children with low birth weight may or may not be preterm, and similarly, infants or young children who are small for gestational age may or may not be preterm.

[0407] In some advantageous embodiments of the invention, the composition or combination is for use infants or young children having a fragile or unbalanced microbiota or dysbiosis of microbiota, such as preterm infants, infants born by Caesarean-section, infants born small for gestational age or with low birth weight, hospitalized infants / young children, infants / young children treated or having been treated by antibiotics, infants / young children sick at birth including necrotizing enterocolitis (NEC), and / or infants / young children suffering or having suffered from gut infection and / or gut inflammation.

[0408] It is indeed foreseen that the composition or combination of the invention may be even more beneficial to infants born with possibly impaired gut microbiota or fragile infants / young children (such as prematurely born infants and / or infants born by C-section). It is also foreseen that the composition or combination of the invention may be even more beneficial to infants / young children exhibiting intestinal disorders (such as diarrhea, infections or colic), especially after birth, for example, during the first 4 weeks after birth.

[0409] In embodiments of the invention, the infants born prematurely or born by caesarean section or born small for gestational age or with low birth weight, or exhibiting unbalanced or abnormal gut microbiota or suffering or having suffered from gut infection and / or gut inflammation, are targeted by the composition or combination of the present invention, and especially when the infants are 0-6 months of age. Without being bound by the theory, it is believed that youngerinfants benefit even more from the composition or combination of the invention, especially when the infants have (or are at risk of having) an unbalanced intestinal microbiota and / or have a fragile health condition (as exemplified by the conditions cited above).

[0410] Nutritional composition

[0411] In some embodiments, the composition or combination of the invention is in the form of a nutritional composition.

[0412] The nutritional composition according to the invention can be for example an infant formula, a starter infant formula, a follow-on or follow-up formula, a growing-up milk, a baby food, an infant cereal composition, a fortifier such as a human milk fortifier, a supplement, a pet food, or a pet food supplement.

[0413] In some preferred embodiments, the composition of the invention is an infant formula, a fortifier or a supplement that may be intended for the first 4 or 6 months of age. In a preferred embodiment, the nutritional composition of the invention is an infant formula. In other preferred embodiments, the nutritional composition of the invention is an infant formula intended for the first 6 months of age. In other preferred embodiments, the nutritional composition of the invention is a starter infant formula.

[0414] In some preferred embodiments, the nutritional composition of the invention is bovine milkbased infant formula. In some more preferred embodiments, the nutritional composition of the invention is bovine milk-based alpha-lactalbumin enriched infant formula.

[0415] In some embodiments, the nutritional composition contains only intact proteins as protein sources.

[0416] In some embodiments, the nutritional composition contains intact and hydrolysed proteins as protein sources.

[0417] In some other embodiments, the nutritional composition of the present invention is a fortifier. The fortifier can be a breast milk fortifier (e.g. a human milk fortifier) or a formula fortifier such as an infant formula fortifier or a follow-on / follow-up formula fortifier.

[0418] When the nutritional composition is a supplement, it can be provided in the form of unit doses. In such cases it is particularly useful to define the amount of oligosaccharides and probiotics in terms of daily dose to be administered to the infant, young child or child.

[0419] When the nutritional composition is a supplement, it may comprise the combination as described herein, and no other additional nutrient on top of the excipients necessary to obtain a stable nutritional composition.The nutritional composition of the present invention can be in solid (e.g. powder), liquid or gelatinous form. In a specific embodiment the nutritional composition is a supplement, wherein the supplement is in powder form and provided in a sachet, preferably a sachet with 0.1 to 20 g per sachet, for example 1 to 10 g per sachet, or in the form of a syrup, preferably a syrup with a total solid concentration of 5 to 75 g / 100 mL (5 to 75% (w / v)). When the supplement is in powder form, it may comprise a carrier. It is however preferred that the supplement is devoid of a carrier. When the supplement is in the form of a syrup, the components are preferably dissolved or suspended in water acidified with citrate. When the nutritional composition is a supplement, it can be free of protein.

[0420] In a particular embodiment the nutritional composition according to the invention is a hypoallergenic composition. In another particular embodiment the composition according to the invention is a hypoallergenic nutritional composition.

[0421] In some preferred embodiments, the nutritional composition is provided in the form of a powder for reconstitution with a liquid such as milk or water. The nutritional composition may have a reconstitution rate of from 100 to 150 g / L, for example 133 g / L.

[0422] In some preferred embodiments, the nutritional composition provided as a liquid or as a powder contains the following oligosaccharides in the following contents, wherein Stage 1 is preferably for infants from 0 to 6 months of age, Stage 2 is preferably for infants from 6 to 12 months of age, Stage 3 is preferably for young children from 1 to 3 of age, Stage 4 is preferably for young children from 3 of age to older, for example to 7 years of age:

[0423]

[0424]

[0425]

[0426]

[0427]

[0428] Other ingredients

[0429] The composition or combination according to the present invention may also comprise any other suitable ingredients.

[0430] Suitably, the composition or combination according to the present invention may also comprise other types of oligosaccharide(s), polysaccharides and / or a fiber(s) and / or a precursor(s) thereof. The other oligosaccharide and / or fiber and / or precursor thereof may be selected from the list comprising human milk oligosaccharides (HMOs), galacto-oligosaccharides (GOS), fructo-oligosaccharides (FOS), xylooligosaccharides (XOS), cello-oligosaccharides (COS), arabinoxylans, arabinans, xylans, inulin, polydextrose, beta-glucans, pectins and any combination thereof and any derived preparations thereof (e.g. partial hydrolysis). They may be in an amount between 0 and 10% by weight of composition.Additional HMOs which may be included in the composition or combination according to the present invention may be selected from the group consisting of lacto-N- fucopentaose (e.g. lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V), lacto-N-fucohexaose, lacto-N-difucohexaose I, fucosyllacto-N-hexaose, fucosyllacto-N-neohexaose, difucosyllacto-N-hexaose I, difucosyllacto-N-neohexaose II, para-lacto-N-neohexaose (para-LNnH), 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, lacto-N-decaose, and any combination thereof.

[0431] In some embodiments, the composition or combination according to the invention comprises at least one additional HMO.

[0432] In other embodiments, the composition or combination according to the present invention is devoid of any further HMOs. Thus, the HMO mixture as described herein may be the only HMOs in the composition or combination of the invention.

[0433] In some embodiments, the composition or combination of the present invention does not comprise at least one further probiotic (or probiotic strain), such as at least one further probiotic bacterial strain.

[0434] In other embodiments, the composition or combination of the present invention can further comprise at least one further probiotic (or probiotic strain), such as at least one further probiotic bacterial strain.

[0435] The 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. In some particular embodiments, the probiotic is a probiotic bacterial strain. In some specific embodiments, it is particularly Bifidobacteria and / or Lactobacilli.

[0436] Suitable probiotic bacterial strains according to the present invention include B. lactis CNCM 1-3446, 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 breve sold by Morinaga under the trade mark B-3, Bifidobacterium breve sold by sold by Yakult under the trade mark BBG-01, Bifidobacterium breve sold by Institut Rosell (Lallemand) under the trademark R0070, 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 1-1225, Streptococcus salivarius DSM 13084 sold by BLIS Technologies Limited of New Zealand under the designation KI2.

[0437] In some embodiments, the composition or combination of the invention comprises Bifidobacterium animalis subsp. lactis CNCM I-3446 or a strain having an Average Nucleotide Identity (ANI) of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% to Bifidobacterium animalis subsp. lactis CNCM I-3446.

[0438] In some embodiments, the composition or combination of the invention comprises Lactobacillus paracasei CNCM 1-2116 or a strain having an Average Nucleotide Identity (ANI) of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% to Lactobacillus paracasei CNCM 1-2116.

[0439] In some embodiments, the composition or combination of the invention comprises Lactobacillus rhamnosus CGMCC 1.3724 or a strain having an Average Nucleotide Identity (ANI) of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% to Lactobacillus rhamnosus CGMCC 1.3724.

[0440] The composition or combination according to the invention may contain from 10e3 to 10e12 cfu of the at least one (further) probiotic strain, more preferably from 10e6 to 10e12 cfu such as from 10e8 to 10e10 cfu of probiotic strain per g of composition or combination on a dry weight basis.

[0441] In one embodiment, the probiotics are viable. In another embodiment, the probiotics are nonreplicating or inactivated. There may be both viable probiotics and inactivated probiotics in some other embodiments. Probiotic components and metabolites can also be added.

[0442] Bifidobacterium animalis subsp. Lactis

[0443] The combination or composition of the invention may further comprise Bifidobacterium animalis subsp. lactis.

[0444] Any suitable Bifidobacterium animalis subsp. lactis (B. lactis) strain may be used in the present invention. Such strains will be well-known to the skilled person. Suitable strains include Bifidobacterium animalis subsp. lactis CNCM I-3446.The Bifidobacterium animalis subsp. lactis may be a strain having at least 99% (suitably, at least 99.9%) AN I to a Bifidobacterium animalis subsp. lactis strain known to the skilled person. Suitably, the Bifidobacterium animalis subsp. lactis 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 Bifidobacterium animalis subsp. lactis CNCM I-3446. Preferably, the Bifidobacterium animalis subsp. lactis has at least 99.9% ANI to Bifidobacterium animalis subsp. lactis CNCM I-3446.

[0445] Bifidobacterium lactis CNCM I-3446 was deposited with the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur (INSTITUT PASTEUR, 25 RUE DU DOCTEUR ROUX, F-75724 PARIS CEDEX 15, FRANCE) by NESTEC S.A. (NESTEC S.A., AVENUE NESTLE 55, CH-1800 VEVEY) according to the Budapest Treaty on 7thJune 2005 receiving the deposit number CNCM I-3446.

[0446] The composition or combination according to the invention may contain from 103to 1012cfu of Bifidobacterium animalis subsp. lactis, more preferably between 107and 1012cfu such as between 108and 101° cfu of Bifidobacterium animalis subsp. lactis per g of composition or combination on a dry weight basis. Suitably, the Bifidobacterium animalis subsp. lactis is administered to the subject in an amount of at least about 106cfu / day, at least about 107cfu / day, or at least about 108cfu / day. Suitably, the Bifidobacterium animalis subsp. lactis is administered to the subject in an amount of about 1012cfu / day or less, about 1011cfu / day or less, or about 101° cfu / day or less.

[0447] The composition or combination according to the invention may contain from 103to 1012cfu of Bifidobacteria (e.g. a combination of Bifidobacterium longum subsp. infantis and Bifidobacterium animalis subsp. lactis) more preferably between 107and 1012cfu such as between 108and 101° cfu of Bifidobacteria (e.g. of a combination of Bifidobacterium longum subsp. infantis and Bifidobacterium animalis subsp. lactis) per g of composition or combination on a dry weight basis. Suitably, the Bifidobacteria (e.g. a combination of Bifidobacterium longum subsp. infantis and Bifidobacterium animalis subsp. lactis) are administered to the subject in an amount of at least about 106cfu / day, at least about 107cfu / day, or at least about 108cfu / day. Suitably, the Bifidobacteria (e.g. a combination of Bifidobacterium longum subsp. infantis and Bifidobacterium animalis subsp. lactis) are administered to the subject in an amount of about 1012cfu / day or less, about 1011cfu / day or less, or about 101° cfu / day or less. In one embodiment, the Bifidobacterium animalis subsp. lactis is viable.

[0448] In another embodiment, the combination or composition according to the present invention also comprises docosahexaenoic acid, arachidonic acid, iron, vitamin B12 and folic acid.In a further embodiment, the combination or composition according to the present invention also comprises docosahexaenoic acid, arachidonic acid, iron, vitamin B12, folic acid, sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositole (PI) and phosphatidylserine (PS).

[0449] In another embodiment, the combination or composition according to the present invention also comprises docosahexaenoic acid, arachidonic acid, iron, vitamin B12, folic acid, sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositole (PI) and phosphatidylserine (PS) in specific amounts.

[0450] In one embodiment, the combination or composition according to the present invention also comprises sphingomyelin in an amount ranging from 300 mg / Kg to 820mg / kg, phosphatidylcholine in an amount 500 mg to 1.3g / Kg, phosphatidylinositol in an amount 400 mg / kg to 800mg / kg, phosphatidylserine in an amount ranging from 200mg / kg to 1000mg / kg, phosphatidylethanolamine in an amount ranging from 250mg / kg to 800mg / kg, iron in an amount ranging from ranging from 3.5mg / 100g to 7mg / 100g, Vitamin B12 in an amount ranging from ranging from 0.1mcg / 100g to 10 mcg / 100g, folic acid in an amount ranging from ranging from 110 mcg / 100g to 400 mcg / 100g and DHA and ARA in an amount 30mg / 100g to 300mg / 100g.The composition according to the invention generally contains a protein source. The protein can be in an amount of from 1.6 to 3 g per 100 kcal, for example about 2 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 / 100kcal or more than 3.6 g / 100kcal (or 3.6 g / 100kcal or more). In some other embodiments the protein amount can be below 2.0 g per 100 kcal, e.g. from 1.8 to 2 g / 100 kcal, or in an amount below 1.8 g per 100 kcal.

[0451] Protein sources based on whey, casein and mixtures thereof may be used as well as protein sources based on soy. As far as whey proteins are concerned, the protein source may be based on acid whey or sweet whey or mixtures thereof and may include alpha-lactalbumin and beta-lactoglobulin in any desired proportions. In some advantageous embodiments the protein source is whey predominant (i.e. more than 50% of proteins are coming from whey proteins, such as 51, 60% or 70%).

[0452] “A2 milk” generally refers to a variety of cow's milk that mostly lacks a form of p-casein proteins called A1, and instead has mostly the A2 form. A2 cow's milk is commercially available from the a2 Milk Company (Auckland, New Zealand). Non-cow milk, including that of humans, sheep, goats, donkeys, yaks, camels, buffalo, and others, also contain mostly A2 p-casein, and so the term "A2 milk" is also used in that context.In one embodiment of the present invention, the protein source predominantly contains A2 p-casein.

[0453] The term “standard SMP” refers to “standard skimmed milk powder” and is derived from milk obtained from mixed herds of cows and thus, comprises multiple variants of p-casein including A1 p -casein and A2 p -casein.

[0454] The term “A2 SMP” refers to “A2 skimmed milk powder” and comprises only A2 p-casein and not A1 p -casein.

[0455] 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. The term “hydrolysed” means in the context of the present invention a protein which has been hydrolysed or broken down into its component amino acids. The proteins may be either fully or partially hydrolysed. It may be desirable to supply partially hydrolysed 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 proteins are required, the hydrolysis process may be carried out as desired and as is known in the art. For example, whey protein hydrolysates may be prepared by enzymatically hydrolysing the whey fraction in one or more steps. If the whey fraction used as the starting material is substantially lactose free, it is found that the protein suffers much less lysine blockage during the hydrolysis process. This enables the extent of lysine blockage to be reduced from about 15% by weight of total lysine to less than about 10% (or to about 10% or less) by weight of lysine; for example about 7% by weight of lysine which greatly improves the nutritional quality of the protein source.

[0456] In an embodiment of the invention at least 70% of the proteins are hydrolysed, preferably at least 80% of the proteins are hydrolysed, such as at least 85% of the proteins are hydrolysed, even more preferably at least 90% of the proteins are hydrolysed, such as at least 95% of the proteins are hydrolysed, particularly at least 98% of the proteins are hydrolysed. In a particular embodiment, 100% of the proteins are hydrolysed.

[0457] In one particular embodiment the proteins of the nutritional composition are hydrolyzed, fully hydrolyzed or partially hydrolyzed. The degree of hydrolysis (DH) of the protein can bebetween 8 and 40, or between 20 and 60 or between 20 and 80 or more than 10, 20, 40, 60, 80 or 90 (or 10, 20, 40, 60, 80 or 90 or more).

[0458] The protein component can alternatively be replaced by a mixture or synthetic amino acid, for example for preterm or low birth weight infants.

[0459] The composition according to the present invention generally contains a carbohydrate source. The carbohydrate can be in an amount of from 5 to 20 g per 100 kcal, for example from 10 to 15 g per 100 kcal. This is particularly preferable in the case where the composition of the invention is an infant formula. In this case, any carbohydrate source conventionally found in infant formulae such as lactose, sucrose, saccharose, maltodextrin, starch and mixtures thereof may be used although one of the preferred sources of carbohydrates is lactose. The composition according to the present invention generally contains a source of lipids. The lipids can be in an amount of from 2 to 8 g per 100 kcal, for example from 5 to 6 g per 100 kcal. This is particularly relevant if the composition of the invention is an infant formula. In this case, the lipid source may be any lipid or fat which is suitable for use in infant formulae. Some suitable fat sources include palm oil, structured triglyceride oil, high oleic sunflower oil and high oleic safflower oil, medium-chain-triglyceride oil. The essential fatty acids linoleic and a-linolenic acid may also be added, as well small amounts of oils containing high quantities of preformed arachidonic acid and docosahexaenoic acid such as fish oils or microbial oils. The fat source may have 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.

[0460] The composition of the invention may also contain all vitamins and minerals understood to be essential in the daily diet and in nutritionally significant amounts. Minimum requirements have been established for certain vitamins and minerals. Examples of minerals, vitamins and other nutrients optionally present in the composition of the invention include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin E, vitamin K, vitamin C, vitamin D, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorous, iodine, iron, magnesium, copper, zinc, manganese, chlorine, potassium, sodium, selenium, chromium, molybdenum, taurine, and L-carnitine. Minerals are usually added in salt form. The presence and amounts of specific minerals and other vitamins will vary depending on the intended population.

[0461] If necessary, the composition of the invention may contain emulsifiers and stabilisers such as soy, lecithin, citric acid esters of mono- and di-glycerides, and the like.

[0462] The composition of the invention may also contain other substances which may have a beneficial effect such as nucleotides, nucleosides, and the like.The composition of the invention may also contain carotenoid(s). In some particular embodiments of the invention, the composition of the invention does not comprise any carotenoid.

[0463] The composition of the invention may comprise from 600 to 700 kcal / L, for example from 650 to 680 kcal / L

[0464] Additional aspects and embodiments of the present invention

[0465] S1. A combination of a human milk oligosaccharide (HMO) mixture, milk fat globule membrane (MFGM) and Bifidobacterium longum subsp. infant is, wherein the HMO mixture comprises or consists of 3'-Galactosyllactose (3’GL), 6'-Galactosyllactose (6’GL), 4'-Galactosyllactose (4’GL), 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3FL) and / or lacto-N-neotetraose (LNnT).S2. The combination according to S1 wherein the combination is administered separately, simultaneously or sequentially, preferably wherein the combination is administered simultaneously.

[0466] 53. The combination according to S1 to S2, wherein the combination is provided in the form of a nutritional composition, preferably wherein the nutritional composition is selected from an infant formula, a starter infant formula, a follow-on or follow-up formula, a baby food, an infant cereal composition, a growing-up-milk, a fortifier such as a human milk fortifier, or a supplement, more preferably wherein the nutritional composition is an infant formula.

[0467] 54. The combination according to any preceding embodiment S1 to S3, wherein the HMO mixture comprises or consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), 3-fucosyllactose (3FL), and lacto-N-neotetraose (LNnT).

[0468] 55. The combination according to any preceding embodiment S1 to S4, wherein the HMO mixture consists or consists essentially of:

[0469] i. 20 wt% to 60 wt% of 2FL, preferably 22 wt% to 55 wt%;

[0470] ii. 4 wt% to 30 wt% of LNT, preferably 6 wt% to 20 wt%;

[0471] iii. 1 wt% to 12 wt % of DFL, preferably 2 wt% to 8 wt%;

[0472] iv. 7 wt% to 23 wt% of 6SL and 3SL combined, preferably 8 wt% to 22 wt%;

[0473] v. 10 wt% to 50 wt% of 3FL, preferably 13 wt% to 46 wt% and

[0474] vi. 3 wt% to 25 wt% of LNnT, preferably 5 wt% to 20 wt%.56. The combination according to any preceding embodiment S1 to S5, wherein the combination is provided in the form of a composition having a total HMO concentration of from 0.5 g / L to 10 g / L, preferably from 1 g / L to 5 g / L, or more preferably from 2 g / L to 3 g / L.

[0475] 57. The combination according to any preceding embodiment S1 to S6, wherein the combination is provided in the form of a composition having a total MOS concentration of from 0.5 g / L to 10 g / L, preferably from 2 g / L to 8 g / L, or more preferably from 4 g / L to 5 g / L.

[0476] 58. The combination according to any preceding embodiment S1 to S7, wherein the Bifidobacterium longum subsp. infantis is Bifidobacterium long urn subsp. infantis LMG 11588 or a strain having an Average Nucleotide Identity (ANI) of at least 95%, preferably at least 99%, more preferably at least 99.9% to Bifidobacterium longum subsp. infantis LMG 11588.

[0477] 59. The combination according to any preceding embodiment S1 to S8, wherein the combination comprises from 103to 1012cfu of Bifidobacterium longum subsp. infantis, preferably from 105to 101° cfu of Bifidobacterium longum subsp. infantis, more preferably from 105to 107cfu of Bifidobacterium longum subsp. infantis per g of combination or composition on a dry weight basis.

[0478] 510. A combination according to any preceding embodiment S1 to S9 for use in preventing and / or treating constipation and / or improving stool consistency and / or stool frequency.

[0479] 511. The combination for use according to S11 , wherein the infant or young child has an age from 0 months to 12 months, preferably from 0 months to 6 months.

[0480] 512. The combination for use according to embodiment S10 to S11, wherein the infant or young child is exclusively formula-fed.

[0481] 513. The combination for use according to any preceding embodiment S10 to S12, for use in reducing the incidence and / or severity of constipation in said infant or young child.

[0482] 514. The combination for use according to any any preceding embodiment S10 to S13, for use in reducing stool hardness in said infant or young child.

[0483] 515. The combination for use according to any preceding embodiment S10 to S14, for use in increasing stool frequency in said infant or young child.

[0484] 516. The combination for use according to any preceding embodiment S10 to S15, wherein the combination prevents and / or treats constipation and / or improves stool consistency and / or stool frequency without any associated gut discomfort or digestive disorders, such as flatulence, bloating, and / or colics.

[0485] 517. The combination according to any of embodiment S1 to S9, for use in for use in preventing and / or treating a viral infection in a subject.

[0486] 518. The combination for use according to embodiment S17, for use in preventing and / or treating a viral infection in a subject, wherein the composition is for use in reducing the risk of developing the viral infection and / or reducing the symptoms associated with the viral infection.519. The composition for use according to embodiemnts S17 or S18, , wherein the viral infection is caused by a virus selected from the group consisting of respiratory syncytial virus (RSV), parainfluenza virus (PIV), influenza virus such as influenza virus A (IVA) and / or influenza virus B (IVB), rhinovirus (RV), adenovirus (ADV), metapneumovirus (MPV), bocavirus (BoV), coronavirus (CoV), myxovirus, herpesvirus, enterovirus (EV), and parachovirus (PeV), or any combination thereof, preferably wherein the viral infection is caused by RSV.

[0487] 520. The combination according to any of embodiment S1 to S9, for use in i) preventing and / or reducing the risk of allergen sensitisation in the respiratory tract and / or ii) preventing and / or reducing the risk of developing a respiratory condition in a subject, and wherein the respiratory condition is a chronic inflammatory disease of the respiratory tract or an allergic respiratory tract disease.

[0488] 521. The combination for use according to embodiment S20, wherein wherein the chronic inflammatory disease of the respiratory tract is asthma or chronic obstructive pulmonary disease (COPD).

[0489] 522. The combination for use according to embodiment S20 or S21, wherein the allergic respiratory tract disease is recurrent wheeze or asthma.

[0490] 523. The combination for use according to embodiment S20, wherein respiratory condition is an Upper Respiratory Tract infection (URTI).

[0491] 524. The combination according to any of embodiments S1 to S9 for use in: i) preventing and / or treating bacterial infections in an infant or a young child; ii) modulating the microbiota of an infant or a young child; and / or iii) preventing and / or treating allergy of an infant or a young child.

[0492] 525. The combination for use according to embodiment S24 wherein the use is for modulating the microbiota of an infant or a young child and administration of said composition results in abundance of Bifidobacteriaceae and / or Bifidobacterium longum subsp. infantis being increased.

[0493] 526. The combination for use according to any of embodiment S24 or S25 wherein the use is for modulating the microbiota of an infant or a young child; and / or preventing and / or treating allergy of an infant or a young child by modulating intestinal short-chain fatty acids (SCFA) production in such infant or young child.

[0494] 527. The combination according to any preceding embodiment S1 to S26 wherein the combination is combination of a human milk oligosaccharide (HMO) mixture, milk fat globule membrane (MFGM) and Bifidobacterium longum subsp. infantis, wherein the HMO mixture comprises or consists of 3'-Galactosyllactose (3’GL), 6'-Galactosyllactose (6’GL), 4'-Galactosyllactose (4’GL), 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyl lactose (3FL)and / or lacto-N-neotetraose (LNnT) and wherein MFGM comprise phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and sphingomyelin.

[0495] 528. The combination according to any preceding embodiment S1 to S26 wherein the combination is combination of a human milk oligosaccharide (HMO) mixture, phospholipids and Bifidobacterium longum subsp. infantis, wherein the HMO mixture comprises or consists of 3'-Galactosyllactose (3’GL), 6'-Galactosyllactose (6’GL), 4'-Galactosyllactose (4’GL), 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3FL) and / or lacto-N-neotetraose (LNnT).

[0496] 529. The combination according to embodiment 28 wherein phospholipids comprise phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and / or sphingomyelin.

[0497] 530. Non-therapeutic use of a combination according to anyone of embodiments S1 to S9 for promotion of gastro intestinal tolerance in a subject.

[0498] 531. A combination of a human milk oligosaccharide (HMO) mixture, and Bifidobacterium longum subsp. infantis, wherein the HMO mixture comprises or consists of 3'-Galactosyllactose (3’GL), 6'-Galactosyllactose (6’GL), 4'-Galactosyllactose (4’GL), 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3FL) and / or lacto-N-neotetraose (LNnT).

[0499] 532. The combination according to S1 wherein the combination is administered separately, simultaneously or sequentially, preferably wherein the combination is administered simultaneously.

[0500] 533. The combination according to S31 to S32, wherein the combination is provided in the form of a nutritional composition, preferably wherein the nutritional composition is selected from an infant formula, a starter infant formula, a follow-on or follow-up formula, a baby food, an infant cereal composition, a growing-up-milk, a fortifier such as a human milk fortifier, or a supplement, more preferably wherein the nutritional composition is an infant formula.

[0501] 534. The combination according to any preceding embodiment S31 to S33, wherein the HMO mixture comprises or consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), 3-fucosyllactose (3FL), and lacto-N-neotetraose (LNnT).

[0502] 535. The combination according to any preceding embodiment S31 to S34, wherein the HMO mixture consists or consists essentially of:i. 20 wt% to 60 wt% of 2FL, preferably 22 wt% to 55 wt%;

[0503] ii. 4 wt% to 30 wt% of LNT, preferably 6 wt% to 20 wt%;

[0504] iii. 1 wt% to 12 wt % of DFL, preferably 2 wt% to 8 wt%;

[0505] iv. 7 wt% to 23 wt% of 6SL and 3SL combined, preferably 8 wt% to 22 wt%;

[0506] v. 10 wt% to 50 wt% of 3FL, preferably 13 wt% to 46 wt% and

[0507] vi. 3 wt% to 25 wt% of LNnT, preferably 5 wt% to 20 wt%.

[0508] 536. The combination according to any preceding embodiment S31 to S35, wherein the combination is provided in the form of a composition having a total HMO concentration of from 0.5 g / L to 10 g / L, preferably from 1 g / L to 5 g / L, or more preferably from 2 g / L to 3 g / L.

[0509] 537. The combination according to any preceding embodiment S31 to S36, wherein the combination is provided in the form of a composition having a total MOS concentration of from 0.5 g / L to 10 g / L, preferably from 2 g / L to 8 g / L, or more preferably from 4 g / L to 5 g / L.

[0510] 538. The combination according to any preceding embodiment S31 to S37, wherein the Bifidobacterium longum subsp. infantis is Bifidobacterium long urn subsp. infantis LMG 11588 or a strain having an Average Nucleotide Identity (ANI) of at least 95%, preferably at least 99%, more preferably at least 99.9% to Bifidobacterium longum subsp. infantis LMG 11588.

[0511] 539. The combination according to any preceding embodiment S31 to S38, wherein the combination comprises from 103to 1012cfu of Bifidobacterium longum subsp. infantis, preferably from 105to 101° cfu of Bifidobacterium longum subsp. infantis, more preferably from 105to 107cfu of Bifidobacterium longum subsp. infantis per g of combination or composition on a dry weight basis.

[0512] 540. A combination according to any preceding embodiment S31 to S39 for use in preventing and / or treating constipation and / or improving stool consistency and / or stool frequency.

[0513] 541. The combination for use according to S40, wherein the infant or young child has an age from 0 months to 12 months, preferably from 0 months to 6 months.

[0514] 542. The combination for use according to embodiment S30 to S41, wherein the infant or young child is exclusively formula-fed.

[0515] 543. The combination for use according to any preceding embodiment S30 to S42, for use in reducing the incidence and / or severity of constipation in said infant or young child.

[0516] 544. The combination for use according to any any preceding embodiment S30 to S43, for use in reducing stool hardness in said infant or young child.

[0517] 545. The combination for use according to any preceding embodiment S30 to S44, for use in increasing stool frequency in said infant or young child.

[0518] 546. The combination for use according to any preceding embodiment S30 to S45, wherein the combination prevents and / or treats constipation and / or improves stool consistency and / or stool frequency without any associated gut discomfort or digestive disorders, such as flatulence, bloating, and / or colics.547. The combination according to any of embodiment S30 to S39, for use in for use in preventing and / or treating a viral infection in a subject.

[0519] 548. The combination for use according to embodiment S47, for use in preventing and / or treating a viral infection in a subject, wherein the composition is for use in reducing the risk of developing the viral infection and / or reducing the symptoms associated with the viral infection.

[0520] 549. The composition for use according to embodiemnts S47 or S48, , wherein the viral infection is caused by a virus selected from the group consisting of respiratory syncytial virus (RSV), parainfluenza virus (PIV), influenza virus such as influenza virus A (IVA) and / or influenza virus B (IVB), rhinovirus (RV), adenovirus (ADV), metapneumovirus (MPV), bocavirus (BoV), coronavirus (CoV), myxovirus, herpesvirus, enterovirus (EV), and parachovirus (PeV), or any combination thereof, preferably wherein the viral infection is caused by RSV.

[0521] 550. The combination according to any of embodiment S31 to S39, for use in i) preventing and / or reducing the risk of allergen sensitisation in the respiratory tract and / or ii) preventing and / or reducing the risk of developing a respiratory condition in a subject, and wherein the respiratory condition is a chronic inflammatory disease of the respiratory tract or an allergic respiratory tract disease.

[0522] 551. The combination for use according to embodiment S50, wherein wherein the chronic inflammatory disease of the respiratory tract is asthma or chronic obstructive pulmonary disease (COPD).

[0523] 552. The combination for use according to embodiment S50 or S51 , wherein the allergic respiratory tract disease is recurrent wheeze or asthma.

[0524] 553. The combination for use according to embodiment S50, wherein respiratory condition is an Upper Respiratory Tract infection (URTI).

[0525] 554. The combination according to any of embodiments S13 to S39 for use in: i) preventing and / or treating bacterial infections in an infant or a young child; ii) modulating the microbiota of an infant or a young child; and / or iii) preventing and / or treating allergy of an infant or a young child.

[0526] 555. The combination for use according to embodiment S54 wherein the use is for modulating the microbiota of an infant or a young child and administration of said composition results in abundance of Bifidobacteriaceae and / or Bifidobacterium longum subsp. infantis being increased.

[0527] 556. The combination for use according to any of embodiment S54 or S55 wherein the use is for modulating the microbiota of an infant or a young child; and / or preventing and / or treating allergy of an infant or a young child by modulating intestinal short-chain fatty acids (SCFA) production in such infant or young child.S57. Non-therapeutic use of a combination according to anyone of embodiments S31 to S39 for promotion of gastro intestinal tolerance in a subject.

[0528] 561. A combination of a human milk oligosaccharide (HMO) mixture, milk fat globule membrane (MFGM), Bifidobacterium longum subsp. inf antis. docosahexaenoic acid, arachidonic acid, iron, vitamin B12, folic acid, sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositole (PI) phosphatidylserine (PS) AND Bifidobacterium animalis subsp. Lactis, wherein the HMO mixture comprises or consists of 3'-Galactosyllactose (3’GL), 6'-Galactosyllactose (6’GL), 4'-Galactosyllactose (4’GL), 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3FL) and / or lacto-N-neotetraose (LNnT).

[0529] 562. The combination according to S61 wherein the combination is administered separately, simultaneously or sequentially, preferably wherein the combination is administered simultaneously.

[0530] 563. The combination according to S61 to S62, wherein the combination is provided in the form of a nutritional composition, preferably wherein the nutritional composition is selected from an infant formula, a starter infant formula, a follow-on or follow-up formula, a baby food, an infant cereal composition, a growing-up-milk, a fortifier such as a human milk fortifier, or a supplement, more preferably wherein the nutritional composition is an infant formula.

[0531] 566. The combination according to any preceding embodiment S61 to S65, wherein the combination is provided in the form of a composition having a total HMO concentration of from 0.5 g / L to 10 g / L, preferably from 1 g / L to 5 g / L, or more preferably from 2 g / L to 3 g / L.

[0532] 567. The combination according to any preceding embodiment S661 to S66, wherein the combination is provided in the form of a composition having a total MOS concentration of from 0.5 g / L to 10 g / L, preferably from 2 g / L to 8 g / L, or more preferably from 4 g / L to 5 g / L.

[0533] 568. The combination according to any preceding embodiment S61 to S67, wherein the Bifidobacterium longum subsp. infantis is Bifidobacterium longum subsp. infantis LMG 11588 or a strain having an Average Nucleotide Identity (ANI) of at least 95%, preferably at least 99%, more preferably at least 99.9% to Bifidobacterium longum subsp. infantis LMG 11588.

[0534] 569. The combination according to any preceding embodiment S61 to S68, wherein the combination comprises from 103to 1012cfu of Bifidobacterium longum subsp. infantis, preferably from 105to 101° cfu of Bifidobacterium longum subsp. infantis, more preferably from 105to 107cfu of Bifidobacterium longum subsp. infantis per g of combination or composition on a dry weight basis.570. A combination according to any preceding embodiment S61 to S69 for use in preventing and / or treating constipation and / or improving stool consistency and / or stool frequency.

[0535] 571. The combination for use according to S70, wherein the infant or young child has an age from 0 months to 12 months, preferably from 0 months to 6 months.

[0536] 572. The combination for use according to embodiment S70 to S71, wherein the infant or young child is exclusively formula-fed.

[0537] 573. The combination for use according to any preceding embodiment S70 to S72, for use in reducing the incidence and / or severity of constipation in said infant or young child.

[0538] 574. The combination for use according to any any preceding embodiment S70 to S73, for use in reducing stool hardness in said infant or young child.

[0539] 575. The combination for use according to any preceding embodiment S70 to S74, for use in increasing stool frequency in said infant or young child.

[0540] 576. The combination for use according to any preceding embodiment S70 to S75, wherein the combination prevents and / or treats constipation and / or improves stool consistency and / or stool frequency without any associated gut discomfort or digestive disorders, such as flatulence, bloating, and / or colics.

[0541] 577. The combination according to any of embodiment S61 to S69, for use in for use in preventing and / or treating a viral infection in a subject.

[0542] 578. The combination for use according to embodiment S77, for use in preventing and / or treating a viral infection in a subject, wherein the composition is for use in reducing the risk of developing the viral infection and / or reducing the symptoms associated with the viral infection.

[0543] 579. The composition for use according to embodiemnts S77 or S78, , wherein the viral infection is caused by a virus selected from the group consisting of respiratory syncytial virus (RSV), parainfluenza virus (PIV), influenza virus such as influenza virus A (IVA) and / or influenza virus B (IVB), rhinovirus (RV), adenovirus (ADV), metapneumovirus (MPV), bocavirus (BoV), coronavirus (CoV), myxovirus, herpesvirus, enterovirus (EV), and parachovirus (PeV), or any combination thereof, preferably wherein the viral infection is caused by RSV.

[0544] 580. The combination according to any of embodiment S61 to S69, for use in i) preventing and / or reducing the risk of allergen sensitisation in the respiratory tract and / or ii) preventing and / or reducing the risk of developing a respiratory condition in a subject, and wherein the respiratory condition is a chronic inflammatory disease of the respiratory tract or an allergic respiratory tract disease.

[0545] 581. The combination for use according to embodiment S70, wherein wherein the chronic inflammatory disease of the respiratory tract is asthma or chronic obstructive pulmonary disease (COPD).582. The combination for use according to embodiment S70 or S71 , wherein the allergic respiratory tract disease is recurrent wheeze or asthma.

[0546] 583. The combination for use according to embodiment S70, wherein respiratory condition is an Upper Respiratory Tract infection (URTI).

[0547] 584. The combination according to any of embodiments S61 to S69 for use in: i) preventing and / or treating bacterial infections in an infant or a young child; ii) modulating the microbiota of an infant or a young child; and / or iii) preventing and / or treating allergy of an infant or a young child.

[0548] 585. The combination for use according to embodiment S74 wherein the use is for modulating the microbiota of an infant or a young child and administration of said composition results in abundance of Bifidobacteriaceae and / or Bifidobacterium longum subsp. infantis being increased.

[0549] 586. The combination for use according to any of embodiment S74 or S75 wherein the use is for modulating the microbiota of an infant or a young child; and / or preventing and / or treating allergy of an infant or a young child by modulating intestinal short-chain fatty acids (SCFA) production in such infant or young child.

[0550] 587. The combination according to any preceding embodiment S61 to S76 wherein the combination is combination of a human milk oligosaccharide (HMO) mixture, milk fat globule membrane (MFGM) and Bifidobacterium longum subsp. infantis, wherein the HMO mixture comprises or consists of 3'-Galactosyllactose (3’GL), 6'-Galactosyllactose (6’GL), 4'-Galactosyllactose (4’GL), 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyl lactose (3FL) and / or lacto-N-neotetraose (LNnT) and wherein MFGM comprise phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and sphingomyelin. 588. The combination according to any preceding embodiment S61 to S76 wherein the combination is combination of a human milk oligosaccharide (HMO) mixture, phospholipids and Bifidobacterium longum subsp. infantis, wherein the HMO mixture comprises or consists of 3'-Galactosyllactose (3’GL), 6'-Galactosyllactose (6’GL), 4'-Galactosyllactose (4’GL), 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3FL) and / or lacto-N-neotetraose (LNnT).

[0551] 589. The combination according to embodiment 78 wherein phospholipids comprise phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and / or sphingomyelin.

[0552] S90. The combination or combination for use according to any of the preceding embodiments wherein the protein source predominantly contains A2 p-casein.S91. The combination or combination for use according to any of the preceding embodiments, wherein it further comprises Bifidobacterium animalis subsp. Lactis, for example Bifidobacterium animalis subsp. lactis CNCM I-3446.

[0553] Manufacture of a composition

[0554] The composition or combination according to the invention may be prepared in any suitable manner. A composition will now be described by way of example.

[0555] For example, a nutritional composition such as an infant formula may be prepared by blending together the protein source, 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 they 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 in the range between about 50°C and about 80°C to aid dispersal of the ingredients. Commercially available liquefiers may be used to form the liquid mixture.

[0556] The oligosaccharide(s) may be added at this stage, especially if the final product is to have a liquid form. If the final product is to be a powder, they may likewise be added at this stage if desired.

[0557] The liquid mixture is then homogenised, for example in two stages.

[0558] The liquid mixture may then be thermally treated to reduce bacterial loads, by rapidly heating the liquid mixture to a temperature in the range between about 80°C and about 150°C for a duration between about 5 seconds and about 5 minutes, for example. This may be carried out by means of steam injection, an autoclave or a heat exchanger, for example a plate heat exchanger.

[0559] Then, the liquid mixture may be cooled to between about 60°C and about 85°C for example by flash cooling. The liquid mixture may then be again homogenised, for example in two stages between about 10 MPa and about 30 MPa in the first stage and between about 2 MPa and about 10 MPa in the second stage. The homogenised mixture may then be further cooled toadd any heat sensitive components, such as vitamins and minerals. The pH and solids content of the homogenised mixture are conveniently adjusted at this point.

[0560] If the final product is to be a powder, the homogenised mixture is transferred to a suitable drying apparatus such as a spray dryer or freeze dryer and converted to powder. The powder should have a moisture content of less than about 5% (or about 5% or less) by weight. The oligosaccharide(s) may also or alternatively be added at this stage by dry-mixing or by blending them in a syrup form of crystals, along with the probiotic strain(s), and the mixture is spray-dried or freeze-dried.

[0561] If a liquid composition is preferred, the homogenised mixture may be sterilised then aseptically filled into suitable containers or may be first filled into the containers and then retorted.

[0562] In another embodiment, the composition of the invention may be a supplement such as a paediatric supplement. 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, cocompounds, 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 conventional pharmaceutical 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.

[0563] 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.

[0564] This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range.

[0565] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between anystated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

[0566] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0567] The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms "comprising", "comprises" and "comprised of" also include the term "consisting of".

[0568] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J.M. and McGee, J.O’D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M.J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D.M. and Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is herein incorporated by reference.

[0569] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0570] The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.EXAMPLES

[0571] Example 1 - Example nutritional composition

[0572] An example of a nutritional composition (e.g. an infant formula) according to the present invention is given in the below table. This composition is given by way of illustration only.

[0573]

[0574]

[0575] In one embodiment of the present invention, the nutritional composition as described in example 1 comprises a source of protein which is enriched in MFGM, for example one alpha lactalbumin enriched whey protein ingredient as above described in an amount comprised between 1.4 and 1.6g / 100 of the dry composition. In such embodiment, the nutritional composition of Example 1 also comprises phospholipids derived from such ingredient.

[0576] Example 2 - randomized, double-blind, controlled trial

[0577] The purpose of this study was to evaluate growth and gastrointestinal (Gl) tolerance in healthy infants fed a new synbiotic-supplemented infant formula. This was a randomized, controlled, double-blind, 2-arm clinical trial. Infants aged >14 to <35 days and exclusively formula-fed were randomized to receive Control Formula or Experimental Formula from enrollment until age 6 months.

[0578] Methods

[0579] Trial population: 240 healthy infants, both male and female, between the ages of 14 and 35 days and with the following characteristics:

[0580] Term birth (> 37 weeks of gestation)

[0581] Normal birth weight (> 2500 g and < 4500 g)

[0582] Exclusively consuming and tolerating a cow’s milk infant formula at time of enrollment No evidence of feeding difficulties or formula intolerance

[0583] No medical conditions that would require special infant formula or that could increase the risk associated with study participation

[0584] No present or previous usage of medications or dietary supplements that could affect study

[0585] Control formula: a bovine milk-based alpha-lactalbumin enriched infant formula.Experimental formula: a bovine milk-based alpha-lactalbumin enriched infant formula as above supplemented with:

[0586] - 2.64 g / L added HMOs (2’-FL, 3-FL, DFL, 3’-SL, 6’-SL, LNnT, LNT)

[0587] - 4.56 g / L MOS (providing 3’-GL, 4’-GL, and 6’-GL)

[0588] 1 x 106colony forming units (CFU) / g B. infantis

[0589] The concentrations of the HMOs in the experimental formula were 1.00 g / L for 2’-FL, 0.385 g / L for 3-FL, 0.121 g / L for DFL, 0.102 g / L for 3’-SL, 0.181 g / L for 6’-SL, 0.348 g / L for LNT, and 0.500 g / L for LNnT.

[0590] Treatment administration: The study formulas were fed orally ad libitum from enrollment until 6 months of age. The experimental and control formulas were provided as powders in 400g cans and were reconstituted in water according to instructions provided on the product label. The study included an optional observational follow-up period until 36 months of age.

[0591] Assessments:

[0592] - Anthropometric measurements at infant age >14 to <35 days (baseline / V1), 1.5 months (V2), 2 months (V3), 3 months (V4), 4 months (V5) and 6 months (V6) (all visits). Children were weighed without clothing or diaper on a calibrated electronic weighing scale and weight recorded to the nearest 10 grams.

[0593] Stool frequency and consistency, Gl symptoms and Gl-related behaviours were recorded in the retrospective 1-day (at baseline) and prospective 3-day Gl Symptom and Behavior Diary for 3 days prior to 1.5 months (V2), 2 months (V3), 3 months (V4), 4 months (V5) and 6 months (V6).

[0594] Stool frequency was calculated as the mean number of stools reported retrospectively over a 1-day period (V1) or prospectively over each 3-day period. For each bowel movement, parent(s) / LAR(s) indicated whether the infant had difficulty in passing stool. Stool consistency was calculated as the mean stool consistency of each stool reported retrospectively over a 1-day period (V1) or prospectively over each 3-day period. Stool consistency was based on a validated 5-point stool scale developed for infants < one year of age: 0=watery, 1=runny, 2=mushy-soft, 3=formed, 4=hard.

[0595] Gl symptom burden index was derived from the IGSQ-13 conducted at each visit - baseline (V1; age >14 to <35 days), age 1.5 months (V2), 2 months (V3), 3 months (V4), 4 months (V5) and 6 months (V6). The IGSQ-13 is an interviewer-assisted questionnaire which assesses an infant’s Gl symptoms over the past week from theparent(s) / LAR(s) perspective. The questionnaire is composed of 13 questions, categorized into 5 symptom domains of stooling, vomiting / spitting-up, crying, fussiness, and flatulence. The scores are summed to calculate an overall index score as a measure of total Gl symptom burden. The values for the IGSQ index score can range from 13 (low Gl burden) to 65 (high Gl burden). The IGSQ is a validated method for assessment of infant Gl-related behaviors (see e.g. Riley AW et al. (2015) Clin Pediatr (Phila) 54, 1167-1174).

[0596] Infant illness and infection outcomes were assessed as part of standard adverse event (AE) reporting. Parent-reported and physician-verified AEs (including serious AEs) were collected from signing of the informed consent form through the post-intervention phone call (P7). The AE information included the type, severity, and seriousness of the event plus the relatedness to the study formulas per the judgement of the study physicians. In addition, medication type and duration (particularly for antibiotics and antipyretics) was also collected continuously during this interval.

[0597] Stool samples were collected at baseline, 2, 4, and 6 months and analyzed for the following endpoints:

[0598] o Fecal metabolic profile including pH and organic acids

[0599] o Fecal markers of gut immunity (secretory immunoglobulin A [slgA], gut inflammation (calprotectin), and gut barrier function (alpha-1 antitrypsin) o Fecal microbiome including compositional and functional aspects

[0600] Statistical : Continuous variables were summarized using the appropriate descriptive statistics, including but not limited to: number of observations (n), mean, standard deviation (SD), median, minimum, and maximum.

[0601] Weight gain was analyzed by ANCOVA correcting for baseline weight, sex, and center. Non-inferiority must be demonstrated in the per-protocol analysis set. Non-inferiority will be concluded if the lower bound of the two-sided 95% confidence interval of the treatment difference between groups is above the non-inferiority margin of -3 g / day. Stool frequency was analyzed by ANCOVA correcting for baseline value, age, sex, and center.

[0602] Stool consistency was calculated as the mean consistency of each stool reported retrospectively over a 1-day period (at baseline) or prospectively over a 3-day period just prior to each study visit. Stool consistency was based on a 5-point stool scale developed for infants < 1 year of age: 0=watery, 1=runny, 2=mushy-soft, 3=formed, 4=hard).

[0603] - All AEs were coded into system organ classes (SOC) and preferred terms (PT) using MedDRA (version 25). For each SOC, the number of events and the number of infantswith at least one event were compared between groups using Fisher’s Exact Test. The same approach was used to compare certain PTs within the “Infections and Infestations” SOC. In addition, the number of infants with > 2 episodes of certain PTs within this same system organ class (SOC) were compared between groups using Poisson Regression.

[0604] Stool endpoints were analyzed as follows:

[0605] o Fecal metabolic profile including pH and organic acids in dry samples were analyzed by ANCOVA correcting for baseline value, sex, and center. Individual short-chain fatty acids were analyzed in absolute terms and also expressed as a percentage of total short-chain fatty acids.

[0606] o Fecal markers slgA, calprotectin, and alpha-1 antitrypsine were measured in dry samples and analyzed by ANCOVA correcting for baseline value, sex, and center

[0607] o For fecal microbiome endpoints, samples were analyzed using a pipeline that yields the taxonomical composition of the microbiome, measures of alpha and beta diversity, and the functional composition of the microbiome. In addition, strain-level microbiome profiling based on single-nucleotide variants was used to track the administered B. infantis LFM11588 probiotic. Comparisons between the two formula groups were performed using a linear (mixed) model correcting for infant age at baseline and center. Multiple testing corrections were performed using the Benjamini-Hochberg method. Genus-level microbiome fecal community types (FCTs) were defined using Dirichlet Multinomial Mixture models and clustered into longitudinal trajectories based on time spent in each FCT.

[0608] Results

[0609] Weight gain velocity

[0610] Weight gain velocity was non-inferior in Experimental Formula-fed infants compared to Control Formula-fed infants. In the per-protocol set, the estimated mean difference in weight gain velocity between Experimental Formula-fed infants (n=107; mean±SD 28.4±6.5 g / d) and Control Formula-fed infants (n=110; 27.4±6.4 g / d) was 1.01 g / d (95% Cl: -0.67, 2.68; p-value for non-inferiority < 0.001).

[0611] There were no significant differences in weight-for-age, length-for-age, weight-for-length, or head circumference-for-age z-scores between groups at any time point.

[0612] Stool consistencyWe observed slightly but statistically significantly softer stools in infants fed Experimental Formula vs. infants fed Control Formula. Stools were generally softer (1.8 vs. 2.0 at 1.5, 2 and 4mo, p<0.0013) in infants fed Experimental Formula vs. infants fed Control Formula. Softer stools were observed after only -19 days of feeding.

[0613]

[0614] Table 1: Mean + SD stool consistency scores in the Full Analysis Set (FAS) based on a 5-point scale (0=watery, 1=runny, 2=mushy-soft, 3=formed, 4=hard). P-values are from an ANCOVA model correcting for baseline value, age, sex and site

[0615] Hard stools may lead to constipation. Therefore, these results indicate that the Experimental Formula promotes softer stools that can help reduce the incidence and severity of constipation in infants.

[0616] Stool frequency

[0617] We observed slightly but statistically significantly more stools per day in infants fed Experimental Formula vs. infants fed Control Formula at 3 months onwards. Stools were more frequent (-2 vs. ~1.5 stools / d after 2 mo, p<0.0457) in infants fed Experimental Formula vs. infants fed Control Formula.

[0618]

[0619] Table 2: Mean + SD stool frequency in the FAS. P-values are from an ANCOVA model correcting for baseline value, age, sex and siteGl tolerance

[0620] Mean IGSQ scores in the FAS were low (<18) in both groups at all time points, indicating good Gl tolerance (Figure 1)

[0621] Adverse event (AE) incidence was similar in both groups (51.4% and 53.5% in EF and CF, respectively). Only 8 serious adverse events (SAEs) were reported (4 events in 4 infants per group). Most AEs were mild and considered unrelated to the product.

[0622] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

[0623] Adverse Events

[0624] 127 infection-related AEs were reported (56 events in the Experimental Formula-fed group compared to 71 events in the Control Formula-fed group. The proportion of infants with infection-related AEs was similar between groups (43 / 109 infants [39%] in the Experimental Formula-fed group compared to 48 / 114 infants [42%] in the Control Formula-fed group). When assessed by PT, fewer upper respiratory tract infection (IIRTI) events were reported in Experimental Formula-fed infants (19 events) compared to Control Formula-fed infants (41 events; p=0.009 by Poisson regression). The number of infants with >2 episodes of IIRTI was lower in the Experimental Formula-fed group (1 [0.9%]) compared to the Control Formula-fed group (12 [10.5%]; p=0.003 by Fisher’s Exact Test). Among remaining PTs within the “Infections and Infestations” SOC, the number of events and infants with events were comparable between groups. Total serious AEs were few and similar between groups (4 events in 4 infants in both groups, none related to the formulas).

[0625] Compared to Control Formula-fed infants, infants fed the synbiotic-supplemented formula had more resilient immune protection as shown by 2 times fewer IIRTI episodes and 91% fewer reoccurrences of IIRTI.

[0626] Microbiome and Gut healthIntra- and inter-individual microbiome diversity indices were different (p<0.0001) in Experimental Formula-fed vs. Control Formula-fed infants from 2-6 months, indicating different microbiome compositions (Figure 2).

[0627] Experimental Formula-fed infants had significantly higher relative and absolute abundance of bifidobacteria driven by infant-type Bifidobacterium species (mainly B. infantis, B. breve, B. longum, B. bifidum) from 2 to 6 months (Figure 3).

[0628] B. infantis dominated in Experimental Formula-fed infants and showed the strongest increase (p<0.0001) (Figure 4).

[0629] From 2 months, B. infantis prevalence (probiotic LMG11588 and endogenous strains) was virtually 100% in Experimental Formula-fed infants, while most Control Formula-fed infants had no detectable B. infantis through 6 months. Infants born via C-section and without older siblings showed the strongest bifidobacteria increase in Experimental Formula-fed infants vs. Control Formula-fed from 2 to 6 months.

[0630] Mucolytic R. gnavus and the opportunistic pathogen C. difficile were higher in Control Formula-fed vs. Experimental Formula-fed infants from 2 -to 6 months (Figure 5).

[0631] These microbiome differences promoted lower fecal pH in Experimental Formula-fed infants from 2 -to 6 months (p<0.0001). Gut health and immune markers were different between groups with lower Alpha-1 Antitrypsin (2, 4, and 6 months; p<0.05), lower calprotectin (4 months, p=0.026), and higher slgA (4 months, p=0.034) in Experimental Formula-fed infants (Figure 6).

[0632] Four fecal community types (FCTs) were identified with Bifidobacterium as the top differentiating genus. Composition at species level highlights FCT3 with higher abundance of infant-type Bifidobacterium species. Four microbiome development trajectories (T1-T4) were identified with significantly more experimental formula (EF) infants showing trajectories T2 and T3 both leading to FCT3 at 6m. Compared to infants in T1 and T4, T3 infants showed consistently higher fecal slgA from 2 to 6m, and T2 infants had higher slgA at 2 and 6m. Similar results were seen with fecal pH. Upper respiratory tract infection (URTI) risk was lower (p=0.039) when B. infantis was detectable.

[0633] Compared to Control Formula, the formula supplemented with a synbiotic blend of HMOs, BMOs and B. Infantis drives an infant-type gut microbiome progression that positively impacts gut and immune function, including enhancing mucosal immunity.(Original in Electronic Form)

[0634] (This sheet is not part of and does not count as a sheet of the international application)

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[0640] 3 / 4

[0641] PCT

[0642] (Original in Electronic Form)

[0643] (This sheet is not part of and does not count as a sheet of the international application)

[0644]

[0645] FOR RECEIVING OFFICE USE ONLY

[0646]

[0647] (Original in Electronic Form)

[0648] (This sheet is not part of and does not count as a sheet of the international application)

[0649] FOR INTERNATIONAL BUREAU USE ONLY

[0650]

Claims

CLAIMS1. A combination of a human milk oligosaccharide (HMO) mixture, bovine milk-derived oligosaccharides (MOS) and Bifidobacterium longum subsp. infantis for use in preventing and / or treating constipation and / or improving stool consistency and / or stool frequency in an infant or a young child, wherein the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosy I lactose (3FL) and / or lacto-N-neotetraose (LNnT).

2. The combination for use according to claim 1, wherein the combination is administered separately, simultaneously or sequentially, preferably wherein the combination is administered simultaneously.

3. The combination for use according to claim 1 or 2, wherein the combination is provided in the form of a nutritional composition, preferably wherein the nutritional composition is selected from an infant formula, a starter infant formula, a follow-on or follow-up formula, a baby food, an infant cereal composition, a growing-up-milk, a fortifier such as a human milk fortifier, or a supplement, more preferably wherein the nutritional composition is an infant formula.

4. The combination for use according to any preceding claim, wherein the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), 3-fucosyllactose (3FL), and lacto-N-neotetraose (LNnT).

5. The combination for use according to any preceding claim, wherein the HMO mixture consists or consists essentially of:i. 20 wt% to 60 wt% of 2FL, preferably 22 wt% to 55 wt%;ii. 4 wt% to 30 wt% of LNT, preferably 6 wt% to 20 wt%;iii. 1 wt% to 12 wt % of DFL, preferably 2 wt% to 8 wt%;iv. 7 wt% to 23 wt% of 6SL and 3SL combined, preferably 8 wt% to 22 wt%;v. 10 wt% to 50 wt% of 3FL, preferably 13 wt% to 46 wt% andvi. 3 wt% to 25 wt% of LNnT, preferably 5 wt% to 20 wt%.

6. The combination for use according to any preceding claim, wherein the combination is provided in the form of a composition having a total HMO concentration of from 0.5 g / L to 10 g / L, preferably from 1 g / L to 5 g / L, or more preferably from 2 g / L to 3 g / L.

7. The combination for use according to any preceding claim, wherein the combination is provided in the form of a composition having a total MOS concentration of from 0.5 g / L to 10 g / L, preferably from 2 g / L to 8 g / L, or more preferably from 4 g / L to 5 g / L.

8. The combination for use according to any preceding claim, wherein the Bifidobacterium longum subsp. infantis is Bifidobacterium longum subsp. infantis LMG 11588 or a strain having an Average Nucleotide Identity (ANI) of at least 95%, preferably at least 99%, more preferably at least 99.9% to Bifidobacterium longum subsp. infantis LMG 11588.

9. The combination for use according to any preceding claim, wherein the combination comprises from 103to 1012cfu of Bifidobacterium longum subsp. infantis, preferably from 105to 101°cfu of Bifidobacterium longum subsp. infantis, more preferably from 105to 107cfu of Bifidobacterium longum subsp. infantis per g of combination or composition on a dry weight basis.

10. The combination for use according to any preceding claim, wherein the infant or young child has an age from 0 months to 12 months, preferably from 0 months to 6 months.

11. The combination for use according to any preceding claim, wherein the infant or young child is exclusively formula-fed.

12. The combination for use according to any preceding claim, for use in reducing the incidence and / or severity of constipation in said infant or young child.

13. The combination for use according to any preceding claim, for use in reducing stool hardness in said infant or young child.

14. The combination for use according to any preceding claim, for use in increasing stool frequency in said infant or young child.

15. The combination for use according to any preceding claim, wherein the combination prevents and / or treats constipation and / or improves stool consistency and / or stool frequency without any associated gut discomfort or digestive disorders, such as flatulence, bloating, and / or colics.

16. The combination for use according to any of the preceding claims, which is a combination of a human milk oligosaccharide (HMO) mixture, Bifidobacterium longum subsp. infantis, wherein the HMO mixture comprises or consists of 3'-Galactosyllactose (3’GL), 6'-Galactosyllactose (6’GL), 4'-Galactosyllactose (4’GL), 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosy I lactose (3FL) and / or lacto-N-neotetraose (LNnT).

17. The combination for use according to any of the preceding claims, which is a combination of a human milk oligosaccharide (HMO) mixture, Bifidobacterium longum subsp. infantis, wherein the HMO mixture comprises or consists of 3'-Galactosyllactose (3’GL), 6'-Galactosyllactose (6’GL), 4'-Galactosyllactose (4’GL), 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), 3-fucosyl lactose (3FL) and lacto-N-neotetraose (LNnT).

18. The combination or combination for use according to any of the preceding embodiments that further comprises a protein source that predominantly contains A2 p-casein.

19. The combination for use according to any of the preceding embodiments, that further comprises Bifidobacterium animalis subsp. Lactis, for example Bifidobacterium animalis subsp. lactis CNCM I-3446.

20. The combination for use according to any of the preceding claims , that further comprises docosahexaenoic acid, arachidonic acid, iron, vitamin B12, folic acid, sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositole (PI) and phosphatidylserine (PS).

21. A combination of which is a combination of a human milk oligosaccharide (HMO) mixture, Bifidobacterium longum subsp. infant is, wherein the HMO mixture comprises or consists of 3'-Galactosyllactose (3’GL), 6'-Galactosyllactose (6’GL), 4'-Galactosyllactose (4’GL), 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3FL) and / or lacto-N-neotetraose (LNnT).

22. A combination of a human milk oligosaccharide (HMO) mixture, bovine milk-derived oligosaccharides (MOS) and Bifidobacterium longum subsp. Infantis, wherein the HMO mixture consists of 2'-fucosy I lactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosy I lactose (3FL) and / or lacto-N-neotetraose (LNnT), that further comprises a protein source that predominantly contains A2 p-casein, that further comprises Bifidobacterium animalis subsp. Lactis, for example Bifidobacterium animalis subsp. lactis CNCM I-3446, and that that further comprises docosahexaenoic acid, arachidonic acid, iron, vitamin B12, folic acid, sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositole (PI) and phosphatidylserine (PS).

23. A combination according to claim 21 or 22, wherein the combination is provided in the form of a nutritional composition, preferably wherein the nutritional composition is selected from an infant formula, a starter infant formula, a follow-on or follow-up formula, a baby food, an infant cereal composition, a growing-up-milk, a fortifier such as a human milk fortifier, or a supplement, more preferably wherein the nutritional composition is an infant formula.

24. A combination of a human milk oligosaccharide (HMO) mixture, milk fat globule membrane (MFGM) and Bifidobacterium longum subsp. infant is, wherein the HMO mixture comprises or consists of 3'-Galactosyllactose (3’GL), 6'-Galactosyllactose (6’GL), 4'-Galactosyllactose (4’GL), 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3FL) and / or lacto-N-neotetraose (LNnT).

25. A combination of a human milk oligosaccharide (HMO) mixture, milk fat globule membrane (MFGM) and Bifidobacterium longum subsp. infantis, wherein the HMO mixture comprises or consists of 3'-Galactosyllactose (3’GL), 6'-Galactosyllactose (6’GL), 4'-Galactosyllactose (4’GL), 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3FL) and / or lacto-N-neotetraose (LNnT) and wherein MFGM comprise phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and sphingomyelin.

26. A combination of a human milk oligosaccharide (HMO) mixture, phospholipids and Bifidobacterium longum subsp. infantis, wherein the HMO mixture comprises or consists of 3'-Galactosyllactose (3’GL), 6'-Galactosyllactose (6’GL), 4'-Galactosyllactose (4’GL), 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3FL) and / or lacto-N-neotetraose (LNnT).

27. The combination according to any of claims 24 to 27 that further comprises a protein source that predominantly contains A2 p-casein.

28. The combination according to any of claims 24 to 28, that further comprises Bifidobacterium animalis subsp. Lactis, for example Bifidobacterium animalis subsp. lactis CNCM 1-3446.

29. The combination to claim 24 , that further comprises docosahexaenoic acid, arachidonic acid, iron, vitamin B12, folic acid, sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositole (PI) and phosphatidylserine (PS).

30. The combination according to any of the claims 24 to 28, wherein the combination is provided in the form of a nutritional composition, preferably wherein the nutritional composition is selected from an infant formula, a starter infant formula, a follow-on or follow-up formula, a baby food, an infant cereal composition, a growing-up-milk, a fortifier such as a human milkfortifier, or a supplement, more preferably wherein the nutritional composition is an infant formula.

31. The combination according to any of the claims 21 to 30 for use in promoting immunity development and / or immune function in a subject, in particular an infant or young child.

32. Non -therapeutic use of a combination according to any of the claims 21 to 30 to promote to immunity development and / or immune function in a subject, in particular an infant or young child.

33. Non -therapeutic use of a combination as described in anyone of claims 1 to 30 for promoting gastrointestinal tolerance in a subject.

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