WO2025233455A1

WO2025233455A1 - Nutritional composition for strenghtening the gut barrier

Info

Publication number: WO2025233455A1
Application number: PCT/EP2025/062624
Authority: WO
Inventors: Fadoua DAOUAD; Ingrid Brunhilde RENES; Noortje IJSSENNAGGER; Jan Knol
Original assignee: Nutricia NV
Current assignee: Nutricia NV
Priority date: 2024-05-08
Filing date: 2025-05-08
Publication date: 2025-11-13
Anticipated expiration: 2026-11-08

Abstract

The invention relates to a nutritional composition for infants, for children as well as a medical nutritional composition, comprising human milk oligosaccharides and Bifidobacterium breve. The invention further relates to the use of said nutritional composition for strengthening the gut epithelial defense and/or the gut barrier function.

Description

NUTRITIONAL COMPOSITION FOR STRENGHTENING THE GUT BARRIER

FIELD OF THE INVENTION

The invention relates to a nutritional composition for infants and children as well as to a medical nutritional composition. The invention further relates to the use of this nutritional composition for strengthening the gut epithelial defense and/or the gut barrier function.

BACKGROUND OF THE INVENTION

Human milk is the uncontested gold standard concerning infant nutrition. However, in some cases breastfeeding is inadequate or unsuccessful for medical reasons or not available because of a choice not to breastfeed. For such situations infant or follow-on formulas have been developed. Commercial infant formulas are commonly used today to provide supplemental or sole source of nutrition early in life. These formulas comprise a range of nutrients to meet the nutritional needs of the growing infant, and typically include fat, carbohydrate, protein, vitamins, minerals, and other nutrients helpful for optimal infant growth and development. Commercial infant formulas are designed to mimic, as closely as possible, the composition and function of human milk.

Human milk contains substantial amounts of non-digestible carbohydrates, known as human milk oligosaccharides (HMOs). Mature human milk contains 5 to 15 g/l of HMOs. It is presumed that more than 200 structurally distinct oligosaccharides are present. The building blocks of human milk oligosaccharides are the monosaccharides D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GIcNAc), L-fucose (Fuc), and sialic acid (N-acetyl neuraminic acid (Neu5Ac). Lactose (Galp1-4Glc) forms the reducing end and can be elongated with N-acetyllactosamine repeat units (Galp1-3/4GlcNAc). Lactose or the polylactosamine backbone can be sialylated in a2-3 and/or a2-6 linkages and/or fucosylated in a1-2, a1-3, and/or a1 -4 linkages. The structural complexity and abundance of these non-digestible oligosaccharides is unique for human milk as in milk of other mammalian species the level of non-digestible oligosaccharides is much lower. The role of HMOs in human milk is to improve the intestinal microbiota by stimulating bifidobacteria and other beneficial lactic acid producing bacteria and thereby inhibiting the growth of potentially pathogenic bacteria. HMOs furthermore inhibit binding of pathogenic micro-organisms to the infant's epithelial cell surface.

In the past, infant formulas did not contain non-digestible oligosaccharides. Subsequently infant formulas were developed, containing prebiotic, non-digestible oligosaccharides to functionally mimic the role of the HMOs. One of the best studied mixtures of such prebiotics is a mixture of galacto-oligosaccharides (GOS) plus long chain fructo-oligosaccharides (IcFOS) in a weight ratio of 9:1. Upon administration of this specific prebiotic mixture to infants, bifidobacteria increase and pathogens decrease in the intestinal microbiota, rendering the microbiota more similar to the microbiota of human milk fed infants (Knol et al, Acta Paediatrica, 2005; 94 (Suppl 449); Knol et al, 31-33. Pediatr Gastroenterol Nutr, Vol. 40, No. 1 , January 2005; WO 2005/039319). Also, formulas containing probiotics have been developed, as well as formulas comprising both prebiotics and probiotics.

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

WO 2023/118510 describes a nutritional composition comprising a mix of Bifidobacterium species and at least one human milk oligosaccharide, wherein the Bifidobacterium species comprise at least a Bifidobacterium bifidum strain able to express at least one extracellular enzyme selected from a fucosidase and a sialidase, and a Bifidobacterium breve strain able to metabolize a saccharide selected from L-fucose and sialic acid, and the human milk oligosaccharide is at least one selected from the group consisting of 2’-fucosyllactose, 3-fucosyllactose, 3’-sialyllactose, and 6’-sialyllactose.

This composition is suggested for reducing the risk of occurrence, preventing and/or treating an intestinal infection, intestinal inflammation and/or diarrhea.

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

ON 113907144 discloses that certain HMOs can prevent infection by Staphylococcus aureus. A wide array of HMO combinations is suggested including the combination of 2’-fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3’-sialyllactose (3’-SL) and 6’-sialyllactose (6’-SL).

US 2014/248415 discloses HMO combinations comprising 2’-FL, LNT, 3’-SL and 6’-SL to be included in infant formula and discloses to optionally include as prebiotic 90% GOS, 10% inulin or FOS. The HMO combination is said to enhance the beneficial effects and efficiency of probiotics.

US 2023/013644 concerns nutritional compositions comprising HMOs for use in providing nutrition to infants at different age stages. Infant formula with the combination of 2’-FL, 3-FL, LNT, 3’-SL, 6’-SL are described. US 2024/139222 discloses various combinations of HMOs and for some at least one probiotic strain of the genus Bifidobacterium can be included. The composition would be useful for preventing disease, disorder or condition associated with one or more of inflammation, immune dysfunction, cancer, allergy or dysbiosis. US 2018/368460 concerns a synthetic mixture of HMOs essentially consisting of LNnT, LNT, 2'-FL, 3'-SL, 6'-SL and either DFL or 3-FL. Optionally, the composition may also contain a long list of ingredients including probiotics. The composition is for preventing and/or treating viral and/or bacterial infections, specifically modulating the indigenous microbiota and improving cognitive function in non-infant humans.

US 2012/171165 describes a composition comprising a neutral human milk oligosaccharide, preferably 2’FL, and relates to the improvement of the gut barrier function in infants. The document discloses that the activity of alkaline phosphatase is a good indicator of the differentiation of the intestinal epithelium.

Still there is a need for infant and young child formula with a mixture of non-digestible oligosaccharides that further improves the intestinal microbiota of infants. Nevertheless, there remains a need for nutritional compositions that reduce the risk of developing gastrointestinal infections or gastro-intestinal inflammatory diseases even further.

SUMMARY OF THE INVENTION

The inventors of the present invention have surprisingly found that nutritional compositions comprising Bifidobacterium breve and a mixture of five human milk oligosaccharides (HMO), synergistically increases the intestinal alkaline phosphatase levels (iALP), both intracellularly and extracellularly, when compared to the control or the B. breve or HMOS mix alone. iALP is a gut mucosal defense enzyme that is critical for protection of the epithelial barrier. iALP expression and function are lost with starvation and are maintained with enteral feeding. iALP has the capacity to detoxify bacterial endotoxin lipopolysaccharide (LPS) and can prevent translocation of active LPS and bacterial invasion across the gut epithelial barrier. Higher expression of iALP is therefore a marker for a stronger gut epithelial defence and/or gut barrier function, which results in a lower risk of developing gastrointestinal infections or gastro-intestinal inflammatory diseases.

Hence a first aspect of the invention pertains to a nutritional composition, not being human milk, comprising digestible carbohydrate, protein, lipid, a mixture of the human milk oligosaccharides (HMO) 2’-fucosyllactose, 3-fucosyllactose, 3’-sialyllactose, 6’-sialyllactose and lacto-N-tetraose, and Bifidobacterium breve for use in one or more of: i) strengthening the gut epithelial defence; ii) strengthening the gut barrier function; iii) preventing intestinal inflammatory diseases; and iv) preventing intestinal infections.

A second aspect of the invention relates to a nutritional composition, not being human milk, comprising digestible carbohydrate, protein, lipid, 0.3 to 4 wt% based on dry weight of a mixture of the human milk oligosaccharides (HMO) 2’-fucosyllactose, 3-fucosyllactose, 3’-sialyllactose, 6’-sialyllactose and lacto-N- tetraose, and 10⁵ to 10¹² colony forming units (cfu) Bifidobacterium breve.

DETAILED DESCRIPTION OF THE INVENTION

In particular the present invention concerns a method for: i) strengthening the gut epithelial defence; ii) strengthening the gut barrier function; iii) preventing intestinal inflammatory diseases; and iv) preventing intestinal infections comprising administration of a nutritional composition comprising digestible carbohydrate, protein, lipid, Bifidobacterium and human milk oligosaccharides (HMOs), to a subject in need thereof, wherein a) the HMOs are a mixture of 2’-fucosyllactose, 3-fucosyllactose, 3’-sialyllactose, 6’-sialyllactose and lacto-N-tetraose, and b) the Bifidobacterium comprises Bifidobacterium breve. For some jurisdictions the invention can be worded as a nutritional composition comprising digestible carbohydrate, protein, lipid, Bifidobacterium and human milk oligosaccharides (HMOs), wherein a) the HMOs are a mixture of 2’-fucosyllactose, 3-fucosyllactose, 3’-sialyllactose, 6’-sialyllactose and lacto-N-tetraose, and b) the Bifidobacterium comprises Bifidobacterium breve, for use in one or more of: i. preventing intestinal inflammatory diseases, and ii. preventing intestinal infections, wherein said nutritional composition is not human milk.

For some jurisdictions the invention can be worded as the use of digestible carbohydrate, protein, lipid Bifidobacterium and human milk oligosaccharides (HMOs), for the manufacture of a nutritional composition, wherein a) the HMOs are a mixture of 2’-fucosyllactose, 3-fucosyllactose, 3’-sialyllactose, 6’-sialyllactose and lacto-N-tetraose, and b) the Bifidobacterium comprises Bifidobacterium breve, for use in one or more of: i. preventing intestinal inflammatory diseases, and ii. preventing intestinal infections,

In particular with regard to i) strengthening the gut epithelial defence; and ii) strengthening the gut barrier function, the present method can be seen as a non-therapeutic method.

The invention thus can also be worded as a non-therapeutic use of a nutritional composition comprising digestible carbohydrate, protein, lipid, Bifidobacterium and human milk oligosaccharides (HMOs), wherein a) the HMOs are a mixture of 2’-fucosyllactose, 3-fucosyllactose, 3’-sialyllactose, 6’-sialyllactose and lacto-N-tetraose, and b) the Bifidobacterium comprises Bifidobacterium breve, for one or more of i) strengthening the gut epithelial defence; and ii) strengthening the gut barrier function, wherein said nutritional composition is not human milk.

In another aspect the invention concerns a nutritional composition, which is not human milk, comprising digestible carbohydrate, protein, lipid, Bifidobacterium breve and human milk oligosaccharides (HMO), wherein a. the HMOS are present in an amount of 0.3 to 4 wt% based on dry weight, b. the HMOS are a mixture of 2’-fucosyllactose, 3-fucosyllactose, 3’-sialyllactose, 6’-sialyllactose and lacto-N-tetraose, and c. the Bifidobacterium breve is present is an amount of 10⁵ to 10¹² colony forming units (cfu) per gram of dry weight of the composition.

In yet even another preferred embodiment, administering a nutritional composition to an infant may be considered non-therapeutic. In those instances, the invention may be worded as defined above by way of a method comprising administering a nutritional composition. For clarity, the method can also be defined as a non-therapeutic method. By definition, the words “non-therapeutic” exclude any therapeutic effect.

Preferably, the use of the present invention is by increasing the intracellular expressed activity of intestinal alkaline phosphatase (iALP) and/or by increasing the extracellular secreted iALP activity.

In a preferred embodiment, the prevention of intestinal inflammatory diseases and/or intestinal infections is by strengthening the gut epithelial defence and/or the gut barrier function.

Preferably, the intestinal inflammatory diseases are selected from enterocolitis, necrotizing enterocolitis (NEC) and inflammatory bowel diseases. Examples of inflammatory bowel diseases are Crohn’s disease and ulcerative colitis.

Preferably, the intestinal infections are infections caused by bacteria, viruses, fungi or parasites. More preferably, the intestinal infections are infections caused by bacteria or viruses. Most preferably, the intestinal infections are bacterial infections.

Preferably, the use of the present invention is in a human subject, more preferably in a human young child of 1 , 2 or 3 years of age, or an infant of 0 up to 12 months of age, more preferably in a human young child of 1 or 2 years of age, or an infant of 0 up to 12 months of age, even more preferably an infant 0-12 months and most preferably an infant of 0-6 months.

Levels of iALP are lower in preterm infants. Preterm infants are infants born before the 37^th week of gestational age. Therefore, the nutritional composition is advantageously for use in these infants born premature.

Allergic infants or allergic young children may benefit from the composition as a strengthened gut barrier function will result in less allergens crossing the intestinal barrier. Therefore, the nutritional composition is advantageously for use in infants or young children that are allergic to food proteins, in particular cow’s milk protein. Subjects with microbial dysbiosis, such as caused by antibiotic treatment or being born by C-section are known to have lowered levels of iALP. Hence, the nutritional composition is advantageously for use in infants or young children that were born via C-section and/or treated with antibiotics.

In an alternative embodiment, the use of the present invention is in an adult human subject.

Bifidobacterium breve

The nutritional composition of the present invention comprises a Bifidobacterium belonging to the genus Bifidobacterium breve.

Bifidobacterium breve is a Gram-positive, anaerobic, branched rod-shaped bacterium. The B. breve according to the present invention preferably has at least 95 % identity of the 16 S rRNA sequence when compared to the type-strain of B. breve ATCC 15700, more preferably at least 97% identity (Stackebrandt & Goebel, 1994, Int. J. Syst. Bacteriol. 44:846-849). Preferred B. breve strains are those isolated from the faeces of healthy human milk-fed infants. Typically, these are commercially available from producers of lactic acid bacteria, but they can also be directly isolated from faeces, identified, characterized, and produced.

Suitable B. breve strains are available. Examples of suitable B. breve strains are B. breve UCC2003 (NCIMB 8807), C50 (CNCM 1-2219), JCM7017, NCFB2258 and NCIMB8815, JCM7019, LMG13208, NCFB2257, NCIMB11815, ATCC 15700, M-16V (BCCM/LMG 23729, Morinaga). Especially preferred is to use B. breve M-16V. Another preferred Bifidobacterium breve to use is Bifidobacterium breve CNCM I- 5177. B. breve CNCM 1-5177 was deposited under the Budapest Treaty at the Collection Nationale de Cultures de Microorganism, at Institut Pasteur, 25 Rue du Dr Roux, Paris, France on 9 March 2017 by Compagnie Gervais Danone. Especially preferred is the B. breve M-16V strain (Morinaga).

The nutritional composition preferably comprises at least 10⁵ cfu B. breve per gram dry weight, more preferably at least 10⁶ cfu, even more preferably at least 10⁷ cfu B. breve per gram dry weight. The nutritional composition preferably contains 10⁵ to 10¹¹ colony forming units (cfu) B. breve per gram dry weight, more preferably 10⁶ to 10¹¹ cfu, even more preferably 10⁷ to 10¹⁰ cfu.

When the nutritional composition is a liquid nutritional composition, preferably a ready-to-drink liquid nutritional composition, the liquid nutritional composition preferably comprises 5.10⁶ to 5.10¹² colony forming units (cfu) probiotic bacteria per 100 ml, more preferably 5.10⁷ to 5.10¹² cfu even more preferably 5.10⁸ to 5.10¹¹ cfu per 100 ml. Based on calories, the nutritional composition preferably comprises 7.5.10⁶ to 7.5.10¹² colony forming units (cfu) probiotic bacteria per 100 kcal, more preferably 7.5.10⁷ to 7.5.10¹² cfu even more preferably 7.5.10⁸ to 7.5.10¹¹ cfu per 100 ml. In terms of doses, the nutritional composition preferably provides between 10⁶ and 10¹¹ cfu probiotic bacteria per serving, more preferably between 10⁷ and 1O¹⁰ cfu probiotic bacteria per serving.

Preferably, the subject receives a daily dosage of 10⁷-10¹² cfu probiotic bacteria for at least 3 consecutive days. More preferably, the subject receives a daily dosage of 10⁸-10¹¹ cfu probiotic bacteria for at least 3 consecutive days.

Human milk oligosaccharides

The nutritional composition comprises human milk oligosaccharides (HMOs). The term “human milk oligosaccharides” or “HMO” as used herein refers to non-digestible oligosaccharides which are present in human breast milk. The HMO comprises a mixture of 2’-fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3'-sialyllactose (3’-SL), and 6'-sialyllactose (6’-SL).

Suitable single HMOs for the preparation of the nutritional composition are commercially available, for example from Kyowa Hakko Bio, Japan; Friesland Campina, The Netherlands; Glycom DSM, Denmark and Chr. Hansen, Denmark. Otherwise, it is well within the reach of the skilled person to obtain HMO by isolation from suitable sources or by chemical synthesis using methods known in the art. A mix of the 5 HMOS is available at Chr. Hansen.

When the nutritional composition is a powdered nutritional composition, the composition preferably comprises 300-4000 mg HMO per 100 g dry weight, more preferably 450-2000 mg HMO per 100 g dry weight.

When the nutritional composition is a ready-to-drink liquid nutritional composition, the composition preferably comprises 20-400 mg HMO per 100 ml, more preferably 30-300 mg HMO per 100 ml and most preferably 40-250 mg HMO per 100 ml HMO.

When expressed in amounts based on calories, preferably the nutritional composition comprises 30-600 mg HMO per 100 kcal, more preferably 45-450 mg HMO per 100 kcal and most preferably 60-375 mg HMO per 100 kcal.

In terms of doses, the nutritional composition preferably provides 40-600 mg HMO per serving, more preferably 50-500 mg HMO per serving.

In terms of doses, the nutritional composition preferably provides a total daily dose of 0.1-10 g HMO, more preferably a total daily dose of 0.2-7 g HMO and most preferably a daily dose of 0.4-4 g HMO. More preferably the HMOs comprises 42 to 62 wt% 2’-FL, 10 to 16 wt% 3-FL, 21 to 31 wt% LNT, 3 to 5 wt% 3’-SL and 4 to 6 wt% 6’-SL, the sum of 2’-FL, 3-FL, LNT, 3’-SL and 6’-SL being 100%.

Preferably the ratio ofB. breve cfu to weight HMOs is 10⁹ to 10¹⁴cfu/gram mixture of HMOs, more preferably 10¹° - 10¹³ cfu/gram HMOs. Such a ratio enables a more optimal effect of the combination of B. breve and the HMOS on the iALP expression.

GOS and FOS

The nutritional composition preferably comprises galacto-oligosaccharide (GOS) and/or fructooligosaccharide (FOS), more preferably the nutritional composition comprises GOS and FOS. GOS and FOS are both non-digestible oligosaccharides which act as a prebiotic.

The GOS is preferably transgalacto-oligosaccharide. A suitable GOS is commercially available, for example VivinalOGOS (FrieslandCampina DOMO). Preferably the GOS is short chain galacto-oligosaccharide (scGOS) with an average degree of polymerization (DP) in the range of 1 to 10, more preferably in the range of 3 to 7.

A suitable FOS is commercially available, for example RaftilinOHP or Raftilose® (Orafti). Preferably the FOS is long chain fructo-oligosaccharide (IcFOS) with an average DP in the range of 10-100, more preferably in the range of 20 to 60. A suitable long chain FOS is Raftilin®HP.

A suitable short chain FOS is commercially available, for example RaftiloseP95 ® (Orafti) or Actilight (Meiji). Short chain FOS (scFOS) is FOS with an average DP in the range of 2-6, more preferably in the range of 2-5.

Preferably a mixture of GOS and FOS is present. Preferably, the weight ratio of GOS to FOS ranges from 100:1 to 1 :10, more preferably from 20:1 to 1 :1 , even more preferably from 7:1 to 10:1 , and most preferably the weight ratio is 9:1 . Preferably these weight ratios apply to scGOS and IcFOS.

In one embodiment, preferably a mixture of scFOS and IcFOS is present. Preferably, the weight ratio of scFOS to IcFOS ranges from 100:1 to 1 :10, more preferably from 20:1 to 1 :1 , even more preferably from 7:1 to 10:1 , and most preferably the weight ratio is 9:1 .

Preferably, the weight ratio of GOS and/or FOS combined to HMO ranges from 20:1 to 1 :10, more preferably from 15:1 to 1 :5 and most preferably from 10:1 to 1 :1 .

Preferably, the nutritional composition comprises 80 mg to 2 g of GOS and/or FOS per 100 ml, more preferably 150 mg to 1 .5 g, most preferably 300 mg to 1 g of GOS and/or FOS per 100 ml. Based on dry weight, the nutritional composition preferably comprises 0.25-20 wt.%, more preferably 0.5- 10 wt.%, and most preferably 1 .5-7.5 wt.% of GOS and/or FOS.

The present nutritional composition comprises either GOS, or FOS, or both GOS and FOS. The amounts for GOS and/or FOS mentioned thus pertain to either GOS if no FOS is present or FOS if no GOS is present or pertain to the sum of GOS plus FOS.

Preferably the ratio of B. breve cfu to weight GOS and/or FOS is 5.10⁹ to 5.10¹⁴ cfu/gram sum of GOS plus FOS, more preferably 5.1O¹⁰ - 5.10¹³ cfu/gram sum of GOS plus FOS.

Digestible carbohydrate

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

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

Protein

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

The source of the protein is preferably selected in such a way that the minimum requirements for essential amino acid content are met and satisfactory growth is ensured. Hence protein sources based on cows' milk proteins such as whey, casein, and mixtures thereof and proteins based on soy, potato or pea are preferred. In case whey proteins are used, the protein source is preferably based on acid whey, sweet whey, whey protein isolate or mixtures thereof. Preferably the nutritional composition comprises at least 3 wt.% casein based on dry weight. Preferably the protein in the nutritional composition is intact and/or non-hydrolyzed.

Preferably the protein in the nutritional composition is hydrolyzed protein and/or free amino acids. Preferably the protein in the nutritional composition is at least 99 wt% hydrolyzed protein based on total protein and/or free amino acids. When hydrolyzed, the protein can be hydrolyzed rice protein, casein or whey protein, preferably hydrolyzed whey protein. Nutritional compositions with such hydrolyzed protein and/or free amino acids are suitable for infants that are allergic to protein, such as cow’s milk protein. Nutritional compositions with free amino acids as protein components are suitable for subjects severely allergic to cow’s milk protein or for subjects having multiple food allergies. Preferably the nutritional composition with at least 99 wt% amino acids based on total protein comprises mixtures of scFOS and IcFOS. As GOS is built from lactose, which is derived from milk, GOS is less suitable for such nutritional compositions for severely allergic subjects for cow’s milk protein.

Lipid

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

The lipid provides preferably 30 to 60% of the total calories of the nutritional composition. More preferably the nutritional composition comprises lipid providing 35 to 55% of the total calories, even more preferably the nutritional composition comprises lipid providing 40 to 50% ofthe total calories. The lipids are preferably present in an amount of 4 to 6 g per 100 kcal. When in liquid form, e.g., as a ready-to-feed liquid, the nutritional composition preferably comprises 2.1 to 6.5 g lipid per 100 ml, more preferably 3.0 to 4.0 g per 100 ml. Based on dry weight, the nutritional composition preferably comprises 10 to 50 wt.%, more preferably 12.5 to 40 wt.% lipid, even more preferably 19 to 30 wt.% lipid.

The lipid preferably comprises vegetable lipid. The presence of vegetable lipids advantageously enables an optimal fatty acid profile, high in polyunsaturated fatty acids and/or more reminiscent to human milk fat. Lipid from mammalian milk alone, e.g., cow’s milk, do not provide an optimal fatty acid profile. The amount of essential fatty acids is too low in mammalian milk.

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

In one preferred embodiment, the nutritional composition comprises 5 to 98 wt.% vegetable lipid based on total lipid, more preferably 10 to 95 wt.%, more preferably 20 to 80 wt.%, even more preferably 25 to 75 wt.%, most preferably 40 to 60 wt.% of vegetable lipid based on total lipid. Preferably, the nutritional composition also comprises non-vegetable lipid. Preferably, said non-vegetable lipid is/are one or more non-vegetable lipid selected from mammalian milk fat, mammalian milk derived lipid as a preferred source of phospholipid, and fish, marine and/or microbial oils as source of LC-PUFA.

Fatty acid composition

SFA relates to saturated fatty acids and/or acyl chains, MUFA relates to mono-unsaturated fatty acid and/or acyl chains, PUFA refers to polyunsaturated fatty acids and/or acyl chains with 2 or more unsaturated bonds; LC-PUFA refers to long chain polyunsaturated fatty acids and/or acyl chains comprising at least 20 carbon atoms in the fatty acyl chain and with 2 or more unsaturated bonds; DHA refers to docosahexaenoic acid and/or acyl chain (22:6, n3); EPA refers to eicosapentaenoic acid and/or acyl chain (20:5 n3); ARA refers to arachidonic acid and/or acyl chain (20:4 n6); DPA refers to docosapentaenoic acid and/or acyl chain (22:5 n3). n3 or omega 3 PUFA refers to polyunsaturated fatty acids and/or acyl chains with 2 or more unsaturated bonds and with an unsaturated bond at the third carbon atom from the methyl end of the fatty acyl chain, n6 or omega 6 PUFA refers to polyunsaturated fatty acids and/or acyl chains with 2 or more unsaturated bonds and with an unsaturated bond at the sixth carbon atom from the methyl end of the fatty acyl chain.

The nutritional composition preferably comprises LA, which refers to linoleic acid and/or acyl chain (18:2 n6). LA is an n6 PUFA and the precursor of n6 LC-PUFA and is an essential fatty acid as it cannot be synthesized by the human body. LA preferably is present in a sufficient amount to promote a healthy growth and development, yet in an amount as low as possible to prevent negative, competitive, effects on the formation of n3 PUFA and a too high n6/n3 ratio. The nutritional composition therefore preferably comprises less than 25 wt.%, more preferably less than 20 wt.%, more preferably less than 15 wt.% LA based on total fatty acids. The nutritional composition preferably comprises at least 5 wt.% LA based on fatty acids, preferably at least 7.5 wt.%, more preferably at least 10 wt.% based on total fatty acids. The nutritional composition preferably comprises ALA, which refers to alpha-linolenic acid and/or acyl chain (18:3 n3). ALA is a n3 PUFA and the precursor of n3 LC-PUFA and is an essential fatty acid as it cannot be synthesized by the human body. Preferably ALA is present in a sufficient amount to promote a healthy growth and development of the infant. The nutritional composition therefore preferably comprises at least 0.5 wt.%, more preferably at least 1 .0 wt.%, more preferably the nutritional composition comprises at least 1 .5 wt.%, even more preferably at least 2.0 wt.% ALA based on total fatty acids. Preferably the nutritional composition comprises less than 10 wt.% ALA, more preferably less than 5.0 wt.% ALA based on total fatty acids.

The weight ratio LA/ALA preferably is well balanced to ensure an optimal n6/n3 PUFA, n6/n3 LC PUFA and DHA/ARA ratio in the cellular membranes. Therefore, the nutritional composition preferably comprises a weight ratio of LA/ALA from 2 to 20, more preferably from 3 to 15, more preferably from 5 to 12, more preferably from 5 to 10. Preferably the n6 PUFA/n3 PUFA weight ratio is from 3 to 20, more preferably from 3 to 15, more preferably from 5 to 12, more preferably from 5 to 10.

Preferably, the nutritional composition comprises n3 LC-PUFA, such as EPA, DPA and/or DHA, more preferably DHA. As the conversion of ALA to DHA may be less efficient in infants, preferably both ALA and DHA are present in the nutritional composition. Preferably the nutritional composition comprises at least 0.05 wt.%, preferably at least 0.1 wt.%, more preferably at least 0.2 wt.%, of DHA based on total fatty acids. Preferably the nutritional composition comprises not more than 2.0, preferably not more than 1 .0 wt.%, of DHA based on total fatty acids.

The nutritional composition preferably comprises ARA. Preferably the nutritional composition comprises at least 0.05 wt.%, preferably at least 0.1 wt.%, more preferably at least 0.2 wt.%, of ARA based on total fatty acids. As the group of n6 fatty acids, especially ARA counteracts the group of n3 fatty acids, especially DHA, the nutritional composition preferably comprises relatively low amounts of ARA. Preferably the nutritional composition comprises not more than 2.0 wt.%, preferably not more than 1 .0 wt.%, of ARA based on total fatty acids. Preferably the weight ratio between DHA and ARA is between 1 to 4/1 , more preferably between 1 to 2/1 , more preferably between 0.6 and 1 .5.

Application

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

The terms as used herein, “infant formula” or “follow-on formula” or “young child formula” refers to compositions that are artificially made or that are synthetic This means that the composition that is administered is not human milk. It also means that the composition that is administered is not native cow’s milk or native milk from another mammal.

In the present context, infant formula refers to nutritional compositions, artificially made, intended for infants of 0 to about 4 to 6 months of age and are intended as a substitute for human milk. Typically, infant formulae are suitable to be used as sole source of nutrition. Such formulae are also known as starter formula. Formula for infants starting for 4 to 6 months of life to 12 months of life are intended to be supplementary feedings to infants that start weaning on other foods. Such formulae are also known as follow-on formulae. Infant formulae and follow-on formulae are subject to strict regulations, for example the EU regulations no. 609/2013 and no. 2016/127. In the present context, young child formula refers to nutritional compositions, artificially made, intended for infants of 12 months to 36 months, which are intended to be supplementary feedings to infants. Such formulae are also known as growing-up milks.

The nutritional composition is preferably an infant formula or follow-on formula and preferably comprises 3 to 7 g lipid/100 kcal, preferably 4 to 6 g lipid/100 kcal, more preferably 4.5 to 5.5 g lipid/100 kcal, preferably comprises 1 .7 to 5 g protein/100 kcal, more preferably 1 .8 to 3.5 g protein/100 kcal, even more preferably 1.8 to 2.1 g protein/100 kcal, most preferably 1 .8 to 2.0 g protein/100 kcal and preferably comprises 5 to 20 g digestible carbohydrate/100 kcal, more preferably 6 to 16 g digestible carbohydrate/100 kcal, and most preferably 10 to 15 g digestible carbohydrate/100 kcal.

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

In one embodiment, the nutritional composition is a powder. Suitably, the nutritional composition is in a powdered form, which can be reconstituted with water or other food grade aqueous liquid, to form a ready- to drink liquid, or is in a liquid concentrate form that should be diluted with water to a ready-to-drink liquid.

In alternative preferred embodiment, the nutritional composition is a medical nutritional product, preferably a medical nutritional product for adults.

A second aspect of the invention relates to a nutritional composition comprising digestible carbohydrate, protein and lipid, as defined herein.

Preferably, all embodiments described herein above in relation to the nutritional composition for use in the first aspect ofthe invention equally apply to the nutritional composition in the second aspect of the invention. In a preferred embodiment, the nutritional composition comprises 14 - 30 g lipid per 100 g dry weight, more preferably 16 - 25 g lipid per 100 g dry weight, and 7 - 16 g protein per 100 g dry weight, more preferably 8 - 12 g protein per 100 g dry weight, and 45 - 80 g digestible carbohydrate per 100 g dry weight, more preferably 55 - 70 g digestible carbohydrate per 100 g dry weight, and 450-2000 mg of a mixture 2’-FL, 3-FL, 3’-SL, 6’-SL and LNT, per 100 g dry weight and 10⁷ to 10¹⁰ cfu B. breve per gram dry weight of the nutritional composition.

In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.

DESCRIPTION OF FIGURES

Figure 1 shows the mean (n=3) secreted iALP activity on day 4 ± SEM for the tested conditions in Example

1 . The iALP activity is shown as the fold-change compared to the iALP activity of the PBS control sample (control) on day 0, which was set to 1. Statistically significant differences are indicated by the horizontal lines above the bars.

Figure 2 shows the mean (n=3) secreted iALP activity on day 4 ± SEM for the tested conditions in Example

2. The iALP activity is shown as the fold-change compared to the iALP activity of the PBS control sample (control) on day 0, which was set to 1 . Statistically significant differences are indicated by the star.

Figure 3 shows the mean (n=3) intracellular expressed iALP activity on day 4 ± SEM for the tested conditions in Example 2. The iALP activity is shown as the enzyme units per gram of protein. Statistically significant differences are indicated by the star.

EXAMPLES

Example 1

An in vitro study was conducted to investigate the effect of B. breve and HMOS in an infant formula matrix on the production of intestinal alkaline phosphatase (iALP) by intestinal cells, both intracellularly expressed and extracellular secreted iALP.

Infant formulas

Two different types of powdered IF’s were tested in these examples. Both were complete standard cow’s milk-based infant formulas having a similar composition, except for the presence of Bifidobacterium breve and/or human milk oligosaccharides.

The IF’s comprised per 100 ml reconstituted formula, 13.6 dry matter, 66 kcal, 1 .3 g protein (intact protein with a casein/whey ratio of 40/60), 7.3 g digestible carbohydrates (mainly lactose), 3.4 g fat and 0.8 g short chain galacto-oligosaccharides (source Vivinal® GOS) and long chain fructo-oligosaccharides (source Raftilin HP®) in a 9/1 w/w ratio, and minerals, vitamins, trace elements and other micronutrients as known in the art and in compliance with directives for infant formula. The fatty acid composition was identical for all IF’s. The fat source of all IF’s comprised vegetable fat, milk lipid, and LC-PUFA containing oil (fish oil and microbial oil).

IFA is a composition without HMO and without probiotic bacteria.

IFB is a composition that comprises 1 .4 wt% HMOS based on dry weight. The HMO blend comprised five types of HMO, said five types of HMO being 2’-FL, 3-FL, LNT, 3’-SL, and 6’-SL in a wt.%, by weight of total HMO weight, of 52%, 13%, 26%, 4% and 5%, respectively (mix Chr. Hansen). In addition, it comprises 1 .10⁸ cfu per g dry weight of the probiotic bacterium Bifidobacterium breve M16-V strain (Morinaga).

In vitro digestion of IPs

The different IFs and a control PBS sample were digested in vitro using an in vitro digestion model (SIM) based on the INFOGEST model (Menard et al., Food chemistry, vol. 240, 2018, 338-345). The different IF’s comprised per 100 ml reconstituted formula 13.6 grams powdered IF product.

The bioreactor comprised at the start of the digestion experiment 35 mL of IF to simulate the ingestion of a 200 mL meal by a 0-6 month-old infant. All other volumes were adjusted proportionally to this volume. The ratio between infant formula to simulated digestive fluids (and the composition thereof) resembled recommendations for digestion model from INFOGEST. The temperature of the bioreactor was set to 37 °C using a water bath.

After the IF reached a temperature of 37 °C, a single shot of simulated saliva fluid (SSF) and simulated gastric fluid (SGF) was added to the bioreactor in order to start the simulation of the gastric phase. The gastric phase lasted 120 minutes during which SGF was continuously added and the pH was gradually lowered following a set curve based on in vitro observations by the addition of 0.25 mL HCI to closely mimic the postprandial infant gastric pH.

After the gastric phase, the pH was increased to 6.5 in 10 minutes by the addition of 1 M NaHCOs to prepare for the simulation of the subsequent intestinal phase. The intestinal phase lasted 180 minutes and was started by a single shot of simulated intestinal fluid (SIF). During the intestinal phase SIF was continuously added and the pH was gradually increased to 7.2 over the course of 180 minutes by the addition of a solution comprising 0.25 M NaHCOs and 0.25 M NaOH. Digesta samples (2 ml) was taken from the bioreactor after 1 hour into the intestinal phase. The samples were visually homogenous, indicating that the samples were representative of the conditions in the bioreactor as a whole. The digesta samples were immediately quenched after collection with 2 ml of sample buffer containing enzyme inhibitor cocktail Pefabloc and Orlistat. After the digesta samples were quenched, the samples were snap frozen using liquid nitrogen.

Cell Culture

A human Caco-2 cell line was used as a model of intestinal epithelium and purchased from ATCC (HTB- 37). Cells (passages 50-70) were maintained in a complete growth medium DMEM (high glucose+ Glutamax, phenol red, 31966021 , Gibco) supplemented with 10% heat-inactivated fetal bovine serum (10270106, Gibco), 1 % penicillin-streptomycin (15140-130, Gibco), 1% non-essential amino acids in MEM (MEM NEAA 100X, 11140-035, Gibco), 1% sodium pyruvate in MEM (100 mM stock, 11360-039, Gibco). The cells were grown in 75 cm² flasks (Nunc EasyFlask, Thermo scientific) in a humid incubator (HeraCell 150, Thermo Scientific) at 37 °C and 5% CO2, and were routinely subcultured after being confluent at 80%, with a change of medium thrice a week after 100% confluence. Cell viability and concentration was measured each week using an automated cell viability analyzer (Vi-Cell XR, Beckman Coulter).

Alkaline phosphatase assay

Caco-2 cells were seeded at 2.5 x 10⁵ cells/mL in 24-well plates (3526, Corning COSTAR®, Corning Inc.) and kept in culture for 16 days, with a change of medium thrice a week.

The digesta samples (of the IF-products and the PBS control) from the in vitro digestion were further diluted in a 1 :32 ratio with culture medium (high glucose+ Glutamax, phenol red, 31966021 , Gibco) supplemented with 1 % BSA, 1% penicillin-streptomycin (15140-130, Gibco), 1 % sodium pyruvate in MEM (100 mM stock, 11360-039, Gibco) prior to addition to the cells which received fresh culture medium just before (final dilution 1 :64). Each condition was tested in triplicate.

Caco-2 cells were incubated with the diluted digesta samples for 4 days, and 20 uL supernatant of each well was collected on each day - day zero (DO) to day 4 (D4) - and tested for secreted alkaline phosphatase activity (Abeam ab83369). After 4 days, cells were lyzed and expressed intracellular iALP levels were measured. In each of the collected samples also the total protein was measured (Pierce BCA Protein Assay kit, Thermo Scientific). The iALP enzyme units were standardized per gram of protein.

Results

The results are depicted in Figure 1 . The iALP activity on the y-axis is shown as a fold-change to the iALP activity of the PBS control sample on DO. The statistically significant differences are indicated in Figure 1 by the horizontal lines above the bars (ANOVA followed by a Least Significant Difference (LSD) test for conditions comparisons (p<0.05)). The combination of B. breve and HMOs induces a higher expression of iALP when compared to the control.

Example 2

The experiment from Example 1 was repeated, but this time four different IFs were compared, to distinguish the effect between HMOS and B. breve:

The amount of B. breve and HMOS, when present, was the same as in example 1. The HMO blend comprised five types of HMO, said five types of HMO being 2’-FL, 3-FL, LNT, 3’-SL, and 6’-SL in a wt.%, by weight of total HMO weight, of 52 wt.%, 13 wt.%, 26 wt.%, 4 wt.% and 5 wt.%, respectively (mix Chr. Hansen). The probiotic Bifidobacterium breve strain was B. breve M16-V (Morinaga), and was present in an amount of 1 .10⁸ cfu per g dry weight.

All other components of the four IF were similar, like in example 1. The IF’s comprised per 100 ml reconstituted formula, 13.6 dry matter, 66 kcal, 1.3 g protein (intact protein with a casein/whey ratio of 40/60), 7.3 g digestible carbohydrates (mainly lactose), 3.4 g fat and 0.8 g short chain galactooligosaccharides (source Vivinal® GOS) and long chain fructo-oligosaccharides (source Raftilin HP®) in a 9/1 w/w ratio, and minerals, vitamins, trace elements and other micronutrients as known in the art and in compliance with directives for infant formula. The fatty acid composition was identical for all IF’s. The fat source of all IF’s was identical and comprised vegetable fat, milk lipid, and LC-PUFA containing oil (fish oil and microbial oil).

Results

The results are depicted in Figure 2 (secreted extracellular iALP on day 4) and Figure 3 (intracellular expressed iALP on day 4) - with on the x-axis the PBS control sample (control) and the different IF’s. The SEM is shown for each condition. For the intracellular expressed iALP the iALP activity on the y-axis is shown in enzyme units standardized per gram of protein. For the extracellularly secreted iALP the iALP activity on the y-axis is shown as a fold-change to the iALP activity of the PBS control sample on DO. The statistically significant differences are indicated in Figure 2 and 3 by the star. The iALP activity, both intracellularly and extracellularly, was similar for control, IF-1 , IF-2 and IF-3. Only I F-4 showed a higher iALP activity, which was significantly different from each of the other tested conditions (ANOVA followed by a Least Significant Difference (LSD) test for conditions comparisons (p<0.05)).

The combination of B. breve and HMO when present in a nutritional composition synergistically increases the intracellular expression of iALP and the secretion of iALP. The increase of expression and secretion of iALP is indicative of enforcement of gut epithelial defence and strengthening of the gut barrier and therefore contributes towards preventing intestinal inflammatory diseases and intestinal infections.

Example 3: Infant formula

Infant formula comprising per 100 ml (obtained from reconstituting 13.7 g powder with water): 66 kcal digestible carbohydrates (mainly lactose): 7.2 g protein (whey protein, casein): 1.3 g lipids: 3.4 g non-digestible oligosaccharides 0.872 g consisting of o 0.7 g GOS/lcFOS in a 9:1 wt/wt ratio o 0.172 g of a mixture of 5 HMDs consisting of 52 wt% 2’-FL, 13 wt% 3-FL, 26 wt% LNT, 4 wt% 3’-SL and 5 wt% 6’-SL about 10⁹ cfu Bifidobacterium breve M16-V micronutrients according to directive for infant formulas

Example 4:

An amino acid based infant formula in powder form, packed with on the package instructions for reconstitution with water and an indication that it is for the use for the dietary management of infants of fl- 12 months that suffer from cow’s milk allergy, multiple food allergies.

About 14.4 g of powder is reconstituted with water to 100 ml of formula.

Per 100 g powder to composition comprises:

- 474 kcal

13.2 g protein equivalent, free amino acids (L-Arg-L-Asp, L-Leu, L-Lys, L-Glu, L-Pro, L-Val, L-lle, Gly, L-Thr, L-Phe , L-Tyr, L-Ser, L-His, L-Ala, L-Cys, L-Trp, L-Met)

50.2 g digestible carbohydrates (mainly glucose syrup)

23.6 g lipid (mainly vegetable lipid and containing microbial oil delivering arachidonic acid, and docosahexaenoic acid)

0.82 g non-digestible oligosaccharide mixture consisting of o 2.74 scFOS (source Raftilose®P95) o 0.29 g IcFOS (source Raftiline®HP) o 0.84 g of a mixture of 5 HMOs consisting of 52 wt% 2’-FL, 13 wt% 3-FL, 26 wt% LNT, 4 wt% 3’-SL and 5 wt% 6’-SL (Chr Hansen)

B. breve M-16V (Morinaga) 10⁸ cfu per g powder minerals, trace elements, vitamins and other micronutrients according to international directives for infant formulas

Claims

1 A nutritional composition, which is not human milk, comprising digestible carbohydrate, protein, lipid, Bifidobacterium breve and human milk oligosaccharides (HMO), wherein a. the HMOS are present in an amount of 0.3 to 4 wt% based on dry weight, b. the HMOS are a mixture of 2’-fucosyllactose, 3-fucosyllactose, 3’-sialyllactose, 6’-sialyllactose and lacto-N-tetraose, and c. the Bifidobacterium breve is present is an amount of 10⁵ to 10¹² colony forming units (cfu) per gram of dry weight of the composition.

2 The nutritional composition according to claim 1 , wherein the composition further comprises 0.5 to 10 wt% of the sum of galacto-oligosaccharides (GOS) and fructo-oligosaccharides (FOS).

3 The nutritional composition according to claim 2, wherein the ratio of B. breve cfu to weight GOS and/or FOS is 5.10⁹ to 5.10¹⁴ cfu/gram sum of GOS plus FOS.

4 The nutritional composition according to any one of claims 1-3, wherein the ratio B. breve cfu to weight HMOs is 10⁹ to 10¹⁴ /gram mixture of HMOs.

5 A nutritional composition comprising digestible carbohydrate, protein, lipid, Bifidobacterium and human milk oligosaccharides (HMOs), wherein a) the HMOS are a mixture of 2’-fucosyllactose, 3-fucosyllactose, 3’-sialyllactose, 6’-sialyllactose and lacto-N-tetraose, and b) the Bifidobacterium comprises Bifidobacterium breve, for use in one or more of: i. preventing intestinal inflammatory diseases, and ii. preventing intestinal infections, wherein said nutritional composition is not human milk, wherein the nutritional composition is selected from an infant formula, a follow-on formula or a growing up milk, and wherein the HMOs are present in an amount of 0.3 to 4 wt% based on dry weight.

6 A non-therapeutic use of a nutritional composition comprising digestible carbohydrate, protein, lipid, Bifidobacterium and human milk oligosaccharides (HMOs), wherein a) the HMOs are a mixture of 2’-fucosyllactose, 3-fucosyllactose, 3’-sialyllactose, 6’-sialyllactose and lacto-N-tetraose, and b) the Bifidobacterium comprises Bifidobacterium breve, for one or more of i) strengthening the gut epithelial defence; and ii) strengthening the gut barrier function, wherein said nutritional composition is not human milk, wherein the nutritional composition is selected from an infant formula, a follow-on formula or a growing up milk, and wherein the HMOs are present in an amount of 0.3 to 4 wt% based on dry weight.

7 The nutritional composition for use according to claim 5 or non-therapeutic use according to claim 6, wherein the preventing intestinal inflammatory diseases, preventing intestinal infections, strengthening the gut epithelial defence and/or strengthening the gut barrier function is by increasing the intracellular expressed activity of intestinal alkaline phosphatase (iALP) and/or by increasing the extracellular secreted iALP activity.

8 The nutritional composition for use according to claim 5 or 7, wherein the preventing intestinal inflammatory diseases and/or preventing intestinal infections is by strengthening the gut epithelial defence and/or the gut barrier function.

9 The nutritional composition for use according to any one of claims 5, 7-8, wherein the intestinal inflammatory diseases are selected from enterocolitis, necrotizing enterocolitis (NEC) and inflammatory bowel diseases.

10 The nutritional composition for use or non-therapeutic use according to any one of claims 5-9, wherein the composition further comprises galacto-oligosaccharide (GOS) and/or fructo-oligosaccharide (FOS).

11 The nutritional composition for use or non-therapeutic use according to any one of the claims 5-10, wherein the Bifidobacterium breve is present is an amount of 10⁵ to 10¹² colony forming units (cfu) per gram dry weight of the composition.

12 The nutritional composition for use or non-therapeutic use according to any one of the claims 5-11 , wherein the use is in an infant or young child, preferably an infant.

13 The nutritional composition for use or non-therapeutic use according to claim 12, wherein the use is in an infant or young child, preferably in an infant, that is born via caesarean section, is born preterm, is treated with antibiotics and/or is allergic to food protein.