WO2025233458A1

WO2025233458A1 - Nutritional composition for strenghtening the gut barrier

Info

Publication number: WO2025233458A1
Application number: PCT/EP2025/062629
Authority: WO
Inventors: Fadoua DAOUAD; Ingrid Brunhilde RENES; Noortje IJSSENNAGGER; Nana BARTKE; 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, children or to a medical nutritional composition. 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

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 lipids are known to have a distinct physical structure composed of large lipid globules with a mode diameter, based on volume, of about 4 pm, existing of a triglyceride core coated by a tri-layer of membranes, the milkfat globule membrane (MFGM). Standard infant formula’s typically have lipid droplets with a mode diameter, based on volume, of about 0.3-0.5pm, due to industrial processing procedures applied to achieve stable products, and the lipid droplets are not surrounded by MFGM, but mostly by milk proteins. Infant formula with lipid globules with an architecture more similar to the lipid globules in human milk have been described (e.g. WO2010/027258 or WO2010/027259).

WO2011/069987 describes a composition comprising a probiotic (e.g. Bifidobacteria lactis) and MFGM, wherein said MFGM enhances the biological effects of said probiotic. Said biological effects comprising promoting immune maturation, reducing inflammation, treating or preventing diseases or infections, enhancing gut comfort, reducing colic, enhancing or regulating sleep or sleep patterns, reducing regurgitation, enhancing digestion, reducing constipation or combinations thereof.

WO2021/152176 describes an infant formula or follow on formula comprising larger lipid globules with a phospholipid coating for modulating timing and outcome of gut maturation in an infant, wherein the timing and outcome of gut maturation in the infant is more similar to the timing and outcome of gut maturation observed in human milk fed infants. WO2023/118510 describes a nutritional composition comprising a mix of Bifidobacterium species and at least one human milk oligosaccharide, wherein a) the Bifidobacterium species comprise at least i. a Bifidobacterium bifidum strain able to express at least one extracellular enzyme selected from a fucosidase and a sialidase, ii. a Bifidobacterium breve strain able to metabolize a saccharide selected from L-fucose and sialic acid, and b) 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 being suggested for reducing the risk of occurrence, preventing and/or treating an intestinal infection, intestinal inflammation and/or diarrhea.

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 the following three ingredients: large phospholipid coated lipid globules, probiotic bacteria and human milk oligosaccharides (HMO), synergistically increase the intestinal alkaline phosphatase levels (iALP), both intracellularly and extracellularly. 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 comprising digestible carbohydrates, protein, lipid, probiotic bacteria and human milk oligosaccharides (HMO), wherein said nutritional composition is not human milk, wherein the lipid is in the form of lipid globules and wherein a. the lipid globules have a mode diameter, based on volume, of at least 1.0 pm; and/or at least 40 vol.% of the lipid globules, based on total lipid volume, have a diameter of 2 to 12 pm; and b. the lipid comprises 0.5 to 20 wt.% mammalian milk derived phospholipids based on total lipids and wherein the lipid globules have a coating on the surface comprising said phospholipids, 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 comprising digestible carbohydrates, protein and lipid, wherein the lipid is in the form of lipid globules and wherein a. the lipid globules have a mode diameter, based on volume, of at least 1.0 pm; and/or at least 40 vol.% of the lipid globules based on total lipid volume have a diameter of 2 to 12 pm; and b. the lipid comprises 0.5 to 20 wt.% mammalian milk derived phospholipids based on total lipids and wherein the lipid globules have a coating on the surface comprising said phospholipids, and wherein the nutritional composition further comprises by dry weight of the composition i. 0.1-10 wt.% HMO; and ii. 0-15 wt.% GOS and/or FOS; and wherein the nutritional composition further comprises 10⁴-10¹⁰ colony forming units (CFU) probiotic bacteria per gram of dry weight of the composition and wherein the nutritional composition is not human milk.

DETAILED DESCRIPTION

A first aspect of the invention thus concerns a nutritional composition comprising digestible carbohydrates, protein, lipid, probiotic bacteria and human milk oligosaccharides (HMO), wherein said nutritional composition is not human milk, wherein the lipid is in the form of lipid globules and wherein a. the lipid globules have a mode diameter, based on volume, of at least 1.0 pm; and/or at least 40 vol.% of the lipid globules, based on total lipid volume, have a diameter of 2 to 12 pm; and b. the lipid comprises 0.5 to 20 wt.% mammalian milk derived phospholipids based on total lipids and wherein the lipid globules have a coating on the surface comprising said phospholipids, 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.

In a preferred embodiment, the invention may also be worded as a method for: i. strengthening the gut epithelial defence; ii. strengthening the gut barrier function; iii. preventing intestinal inflammatory diseases; and/or iv. preventing intestinal infections; said method comprising administration of a nutritional composition comprising digestible carbohydrates, protein, lipid, probiotic bacteria and human milk oligosaccharides (HMO), wherein said nutritional composition is not human milk, wherein the lipid is in the form of lipid globules and wherein a. the lipid globules have a mode diameter, based on volume, of at least 1.0 pm; and/or at least 40 vol.% of the lipid globules, based on total lipid volume, have a diameter of 2 to 12 pm; and b. the lipid comprises 0.5 to 20 wt.% mammalian milk derived phospholipids based on total lipids and wherein the lipid globules have a coating on the surface comprising said phospholipids.

In another preferred embodiment, the invention may also be worded as the use of digestible carbohydrates, protein and lipid in the manufacture of a nutritional composition for: i. strengthening the gut epithelial defence; ii. strengthening the gut barrier function;

Hi. preventing intestinal inflammatory diseases; and/or iv. preventing intestinal infections; said nutritional composition comprising digestible carbohydrates, protein, lipid, probiotic bacteria and human milk oligosaccharides (HMO), wherein said nutritional composition is not human milk, wherein the lipid is in the form of lipid globules and wherein a. the lipid globules have a mode diameter, based on volume, of at least 1.0 pm; and/or at least 40 vol.% of the lipid globules, based on total lipid volume, have a diameter of 2 to 12 pm; and b. the lipid comprises 0.5 to 20 wt.% mammalian milk derived phospholipids based on total lipids and wherein the lipid globules have a coating on the surface comprising said phospholipids.

In yet another preferred embodiment, the invention may also be worded as the use of a nutritional composition comprising digestible carbohydrates, protein, lipid, probiotic bacteria and human milk oligosaccharides (HMO), wherein said nutritional composition is not human milk, wherein the lipid is in the form of lipid globules and wherein a. the lipid globules have a mode diameter, based on volume, of at least 1.0 pm; and/or at least 40 vol.% of the lipid globules, based on total lipid volume, have a diameter of 2 to 12 pm; and b. the lipid comprises 0.5 to 20 wt.% mammalian milk derived phospholipids based on total lipids and wherein the lipid globules have a coating on the surface comprising said phospholipids, for: i. strengthening the gut epithelial defence; ii. strengthening the gut barrier function;

Hi. preventing intestinal inflammatory diseases; and/or iv. preventing intestinal infections. 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.

The term “preventing” as used herein refers to stopping, delaying or reducing the incidence/severity of a disease, before the disease occurs.

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 intestinal infections.

Preferably, the use of the present invention is in a human subject, more preferably in a human infant aged 0-36 months, even more preferably a human infant aged 0-24 months, yet even more preferably a human infant aged 0-12 months and most preferably a human infant aged 0-6 months.

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

Lipid globule size

The lipid is present in the nutritional composition in the form of lipid globules. When the nutritional composition is in liquid form, these lipid globules are emulsified in the aqueous phase. Alternatively, when the nutritional composition is in powder form, the lipid globules are present in the powder and the powder is suitable for reconstitution with water or another food grade aqueous phase. The lipid globules comprise a core and a surface.

The lipid globules in the nutritional composition preferably have mode diameter, based on volume, of at least 1 .0 pm, more preferably at least 2.0 pm, even more preferably at least 3.0 pm, and most preferably at least 4.0 pm. Preferably, the lipid globules have a mode diameter, based on volume, between 1.0 and 10 pm, more preferably between 2.0 and 8.0 pm, even more preferably between 3.0 and 7.0 pm, and most preferably between 4.0 pm and 6.0 pm.

Alternatively, or preferably in addition, the size distribution of the lipid globules is preferably in such a way that at least 45 volume % (vol.%), more preferably at least 55 vol.%, even more preferably at least 65 vol.%, and most preferably at least 75 vol.% of the lipid globules have a diameter between 2 and 12 pm. In a more preferred embodiment, at least 45 vol.%, preferably at least 55 vol.%, more preferably at least 65 vol.%, and most preferably at least 75 vol.% of the lipid globules have a diameter between 2 and 10 pm. In an even more preferred embodiment, at least 45 vol.%, more preferably at least 55 vol.%, yet even more preferably at least 65 vol.%, and most preferably at least 75 vol.% of the lipid globules have a diameter between 4 and 10 pm. Preferably less than 5 vol.% of the lipid globules have a diameter above 12 pm.

The volume percentage of lipid globules is based on volume of total lipid. The mode diameter relates to the diameter which is the most present based on volume of total lipid, or the peak value in a graphic representation, having on the X-axis the diameter and on the Y-axis the volume (%).

The volume of the lipid globules and its size distribution can suitably be determined using a particle size analyzer such as a Mastersizer 2000 (Malvern Instruments, Malvern, UK), for example by the method described in Michalski et al., 2001 , Lait 81 : 787-796.

Phospholipid

The lipid in the nutritional composition comprises 0.5 to 20 wt.% phospholipids based on total lipid and the lipid globules have a coating on the surface comprising said phospholipids. Preferably, the nutritional composition comprises 0.6 to 10 wt.%, more preferably 0.7 to 8 wt.%, even more preferably 0.8 to 6 wt.%, and most preferably 1 to 5 wt.% phospholipids based on total lipid.

Phospholipids are amphipathic of nature and include glycerophospholipids and sphingomyelin. By ‘coating’ is meant that the outer surface layer of the lipid globules comprises phospholipid, whereas phospholipid is virtually absent in the core of the lipid globule. A suitable way to determine whether phospholipid is located on the surface of lipid globules is confocal laser scanning microscopy or transmission electron microscopy; see for instance Gallier et al. (A novel infant milk formula concept: Mimicking the human milk fat globule structure, Colloids and Surfaces B: Biointerfaces, 136 (2015), 329-339).

The nutritional composition preferably comprises glycerophospholipids. Examples of glycerophospholipids are phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylglycerol (PG). Preferably the nutritional composition comprises one or more of PC, PS, PI and PE, more preferably the nutritional composition comprises at least PC.

The nutritional composition preferably comprises sphingomyelin. Sphingomyelins have a phosphorylcholine or phosphorylethanolamine molecule esterified to the 1 -hydroxy group of a ceramide. They are classified as phospholipid as well as sphingolipid, but are not classified as a glycerophospholipid nor as a glycosphingolipid. Preferably the nutritional composition comprises 0.05 to 10 wt.% sphingomyelin based on total lipid, more preferably 0.1 to 5 wt.%, even more preferably 0.2 to 2 wt.%. Preferably the nutritional composition comprises at least 5 wt.%, more preferably 5 to 40 wt.% sphingomyelin based on total phospholipid, more preferably 10 to 35 wt.%, even more preferably 15 to 35 wt.% sphingomyelin, based on total phospholipid.

The nutritional composition preferably comprises glycosphingolipids. The term glycosphingolipids in the present context particularly refers to glycolipids with an amino alcohol sphingosine. The sphingosine backbone is O-linked to a charged head-group such as ethanolamine, serine or choline backbone. The backbone is also amide linked to a fatty acyl group. Glycosphingolipids are ceramides with one or more sugar residues joined in a beta-glycosidic linkage at the 1-hydroxyl position, and include gangliosides. Preferably the nutritional composition contains gangliosides, more preferably at least one ganglioside selected from the group consisting of GM3 and GD3. Preferably the nutritional composition comprises 0.1 to 10 wt.% glycosphingolipids based on total lipid, more preferably 0.5 to 5 wt.%, even more preferably 2 to 4 wt.% glycosphingolipids, based on total lipid.

The nutritional composition preferably comprises cholesterol. The nutritional composition preferably comprises at least 0.005 wt.% cholesterol based on total lipid, more preferably at least 0.02 wt.%, even more preferably at least 0.05 wt.%, and most preferably at least 0.1 wt.% cholesterol based on total lipid. Preferably the amount of cholesterol does not exceed 10 wt.% based on total lipid, more preferably does not exceed 5 wt.%, most preferably does not exceed 1 wt.% of cholesterol based on total lipid in the nutritional composition.

Preferred sources for providing the phospholipid, glycosphingolipid and/or cholesterol are egg lipids, milk fat, buttermilk fat and butter serum fat (such as beta serum fat). Another preferred source for phospholipid, particularly PC, is soy lecithin and/or sunflower lecithin.

The nutritional composition preferably comprises phospholipid derived from mammalian milk. Preferably the nutritional composition comprises phospholipid and glycosphingolipid derived from mammalian milk. Preferably also cholesterol is derived from mammalian milk. The nutritional composition preferably comprises phospholipid, glycosphingolipid and/or cholesterol derived from mammalian milk of cows, mares, sheep, goats, buffalos, horses and camels. More preferably the nutritional composition comprises phospholipid, glycosphingolipid and/or cholesterol derived from cow’s milk.

Phospholipid derived from mammalian milk includes preferably phospholipid that is derived from milk lipid, cream lipid, cream serum lipid, butter serum lipid (beta serum lipid), whey lipid, cheese lipid and/or buttermilk lipid. Buttermilk lipid is typically obtained during the manufacture of buttermilk. Butter serum lipid or beta serum lipid is typically obtained during the manufacture of anhydrous milk fat from butter. More preferably the phospholipid, glycosphingolipid and/or cholesterol is derived from whey, e.g., a whey protein concentrate. Suitable commercially available sources for phospholipid from milk are BAEF, SM2, SM3 and SM4 powder of Corman, Salibra of Glanbia, Vivinal MFGM of FrieslandCampina and LacProdan MFGM- 10 or PL20 from Aria.

The use of phospholipid from mammalian milk fat advantageously comprises the use of milk fat globule membranes, which are more reminiscent to the situation in human milk. The concomitant use of phospholipid derived from mammalian milk and triglycerides derived from vegetable lipids therefore enables the manufacture of coated lipid globules with a coating more similar to human milk, while at the same time providing an optimal fatty acid profile.

Preferably the phospholipid is derived from mammalian milk, more preferably derived from or forms part of milk fat globule membrane (MFGM). Preferably the phospholipid is derived from cow’s milk, more preferably derived from or forms part of cow’s MFGM.

Preferably the nutritional composition comprises phospholipid and glycosphingolipid and more preferably the weight ratio of phospholipid : glycosphingolipid is from 2:1 to 12:1 , more preferably from 2:1 to 10:1 and even more preferably 2:1 to 5:1 .

Methods for obtaining lipid globules with an increased size and/or coating with phospholipid are for example described in WO2010/027258 and WO2010/027259.

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

The lipid preferably comprises vegetable lipids. 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. Lipids 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 lipids based on total lipids, 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 lipids based on total lipids. Preferably, the nutritional composition also comprises non-vegetable lipids. Preferably, said non-vegetable lipids are one or more non-vegetable lipids 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 1/4 to 4/1 , more preferably between 1/2 to 2/1 , more preferably between 0.6 and 1 .5. Probiotics

The nutritional composition comprises probiotic bacteria. The term “probiotic bacteria” as used herein refers to live beneficial bacteria that provide health benefits when consumed, generally by improving or restoring the gut microbiota. The term “gut microbiota” as used herein refers to all microorganisms, including bacteria, archaea, virus, and fungi, that are found in the digestive tract of a human subject.

The probiotic bacteria are preferably selected from Lactobacillus, Bifidobacterium and combinations thereof, more preferably the probiotic bacteria comprise Bifidobacterium, even more preferably the probiotic bacteria are Bifidobacterium, yet even more preferably the probiotic bacteria are Bifidobacterium breve and most preferably the probiotic bacteria are the strain B. breve M-16V (Morinaga).

The probiotic bacteria are preferably provided in therapeutically effective amounts. The nutritional composition preferably contains between 10⁴ and 1O¹⁰ colony forming units (cfu) probiotic bacteria per gram dry weight of the present composition, more preferably between 10⁵ and 10⁹, and most preferably between 10⁶ and 10⁸ CFU probiotic bacteria per gram dry weight of the nutritional composition.

When the nutritional composition is a liquid nutritional composition, preferably a ready-to-drink liquid nutritional composition, the liquid nutritional composition preferably comprises 10⁶ and 10¹⁰ colony forming units (cfu) probiotic bacteria per 100 ml, more preferably between 10⁷ and 10⁹ CFU per 100 ml.

In terms of doses, the nutritional composition preferably provides between 10® and 10¹¹ CFU probiotic bacteria per serving, more preferably between 10⁷ and 10¹° CFU probiotic bacteria per serving.

Preferably, the use is in a subject and 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 (HMO). The term “human milk oligosaccharides” or “HMO” as used herein refers to non-digestible oligosaccharides which are present in human breast milk. Human breast milk comprises two types of carbohydrates: lactose and HMO. HMO are the third most abundant component of human breast milk, after lactose and lipids. Human breast milk contains three major HMO types: fucosylated HMO, sialylated HMO and N-acetylated HMO.

Suitable HMO 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.

Preferably, the wt. ratio of HMO to mammalian milk derived phospholipids in the nutritional composition is between 1 :10 to 30:1 , more preferably between 1 :5 to 20:1 , and most preferably between 1 :1 to 10:1 .

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 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 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.

The HMO in the nutritional composition is preferably selected from 2’-fucosyllactose (2’FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), para-lacto-N-neohexaose (para-LNnH), sialic acid, 3' sialyllactose (3’SL), 6' sialyllactose (6’SL), difucosyllactose (DFL), lacto-N-fucopentaose, lacto-N- fucohexaose, lacto-N-difucohexaose, sialyl-lacto-N-tetraose (LSTa), sialyl-lacto-N-tetraose b (LSTb), sialyl- lacto-N-tetraose c (LSTc), disialyllacto-N-tetraose (DSLNT), lacto-N-neodifucohexaose (LNnDFH I), fucosyllacto-N-hexaose, fucosyllacto-N-neohexaose, difucosyllacto-N-hexaose I, difuco-lacto-N- neohexaose, difucosyllacto-N-neohexaose I, difucosyllacto-N-neohexaose II, fucosyl-para-Lacto-N- hexaose, and tri-fuco-para-Lacto-N-hexaose I and combinations thereof.

The HMO preferably comprises at least 2 types of HMO, more preferably at least 3 types of HMO, even more preferably at least 4 types of HMO, and most preferably at least 5 types of HMO. Preferably, these types of HMO are selected from 2’-fucosyllactose (2’FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), para-lacto-N-neohexaose (para-LNnH), sialic acid, 3' sialyllactose (3’SL), 6' sialyllactose (6’SL), difucosyllactose (DFL), lacto-N-fucopentaose, lacto-N-fucohexaose, lacto-N- difucohexaose, sialyl-lacto-N-tetraose (LSTa), sialyl-lacto-N-tetraose b (LSTb), sialyl-lacto-N-tetraose c (LSTc), disialyllacto-N-tetraose (DSLNT), lacto-N-neodifucohexaose (LNnDFH I), fucosyllacto-N-hexaose, fucosyllacto-N-neohexaose, difucosyllacto-N-hexaose I, difuco-lacto-N-neohexaose, difucosyllacto-N- neohexaose I, difucosyllacto-N-neohexaose II, fucosyl-para-Lacto-N-hexaose, and tri-fuco-para-Lacto-N- hexaose I and combinations thereof.

In a preferred embodiment, the HMO is selected from 2’FL, 3-FL, DFL, LNT, LNnT, 3’SL, 6’SL, and combinations thereof. More preferably the HMO is selected from 2’FL, 3-FL, LNT, 3’SL, 6’SL and combinations thereof.

In a particularly preferred embodiment, the nutritional composition comprises 5 types of HMO, said 5 types of HMO being 2’FL, 3-FL, LNT, 3’SL, and 6’SL. More preferably, the HMO comprises 40-60 wt.% 2’FL, 10- 20 wt.% 3-FL, 20-30 wt.% LNT, 2-7 wt.% 3’SL, and 4-8 wt.% 6’SL based on total HMO weight.

In alternative preferred embodiment, the HMO comprise at least 40 wt.%, more preferably 45-100 wt.%, and most preferably 50-90 wt.% of 2’FL based on total HMO weight.

In yet another alternative preferred embodiment, the HMO comprises a combination of 2’FL and LNnT, more preferably the HMO consists of the combination of 2’FL and LNnT. Preferably, the HMO comprises 60-90 wt.% 2’FL and 10-40 wt.% LNnT based on total HMO weight, more preferably the HMO comprises 65-85 wt.% 2’FL and 15-35 wt.% LNnT and most preferably the HMO comprises 70-80 wt.% 2’FL and 20- 30 wt.% LNnT.

GOS and FOS

The nutritional composition preferably comprises galacto-oligosaccharides (GOS) and/or fructooligosaccharides (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 are preferably transgalacto-oligosaccharides. A suitable GOS is commercially available, for example VivinalOGOS (FrieslandCampina DOMO). Preferably the GOS are short chain galactooligosaccharides (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 are long chain fructo-oligosaccharides (IcFOS) with an average DP in the range of 10-100, more preferably in the range of 20 to 60. 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 ratio’s apply to scGOS and IcFOS (sc = short chain; Ic = longchain).

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/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.

In a preferred embodiment, the nutritional composition comprises by dry weight of the composition:

- 10-50 wt.% lipids, wherein said lipids comprise 0.5 to 20 wt.% mammalian milk derived phospholipids based on total lipids;

- 0.1-10 wt.% HMO;

- 0-15 wt.% GOS and/or FOS; and wherein the nutritional composition further comprises 1 O⁴-1 O¹⁰ colony forming units (CFU) Bifodobacterium per gram of dry weight of the composition.

More preferably, the nutritional composition comprises by dry weight of the composition:

- 20-40 wt.% lipids, wherein said lipids comprise 1 to 5 wt.% mammalian milk derived phospholipids based on total lipids;

- 0.5-4 wt.% HMO;

- 1-10 wt.% GOS and/or FOS; and wherein the nutritional composition further comprises 1 O⁴-1 O¹⁰ colony forming units (CFU) Bifidobacterium breve per gram of dry weight of the composition.

Digestible carbohydrates

The nutritional composition comprises digestible carbohydrates. The digestible carbohydrates preferably provide 30 to 80% of the total calories of the nutritional composition. Preferably the digestible carbohydrates provide 40 to 60% of the total calories. Based on calories the nutritional composition preferably comprises of 5 to 20 g of digestible carbohydrates 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 carbohydrates.

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 glycemic 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.

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 formulae 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. It was found that lipid globules maintained their size and coating when reconstituted.

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

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 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 star.

• Figure 2 shows the mean (n=3) intracellular expressed iALP activity on day 4 ± SEM for the tested conditions in Example 1. The iALP activity is shown as the enzyme units per gram of protein. Statistically significant differences are indicated by the star.

• Figure 3a and 3b show the mean (n=3) secreted iALP activity on day 3 (3a - left) and day 4 (3b - right) ± 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 horizontal lines above the bars.

EXAMPLES

Preparation of infant formulas (IF)

Five types of powdered IF’s were tested in these examples. All were complete standard cow’s milk-based infant formulas having a similar composition, except for the presence of milk derived phospholipids, probiotic bacteria, human milk oligosaccharides and/or the lipid globule size and coating (see Table 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 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, in terms of saturated, monounsaturated and polyunsaturated fatty acids, and in n3- and n6-PUFA content. The fat source of all IF’s comprised vegetable fat (blend of low erucic acid rape seed oil, coconut oil, high oleic sunflower oil, sunflower oil), bovine anhydrous milk fat, and LC-PUFA containing oil (fish oil and microbial oil). In addition, IF-A, IF-B, IF-C and I F-1 comprised a whey protein concentrate enriched in MFGM (Lacprodan MFGM-10, Aria), which provided additional milk fat including phospholipids (milk phospholipid was about 1 .5 wt.% based on total lipid of the IF).

The lipid globules of IF-A, IF-B, IF-C and I F-1 had a coating on the surface comprising mammalian milk phospholipids. These particular IF’s were prepared following a production process as described in WO2013/135739. The particle size distribution is indicated in Table 1 below. IF-D was produced according to a standard production process known in the art for producing infant formula.

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.

Table 1 - Composition of the IF’s

Example 1

An in vitro study was conducted to investigate the effect of the IF products described herein above on the production of intestinal alkaline phosphatase (iALP) by intestinal cells, both intracellularly expressed and extracellular secreted iALP.

In vitro digestion of I Fs

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 were reconstituted by dispersing 13.6 grams of powdered IF product in 90 ml lukewarm water and shaking until dispersed.

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 (secreted extracellular iALP on day 4) and Figure 2 (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 1 and 2 by the star. The iALP activity, both intracellularly and extracellularly, was similar for control, IF-A, IF-B and IF-C. Only I F-1 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, HMO and large phospholipid coated lipid globules 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 2

The experiment from Example 1 was repeated, but this time IF-D was compared to I F-1 and only secreted iALP was measured on day 3 and day 4.

Results

The results are depicted in Figures 3a (day 3) and 3b (day 4). 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 Figures 3a and 3b 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 HMO - with (IF-1) or without (IF-D) the presence of large phospholipid coated lipid globules - induces a higher expression of iALP. The combination of B. Breve, HMO and large phospholipid coated lipid globules (IF-1) has a stronger and earlier effect on iALP, compared to IF-D.

Claims

1. A nutritional composition comprising digestible carbohydrates, protein, lipid, probiotic bacteria and human milk oligosaccharides (HMO), wherein said nutritional composition is not human milk, wherein the lipid is in the form of lipid globules and wherein a. the lipid globules have a mode diameter, based on volume, of at least 1 .0 pm; and/or at least 40 vol.% of the lipid globules, based on total lipid volume, have a diameter of 2 to 12 pm; and b. the lipid comprises 0.5 to 20 wt.% mammalian milk derived phospholipids based on total lipids and wherein the lipid globules have a coating on the surface comprising said phospholipids, for use in one or more of: i. strengthening the gut epithelial defence; ii. strengthening the gut barrier function;

Hi. preventing intestinal inflammatory diseases; and iv. preventing intestinal infections.

2. The nutritional composition for use according to claim 1 , wherein the probiotic bacteria comprise Bifidobacterium.

3. The nutritional composition for use according to claim 1 or 2, wherein the composition further comprises galacto-oligosaccharides (GOS) and/or fructo-oligosaccharides (FOS).

4. The nutritional composition for use according to any of the preceding claims, wherein the composition comprises at least two types of HMO.

5. The nutritional composition for use according to any of the preceding claims, wherein the HMO is selected from 2’-fucosyllactose (2’FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), lacto-N- neotetraose (LNnT), para-lacto-N-neohexaose (para-LNnH), sialic acid, 3' sialyllactose (3’SL), 6' sialyllactose (6’SL), difucosyllactose (DFL), lacto-N-fucopentaose, lacto-N-fucohexaose, lacto-N- difucohexaose, sialyl-lacto-N-tetraose (LSTa), sialyl-lacto-N-tetraose b (LSTb), sialyl-lacto-N-tetraose c (LSTc), disialyllacto-N-tetraose (DSLNT), lacto-N-neodifucohexaose (LNnDFH I), fucosyllacto-N- hexaose, fucosyllacto-N-neohexaose, difucosyllacto-N-hexaose I, difuco-lacto-N-neohexaose, difucosyllacto-N-neohexaose I, difucosyllacto-N-neohexaose II, fucosyl-para-Lacto-N-hexaose, and tri-fuco-para-Lacto-N-hexaose I, and combinations thereof.

6. The nutritional composition for use according to any of the preceding claims, wherein the HMO is selected from 2’FL, 3-FL, DFL, LNT, LNnT, 3’SL, 6’SL and combinations thereof.

7. The nutritional composition for use according to any of the preceding claims, wherein the nutritional composition comprises by dry weight of the composition:

- 0.1-10 wt.% HMO;

- 0-15 wt.% GOS and/or FOS; and wherein the nutritional composition further comprises 1O⁴-1O¹⁰ colony forming units (CFU) Bifidobacterium per gram of dry weight of the composition.

8. The nutritional composition for use according to any of the preceding claims, wherein the use is by increasing the intracellular expressed activity of intestinal alkaline phosphatase (iALP) and/or by increasing the extracellular secreted iALP activity.

9. The nutritional composition for use according to any of the preceding claims, wherein the prevention of intestinal inflammatory diseases and/or intestinal infections is by strengthening the gut epithelial defence and/or the gut barrier function.

10. The nutritional composition for use according to any of the preceding claims, wherein the intestinal inflammatory diseases are selected from enterocolitis, necrotizing enterocolitis (NEC) and inflammatory bowel diseases.

11 . The nutritional composition for use according to any of the preceding claims, wherein the mammalian milk derived phospholipids are derived from or form part of milk fat globule membrane (MFGM).

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

13. The nutritional composition for use according to any of claims 1-11 , wherein the nutritional composition is a medical nutritional composition.

14. A nutritional composition comprising digestible carbohydrates, protein and lipid, wherein the lipid is in the form of lipid globules and wherein a. the lipid globules have a mode diameter, based on volume, of at least 1 .0 pm; and/or at least 40 vol.% of the lipid globules based on total lipid volume have a diameter of 2 to 12 pm; and b. the lipid comprises 0.5 to 20 wt.% mammalian milk derived phospholipids based on total lipids and wherein the lipid globules have a coating on the surface comprising said phospholipids, and wherein the nutritional composition further comprises by dry weight of the composition i. 0.1-10 wt.% HMO; and ii. 0-15 wt.% GOS and/or FOS; and wherein the nutritional composition further comprises 1O⁴-1O¹⁰ colony forming units (CFU) probiotic bacteria per gram of dry weight of the composition and wherein the nutritional composition is not human milk.

15. The nutritional composition according to claim 14, wherein the wt. ratio of HMO to mammalian milk derived phospholipids is between 1 :10 to 30:1.