WO2025233459A1

WO2025233459A1 - Nutritional composition for preventing intestinal barrier disruption

Info

Publication number: WO2025233459A1
Application number: PCT/EP2025/062630
Authority: WO
Inventors: Gabriël THOMASSEN; Evan Abrahamse; Ingrid Brunhilde RENES; Nana BARTKE
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 present invention relates to a nutritional composition comprising digestible carbohydrates, protein and lipid, wherein the lipid is in the form of larger lipid globules with a coating comprising phospholipids for use in preventing and/or treating intestinal barrier disruption in a human subject.

Description

NUTRITIONAL COMPOSITION FOR PREVENTING INTESTINAL BARRIER DISRUPTION

FIELD OF THE INVENTION

The present invention is in the field of nutritional compositions, which have a beneficial effect on intestinal barrier function.

BACKGROUND OF THE INVENTION

The intestinal barrier is a critical interface of the human body - on the one hand it provides for a strong defence against foreign substances while on the other hand it regulates the absorption of essential nutrients, water and electrolytes. The intestinal barrier is composed of multiple layers. The outer one comprises the mucus layer, the commensal gut microbiota and defence proteins such as antimicrobial proteins (AMPs) and secretory immunoglobulin A (slgA). Intestinal epithelial cells (lECs) are the middle layer, while the inner part is composed of e.g. immune cells of innate and adaptive immunity.

WO2021/152176 describes nutritional compositions comprising lipid globules that are larger than the lipid globules in conventional infant milk formulas. Said lipid globules also comprise a coating comprising milk derived phospholipids. This nutritional composition has a beneficial effect on gut maturation, i.e. the consumption of the nutritional composition results in a gut maturation timing and outcome that is more similar to the timing and outcome of gut maturation in a breastfed infant. The gastrointestinal tract experiences rapid growth and maturation following birth wherein the adequate digestive function develops, a functional gut barrier is established, a gut microbiota develops and the immune functionality develops. This means that during the first weeks of life all mammals have an immature intestine.

Postnatally, and also after the gut has matured and an intestinal barrier has been established, the normal healthy gut is constantly challenged by daily contact with partially digested food products, digestive agents such as bile acids, gut microbiota, pathogens, antibiotics, xenobiotics, and pollutants.

Chronic disruption of intestinal barrier function and gut homeostasis has been linked to necrotizing enterocolitis (NEC), inflammatory bowel diseases (IBD), celiac disease, irritable bowel syndrome, and colorectal cancer.

Therefore it is crucial to look for potential precautions against (chronic) intestinal barrier disruption and to promote the upkeep and normal maintenance of the intestinal barrier function in response to damage or wounding of the intestinal barrier.

Nutritional compositions have been described in the art that have an effect on intestinal barrier function and integrity. W02005/122790 describes a method for stimulating barrier integrity by administering a composition comprising eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (ARA), and at least two distinct oligosaccharides. The oligosaccharides act indirectly by being fermented to short chain fatty acids (SCFA) by the intestinal microbiota. The SCFA are the active ingredient that support the intestinal barrier function.

WO2013/016111 discloses nutritional compositions with galacto-oligosaccharides for preventing injury and/or improving the healing of the gastrointestinal tract. A mix of galacto-oligosaccharides is tested.

In W02020/013683 a nutritional composition is described which comprises 2’-fucosyllactose for use in the treatment of virus induced intestinal barrier disruption.

W02020/229690 describes the use of beta 1 ,3-galactosyllatose, another HMO, for improving the gut barrier function and discloses the potential for administration to subjects at risk of gut barrier permeability such as infants and young children.

WO2023/275391 describes a nutritional composition comprising:

• at least 45 wt.% oleic acid based on total fatty acids,

• no more than 10 wt.% palmitic acid based on total fatty acids,

• a wt./wt. ratio of palmitic acid : oleic acid of no more than 1 :4.5, and

• at least 0.05 wt.% betal ,3'-galactosyllactose based on total dry weight of the composition, for use treating and/or preventing intestinal barrier disruption.

US2022/0248738 describes compositions and uses of milk fat globule membrane complex (MFGM)- enriched complexes (e.g. liposomes) to store, protect and deliver commensal bacteria, milk oligosaccharides, and/or natural or recombinant proteins and enzymes to the gut of a mammal. These complexes are given to a mammal in need of intestinal maturation or repair or in cases where the intestinal microbiome may have been disrupted.

Anderson et al., 2018, PLOS one, ‘Bovine dairy complex lipids improve in vitro measures of small intestinal epithelial barrier integrity’ describes the effect of diary lipid fractions on in vitro measures of intestinal barrier integrity, including trans-epithelial electrical resistance (TEER), mannitol flux, and confocal microscopy of key intracellular tight junction proteins. The effects of the lipid fractions on both unchallenged cells and those challenged with permeability-inducing compounds tumour necrosis factor alpha (TNFa) and lipopolysaccharide (LPS) were examined.

SUMMARY OF THE INVENTION

The inventors investigated the effects of an experimental nutritional composition comprising a lipid component with larger-than-conventional phospholipid coated lipid globules on a human intestinal organoid model. An in vitro digestion model closely mimicking digestive conditions in real life was used to obtain a digested version of the nutritional composition encountered by the intestinal barrier. A comparison was carried out to a control composition with a lipid component in the form of small lipid globules coated with protein with or without the presence of separately added milk fat globule membrane (MFGM), which is a source of milk-derived phospholipids. The nutritional compositions were similar in caloric content, as well as in total fat, carbohydrate and protein content.

In the human intestinal organoid model, cell layer resistance was evaluated with a technology using electric cell-substrate impedance sensing (ECIS), wherein resistance is correlated with cell layer/barrier integrity. The inventors surprisingly found that the resistance after wounding of the intestinal organoid culture in the presence of the digested experimental nutritional composition showed a faster recovery. The re-establishment is likely achieved by migration of existing cells, via a process called epithelial restitution.

The current invention thus provides a nutritional composition comprising digestible carbohydrates, protein and lipid, wherein the lipid is in the form of lipid globules, wherein a. said lipid globules have a volume-weighed mode diameter of at least 1 .0 pm; and/or at least 45 vol.%, based on total lipid volume, of the lipid globules have a diameter between 2 and 12 pm; and b. the lipid comprises 0.5 to 20 wt.% phospholipids based on total lipid and wherein the lipid globules have a coating comprising phospholipids, for use in preventing and/or treating intestinal barrier disruption in a human subject.

The consumption of this nutritional composition thus may be able to help to break the vicious cycle of injury and inflammation that characterizes conditions such as NEC, inflammatory bowel diseases and irritable bowel syndrome.

DETAILED DESCRIPTION OF THE INVENTION

The invention thus concerns a nutritional composition comprising digestible carbohydrates, protein and lipid, wherein the lipid is in the form of lipid globules, wherein a. said lipid globules have a volume-weighed mode diameter of at least 1 .0 pm; and/or at least 45 vol.%, based on total lipid volume, of the lipid globules have a diameter between 2 and 12 pm; and b. the lipid comprises 0.5 to 20 wt.% phospholipids based on total lipid and wherein the lipid globules have a coating comprising phospholipids, for use in preventing and/or treating intestinal barrier disruption in a human subject.

In a preferred embodiment, the invention may also be worded as a method for preventing and/or treating intestinal barrier disruption in a human subject, wherein the method comprises administration of a nutritional composition to said subject, said nutritional composition comprising digestible carbohydrates, protein and lipid, wherein the lipid is in the form of lipid globules, wherein a. said lipid globules have a volume-weighed mode diameter of at least 1 .0 pm; and/or at least 45 vol.%, based on total lipid volume, of the lipid globules have a diameter between 2 and 12 pm; and b. the lipid comprises 0.5 to 20 wt.% phospholipids based on total lipid and wherein the lipid globules have a coating comprising 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 preventing and/or treating intestinal barrier disruption in a human subject, said nutritional composition comprising digestible carbohydrates, protein and lipid, wherein the lipid is in the form of lipid globules, wherein a. said lipid globules have a volume-weighed mode diameter of at least 1 .0 pm; and/or at least 45 vol.%, based on total lipid volume, of the lipid globules have a diameter between 2 and 12 pm; and b. the lipid comprises 0.5 to 20 wt.% phospholipids based on total lipid and wherein the lipid globules have a coating comprising phospholipids.

In yet another preferred embodiment, the invention may also be worded as the use of a nutritional composition for preventing and/or treating intestinal barrier disruption in a human subject, wherein the use comprises administration of a nutritional composition to said subject, said nutritional composition comprising digestible carbohydrates, protein and lipid, wherein the lipid is in the form of lipid globules, wherein a. said lipid globules have a volume-weighed mode diameter of at least 1 .0 pm; and/or at least 45 vol.%, based on total lipid volume, of the lipid globules have a diameter between 2 and 12 pm; and b. the lipid comprises 0.5 to 20 wt.% phospholipids based on total lipid and wherein the lipid globules have a coating comprising phospholipids.

In yet another embodiment, administering a nutritional composition to a human subject 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 “intestinal barrier” as used herein refers to an established intestinal barrier that is in a mature state.

The term “intestinal barrier disruption” as used herein refers to any damage or wounding of the intestinal barrier that interferes with proper functioning of the intestinal barrier and results in reduced intestinal barrier integrity. The term “preventing intestinal barrier disruption” as used herein refers to promoting intestinal conditions wherein the intestinal barrier is in optimal condition and thereby less vulnerable to damage or wounding.

The term “treating intestinal barrier disruption” as used herein refers to promoting intestinal conditions where the intestinal barrier can recover relatively faster after being exposed to damage or wounding.

Rath et al., 2023, DOI: 10.1080/17474124.2023.2242772, describes the link between disrupted intestinal barrier integrity and inflammatory bowel diseases.

Hanning et al., 2021 , DOI: 10.1177/1756284821993586, describes a link between disrupted intestinal barrier integrity and irritable bowel disease.

Piena-Spoel etal., 2001 , DOI: 10.1053/jpsu.2001 .22288, describes the disturbed intestinal integrity and thereby increased intestinal permeability, as symptoms of necrotizing enterocolitis (NEC) in infants.

Preferably, the human subject is at risk of intestinal barrier disruption.

In one preferred embodiment, the human subject at risk of intestinal barrier disruption is selected from human subjects exposed to: i. antibiotics; ii. intestinal pathogens;

Hi. high fat diet; iv. western style diet; and/or v. alcohol.

More preferably, the human subject at risk of intestinal barrier disruption is selected from human subjects exposed to: i. antibiotics; ii. high fat diet;

Hi. western style diet; and/or iv. alcohol.

Most preferably, the human subject at risk of intestinal barrier disruption is selected from human subjects exposed to a high fat diet and/or western style diet.

Preferably, if the human subject is at risk of intestinal barrier disruption due to exposure to intestinal pathogens, the intestinal pathogens are preferably bacteria, more preferably the intestinal pathogens are bacteria selected from Clostridioides difficile, Salmonella typhimurium, Vibrio cholerae and combinations thereof.

Preferably, the nutritional composition is a medical nutritional composition In a preferred embodiment, the human subject is an adult human subject. Preferably, the use is for preventing or treating inflammatory bowel disease, celiac disease, irritable bowel syndrome, colorectal cancer or combinations thereof in the adult human subject. More preferably, the use is for preventing or treating inflammatory bowel disease, irritable bowel syndrome or combinations thereof, in the adult human subject.

In another preferred embodiment, the human subject is an infant, preferably the human subject is a preterm infant. Preferably, the use is for preventing or treating NEC in an infant, more preferably for preventing or treating NEC in a preterm infant.

Preferably, the infant is aged 0-36 months, more preferably 0-24 months, even more preferably 0-12 months and most preferably 0-6 months.

Preferably, the nutritional composition is selected from infant formula, follow-on formula, young child formula, and human milk fortifier, more preferably the nutritional composition is selected from infant formula, follow-on formula and human milk fortifier, and most preferably the nutritional composition is selected from infant formula and follow-on formula.

The terms as used herein, “infant formula” or “follow-on formula” or “young child formula” or “human milk fortifier”, refers to nutritional compositions that are artificially made orthat 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.

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 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 milk fat globule membrane (MFGM) or provided as part of MFGM. Preferably the phospholipid is derived from cow’s milk lipid, more preferably derived from cow’s MFGM or provided as 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 100 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 nonvegetable 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.

Faty 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 in order 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 in order 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, most preferably from 5 to 10.

Preferably, the nutritional composition further comprises LCPUFA’s, more preferably one or more LCPUFA’s selected from DHA, DPA, EPA, and ARA, most preferably LCPUFA’s selected from DHA and/or ARA.

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.

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.

Non digestible carbohydrates

The nutritional composition preferably comprises non-digestible oligosaccharides. Preferably the nutritional composition comprises non-digestible oligosaccharides with a degree of polymerization (DP) between 2 and 250, more preferably 3 and 60. Preferably the nutritional composition comprises fructo-oligosaccharides, galacto-oligosaccharides and/or galacturonic acid oligosaccharides, more preferably fructo-oligosaccharides and/or galactooligosaccharides, even more preferably galacto-oligosaccharides, most preferably transgalactooligosaccharides. In a preferred embodiment the nutritional composition comprises a mixture of galactooligosaccharides and fructo-oligosaccharides, more preferably transgalacto-oligosaccharides and fructo-oligosaccharides. Suitable non-digestible oligosaccharides are for example VivinalOGOS (FrieslandCampina DOMO), RaftilinOHP or Raftilose® (Orafti).

Preferably, the nutritional composition comprises 80 mg to 2 g non-digestible oligosaccharides per 100 ml, more preferably 150 mg to 1.5 g, even more preferably 300 mg to 1 g non-digestible oligosaccharides per 100 ml. Based on dry weight, the nutritional composition preferably comprises 0.25 wt.% to 20 wt.%, more preferably 0.5 wt.% to 10 wt.%, even more preferably 1 .5 wt.% to 7.5 wt.% of non-digestible oligosaccharides.

Formula

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 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 element 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 THE FIGURES

Figure 1: ECIS wound healing model with intestinal organoids - The resistance values are shown from 1 h before till 15 h after wounding (t = 0 h) of the intestinal barrier by an electric current in a 2D intestinal organoid model. The resistance is normalized to 1 h before wounding. The data indicates statistically significant differences - using RM two-way ANOVA statistics - between nutritional composition 1 and A at 10 and 15 h after wounding.

Figure 2: ECIS wound healing model with intestinal organoids - The area under the curve resistance values are shown from 1 h before till 15 h after wounding (t = Oh) of the intestinal barrier by an electric current in a 2D intestinal organoid model. The area under the curve (AUC) is based on the data curves from time point -1 h to 15 h obtained from Figure 1. Indicated significance between the values plotted for the nutritional composition 1 and A are obtained with a unpaired t-test on the data of the cumulative curves with and without Welch’s correction for equal SD of populations.

EXAMPLES

Tested compositions

Three nutritional compositions and a human milk sample were compared. Nutritional composition A (comparative) corresponds to an infant milk formula that was prepared in the conventional way with a high pressure homogenization, resulting in small lipid globules and mainly protein in the coating. Nutritional composition B (comparative) is prepared in a similar way as Nutritional composition A, except that a source of milk-derived phospholipids (MFGM) was added after the emulsification step has taken place. Nutritional composition 1 (according to invention) is similar to nutritional composition B except that a different preparation process was applied, the source of milk-derived phospholipids was added before emulsification and the nutritional composition was prepared in a similar way as described in example 1 B of WO2010/027259.

Powdered nutritional composition A comprised per 100 ml ready to drink formula 66 kcal, 1 .3 g protein (whey protein and casein in a 6/4 w/w ratio), 7.3 g digestible carbohydrates (mainly lactose), 3.4 g fat and 0.8 g short chain galacto-oligosaccharides (source VivinalOGOS) and long chain fructooligosaccharides (source RaftilinHP®) 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. About 13.6 g of the powdered nutritional composition was reconstituted in 100 ml water to obtain a ready-to-drink infant formula. The fat component comprised a 1 :1 blend of vegetable fat (blend of palm oil, low erucic acid rape seed oil, coconut oil, high oleic sunflower oil, sunflower oil) and anhydrous milk fat, and further about 1 .5 wt.% of an LC-PUFA premix (fish oil and microbial oil). The lipid comprised lipid globules having a mode diameter, based on volume, of about 0.4 pm, and the volume % of lipid globules with a mode between 2 and 12 pm was below 10 vol.%. Nutritional composition B was similar to nutritional composition A. The following differences are noted: the nutritional composition further comprised about 0.8 g of a whey concentrate enriched in MFGM per 100 ml of ready to drink formula - this resulted in the presence of milk-derived phospholipids at about 1.5 wt.% based on total lipid in the nutritional composition. The phospholipids were not present in the coating of the lipid globules.

Nutritional composition 1 was similar to nutritional composition B. The lipid comprised lipid globules with a mode diameter, based on volume, of about 4 pm, and the vol.% of lipid globules with a mode between 2 and 12 pm was above 45 vol.%. The milk-derived phospholipids were present in the same amount as in nutritional composition B, but were present in the coating on the lipid globules.

The fatty acid composition was very similar between the three nutritional compositions, more specifically the levels of saturated, mono-unsaturated and poly-unsaturated fatty acids, and in n3- and n6-PUFA content were similar.

Fresh human milk samples were obtained from volunteers in the Netherlands, the obtained samples were stored at -80°C before use in the experiment.

Example 1 - In vitro dynamic lipid digestion of nutritional compositions

Each of the samples (125 mL) (in ready-to-drink format for the infant formula’s) were digested in vitro using a semi dynamic lipid digestion model, a tiny-TIM setup (The TIM company, Delft, The Netherlands). The tiny-TIM setup consists of an advanced gastric compartment which allows realistic simulation of gastric morphology and motility, coupled via a peristaltic valve to a small intestinal compartment which simulates the transit through the small intestine. Using the tiny-TIM dynamic infant gastrointestinal conditions were simulated for a total of 300 minutes.

First, the nutritional compositions were introduced to the gastric compartment and the contents of the ‘stomach’ were gradually acidified. The contents of the gastric compartment were then emptied into the intestine compartment with a GE % = 60 min. Bile and pancreatic enzymes were mixed with the lipids emptied from the stomach and lipid digestion subsequently took place. The contents collected from the intestinal compartment between 30-45 min intestinal digestion were filtered (<0.05 pm) to obtain a permeate comprising the bio-accessible lipids and the permeate was stored at -20°C (the permeate will be referred to as digested nutritional composition).

Example 2 - ECIS model - cell layer resistance after wounding

The effect of the digested nutritional compositions - as described in Example 1 - on cell layer resistance after wounding was investigated. Electric cell-substrate impedance sensing (ECIS) was used as a method to study the cell layer behaviours in an organoid tissue culture. In the ECIS model, resistance is a measure for cell layer / barrier integrity. Wounding (by an electric current) induces a gap in the cell layer, causing the resistance to drop. 2D human early life intestinal organoids containing a mix of different specialized and non-specialized epithelial cells similar to in vivo were seeded and cultured on ECIS chips until a monolayer was established. The intestinal organoids are representative for the cell/tissue structure of the intestinal tract, originating from human intestinal tissue and showing similar tissue composition and characteristics as “real” human intestinal tissue.

Once the monolayer was established the treatment was started with respective interventions (in quadruplicate per intervention). This corresponds to time point -5 h. The digested nutritional compositions from example 1 were diluted 1 :20 in cell culture medium, and the former cell culture media were replaced with the media comprising the digested nutritional composition. 5 hours after the start of the intervention, corresponding to time point 0 h, the intestinal organoid monolayer on the ECIS chip was wounded by an electric current which led to a drop in resistance. The gradual increase of the resistance, compared to t = -1 h, was measured over 15 hours (Figure 1) and the area under the curve (AUC) from time point -1 h to 15 h is shown in Figure 2. The recovery of resistance for digested nutritional composition 1 treated organoids is very fast, reaching pre-wounding resistance values already after 5 hours and then increasing further until 10 hours before it plateaus. Human milk also reaches pre-wounding resistance values after 5 hours and at 15 hours human milk-induced resistance values are comparable to nutritional composition 1 . The resistance of digested control composition- treated organoids only increased slowly after wounding.

Re-establishment of the resistance after wounding indicates recovery of the cell layer / barrier and is in this model likely achieved by migration of existing cells, as mammalian cells divide only approximately once per 24 hours. Cell migration in the gut is described to be relevant for epithelial homeostasis, tissue maintenance (normal tissue renewal), wound repair and morphogenesis.

The results are indicative for the specific effect of the phospholipid coated large lipid globules on recovering the intestinal barrier function and treating and/or preventing the intestinal barrier disruption under challenged conditions.

Claims

1. A nutritional composition comprising digestible carbohydrates, protein and lipid, wherein the lipid is in the form of lipid globules, wherein i. said lipid globules have a volume-weighed mode diameter of at least 1 .0 pm; and/or at least 45 vol.%, based on total lipid volume, of the lipid globules have a diameter between 2 and 12 pm; and ii. the lipid comprises 0.5 to 20 wt.% phospholipids based on total lipid and wherein the lipid globules have a coating comprising phospholipids, for use in preventing and/or treating intestinal barrier disruption in a human subject.

2. The nutritional composition for use according to claim 1 , wherein the human subject is at risk of intestinal barrier disruption.

3. The nutritional composition for use according to claim 2, wherein the human subject at risk of intestinal barrier disruption is selected from human subjects exposed to: i. antibiotics; ii. intestinal pathogens;

Hi. high fat diet; iv. western style diet; and/or v. alcohol.

4. The nutritional composition for use according to claim 3, wherein the human subject at risk of intestinal barrier disruption is selected from human subjects exposed to: i. antibiotics; ii. high fat diet;

Hi. western style diet; and/or iv. alcohol.

5. The nutritional composition for use according to claim 3 or 4, wherein the human subject at risk of intestinal barrier disruption is selected from human subjects exposed to a high fat diet and/or western style diet.

6. The nutritional composition for use according to claim 3, wherein the human subject is exposed to intestinal pathogens, wherein the intestinal pathogens are bacteria, preferably bacteria selected from Clostridioides difficile, Salmonella typhimurium, Vibrio cholerae and combinations thereof.

7. The nutritional composition for use according to any one of the preceding claims, wherein the nutritional composition is a medical nutritional composition.

8. The nutritional composition for use according to any one of the preceding claims, wherein the human subject is an adult.

9. The nutritional composition for use according to claim 8, wherein the use is for preventing or treating inflammatory bowel disease, celiac disease, irritable bowel syndrome, colorectal cancer or combinations thereof in the adult human subject,.

10. The nutritional composition for use according to any one of claims 1-7, wherein the human subject is an infant, preferably a pre-term infant.

11 . The nutritional composition for use according to claim 10, wherein the use is for preventing or treating necrotizing enterocolitis (NEC) in an infant.

12. The nutritional composition for use according to claim 10 or 11 , wherein the nutritional composition is selected from infant formula, follow-on formula, young child formula, and human milk fortifier.

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

14. The nutritional composition for use according to any one of the preceding claims, wherein the nutritional composition further comprises non-digestible oligosaccharides, preferably the nutritional composition comprises galacto-oligosaccharides and/or fructo-oligosaccharides.

15. The nutritional composition for use according to any one of the preceding claims, wherein the nutritional composition further comprises LCPUFA’s, preferably one or more LCPUFA’s selected from DHA, DPA, EPA, and ARA.