Improvement of barrier integrity

󰀅󰀛&󰀋󰀋󰀍󰀌󰀋󰀋󰀐󰀍󰀋󰀗󰀍󰀅

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EP2 100 520A2

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(43)Date of publication:

EUROPEAN PATENT APPLICATION

(51)Int Cl.:A23L1/29(2006.01)A23L1/30(2006.01)16.09.2009Bulletin2009/38

(21)Application number: 09158304.7(22)Date of filing: 22.06.2004(84)Designated Contracting States:

AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PL PT RO SE SI SK TR

•Koetsier, Marleen Antoinette8161 XJ, Epe (NL)•Beermann, Christopher36100 Petersberg (DE)•Stahl, Bernd

DE-61191, Rosbach-Rodheim (DE)(62)Document number(s) of the earlier application(s) in

accordance with Art. 76 EPC: 07108969.2 / 1 815 75504748674.1 / 1 672 987

(74)Representative: Swinkels, Bart Willem

Nederlandsch Octrooibureau J. W. Frisolaan 13

2517 JS Den Haag (NL)Remarks:

This application was filed on 21-04-2009 as a divisional application to the application mentioned under INID code 62.

(71)Applicant: N.V. Nutricia

2712 HM Zoetermeer (NL)(72)Inventors:

•van Tol, Eric Alexander Franciscus6824 MZ, Arnhem (NL)•Willemsen, Linette Eustachia Maria3522 AJ, Utrecht (NL)(54)(57)

Improvement of barrier integrity

(EPA), docosahexaenoic acid (DHA) and arachidonic ac-id (ARA),; and at least two distinct oligosaccharides.

The invention concerns a method for stimulating

barrier integrity in a mammal by administering to a mam-mal a composition comprising: eicosapentaenoic acid

EP2 100 520A2Printed by Jouve, 75001 PARIS (FR)

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Description

FIELD OF THE INVENTION

5[0001]The present invention relates to a method for improving intestinal barrier integrity and a composition suitablefor use in such method.

BACKGROUND OF THE INVENTION

101520[0002]The gastrointestinal epithelium normally functions as a selective barrier permitting the absorption of nutrients,electrolytes and water and preventing the exposure to dietary and microbial antigens, including food allergens. Thegastrointestinal epithelium limits the passage of antigens to the systemic circulation, that may be causing inflammatoryreactions, e.g. allergic reactions. As the incidence of allergy, particularly food allergy, is increasing, many researchgroups search for (preventive) cures for these ailments.

[0003]EP1272058 describes a composition containing indigestible oligosaccharides for improving tight junction toreduce intestinal permeability and reducing allergic reaction. The composition may comprise LC-PUFA’s (long chain-polyunsaturated faty acids).

[0004]EP 745001 describes a combination of indigestible oligosaccharides and n-3 and n-6 fatty acids for treatmentulcerative colitis.

[0005]Usami et al (Clinical Nutrition 2001, 20(4): 351-359) describe the effect of eicosapentaenoic acid (EPA) on tightjunction permeability in intestinal monolayer cells. In their hands, EPA was found to increase permeability, indicatingthat EPA is unsuitable to improve intestinal barrier integrity.

[0006]The prior art formulations are not optimally suited for improving barrier integrity.SUMMARY OF THE INVENTION

[0007]The present invention provides a combination of selected long chain polyunsaturated fatty acids (LC-PUFA’s)and selected oligosaccharides. The present combination of LC-PUFA’s and oligosaccharides effectively improves barrierintegrity, by synergistically improving intestinal permeability and mucus production, and is particularly suitable for im-proving barrier integrity in human infants.

[0008]It was surprisingly found that selected LC-PUFA’s effectively reduce epithelial paracellular permeability. Incontrast to what Usami et al (Clinical Nutrition 2001, 20(4): 351-359) have reported, the present inventors found thatC18 and C20 polyunsaturated fatty acids, particularly eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) andarachidonic acid (ARA), are capable of effectively reducing intestinal tight junction permeability.

[0009]In addition to the LC-PUFAs, the present composition contains oligosaccharides. The selected oligosaccharidesimprove the barrier integrity by stimulating the production of the mucus, which results in an increased mucus layerthickness. It is believed this effect is caused by the effects of the distinct oligosaccharides on the short chain fatty acid(SCFA) production. Hence, when enterally administered to a mammal, the present combination of LC-PUFA and indi-gestible oligosaccharides synergistically improve barrier integrity and/or synergistically reduce intestinal permeability bysimultaneous reduction of tight junction permeability and stimulation of mucus production.

[0010]In a further aspect, the present composition improves the quality of the intestinal mucus layer. The mucus layercomprises mucins. Mucins are high molecular mass glycoproteins that are synthesized and secreted by goblet cells.They form a gel-like layer on the mucosal surface, thereby improving barrier integrity. The mucus layer comprises differenttypes of mucins, e.g. acid, neutral and sulphonated mucins. An increased heterogeneity of the mucus layer is believedto improve barrier functionality.

[0011]The present composition preferably comprises at least two different oligosaccharides, which influence themucosal architecture and advantageously influence mucin heterogeneity in the mucus layer, either directly or by changingthe intestinal flora. Each different selected oligosaccharide is believed to have a different effect on mucus quantity andquality. Moreover, the two distinct oligosaccharides are also able to stimulate quality of mucus as reflected by the degreeof sulphation through their synergistic stimulation of SCFA production. It was surprisingly found by the present inventorsthat a mixture of two different oligosaccharides according to the present invention synergistically stimulates acetateproduction. It was also found by the present inventors mucus production is dependent on acetate production.

[0012]The present composition is preferably further improved by providing both long- and short-chain oligosaccha-rides. The supply of different chain lengths results in stimulation of mucus production in different parts of the ileum andcolon. The short chain oligosaccharides (typically with a degree of polymerisation (DP) of 2,3,4 or 5) stimulate mucinproduction in the proximal colon and/or distal ileum, while the oligosaccharides with longer chain lengths (preferablywith a degree of polymerisation (DP) of more than 5 up to 60) are believed to stimulate mucin production in the moredistal parts of the colon.

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[0013]Even further improvements can be achieved by providing the at least two different oligosaccharides both asshort-chain and long-chain oligosaccharides. These preferred embodiments all contribute to further improved barrierintegrity throughout the ileum and/or colon.

[0014]Furthermore, it was surprisingly found that EPA, DHA and ARA were able to reduce the harmful effects ofinterleukin 4 (IL-4) on intestinal permeability. IL-4 is a cytokine which is secreted in increased amounts by mucosal T-cells in certain patients and induces intestinal permeability. Hence the present invention also provides for a method forthe treatment and/or prevention of diseases wherein intestinal IL-4 concentration is increased, such as allergy, particularlyatopic dermatitis.

DETAILED DESCRIPTION OF THE INVENTION[0015]

The present invention relates to a nutritional composition comprising:51015a) EPA, DHA and ARA, wherein the content of long chain polyunsaturated fatty acid with 20 and 22 carbon atomsdoes not exceed 15 wt.% of the total fat content; and

b) at least two distinct oligosaccharides, wherein the two distinct oligosaccharides have a homology in monose unitsbelow 90 %.

[0016]This composition can be advantageously used in a method for stimulating intestinal barrier integrity, said methodcomprising administering to a mammal said composition.Polyunsaturated fatty acids

0017]The present inventors surprisingly found that eicosapentaenoic acid (EPA, n-3), docosahexaenoic acid (DHA,[

n-3) and arachidonic acid (ARA, n-6) effectively reduce intestinal tight junction permeability. Hence the present compo-sition, which is particularly suitable for improving intestinal barrier integrity, comprises EPA, DHA and ARA.

[0018]The present inventors found that lower concentration of LC-PUFA’s, were effective in reducing tight junctionpermeability (see Examples vs. Usami et al). Hence, the content of LC-PUFA with 20 and 22 carbon atoms in the presentcomposition, preferably does not exceed 15 wt.% of the total fat content, preferably does not exceed 10 wt.%, evenmore preferably does not exceed 5 wt.% of the total fat content. Preferably the present composition comprises at least0.1 wt.%, preferably at least 0.25 wt, more preferably at least 0.5 wt.%, even more preferably at least 0.75 wt.% LC-PUFA with 20 and 22 carbon atoms of the total fat content. For the same reason, the EPA content preferably does notexceed 5 wt.% of the total fat, more preferably does not exceed 1 wt.%, but is preferably at least 0.05 wt%, morepreferably at least 0.1 wt.% of the total fat. The DHA content preferably does not exceed 5 wt.%, more preferably doesnot exceed 1 wt.%, but is at least 0.1 wt% of the total fat. As ARA was found to be particularly effective in reducing tightjunction permeability, the present composition comprises relatively high amounts, preferably at least 0.1 wt.%, evenmore preferably at least 0.25 wt.%, most preferably at least 0.5 wt.% of the total fat. The ARA content preferably doesnot exceed 5 wt.%, more preferably does not exceed 1 wt.% of the total fat. In the present ARA containing enteralcomposition, EPA and DHA are advantageously added to balance the action of ARA, e.g. reduce the potential proin-flammatory action of ARA metabolites. Excess metabolites from ARA may cause inflammation. Hence, the presentcomposition preferably comprises ARA, EPA and DHA, wherein the weight ratio ARA/DHA preferably is above 0.25,preferably above 0.5, even more preferably above 1. The ratio is preferably below 25. The weight ratio ARA/EPA ispreferably between 1 and 100, more preferably between 5 and 20.

[0019]The present composition preferably comprises between 5 and 75 wt.% polyunsaturated fatty acids based ontotal fat, preferably between 10 and 50 wt.%.

[0020]If the present composition is used as an infant formula (e.g. a method for feeding an infant, said methodcomprising administering the present composition to an infant), the content of LC-PUFA, particularly the LC-PUFA with20 and 22 carbon atoms, preferably does not exceed 3 wt.% of the total fat content as it is desirable to mimic humanmilk as closely as possible. For the same reason, the omega-3 LC-PUFA content preferably does not exceed 1 wt.% ofthe total fat content; the omega-6 LC-PUFA content preferably does not exceed 2 wt.% of the total fat content; the ARA(omega-6) content is preferably below 1 wt.% of the total fat content; and/or the weight ratio EPA/DHA is preferably 1or lower, more preferably below 0.5.

[0021]The LC-PUFA with 20 and 22 carbon atoms may be provided as free fatty acids, in triglyceride form, in phos-pholipid form, or as a mixture of one of more of the above. The present composition preferably comprises at least oneof ARA and DHA in phospholipid form.

[0022]The present nutritional composition preferably also provides omega-9 (n-9) fatty acid (preferably oleic acid, 18:1), to provide sufficient nutrition. Preferably the present composition provides at least 15 wt.% n-9 fatty acid based onthe weight of the total fatty acids, more preferably at least 25 wt%. The content of n-9 fatty acids is preferably below 80 wt.%.

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Oligosaccharides

[0023]Suitable oligosaccharides according to the invention are saccharides which have a degree of polymerisation(DP) of at least 2 monose units, which are not or only partially digested in the intestine by the action of acids or digestiveenzymes present in the human upper digestive tract (small intestine and stomach), but which are fermentable by thehuman intestinal flora. The term monose units refers to units having a closed ring structure, preferably hexose, e.g. thepyranose or furanose forms. The degree of polymerisation of the oligosaccharide is typically below 60 monose units,preferably below 40, even more preferably below 20.

[0024]The present composition comprises at least two different oligosaccharides, wherein the oligosaccharides havea homology in monose units below about 90%, preferably below 50%, even more preferably below 25%, even morepreferably below 5%. The term \"homology\" as used in the present invention is the cumulative of the percentage of samemonose units in the different oligosaccharides. For example, oligosaccharide 1 (OL1) has the structure fruc-fruct-glu-gal, and thus comprises 50% fruc, 25% gal and 25% glu. Oligosaccharide 2 (OL2) has the structure fruc-fruc-glu, andthus comprises 66% fruc, 33% glu. The different oligosaccharides thus have a homology of 75% (50% fruc + 25% glu).[0025]In a preferred embodiment, the present composition comprises galactooligosaccharides and at least one se-lected from the group consisting of fructooligosaccharides and inulin.

[0026]Each of the present oligosaccharides preferably comprises at least 66%, more preferably at least 90% monoseunits selected from the group consisting of mannose, arabinose, fructose, fucose, rhamnose, galactose, β-D-galacto-pyranose, ribose, glucose, xylose, uronic acid and derivatives thereof, calculated on the total number of monose unitscontained therein.

[0027]According to a further embodiment at least one of the oligosaccharides of the present composition is selectedfrom the group consisting of fructans, fructooligosaccharides, indigestible dextrins galactooligosaccharides (includingtransgalactooligosaccharides), xylooligosaccharides, arabinooligosaccharides, glucooligosaccharides, mannooligosac-charides, fucooligosaccharides, acidic oligosaccharides (see below, e.g. uronic acid oligosaccharides such as pectinhydrolysate) and mixtures thereof. Preferably the present composition comprises at least one, preferably at least two,of the oligosaccharides selected from the group consisting of fructooligosaccharides or inulin, galactooligosaccharidesand pectin hydrolysate.

[0028]For good mucus quantity and quality, the present composition preferably comprises at least one oligosaccharide,which comprises at least 66% galatose or fructose as a monose unit. In a preferred embodiment the compositioncomprises at least one oligosaccharide which comprises at least 66% galatose as a monose unit and at least oneoligosaccharide which comprises at least 66% fructose as a monose unit. In a particularly preferred embodiment, thepresent composition comprises galactooligosaccharide and an oligosaccharide selected from the group consisting offructooligosaccharides and inulin. Fructooligosaccharides stimulate sulfomucin production in the distal colon of humanflora-associated rats (Kleessen et al, (2003) Brit J Nutr 89:597-606) and galactooligosaccharides stimulate the acidmucin production (Meslin et al, Brit. J.Nutr (1993), 69: 903-912)).

[0029]For further improvement of mucus layer thickness over the whole area of the colon, at least 10 wt.% of theoligosaccharides in the present composition has a DP of 2 to 5 (i.e. 2, 3, 4 and/or 5) and at least 5 wt.% has a DP of 10to 60. Preferably at least 50 wt.%, more preferably at least 75 wt.% of the oligosaccharides have a DP of 2 to 9 (i.e. 2,3, 4, 5, 6, 7, 8, and/or 9), because these are believed to work throughout the ileum and proximal and middle parts of thecolon and because the weight percentage of oligosaccharides that needs to be incorporated in the composition to achievethe desired effect is reduced.

[0030]Preferably the weight ratios:a. (oligosaccharides with DP 2 to5): (oligosaccharides with DP 6,7,8 and/or 9) > 1; and

b. (oligosaccharides with DP 10 to60): (oligosaccharides with DP 6,7,8 and/or 9) > 1 are both above 1.

[0031]Preferably both weight ratios are above 2, even more preferably above 5.

[0032]For even further improvement of mucus layer thickness and quality over the whole area of the colon, preferablyeach of the at least two different oligosaccharides are provided in different chain lengths, preferably at least 10 wt.% ofeach oligosaccharide based on the total weight of the respective oligosaccharide has a DP of 2 to 5 (i.e. 2, 3, 4 and/or5) and at least 5 wt.% has a DP between 10 and 60. Preferably at least 50 wt.%, more preferably at least 75 wt.% ofthe oligosaccharide based on the total weight of that oligosaccharides has a DP between 2 and 10, because these arebelieved to work throughout in the ileum and proximal and middle parts of the colon.Acidic oligosaccharides

[0033]To further improve barrier integrity, the present composition preferably includes acidic oligosaccharides witha DP between 2 and 60. The term acid oligosaccharide refers to oligosaccharides comprising at least one acidic group

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selected from the group consisting of N-acetylneuraminic acid, N-glycoloylneuraminic acid, free or esterified carboxylicacid, sulfuric acid group and phosphoric acid group. The acidic oligosaccharide preferably comprises uronic acid units(i.e. uronic acid polymer), more preferably galacturonic acid units. The acid oligosaccharide may be a homogeneous orheterogeneous carbohydrate. Suitable examples are hydrolysates of pectin and/or alginate. In the intestinal tract, theuronic acid polymers are hydrolysed to uronic acid monomers, which stimulate production of intestinal acetate, whichin turn stimulates intestinal mucus secretion (Barcelo et al., Gut 2000; 46:218-224).Preferably the acid oligosaccharide has the structure I below, wherein the terminal hexose (left) preferably comprisesa double bond. The hexose units other than the terminal hexose unit(s) are preferably uronic acid units, even morepreferably galacturonic acid units. The carboxylic acid groups on these units may be free or (partly) esterified, andpreferably at least 10% is methylated (see below).510152025wherein:R is preferably selected from the group consisting of hydrogen, hydroxy or acid group, preferably hydroxy; andat least one selected from the group consisting of R2, R3, R4 and R5 represents N-acetylneuraminic acid, N-gly-coloylneuraminic acid, free or esterified carboxylic acid, sulfuric acid group and phosphoric acid group, and theremaining of R2, R3, R4 and R5 representing hydroxy and/or hydrogen. Preferably one selected from the groupconsisting of R2, R3, R4 and R5 represents N-acetylneuraminic acid, N-glycoloylneuraminic acid, free or esterifiedcarboxylic acid, sulfuric acid group or phosphoric acid group, and the remaining represent hydroxy and/or hydrogen.Even more preferably one selected from the group consisting of R2, R3, R4 and R5 represents free or esterifiedcarboxylic acid and the remaining of R2, R3, R4 and R5 representing hydroxy and/or hydrogen; and

n is an integer and refers to a number of hexose units (see also Degree of Polymerisation, below), which may beany hexose unit. Suitably n is an integer between 1-5000. Preferably the hexose unit(s) is a uronic acid unit.Most preferably R1, R2 and R3 represent hydroxy, R4 represent hydrogen, R5 represents carboxylic acid, n is any numberbetween 1 and 250, preferably between 1 and 10 and the hexose unit is galacturonic acid.

[0034]The detection, measurement and analyses of the preferred acid oligosaccharides as used in the present methodare given in applicants earlier patent application relating to acid oligosaccharides, i.e. WO 0/160378.

[0035]For stimulation improvement of mucus layer thickness over the whole area of the colon, the present compositionpreferably comprises at least 10 wt.% acid oligosaccharides with a DP of 2 to 5 (i.e. 2, 3, 4 and/or 5) and at least 5 wt.% acid oligosaccharides with a DP between 10 and 60, said wt.% being based on the total weight of the oligosaccharides.[0036]The acid oligosaccharides used in the invention are preferably prepared from pectin, pectate, alginate, chon-droitine, hyaluronic acids, heparine, heparane, bacterial carbohydrates, sialoglycans, fucoidan, fucooligosaccharides orcarrageenan, more preferably from pectin and/or alginate.Content of oligosaccharide

0037]When in ready-to-feed liquid form, the present composition preferably comprises 0.1 to 100 grams indigestible[

oligosaccharide per liter, more preferably between 0.5 and 50 grams per liter even more preferably between 1 and 25grams per liter. A too high content of oligosaccharides may cause discomfort due to excessive fermentation, while avery low content may result in an insufficient mucus layer.The weight ratio of the at least two different oligosaccharides is preferably between 1 and 10, more preferably between1 and 5. These weight ratios stimulate mucin production of different types at different sites in the intestine optimally.

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[0038]The oligosaccharide is preferably included in the present composition according to the invention in an amountexceeding 0.1 wt.%, preferably exceeding 0.2 wt.%, more preferably exceeding 0.5 wt.% and even more preferablyexceeding 1 wt.% based on the total dry weight of the composition. The present composition preferably has an oligosac-charide content below 20-wt.%, more preferably below 10-wt.% even more preferably below 5-wt.%.

[0039]Addition of nucleotides and/or nucleosides to the present composition further improves gut mucosal barrierfunction, particularly as it inhibits and/or or reduces the incidence of bacterial translocation and decreases intestinalinjury. Hence, the present composition preferably also comprises between 1 and 500 mg nucleosides and/or nucleotidesper 100 gram of the dry formula, even more preferably between 5 and 100 mg.Application

[0040]The present composition can be advantageously used in a method for improving barrier integrity in mammals,particularly humans. The present composition can also be advantageously used in a method for the treatment or pre-vention of diseases associated with reduced barrier integrity, said method comprising administering to a mammal thepresent composition. The present composition is preferably administered orally.

[0041]For the ill and infants, the present composition is preferably combined with complete nutrition, including protein,carbohydrate and fat. The present composition is advantageously administered to infants with the age between 0 and2 years. The composition may be administered to patients which suffer from an impaired barrier integrity and healthypatients. The present composition is advantageously used in a method for providing the nutritional requirements of apremature infant (an infant born before 37 weeks gestation).

[0042]The present composition can also be advantageously used in a method for treatment and/or prevention ofintestinal damage by administering the present composition to the patient prio to or after a medical treatment, whichmay cause intestinal damage. Such medical treatment may for example be surgery or enteral medicine treatment (e.g.antibiotic, analgesic, NSAID, chemotherapeutic agents etc).

[0043]The present composition can also be advantageously used to treat or prevent diseases wherein intestinalbarrier disruption is underlying the development of the course of the disease, e.g. in a method for the treatment orprevention of chronic inflammatory diseases, particularly inflammatory bowel disease (IBD), irritable bowel syndrome(IBS), celiac disease, pancreatitis, hepatitis, arthritis or diabetes. Furthermore, the invention can be used in a methodfor providing nutrition to patients which have undergone or are undergoing abdominal surgery and patients that experiencepostoperative dysfunction of the gut and/or malnourished patients.

[0044]In a further embodiment of the invention the present composition is advantageously administered to patientssuffering from acquired immune deficiency syndrome (AIDS) and/or patients which are infected with the human immu-nodeficiency virus (HIV), e.g. in a method for the treatment of AIDS and/or HIV infection. Said method comprises theoral administration of the present composition, preferably combined with nutrients selected from the group consisting ofcarbohydrate, protein and fat.

[0045]Furthermore, the invention can also be used to treat or prevent complications resulting from reduced barrierintegrity, particularly in a method for the treatment and/or prevention of diarrhea, particularly infant diarrhea. Due to thereduced incidence in infant diarrhea, the present composition can also be advantageously used to reduce diaper rash.[0046]Administering the present composition reduces passage of dietary and microbial antigens, particularly foodallergens, from the intestinal lumen into the mucosal or systemic circulation, and hence can be advantageously used ina method for the treatment or prevention of allergy and/or allergic reaction, particularly in a method for the treatment orprevention of food allergy, e.g. allergic reaction resulting from the ingestion of foodstuff.

[0047]It was also found by the present inventors that EPA, DHA and/or ARA are capable of reducing the effects ofIL-4 on intestinal permeability. Hence, one aspect of the present invention provides for a method for the treatment and/orprevention of diseases wherein intestinal IL-4 concentration is increased (e.g. allergic diseases), said method comprisingadministering an LC-PUFA preferably selected from the group consisting of EPA, DHA and ARA, preferably combinedwith the present selected oligosaccharides. Hence, the present composition can also be advantageously used in amethod for the treatment of atopic dermatitis.

[0048]Since the barrier function of newborns has not been fully developed, the present composition can be advan-tageously administered to young infants, i.e. infants with the age between 0 and 6 months. The composition may beadministered to the infant in the form of an infant formula without human milk or admixed with human milk. Hence thepresent invention also provides for a formula feed comprising human milk and the present composition. The compositionsincluding human milk and the present composition are particularly suitable for feeding premature infants.

[0049]The present composition is preferably provided as a packaged powder or packaged ready-to-feed formula. Toprevent spoilage of the product, packaging size of ready-to-feed formula preferably does not exceed one serving, e.g.preferably does not exceed 500 ml; and packaging size of the present composition in powder form preferably does notexceed 250 servings. Suitable packaging sizes for the powder are 2000 grams or less, preferably per 1000 grams or less.[0050]The packaged products provided with labels that explicitly or implicitly direct the consumer towards the use of

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said product in accordance with one or more of the above or below purposes, are encompassed by the present invention.Such labels may for example make reference to the present method for preventing allergic reaction to food allergensby including wording like \"reduced food sensitivity\wording. Similarly, reference to the present method for treating and/or preventing allergy may be made by incorporatingterminology equivalent to \"improved resistance\" or \"reduced sensitivity\".Formula’s

[0051]It was found that the present composition can be advantageously applied in food, such as baby food and clinicalfood. Such food preferably comprises lipid, protein and carbohydrate and is preferably administered in liquid form. Theterm \"liquid food\" as used in the present invention includes dry food (e.g. powders) which are accompanied with instruc-tions as to admix said dry food mixture with a suitable liquid (e.g. water).

[0052]Hence, the present invention also relates to a nutritional composition which preferably comprises between 5and 50 en% lipid, between 5 and 50 en% protein, between 15 and 90 en% carbohydrate and the present combinationof oligosaccharides and LC-PUFA’s. Preferably the present nutritional composition preferably contains between 10 and30 en% lipid, between 7.5 and 40 en% protein and between 25 and 75 en% carbohydrate (en% is short for energypercentage and represents the relative amount each constituent contributes to the total caloric value of the preparation).[0053]Preferably a combination of vegetable lipids and at least one oil selected from the group consisting of fish oiland omega-3 vegetable, algae or bacterial oil is used.

[0054]The proteins used in the nutritional preparation are preferably selected from the group of non-human animalproteins (such as milk proteins, meat proteins and egg proteins), vegetable proteins (such as soy protein, wheat protein,rice protein, and pea protein), free amino acids and mixtures thereof. Cow milk derived nitrogen source, particularly cowmilk protein proteins such as casein and whey proteins are particularly preferred.

[0055]A source of digestible carbohydrate may be added to the nutritional formula. It preferably provides about 40%to about 80% of the energy of the nutritional composition. Any suitable (source of) carbohydrate may be used, for examplesucrose, lactose, glucose, fructose, corn syrup solids, and maltodextrins, and mixtures thereof.

[0056]The present composition is preferably used as an infant formula and preferably contains 7.5 to 12.5 energy %protein; 40 to 55 energy % carbohydrates; and 35 to 50 energy % fat. As the present composition is suitably used toreduce the allergic reaction in an infant, the protein of the infant formula is preferably selected from the group consistingof hydrolyzed milk protein (e.g. hydrolyzed casein or hydrolyzed whey protein), vegetable protein and/or amino acids.The use of these proteins further reduced the allergic reactions of the infant.

[0057]Stool irregularities (e.g. hard stools, insufficient stool volume, diarrhoea) is a major problem in many babiesand ill subjects that receive liquid foods. It was found that stool problems may be reduced by administering the presentoligosaccharides in liquid food which have an osmolality between 50 and 500 mOsm/kg, more preferably between 100and 400 mOsm/kg.

[0058]In view of the above, it is also important that the liquid food does not have an excessive caloric density, howeverstill provides sufficient calories to feed the subject. Hence, the liquid food preferably has a caloric density between 0.1and 2.5 kcal/ml, even more preferably a caloric density of between 0.5 and 1.5 kcal/ml, most preferably between 0.6and 0.8 kcal/ml.EXAMPLES

Example 1: Effect of LC-PUFA on barrier integrity

455101520253035405055[0059]Monolayers (MC) of intestinal epithelial cell lines T84 (American Type Culture Collection (ATTC), Manassas,USA) were cultured on transwell filters (Coming, Costar BV, The Netherlands) allowing both mucosal and serosalsampling and stimulation of human intestinal epithelial cells. Two weeks post confluency the monolayers were incubatedin the luminal compartment with polyunsaturated fatty acids ARA (arachidonic acid; 5,8,11,14-eicosatetraenoic acid),DHA (cis-4,7,10,13,16,19 docosahexaenoic acid), EPA (eicosapentaenoic acid) or control palmitic (C 16:0) acid (Palm)(Sigma, St. Louis, USA). The latter procedure was chosen to mimic the in vivo administration route of the dietarycompounds. Cells were incubated with ARA, DHA, EPA, or palmitic acid for 0, 24, 48 and 72 hr at different concentrations(10 PM and 100 PM). Experiments were performed to evaluate basal barrier integrity. The epithelial barrier function wasdetermined by measuring the transepithelial resistance (TER, Ω.cm2) was measured by epithelial volt-ohm meter (EVOM;World Precision Instruments, Germany) and permeability for 4kD FITC dextran (paracellular permeability marker, Sigma,USA). Resistance (. Epithelial permeability for 4 kDa FITC-dextran was determined as follows. Prior to dextran fluxesthe medium was refreshed with culture medium without phenol red for one hour followed by addition of 5 Pl (stock 100mg/ml) 4 kDa FITC-dextran to the lumenal compartment. After 30 min incubation 100 Pl sample was collected from theserosal compartment and the fluorescent signal measured at excitation wavelength 485 nm and emission 520 nm

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(FLUOstar Galaxy®, BMG Labtechnologies, USA). FITC-dextran fluxes were calculated as pmol FITC-dextran/cm2/h.Statistical analyses were performed using the ANOVA (SPSS version 10).Results of the effect of fatty acids (100 PM) on spontaneous barrier integrity after 72 hr incubation are given in Table 1.Table 1 shows that the LC-PUFA’s ARA, EPA and DHA reduce the molecular flux and improve epithelial resistance. Incontrast the control experiments show that palmitic acid has the opposite effects, i.e. compromises barrier integrity.These results are indicative for the advantageous use of EPA, DHA and ARA, and in particularly ARA in the compositionaccording to the present invention and for use in a method according to the present invention, e.g. in a method forimproving barrier integrity. These result further support the synergistic effects of the present combination of fatty acidsand indigestible oligosaccharides.Figure 1 shows the time and dose (10PM and 100PM) dependent effects of various fatty acids (palmitic acid, DHA, GLA,and AA) on basal barrier integrity (TER). Figure 1 shows that the LC-PUFA’s AA, DHA, and GLA, improve the epithelialbarrier integrity as reflected by increased resistance (TER). These results are indicative for the advantageous use ofEPA, DHA, GLA and ARA, in particularly ARA, in the composition according to the present invention and for use in amethod according to the present invention, i.e. in a method for improving barrier integrity. These results further supportthe synergistic effects of the present combination of fatty acids and indigestible oligosaccharides.Table 1

Ingredient (LC-PUFA)

2051015Flux79161722865

Resistance (TER)

1090831157418161493

ControlPalmitic acidDHAARA

25EPA

Example 2: Effect of LC-PUFA on IL-4 mediated barrier disruption

3035404550[0060]Monolayers (MC) of intestinal epithelial cell lines T84 (ATCC, USA) were cultured on transwell filters (Coming,Costar BV, The Netherlands) allowing both mucosal and serosal sampling and stimulation of human intestinal epithelialcells. Two weeks post confluency the monolayers were incubated in the presence of IL-4 (2 ng/ml, serosal compartment,Sigma, USA) with or without polyunsaturated fatty acids ARA, DHA, GLA, EPA, or control palmitic acid (10 PM or 100PM, mucosal compartment, Sigma, St. Louis, USA). Cells were pre-incubated with ARA, DHA, EPA, or palmitic acid for48 hr prior to the IL-4 incubation. The co-incubation of PUFA’s and palmetic acid with IL-4 was continued for another48 hr; while culture medium and additives were changed every 24 hr. The epithelial barrier function was determined bymeasuring the transepithelial resistance (TER) and permeability as described in example 1. Statistical evaluation wasperformed as described in example 1.

Results of the effect of ARA, DHA, EPA and palmitic acid (100 PM) on IL-4 mediated barrier disruption are given in Table2. Table 2 shows that the LC-PUFA’s ARA, DHA and EPA inhibit the increased flux caused by IL-4. In contrast palmeticacid had a detrimental effect and decreased barrier disruption compared to control. These results are indicative for theadvantageous use of ARA, DHA, and EPA in clinical and infant nutrition formulations to prevent or reduce IL-4 mediatedbarrier disruption, e.g. as occurs in food or cows milk allergy. These result further support the synergistic effects of thepresent combination of fatty acids and indigestible oligosaccharides.

[0061]Figure 2 gives the time and dose (10PM and 100PM) dependent protective effects of various FA’s (palmiticacid, DHA, GLA, and AA) on IL-4 mediated barrier destruction (Flux). Figure 2 shows that ARA, DHA and GLA protectagainst IL-4 mediated barrier disruption as reflected by decreased 4kD dextran flux. These results are indicative for theadvantageous use of ARA, DHA and GLA in clinical and infant nutrition formulations to prevent or reduce IL-4 mediatedbarrier disruption, e.g. as occurs in food or cows milk allergy. These result further support the synergistic effects of thepresent combination of fatty acids and indigestible oligosaccharides.Table 2

Ingredient (LC-PUFA)

IL-4 Flux582777

IL-4 TER374321

55ControlPalmitic acid

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(continued)

Ingredient (LC-PUFA)DHA

5IL-4 Flux271218228

IL-4 TER547636539

ARAEPA

Example 3: Effect of oligosaccharides on acetate production

101520253035[0062]Micro-organisms were obtained from fresh faeces from bottle fed babies. Fresh faecal material from babiesranging 1 to 4 month of age was pooled and put into preservative medium within 2 h. As substrate either prebiotics(TOS; TOS/inulin (HP) mixture in a 9/1 (w/w) ratio; inulin; oligofructose(OS)/inulin mixture in a 1/1 (w/w) ratio, or none(blanc) were used. The transgalactooligosaccharides (TOS) were obtained from Vivinal GOS, Borculo Domo Ingredients,Zwolle, The Netherlands and comprises as indigestible oligosaccharides: 33 wt.% disaccharides, 39 wt.% trisaccharides,18 wt.% tetrasaccharides, 7 wt.% pentasaccharides and 3 wt.% hexa-, hepta- en octasaccharides. The inulin (HP) Oraftiactive food ingredients, Tienen, Belgium, i.e. Raftiline HP®, with an average DP of 23.Media: McBain & MacFarlanemedium: buffered peptone water 3.0g/l, yeast extract 2.5 g/l. mucin (brush borders) 0.8 g/l, tryptone 3.0g/l, L-Cysteine-HCl 0.4 g/l, bile salts 0.05 g/l, K2HPO4.3H2O 2.6 g/l, NaHCO3 0.2 g/l, NaCl 4.5 g/l, MgSO4.7H2O 0.5 g/l, CaCl2 0.228g/l, FeSO4.7H2O 0.005 g/l. Fill 500 ml Scott bottles with the medium and sterilized 15 minutes at 121 °C. Bufferedmedium: K2HPO4.3H2) 2.6 g/l, NaHCO3 0.2 g/l, NaCl 4.5 g/l, MgSO4.7H2O, 0.5 g/l, CaCl2 0.228 g/l, FeSO4.7H2O0.005 g/l. Adjust to pH 6.3 󰀙 0.1 with K2HPO4 or NaHCO3. Fill 500ml Scott bottles with the medium and sterilized 15minutes at 121 °C.Preservative medium: Buffered peptone 20.0 g/l, L-Cysteine-HCl 0.5 g/l, Sodium thioglycollate 0.5 g/l, resazurine tablet1 per litre, adjust to pH 6.7 󰀙 0.1 with 1 M NaOH or HCl. Boiled in microwave. Serum bottles were filled with 25 mlmedium and sterilized for 15 minutes at 121 °C.Fresh faecal samples were mixed with preservative medium and stored for several hours at 4 °C. The preserved solutionof faeces was centrifuged at 13,000 rpm for 15 minutes, supernatant removed and faeces mixed with McBain & MacFarlane medium in a weight ratio of 1:5. Of this faecal suspension 3 ml were combined with 85 mg glucose or prebioticor with no addition (blanc) in a bottle and mixed thoroughly. A t=0 sample was withdrawn (0.5 ml). 2.5 ml of the resultingsuspension is brought in a dialysis tube in a 60 ml bottle filled with 60 ml of the buffered medium. The bottle was closedwell and incubated at 37°C. Samples were taken from the dialysis tube (0.2 ml) or dialysis buffer (1.0 ml) with a hypodermicsyringe after 3, 24, and 48 hours and immediately put it on ice to stop fermentation. The experiment was carried outusing the following samples:1) 85mg TOS2) 85 mg inulin

3) 85mg TOS/inulin in a ratio of 9/1 (w/w) and4) 85mg OS/inulin in a ratio of 1/1 (w/w).

[0063]SCFA (acetate, propionate, butyrate) were quantitated using a Varian 3800 gas chromatograph (GC) (VarianInc., Walnut Creek, U.S.A.) equipped with a flame ionisation detector. 0.5 Pl of the sample was injected at 80 °C in thecolumn (Stabilwax, 15 x 0.53 mm, film thickness 1.00 Pm, Restek Co., U.S.A.) using helium as a carrier gas (3.0 psi).After injection of the sample, the oven was heated to 160 °C at a speed of 16 °C/min, followed by heating to 220 °C ata speed of 20 °C/min and finally maintained at 220 °C for 1.5 minutes. The temperature of the injector and detector was200 °C. 2-ethylbytyric acid was used as an internal standard.Figure 3 depicts the absolute (Figure 3A) and relative SCFA profile (Figure 3B) resulting from fermenting the differentoligosaccharides. Figure 3A shows that a mixture of two different oligosaccharides (TOS/Inulin), wherein the two distinctoligosaccharides have a homology in monose units below 90 and a different chain length results in a significantly andsynergistically increased amount of SCFA (particularly acetate) per gram fiber than single components. Figure 3B showsthat the addition of a combination of TOS/Inulin favored a higher proportion of the beneficial acetate (B). The acetateproduction in vivo translates to improved mucus production by goblet cells and a measure for intestinal mucus layerthickness (see example 4). These results are indicative for the advantageous use of the present composition.Example 4: Effects of SCFA on mucus production.[0064]

Monolayers of intestinal epithelial T84 cells (ATCC, USA) cells were cultured in 24 or 96 wells tissue culture

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plates (Corning B.V.). T84 were incubated with the short chain fatty acids acetate, proprionate and butyrate (SCFA,Merck, USA) for 24 h in a concentration range of 0.025-4.0 mM. Supernatants and/or cells were collected and MUC-2(mucin) expression determined. A dotblot technique was used to determine MUC-2 expression in the cell cultures, sincemucins are extremely large glycoproteins (over 500 kDa) which makes them difficult to handle in western blotting tech-niques. The method was validated using pre-immune serum (T84 stained negative), CCD-18Co (ATCC, USA) negativecontrol cells and bovine serum albumin (BSA). Cell samples were collected in Laemmli (protein isolation buffer) andprotein determination performed using a microprotein assay (Biorad, USA) according to the manufacturers protocol.Samples (0.3-0.7-1.0 Pg/2 Pl) were dotted on nitrocellulose membranes (Schleicher & Schuell, Germany). Membraneswere blocked in TBST/5% Protivar (Nutricia, The Netherlands) followed by 1 h incubation with anti-MUC-2 antibody(kindly donated by Dr. Einerhand, Erasmus University, Rotterdam, The Netherlands). After washing, blots were incubatedwith goat anti-rabbit-HRP (Santacruz Biotechnology, USA) and for substrate detection ECL (Roche Diagnostics, TheNetherlands) was used. Densitometry was performed using the Lumi-Imager (Boehringer Mannheim B.V., The Nether-lands) and the signal was expressed in light units (BLU). BLU’s were also expressed relative to control incubations(%BLU). To compare the stimulatory effect of SCFA on MUC-2 expression basal MUC-2 expression levels were deducted.Figure 4 shows the differential effects of SCFA (acetate, proprionate, butyrate) on MUC-2 expression in intestinal epithelialcells (MC T84) and epithelial-mesenchymal cell co-cultures (CC T84). Figure 2 also shows that acetate is more potentin stimulating MUC-2 expression (mucus production) as compared to propionate and butyrate. Hence, the presentcombination of oligosaccharides (which was shown to increase acetate production (see example 3)) is particularly usefulfor stimulating mucus production and can be advantageously used in a method for stimulating barrier integrity.Example 5: Infant milk formula I

[0065]Ingredients (per liter), energy 672 Kcal; Protein 15 g; Whey: Casein ratio 60:40; Fat 36 g; Carbohydrate 72 g;Vitamin A 750 RE; Mixed natural carotids 400 IU; Vitamin D 10.6 mcg; Vitamin F 7.4 mg; Vitamin K 67.0 mcg; VitaminB.sub.1 (thiamin) 1000 mcg; Vitamin B.sub.2 (riboflavin) 1500 mcg; Vitamin B.sub.6 (pyridoxine) 600 mcg; Vitamin B.sub.12 (cyanacobalmine) 2.0 mcg; Niacin 9.0 mcg; Folic Acid 80 mcg; Pantothenic Acid 3000 mcg; Biotin 90 mcg; VitaminC (ascorbic acid) 90 mg; Choline 100 mg; Inositol 33 mg; Calcium 460 Mg; Phosphorous 333 Mg; Magnesium 64 Mg;Iron 8.0 Mg; Zinc 6.0 Mg; Manganese 50 mcg; Copper 560 mcg; Iodine 100 mcg; Sodium 160 mg; Potassium 650 mg;Chloride 433 mg and Selenium 14 mcg; wherein the fat content provides includes 3 gram fish oil and 3 grams 40%arachidonic acid oil (DSM Food Specialties, Delft, Netherlands); further comprising 4 gram transgalactooligosaccharidesElix’or™ (Borculo Domo Ingredients, Netherlands) and 4 gram Raftiline™ (Orafti Active Food Ingredients, Belgium).

51015202530Claims

351.A nutritional composition comprising:a. EPA, DHA and ARA, wherein the content of long chain polyunsaturated fatty acid with 20 and 22 carbonatoms does not exceed 15 wt.% of the total fat content; and

b. at least two distinct oligosaccharides (OL1 and OL2), wherein the two distinct oligosaccharides have ahomology in monose units below 90 %.

402.

45The composition according to claim 1 further comprising:c. acidic oligosaccharides with a DP between 2 and 60, wherein the acidic oligosaccharides comprises at leastone acidic group selected from the group consisting of N-acetylneuraminic acid, N-glycoloylneuraminic acid,free or esterified carboxylic acid, sulfuric acid group and phosphoric acid group.

3.

50The composition according to claim 2 further comprising:d. between 1 and 500 mg nucleosides and/or nucleotides per 100 gram dry formula.

4.

55The composition according to claim 1-3 comprising galactooligosaccharide and a fructan selected from the groupconsisting of fructooligosaccharides, inulin and mixtures thereof.

The composition according to claim 1-4 wherein at least 10 wt.% of the oligosaccharide has a degree of polymerisation(DP) of 2 to 5 and at least 5 wt.% has a DP of between 10 and 60.

5.

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6.

The composition according to any one claims 1-5, further comprising an acidic oligosaccharide, preferably an uronicacid polymer with a DP between 2 and 60.

The composition according to any one of claims 1-6, comprising 7.5 to 12.5 energy % protein; 40 to 55 energy %carbohydrates; and 35 to 50 energy % fat, wherein said protein comprises a member selected from the groupconsisting of hydrolyzed milk protein, vegetable protein and/or amino acids.

The composition according to any one of claims 1-7, said composition having a caloric content of 0.6 to 0.8 kcal/ml;an osmolality of 50 to 500 mOsm/kg; and a viscosity below 50 mPas.

The composition according to any one of claims 1-8, suitable for feeding an infant, wherein:a. the content of long chain polyunsaturated fatty acid is below 3 wt.% of the total fat content;b. the omega-3 long chain polyunsaturated fatty acid is below 1 wt.% of the total fat content;c. the omega-6 long chain polyunsaturated fatty acid is below 2 wt.% of the total fat content;d. the ARA content is below 1 wt.% of the total fat content; ande. the ratio EPA/DHA is 1 or below.

10.The composition according to any one of claims 1-9, for use as a medicament.

207.

58.

109.

1511.Use of a composition according to any one of the claims 1-9 for manufacture of a composition for administration to

an infant with the age between 0 and 2 years.12.Use of a composition according to any one of claims 1-9 for the manufacture of a composition for use in a method

for the treatment or prevention of allergy, said method comprising administering to a mammal the compositionaccording to any one of claims 1-9.13.Use of a composition according to any one of claims 1-9 for the manufacture of a composition for use in a method

for the treatment or prevention of diarrhea, said method comprising administering to a mammal the compositionaccording to any one of claims 1-9.14.Use of a composition according to any one of claims 1-9 for the manufacture of a medicament for use in a method

to reduce diaper rash, said method comprising administering to a mammal the composition according to any oneof claims 1-9.

35253015.Use of a composition according to any one of claims 1-9 for the manufacture of a composition for

a. providing the nutritional requirements of a premature infant;

b. the treatment or prevention of chronic inflammatory diseases, particularly inflammatory bowel disease, irritablebowel disease, celiac disease, pancreatitis, hepatitis, arthritis or diabetes;

c. For providing nutrition to patients which have undergone abdominal surgery and patients that experiencepostoperative dysfunction of the gut and/or malnourished patients;

d. administration to patients suffering from acquired immune deficiency syndrome and/or patients which areinfected with the human immunodeficiency virus;

e. treat or prevent complications resulting from reduced barrier integrity, particularly prevention of diarrhea;f. treatment or prevention of allergy;

g. treatment and/or prevention of diseases wherein intestinal IL-4 concentration is increased; orh. stimulating intestinal barrier integrity.

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the Europeanpatent document. Even though great care has been taken in compiling the references, errors or omissions cannot beexcluded and the EPO disclaims all liability in this regard.Patent documents cited in the description•EP 1272058 A [0003]•

•

EP 745001 A [0004]

Non-patent literature cited in the description•Usami et al. Clinical Nutrition, 2001, vol. 20 (4),•351-359 [0005] [0008]

•

Kleessen et al. Brit J Nutr, 2003, vol. 89, 597-606•

[0028]

16

WO 0160378 A [0034]

Meslin et al. Brit. J.Nutr, 1993, vol. 69, 903-912[0028]

Barcelo et al. Gut, 2000, vol. 46, 218-224 [0033]