Improvement of intestinal barrier integrity

Background of the invention

The gastrointestinal epithelium usually functions as a selective barrier allowing the absorption of nutrients, electrolytes and water, and preventing exposure to food and microbial antigens, including food allergens. The gastrointestinal epithelium limits the passage of antigens to the systemic circulation, which can cause inflammatory reactions, e.g. allergic reactions. Due to the fact that the incidence of allergies, especially food allergies, is increasing, many research groups are looking for (preventive) treatment for these ailments.

EP1272058 describes a composition containing indigestible oligosaccharides for improving tight junctions to reduce intestinal permeability and reduce an allergic reaction. The composition may contain LC-PUFA (long chain polyunsaturated fatty acids).

EP 745001 describes a combination of indigestible oligosaccharides and n-3 and n-6 ​​fatty acids for the treatment of ulcerative colitis.

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Usami et al. (Clinical Nutrition 2001, 20 (4): 351-359) describe the effect of eicosapentaenoic acid (EPA) on tight junction permeability in monolayer of intestinal cells. They found that EPA increases permeability, indicating that EPA is unsuitable for improving intestinal barrier integrity.

Formulations known in the art are not optimally adapted to improve barrier integrity.

Summary of the invention

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

It has been surprisingly found that selected LC-PUFAs effectively reduce epithelial intracellular permeability. Contrary to what Usami et al. (Clinical Nutrition 2001, 20 (4): 351-359) described, the present inventors have found that C18 and C20 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and

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EP 1 672 987 B1 (ARA), are able to effectively permeate intestinal mucus joints. It is thought to be influenced by various arachidonic reduction strict.

In addition to LC-PUFA, the present composition contains oligosaccharides. Selected oligosaccharides improve barrier integrity by stimulating mucus production, which results in a thickening of the layer that this effect is due to oligosaccharides for the production of short chain fatty acids (SCFA). Consequently, when enterally administered to a mammal, the present combination of LC-PUFA and indigestible oligosaccharides synergistically improves barrier integrity and / or synergistically reduces intestinal permeability by simultaneously reducing tight junction permeability and stimulating mucus production.

In a further aspect, the present composition improves the quality of the intestinal mucus layer. The mucus layer contains mucins. Mucins are high molecular weight glycoproteins that are synthesized and secreted by goblet cells. They form a gel-like layer on the mucosal surface, thereby improving barrier integrity. The slack layer contains different types of mucins, e.g. acid, neutral and mucins

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Sulphonated EP 1 672 987 B1. It is believed that the increased heterogeneity of the mucus layer improves barrier functionality.

The present composition preferably contains at least two different oligosaccharides that affect mucus architecture and favorably affect mucin heterogeneity in the mucus layer, either directly or by altering the intestinal flora. It is believed that each different selected oligosaccharide has a different effect on the quality and quantity of mucus. In addition, two different oligosaccharides are also able to stimulate mucus quality, which is reflected in the degree of sulfonation, through their synergistic stimulation of SCFA production. The present inventors have surprisingly found that the mixture of two different oligosaccharides of the present invention synergistically stimulates acetate production. The present inventors have also surprisingly found that mucus production is dependent on acetate production.

The present composition is preferably more improved by long chain as well as providing both and short chain oligosaccharides. Providing molecules with different chain lengths results in stimulation of mucus production in various parts of the ileum and colon. Short-chain oligosaccharides (usually grade

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Polymerization (DP) of 2, 3, 4 or 5) stimulates the production of mucins in the proximal colon and / or distal ileum, while longer chain oligosaccharides (preferably with a degree of polymerization (DP) are considered) more than 5 to 60) stimulate the production of mucins in the more distal parts of the colon.

Even greater improvement can be achieved by providing at least two different oligosaccharides, both short and long chain oligosaccharides. These preferred embodiments contribute to further improving barrier integrity throughout the ileum and / or colon.

In addition, it was surprisingly found that EPA, DHA and ARA were able to reduce the harmful effects of interleukin 4 (IL-4) on intestinal permeability. IL4 is a cytokine that is secreted in increased amounts by mucosal T cells in some patients and induces intestinal permeability.

The present invention therefore provides a method for the treatment and / or prevention of diseases in which the concentration of IL-4 in the intestine is increased, such as allergies and in particular atopic dermatitis.

Detailed description of the invention

The present invention relates to a nutritional composition comprising:

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a) EPA, DHA and ARA, wherein the content of long chain polyunsaturated fatty acids with 20 and 22 carbon atoms does not exceed 15 wt. total fat content; and

b) at least two different oligosaccharides, wherein the two different oligosaccharides have homology in the monose unit of less than 90%.

The composition may advantageously be used in a method of stimulating intestinal barrier integrity, the method comprising administering to the mammal said composition.

Polyunsaturated fatty acids

The present inventors have 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. Thus, the present composition, which is particularly suitable for improving intestinal barrier integrity, contains EPA, DHA and ARA.

The present inventors have found that a lower concentration of LC-PUFA was effective in reducing tight junction permeability (see Examples for Usami et al.). Thus, the LC-PUFA content of 20 and 22 carbon atoms in the present composition preferably does not exceed 15 wt. total fat content,

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<td>favorably</td><td>no</td><td>exceeds</td><td> 10</td><td>wt%</td><td>, yet</td>

<td>preferably</td><td>no</td><td>exceeds</td><td> 5</td><td>wt%</td><td>the total</td>

<td>content</td><td colspan="3">fat. favorably</td><td>this</td><td>composition</td>

contains at least 0.1 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 total fat content. For the same reason, the EPA content preferably does not exceed 5 wt. total fat, more preferably it does not exceed 1 wt.%, but is preferably at least 0.05 wt.%, more preferably at least 0.1 wt.% total fat. The DHA content preferably does not exceed 5 wt.%, More preferably does not exceed 1 wt.%, But is at least 0.1 wt. total fat. Since ARA has been found to be particularly effective in reducing tight junction permeability, the present composition contains relatively high amounts, preferably at least 0.1 wt.%, Even more preferably at least 0.25 wt.%, Most preferably at least 0.5 wt.% . total fat. The ARA content preferably does not exceed 5 wt.%, More preferably does not exceed 1 wt. all of the fat. In the present intestinal composition containing ARA, EPA and DHA are preferably added to balance the effects of ARA, e.g. to reduce the potentially proinflammatory effects of ARA metabolites.

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Excess metabolites from ARA can cause inflammation. Thus, the present composition preferably comprises ARA, EPA and DHA, wherein the ARA / DHA weight ratio is preferably above 0.25, preferably above 0.5, even more preferably above 1. The ratio is preferably below 25. The weight ratio ARA / EPA is preferably between 1 and 100, more preferably between 5 and 20.

The present composition preferably contains between 5 and 75 wt. polyunsaturated fatty acids based on total fat, preferably between 10 and 50 wt.

When the present composition is used as an infant formula (e.g. a method of feeding an infant, the method comprising administering the present composition to the infant), the LC-PUFA content, especially the LC-PUFA with 20 and 22 carbon atoms, preferably does not exceed 3 wt% total fat, since it is desirable to imitate human milk as soon as possible. For the same reason, the omega-3 LC-PUFA content preferably does not exceed 1 wt. total fat content; the omega-6 LC-PUFA content preferably does not exceed 2 wt. total fat content; the ARA (omega-6) content is preferably below 1 wt. total fat content; and / or the weight ratio EPA / DHA is preferably 1 or less, more preferably below 0.5.

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LC-PUFA with 20 and 22 carbon atoms can be provided as free fatty acids, in the form of triglycerides, in the form of phospholipids, or as a mixture of any of the foregoing. The present composition preferably contains at least one of ARA and DHA in the form of phospholipids.

The present nutritional composition preferably also contains omega-9 (n-9) fatty acid (preferably oleic acid, 18: 1) to provide adequate nutrition. Preferably the present composition provides at least 15 wt. n-9 fatty acid based on the weight of all fatty acids, more preferably at least 25 wt. The content of n-9 fatty acids is preferably below 80 wt.

oligosaccharides

Suitable oligosaccharides of the invention are saccharides with a degree of polymerization (DP) of at least 2 monose units, which are not only partially digested in the intestine by the action of acids or digestive enzymes present in the upper human digestive system (small intestine and stomach), but which are fermented by human intestinal flora. The term monose units refers to units with a closed ring structure, preferably hexose, e.g. forms of pyranose or furanose. The degree of polymerization of the oligosaccharide is usually below

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Monose units, preferably below 40, even more preferably below 20.

The present composition contains at least two different oligosaccharides, the oligosaccharides having homology in monose units below about 90%, preferably below 50%, more preferably below 25%, still preferably below 5%. The term "homology" as used in the present invention means the cumulative percentage of the same monose units in different oligosaccharides. For example, oligosaccharide 1 (OL1) has a fruc-fruct-glu-gal structure and therefore contains 50% fruc, 25% gal and 25% glu. Oligosaccharide 2 (OL2) has a fruc-fruc-glu structure and therefore contains 66% fruc, 33% glu. The different oligosaccharides therefore have a homology of 75% (50% fruc + 25% glu).

In a preferred embodiment, the present composition comprises galactooligosaccharides and at least one selected from the group consisting of fructooligosaccharides and inulin.

Each of the present oligosaccharides preferably contains at least 66%, more preferably at least 90% of monose units selected from the group consisting of mannose, arabinose, fructose, fucose, rhamnose, galactose, β-D-galactopyranose, ribose, glucose, xylose, uronic acid and their derivatives, calculated

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EP 1 672 987 B1 relative to the total number of monose units contained therein.

According to a further embodiment, at least one of the oligosaccharides of the present composition is selected from the group consisting of fructans, fructooligosaccharides, indigestible dextrin galactooligosaccharides (including transgalphooligosaccharides, xylooligosaccharides, oligosaccharides, glucosaccharides, uronic acid oligosaccharides (such as pectin hydrolyzate) and mixtures thereof. Preferably the present composition contains at least one, preferably at least two, of oligosaccharides selected from the group consisting of fructooligosaccharides or inulin, galactooligosaccharides and pectin hydrolyzate.

For good mucus quantity and quality, the present composition preferably contains at least one oligosaccharide, which contains at least 66% galactose or fructose as a monose unit. In a preferred embodiment, the composition comprises at least one oligosaccharide that contains at least 66% galactose as a monose unit and at least one oligosaccharide that contains at least 66% fructose as a monose unit. In particularly favorable

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In an embodiment, the present composition comprises galactooligosaccharide and an oligosaccharide selected from the group consisting of fructooligosaccharides and inulin. Fructooligosaccharides stimulate the production of sulfomucin in the distal colon in rats with human flora (Kleessen et al., (2003) Brit J Nutr 89: 597-606) and galactooligosaccharides stimulate the production of acid mucins (Meslin et al., Brit. J. Nutr (1993) ), 69: 903912)).

In order to further improve the thickness of the slack layer throughout the entire colon, at least 10 wt. oligosaccharides present in the present composition have a DP of 2 to 5 (i.e. 2, 3, 4 and / or 5) and at least 5 wt. has a DP of 10 to 60. Preferably at least 50 wt.%, more preferably at least 75 wt. oligosaccharides have a DP of 2 to 9 (i.e. 2, 3, 4, 5, 6, 7, 8, and / or 9) because they are thought to act throughout the ileum and the proximal and middle colon, and because the weight percentage of oligosaccharides to be included to the composition to achieve the desired effect is reduced.

Preferably weight ratios:

a. (oligosaccharides with DP 2 to 5): (oligosaccharides with DP 6, 7, 8 and / or 9)&gt; 1; and

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b. (oligosaccharides with DP 10 to 60): (oligosaccharides with DP 6, 7, 8 and / or 9)&gt; 1 are both above 1.

Preferably, both weight ratios are above 2, more preferably above 5.

In order to further improve the thickness of the slag layer and the amount throughout the colon region, preferably each of at least two different oligosaccharides is provided in a form with different chain lengths, preferably at least 10 wt. each oligosaccharide relative to the total weight of the respective oligosaccharide has a DP of 2 to 5 (i.e. 2, 3, 4 and / or 5) and at least 5 wt. has a DP between 10 and 60. Preferably at least 50 wt.%, more preferably at least 75 wt. oligosaccharides based on the total weight of these oligosaccharides have a DP between 2 and 10 because they are thought to act throughout the ileum and the proximal and mid-colon.

Acid oligosaccharides

To further improve barrier integrity, the present composition preferably contains acid oligosaccharides with a DP between 2 and 60. The term acid oligosaccharides refers to oligosaccharides containing at least one acid group selected from the group consisting of N-acetylneuraminic, N-glycolyloneuraminic, free or esterified

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Carboxylic acid, sulfuric acid group and phosphoric acid group. The acid oligosaccharide preferably contains uronic acid units (i.e. uronic acid polymer), more preferably glacturonic acid units. The acid oligosaccharide may be a homogeneous or heterogeneous carbohydrate. Suitable examples are pectin and / or alginate hydrolysates. In the intestinal tract, uronic acid polymers are hydrolysed to uronic acid monomers that stimulate the production of intestinal acetate, which in turn stimulates the secretion of intestinal mucus (Barcelo et al., Gut 2000; 46: 218224).

Preferably the acid oligosaccharide has a structure I below in which the final hexose (left side) preferably contains a double bond. The hexose units other than the final hexose unit (s) are preferably uronic acid units, even more preferably galacturonic acid units. The carboxylic acid groups on these units may be free or (partly) esterified, and preferably at least 10% is methylated (see below).

Structure I: Polymeric acid oligosaccharide

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<img file="PL1672987T3_D0001.tif" />

R is preferably selected from the group consisting of a hydrogen atom, a hydroxyl or acid group, preferably a hydroxyl group, and at least one selected from the group consisting of R2, R3, R4 and R5 is N-ethyluraminic acid, N-glycolylneuraminic acid, free or esterified carboxylic acid, sulfuric acid group and phosphoric acid group, and the remaining R2, R3, R4 and R5 are a hydroxyl group and / or a hydrogen atom. Preferably one selected from the group consisting of R2, R3, R4 and R5 is N-acetylneuraminic acid, N-glycolyloneuraminic acid, free or esterified carboxylic acid, sulfuric acid group or phosphoric acid group, and the remaining ones are hydroxyl group and / or hydrogen atom . Even more preferably one selected from the group consisting of R2, R3, R4 and R5 is a free or esterified carboxylic acid and the remaining R2, R3, R4 and R5 are a hydroxyl group and / or a hydrogen atom; an is an integer and refers to the number of hexose units (see also Polymerization degree, below), which can be any

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EP 1 672 987 B1 unit of hexose. Suitably n is an integer between 1-5000. Preferably the hexose unit (s) is a uronic acid unit.

Most preferably R1, R2 and R3 are hydroxyl, R4 is hydrogen, R5 is carboxylic acid, n is any integer between 1 and 250, preferably between 1 and 10 and the hexose unit is galacturonic acid.

The detection, measurement and analysis of preferred acid oligosaccharides used in the present method are given in the prior patent application of the present applicants regarding acid oligosaccharides, i.e. WO 0/160378.

To stimulate the improvement of slack layer thickness

<td>in all</td><td colspan="2">region</td><td colspan="2">present colon</td><td>composition</td>

<td>favorably</td><td colspan="2">It contains</td><td>at least</td><td>10 wt.</td><td>sour</td>

<td colspan="2">oligosaccharides</td><td>about DP</td><td>of 2</td><td>up to 5 (i.e.</td><td> . 2, 3, 4</td>

<td>and / or 5)</td><td>and</td><td>What</td><td>at least 5</td><td>wt%</td><td>sour</td>

<td colspan="2">oligosaccharides</td><td>about DP</td><td>between 10 and</td><td colspan="2">60, wt.%</td>

is based on the total weight of oligosaccharides.

The acid oligosaccharides used in the invention are preferably prepared from pectin, pectinate, alginate, chondroitin, hyaluronic acids, heparin, heparan, bacterial carbohydrates, sialoglycans, fucoidan, fucooligosaccharides or carrageenan, more preferably pectin and / or alginate.

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Oligosaccharide content

In the case of a ready-to-eat liquid form, the present composition preferably contains 0.1 to 100 gram of indigestible oligosaccharide per liter, preferably between 0.5 and 50 gram per liter, even more preferably between 1 and 25 gram per liter. Too high oligosaccharide content can cause discomfort due to excessive fermentation, while very low content may result in an insufficient mucus layer.

The weight ratio of at least two different oligosaccharides is preferably between 1 and 10, more preferably between 1 and 5. These weight ratios optimally stimulate the production of different types of mucins at different places in the intestine.

The oligosaccharide is preferably included in the present composition according to the invention in an amount exceeding 0.1% by weight, preferably exceeding 0.2% by weight, even more preferably exceeding 0.5% by weight, and even more preferably exceeding 1% by weight. relative to the total dry weight of the composition. In the present composition, the oligosaccharide content is preferably below 20 wt.%, More preferably below 10 wt.%, Even more preferably below 5 wt.

The addition of nucleotides and / or nucleosides to the present composition further improves

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The functioning of the mucosa in particular because of the intestinal barrier in that it inhibits and / or reduces the incidence of bacterial translocation and reduces intestinal damage. Thus, the present composition preferably also contains between 1 and 500 mg nucleosides and / or nucleotides per 100 gram dry preparation, more preferably between 5 and 100 mg.

Use

The present composition can be advantageously used in a method of improving barrier integrity in mammals, especially humans. The present composition can also be advantageously used in a method of treating or preventing diseases associated with reduced barrier integrity, the method comprising administering to the mammal the present composition. The present composition is preferably administered orally.

In the case of patients and babies, the present composition is preferably combined with complete nutrition containing protein, carbohydrate and fat. The present composition is preferably administered to young children between 0 and 2 years of age. The composition can be administered to patients suffering from barrier integrity disorder and to healthy patients. The present composition is preferably used in a method of providing nutritional requirements for premature babies (infants born before 37 weeks of gestation).

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The present composition can also be advantageously used in the treatment and / or prevention of intestinal damage by administering the present composition to a patient before or after a medical procedure that can cause intestinal damage. Such a medical procedure may be, for example, surgery or enteric drug treatment (e.g., antibiotic, anesthetic drug, NSAID, chemotherapeutic agents, etc.).

The present composition can also be advantageously used for the treatment or prevention of diseases in which the disruption of the intestinal barrier underlies the development of the disease, e.g. in the method of treatment or prevention of chronic inflammatory diseases, especially inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), celiac disease, pancreatitis, hepatitis, arthritis or diabetes. In addition, the invention may be used in a method of providing nutrition to patients who have undergone or undergo abdominal surgery and patients who experience postoperative bowel dysfunction and / or malnourished patients.

In a further embodiment of the invention, the present composition is preferably administered to patients suffering from acquired immune deficiency syndrome (AIDS) and / or patients who are infected with human virus

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Immunodeficiency (HIV), e.g. in the method of treatment of AIDS and / or HIV infection. Such a method involves the oral administration of the present composition, preferably in combination with nutrients selected from the group consisting of carbohydrate, protein and fat.

In addition, the invention can also be used to treat or prevent complications due to reduced barrier integrity, especially in the method of treatment and / or prevention of diarrhea, especially diarrhea in infants. Due to the reduced incidence of diarrhea in infants, the present composition can also be advantageously used to reduce diaper rash.

Administration of the present composition reduces the passage of food and microbial antigens, especially food allergens, from the intestinal lumen to the mucosal or systemic circulation, and therefore can be advantageously used in a method of treatment or prevention of allergies and / or allergic reactions, especially in a method of treatment or prevention of food allergies. e.g. an allergic reaction arising from the consumption of a food product.

The present inventors have found that EPA, DHA and / or ARA are able to reduce the effects of IL-4 on intestinal permeability. Thus, one aspect of the present invention provides a method of treatment and / or

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Prophylaxis of diseases in which intestinal IL-4 concentration is increased (e.g. allergic diseases), the method comprising administering LC-PUFA preferably selected from the group consisting of EPA, DHA and ARA, preferably in combination with with these selected oligosaccharides. Thus, the present composition can also be advantageously used in a method of treating atopic dermatitis.

Because the functioning of the neonatal barrier is not fully developed, the present composition can be advantageously administered to young infants, i.e. infants between 0 and 6 months of age. The composition may be administered to infants in the form of an infant formula without human milk or in a form mixed with human milk. Thus, the present invention also provides a nutritional preparation comprising human milk and the present composition. Compositions containing human milk and the present composition are particularly suitable for feeding premature babies.

The present composition is preferably provided in the form of a powder in the package or a ready-to-eat preparation in the package. To avoid deterioration of the product, the packaging size of the ready-to-eat preparation does not exceed one serving, e.g. preferably does not exceed 500 ml; and the size of this package

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For example, refer to a powder composition preferably does not exceed 250 servings. Suitable portion sizes for the powder are 2000 gram or less, preferably per 1000 gram or less.

Also included in the present invention are packaged products provided with labels that directly or indirectly direct the consumer to use the product in accordance with one or more of the foregoing purposes. Such labels may, for the prophylaxis of the allergic response to the method of intestinal allergens ", similar wording.

by incorporating a wording such as "reduced food sensitivity", "improves tolerance, improved food tolerance," or Similarly, references to this method of treatment and / or prevention of allergies can be made by introducing terminology equivalent to "increased immunity" or " reduced sensitivity ".

preparations

It has been found that the present composition can be advantageously used in foods such as baby foods and clinical foods. Such food preferably contains fat, protein and carbohydrate, and is preferably administered in liquid form. The term "liquid food" as used in the present invention includes dry

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Food (e.g. powders), which is accompanied by instructions on how to mix said dry food mixture with a suitable liquid (e.g. water).

Thus, the present invention also relates to a nutritional composition which preferably contains between 5 and 50% en from fat, between 5 and 50% en from protein, between 15 and 90% en from carbohydrate and the present combination of oligosaccharides and LC-PUFA. Preferably the present nutritional composition preferably contains between 10 and 30% en from fat, between 7.5 and 40% en from protein and between 25 and 75% en from carbohydrate (% en is the short form for the percentage of energy and reflects the relative amount of each component as contributes to the total caloric value of the preparation).

Preferably, a combination of vegetable fats and at least one oil selected from the group consisting of fish oil and omega-3 oil of plant origin, algae, or bacteria is used.

The proteins used in the nutritional preparation are preferably selected from the group of non-human animal proteins (such as milk proteins, meat proteins and egg proteins), vegetable proteins (such as soy protein, wheat protein, rice protein, pea protein), free amino acids and mixtures thereof. Source of nitrogen derived from cow's milk, especially protein from

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Cow's milk proteins such as casein and whey proteins are particularly preferred.

A source of digestible carbohydrate can be added to the nutritional preparation. It preferably provides about 40% to about 80% of the energy of the nutritional composition. Any suitable (source) carbohydrate may be used, for example, sucrose, lactose, glucose, fructose, corn syrup and maltodextrin solid ingredients, and mixtures thereof.

The present composition is preferably used as an infant formula and preferably contains 7.5 to

12.5% ​​of energy from protein; 40 to 55% of energy from carbohydrates; and 35 to 50% energy from fat. Because the present composition is appropriately used to reduce the allergic reaction in infants, the protein in the infant formula is preferably selected from the group consisting of hydrolyzed milk protein (e.g. hydrolyzed casein or hydrolyzed whey protein), vegetable protein and / or amino acids. The use of these proteins further reduces allergic reactions in infants.

Stool abnormalities (e.g., hard stools, insufficient stool volume, diarrhea) are a major problem for many children and the sick who receive liquid food. It has been found that these stool problems can be reduced by administration

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Of the present oligosaccharides in liquid food whose osmolarity is between 50 and 500 mOsm / kg, more preferably between 100 and 400 mOsm / kg.

In light of the above, it is also important that liquid food does not have excessive caloric density, however, it still provides enough calories to feed the individual. Thus, liquid food preferably has a caloric density between 0.1 and 2.5 kcal / ml, more preferably a caloric density between 0.5 and

1.5 kcal / ml, most preferably between 0.6 and 0.8 kcal / ml.

Examples

Example 1: Effect of LC-PUFA on barrier integrity

Monolayers (MC) of the T84 intestinal epithelial cell line (American Type Culture Collection (ATTC),

Manassas,

USA) were cultured on transwell wells (Corning, Costar BV, The Netherlands) allowing both mucosal and serous samples to be sampled and stimulating human intestinal epithelial cells. Two weeks after confluence, the monolayers were incubated in the lumenal range with polyunsaturated fatty acids

ARA (arachidonic acid;

eicosatetraenoic acid), DHA (5,8,11,14cis-4,7,10,13,16,19 docosahexaenoic acid), EPA (eicosapentaenoic acid) or

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Control palmitic acid (C 16: 0) (Palm) (Sigma, St. Louis, USA). The last procedure was chosen to imitate the route of administration of nutrients in vivo. Cells were incubated with ARA, DHA, EPA, or palmitic acid for 0, 24, 48 and 72 hours. at various concentrations of μΜ

100 μΜ)

conducted

Instruments, Germany) experiments to assess the initial barrier integrity. Epithelial barrier function was determined by measuring transepithelial resistance (TER, Ω.σζκι) using an epithelial volt-ohm meter (EVOM; World Precision permeability for 4kD FITC dextran (para-cell permeability marker, Sigma, USA). Resistance (. Epithelial permeability for 4 kDa FITC-dextran was determined as follows. Prior to the dextran flow, the medium was refreshed with culture medium without phenol red for one hour and then added 5 µL (100 mg / mL initial) of 4 kDa FITCdextran to the lumenal incubation interval 100 µl serum sample was collected and the fluorescence signal measured at excitation wavelength of 485 nm and emission 520 nm (FLUOstar Galaxy®, BMG Labtechnologies, USA). FITC-dextran flows were calculated as pmol FITCdextran / cm²/ Hr. Statistical analyzes were performed using ANOVA (SPSS version 10).

After 30 min range

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Results regarding the effect of fatty acids (100 μΜ) on spontaneous barrier integrity after 72 hours. incubations are given in Table 1. Table 1 shows that LCPUFA: ARA, EPA and DHA reduce particle flow and improve epithelial resistance. In contrast, control experiments show that palmitic acid exerts opposite effects, i.e., reduces barrier integrity. These results are indicative of the beneficial use of EPA, DHA and ARA, especially ARA in the composition of the present invention, and for use in the method of the present invention, e.g. in a method of improving barrier integrity. These results also support the synergistic effects of this combination of fatty acids and indigestible oligosaccharides.

Figure 1 shows the effects of different fatty acids over time and dose (10 gM and 100 gM) (palmitic acid, DHA, GLA, and AA) on initial barrier integrity (TER). Figure 1 shows that LCPUFA: AA, DHA, and GLA improve epithelial barrier integrity expressed by increased resistance (TER). These results are indicative of the beneficial use of EPA, DHA, GLA and ARA, especially ARA, in the composition of the present invention, and for use in the method of the present invention, i.e. in a method of improving barrier integrity. These results represent

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EP 1 672 987 B1 furthermore supports the synergistic effects of the present combination of fatty acids and indigestible oligosaccharides.

Table 1

<td>Ingredient (LC-PUFA)</td><td rowspan="2">Flow</td><td rowspan="2">Resistance (TER)</td>

<td></td>

<td>Control</td><td> 79</td><td> 1090</td>

<td>Palmitic acid</td><td> 161</td><td> 831</td>

<td>DHA</td><td> 72</td><td> 1574</td>

<td>ARA</td><td> 28</td><td> 1816</td>

<td>EPA</td><td> 65</td><td> 1493</td>

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

Monolayers (MC) of the T84 intestinal epithelial cell line (ATCC, USA) were grown on transwell wells (Corning, Costar BV, The Netherlands) allowing both mucosal and serosal samples to be taken and stimulating human intestinal epithelial cells. Two weeks after confluence, the monolayers were incubated in the presence of IL-4 (2 ng / ml, serum compartment, Sigma, USA) with or without polyunsaturated fatty acids ARA, DHA,

GLA, EPA, or control palmitic acid (10 μΜ or 100 gM, mucosal compartment, Sigma, St. Louis,

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USA). Cells were preincubated with ARA, DHA, EPA, or palmitic acid for 48 hours. before incubation with IL-4. Co-incubation of PUFA with palmitic acid and IL-4 was continued for the next 48 hours, while the culture medium and additives were changed every 24 hours. Epithelial barrier function was determined by measuring transepithelial resistance (TER) and permeability as described in Example 1. Statistical evaluation was performed as described in Example

1.

The results regarding the effect of ARA, DHA, EPA and palmitic acid (100 μΜ) on IL-4-dependent barrier disruption are given in Table 2. Table 2 shows that LCPUFA: ARA, DHA and EPA inhibit the increased flow caused by IL-4. In contrast, palmitic acid exerted a detrimental effect and reduced barrier disruption compared to control. These results are indicative of the beneficial use of ARA, DHA and EPA in clinical formulations and nutritional formulas for children for the prevention or reduction of IL-4-dependent barrier disruption, e.g. as in food allergy or cow's milk allergy. These results also support the synergistic effects of this combination of fatty acids and indigestible oligosaccharides.

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Figure 2 shows time and dose dependent (10 μΜ and 100 μΜ) protective effects of various FA (palmitic acid, DHA, GLA and AA) on IL-4 dependent barrier destruction (flow). Figure 2 shows that

ARA, DHA and GLA protect against IL-4-dependent barrier disruption, which is reflected by the reduced 4kD flow of dextran. These results are indicative of the beneficial use of ARA, DHA and GLA in clinical and nutritional formulations for children for prevention or reduction of IL-4 dependent barrier disruption, e.g. as in food allergy or cow's milk allergy. These results also support the synergistic effects of this combination of fatty acids and indigestible oligosaccharides.

Table 2

<td>Ingredient (LC-PUFA)</td><td>IL-4 flow</td><td>IL-4 TER</td>

<td>Control</td><td> 582</td><td> 374</td>

<td>Palmitic acid</td><td> 777</td><td> 321</td>

<td>DHA</td><td> 271</td><td> 547</td>

<td>ARA</td><td> 218</td><td> 636</td>

<td>EPA</td><td> 228</td><td> 539</td>

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Example 3: Effect of oligosaccharides on acetate production

Microorganisms were obtained from fresh stools from bottle-fed children. Fresh stool material from children aged 1 to 4 months was combined and placed in the preservation medium within 2 hours. Prebiotics (TOS; TOS / inulin (HP) ratio 9/1 (w / w)); inulin; oligofructose (OS) / inulin ratio 1/1 (w / w), or nothing (blind).

Transgalactooligosaccharides (TOS) were obtained from Vivinal GOS, Borculo Domo Ingredients, Zwolle, The Netherlands and they contain as indigestible oligosaccharides: 33 wt. disaccharides, 39 wt. trisaccharides, 18 wt. tetrasaccharides, 7 wt. pentasaccharides and 3 wt. hexa-, hepta- and octasaccharides. Inulin active ingredients (HP) Orafti, Tienen, Belgium, i.e. Raftiline HP®, with an average DP of 23. Media: McBain &amp; MacFarlane medium: buffered peptone water 3.0 g / l, yeast extract 2.5 g / l, mucin (brushes) 0.8 g / l, trypton 3.0 g / l, L-cysteine HCl 0.4 g / l, bile salts 0.05 g / l, K2HP04.3H20 2.6 g / 1, NaHCO3 0.2 g / l, NaCl 4.5 g / l, MgSO4.7H2O 0.5 g / l, CaCl2 0.228 g / l, FeSO4.7H20 0.005 g / l. Fill 500 ml Scott bottles with the medium and sterilize 15 minutes at 121 ° C. Buffered medium: K2HPO4.3H2O 2.6 g / l, NaHCO3 0.2 g / l,

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NaCl 4.5 g / l, MgSO4.7 H2O, 0.5 g / l, CaCl2 0.228 g / l, FeSO4.7H2O 0.005 g / l. Adjust to pH 6.3 ± 0.1 using K2HPO4 or NaHCO3. Fill 500 ml Scott bottles with the medium and sterilize 15 minutes at 121 ° C. Preservation medium: Buffer peptone 20.0 g / l, Lcysteine-HCl 0.5 g / l, sodium thioglycolate 0.5 g / l, 1 resazurin tablet per liter, adjust to pH 6.7 ± 0.1 using 1 M NaOH or HCl. Boil in a microwave. Serum bottles were filled with 25 ml medium and sterilized for 15 minutes at 121 ° C.

Fresh stool samples were mixed with preservative medium and stored for several hours at 4 ° C. The preserved stool solution was centrifuged at 13,000 rpm for 15 minutes, the supernatant removed and the stools mixed with McBain &amp; Mac Farlane medium in a 1: 5 weight ratio. From this suspension, 3 ml of stool was combined with 85 mg glucose or prebiotic, or with nothing (blind) in the bottle and mixed thoroughly. At t = 0 a sample (0.5 ml) was taken. 2.5 ml of the suspension thus formed was introduced into the dialysis tube in a 60 ml bottle filled with 60 ml. The bottle was well closed and buffered medium. incubated at 37 ° C tubes

Samples were taken from dialysis (0.2 ml) or dialysis buffer (1.0 ml) syringe with subcutaneous injection after 3, 24 and 48 hours, and immediately placed on ice for

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EP 1 672 987 B1 complete fermentation. The experiment was carried out using the following samples:

1) 85 mg TOS

2) 85 mg of inulin

3) 85 mg TOS / inulin in a ratio of 9/1 (w / w) and

4) 85 mg OS / inulin in a 1/1 ratio (w / w).

SCFA (acetate, propionate, butyrate) was quantified using a Varian 3800 (GC) gas chromatograph (Varian Inc., Walnut Creek, USA) equipped with an ionization flame detector. 0.5 g of sample was injected at 80 ° C onto a column (Stabilwax, 15 x 0.53 mm, layer thickness 1.00 gm, Restek Co., USA) using helium as carrier gas (3.0 psi). After sample injection, the oven was heated to 160 ° C at 16 ° C / min, and then heated to 220 ° C at 20 ° C / min. and finally held at 220 ° C for 1.5 minutes. The temperature of the dosing device (injector) and detector was 200 ° C. 2-ethylbutyric acid was used as the internal standard.

Figure 3 shows the absolute (Figure 3A) and relative SCFA profile (Figure 3B) formed by fermentation of various oligosaccharides. Figure 3A shows that a mixture of two different oligosaccharides (TOS / inulin), with two different oligosaccharides having homology of monose units below 90 and

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The different chain lengths result in a significantly and synergistically increased amount of SCFA (especially acetate) per gram of fiber compared to the ingredients alone. Figure 3B shows that the addition of the TOS / inulin combination promoted a higher proportion of preferred acetate (B). In vivo acetate production leads to improved mucus production by goblet cells and an intestinal mucus layer thickness gauge (see example 4). These results are indicative of the beneficial use of the present composition.

Example 4: Effects of SCFA on mucus production. Monolayer of intestinal epithelial T84 cells (ATCC, USA) were grown in 24 or 96 well tissue culture plates (Corning BV). T84 was incubated with acetate, propionate and butyrate of short chain fatty acids (SCFA, Merck, USA) for 24 hours. at a concentration in the range of 0.025-4.0 mM. Supernatants and / or cells were collected and MUC-2 (mucin) expression was determined. The dotblot technique was used to determine MUC-2 expression in cell cultures because mucins are extremely large glycoproteins (over 500 kDa), which makes them difficult to manipulate in western hybridization techniques. The method was checked using pre-serum (T84 stained negatively), negative control cells

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CCD-18Co (ATCC, USA) and bovine serum albumin (BSA). Cell samples were collected in Laemmli (protein isolation buffer) and the protein was measured using protein micro-assay (Biorad, USA) according to the manufacturer's protocol. Samples (0.3-0.7-1.0 ąg / 2 ąl) were transferred to nitrocellulose membranes (Schleicher &amp; Schuell, Germany). Membranes were blocked in TBST / 5% Protivar (Nutricia, The Netherlands) and then incubated for 1 hour. with the anti-MUC-2 antibody (supplied eagerly by Dr. Einerhand, Erasmus University, Rotterdam, Netherlands). After washing, blots were incubated with goat anti-rabbit-HRP (Santacruz Biotechnology, USA) and used to detect ECL substrate (Roche Diagnostics, The Netherlands). Densitometry was performed using Lumi-Imager (Boehringer Mannheim BV, The Netherlands) and the signal was expressed in light units (BLU). BLUs were also expressed relative to control incubations (% BLU). Baseline MUC-2 expression levels were subtracted to compare the stimulating effect of SCFA on MUC-2 expression.

Figure 4 shows the varying effects of SCFA (acetate, proprionate, butyrate) on MUC-2 expression in intestinal epithelial cells (MC T84) and epithelial mesenchymal cell co-cultures (CC T84). Figure 2 also shows that acetate more strongly stimulates MUC-2 expression (mucus production) compared to propionate and

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Butyrate. Thus, the present combination of oligosaccharides (which has been shown to stimulate acetate production (see example 3)) is particularly useful for stimulating mucus production and can be advantageously used in a method of stimulating barrier integrity.

Example 5: Infant Milk Preparation I Ingredients (per liter), energy 672 Kcal; 15 g protein; Whey ratio: casein 60:40; fat 36 g; carbohydrates 72 g; vitamin A 750 RE; mixed natural carotides 400 IU; vitamin D 10.6 mcg; Vitamin F 7.4 mg; vitamin K 67.0 mcg; vitamin B.sub.1 (thiamine) 1000 mcg; vitamin B.sub.2 (riboflavin) 1500 mcg; vitamin B.sub.6 (pyridoxine) 600 mcg; vitamin

B.sub.12 (cyanocobalmine) 2.0 mcg; niacin 9.0 mcg; 80 mcg folic acid; pantothenic acid 3000 mcg; biotin 90 mcg; vitamin C (ascorbic acid) 90 mg; choline 100 mg; inositol 33 mg; calcium 460 Mg; phosphorus 333 Mg; magnesium 64 Mg; iron 8.0 Mg; 6.0 Mg zinc; manganese 50 mcg; copper 560 mcg; iodine 100 mcg; 160 mg sodium; potassium 650 mg; 433 mg chloride and 14 mcg selenium; the fat content contains 3 grams of fish oil and 3 grams of 40% arachidonic acid oil (DSM Food Specialties, Delft, The Netherlands); in addition, containing 4 grams of Elix'or ™ transgalactooligosaccharides (Borculo Domo

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Ingredients, Netherlands) and 4 we play Raftiline^TM (Orafti Active Food Ingredients, Belgium).