Improvement of intestinal barrier integrity

ES 2 285 484 T3

DESCRIPTION

Improved integrity of the intestinal barrier.

Field of the invention

The present invention relates to a method for improving the integrity of the intestinal barrier and to a composition suitable for use in such a method.

Background of the invention

The gastrointestinal epithelium normally functions as a selective barrier that allows the absorption of nutrients, electrolytes, and water and that prevents exposure to dietary and microbial antigens, including food allergens. The gastrointestinal epithelium limits the passage of antigens into the systemic circulation, which can cause inflammatory reactions, for example allergic reactions. Since the incidence of allergies, particularly food allergies, is increasing, many research groups seek (preventive) cures for these ailments.

EP1272058 describes a composition containing indigestible oligosaccharides for the enhancement of tight junction to reduce intestinal permeability and reduction of allergic reaction. The composition may comprise PCFA (long chain polyunsaturated fatty acids).

EP745001 describes a combination of indigestible oligosaccharides and n-3 and n-6 ​​fatty acids for the treatment of ulcerative colitis.

Usami et al (Clinical Nutrition 2001, 20 (4): 351-359) describe the effect of eicosapentaenoic acid (EPA) on tight junction permeability in intestinal monolayer cells. According to them, EPA was found to increase permeability, indicating that EPA is not suitable for improving the integrity of the intestinal barrier.

Prior art formulations are not optimally suitable for improving barrier integrity. Summary of the invention

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

Surprisingly, it was found that selected AGPCL effectively reduce epithelial paracellular permeability. In contrast to what Usami et al (Clinical Nutrition 2001, 20 (4): 351-359) have reported, the present inventors found that C18 and C20 polyunsaturated fatty acids, in particular eicosapentaenoic acid (EPA), docosahexaenoic acid (ADH) and arachidonic acid (AA), are able to effectively reduce the permeability of the intestinal tight junction.

In addition to the AGPCL, the present composition contains oligosaccharides. Selected oligosaccharides improve barrier integrity by stimulating mucus production, resulting in increased mucus layer thickness. This effect is believed to be caused by the effects of different oligosaccharides on the production of short chain fatty acids (SCFA). Thus, when administered enterally to a mammal, the present combination of AGPCL and indigestible oligosaccharides synergistically improves barrier integrity and / or synergistically reduces intestinal permeability by simultaneously reducing tight junction permeability and stimulating of mucus production.

In another aspect, the present composition improves the quality of the intestinal mucus layer. The mucus layer comprises mucins. Mucins are high molecular mass glycoproteins that are synthesized and secreted by goblet cells. These form a gel-like layer on the mucosal surface, thereby improving the integrity of the barrier. The mucus layer comprises different types of mucins, for example acidic, neutral and sulfonated mucins. Increased heterogeneity of the mucus layer is believed to enhance the functionality of the barrier.

The present composition preferably comprises at least two different oligosaccharides, which influence the mucosal architecture and advantageously influence the heterogeneity of the mucin in the mucus layer, either directly or by changing the intestinal flora. Each different selected oligosaccharide is believed to have a different effect on the quantity and quality of mucus. Likewise, the two different oligosaccharides are also capable of stimulating mucus quality as reflected by the degree of sulfation through their synergistic stimulation of SCFA production. Surprisingly, it was discovered by the present inventors that a mixture of two different oligosaccharides according to the present invention synergistically stimulates acetate production. It was also discovered by the present inventors that mucus production is dependent on acetate production.

The present composition is further improved preferably by providing both long chain and short chain oligosaccharides. Supply of different chain lengths results in stimulation of production

ES 2 285 484 T3 of mucus in different parts of the ileum and colon. Short chain oligosaccharides (typically with a degree of polymerization (GP) of 2,3,4 or 5) stimulate mucin production in the proximal colon and / or distal ileum, whereas oligosaccharides with chain lengths Longer periods (preferably with a degree of polymerization (GP) of more than 5 to 60) stimulate mucin production in the more distal parts of the colon.

Still other improvements can be achieved by providing the at least two different oligosaccharides as both short-chain and long-chain oligosaccharides. These preferred embodiments all contribute to the most improved barrier integrity for the ileum and / or the colon.

Furthermore, it was surprisingly found that EPA, ADH and AA are capable of reducing the deleterious effects of interleukin 4 (IL-4) on intestinal permeability. IL-4 is a cytokine that is secreted in increased amounts by mucosal T cells in certain patients and induces intestinal permeability. Therefore, the present invention also provides a method for the treatment and / or prevention of diseases where the intestinal IL-4 concentration is increased, such as allergy, in particular atopic dermatitis.

Detailed description of the invention

The present invention relates to a nutritional composition comprising:

a) EPA, ADH and AA, where the content of long chain polyunsaturated fatty acid with 20 and 22 carbon atoms does not exceed 15% by weight of the total fat content; Y

b) at least two different oligosaccharides, where the two different oligosaccharides have homology in monose units below 90%.

This composition can be used advantageously in a method for stimulating the integrity of the intestinal barrier, said method comprising the administration of said composition to a mammal.

Polyunsaturated fatty acids

Surprisingly, the present inventors found that eicosapentaenoic acid (EPA, n-3), docosahexaenoic acid (ADH, n-3) and arachidonic acid (AA, n-6) effectively reduce the permeability of the intestinal tight junction. Therefore, the present composition, which is especially suitable for improving the integrity of the intestinal barrier, comprises EPA, ADH and AA.

The present inventors found that the lower concentration of AGPCL was effective in reducing tight junction permeability (see examples vs. Usami et al). Therefore, the content of C-20 and 22-C-AGPCL in the present composition, preferably does not exceed 15% by weight of the total fat content, preferably does not exceed 10% by weight, even more preferably does not exceed 5 % by weight of the total fat content. Preferably, the present composition comprises at least 0.1% by weight, preferably at least 0.25% by weight, more preferably at least 0.5% by weight, even more preferably at least 0.75% by weight of AGPCL with 20 and 22 carbon atoms of the total fat content. For the same reason, the EPA content preferably does not exceed 1% by weight, but is preferably at least 0.05% by weight, more preferably at least 01% by weight of the total fat. The content of ADH preferably does not exceed 5% by weight, more preferably does not exceed 1% by weight, but is at least 0.1% by weight of the total fat. Since AA was found to be especially effective in reducing tight junction permeability, the present composition comprises relatively high amounts, preferably at least 0.1% by weight, even more preferably at least 0.25% by weight, most preferably at least 0.5% by weight of the total fat. The AA content preferably does not exceed 5% by weight, more preferably does not exceed 1% by weight of the total fat. In the present enteral composition containing AA, EPA and ADH are advantageously added to balance the action of AA, eg, reduce the potential pro-inflammatory action of AA metabolites. Excess metabolites of AA can cause inflammation. Therefore, the present composition preferably comprises AA, EPA and ADH, where the AA / ADH weight ratio is preferably above 0.25, preferably above 0.5, even more preferably above 1. The ratio it is preferably below 25. The AA / AEP weight ratio is preferably between 1 and 100, more preferably between 5 and 20.

The present composition preferably comprises between 5 and 75% by weight of polyunsaturated fatty acids based on total fat, preferably between 10 and 50% by weight.

If the present composition is used as an infant formula (for example, a method of feeding a child, said method comprising administering the present composition to a child), the content of AGPCL, in particular AGPCL with 20 and 22 atoms carbon, preferably not more than 3% by weight of the total fat content, as is desirable to mimic human milk as closely as possible. For the same reason, the omega-3 SFFA content preferably does not exceed 1% by weight of the total fat content; the omega-6 AGPCL content preferably does not exceed 2% by weight of the total fat content; the AA (omega6) content is preferably below 1% by weight of the total fat content; and / or the AEP / ADH weight ratio is preferably 1 or less, more preferably below 0.5.

ES 2 285 484 T3

The C 20 and 22 PCFA can be provided as free fatty acids, in triglyceride form, in phospholipid form, or as a mixture of one of more of the above. The present composition preferably comprises at least one of AA and ADH in phospholipid form.

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% by weight of n-9 fatty acid based on the weight of total fatty acids, more preferably at least 25% by weight. The n-9 fatty acid content is preferably below 80% by weight.

Oligosaccharides

Suitable oligosaccharides according to the invention are saccharides that have a degree of polymerization (GP) of at least 2 monose units, which are not or are only partially digested in the intestine by the action of digestive acids or enzymes present in the upper digestive tract human (small intestine and stomach), but which are fermentable by human intestinal flora. The term "monose units" refers to units having a closed ring structure, preferably hexose, for example, the pyranose or furanose forms. The degree of polymerization of the oligosaccharide is usually below 60 monose units, preferably below 40, even more preferably below 20.

The present composition comprises at least two different oligosaccharides, where the oligosaccharides have a homology in monose units below about 90%, preferably below 50%, even more preferably below 25%, even more preferably below 5%. The term "homology" as used in the present invention is the cumulative percentage of the same monose units in the different oligosaccharides. For example, oligosaccharide 1 (OL1) has the structure fruc-fruct-glu-gal, and therefore comprises 50% fruc; gal at 25% and glu at 25%. Oligosaccharide 2 (OL2) has the structure fruc-fruc-glu, and therefore comprises 66% fruc, 33% glu. The different different oligosaccharides therefore have 75% homology (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 oligosaccharides present preferably comprises at least 66%, more preferably at least 90% of monose units selected from the group consisting of mannose, arabinose, fructose, fucose, rhamnose, galactose, jd -D-galactopyranose, ribose, glucose, xylose, uronic acid and derivatives thereof, calculated on the total number of monose units contained therein.

According to another embodiment, at least one of the oligosaccharides of the present composition is selected from the group consisting of fructans, fructooligosaccharides, galactooligosaccharides of indigestible dextrins (including transgalactooligosaccharides), xylooligosaccharides, mannooligosaccharides, glucosaccharides, , fucooligosaccharides, acidic oligosaccharides (see below, for example, uronic acid oligosaccharides such as pectin hydrolyzate) 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, galactooligosaccharides and pectin hydrolyzate.

For good quantity and quality of mucus, the present composition preferably comprises at least one oligosaccharide, which comprises at least 66% galactose or fructose as monose unit. In a preferred embodiment, the composition comprises at least one oligosaccharide comprising at least 66% galactose as monose unit and at least one oligosaccharide comprising at least 66% fructose as monose unit. In an especially preferred embodiment, the present composition comprises galactooligosaccharides and an oligosaccharide selected from the group consisting of fructooligosaccharides and inulin. Fructooligosaccharides stimulate sulfomucine production in the distal colon of rats associated with human flora (Kleessen et al, (2003) Brit J Nutr 89: 597-606) and galactooligosaccharides stimulate acid mucin production (Meslin et al, Brit J.Nutr (1993), 69: 903-912)).

To further improve the thickness of the mucus layer over the entire colon area, at least 10% by weight of the oligosaccharides in the present composition have a GP of 2 to 5 (i.e. 2, 3, 4 and / or 5) and at least 5% by weight has a GP of 10 to 60. Preferably at least 50% by weight, more preferably at least 75% by weight of the oligosaccharides have a GP of 2 to 9 (i.e. 2, 3,4, 5, 6, 7, 8; and / or 9 ), because it is believed that they work throughout the ileum and proximal and middle parts of the colon and because the percentage by weight of oligosaccharides that needs to be incorporated into the composition to achieve the desired effect is reduced.

Preferably the weight ratios:

to. (oligosaccharides with GP 2 to 5): (oligosaccharides with GP 6, 7, 8 and / or 9)> 1; Y

b. (oligosaccharides with GP 10 to 60): (oligosaccharides with GP 6, 7, 8 and / or 9)> 1 are both above 1.

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

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To further improve the thickness and quality of the mucus layer throughout the colon area, preferably each of the at least two different oligosaccharides are provided in different chain lengths, preferably at least 10% by weight of each oligosaccharide based on the total weight of the respective oligosaccharide it has a GP of 2 to 5 (ie 2, 3, 4 and / or 5) and at least 5% by weight has a GP between 10 and 60. Preferably at least 50% by weight, more preferably at least 75% by weight of the oligosaccharide based on the total weight of those oligosaccharides has a GP between 2 and 10, because these are believed to work throughout the ileum and proximal parts and colon stockings. Acidic oligosaccharides

To further enhance the integrity of the barrier, the present composition preferably includes acidic oligosaccharides with a GP between 2 and 60. The term acidic oligosaccharide refers to oligosaccharides comprising at least one acidic group selected from the group consisting of N-acetylneuraminic acid. , N-glycolloylneuraminic acid, free or esterified carboxylic acid, sulfuric acid group and phosphoric acid group. The acidic oligosaccharide preferably comprises uronic acid units (ie, uronic acid polymer), more preferably galacturonic acid units. The acidic oligosaccharide can be a homogeneous or heterogeneous carbohydrate. Suitable examples are pectin and / or alginate hydrolysates. In the intestinal tract, uronic acid polymers are hydrolyzed to uronic acid monomers, which stimulate intestinal acetate production, which in turn stimulates intestinal mucus secretion (Barcelo et al., Gut 2000; 46: 218 -224).

Preferably, the acidic oligosaccharide has structure I below, where the terminal hexose (left) preferably comprises a double bond. Hexose units other than the terminal hexose unit (s) are preferably uronic acid units, even more preferably galacturonic acid units. The carboxylic acid groups on these units can be free or (partially) esterified, and preferably at least 10% is methylated (see below).

Structure I

Polymeric acid oligosaccharide

<img file="ES2285484T3_D0001.tif" />

where:

R is preferably selected from the group consisting of the group hydrogen, hydroxy or acid, preferably hydroxy; and at least one selected from the group consisting of R2, R3, R4 and R5 represents the N-acetylneuraminic acid, the N-glycolyneuraminic acid, the free or esterified carboxylic acid, the sulfuric acid group and the phosphoric acid group, and the rest of R2, R3, R4 and R5 representing hydroxy and / or hydrogen. Preferably, one selected from the group consisting of R2, R3, R4 and R5 represents N-acetylneuraminic acid, N-glycolloylneuraminic acid, free or esterified carboxylic acid, sulfuric acid group or phosphoric acid group, and the remainder representing hydroxy and / or hydrogen.

Even more preferably, one selected from the group consisting of R2, R3, R4 and R5 represents free or esterified carboxylic acid and the remainder 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 Polymerization, below), which can be any 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 represents hydrogen, R5 represents carboxylic acid, n is any number between 1 and 250, preferably between 1 and 10, and the hexose unit is galacturonic acid.

The detection, measurement and analysis of the preferred acidic oligosaccharides as used in the present method are given in applicants' earlier patent application relating to acidic oligosaccharides, ie WO 0/160378.

For enhancing the stimulation of the mucus layer thickness over the entire colon area, the present composition preferably comprises at least 10% by weight of acidic oligosaccharides with a GP of 2 to 5 (i.e. 2, 3, 4 and / or

ES 2 285 484 T3

5) and at least 5% by weight of acidic oligosaccharides with a GP between 10 and 60, said% by weight being based on the total weight of the oligosaccharides.

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

Oligosaccharide content

When in ready-to-deliver liquid form, the present composition preferably comprises 0.1 to 100 grams of indigestible oligosaccharide per liter, more preferably between 0.5 and 50 grams per liter even more preferably between 1 and 25 grams per liter. Too high an oligosaccharide content can cause discomfort due to over fermentation, while too low a content can result in insufficient mucus layer.

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

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, more preferably exceeding 0.5% by weight, and even more preferably it exceeds 1% by weight based on the total dry weight of the composition. The present composition preferably has an oligosaccharide content below 20% by weight, more preferably below 10% by weight, even more preferably below 5% by weight.

The addition of nucleotides and / or nucleosides to the present invention further improves the function of the mucosal barrier of the intestine, especially since it inhibits and / or reduces the incidence of bacterial translocation and decreases intestinal injury. Therefore, the present composition also preferably comprises between 1 and 500 mg. of nucleosides and / or nucleotides per 100 grams of the dry formula, even more preferably between 5 and 100 mg.

App

The present composition can be advantageously used in a method to improve the integrity of the barrier in mammals, in particular humans. The present composition may also be advantageously used in a method for the treatment or prevention of diseases associated with reduced barrier integrity, said method comprising administration to a mammal of the present composition. The present composition is preferably administered orally.

For the sick and children, the present composition is preferably combined with complete nutrition, including protein, carbohydrate and fat. The present composition is advantageously administered to children with the age between 0 and 2 years. The composition can be administered to patients suffering from impaired barrier integrity and to healthy patients. The present composition is advantageously used in a method for providing the nutritional requirements of a premature infant (a child born before 37 weeks of gestation).

The present composition can also be used advantageously in a method for the treatment and / or prevention of intestinal damage by administering the present composition before or after a medical treatment that may cause intestinal damage. Such medical treatment may for example be surgical or enteral medicine treatment (eg antibiotic, analgesic, NSAIDs, chemotherapeutic agents, etc.).

The present composition can also be advantageously used to treat or prevent diseases where the breakdown of the intestinal barrier underlies the development of the disease course, for example, in a method for the treatment or prevention of chronic inflammatory diseases, in particular inflammatory disease bowel disease (IBD), irritable bowel syndrome (IBS), celiac disease, pancreatitis, hepatitis, arthritis, or diabetes. Also, the invention can be used in a method for providing nutrition to patients who have undergone or are undergoing abdominal surgery and patients experiencing postoperative bowel dysfunction and / or malnourished patients.

In another embodiment of the invention, the present composition is advantageously administered to patients suffering from acquired immunodeficiency syndrome (AIDS) and / or patients who are infected with the human immunodeficiency virus (HIV), for example, in a method for the treatment of AIDS and / or HIV infection. Said method comprises the oral administration of the present composition, preferably combined with nutrients selected from the group consisting of carbohydrate, protein and fat.

Furthermore, the invention can also be used to treat or prevent complications resulting from reduced barrier integrity, in particular in a method for the treatment and / or prevention of diarrhea, in particular diarrhea in children. Due to the reduced incidence of diarrhea in children, the present composition can also be advantageously used to reduce diaper chafing.

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Administration of the present composition reduces the passage of dietary and microbial antigens, in particular food allergens, from the intestinal lumen into the mucosal or systemic circulation, and therefore can be advantageously used in a method for the treatment or prevention of allergy and / or allergic reaction, in particular in a method for the treatment or prevention of food allergy, for example, allergic reaction resulting from ingestion of food product.

It was also discovered by the present inventors that EPA, ADH and / or AA are capable of reducing the effects of IL-4 on intestinal permeability. Therefore, one aspect of the present invention provides a method for the treatment and / or prevention of diseases where the concentration of intestinal IL-4 is increased (for example, allergic diseases), said method comprising the administration of a preferably selected AGPCL from the group consisting of EPA, ADH and AA, preferably combined with the selected oligosaccharides present. Therefore, the present composition can also be advantageously used in a method for the treatment of atopic dermatitis.

Since the newborn barrier function has not been fully developed, the present composition can advantageously be administered to young children, that is, children with the age between 0 and 6 months. The composition can be administered to the infant in the form of an infant formula without human milk or mixed with human milk. Therefore, the present invention also provides a formula feed comprising human milk and the present composition. Compositions including human milk and the present composition are especially suitable for feeding premature infants.

The present composition is preferably intended as a packaged powder or ready-to-supply packaged formula. To prevent deterioration of the product, the package size of the ready-to-supply formula preferably does not exceed one serving, for example, preferably does not exceed 500 ml; and the packaging size of the composition present in powder form preferably does not exceed 250 portions. Suitable pack sizes for the powder are 2,000 grams or less, preferably per 1,000 grams or less.

Packaged products provided with labels that explicitly or implicitly direct the consumer towards the use of said product in accordance with one or more of the above or below objectives are encompassed by the present invention. Such labels may for example refer to the present method of preventing allergic reaction to food allergens by including expressions such as "reduced food sensitivity", "improved intestinal tolerability", "improved food tolerance" or the like. Similarly, the present method of treating and / or preventing allergy can be referred to by incorporating terminology equivalent to "improved resistance" or "reduced sensitivity."

Formulas

It was found that the present composition can be advantageously applied in food, such as baby food and clinical food. Such food preferably comprises lipid, protein and carbohydrate and is preferably administered in liquid form. The term "liquid food" as used in the present invention includes dry food (eg powders), which are accompanied by instructions for mixing said dry food mixture with a suitable liquid (eg water).

Therefore, the present invention also relates to a nutritional composition that preferably comprises between 5 and 50% in lipid, between 5 and 50% in protein, between 15 and 90% in carbohydrate and the present combination of oligosaccharides and AGPCL. Preferably, the present nutritional composition contains between 10 and 30% in lipid, between 7.5 and 40% in protein and between 25 and 75% in carbohydrate (% in is the abbreviation for the percentage of energy and represents the relative amount, each constituent contributes to the total caloric value of the preparation).

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

The proteins used in the nutritional preparation are preferably selected from the group of non-human animal proteins (such as dairy 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. The nitrogen source derived from cow's milk, in particular cow's milk protein proteins such as casein and whey proteins are especially preferred.

A digestible carbohydrate source can be added to the nutritional formula. Preferably, it provides about 40% to about 80% of the energy of the nutritional composition. Any suitable carbohydrate (source) can be used, for example, sucrose, lactose, glucose, fructose, corn syrup solids, and maltodextrins, and mixtures thereof.

The present composition is preferably used as infant formula and preferably contains 7.5 to 12.5% ​​protein energy; 40 to 55% energy from carbohydrates; and 35 to 50% energy from fat. Since the present composition is suitably used to reduce the allergic reaction in a child, the protein of the infant formula is preferably selected from the group consisting of hydrolyzed milk protein (for example,

ES 2 285 484 T3 hydrolyzed casein or hydrolyzed whey protein), vegetable protein and / or amino acids. The use of these proteins further reduced the child's allergic reactions.

Bowel irregularities (eg, hard bowel movements, insufficient stool volume, diarrhea) are a major problem in many infants and sick individuals who are given liquid foods. It was found that deposition problems can be reduced by administering the oligosaccharides present in liquid food having an osmolality between 50 and 500 mOsm / kg, more preferably between 100 and 400 mOsm / kg.

In view of the above, it is also important that the liquid food does not have an excessive caloric density, yet it still provides enough calories to feed the individual. Therefore, the liquid food preferably has a caloric density between 0.1 and 2.5 kcal / ml, even more preferably a caloric density between 0'S and 1.5 kcal / ml, most preferably between 0.6 and 0.8 kcal / ml.

Examples

Example 1

Effect of AGPCL on barrier integrity

Monolayers (MC) of T84 intestinal epithelial cell lines (American Type Culture Collection, American Type Culture Collection (ATTC), Manassas, USA) were cultured on transwell filters (Corning, Costar BV, The Netherlands) allowing both mucosal and serous sampling. as the stimulation of human intestinal epithelial cells. Two weeks after confluence, the monolayers were incubated in the luminal compartment with polyunsaturated fatty acids AA (arachidonic acid; 5, 8, 11, 14-eicosatetraenoic acid), ADH (cis-4, 7, 10, 13, 16 acid , 19 docosahexaenoic), EPA (eicosapentaenoic acid) or control (palm) palmitic acid (C 16: 0) (Sigma, St. Louis, USA). The latter procedure was chosen to mimic the route of in vivo administration of the dietary compounds. Cells were incubated with AA, ADH, EPA, or palmitic acid for 0.24, 48 and 72 hours at different concentrations (10 pM and 100 pM). Experiments were performed to assess the integrity of the basal barrier. The function of the epithelial barrier was determined by measuring the transepithelial resistance (TEN, Ω cm²) was measured by epithelial volt-ohm meter (EVOM; World Precision Instruments, Germany), and the permeability for dextran-FITC of 4 kDa (marker of paracellular permeability, Sigma, USA). Resistance (epithelial permeability for dextran-4 kDa FITC was determined as follows. Prior to the dextran flows, the medium was refreshed with culture medium without phenol red for one hour, followed by the addition of 5 µl (100 mg / ml stock) of 4 kDa dextran-FITC to the lumenal compartment. After 30 minutes of incubation, 100 µl of sample was collected from the serous compartment and the fluorescent signal was measured at excitation wavelength of 485 nm and emission wavelength 520 nm (FLUOstar Galaxy^®, BMG Labtechnologies, USA). The dextran-FITC fluxes were calculated as pmol dextran-FITC / cm²/ h. Statistical analyzes were performed using ANOVA (SPSS version 10).

Table 1 gives the results of the effect of fatty acids (100 pM) on the integrity of the spontaneous barrier after 72 hours of incubation. Table 1 shows that AGPCL AA, AEP and ADH reduce molecular flux and improve epithelial resistance. On the contrary, control experiments show that palmitic acid has the opposite effects, that is, it compromises the integrity of the barrier. These results are indicative for the advantageous use of EPA, ADH and AA, and in particular AA in the composition according to the present invention and for use in a method according to the present invention, for example in a method to improve the integrity of the barrier. This result further supports the synergistic effects of the present combination of indigestible oligosaccharides and fatty acids.

Figure 1 shows the time and dose dependent effects (10 pM and 100 pM) of various fatty acids (palmitic acid, ADH, FFA, and AA) on basal barrier integrity (RTE). Figure 1 shows that AGPCL AA, ADH, and AGL enhance the integrity of the epithelial barrier as reflected by increased resistance (RTE). These results are indicative for the advantageous use of AEP, ADH, AGL and AA, in particular AA, in the composition according to the present invention and for use in a method according to the present invention, that is, in a method to improve the integrity of the barrier. These results further support the synergistic effects of the present combination of indigestible oligosaccharides and fatty acids.

TABLE 1

<td>Ingredient (AGPCL)</td><td>Flow</td><td>Resistance (RTE)</td>

<td>Control</td><td> 79</td><td> 1.090</td>

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

<td>ADH</td><td> 72</td><td> 1.574</td>

<td>AA</td><td> 28</td><td> 1.816</td>

<td>AEP</td><td> 65</td><td> 1.493</td>

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Example 2

Effect of AGPCL on IL-4-mediated barrier breakdown

Monolayers (MC) of T84 intestinal epithelial cell lines (ATCC, USA) were cultured on transwell filters (Corning, Costar bV, The Netherlands) allowing both mucosal and serous sampling and stimulation of human intestinal epithelial cells. Two weeks after confluence, the monolayers were incubated in the presence of IL-4 (2 ng / ml, serous compartment, Sigma, USA) with or without polyunsaturated fatty acids AA, ADH, AGL, EPA or control palmitic acid ( 10 pM or 100 pM, mucosal compartment, Sigma, St. Louis, USA). Cells were preincubated with AA, ADH, EPA, or palmitic acid for 48 hours prior to IL-4 incubation. Co-incubation of AGP and palmitic acid with IL-4 was continued for another 48 hours; while the culture medium and additives were changed every 24 hours. The epithelial barrier function was determined by measuring transepithelial resistance (TEN) and permeability as described in example 1. Statistical evaluation was carried out as described in example 1.

The results of the effect of AA, ADH, EPA and palmitic acid (100 pM) on IL-4 mediated barrier breakdown are given in Table 2. Table 2 shows that AGPCL AA, ADH and EPA inhibit increased flux. caused by IL-4. In contrast, palmitic acid had a detrimental effect and decreased barrier breakdown compared to the control. These results are indicative for the advantageous use of AA, ADH, and EPA in clinical and infant nutrition formulations to prevent or reduce IL-4-mediated barrier breakdown, for example, as occurs in cow's milk allergy. or to food. This result further supports the synergistic effects of the present combination of indigestible oligosaccharides and fatty acids.

Figure 2 gives the time and dose dependent protective effects (10 pM and 100 pM) of various fatty acids (palmitic acid, ADH, FFA and AA) on IL-4 mediated barrier destruction (flux). Figure 2 shows that AA, ADH and AGL protect against IL-4 mediated barrier breakdown as reflected by decreased 4 kD dextran flux. These results are indicative for the advantageous use of AA, ADH and AGL in infant and clinical nutrition formulations to prevent or reduce IL-4 mediated barrier breakdown, for example, as occurs in allergy to milk from cow or food. These results further support the synergistic effects of the present combination of indigestible fatty acids and oligosaccharides.

TABLE 2

<td>Inq network entity (AGPCL)</td><td>IL-4 flow</td><td>IL-4 RTE</td>

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

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

<td>ADH</td><td> 271</td><td> 547</td>

<td>AA</td><td> 218</td><td> 636</td>

<td>AEP</td><td> 228</td><td> 539</td>

Example 3

Effect of oligosaccharides on acetate production

Microorganisms were obtained from fresh feces of bottle-fed infants. Fresh fecal material from infants between 1 and 4 months of age was collected and placed in a preservative medium within 2 hours. As substrate, prebiotics (TOS; mixture of TOS / inulin (HP) in a ratio of 9/1 (w / w); inulin; mixture of oligofructose (OS) linulin in a ratio of 1/1 (w / w), or none (white). Transgalactooligosaccharides (TOS) were obtained from Vivinal GOS, Borculo Domo Ingredients, Zwolle, The Netherlands and comprise as indigestible oligosaccharides: 33% by weight disaccharides, 39% by weight trisaccharides, 18% by weight tetrasaccharides, 7% pentasaccharides by weight and hexa, hepta and octasaccharides at 3% by weight. Orafti Inulin Active Food Ingredients (HP), Have, Belgium i.e Raftiline HP^®, with a mean GP of 23. Media: McBain &amp; MacFarlane medium: 3.0 g / l of buffered peptone water, 2.5 g / l of yeast extract. 0.8 g / l mucin (brush edges), 3.0 g / l tryptone, 0.4 g / l L-cysteine-HCl, 0.05 g / l bile salts, 2 ' 6 g / l K₂HPO₄.3H₂0.2 g / l NaHCO₃, 4.5 g / l NaCl, 0.5 g / l MgSO₄.7H₂0.228 g / l CaCl₂, 0.005 g / l FeSO₄.7H₂O. 500 ml Scott bottles are filled with medium and sterilized for 15 minutes at 121 ° C. Buffered medium: 2.6 g / l of K₂HPO₄.3H₂, 0.2 g / l NaHCO₃, 4.5 g / l NaCl, 0.5 g / l MgSO₄.7H₂0.228 g / l CaC_!2, 0.005 g / l FeSO₄.7H₂O. Adjusts to pH 6.3 ± 0.1 with K₂HPO₄ or NaHCO₃. 500 ml Scott bottles are filled with the medium and sterilized for 15 minutes at 121 ° C. Preservative medium: 20'0 g / l of buffered peptone, 0.5 g / l of L-cysteine-HCl, 0.5 g / l of sodium thioglycollate, 1 tablet of

ES 2 285 484 T3 resazurin per liter, is adjusted to pH 6.7 ± 0.1 with 1 M NaOH or HCl. It is boiled in the microwave. The serum bottles were filled with 25 ml of medium and sterilized for 15 minutes at 121 ° C.

Fresh fecal 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 was removed and the stool was mixed with McBain &amp; MacFarlane medium in a 1: 5 weight ratio. Of this fecal suspension, 3 ml were combined with 85 mg of glucose or prebiotic or without addition (blank) in a bottle and were mixed well. A sample t = 0 (0.5 ml) was withdrawn. 2.5 ml of the resulting suspension is taken to a dialysis tube in a 60 ml bottle filled with 60 ml of the buffered medium. The bottle was tightly closed and incubated at 37 ° C. Samples were taken from the dialysis tube (02 ml) or dialysis buffer (1.0 ml) with a hypodermic syringe after 3, 24, and 48 hours and were immediately placed on ice to stop fermentation. The experiment was carried out using the following samples:

1) 85 mg TOS

2) 85 mg of inulin

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

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

SCFA (acetate, propionate, butyrate) were quantified using a Varian 3800 (GC) gas chromatograph (Varian Inc., Walnut Creek, USA) equipped with a flame ionization detector. 0.5 μl of the sample was injected at 80 ° C into the column (Stabilwax, 15 x 0.53 mm, film thickness 1.00 pm, Restek Co., USA) using helium as carrier gas (3.0 lb / in²). After injection of the sample, the oven was heated to 160 ° C at a rate of 16 ° C / min, followed by heating to 220 ° C at a rate of 20 ° C / min and finally maintained at 220 ° C. for 1.5 minutes. The injector and detector temperature was 200 ° C. 2-Ethylbityric acid was used as an internal standard.

Figure 3 represents the absolute (Figure 3A) and relative (Figure 3B) SCFA profile resulting from fermenting the different oligosaccharides. Figure 3A shows that a mixture of two different oligosaccharides (TOS / inulin), where the two different oligosaccharides have a homology in monose units below 90 and a different chain length results in a significantly and synergistically increased amount of SCFA ( in particular, acetate) per gram of fiber than simple components. Figure 3B shows that the addition of a combination of TOS / inulin favored a higher proportion of the beneficial acetate (B). In vivo acetate production results in improved mucus production by goblet cells and a measure for the thickness of the intestinal mucus layer (see Example 4). These results are indicative for the advantageous use of the present composition.

Example 4

Effects of AGCC on mucus production

Intestinal epithelial T84 cell monolayers (ATCC, USA) were grown in 24 or 96 well tissue culture plates (Corning BV). T84 were incubated with the short chain fatty acids acetate, proprionate and butyrate, (CCFA, Merck, USA) for 24 hours in a concentration range of 0Ό25-4Ό mM. The supernatants and / or cells were collected and the expression of MUC-2 (mucin) was determined. A dotblot technique was used to determine MUC-2 expression in cell cultures, since mucins are extremely large glycoproteins (above 500 a), which makes them difficult to handle in western blot techniques. The method was validated using preimmune serum (negative stained T84), CCD18Co negative control cells (ATCC, USA) and bovine serum albumin (ASB). Cell samples were collected in Laemmli (protein isolation buffer) and protein determination was carried out using a microprotein assay (Biorad, USA) according to the manufacturers protocol. Samples (0.3-0.7-1'0 pg / 2 pl) were spotted on nitrocellulose membranes (Schleicher &amp; Schuell, Germany). The membranes were blocked in TBST / Protivar 5% (Nutricia, The Netherlands) followed by a 1 hour incubation with anti MUC-2 antibody (kindly donated by Dr. Einerhand, Erasmus University, Rotterdam, The Netherlands). After washing, spots were incubated with goat anti-rabbit HRP (Santacruz Biotechnology, USA) and ECL (Roche Diagnostics, The Netherlands) was used for substrate detection. Densitometry was performed using the Lumi-Imager (Boehringer Mannheim BV, The Netherlands) and the signal was expressed in illumination units (BLU, backlight unit). BLUs were also expressed relative to control incubations (% BLU). To compare the stimulatory effect of SCFA on MUC-2 expression, basal levels of MUC-2 expression were deduced. Figure 4 shows the differential effects of SCFA (acetate, proprionate, butyrate) on MUC-2 expression in intestinal epithelial cells (MC T84) and mesenchymal epithelial cell cocultures (CC T84). Figure 2 also shows that acetate is more potent in stimulating MUC-2 expression (mucus production) than compared to propionate and butyrate. Therefore, the present combination of oligosaccharides (which was shown to increase acetate production (see example 3)) is particularly useful in stimulating mucus production and can be used to advantage in a method to promote completeness. of the barrier.

ES 2 285 484 T3

Example 5

Children's milk formula

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; vitamin B. sub. 1 (thiamine) 1,000 mcg; vitamin B. sub. 2 (riboflavin) 1,500 mcg; vitamin B. sub. 6 (pyridoxine) 600 mcg; Vitamin B. sub 12 (cyanocobalamin) 2.0 mcg; niacin 9.0 mcg; folic acid 80 mcg; 3,000 mcg pantothenic acid; biotin 90 mcg; vitamin C (ascorbic acid) 90 mcg; choline 100 mg; inositol 33 mg; calcium 460 Mg; 333 Mg phosphorus; magnesium 64 Mg; iron 8.0 Mg; zinc 6.0 Mg; manganese 50 mcg; copper 560 mcg; 100 mcg iodine; sodium 160 mg; potassium 650 mg; chloride 433 mg and selenium 14 mcg; where the fat content includes 3 grams of fish oil and 3 grams of 40% arachidonic acid oil (DSM Food Specialties, Delft, The Netherlands); further comprising 4 grams of Elix'or ™ transgalactooligosaccharides (Borculo Domo Ingredients, The Netherlands) and 4 grams of Raftiline ™ (Orafti Active Food Ingredients, Belgium).