JP5502817B2 - Improving barrier integrity - Google Patents

Description

  The present invention relates to methods for improving intestinal barrier integrity and compositions suitable for use in such methods.

  The gastrointestinal epithelium normally functions as a selective barrier that allows absorption of nutrients, electrolytes, and water and prevents exposure to dietary and microbial antigens, including food allergens. The gastrointestinal epithelium restricts the passage of antigens that can cause inflammatory reactions, such as allergic reactions, into the systemic circulation. Due to the increasing incidence of allergies, especially dietary allergies, many research groups are looking for (preventive) treatments for these diseases.

EP1272058 describes a composition comprising indigestible oligosaccharides for improving tight junctions to reduce intestinal permeability and for reducing allergic reactions. The composition may comprise LC-PUFA's (long chain-polyunsaturated fatty acids).
EP 745001 describes indigestible oligosaccharides and combinations of n-3 and n-6 fatty acids for the treatment of ulcerative colitis.

  Usami et al. Describe the effect of eicosapentaenoic acid (EPA) on tight junction permeability in intestinal monolayers (Clinical Nutrition 2001, 20 (4): 351-359). Their studies have shown that EPA increases permeability, indicating that EPA is not suitable for improving intestinal barrier integrity.

  Prior art formulations are not optimal for improving barrier integrity.

  The present invention provides combinations of specific long chain polyunsaturated fatty acids (LC-PUFA's) and specific oligosaccharides. The LC-PUFA's and oligosaccharide combination synergistically improves barrier integrity and improves barrier integrity in human infants by improving intestinal permeability and mucus production. Especially suitable.

  It is a surprising discovery that certain LC-PUFA's effectively reduce epithelial paracellular permeability. In contrast to the report by Usami et al. (Clinical Nutrition 2001, 20 (4): 351-359), we have identified C18 and C20 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA), docosahexaenoic acid. It has been discovered that (DHA) and arachidonic acid (ARA) can effectively reduce the permeability of intestinal tight junctions.

  In addition to LC-PUFAs, the composition includes oligosaccharides. Certain oligosaccharides improve the integrity of the barrier by stimulating the production of mucus and consequently increasing the thickness of the mucus layer. This effect is believed to be caused by the effect of different oligosaccharides in the production of short chain fatty acids (SCFA). Therefore, when administered enterally to mammals, the inventive combination of LC-PUFA and indigestible oligosaccharides synergistically acts as a barrier by reducing the permeability of concomitant tight junctions and stimulating mucosal production. Improves integrity and / or synergistically reduces intestinal permeability.

  In a further aspect, the composition improves the quality of the intestinal mucosal layer. The mucosal layer contains mucin. 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 the integrity of the barrier. The mucosal layer contains different types of mucins, for example, acidic, neutral and sulfonated mucins. Increased mucosal layer heterogeneity is believed to improve barrier integrity.

  The composition preferably comprises at least two different oligosaccharides, which affect the structure of the mucosa, either directly or by altering the intestinal flora, and are advantageously mucin-free in the mucin layer. Affects uniformity. Each different specific oligosaccharide is believed to have different effects on the quantity and quality of the mucosa. Moreover, the two different oligosaccharides can also stimulate mucosal quality as reflected in the degree of sulfation through the synergistic stimulation of SFCA generation. It is surprising that the inventors have discovered that a mixture of two different oligosaccharides according to the present invention synergistically stimulates acetate formation. It was also discovered by the present inventors that mucosal formation depends on acetate formation.

  The composition is preferably further improved by providing both long and short oligosaccharides. The provision of different chain lengths results in stimulation of mucosal production in different parts of the ileum and large intestine. Short-chain oligosaccharides (typically with a degree of polymerization (DP) of 2, 3, 4 or 5) stimulate mucin production in the proximal and / or distal ileum, while (preferably greater than 5) Long-chain oligosaccharides (with a degree of polymerization (DP) up to 60) are thought to stimulate mucin production at more distal sites in the large intestine.

  Even further improvements can be achieved by providing at least two different oligosaccharides as both short and long oligosaccharides. All of these preferred embodiments contribute to further improvement of barrier integrity throughout the ileum and / or large intestine.

  Furthermore, it has surprisingly been found that EPA, DHA, and ARA can reduce the deleterious effects of interleukin 4 (IL-4) on intestinal permeability. IL-4 is a cytokine that is secreted in large amounts by the mucosal T cells of certain patients and causes intestinal permeability. Therefore, the present invention also provides a method for the treatment and / or prevention of diseases with increased intestinal IL-4 levels, such as allergies, particularly atopic dermatitis.

FIG. 1 is a graph showing the time and concentration (10 μM and 100 μM) dependent effects of various fatty acids (palmitic acid, DHA, GLA and AA) on basal barrier integrity (TER). FIG. 1 is a graph showing the time and concentration (10 μM and 100 μM) dependent effects of various fatty acids (palmitic acid, DHA, GLA and AA) on basal barrier integrity (TER). FIG. 2 is a graph showing the time and concentration (10 μM and 100 μM) dependent protection effects of various fatty acids (palmitic acid, DHA, GLA and ARA) on IL-4 mediated barrier breakage (flux). is there. FIG. 2 is a graph showing the time and concentration (10 μM and 100 μM) dependent protection effects of various fatty acids (palmitic acid, DHA, GLA and ARA) on IL-4 mediated barrier breakage (flux). is there. FIG. 3 is a graph showing absolute (FIG. 3A) and relative (FIG. 3B) SCFA profiles from different oligosaccharide fermentations. FIG. 3 is a graph showing absolute (FIG. 3A) and relative (FIG. 3B) SCFA profiles from different oligosaccharide fermentations. FIG. 4 is a graph showing the different effects of SCFA (acetate, propionate, butyrate) on MUC-2 expression in intestinal epithelial cells (MC T84) and epithelial-mesenchymal tissue cell co-culture (CC T84). It is.

Detailed Description of the Invention

The present invention is a nutritional composition comprising:
including EPA, DHA and ARA such that the content of long chain polyunsaturated fatty acids having 20 and 22 carbon atoms does not exceed 15% by weight of the total fat content; and
b. relates to a composition comprising at least two different oligosaccharides having a homology of less than 90% in monosaccharide units.

  The composition can be advantageously used in a method for stimulating intestinal barrier integrity, comprising administering the composition to a mammal.

[Polyunsaturated fatty acid]
The inventors surprisingly found that eicosapentaenoic acid (EPA, n-3), docosahexaenoic acid (DHA, n-3), and arachidonic acid (ARA, n-6) provide intestinal tight junction permeability. Found to effectively reduce. The composition is therefore particularly suitable for improving the integrity of the intestinal barrier and comprises EPA, DHA and ARA.

  The inventors have found that low concentrations of LC-PUFA's are effective in reducing tight junction permeability (see Examples and Usami et al. Results). Therefore, the content of LC-PUFA having 20 and 22 carbon atoms in the composition preferably does not exceed 15% by weight of the total fat content, preferably does not exceed 10% by weight, more preferably the total fat content. Does not exceed 5% by weight. Preferably, the composition comprises LC-PUFA having 20 and 22 carbon atoms, at least 0.1%, preferably at least 0.25%, more preferably at least 0.5%, even more preferably at least 0.75% by weight of total fat. Included in%. For the same reason, the EPA content preferably does not exceed 5% by weight of the total fat content, more preferably does not exceed 1% by weight, but preferably it is at least 0.05% by weight of the total fat content, more preferably At least 0.1% by weight. The DHA content is preferably not more than 5% by weight of the total fat content, more preferably not more than 1% by weight, but at least 0.1% by weight. Since ARA has been found to be particularly effective in reducing tight junction permeability, the composition comprises a relatively high amount, preferably 0.1% by weight of total fat, more preferably at least 0.25% by weight. Most preferably at least 0.5% by weight. The ARA content preferably does not exceed 5% by weight of the total fat content, more preferably does not exceed 1% by weight. In enteral compositions containing ARA, EPA and DHA are conveniently added to balance the effects of ARA, for example, to reduce the potential pro-inflammatory effects of ARA metabolism. Excess metabolites from ARA can cause inflammation. Therefore, the present composition preferably comprises ARA, EPA and DHA, the weight ratio of ARA / DHA is preferably greater than 0.25, preferably greater than 0.5, and even more preferably greater than 1. The ratio is preferably less than 25. The weight ratio of ARA / EPA is preferably between 1 and 100, more preferably between 5 and 20.

  The composition preferably comprises from 5 to 75% by weight of polyunsaturated fatty acids, preferably from 10 to 50% by weight, based on the total fat content.

  When the composition is used as an infant formula (eg, a method for feeding an infant, the method comprising administering the composition to the infant), particularly LC-PUFA, The content of LC-PUFA having 20 and 22 carbon atoms is preferably not more than 3% by weight of the total fat content, as it is desired to resemble human milk as much as possible. For similar reasons, the omega-3 LC-PUFA content preferably does not exceed 1% by weight of the total fat content; the omega-6 LC-PUFA content preferably does not exceed 2% by weight of the total fat content; The (omega-6) content is preferably less than 1% by weight of the total fat content; and / or the weight ratio of EPA / DHA is preferably less than 1 and more preferably less than 0.5.

LC-PUFAs having 20 and 22 carbon atoms may be provided as free fatty acids in the form of triglycerides, in the form of phospholipids, or as a mixture of one or more of the above forms. The composition preferably comprises at least one ARA and DHA in the form of phospholipids.
The nutritional composition also preferably provides omega-9 (n-9) fatty acids (preferably oleic acid, 18: 1) to provide sufficient nutrition. Preferably, the composition provides at least 15 wt% n-9 fatty acids, more preferably at least 25 wt%, based on the weight of total fatty acids. The content of n-9 fatty acids is preferably less than 80% by weight.

[oligosaccharide]
Suitable oligosaccharides according to the invention have a degree of polymerization of at least two monosaccharide units and are not digested or partially digested in the intestine by the action of acids or digestive enzymes present in the upper digestive tract (small intestine and stomach) of humans. It is a sugar that can only be digested but can be fermented by human intestinal flora. The term monosaccharide unit means a unit having a closed ring structure, preferably a hexose such as pyranose or furanose form. The degree of polymerization of oligosaccharides is typically less than 60 monosaccharide units, preferably less than 40 monosaccharide units, and more preferably less than 20 monosaccharide units.

  The composition comprises at least two different oligosaccharides, the oligosaccharides having a homology of less than about 90% in monosaccharide units, preferably less than 50%, more preferably less than 25%, Preferably it has less than 5% homology. The term “homology” as used in the present invention is the cumulative percentage of the same monosaccharide unit in different oligosaccharides. For example, oligosaccharide 1 (OL1) has the structure of fructose-fructose-glucose-galactose and thus contains 50% fructose, 25% galactose and 25% glucose. Oligosaccharide 2 (OL2) has the structure of fructose-fructose-glucose and therefore contains 66% fructose and 33% glucose. These different oligosaccharides therefore have 75% homology (50% fructose + 25% glucose).

  In a preferred embodiment, the composition comprises galactooligosaccharide and at least one selected from the group consisting of fructooligosaccharide and inulin.

  Each oligosaccharide is preferably at least 66%, more preferably at least 90% mannose, arabinose, fructose, fucose, rhamnose, galactose, β-D, calculated by the total number of monosaccharide units contained therein. -Containing monosaccharide units selected from the group consisting of galactopyranose, ribose, glucose, xylose, uronic acid and derivatives thereof.

  According to a further embodiment, the at least one oligosaccharide of the composition comprises fructans, fructooligosaccharides, indigestible dextrin galactooligosaccharides (including transgalactooligosaccharides), xylo-oligosaccharides, arabino-oligosaccharides, gluco-oligosaccharides, manno-oligosaccharides , Fuco-oligosaccharides, acidic oligosaccharides (see below, eg uronic oligosaccharides such as pectin hydrolysates) and mixtures thereof. Preferably, the composition comprises at least one, preferably at least two, oligosaccharides selected from the group consisting of fructooligosaccharides or inulin, galactooligosaccharides and pectin hydrolysates.

  For good mucosal quantity and quality, the composition preferably comprises at least one oligosaccharide comprising at least 66% galactose or fructose as monosaccharide units. In a preferred embodiment, the composition comprises at least one oligosaccharide comprising at least 66% galactose as a monosaccharide unit and at least one oligosaccharide comprising at least 66% fructose as a monosaccharide unit. In particularly preferred embodiments, the composition comprises a galactooligosaccharide and an oligosaccharide selected from the group consisting of fructooligosaccharide and inulin. Fructooligosaccharides stimulate sulfomucin production in the proximal colon of human flora-related rats (Kleessen et al, (2003) Brit J Nutr 89: 597-606), and galactooligosaccharides stimulate acidic mucin production (Meslin et al, Brit. J. Nutr (1993), 69: 903-912).

  In order to improve the thickness of the mucosal layer over the entire area of the large intestine, at least 10% by weight of the oligosaccharides in the composition has a DP of 2 to 5 (ie 2, 3, 4, and / or 5); At least 5% by weight has a DP of 10 to 60. Preferably at least 50% by weight, more preferably at least 75% by weight of the oligosaccharide has a DP of 2 to 9 (ie 2, 3, 4, 5, 6, 7, 8, and / or 9). Is believed to function through the ileum and the proximal and intermediate large intestines, and because the weight percentage of oligosaccharides that need to be incorporated into the composition to achieve the desired effect is reduced. It is.

Preferred weight ratio:
(oligosaccharides with DP of 2 to 5): (oligosaccharides with DP of 6, 7, 8 and / or 9)>1; and
b. (Oligosaccharide with DP of 10 to 60): (Oligosaccharide with DP of 6, 7, 8 and / or 9)> 1
Are both greater than 1.

  Preferably, both weight ratios are greater than 2, more preferably greater than 5.

  For further improvement of the thickness and quality of the mucosal layer over the entire area of the large intestine, preferably each of the at least two different oligosaccharides is provided with a different chain length, preferably the total weight of each oligosaccharide And at least 10% by weight of each oligosaccharide has 2 to 5 (ie 2, 3, 4, and / or 5) DP and at least 5% by weight has 10 to 60 DP, based on . Based on the total weight of the oligosaccharide, preferably at least 50% by weight, more preferably at least 75% by weight of the oligosaccharide has a DP between 2 and 10, which includes the ileum and proximal and intermediate This is because it is considered to function through the large intestine.

[Acid oligosaccharide]
In order to further improve the integrity of the barrier, the composition preferably comprises an acidic oligosaccharide having a DP of 2 to 60. The term acidic oligosaccharide refers to an oligo that contains at least one acidic group selected from the group consisting of N-acetylneuraminic acid, N-glycoylneuraminic acid, free or ester carboxylic acid, sulfate group and phosphate group. Means sugar. The acidic oligosaccharide preferably comprises uronic acid units (ie uronic acid polymer), more preferably galacturonic acid units. Acidic oligosaccharides can be uniform or heterogeneous carbohydrates. Suitable examples are pectin and / or alginate hydrolysates. In the gastrointestinal tract, uronic acid polymers are hydrolyzed into uronic acid monomers, which stimulate intestinal acetate formation and then intestinal mucosal secretion (Barcelo et al., Gut 2000; 46 : 218-224).

  Preferably, the acidic oligosaccharide has the following structure I, and this terminal hexose (left) preferably has a double bond. The hexose units other than the terminal hexose units are preferably uronic acid units, and more preferably galacturonic acid units. The carboxylic acid groups in these units may be free or (partially) esterified, preferably at least 10% methylated (see below).

Structure I: Polymerized acidic oligosaccharide

put it here:
R is preferably selected from the group consisting of hydrogen, a hydroxyl group or an acidic group, preferably a hydroxyl group; and
At least one selected from the group consisting of R ₂ , R ₃ , R ₄ and R ₅ is N-acetylneuraminic acid, N-glycoloylneuraminic acid, free or ester carboxylic acid, sulfate group and phosphoric acid The remainder of R ₂ , R ₃ , R ₄ and R ₅ represents a hydroxyl group and / or hydrogen. Preferably, one selected from the group consisting of R ₂ , R ₃ , R ₄ and R ₅ is N-acetylneuraminic acid, N-glycoloylneuraminic acid, free or ester carboxylic acid, sulfate group and Represents a phosphate group, and the remainder represents a hydroxyl group and / or hydrogen. More preferably, one selected from the group consisting of R _2, R _3, R ₄ and R ₅ represents free or esterified carboxylic acid and the remaining R _2, R _3, R ₄ and R ₅ are hydroxyl And / or represents hydrogen; and
n is an integer and represents the number of hexose units (see also degree of polymerization, see below), which may be any hexose unit. A suitable n is an integer from 1 to 5000. Preferably, the hexose unit is a uronic acid unit.

Most preferably R ₁ , R ₂ and R ₃ represent hydroxy, R ₄ represents hydrogen, R ₅ represents a carboxylic acid, n is any number between 1 and 250, preferably 1 to 10 Number, the hexose unit is galacturonic acid.

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

  In order to stimulate an improvement in the thickness of the mucosal layer over the entire area of the large intestine, the composition is preferably at least 10% by weight of 2 to 5 (ie 2, 3, 4 and / or 5) acidic oligosaccharides having a DP and at least 5% by weight acidic oligosaccharides having a DP of 10 to 60.

  The acidic oligosaccharide used in the present invention is preferably made from pectin, pectate, alginate, chondroitin, hyaluronic acid, heparin, heparane, bacterial carbohydrate, sialoglican, fucoidan, fucooligosaccharide or carrageenan, More preferably it is made from pectin and / or alginate.

[Oligosaccharide content]
In the form of a ready-to-take liquid, the composition preferably comprises 0.1 to 100 grams of indigestible oligosaccharides per liter, more preferably 0.5 to 50 grams per liter, and more preferably per liter. Contains 1 to 25 grams. An oligosaccharide content that is too high may cause discomfort due to excessive fermentation, while a very low content may result in an insufficient mucosal layer.

  The weight ratio of the at least two different oligosaccharides is preferably between 1 and 10, more preferably between 1 and 5. This weight ratio optimally stimulates different types of mucin production at different sites in the intestine.

  Oligosaccharides are preferably included in the composition according to the invention in an amount of more than 0.1% by weight, preferably more than 0.2% by weight, more preferably, based on the total dry weight of the composition. The amount is more than 0.5% by weight, more preferably more than 1% by weight. The composition preferably has an oligosaccharide content of less than 20% by weight, more preferably less than 10% by weight, even more preferably less than 5% by weight.

  The addition of nucleotides and / or nucleosides to the composition further improves the function of the intestinal mucosal barrier, in particular, inhibits and / or reduces the incidence of bacterial metastasis and reduces intestinal damage. Therefore, the composition also preferably comprises 1 to 500 mg, more preferably 5 to 100 mg of nucleosides and / or nucleotides per 100 grams of dry formulation.

[application]
The composition can be advantageously used in a method for improving barrier integrity in mammals, particularly humans. The composition can also be advantageously used in a method for the treatment or prevention of a disease associated with reduced barrier integrity, the method comprising administering the composition to a mammal. The composition is preferably administered orally.

  For illness and infancy, the composition is preferably combined with complete nutrition, including protein, carbohydrates and fat. The composition is conveniently administered to infants from 0 to 2 years of age. The composition may be administered to patients with impaired barrier integrity and healthy patients. The composition is advantageously used in a method for providing nutritional requirements for preterm infants (infants born before 37 weeks of gestation).

  The composition may also cause intestinal damage in a method for treating and / or preventing intestinal damage by administering the composition to a patient prior to or after medical treatment. Can be used conveniently. Such medical treatment may be, for example, surgery or enteral medicine treatment (eg, antibiotics, analgesics, NSAIDs, chemotherapeutics, etc.).

  The composition can also advantageously be used for the treatment or prevention of diseases where intestinal barrier breakage is underlying the development of the course of the disease, e.g. chronic inflammatory diseases, in particular inflammatory bowel diseases ( may be used in methods for the treatment or prevention of inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), childhood celiac disease, pancreatitis, hepatitis, arthritis or diabetes it can. Moreover, the present invention provides nutrition to patients who have or have undergone abdominal surgery and to patients experiencing post-intestinal dysfunction and / or to malnourished patients Can be used in the process.

  In a further embodiment of the invention, the composition is conveniently applied to a patient suffering from acquired immune deficiency syndrome (AIDS), for example, in a method for the treatment of AIDS and / or HIV infection, and / or Administered to patients infected with human immunodeficiency virus (HIV). Such a method comprises oral administration of the composition, preferably in combination with a nutrition selected from the group consisting of carbohydrates, proteins and fats.

  Moreover, the present invention can also be used to treat or prevent complications due to reduced barrier integrity, and in particular, a method for the treatment and / or prevention of diarrhea, especially infancy diarrhea. Can be used. Due to the reduced incidence of diarrhea in infancy, the composition can be advantageously used to reduce diaper rash.

  Administering the composition reduces the passage of dietary and microbial antigens, particularly food allergens, from the intestinal lumen into the mucosal or systemic circulation and is therefore preferably allergic and / or allergic reaction It can be advantageously used in a method for treatment or prevention, in particular it can be advantageously used in a method for the treatment or prevention of food allergies, for example allergic reactions caused by food intake.

  The inventors have also discovered that EPA, DHA and / or ARA can reduce 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 (eg, allergic diseases) in which intestinal IL-4 levels are increased, preferably comprising EPA, DHA and It comprises administering LC-PUFA selected from the group consisting of ARA, preferably in combination with certain specific oligosaccharides. Therefore, the present composition can also be advantageously used in a method for the treatment of atopic dermatitis.

  Since the neonatal barrier function is not fully developed, the composition can be conveniently administered to young infants, ie infants 0 to 6 months old. The composition may 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 milk diet comprising human milk and the composition. Compositions comprising human milk and the present composition are particularly suitable for feeding premature infants.

  The composition is preferably provided as a packaged powder or a packaged ready-to-use formulation. To prevent product degradation, the ready-to-take formulation packaging size does not exceed one serving, for example 500 ml; the packaging size according to the invention in powder form is 250 servings Not exceed. Suitable packaging sizes for powders are 2000 grams or less, preferably 1000 grams or less.

  Packaged products that are labeled explicitly or implicitly to the consumer for use of the product in accordance with one or more of the above or below objectives are encompassed by the present invention. Such a label inhibits allergic reactions to food allergens, for example by including “reduced food sensitivity”, “improves intestinal tolerance”, “improved food tolerance” or similar terms Reference may be made to the present method. Similarly, reference to the present method for the treatment and / or prevention of allergies may be made by incorporating a term equivalent to “improved resistance” or “reduced sensitivity”.

[Formulated milk]
The composition can be advantageously applied to meals, such as infant foods and clinical foods. Such a meal preferably comprises lipids, proteins and carbohydrates and is preferably administered in liquid form. As used herein, the term “liquid meal” includes a dry meal (eg, flour), accompanied by instructions indicating that the dry meal mixture is mixed with a suitable liquid (eg, water). .

  Therefore, the present invention also preferably relates to a nutritional composition comprising 5 to 50 en% lipid, 5 to 50 en% protein, 15 to 90 en% carbohydrate and this combination of oligosaccharides and LC-PUFA's. Preferably, the nutritional composition preferably comprises 10 to 30 en% lipid, 7.5 to 40 en% protein and 25 to 75 en% carbohydrate (en% is an abbreviation for percent energy and the individual ingredients are Represents the relative amount contributing to the total caloric value of the formulation).

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

  Proteins used in nutritional formulations are preferably non-human animal proteins (milk protein, meat protein and egg protein), vegetable proteins (soy protein, wheat protein, rice protein, and pea protein), free Selected from the group of amino acids and mixtures thereof. Especially preferred are milk proteins and whey proteins such as milk from nitrogen sources, especially casein.

  Digestible carbohydrate ingredients may be added to the nutritional formula. It preferably provides about 40% to about 80% of the energy of the nutritional composition. Any suitable carbohydrate may be used, such as sucrose, lactose, glucose, fructose, corn syrup solids, and maltodextrins, and mixtures thereof.

  The composition is preferably used as an infant formula and preferably comprises 7.5 to 12.5 energy% protein; 40 to 55 energy% carbohydrates; and 35 to 50 energy% fat. Since the composition is suitably used for reducing allergic reactions in infants, the protein of infant formula is preferably hydrolyzed milk protein (eg hydrolyzed casein or hydrolyzed Whey protein), vegetable protein and / or amino acid. The use of these proteins further reduced allergic reactions in infants.

  Stool irregularities (eg, hard stool, insufficient stool volume, diarrhea) are a major problem for many infants and sick patients who receive a liquid diet. It has been found that stool problems may be reduced by administration of the oligosaccharides with a liquid diet having an osmolality of 50 to 500 mOsm / kg, more preferably 100 to 400 mOsm / kg.

  In view of the above, it is also important that the liquid diet does not have excessive caloric density, but still provides enough calories to give the patient. For this reason, the liquid food preferably has a caloric density of 0.1 to 2.5 kcal / ml, more preferably 0.5 to 1.5 kcal / ml, most preferably 0.6 to 0.8 kcal / ml.

[Example 1]: Effect of LC-PUFA on barrier integrity A monolayer (MC) of the intestinal epithelial cell line T84 (American Type Culture Collection (ATTC), Manassas, USA) was transformed into transwell filters ( Corning, Costar BV, the Netherlands) to enable both mucosal and serosal sampling and stimulation of human intestinal epithelial cells. Two weeks after becoming confluent, the monolayer was separated into 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 (C16: 0) acid (Palm) (Sigma, St. Louis, USA). The latter procedure was chosen to mimic the in vivo route of administration of dietary compounds. Cells were cultured with ARA, DHA, EPA, or palmitic acid at different concentrations (10 μM and 100 μM) for 0, 24, 48 and 72 hours. Experiments were performed to assess basal barrier integrity. Epithelial barrier function was determined by measuring transepithelial resistance (TER, Ω.cm ² ) and permeability of 4 kD FITC dextran (paracellular permeability marker, Sigma, USA). The transepithelial resistance was measured with an epithelial volt-ohm meter (EVOM; World Precision Instruments, Germany). The epithelial permeability of 4kD FITC dextran was determined as follows. Prior to dextran flux, the medium was replaced with culture medium without phenol for 1 hour followed by the addition of 5 μl (stock 100 mg / ml) of 4 kDa FITC-dextran to the luminal fraction. After 30 minutes of incubation, a 100 μl sample was collected from the serosa fraction and the fluorescence signal was measured at an excitation wavelength of 485 nm and an emission wavelength of 520 nm (FLUOstar Galaxy®, BMG Labtechnologies, USA). FITC-dextran flux was calculated as pmol FITC-dextran / cm ² / h. Statistical analysis was performed using ANOVA (SPSS version 10).

  The results of the effect of fatty acids (100 μl) on spontaneous barrier integrity after 72 hours of culture are shown in Table 1. Table 1 shows that LC-PUFA's ARA, EPA and DHA reduce molecular flux and improve epithelial resistance. In contrast, control experiments show that palmitic acid has the opposite effect, ie impairs the barrier integrity. These results show the advantageous use of EPA, DHA and ARA, in particular ARA, in the composition according to the invention and indicate their use in the method according to the invention, for example for improving the integrity of the barrier. . These results further support the synergistic effect of this combination of fatty acids and indigestible oligosaccharides.

FIG. 1 shows the time and concentration (10 μM and 100 μM) dependent effects of various fatty acids (palmitic acid, DHA, GLA and AA) on basal barrier integrity (TER). FIG. 1 shows that LC-PUFA's AA, DHA, and GLA improve epithelial barrier integrity, as reflected in increased resistance (TER). These results show the advantageous use of EPA, DHA, GLA and ARA, in particular ARA, in the composition according to the invention, and the use in the method according to the invention, i.e. for improving the integrity of the barrier. Show. These results further support the synergistic effect of this combination of fatty acids and indigestible oligosaccharides.

[Example 2]: Effect of LC-PUFA on IL-4 mediated barrier breakage Monolayer (MC) of intestinal epithelial cell line T84 (ATTC, USA) was transformed into transwell filters (Corning , Costar BV, the Netherlands) to enable both mucosal and serosal sampling and stimulation of human intestinal epithelial cells. Two weeks after becoming confluent, the monolayer is subjected to polyunsaturated fatty acids ARA, DHA, GLA, EPA or control in the presence of IL-4 (2 ng / ml, serosal fraction, Sigma, USA) Cultured with or without palmitic acid (10 μM or 100 μM, mucosal fraction, Sigma, St. Louis, USA). Cells were precultured in the presence of ARA, DHA, EPA or palmitic acid for 48 hours prior to IL-4 culture. Co-culture of IL-4 with PUFA's and palmitic acid was continued for an additional 48 hours; while culture medium and additives were changed every 24 hours. The function of the epithelial barrier 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 of the effects of ARA, DHA, EPA and palmitic acid (100 μM) on IL-4 mediated barrier failure are shown in Table 2. Table 2 shows that LC-PUFA's ARA, DHA and EPA inhibit the increase in flux caused by IL-4. In contrast, palmitic acid had a detrimental effect and reduced barrier breakage compared to control. These results show the advantageous use of ARA, DHA and EPA in clinical and infant nutritional formulations to prevent or reduce IL-4 mediated barrier breaks such as food or milk allergies ing. These results further support the synergistic effect of this combination of fatty acids and indigestible oligosaccharides.

FIG. 2 shows the time and concentration (10 μM and 100 μM) dependent protection effects of various fatty acids (palmitic acid, DHA, GLA and ARA) on IL-4 mediated barrier breakage (flux). . FIG. 2 shows that ARA, DHA and GLA protect against IL-4 mediated barrier failure, as reflected in the decrease in 4 kD dextran flux. These results show the advantageous use of ARA, DHA and GLA in clinical and infant nutritional formulations to prevent or reduce IL-4 mediated barrier breaks such as food or milk allergies ing. These results further support the synergistic effect of this combination of fatty acids and indigestible oligosaccharides.

Example 3: Effect of oligosaccharides on acetate production Microorganisms were obtained from fresh stool of infants raised in milk (artificial nutrition). Fresh stool material taken from infants between 1 and 4 months of age was pooled and placed in storage medium for 2 hours. Prebiotics (TOS; ratio of TOS / inulin (HP) mixture 9/1 (w / w); inulin; ratio of oligofructose (OS) / inulin mixture 1/1 (w / w)) as substrate Or nothing was used (blank). Transgalactooligosaccharides (TOS) were obtained from Vivinal GOS, Borculo Domo Ingredients (Zwolle, The Netherlands) as indigestible oligosaccharides: 33% disaccharides, 39% trisaccharides, 18% tetrasaccharides 7% by weight pentasaccharide and 3% by weight hexa-, 7-, octasaccharide. Inulin (HP) is obtained as Raftiline HP® from Orafti active food ingredients (Tien, Belgium) with an average DP of 23. Medium: McBain & MacFarlane Medium: buffered peptone water 3.0 g / l, yeast extract 2.5 g / l, mucin (brush borders) 0.8 g / l, tryptone 3.0 g / l, L-cysteine-HCl 0.4 g / l, Bile salt 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 0.228 g / l, FeSO4.7H2O 0.005 g / l. Fill a 500 ml Scott bottle with medium and sterilize at 121 ° C for 15 minutes. Buffer medium: 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, FeSO4.7H2O 0.005 g / l. Adjust to pH 6.3 ± 0.1 with K2HPO4 or NaHCO3. Fill a 500 ml Scott bottle with medium and sterilize at 121 ° C for 15 minutes. Storage medium: Buffered peptone 20.0 g / l, L-cysteine-HCl 0.5 g / l, sodium thioglycolate 0.5 g / l, 1 resazurin tablet per liter. Adjust to pH 6.7 ± 0.1 with 1M NaOH or HCl. Boil in a microwave. Serum bottles were filled with 25 ml of medium and sterilized at 121 ° C. for 15 minutes.

  Fresh stool samples were mixed with storage medium and stored at 4 ° C. for several hours. The fecal stock solution was centrifuged at 13,000 rpm for 15 minutes, the supernatant was removed, and the feces were mixed with McBain & MacFarlane medium at a weight ratio of 1: 5. 3 ml of this stool suspension was mixed well in a bottle with 85 mg glucose or prebiotic or nothing added (blank). A sample at t = 0 was removed (0.5 ml). 2.5 ml of the remaining suspension was transferred to a dialysis tube and placed in a 60 ml bottle filled with 60 ml of buffer medium. The bottle was closed tightly and incubated at 37 ° C. Samples were taken after 3, 24, and 48 hours from dialysis tubes (0.2 ml) or dialysis buffer (1.0 ml) using hypodermic syringes and immediately placed on ice to stop fermentation. This experiment was performed using the following samples.

1) 85mg TOS
2) 85mg inulin
3) Ratio of 85mg TOS / Inulin 9/1 (w / w), and
4) Ratio of 85mg OS / inulin 1/1 (w / w).

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

  FIG. 3 shows absolute (FIG. 3A) and relative (FIG. 3B) SCFA profiles from different oligosaccharide fermentations. FIG. 3A shows that a mixture of two different oligosaccharides (TOS / inulin) with homology at monosaccharide units of less than 90 and different chain lengths (TOS / inulin) is more SCFA per gram of fiber (especially than single component) It represents a significant and synergistic increase in the amount of acetate). FIG. 3B shows that the addition of the TOS / inulin combination results in a higher ratio of beneficial acetate (B). In vivo acetate production translates to improved mucosal production by goblet cells and the extent of intestinal mucosal layer thickness (see Example 4). These results show an advantageous use of the composition.

[Example 4]: Effect of SCFA on mucosal production Monolayer intestinal epithelial T84 cells (ATCC, USA) were cultured in 24- or 96-well tissue culture plates (Corning BV). T84 was cultured for 24 hours with short chain fatty acid acetate, propionate and butyrate in a concentration range of 0.025-4.0 mM. The supernatant and / or cells were collected and MUC-2 (mucin) expression was detected. Since mucin is a very large glycoprotein (greater than 500 kDa) and difficult to handle by Western blotting technology, MUC-2 expression in cell culture was detected by dot blot technology. This method was confirmed using preimmune serum (T84 negatively stained), CCD-18Co (ATCC, USA) negative control cells and bovine serum albumin (BSA). Cell samples were collected with Laemmli (protein isolation buffer) and protein detection was performed using a microprotein assay (Biorad, USA) according to the instructions. Samples (0.3-0.7-1.0 μg / 2 μl) were doted onto a nitrocellulose membrane (Schleicher & Schuell, Germany). Membranes were blocked with TBST / 5% Protivar (Nutricia, The Netherlands) followed by 1 hour incubation with anti-MUC-2 antibody (kindly courtesy of Dr. Einerhand, Erasmus University, The Netherlands). After washing, the blot was incubated with goat anti-rabbit-HRP (Santacruz Biotechnology, USA) and ECL (Roche Diagnostics, The Netherlands) was used for substrate detection. Concentration measurements were performed using a Lumi-Imager (Boehringer Mannheim BV, The Netherlands) and signals were expressed in light units (BLU). BLU's were also expressed relative to control incubations (% BLU). To compare the effects of SCFA stimulation on MUC-2 expression, the basic MUC-2 expression values were subtracted.

  FIG. 4 shows the different effects of SCFA (acetate, propionate, butyrate) on MUC-2 expression in intestinal epithelial cells (MC T84) and epithelial-mesenchymal cell co-culture (CC T84). Yes. FIG. 2 also shows that acetate stimulates MUC-2 expression (mucosal production) more strongly than propionate, butyrate. Therefore, this combination of oligosaccharides (showing enhanced acetate production (see Example 3)) is particularly useful for stimulating mucosal production and can be advantageously used in methods for stimulating barrier integrity .

[Example 5]: Infant formula I
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) 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; vitamin C (ascorbic acid) 90 mg; choline 100 mg; inositol 33 mg; calcium 460 Mg; Phosphorous acid 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 ion 433 mg and Selenium 14 mcg; Fat here Content is 3 grams of fish oil and 40% arachi 3 grams of acid (DSM Food Specialties, Delft, The Netherlands); 4 grams of transgalactooligosaccharide Elix'or ™ (Borculo Domo Ingredients, Netherlands) and 4 grams of Raftiline ™ (Orafti Active Food Ingredients, Belgium) )including.

Claims (7)

Use of polyunsaturated fatty acids to produce a composition for use in a method for improving intestinal barrier integrity by reducing tight junction permeability and simultaneously stimulating mucosal formation. The method comprises administering to a mammal a composition containing a and b below:
a. Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (ARA); where the content of long chain polyunsaturated fatty acids having 20 and 22 carbon atoms is 15% by weight of the total fat content Not exceeding; and
b. Fructans selected from the group consisting of galactooligosaccharides and fructooligosaccharides, inulin and mixtures thereof. A nutritional composition comprising:
including EPA, DHA and ARA such that the content of long chain polyunsaturated fatty acids having 20 and 22 carbon atoms does not exceed 15% by weight of the total fat content; and
b. A composition comprising galactooligosaccharides and fructans selected from the group consisting of fructooligosaccharides, inulin and mixtures thereof.   3. The composition of claim 2, wherein at least 10% by weight galactooligosaccharide and fructan have a degree of polymerization (DP) of 2 to 5 and at least 5% by weight have a DP between 10 and 60. Composition. 4. A composition according to any one of claims 2 or 3 , comprising 7.5 to 12.5 energy% protein; 40 to 55 energy% carbohydrate; and 35 to 50 energy% fat. A composition comprising a member selected from the group consisting of hydrolyzed milk protein, vegetable protein and / or amino acid. 5. A composition according to any one of claims 2 to 4 , wherein the composition has a caloric content of 0.6 to 0.8 kcal / ml; a osmolality of 50 to 500 mOsm / kg; and A composition having a viscosity of less than 50 mPas. 6. A composition according to any one of claims 2 to 5 , which is suitable for feeding an infant:
a. the content of long-chain polyunsaturated fatty acids is less than 3% by weight of the total fat content;
b. Omega-3 long chain polyunsaturated fatty acid is less than 1% by weight of total fat content;
c. Omega-6 long chain polyunsaturated fatty acid is less than 2% by weight of total fat content;
d. the ARA content is less than 1% by weight of the total fat content; and
e. A composition having an EPA / DHA ratio of 1 or less. 7. A composition according to any one of claims 2 to 6 for use as a medicament.

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