CN101693103A

CN101693103A - Improvement of completeness of intestinal barrier

Info

Publication number: CN101693103A
Application number: CN200910209324A
Authority: CN
Inventors: E·A·F·文托; L·E·M·威廉姆森; M·A·克齐尔; C·比尔曼; B·斯坦尔
Original assignee: Nutricia NV
Current assignee: Nutricia NV
Priority date: 2004-06-22
Filing date: 2004-06-22
Publication date: 2010-04-14
Anticipated expiration: 2024-06-22
Also published as: CN101693103B

Abstract

The invention relates to an improvement of completeness of an intestinal barrier, in particular to a method for exciting the barrier completeness of mammalian by applying a composition containing eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (ARA) and at least two different oligosaccharides to the mammalians.

Description

The improvement of completeness of intestinal barrier

The application is that application number is 200480043417.0, and the applying date is on June 22nd, 2004, and denomination of invention is divided an application for the Chinese invention patent application of " improvement of completeness of intestinal barrier ".

Technical field

The compositions that the present invention relates to improve the method for completeness of intestinal barrier and be applicable to the method.

Background technology

Gastrointestinal epithelial serves as the selectivity barrier usually---and it allows to absorb nutrient, electrolyte and water and prevent to be exposed to diet and microbial antigen, comprises food allergen.The gastrointestinal epithelial limited antigen enters in the body circulation, and the latter can cause inflammatory reaction, for example anaphylaxis.Because irritated, particularly the incidence rate of food anaphylaxis grows with each passing day, (preventative) treatment of these diseases is all being sought by many research institutions.

EP1272058 has described and has been used to improve tight connection to reduce the intestinal permeability and to reduce the anaphylactoid compositions that comprises non-fermentable dietary fibers.Said composition can comprise LC-PUFA ' s (long-chain-poly-unsaturated fatty acid).

EP 745001 has described non-fermentable dietary fibers and n-3 and n-6 fatty acid in conjunction with being used for the treatment of ulcerative colitis.

(Clinical Nutrition 2001,20 (4): 351-359) described eicosapentaenoic acid (EPA) to closely connecting infiltrative influence in the intestinal cell monolayer for people such as Usami.In their research, EPA it is found that and improved permeability, this means that EPA is not suitable for improving completeness of intestinal barrier.

The prior art preparation is not to be suitable for improving barrier integrity most.

Summary of the invention

The combination of the oligosaccharide that the invention provides the poly-unsaturated fatty acid of selected long-chain (LC-PUFA ' s) and select.The combination of LC-PUFA ' s of the present invention and oligosaccharide improves intestinal permeability and mucus generation and has effectively improved barrier integrity by collaborative, and is particularly suitable for improving the barrier integrity of human infant.

Be surprised to find that selected LC-PUFA ' s effectively reduces the other permeability of epithelial cell.With people (Clinical Nutrition 2001 such as Usami, 20 (4): that 351-359) has discussed is opposite, the inventor finds, C18 and C20 gather unsaturated fatty acid, particularly eicosapentaenoic acid (EPA), docosahexenoic acid (DHA) and arachidonic acid (ARA) can effectively be reduced intestinal and closely be connected permeability.

Except LC-PUFAs, this compositions also comprises oligosaccharide.Selected oligosaccharide has improved barrier integrity by stimulating mucus to generate, and this has improved rete malpighii thickness.This effect is considered to be caused by the influence that different oligosaccharide generate short-chain fatty acid (SCFA).Therefore, when being administered to mammal through intestinal and going up, the combination of LC-PUFA of the present invention and non-fermentable dietary fibers closely is connected permeability and stimulates mucus to generate by reducing simultaneously, works in coordination with to have improved barrier integrity and/or work in coordination with and reduced the intestinal permeability.

Aspect further, this compositions has been improved the quality of casing slime layer.Rete malpighii comprises mucin.Mucin is the high molecular glycoprotein by the synthetic justacrine of goblet cell.They form the gel layer on mucomembranous surface, improve barrier integrity thus.Rete malpighii comprises dissimilar mucin, for example acid, neutrality and sulfonation mucin.The heterogeneity of the raising of rete malpighii is considered to improve barrier function.

This compositions preferably comprises at least two kinds of different oligosaccharide, and they directly or by changing intestinal flora influence the mucin heterogeneity that the mucosa structure also advantageously influences rete malpighii.Every kind of different selected oligosaccharide is considered to that mucus amount and quality are had Different Effects.In addition, two kinds of different oligosaccharide can also promote the mucus quality that embodies as state of cure (vulcanization) by they collaborative generations that stimulates SCFA.The inventor is surprised to find that the mixture of two kinds of different oligosaccharide of the present invention is worked in coordination with has stimulated the acetate generation.The inventor finds that also mucus generates and depends on that acetate generates.

Preferably by providing long-chain and short chain oligosaccharide further to improve this compositions.Provide different chain length to cause stimulating mucus to generate at the different parts of ileum and colon.Short chain oligosaccharide (having 2,3,4 or 5 the degree of polymerization (DP) usually) stimulates mucus to generate in proximal colonic and/or far-end ileum place, and the oligosaccharide that has than long-chain long (preferably having the degree of polymerization (DP) greater than 5 to 60) is considered to stimulate mucus to generate at the more distal site of colon.

By at least two kinds of different oligosaccharide are provided---short chain and long-chain oligosaccharide, can realize further improving.These preferred embodiments all help further to improve the barrier integrity in whole ileum and/or the colon.

In addition, be surprised to find that EPA, DHA and ARA can reduce interleukin-4 (IL-4) to the infiltrative adverse effect of intestinal.IL-4 is excretory and bring out the infiltrative cytokine of intestinal with the amount that improves by mucosa T-cell in some patient's body.Therefore, the present invention also provides the method that improves diseases associated (for example anaphylaxis, particularly atopic dermatitis) with intestinal IL-4 concentration that treats and/or prevents.

Detailed Description Of The Invention

The present invention relates to alimentation composition, it comprises:

A) EPA, DHA and ARA, the content that wherein contains the poly-unsaturated fatty acid of long-chain of 20 and 22 carbon atoms is no more than the 15wt% of total lipid content; With

B) at least two kinds of different oligosaccharide, wherein these two kinds of different oligosaccharide have and are lower than 90% monosaccharide unit homology.

This compositions can be advantageously utilised in the method that excites completeness of intestinal barrier, and described method comprises the described compositions of administration.

Poly-unsaturated fatty acid

The inventor is surprised to find that, eicosapentaenoic acid (EPA, n-3), docosahexenoic acid (DHA, n-3) and arachidonic acid (ARA n-6) effectively reduces intestinal and closely connects permeability.Therefore, this compositions that is particularly suitable for improving completeness of intestinal barrier comprises EPA, DHA and ARA.

The inventor finds that the LC-PUFA ' s of low concentration can effectively reduce tight connection permeability (referring to people such as embodiment vs.Usami).Therefore, the content of LC-PUFA in this compositions that contains 20 and 22 carbon atoms preferably is no more than the 15wt% of total lipid content, preferably is no more than the 10wt% of total lipid content, preferably is no more than 5wt% again.Preferably, compositions of the present invention comprises the 0.1wt% at least that accounts for total lipid content, preferred 0.25wt% at least, more preferably 0.5wt% at least, the preferred again LC-PUFA that contains 20 and 22 carbon atoms of 0.75wt% at least.For the same reason, EPA content preferably is no more than the 5wt% of total fat, more preferably no more than 1wt%, but is preferably the 0.05wt% at least of total fat, more preferably 0.1wt% at least.DHA content preferably is no more than the 5wt% of total fat, more preferably no more than 1wt%, but is at least 0.1wt%.Because ARA it is found that and reduced tight connection permeability especially effectively, this compositions comprises relative higher amount, is preferably the 0.1wt% at least of total fat, preferred 0.25wt% at least again, most preferably 0.5wt% at least.ARA content preferably is no more than the 5wt% of total fat, more preferably no more than 1wt%.Of the present invention contain ARA in the intestinal compositions, advantageously add EPA and DHA effect with balance ARA, for example reduce the metabolic potential short inflammatory effects of ARA.The excess metabolism thing of ARA may cause inflammation.Therefore, this compositions preferably comprises ARA, EPA and DHA, and wherein wt preferably is higher than 0.25 than ARA/DHA, preferably is higher than 0.5, more preferably is higher than 1.This ratio preferably is lower than 25.Weight ratio ARA/EPA is preferably 1 to 100, and more preferably 5 to 20.

This compositions preferably comprise account for total fat 5 to 75wt%, preferred 10 to 50wt% poly-unsaturated fatty acid.

If this compositions is as the infant formula (method of feeding infant for example, described method comprises to this compositions of infant feeding), LU-PUFA, the content that particularly contains the LC-PUFA of 20 and 22 carbon atoms preferably is no more than the 3wt% of total lipid content, because need as far as possible closely to simulate human milk.For the same reason, Ω-3 LC-PUFA content preferably is no more than the 1wt% of total lipid content; Ω-6 LC-PUFA content preferably is no more than the 2wt% of total lipid content; ARA (Ω-6) content preferably is lower than the 1wt% of total lipid content; And/or weight ratio EPA/DHA is preferably 1 or lower, is more preferably less than 0.5.

The LC-PUFA that contains 20 and 22 carbon atoms can be used as free fatty, with the triglyceride form, provide with phospholipid form or as the mixture of one or more above-mentioned forms.This compositions preferably comprises the ARA and the DHA of at least a phospholipid form.

This alimentation composition preferably also provides Ω-9 (n-9) fatty acid (preferred oleic acid, 18: 1), so that enough nutrition to be provided.Preferably, this compositions provides the n-9 fatty acid of the 15wt% at least that accounts for total fatty acids weight, more preferably 25wt% at least.The content of n-9 fatty acid preferably is lower than 80wt%.

Oligosaccharide

Suitable oligosaccharide of the present invention is the saccharide of the degree of polymerization (DP) with at least 2 monosaccharide units, it can or only partly not digest by the acid of existence in the human upper digestive tract (small intestinal stomach function regulating) or the enteral that acts on of digestive enzyme, but it can be fermented by human intestinal flora.The term monosaccharide unit is meant the unit with closed-loop structure, preferred hexose, for example pyranose or furanose form.The degree of polymerization of oligosaccharide is usually less than 60 monosaccharide units, preferably is lower than 40, preferably is lower than 20 again.

This compositions comprises at least two kinds of different oligosaccharide, and wherein oligosaccharide has and is lower than approximately 90%, preferably is lower than 50%, preferably is lower than 25% again, preferably is lower than 5% monosaccharide unit homology again.The term that uses among the present invention " homology " is the percentage ratio accumulation of identical monosaccharide unit in the different oligosaccharide.For example, oligosaccharide 1 (OL1) has structure fructose (fruc)-fructose (fruct)-glucose (glu)-galactose (gal), and comprises 50% fructose thus, 25% galactose and 25% glucose.Oligosaccharide 2 (OL2) has structure fructose-fructose-glucose, and comprises 66% fructose thus, 33% glucose.Therefore different oligosaccharide has 75% homology (50% fructose+25% glucose).

In preferred embodiments, this compositions comprises oligomeric galactose and is selected from least a of oligofructose and inulin.

This oligosaccharide preferably comprises at least 66% separately, and more preferably at least 90% (calculating on the basis of contained therein monosaccharide unit total amount) is selected from the monosaccharide unit of mannose, arabinose, fructose, fucose, rhamnose, galactose, β-D-galactopyranose, ribose, glucose, xylose, alduronic acid and derivant thereof.

According to further embodiment, at least a oligosaccharide of this compositions is selected from levan, oligofructose, difficult digestion dextrin oligomeric galactose (comprising transgalactooligosac,harides), oligomeric xylose, oligomeric arabinose, oligomeric glucose, Oligomeric manna sugar, oligomeric fucose, acidic oligomer sugar and (sees below, for example uronic acid oligosaccharides, for example pectin hydrolysate) and their mixture.Preferably, this compositions comprises at least a, preferred at least two kinds of oligosaccharide that are selected from oligofructose or inulin, oligomeric galactose and pectin hydrolysate.

For good mucus amount and quality, this compositions preferably comprises at least a oligosaccharide that comprises at least 66% galactose or fructose as monosaccharide unit.In preferred embodiments, said composition comprises at least a oligosaccharide and at least a oligosaccharide that comprises at least 66% fructose as monosaccharide unit that comprises at least 66% galactose as monosaccharide unit.In particularly preferred embodiments, this compositions comprises galactooligosacchariwith and the oligosaccharide that is selected from oligofructose and inulin.Oligofructose generates people (2003) Brit J Nutr 89:597-606 such as () Kleessen at the far-end colon moderate stimulation sulfomucin of the relevant rat of human flora, oligomeric galactose has stimulated sour mucin to generate (people such as Meslin, Brit.J.Nutr (1993), 69:903-912).

In order further to improve the thickness of rete malpighii on the whole colon regions, in this compositions at least the oligosaccharide of 10wt% have 2 to 5 DP (promptly 2,3,4 and/or 5), and 5wt% has 10 to 60 DP at least.Preferably, at least 50wt%, more preferably the oligosaccharide of 75wt% has 2 to 9 (promptly at least, 2,

DP

3,4,5,6,7,8 and/or 9), because these are considered to play a role at whole ileum with in the near-end and the middle part of colon, and reduced to realizing that required effect need incorporate the percentage by weight of the oligosaccharide in the compositions into.

Preferred weight ratio:

A. (DP is 2 to 5 oligosaccharide): (DP is 6,7,8 and/or 9 oligosaccharide)＞1; With

B. (DP is 10 to 60 oligosaccharide): (DP is 6,7,8 and/or 9 oligosaccharide)＞1,

Both are all greater than 1.

Preferred two kinds of weight ratios are all greater than 2, more preferably all greater than 5.

In order further to improve rete malpighii thickness and the quality on the whole colon regions, preferably provide at least two kinds of different oligosaccharide with different chain length, based on the gross weight of oligosaccharide separately, the preferred 10wt% at least of every kind of oligosaccharide has 2 to 5 DP (promptly, 2,3,4 and/or 5), and at least 5wt% has 10 to 60 DP.Based on this oligosaccharide gross weight, the preferably 50wt% at least of this oligosaccharide, more preferably 75wt% has 2 to 10 DP at least, because these are considered to play a role at whole ileum with in the near-end and the middle part of colon.

Acidic oligomer sugar

In order further to improve barrier integrity, it is 2 to 60 acidic oligomer sugar that this compositions preferably includes DP.Term acidic oligomer sugar is meant and comprises the oligosaccharide that at least one is selected from the acidic group of N-acetyl neuraminic acid, N-glycolyl neuraminic acid, free or esterification carboxylic acid, sulfate group and phosphate group.Acidic oligomer sugar preferably comprises uronic acid unit (being uronic acid polymer), more preferably galacturonic acid acid unit.Acidic oligomer sugar can be homogenizing or heterogeneous carbohydrate.Suitable example is the hydrolyzate of pectin and/or alginate.In intestinal, uronic acid polymer is hydrolyzed into the alduronic acid monomer, this has stimulated the generation of enteral acetate, its stimulated again the casing slime secretion (people such as Barcelo, Gut 2000; 46:218-224).

Preferably, acidic oligomer sugar has following structure I, and wherein terminal hexose (left side) preferably comprises two keys.Hexose beyond the terminal hexose units is preferably uronic acid unit, more preferably the galacturonic acid acid unit.Hydroxy-acid group on these unit can be for free or (part) esterification, and preferred at least 10% methylate (seeing below).

Structure I: polymerizable acidic oligosaccharide

Wherein:

R is preferably selected from hydrogen, hydroxyl or acidic group, preferred hydroxyl; And

R ₂, R ₃, R ₄And R ₅In at least one represent N-acetyl neuraminic acid, N-glycolyl neuraminic acid, free or esterification carboxylic acid, sulfate group and phosphate group, and R ₂, R ₃, R ₄And R ₅In all the other group representation hydroxy and/or hydrogen.Preferably, R ₂, R ₃, R ₄And R ₅In one represent N-acetyl neuraminic acid, N-glycolyl neuraminic acid, free or esterification carboxylic acid, sulfate group or phosphate group, and all the other group representation hydroxy and/or hydrogen.More preferably, R ₂, R ₃, R ₄And R ₅In a free or esterification carboxylic acid of representative, and R ₂, R ₃, R ₄And R ₅In all the other group representation hydroxy and/or hydrogen; And

N is integer and is meant hexose number (degree of polymerization also vide infra) that it can be any hexose.Suitably, n is the integer of 1-5000.Preferably, hexose is a uronic acid unit.

Most preferably, R ₁, R ₂And R ₃Representation hydroxy, R ₄Represent hydrogen, R ₅Represent carboxylic acid, n is 1 to 250, any number of preferred 1 to 10, and hexose is a galacturonic acid.

Detection, measurement and the analysis of the preferred acidic oligosaccharide that uses in this method provides in the patent application formerly (being WO 0/160378) of applicant about acidic oligomer sugar.

Stimulation for rete malpighii thickness on the whole colon regions improves, this compositions preferably comprise the acidic oligomer sugar of (promptly 2,3,4 and/or 5) that is 2 to 5 of 10wt%DP at least and at least 5wt%DP be 10 to 60 acidic oligomer sugar, described wt% is based on the gross weight of oligosaccharide.

Acidic oligomer sugar used among the present invention is more preferably made by pectin and/or alginate preferably by pectin, pectate, alginate, chrondroitin, hyaluronic acid, heparin, heparin (heparane), antibacterial carbohydrate, sialic saccharide (sialoglycans), fucoidan, oligomeric fucose (fucooligosaccharides) or carrageenin.

Oligosaccharide content

When in instant liquid form, this compositions comprises 0.1 to 100 gram indigestible oligosaccharide for preferred every liter, more preferably every liter 0.5 to 50 gram, preferred more every liter 1 to 25 gram.Too high oligosaccharide content may cause discomfort owing to excessive fermentation, and low-down content may cause the rete malpighii deficiency.

The weight ratio of at least two kinds of different oligosaccharide is preferably 1 to 10, and more preferably 1 to 5.The dissimilar diverse location of mucin in intestinal of the most suitable stimulation of these weight ratios generates.

Preferably comprise the 0.1wt% that surpasses that accounts for the compositions gross dry weight in of the present invention the compositions, preferably surpass 0.2wt%, more preferably surpass 0.5wt%, preferably surpass the oligosaccharide of 1wt% again.This compositions preferably has the 20wt% of being lower than, and more preferably less than 10wt%, preferably is lower than the oligosaccharide content of 5wt% again.

In this compositions, add nucleotide and/or nucleoside and further improved the intestinal mucosal barrier function, particularly because its inhibition and/or reduced the incidence rate of bacterial translocation and reduced damage of intestines.Therefore, also preferred per 100 grams of this compositions are done to fill a prescription and are comprised 1 to 500 milligram of nucleoside and/or nucleotide, more preferably 5 to 100 milligrams.

Use

This compositions can be advantageously utilised in improves mammal, in the method for the interior barrier integrity of particularly human body.This compositions also can be advantageously utilised in the treatment or prevention method with the barrier integrity diseases associated that reduces, and described method comprises this compositions of administration.This compositions preferred oral.

For patient and baby, this compositions preferably combines with complete nutrition (comprising protein, carbohydrate and fat).This compositions is advantageously used in 0 to 2 years old big baby.Said composition can be used for impaired patient of barrier integrity and healthy patients.This compositions is advantageously utilised in the method for the nutritional need that premature infant (baby of birth before the conceived expiration of 37 weeks) is provided.

This compositions also can be advantageously utilised in the method that treats and/or prevents damage of intestines by before or after the medical therapy that may cause damage of intestines the patient being used this compositions.This medical therapy can be, for example, and surgery or through intestinal Drug therapy (for example antibiotic, analgesic, NSAID, chemotherapeutant, or the like).

This compositions also can be advantageously used in treatment or prevent following disease---and its midgut barrier destroys the basis that constitutes this disease process development, for example be used in the method for treatment or prophylaxis of chronic, particularly inflammatory bowel (IBD), irritable bowel syndrome (IBS), celiac disease, pancreatitis, hepatitis, arthritis or diabetes.In addition, the present invention also can be used on to or carry out the patient of abdominal surgery and postoperative bowel dysfunction patient and/or malnutrition patient and provide in the method for nutrition.

In further embodiment of the present invention, this compositions is advantageously used in acquired immune deficiency syndrome (AIDS) (AIDS) patient and/or infects the patient of HIV (human immunodeficiency virus) (HIV), for example, is used in the method for treatment AIDS and/or HIV infection.Described method comprises oral compositions, preferably combines with the nutrient that is selected from carbohydrate, protein and fat.

In addition, the complication that the present invention also can be used for treating or prevents to be caused by the barrier integrity that reduces particularly is used in and treats and/or prevents diarrhoea, particularly in the method for infantile diarrhea.Because the reduction of infantile diarrhea incidence rate, this compositions also can be advantageously used in the reduction diaper rash.

Use this compositions and reduced diet and microbial antigen, particularly food allergen enters mucosa or the body circulation from enteric cavity, and therefore can be advantageously utilised in treatment or Polyglucan disease and/or the anaphylactoid method, particularly be used in treatment or prevention phagopyrism, for example in the anaphylactoid method that causes by food intake.

The inventor finds that also EPA, DHA and/or ARA can reduce IL-4 to the infiltrative influence of intestinal.Therefore, one aspect of the present invention provides the method that treats and/or prevents that improves diseases associated (for example anaphylaxis) with intestinal IL-4 concentration, described method comprises using and is preferably selected from EPA, DHA and ARA, preferably with the bonded LC-PUFA of the selected oligosaccharide of the present invention.Therefore, the present invention also can be advantageously utilised in the method for treatment atopic dermatitis.

Because neonatal barrier function is not completed into as yet, this compositions can be advantageously used in young infant, promptly 0 to 6 months big babies.Said composition can be used for the baby with no human milk or with the blended infant formula form of human milk.Therefore, the present invention also provides the formulations that comprises human milk and this compositions.The compositions that comprises human milk and this compositions is specially adapted to feeding premature babies.

This compositions preferably provides as packing powder or packaged instant prescription.In order to prevent damage of product, the Package size of instant prescription preferably is no more than once gives appetite, for example, preferably is no more than 500 milliliters; The Package size of this compositions of powder type preferably is no more than 250 times and gives appetite.The suitable package of powder is of a size of 2000 grams or lower, is preferably 1000 grams or lower.

Have clearly or implicitly indicate consumer according to one or more above-mentioned or following purpose use the packaging product of the label of described product to comprise in the present invention.This label can for example be quoted the anaphylactoid method of prevention of the present invention to food allergen by the wording or the similar wording that comprise " food sensitivity of reduction ", " improving the intestinal toleration ", " improved food toleration " and so on.Similarly, can quote the hypersensitive method that treats and/or prevents of the present invention by comprising the term suitable with " improved repellence " or " sensitivity of reduction ".

Preparation

This compositions it is found that and can be advantageously used in food, for example in pablum and the clinical food.This food preferably comprises lipid, protein and carbohydrate, and preferably uses with liquid form.Term used herein " supping " comprises dry food (for example powder), described dried foods mixture can be mixed with suitable liquid (for example water) according to description.

Therefore, the invention still further relates to and preferably comprise 5 to 50en% lipids, 5 to 50en% protein, the alimentation composition of the combination of 15 to 90en% carbohydrates and oligosaccharide of the present invention and LC-PUFA ' s.Preferably, this alimentation composition preferably comprises 10 to 30en% lipids, 7.5 to 40en% protein and 25 to 75en% carbohydrates (en% is the abbreviation of energy percentage, represents the Relative Contribution amount of every kind of composition to the goods gross calorific value).

Preferably, use vegetable lipid and at least a combination that is selected from fish oil and Ω-3 plant, algae or antibacterial oil.

Used protein is preferably selected from inhuman animal protein (for example lactoprotein, meat albumen and egg albumen), phytoprotein (for example soybean protein, glutelin, rice protein and Semen Pisi sativi protein), free amino acid and their mixture in this dietetic product.The cow's milk protein of Lac Bovis seu Bubali deutero-nitrogenous source, particularly casein and lactalbumin and so on is preferred especially.

Can in nutrient formulation, add the digestible carbohydrate source.It preferably provides about 40% to about 80% energy of alimentation composition.Can use any suitable carbohydrate (source), for example sucrose, lactose, glucose, fructose, corn-syrup solids and maltodextrin and their mixture.

This compositions is preferably used as infant formula and is also preferably comprised 7.5 to 12.5 energy % protein; 40 to 55 energy % carbohydrates; With 35 to 50 energy % fat.Because this compositions is applicable to the anaphylaxis that reduces the baby, the protein of infant formula is preferably selected from lactoalbumin hydrolysate (for example caseinhydrolysate or lact albumin hydrolysate), vegetable protein and/or aminoacid.These proteinic uses have further reduced baby's anaphylaxis.

Irregular defecation (for example, feces is stiff, just quantity not sufficient, diarrhoea) is the patient's of many babies of puzzlement and edible supping a major issue.Discovery can more preferably be used oligosaccharide of the present invention and alleviate the defecation problem by having 50 to 500mOsm/kg in the supping of 100 to 400mOsm/kg osmolality.

Consider the problems referred to above, important also comprises, supping does not have undue heat density but still the heat that is enough to support object is provided.Therefore, supping preferably has the heat density of 0.1 to 2.5 kcal/ml, more preferably the heat density of 0.5 to 1.5 kcal/ml, the most preferably heat density of 0.6 to 0.8 kcal/ml.

Description of drawings

Fig. 1 has shown time and dosage (10 μ Ms and 100 μ Ms) the dependency effect of various fatty acids (Palmic acid, DHA, GLA and AA) to basic barrier integrity (TER).

Fig. 2 has shown that various FA ' s (Palmic acid, DHA, GLA and ARA) destroy the time and dosage (10 μ M and 100 μ M) the dependency protective effect of (flux) to the barrier of IL-4 mediation.

Fig. 3 described to make different oligosaccharide fermentations and absolute (Fig. 3 A) scheme (Fig. 3 B) with relative SCFA.

Fig. 4 has shown the influence that SCFA (acetate, propionate, butyrate) expresses MUC-2 in enterocyte (MCT84) and epithelium mesenchymal cell coculture (CC T84).

The specific embodiment

Embodiment

Embodiment 1:LC-PUFA is to the influence of barrier integrity

With monolayer (MC) enterocyte is T84 (American Type Culture Collection (ATTC), Manassas, the U.S.) go up cultivation at the transwell filter (Corning, Costar BV, Holland) that can carry out sampling of mucosa and serous coat and stimulating human enterocyte.Converge two weeks of back, these monolayers are used poly-unsaturated fatty acid ARA (arachidonic acid in chamber (luminal compartment); 5,8,11,14-eicosatetraenoic acid), DHA (cis-4,7,10,13,16,19 docosahexenoic acid), EPA (eicosapentaenoic acid) or tester Petiolus Trachycarpi (C 16:0) acid (Palm) (Sigma, St.Louis, U.S.) cultivates.The program of selecting the latter is with route of administration in the body of simulation diet chemical compound.Cell was cultivated 0,24,48 and 72 hour with variable concentrations (10 μ M and 100 μ M) with ARA, DHA, EPA or Palmic acid.Experimentize to evaluate and test basis (basal) barrier integrity.By using epithelium voltage resistance meter (EVOM; World Precision Instruments, Germany) record transepithelial electrical resistance (TER, Ω .cm ²) and the permeability of 4kD FITC glucosan (the other permeability labelling of cell, Sigma, the U.S.), determine the epithelium barrier function.Following mensuration resistance (the epithelium permeability of 4kDa FITC glucosan).Flow out (fluxes) before at glucosan, culture medium with not containing phenol red culture media supplemented 1 hour, is added 5 microlitres (reserves 100 mg/ml) 4kDa FITC glucosan then in chamber (lumenal compartment).After 30 minutes cultivate, from the serous coat compartment, collect 100 microlitre samples and measure fluorescence signal (FLUOstar with 485 nanometer excitation wavelengths and 520 nanometer emission wavelengths

BMG Labtechnologies, the U.S.).With pmolFITC-glucosan/square centimeter/hour calculating FITC-glucosan flux.Use ANOVA (SPSS version 10) to carry out statistical analysis.

After cultivating 72 hours, fatty acid (100 μ M) is listed in the table 1 influence of spontaneous barrier integrity.Table 1 shows that LU-PUFA ' s ARA, EPA and DHA have reduced molecular flux and improved epithelium resistance.On the contrary, control experiment shows that Palmic acid has adverse effect, has promptly damaged barrier integrity.These results show, EPA, DHA and ARA, particularly ARA in compositions of the present invention favourable purposes and in method of the present invention, the purposes in the method for improving barrier integrity for example.These results further prove the cooperative effect of the combination of fatty acid of the present invention and indigestible oligosaccharide.

Fig. 1 has shown time and dosage (10 μ Ms and 100 μ Ms) the dependency effect of various fatty acids (Palmic acid, DHA, GLA and AA) to basic barrier integrity (TER).Fig. 1 shows that shown in the resistance (TER) that improves, LC-PUFA ' s AA, DHA and GLA have improved the epithelium barrier integrity.These results show EPA, DHA, GLA and ARA, particularly ARA in compositions of the present invention favourable purposes and in method of the present invention, the purposes in improving the method for barrier integrity for example.These results further prove the cooperative effect of the combination of fatty acid of the present invention and indigestible oligosaccharide.

Table 1

? Composition (LC-PUFA)	?? Flux	? Resistance (TER)
? Composition (LC-PUFA)	?? Flux	? Resistance (TER)	Tester	??79	?1090
Palmic acid	??161	?831	Tester	??79	?1090
Palmic acid	??161	?831	?DHA	??72	?1574
?ARA	??28	?1816	?DHA	??72	?1574
?ARA	??28	?1816	?EPA	??65	?1493

Embodiment 2:LC-PUFA is to the destructive influence of barrier of IL-4 mediation

With monolayer (MC) enterocyte is that T84 (ATTC, the U.S.) goes up cultivation at the transwell filter (Corning, CostarBV, Holland) that can carry out sampling of mucosa and serous coat and stimulating human enterocyte.Converge the back two weeks, with these monolayers at IL-4 (2 nanograms/milliliter, serous coat compartment, Sigma, U.S.) exist down and containing or do not containing poly-unsaturated fatty acid ARA, DHA, GLA, EPA or contrast Palmic acid (10 μ M or 100 μ M, mucosa compartment, Sigma, St.Louis, the U.S.) situation under cultivate.Before IL-4 cultivates, cell was cultivated 48 hours with ARA, DHA, EPA or Palmic acid are pre-.The common cultivation of PUFA ' s and Palmic acid and IL-4 continues 48 hours again; Changed culture medium and additive in per 24 hours.Determine the epithelium barrier function by measuring transepithelial electrical resistance (TER) and permeability as described in example 1 above.Add up evaluation and test as described in example 1 above.

ARA, DHA, EPA and Palmic acid (100 μ M) the results are shown in the table 2 the destructive influence of barrier of IL-4 mediation.Table 2 shows that LC-PUFA ' s ARA, DHA and EPA have suppressed the flux raising that IL-4 causes.On the contrary, Palmic acid has adverse effect and compares with tester and reduced barrier destruction.These results show that ARA, DHA and EPA prevent or reduce the favourable purposes of the barrier destruction (destroying as the barrier that takes place) of IL-4 mediation in clinical and infant nutrition prescription in food or milk allergy.These results further prove the cooperative effect of the combination of fatty acid of the present invention and indigestible oligosaccharide.

Fig. 2 has shown that various FA ' s (Palmic acid, DHA, GLA and ARA) destroy the time and dosage (10 μ M and 100 μ M) the dependency protective effect of (flux) to the barrier of IL-4 mediation.Fig. 2 shows that shown in the 4kD glucosan flux that reduces, the barrier that ARA, DHA and GLA have suppressed the IL-4 mediation destroys.These results show that ARA, DHA and EPA prevent or reduce the favourable purposes of the barrier destruction (destroying as the barrier that takes place) of IL-4 mediation in clinical and infant nutrition prescription in food or milk allergy.These results further prove the cooperative effect of the combination of fatty acid of the present invention and indigestible oligosaccharide.

Table 2

? Composition (LC-PUFA)	?? The IL-4 flux	?? IL-4TER
? Composition (LC-PUFA)	?? The IL-4 flux	?? IL-4TER	Tester	??582	??374
Palmic acid	??777	??321	Tester	??582	??374
Palmic acid	??777	??321	?DHA	??271	??547
?ARA	??218	??636	?DHA	??271	??547
?ARA	??218	??636	?EPA	??228	??539

Embodiment 3: the influence that oligosaccharide generates acetate

Microorganism is available from the baby's of bottle feeding fresh excreta.To compile from 1 to 4 months big babies' fresh excreta material and in anticorrosion medium, place 2 hours.As substrate, use prebiotics (TOS); TOS/ inulin (HP) mixture of 9/1 (w/w) ratio; Inulin; Oligofructose (the OS)/inulin mixture of 1/1 (w/w) ratio, or what does not use (blank).Transgalactooligosac,harides (TOS) is available from Vivinal GOS, Borculo DomoIngredients, Zwolle, Holland, and comprise 33wt% disaccharide, 39wt% trisaccharide, 18wt% tetrose, 7wt% pentasaccharides and 3wt% six-, seven-en, eight sugar be as indigestible oligosaccharide.Inulin (HP) Orafti Active Food Ingredients, Tienen, Belgium, i.e. Raftiline

Has 23 average DP.Medium: McBain﹠amp; Macfarlane medium: buffering peptone water 3.0 grams per liters, yeast extract 2.5 grams per liters, mucin (brush border) 0.8 grams per liter, tryptone 3.0 grams per liters, L-cysteine-HCl 0.4 grams per liter, bile salts 0.05 grams per liter, K ₂HPO ₄.3H ₂O 2.6 grams per liters, NaHCO ₃0.2 grams per liter, NaCl 4.5 grams per liters, MgSO ₄.7H ₂O 0.5 grams per liter, CaCl ₂0.228 grams per liter, FeSO ₄.7H ₂O 0.005 grams per liter.The medium of in 500 milliliters of Scott bottles, packing into, and 121 ℃ of sterilizations 15 minutes.Buffer medium: K ₂HPO ₄.3H ₂O 2.6 grams per liters, NaHCO ₃0.2 grams per liter, NaCl 4.5 grams per liters, MgSO ₄.7H ₂O 0.5 grams per liter, CaCl ₂0.228 grams per liter, FeSO ₄.7H ₂O 0.005 grams per liter.Use K ₂HPO ₄Or NaHCO ₃Be adjusted to pH 6.3 ± 0.1.The medium of in 500 milliliters of Scott bottles, packing into, and 121 ℃ of sterilizations 15 minutes.

Anticorrosion medium: buffering peptone water 20.0 grams per liters, L-cysteine-HCl 0.5 grams per liter, THIOGLYCOLLIC ACID are received 1/ liter of salt 0.5 grams per liter, "diazoresorcinol" (resazurine) sheet, are adjusted to pH 6.7 ± 0.1 with 1M NaOH or HCl.In microwave, seethe with excitement.25 mL media of in serum bottle, packing into and 121 ℃ the sterilization 15 minutes.

Fresh excreta sample and anticorrosion medium mixed be incorporated in 4 ℃ and preserve a few hours.With 13, centrifugal 15 minutes of 000rpm removes supernatant with the listerine of feces, and with feces and McBain﹠amp; Mac Farlane medium mixes with 1: 5 weight ratio.With 3 milliliters of this feces suspensions in bottle with 85 milligrams of glucoses or prebiotics or do not merge and fully mix with any additive (blank).When t=0, extract sample (0.5 milliliter).2.5 milliliters of gained suspensions are introduced in the dialysis tube in 60 milliliters of bottles that 60 milliliters of buffer mediums are housed.Should bottle fully sealing and 37 ℃ of cultivations.Extracted sample with hypodermic syringe from dialysis tube (0.2 milliliter) or dialysis buffer agent (1.0 milliliters) after 3,24 and 48 hours, existing side by side soon, it places on ice to stop fermentation.Use following sample to experimentize:

1) 85 milligrams of TOS

2) 85 milligrams of inulin

3) 85 milligrams of ratios TOS/ inulin that is 9/1 (w/w) and

4) 85 milligrams of OS/ inulin that ratio is 1/1 (w/w).

Use is furnished with the amount of Varian 3800 gas chromatograies (GC) (VarianInc., Walnut Creek, the U.S.) the mensuration SCFA (acetate, propionate, butyrate) of flame ionisation detector.Use helium to do carrier gas (3.0psi), in post (Stabilwax, 15 * 0.53 millimeters, 1.00 microns of thickness, Restek Co., the U.S. .), inject 0.5 microlitre sample at 80 ℃.After the injected sample, this stove is heated to 160 ℃ with 16 ℃/minute speed, is heated to 220 ℃ with 20 ℃/minute speed then, kept 1.5 minutes at 220 ℃ at last.The temperature of syringe and detector is 200 ℃.Use 2 Ethylbutanoic acid to make interior mark.

Fig. 3 described to make different oligosaccharide fermentations and absolute (Fig. 3 A) scheme (Fig. 3 B) with relative SCFA.Fig. 3 A shows, the mixture of two kinds of different oligosaccharide (TOS/ inulin) (wherein two kinds of different oligosaccharide have the monosaccharide unit homology and different chain lengths less than 90) causes comparing obviously and the amount of the SCFA (particularly acetate) of the every gram fiber that improves synergistically with individual components.Fig. 3 B shows that the compositions of adding the TOS/ inulin helps producing more a high proportion of useful acetate (B).Acetate output becomes in live body by the mucus output of goblet cell raising and a kind of measurement standard (referring to embodiment 4) of casing slime layer thickness.These results show the favourable purposes of this compositions.

The influence that embodiment 4:SCFA generates mucus

Monolayer enteric epithelium T84 cell (ATTC, the U.S.) is cultivated in 24 or 96 hole tissue culturing plates (Corning B.V.).T84 was cultivated 24 hours with the concentration range of 0.025-4.0mM with short-chain fatty acid acetate, propionate and butyrate (SCFA, Merck, the U.S.).Collect supernatant and/or cell and measure the expression of MUC-2 (mucin).Use the MUC-2 in the dot blotting technical measurement cell culture to express, because mucin is great glycoprotein (surpassing 500kDa), this makes them be difficult to operation in western blotting technique.Use pre-immune serum (T84 dye negative thing), CCD-18Co (ATCC, the U.S.) negative control cell and bovine serum albumin (BSA) to confirm this method.Collecting cell sample and use micro protein checking method (Biorad, the U.S.) to carry out protein determination in Laemmli (Separation of Proteins buffer agent) according to the indication of manufacturer.At NC Nitroncellulose film (Schleicher﹠amp; Schuell, Germany) go up with a labelling sample (0.3-0.7-1.0 microgram/2 microlitres).With film sealing in TBST/5%Protivar (Nutricia, Holland), use then to resist-MUC-2 antibody (Dr.Einerhand, Erasmus University, Rotterdam, Holland grants with open arms) cultivated 1 hour.After the washing, trace is cultivated with goat-anti rabbit-HRP (Santacruz Biotechnolog, the U.S.), detected, use ECL (Roche Diagnostics, Holland) for substrate.Use Lumi-Imager (BoehringerMannheim B.V., Holland) to carry out density measurement also with light unit (BLU) expression signal.Also relatively represent BLU ' s (%BLU) with contrast culture.In order to compare SCFA, deduct basic MUC-2 expression to the excitation that MUC-2 expresses.

Fig. 4 has shown the influence that SCFA (acetate, propionate, butyrate) expresses MUC-2 in enterocyte (MCT84) and epithelium mesenchymal cell coculture (CC T84).Fig. 2 shows that also acetate is compared with butyrate with propionate, and is more effective aspect stimulation MUC-2 expression (mucus generation).Therefore, oligosaccharide combination of the present invention (it shows and has improved acetate output (referring to embodiment 3)) is particularly useful for stimulating mucus to generate and can be advantageously utilised in the method that excites barrier integrity.

Embodiment 5: the baby milk formula I

Composition (every liter), 672 kilocalories of energy; Protein 15 grams; Milk surum: casein ratio 60: 40; Fat 36 grams; Carbohydrate 72 grams; Vitamin A 750 RE; Mix carotene (carotids) 400IU; Vitamin D 10.6 micrograms; 7.4 milligrams of vitaminFs; Vitamin K 67.0 micrograms; Vitamin B1 (thiamine) 1000 micrograms; Vitamin B2 (riboflavin) 1500 micrograms; Vitamin B6 (pyridoxol) 600 micrograms; Vitamin B12 (cyanacobalmine) 2.0 micrograms; Nicotinic acid 9.0 micrograms; Folic acid 80 micrograms; Pantothenic acid 3000 micrograms; Biotin 90 micrograms; 90 milligrams of vitamin Cs (ascorbic acid); 100 milligrams in choline; 33 milligrams of inositols; 460 milligrams of calcium; 333 milligrams in phosphorus; 64 milligrams in magnesium; 8.0 milligrams of ferrum; 6.0 milligrams on zinc; Manganese 50 micrograms; Copper 560 micrograms; Iodine 100 micrograms; 160 milligrams in sodium; 650 milligrams in potassium; 433 milligrams of chlorides and selenium 14 micrograms; Wherein fat content comprises 3 gram fish oil and 3 gram 40% arachidonic oil (DSM Food Specialities, Delft, Holland); Further comprise 4 gram transgalactooligosac,harides acid Elix ' or ^TM(Borculo Domo Ingredients, Holland) and 4 gram Raftiline ^TM(Orafti Active Food Ingredients, Belgium).

Claims

1. infant formula composition, it comprises the protein of 7.5 to 12.5 energy %; The carbohydrate of 40 to 55 energy %; With the fat of 35 to 50 energy %, wherein said protein comprise lactoprotein, vegetable protein and/or amino acid whose a member of being selected from hydrolysis and

A.EPA, DHA and ARA, the content that wherein contains the poly-unsaturated fatty acid of long-chain of 20 and 22 carbon atoms is no more than the 15wt% of total lipid content; With

B. at least two kinds of different oligosaccharide (OL1 and OL2), wherein these two kinds of different oligosaccharide have and are lower than 90% monosaccharide unit homology; With

C. per 100 grams are done 1 to 500 milligram of nucleoside and/or nucleotide that prescription comprises.

2. compositions according to claim 1, wherein said protein are the lactoprotein of hydrolysis.

3. compositions according to claim 2, wherein said protein are caseinhydrolysate or lact albumin hydrolysate.

4. infant formula composition, it comprises the protein of 7.5 to 12.5 energy %; The fat of the carbohydrate of 40 to 55 energy % and 35 to 50 energy %, wherein said protein be aminoacid and/or vegetable protein and

B. at least two kinds of different oligosaccharide (OL1 and OL2), wherein these two kinds of different oligosaccharide have and are lower than 90% monosaccharide unit homology.

5. according to the described compositions of claim 1-4, it comprises oligomeric galactose and the levan that is selected from oligofructose, inulin and composition thereof.

6. according to the described compositions of claim 1-5, wherein at least the oligosaccharide of 10wt% have 2 to 5 the degree of polymerization (DP) and at least 5wt% have 10 to 60 DP.

7. according to each described compositions of claim 1-6, further comprise acidic oligomer sugar, preferred DP is 2 to 60 uronic acid polymer.

8. according to each described compositions of claim 1-7, described compositions has the energy content of 0.6 to 0.8 kcal/ml; 50 to 500mOsm/kg osmolality; With the viscosity that is lower than 50mPas.

9. according to each described compositions of claim 1-8, as medicament.

10. the purposes that is used for 0 to 2 years old baby's compositions according to each described compositions of claim 1-8 in preparation.

11. be used for preparation treatment or Polyglucan disease according to each described compositions of claim 1-8, the purposes of the medicine of particularly treating or preventing to use in the method for phagopyrism, described method comprises administration according to each described compositions of claim 1-8.