Nutritional composition comprising 2'fucosyllactose and dietary butyric acid

Abstract

The present invention relates to a nutritional composition for infants or young children comprising 2'fucosyllactose, dietary butyric acid and optionally 3 'galactosyllactose.

Description

[0001] This application is a divisional application of an invention patent application with an application date of June 4, 2020, application number 202080005720.0, and invention name “Nutritional composition containing 2'-fucosyllactose and dietary butyric acid”. Technical Field

[0002] The present invention relates to the field of infant formula and improving intestinal health. Background of the Invention

[0004] Human milk is a preferred baby food. Human milk provides several biologically active factors that are beneficial to the relatively immature immune system and intestinal health in the early stage of neonatal life. Human milk-fed infants have a lower incidence of infection than formula-fed infants. Many components in human milk, including immunoglobulins (such as sIgA), interleukins (IL) -1, IL-6, IL-8, IL-10, interferon-γ (IFN-γ), immune active cells, transforming growth factor-β (TGF-β), lactoferrin, nucleotides and human milk oligosaccharides (HMOs), are considered to be involved in the protective effect of resisting pathogen infection. In addition, intestinal maturation and microbial flora development in human milk-fed infants are considered to be optimal.

[0005] However, breastfeeding an infant is not always possible or desirable. In such cases, infant formula or follow-on formula is a good alternative. In order to simulate the beneficial effects of human milk as closely as possible, these formulas should have an optimal composition.

[0006] WO 2005 / 122790 discloses a method of stimulating barrier integrity by administering a composition comprising eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (ARA) and at least two different oligosaccharides. The oligosaccharides act indirectly through fermentation by the intestinal microbiota into short chain fatty acids (SCFA).

[0007] WO 2016 / 013935 discloses the use of indigestible oligosaccharides in the preparation of a composition for providing nutrition to infants at increased risk of suffering from food allergies. Preferably, infants are at increased risk of exposure to trichothecene mycotoxins, for example by consuming many cereals. In an embodiment, Vivinal GOS is a source of galacto-oligosaccharides.

[0008] WO 2006 / 115412 relates to a liquid nutritional composition for improving intestinal barrier function and for preventing allergies. The composition comprises lipids having short-chain fatty acyl chains and non-digestible fermentable carbohydrates.

[0009] WO 2004 / 112509 discloses a composition for inducing a pattern of intestinal barrier maturation similar to that observed with breastfeeding. The composition helps to improve intestinal barrier maturation, for example during neonatal stress. It is disclosed that maternal separation in rats increases intestinal permeability, and a blend containing LC-PUFA, Lactobacillus paracasei and indigestible oligosaccharides can restore intestinal permeability to normal levels.

[0010] There remains a need for improved infant formulas and compositions for young children that more closely resemble human milk in structure and function. Summary of the invention

[0011] The inventors have found that the combination of the nutritional ingredients 2'-FL and dietary butyrate has a beneficial effect on intestinal barrier function. It has also been found that the immune system and microbiota respond differently when both 2'-FL and dietary butyrate are present than when only one of these ingredients is present. A mixture of 2'-FL and dietary butyrate has been shown to have a beneficial effect on alkaline phosphatase expression, indicating improved maturation of intestinal barrier function and improved defense against enteric pathogens. The presence of 3'-GL further improves these effects. Therefore, a nutritional composition comprising dietary butyrate and 2'-FL and preferably additionally 3'-GL will have an improved effect on infant health.

[0012] List of implementation plans

[0013] 1. A nutritional composition for infants or young children, comprising

[0014] a.2'-fucosyllactose, and

[0015] b. Dietary butyrate.

[0016] 2. The nutritional composition according to embodiment 1, further comprising 3' galactosyl lactose.

[0017] 3. The nutritional composition according to any one of the preceding embodiments, wherein the composition is at least partially fermented by lactic acid producing bacteria and comprises 0.1 to 1.5 wt. % of the total lactic acid and lactate, based on the dry weight of the nutritional composition, and wherein at least 90 wt. % of the total lactic acid and lactate is L-lactic acid and L-lactate.

[0018] 4. The nutritional composition according to any one of the preceding embodiments, wherein the composition further comprises LC-PUFA selected from DHA, ARA and EPA, preferably DHA and EPA, preferably DHA, EPA and ARA, wherein the nutritional composition preferably comprises at least 1% by weight of the sum of DHA, ARA and EPA, based on the total fatty acids.

[0019] 5. The nutritional composition according to any one of the preceding embodiments, wherein the formula further comprises galacto-oligosaccharides and / or fructo-oligosaccharides.

[0020] 6. The nutritional composition according to any one of the preceding embodiments, wherein the nutritional composition is selected from an infant formula, a follow-on formula or a toddler formula, preferably an infant formula.

[0021] 7. The nutritional composition according to any one of the preceding embodiments, wherein the composition comprises (i) 0.02 to 1 gram of 2'-FL / 100 ml of the nutritional composition; (ii) 0.15 to 7.5 wt % on a dry weight basis; and / or (iii) 0.03 to 1.5 g / 100 kcal.

[0022] 8. The nutritional composition according to any one of the preceding embodiments, wherein the nutritional composition comprises (i) 0.010 to 0.250 g 3'-GL / 100 ml; (ii) 0.075 to 2 wt % on a dry weight basis; and / or (iii) 0.015 to 0.4 g / 100 kcal.

[0023] 9. The nutritional composition according to any of the preceding embodiments, wherein the nutritional composition comprises (i) 0.3 to 5 wt % dietary butyrate, based on total fatty acids; (ii) 10 mg to 175 mg / 100 ml; (iii) 15 to 250 mg / 100 kcal; and / or (iv) 0.075 to 1.3 wt %, based on the dry weight of the nutritional composition.

[0024] 10 The nutritional composition according to any one of the preceding embodiments, wherein the nutritional composition comprises (i) 0.2 to 5 g total galacto-oligosaccharide and fructo-oligosaccharide / 100 ml; and / or (ii) 0.3 to 7.5 g / 100 kcal, 1.5 to 35 wt %, based on dry weight.

[0025] 11. A nutritional composition according to any one of the preceding embodiments, for improving intestinal barrier function and / or for improving the immune system and / or for improving intestinal microbiota and / or for treating or preventing infection.

[0026] 12. The nutritional composition according to any one of the preceding embodiments, for use in treating or preventing allergies, preferably for inducing oral tolerance to allergens.

[0027] 13. The nutritional composition for use according to embodiment 11 or 12, wherein the nutritional composition is administered to an infant or a young child, preferably an infant.

[0028] 14. The nutritional composition according to any one of embodiments 1-10 or the nutritional composition for use according to any one of embodiments 11-13, for providing nutrition to an infant. DETAILED DESCRIPTION

[0029] The present invention relates to a nutritional composition for infants or young children comprising

[0030] a.2'-fucosyllactose, and

[0031] b. Dietary butyrate.

[0032] In a preferred embodiment, the nutritional composition further comprises 3' galactosyl lactose.

[0033] In another or further preferred embodiment, the nutritional composition is at least partially fermented by lactic acid producing bacteria and comprises 0.1 to 1.5 wt.-% total lactic acid and lactate, based on the dry weight of the nutritional composition, and wherein at least 90 wt.-% of the total lactic acid and lactate is L-lactic acid and L-lactate.

[0034] The invention also relates to the nutritional composition for use as a medicament, preferably for treating, preventing and / or alleviating diseases and / or conditions. The nutritional composition is preferably used for improving the intestinal barrier function, for improving the immune system, for improving the intestinal microbiota, for treating or preventing infections, in particular intestinal infections, and / or for treating or preventing allergies, preferably for inducing oral tolerance to allergens.

[0035] This aspect of the invention can also be expressed as the use of the nutritional composition in the preparation of a medicament for treating, preventing and / or alleviating a disease and / or condition, preferably for treating a disease. The use of the nutritional composition is preferably for improving the intestinal barrier function, for improving the intestinal microbiota, for treating or preventing infections, especially intestinal infections, and / or for treating or preventing allergies, preferably for inducing oral tolerance to allergens.

[0036] This aspect of the invention can also be expressed as the use of the nutritional composition for treating, preventing and / or alleviating diseases and / or conditions. The use of the nutritional composition is preferably for improving the intestinal barrier function, for improving the intestinal microbiota, for treating or preventing infections, in particular intestinal infections, and / or for treating or preventing allergies, preferably for inducing oral tolerance to allergens.

[0037] This aspect of the invention can also be expressed as a method for treating, preventing and / or alleviating a disease and / or condition, comprising administering the composition to a subject in need thereof. The method is preferably used to improve intestinal barrier function, to improve intestinal microbiota, to treat or prevent infection, in particular intestinal infection, and / or to treat or prevent allergy, preferably to induce oral tolerance to allergens.

[0038] definition

[0039] In the context of the present invention, the term "prevention" means "reducing the risk of (occurring)" or "reducing the severity". The term "preventing a condition" also includes "treating a person at (increased) risk of said condition".

[0040] In this document and in the claims thereof, the verb "to comprise" and its conjugations are used in its non-limiting sense, meaning that items following the word are included, but items not specifically mentioned are not excluded. Furthermore, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that one and only one of the element be present. Thus, the indefinite article "a" or "an" generally means "at least one".

[0041] 2'-fucosyllactose

[0042] The nutritional composition of the present invention comprises 2'-fucosyllactose (2-'FL). 2'-FL was found to improve the intestinal barrier function. 2'-FL was also found to improve the immune system. Fucosyllactose (FL) is a non-digestible oligosaccharide present in human milk. It is not present in cow's milk. It consists of three monosaccharide units linked together: fucose, galactose and glucose. Lactose is a galactose unit linked to a glucose unit via a β1,4 bond. The fucose unit is linked to a galactose unit of a lactose molecule via an α1,2 bond (2'-fucosyllactose, 2'-FL) or to a glucose unit of lactose via an α-1,3 bond (3-fucosyllactose, 3-FL).

[0043] 2'FL, preferably α-L-Fuc-(1→2)-β-D-Gal-(1→4)-D-Glc, is commercially available, for example from Sigma-Aldrich. Alternatively, it can be isolated from human milk, for example as described in Andersson & Donald, 1981, J Chromatogr. 211: 170-1744, or produced by genetically modified microorganisms, for example as described in Albermann et al., 2001, Carbohydrate Res. 334: 97-103.

[0044] Preferably, the nutritional composition of the present invention comprises 10 mg to 1 g 2'FL / 100 ml, more preferably 20 mg to 0.5 g, even more preferably 40 mg to 0.2 g 2'-FL / 100 ml. Based on dry weight, the nutritional composition of the present invention preferably comprises 0.075 wt% to 7.5 wt% 2'FL, more preferably 0.15 wt% to 3.75 wt% 2'FL, even more preferably 0.3 wt% to 1.5 wt% 2'-FL. Based on energy, the nutritional composition of the present invention preferably comprises 0.015 to 1.5 g 2'-FL / 100 kcal, more preferably 0.03 to 0.075 g 2'-FL / 100 kcal, even more preferably 0.06 to 0.3 g 2'-FL / 100 kcal. Smaller amounts of fucosyllactose are less effective in stimulating the immune system or improving intestinal barrier function, while too high an amount will result in an unnecessarily high cost of the product.

[0045] Dietary butyrate

[0046] The nutritional composition of the present invention contains dietary butyric acid. Butyric acid was found to improve intestinal barrier function. The nutritional composition preferably comprises 0.3 to 5 wt. %, preferably 0.6 to 5 wt. %, even more preferably 1 to 5 wt. % butyric acid, based on the weight of the total fatty acyl chains. The nutritional composition of the present invention preferably contains tributyrin (i.e. a triglyceride having 3 butyric acid chains ester-bonded to a glycerol backbone).

[0047] Preferably, the nutritional composition contains 0.075 to 1.3 wt. %, preferably 0.15 to 1.3 wt. % and more preferably 0.25 to 1.3 wt. % butyric acid, based on the dry weight of the composition. Alternatively, the nutritional composition comprises 0.015 to 0.25 g butyric acid / 100 kcal, preferably 0.03 to 0.25 g butyric acid / 100 kcal and more preferably 0.05 to 0.25 g butyric acid / 100 kcal. When the nutritional composition is liquid, the composition preferably contains 0.01 to 0.175 g butyric acid / 100 ml, more preferably 0.02 to 0.175 g butyric acid / 100 ml and more preferably 0.035 to 0.175 g butyric acid / 100 ml. Human milk is known to contain very low levels of butyric acid, in particular <0.1 wt. %, based on total fatty acids.

[0048] Dietary butyric acid can be provided by any suitable source known in the art. Non-limiting sources of dietary butyric acid include fats and derivatives of animal origin, such as but not limited to milk, milk fat, butter fat, butter oil, butter, buttermilk, butter serum, cream; microbial fermentation derivatives, such as but not limited to yogurt and fermented buttermilk; and seed oil products derived from plant sources, such as pineapple and / or pineapple oil, almonds and / or almond oil, barley, oats, brown rice, bran, mung beans, legumes, green leafy vegetables, apples, kiwis, oranges. In some embodiments, dietary butyric acid is synthetically produced. The preferred source of dietary butyric acid is ruminant milk fat, preferably bovine milk fat.

[0049] In embodiments where dietary butyrate is produced synthetically, the chemical structure of the dietary butyrate may be modified as desired. In addition, the synthetically produced dietary butyrate may be purified by any means known in the art to prepare a purified dietary butyrate additive that can be incorporated into the nutritional compositions disclosed herein. Dietary butyrate may be provided by butyrate esters in dairy lipid and / or triglyceride bound form.

[0050] In some embodiments, dietary butyrate may include butyrates, such as sodium butyrate, potassium butyrate, calcium butyrate, magnesium butyrate, and combinations thereof. In certain embodiments, dietary butyrate includes a suitable butyrate that has been coated with one or more fats or lipids. In certain embodiments where the dietary butyrate includes a fat-coated butyrate, the nutritional composition may be a dry powder composition into which the dietary butyrate is incorporated. Preferably, the dietary butyrate is provided as part of a triglyceride. This is advantageous because butyric acid is volatile (and foul-smelling) when provided in free form or in salt form. In triglyceride form, butyric acid is released in and after the stomach due to the action of lipase.

[0051] In a preferred embodiment, the weight ratio of 2'-FL to dietary butyrate ranges from 10:1 to 1:10, preferably from 5:1 to 1:5, more preferably from 3:1 to 1:3.

[0052] 3'-galactosyl lactose

[0053] The nutritional composition of the present invention preferably comprises 3'-galactosyl lactose. Preferably, 3'-galactosyl lactose is the trisaccharide Gal-(β 1,3)-Gal-(β 1,4)-Glc. In the context of the present invention, all references to 3-'GL refer to β1,3'-galactosyl lactose or β3'GL, unless explicitly stated otherwise. The trisaccharide can be administered in the form of a suitable matrix or nutritional composition. The trisaccharide can be, for example, part of a mixture of galacto-oligosaccharides (GOS), preferably β-galacto-oligosaccharides (βGOS). It was found that β3'-GL improves intestinal barrier function.

[0054] The nutritional composition of the present invention preferably comprises 0.07 to 3.75% by weight of Gal(β 1-3)-Gal(β 1-4)-Glc, based on the dry weight of the nutritional composition. In a preferred embodiment, the nutritional composition comprises 0.07 to 0.375% by weight of Gal(β 1-3)-Gal(β 1-4)-Glc, based on the dry weight of the nutritional composition. In another preferred embodiment, the nutritional composition comprises 1.125 to 1.725% by weight of Gal(β 1-3)-Gal(β 1-4)-Glc, based on the dry weight of the nutritional composition.

[0055] The nutritional composition of the present invention preferably comprises 15 to 750 mg Gal (β 1-3) -Gal (β 1-4) -Glc / 100 kcal nutritional composition. In a preferred embodiment, the nutritional composition comprises 15 to 75 mg Gal (β 1-3) -Gal (β 1-4) -Glc / 100 kcal nutritional composition. In another preferred embodiment, the nutritional composition comprises 225 to 375 mg Gal (β 1-3) -Gal (β 1-4) -Glc / 100 kcal nutritional composition.

[0056] The nutritional composition of the present invention preferably comprises 10 to 500 mg Gal (β 1-3) -Gal (β 1-4) -Glc / 100 ml nutritional composition. In a preferred embodiment, the nutritional composition comprises 10 to 50 mg Gal (β 1-3) -Gal (β 1-4) -Glc / 100 ml nutritional composition. In another preferred embodiment, the nutritional composition comprises 150 to 250 mg Gal (β 1-3) -Gal (β 1-4) -Glc / 100 ml nutritional composition. It is known that human milk contains low levels of 3'GL, in particular an average of no more than 5 mg / 100 ml.

[0057] The combination of 2'-FL, butyric acid and 3'-GL has a further improved effect on health, in particular on improving the intestinal barrier function, on improving the immune system, on improving the intestinal microbiota and / or on treating or preventing infections, in particular intestinal infections.

[0058] In a preferred embodiment, the weight ratio of 2'-FL to 3'-GL ranges from 10:1 to 1:10, preferably from 5:1 to 1:5, more preferably from 3:1 to 1:3.

[0059] Oligosaccharides

[0060] If present, β1,3'-galactosyl lactose may be part of a mixture of galacto-oligosaccharides (GOS), preferably β-galacto-oligosaccharides (BGOS). In particular, it is also advantageous to add GOS to the nutritional composition of the present invention in addition to β1,3'-galactosyl lactose (β3'-GL). A mixture of GOS of different sizes and bonds has an increased beneficial effect on the microbiota and improves the production of short-chain fatty acids, which in turn has a further improved effect on the immune system and / or the treatment or prevention of infections, in particular intestinal infections. The presence of GOS other than β3'-GL has an additional effect on the intestinal barrier function in the large intestine and the end of the small intestine, while β3'-GL is also - and usually - effective in the small intestine. Therefore, the addition of 3'-GL and GOS has a further improved effect on health, in particular on improving the intestinal barrier function, on improving the immune system, on improving the intestinal microbiota and / or on treating or preventing infections, in particular intestinal infections.

[0061] In the context of the present invention, a suitable way to form GOS is to treat lactose with β-galactosidase. Depending on the specificity of the enzyme used, a galactose unit is produced by the hydrolysis of lactose and is coupled to another lactose unit via a β bond to form a trisaccharide. A galactose unit can also be coupled to another single galactose unit to form a disaccharide. Subsequent galactose units are coupled to form oligosaccharides. Most of the oligosaccharides formed in this way have a degree of polymerization (DP) of less than 7. Depending on the enzyme, these bonds between the galactose residues can be mainly β1,4', β1,6' or β1,3'.

[0062] A suitable method for preparing β1,6' and / or β1,4' GOS is to use β-galactosidase from Bacillus circulans. A commercially available source of BGOS is Vivinal-GOS purchased from Friesland Campina Domo (Amersfoort, The Netherlands). Vivinal-GOS includes BGOS, which has mainly DP 2-8 (peak at DP3) and has mainly β1,4' bonds and β1,6' bonds, wherein β1,4' bonds are more predominant. β1,4'-galactosyl lactose and β1,6'-galactosyl lactose can be enriched or purified from these GOS mixtures, as known in the art, for example by size exclusion chromatography. Other commercially available sources of BGOS having predominantly β1,4' and / or β1,6' linkages are Oligomate 55 and 50 from Yakult, and Cup Oligo from Nissin Sugar. Alternatively, β1,4'-galactosyl lactose and β1,6'-galactosyl lactose are commercially available as single components (Carbosynth).

[0063] A suitable method for preparing β1,3'GOS is to use a β-galactosidase from Streptococcus thermophilus (S. thermophilus). Particularly suitable is the use of a β-galactosidase from strains CNCM I-1470 and / or CNCM I-1620 in the method disclosed in Example 4 of FR2723960 or Example 6 of EP0778885. Streptococcus thermophilus CNCM I-1620 was deposited by Compagnie Gervais Danone on August 23, 1995 at the Collection Nationale de Cultures de Microorganisms van Institute Pasteur, Paris, France, under the Budapest Treaty. Strain Streptococcus thermophilus CNCM I-1620 is also known as strain Streptococcus thermophilus ST065. Streptococcus thermophilus CNCM I-1470 was deposited by Compagnie Gervais Danone under the Budapest Treaty on August 25, 1994 at the Collection Nationale de Cultures de Microorganisms van Institute Pasteur, Paris, France. The composition of this GOS is also described in more detail in LeForestier et al., 2009 Eur J Nutr, 48: 457-464. Both strains are also disclosed in WO 96 / 06924. Another commercially available GOS enriched in β1,3 galacto-oligosaccharides and β1,6 galacto-oligosaccharides is Bimuno from Clasado, or Purimune from GTC Nutrition. β1,6'-galactosyl lactose and β1,3'-galactosyl lactose can be enriched or purified from these GOS mixtures known in the art, for example by size exclusion chromatography. Alternatively, pure β1,3'-galactosyl lactose is commercially available (Carbosynth).

[0064] GOS, including BGOS, are indigestible. Human digestive enzymes (including human lactase) cannot hydrolyze GOS. Therefore, GOS reach the large intestine intact when consumed and are available for fermentation by intestinal microflora.

[0065] Preferably, the nutritional composition comprises at least 250 mg GOS / 100 ml, more preferably at least 400, even more preferably at least 600 mg / 100 ml. Preferably, the nutritional composition comprises not more than 2500 mg GOS / 100 ml, preferably not more than 1500 mg, more preferably not more than 1000 mg. More preferably, the nutritional composition of the present invention comprises GOS in an amount of 250 to 2500 mg / 100 ml, even more preferably 400 to 1500 mg / 100 ml, even more preferably 600 to 1000 mg / 100 ml.

[0066] Preferably, the nutritional composition comprises at least 1.75 wt. %, more preferably at least 2.8 wt. %, even more preferably at least 4.2 wt. % GOS based on the dry weight of the total composition, all based on the dry weight of the total composition. Preferably, the nutritional composition comprises no more than 17.5 wt. %, more preferably no more than 10.5 wt. %, even more preferably no more than 7 wt. % GOS based on the dry weight of the total composition. The nutritional composition of the present invention preferably comprises GOS in an amount of 1.75 to 17.5 wt. %, more preferably 2.8 to 10.5 wt. %, most preferably 4.2 to 7 wt. %, all based on the dry weight of the total composition.

[0067] Preferably, the nutritional composition according to the present invention comprises at least 0.35 g GOS / 100 kcal, more preferably at least 0.6 g, even more preferably at least 0.8 g / 100 kcal. Preferably, the nutritional composition comprises not more than 3.7 g GOS / 100 kcal, preferably not more than 2.5 g / 100 kcal, more preferably not more than 1.5 g / 100 kcal. More preferably, the nutritional composition according to the present invention comprises GOS in an amount of 0.35 to 3.7 g / 100 kcal, even more preferably 0.6 to 2.5 g / 100 ml, even more preferably 0.8 to 1.5 g / 100 ml.

[0068] Smaller amounts result in a less effective composition, while the presence of higher amounts of GOS may result in side effects such as osmotic disturbance, abdominal pain, bloating, gas formation, and / or flatulence.

[0069] As defined in the nutritional composition of the present invention, the total amount of GOS includes the amount of β1,3'-galactosyl lactose.

[0070] In a preferred embodiment, nutritional composition comprises 0.25 to 2.5g galacto-oligosaccharide / 100ml, and wherein the galacto-oligosaccharide of 10mg to 500mg / 100ml is Gal(β 1-3)-Gal(β 1-4)-Glc.In another preferred embodiment, nutritional composition comprises 0.25 to 2.5g galacto-oligosaccharide / 100ml, and wherein the amount of Gal(β 1-3)-Gal(β 1-4)-Glc is greater than 20 % by weight, based on total galacto-oligosaccharide.In another preferred embodiment, nutritional composition comprises 0.25 to 2.5g galacto-oligosaccharide / 100ml, and wherein the amount of Gal(β 1-3)-Gal(β 1-4)-Glc is 10-500mg / 100ml. In another preferred embodiment, the nutritional composition comprises 0.25 to 2.5 g galacto-oligosaccharides / 100 ml, wherein the amount of Gal(β1-3)-Gal(β1-4)-Glc is greater than 20 wt %, based on total galacto-oligosaccharides, and wherein the amount of Gal(β1-3)-Gal(β1-4)-Glc is 150 to 250 mg / 100 ml.

[0071] In another preferred embodiment, the nutritional composition comprises 0.25 to 2.5 g galacto-oligosaccharides / 100 ml, wherein the amount of Gal(β 1-3)-Gal(β 1-4)-Glc is 10 to 50 mg / 100 ml.

[0072] In the GOS formulation, the amount of β1,3'-galactosyl lactose preferably ranges from 60-65% by weight based on total galacto-oligosaccharides (excluding lactose, galactose and glucose). Other preferred sources of β1,3'-galactosyl lactose include Bimuno (Clasado) or Purimune (GTC Nutrition).

[0073] Preferably - as further explained below - the nutritional composition of the present invention further comprises fructooligosaccharides (FOS).

[0074] Fermented composition

[0075] The nutritional composition of the present invention is preferably at least partially fermented. A partially fermented nutritional composition comprises a composition at least partially fermented by lactic acid producing bacteria. It has been shown that partially fermented formulas have a protective effect on maintaining intestinal permeability when exposed to physiological or psychological stress.

[0076] Fermentation is preferably carried out during the preparation process of the nutritional composition. Preferably, the nutritional composition does not contain a large amount of live bacteria in the final product, and this can be achieved by heat inactivation after fermentation or inactivation by other means. Preferably, the fermented composition is a milk-derived product, which is a milk substrate fermented by lactic acid producing bacteria, wherein the milk substrate comprises at least one selected from the following: milk, whey, whey protein, whey protein hydrolysate, casein, casein hydrolysate or a mixture thereof. Suitably, nutritional compositions comprising fermented compositions and non-digestible oligosaccharides and methods for their preparation are described in WO 2009 / 151330, WO 2009 / 151331 and WO 2013 / 187764.

[0077] The fermented composition preferably comprises bacterial cell fragments, such as glycoproteins, glycolipids, peptidoglycans, lipoteichoic acid (LTA), lipoproteins, nucleotides and / or capsular polysaccharides. It is advantageous to directly use a fermented composition comprising inactivated bacteria and / or cell fragments as a part of the final nutritional product, because this will result in a higher concentration of bacterial cell fragments. When using commercial preparations of lactic acid bacteria, they are usually washed, and the material is separated from the aqueous growth medium comprising bacterial cell fragments, thereby reducing or eliminating the presence of bacterial cell fragments. In addition, during fermentation and / or other interactions of lactic acid bacteria with milk substrates, other bioactive compounds can be formed, such as short-chain fatty acids, bioactive peptides and / or oligosaccharides and other metabolites, which can also lead to intestinal microbial functions more similar to the intestinal microbial functions of breast-fed infants. Such bioactive compounds produced by lactic acid bacteria during fermentation can also be referred to as post-biotics. It is believed that the composition comprising such post-biotics is advantageously closer to breast milk, because breast milk is not a clean synthetic formula, but contains metabolites, bacterial cells, cell fragments, etc. Therefore, it is believed that fermented compositions, in particular fermented milk-derived products, have an improved effect in preventing premature intestinal maturation in infants and inducing an intestinal maturation pattern in infants that is more similar to the intestinal maturation pattern observed in breastfed infants compared to non-fermented milk-derived products without lactic acid-producing bacteria or with only added lactic acid-producing bacteria.

[0078] Preferably, based on dry weight, the final nutritional composition comprises 5 to 97.5 wt %, more preferably 10 to 90 wt %, more preferably 20 to 80 wt %, even more preferably 25 to 60 wt % of the fermented composition. The level of the sum of lactic acid and lactate in the final nutritional composition can be used as a way to specify that the final nutritional composition comprises at least a partially fermented composition and a specified fermentation degree, because this is the metabolic end product produced by lactic acid bacteria during fermentation. The final nutritional composition of the present invention preferably comprises 0.1 to 1.5 wt % of the sum of lactic acid and lactate, more preferably 0.15 to 1.0 wt %, even more preferably 0.2 to 0.5 wt %, based on the dry weight of the composition. Alternatively, the nutritional composition comprises 0.02 to 0.3 g of the sum of lactic acid and lactate / 100 kcal, preferably 0.03 to 0.2 of the sum of lactic acid and lactate / 100 kcal, preferably 0.04 to 0.1 of the sum of lactic acid and lactate / 100 kcal. Alternatively, when the composition is liquid, the sum of lactic acid and lactate is 0.0125 to 0.2 g / 100 ml, preferably 0.02 to 0.125 g / 100 ml, preferably 0.03 to 0.07 g / 100 ml.

[0079] Preferably, at least 50% by weight, even more preferably at least 90% by weight of the sum of lactic acid and lactate is in the form of the L(+)-isomer. Therefore, in one embodiment, the sum of L(+)-lactic acid and L(+)-lactate is greater than 50% by weight, more preferably greater than 90% by weight, based on the sum of total lactic acid and lactate. Herein, L(+)-lactate and L(+)-lactic acid are also referred to as L-lactate and L-lactic acid.

[0080] The combination of 2'-FL, butyric acid, optionally 3'-GL and a partially fermented formula has a further improved effect on health, in particular on improving the intestinal barrier function, on improving the immune system, on improving the intestinal microbiota and / or on treating or preventing infections, in particular intestinal infections.

[0081] LCPUFA .

[0082] The nutritional composition of the present invention preferably comprises long-chain polyunsaturated fatty acids (LC-PUFA). LC-PUFA is a fatty acid or fatty acyl chain having a length of 20 to 24 carbon atoms, preferably 20 or 22 carbon atoms, which contains two or more unsaturated bonds. Preferably, the nutritional composition comprises at least one, preferably two, more preferably three LC-PUFAs selected from docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and arachidonic acid (ARA). It has been found that these LC-PUFA improve intestinal barrier function and can therefore be particularly advantageously combined with 2-'FL, butyric acid and optionally 3'-GL to further improve the intestinal barrier. This combination has unexpected beneficial effects and preferably works synergistically. Preferably, the nutritional composition comprises an increased amount of such LC-PUFA. Current infant formulas, when they contain these LC-PUFAs, typically have a sum of DHA, ARA and EPA in an amount of 0.4 to 0.9% by weight, based on total fatty acids. In the nutritional composition of the present invention, the amount of these LC-PUFAs is preferably greater than 1% by weight, preferably greater than 1.1% by weight, based on the total fatty acids. Preferably, the amount of these LC-PUFAs is not greater than 15% by weight, preferably not greater than 5% by weight, preferably not greater than 2.5% by weight, based on the total fatty acids. It is further preferred that the amount of these LC-PUFAs ranges from 1 to 15% by weight, preferably 1.1 to 5% by weight, more preferably 1.5 to 2.5% by weight, based on the total fatty acids. This is considered to be the optimal range for improving the intestinal barrier function in infant formulas.

[0083] Preferably, the amount of DHA is at least 0.4, preferably at least 0.5 wt%, based on the total fatty acids. Preferably, the amount of DHA is not more than 1 wt%, preferably not more than 0.7 wt%, based on the total fatty acids. Preferably, the nutritional composition comprises DHA in an amount of at least 0.5 wt%, preferably at least 0.7 wt%, more preferably at least 1 wt%, based on the total fatty acids. Preferably, the nutritional composition comprises DHA in an amount of 0.4 to 1 wt%, more preferably 0.5 to 0.7 wt%.

[0084] Preferably, the nutritional composition comprises EPA in an amount of at least 0.09 wt.%, preferably at least 0.1 wt.% and preferably not more than 0.4 wt.%, more preferably not more than 0.1 wt.%, based on total fatty acids. Preferably, the nutritional composition comprises EPA in an amount of 0.09 to 0.4 wt.%, more preferably 0.1 to 0.2 wt.%.

[0085] Preferably, the nutritional composition comprises at least 0.25 wt%, more preferably at least 0.5 wt% and preferably not more than 1 wt% ARA, based on total fatty acids. Preferably, the nutritional composition comprises 0.4 to 1 wt%, more preferably 0.5 to 0.7 wt% ARA.

[0086] Preferably, the nutritional composition comprises DHA in an amount of 0.4 to 1.0% by weight based on total fatty acids, and EPA in an amount of 0.09 to 0.4% by weight based on total fatty acids. More preferably, the nutritional composition comprises DHA in an amount of 0.5 to 0.7% by weight based on total fatty acids, and EPA in an amount of 0.1 to 0.2% by weight based on total fatty acids. It is particularly preferred that the nutritional composition comprises DHA in an amount greater than 0.5% by weight based on total fatty acids, and EPA in an amount greater than 0.1% by weight based on total fatty acids. Preferably, the nutritional composition comprises DHA, EPA and ARA in an amount of 0.4 to 1.0% by weight, 0.09 to 0.4% by weight and 0.25 to 1.0% by weight based on total fatty acids, respectively. More preferably, the nutritional composition comprises DHA, EPA and ARA in an amount of 0.5 to 0.7% by weight, 0.1 to 0.2% by weight and 0.5 to 0.7% by weight based on total fatty acids, respectively.

[0087] Preferably, the nutritional composition comprises 20 to 50 mg / 100 kcal of DHA and 4.3 to 10.8 mg / 100 kcal of EPA. More preferably, the nutritional composition comprises 25 to 33.5 mg / 100 kcal of DHA and 5.4 to 7.2 mg / 100 kcal of EPA. Most preferably, the nutritional composition comprises about 25 mg / 100 kcal of DHA and about 5.4 mg / 100 kcal of EPA. In these embodiments, ARA is optionally present. If present, the amount of ARA is preferably 12.5 to 50 mg, more preferably 25 to 33.5 mg and most preferably about 25 mg / 100 kcal. Preferably, the weight ratio of DHA / ARA is 0.9 to 2.

[0088] Preferably, the weight ratio of DHA / EPA / ARA is 1:(0.19-0.7):(0.9-2.0). Such amounts and / or ratios of DHA, EPA and ARA are optimal for further improving the intestinal barrier function, further improving the intestinal microbiota and / or treating or preventing infections, in particular intestinal infections. LC-PUFAs can be provided as free acids, in the form of triglycerides, in the form of diglycerides, in the form of monoglycerides, in the form of phospholipids or as a mixture of one or more of the above. Suitable sources of these LC-PUFAs are, for example, fish oils and oils from Mortierella alpina.

[0089] Preferably, the nutritional composition of the present invention comprises lipids, wherein the lipids comprise LC-PUFAs selected from DHA, EPA and ARA, and wherein the sum of DHA, EPA and ARA is at least 1% by weight based on total fatty acids, and wherein the lipids comprise DHA in an amount of 0.4 to 1.0% by weight based on total fatty acids, EPA in an amount of 0.09 to 0.4% by weight based on total fatty acids, and ARA in an amount of 0.25 to 1% by weight based on total fatty acids. In this embodiment, it is further preferred that the lipids comprise DHA in an amount of 0.5 to 0.7% by weight based on total fatty acids, EPA in an amount of 0.1 to 0.2% by weight based on total fatty acids, and ARA in an amount of 0.5 to 0.7% by weight based on total fatty acids. More preferably, lipids comprise at least 0.5% by weight of DHA, at least 0.1% by weight of EPA, and at least 0.5% by weight of ARA, all based on total fatty acids.

[0090] The combination of 2'-FL, butyrate, optionally 3'-GL and LC-PUFA, in particular EPA, DHA and / or ARA, has a further improving effect on health, in particular on improving the intestinal barrier function, on improving the immune system, on improving the intestinal microbiota and / or on treating or preventing infections, in particular intestinal infections.

[0091] Nutritional composition

[0092] The nutritional composition of the present invention is not human milk.

[0093] The nutritional composition of the present invention is for infants or young children.

[0094] The nutritional composition of the present invention preferably comprises lipid, protein and carbohydrate, and is preferably administered in liquid form. The nutritional composition of the present invention can also be in the form of dry food, preferably in powder form, with instructions for mixing the dry food, preferably powder, with a suitable liquid, preferably water. Therefore, the nutritional composition of the present invention can be in the form of a powder, suitable for reconstituted with water to provide a ready-to-drink nutritional composition, preferably a ready-to-drink infant formula, a follow-up formula or a toddler formula, more preferably a ready-to-drink infant formula or a follow-up formula. The nutritional composition of the present invention preferably comprises other fractions, such as vitamins, minerals, trace elements and other micronutrients, so that it becomes a complete nutritional composition. Preferably, according to international directives, infant formulas and follow-up formulas comprise vitamins, minerals, trace elements and other micronutrients.

[0095] The nutritional composition of the present invention preferably comprises lipids, proteins and digestible carbohydrates, wherein lipids provide 25 to 65% of the total calories, proteins provide 6.5 to 16% of the total calories, and digestible carbohydrates provide 20 to 80% of the total calories. Preferably, in the nutritional composition of the present invention, lipids provide 30 to 55% of the total calories, proteins provide 7 to 9% of the total calories, and digestible carbohydrates provide 35 to 60% of the total calories. To calculate the total calorie % of protein, the sum of the energy provided by protein, peptides and amino acids needs to be taken into account.

[0096] Preferably, lipid provides 3 to 7 g lipid / 100 kcal, preferably 3.5 to 6 g / 100 kcal, protein provides 1.6 to 4 g / 100 kcal, preferably 1.7 to 2.3 g / 100 kcal, and digestible carbohydrates provide 5 to 20 g / 100 kcal, preferably 8 to 15 g / 100 kcal of the nutritional composition. Preferably, the nutritional composition of the present invention comprises lipids providing 3.5 to 6 g / 100 kcal, proteins providing 1.7 to 2.3 g / 100 kcal, and digestible carbohydrates providing 8 to 15 g / 100 kcal of the nutritional composition.

[0097] Preferably, lipid provides 2.5 to 6.5 g lipid / 100 ml, preferably 2.5 to 4 g / 100 ml, protein provides 1 to 3 g / 100 ml, preferably 1 to 1.5 g / 100 ml, and digestible carbohydrates provide 3 to 13 g / 100 ml, preferably 5 to 10 g / 100 ml of the nutritional composition. Preferably, the nutritional composition of the present invention comprises lipids providing 2.0 to 6.5 g / 100 ml, proteins providing 1 to 3 g / 100 ml, and digestible carbohydrates providing 5 to 10 g / 100 ml of the nutritional composition.

[0098] Preferably, the lipids represent 15 to 45 wt%, preferably 20 to 30 wt%, based on the dry weight of the composition; the proteins represent 8 to 20 wt%, preferably 8.5 to 11.5 wt%, based on the dry weight of the composition; and the digestible carbohydrates represent 25 to 90 wt%, preferably 40 to 75 wt%, based on the dry weight of the composition. Preferably, the nutritional composition of the present invention comprises 20 to 30 wt% lipids, 8.5 to 11.5 wt% protein and 40 to 75 wt% digestible carbohydrates, all based on the dry weight of the composition.

[0099] The composition of the present invention preferably comprises lipid. Preferably, the composition of the present invention comprises at least one lipid selected from plant lipids. Preferably, the composition of the present invention comprises a combination of plant lipids and at least one oil selected from fish oil, algae oil, fungal oil and bacterial oil. The lipid of the nutritional composition of the present invention preferably provides 3 to 7g / 100kcal nutritional composition, preferably lipid provides 3.5 to 6g / 100kcal. When in liquid form, for example as a ready-to-eat liquid, the nutritional composition preferably comprises 2.0 to 6.5g lipid / 100ml, more preferably 2.5 to 4.0g / 100ml. Based on dry weight, the nutritional composition of the present invention preferably comprises 15 to 45% by weight, more preferably 20 to 30% by weight of lipid. Preferably, the nutritional composition of the present invention comprises at least one, preferably at least two, selected from the following lipid sources: rapeseed oil (such as rapeseed oil (colza oil), low erucic acid rapeseed oil and Canadian rapeseed (canola) oil), high oleic sunflower oil, high oleic safflower oil, olive oil, sophora flavescens oil (marineoil), microbial oil, coconut oil, palm kernel oil.

[0100] The nutritional composition of the present invention preferably comprises protein. The protein used in the nutritional composition is preferably selected from non-human animal proteins, preferably milk proteins, vegetable proteins, such as preferably soy proteins and / or rice proteins, and mixtures thereof. The nutritional composition of the present invention preferably contains casein and / or whey protein, more preferably bovine whey protein and / or bovine casein. Therefore, in one embodiment, the protein in the nutritional composition of the present invention comprises a protein selected from whey protein and casein, preferably whey protein and casein, preferably whey protein and / or casein from cow's milk. Preferably, based on the total protein, the protein comprises less than 5% by weight of free amino acids, dipeptides, tripeptides or hydrolyzed proteins. The nutritional composition of the present invention preferably comprises casein and whey protein, and the weight ratio of casein: whey protein is 10:90 to 90:10, more preferably 20:80 to 80:20, even more preferably 35:65 to 55:45.

[0101] In one embodiment, the protein used in the nutritional composition comprises hydrolyzed protein, preferably the protein used in the nutritional composition is hydrolyzed protein or in other words consists of hydrolyzed protein. The hydrolyzed protein may also contain free amino acids. Preferably, the hydrolyzed protein comprises hydrolyzed whey protein. In one embodiment, the protein used in the nutritional composition is free amino acids or in other words consists of free amino acids. Therefore, in a preferred embodiment, the nutritional composition of the present invention comprises 2'-FL and dietary butyric acid and optionally 3'GL, and also comprises hydrolyzed protein and / or free amino acids. Such a composition is preferably used for preventing or treating allergies, more preferably for preventing or treating cow's milk protein allergy.

[0102] The weight % protein is calculated based on the dry weight of the nutritional composition according to the invention by measuring the total nitrogen according to the Kjeldahl-method and using a conversion factor of 6.38 in the case of casein or 6.25 for proteins other than casein. The term "protein" or "protein component" as used in the present invention refers to the sum of proteins, peptides and free amino acids.

[0103] The nutritional composition of the present invention preferably comprises 1.6 to 4.0g protein / 100kcal nutritional composition, preferably 11.7 to 2.3g / 100kcal nutritional composition. Based on the total calorie meter, too low a protein content can lead to insufficient growth and development of infants and young children. Too high an amount can bring a metabolic burden to the kidneys of infants and young children, for example. When in liquid form, as a ready-to-feed liquid, the nutritional composition preferably comprises 1.0 to 3.0g, more preferably 1.0 to 1.5g protein / 100ml. Based on dry weight, the nutritional composition of the present invention preferably comprises 8 to 20% by weight, more preferably 8.5 to 11.5% by weight of protein, based on the dry weight of the total nutritional composition.

[0104] The nutritional composition of the present invention preferably comprises digestible carbohydrates providing 5 to 20 g / 100 kcal, preferably 8 to 15 g / 100 kcal. Preferably, in the nutritional composition of the present invention, the amount of digestible carbohydrates is 25 to 90 weight %, more preferably 8.5 to 11.5 weight %, based on the total dry weight of the composition. Preferred digestible carbohydrates are lactose, glucose, sucrose, fructose, galactose, maltose, starch and maltodextrin. Lactose is the main digestible carbohydrate present in human milk. The nutritional composition of the present invention preferably comprises lactose. Preferably, the nutritional composition of the present invention does not comprise a large amount of carbohydrates other than lactose. Compared with digestible carbohydrates such as maltodextrin, sucrose, glucose, maltose and other digestible carbohydrates with a high glycemic index, lactose has a lower glycemic index and is therefore preferred. The nutritional composition of the present invention preferably comprises digestible carbohydrates, wherein at least 35 wt. %, more preferably at least 50 wt. %, more preferably at least 60 wt. %, more preferably at least 75 wt. %, even more preferably at least 90 wt. %, most preferably at least 95 wt. % of the digestible carbohydrates are lactose. Based on dry weight, the nutritional composition of the present invention preferably comprises at least 25 wt. % lactose, preferably at least 40 wt. %, more preferably at least 50 wt. % lactose.

[0105] The nutritional composition of the present invention preferably comprises non-digestible oligosaccharides (NDO). The term "oligosaccharide" as used herein refers to a sugar having a degree of polymerization (DP) of 2 to 250, preferably a DP of 2 to 100, more preferably 2 to 60, and even more preferably 2 to 10. If an oligosaccharide having a DP of 2 to 100 is included in the nutritional composition of the present invention, this will result in that the composition may contain oligosaccharides having a DP of 2 to 5, a DP of 50 to 70, and / or a DP of 7 to 60. The term "non-digestible oligosaccharide" (NDO) as used in the present invention refers to an oligosaccharide that is not digested in the intestine by the action of acids or digestive enzymes present in the upper digestive tract of a person, such as the small intestine and the stomach, but is preferably fermented by the human intestinal microbiota. For example, sucrose, lactose, maltose, and maltodextrin are considered digestible.

[0106] Preferably, the non-digestible oligosaccharides of the present invention are soluble. The term "soluble" as used herein when referring to polysaccharides, fibers or oligosaccharides means that the material is at least soluble according to the method described by L Prosky et al., J. Assoc. Off. Anal. Chem. 71, 1017-1023 (1988).

[0107] β1,3'-galactosyl lactose can be present in the nutritional composition of the present invention in the form of itself or as part of a mixture of galacto-oligosaccharides (GOS), preferably β-galacto-oligosaccharides (BGOS). In a preferred embodiment, β1,3'-galactosyl lactose is present as part of a mixture of galacto-oligosaccharides. In one embodiment, the amount of Gal(β1-3)-Gal(β1-4)-Glc is greater than 20% by weight, based on total galacto-oligosaccharides.

[0108] Preferably, the nutritional composition of the present invention also comprises oligofructose (FOS). The term "oligofructose" used in the present invention refers to a carbohydrate composed of more than 50%, preferably more than 65% fructose units based on monomer subunits, wherein at least 50%, more preferably at least 75%, even more preferably at least 90% of the fructose units are linked together by β-glycosidic bonds, preferably β-2,1 glycosidic bonds. Glucose units may be present at the reducing end of the fructose unit chain. Preferably, the DP or average DP of oligofructose is in the range of 2 to 250, more preferably 2 to 100, even more preferably 10 to 60. The term "oligofructose" includes levan, hydrolyzed levan, inulin, hydrolyzed inulin and synthetic oligofructose. Preferably, the preparation comprises short-chain oligofructose with an average degree of polymerization (DP) of 3 to 6, more preferably hydrolyzed inulin or synthetic oligofructose. Preferably, the preparation comprises long-chain oligofructose with an average DP greater than 20. Preferably, the preparation comprises short-chain oligofructose and long-chain oligofructose simultaneously.Fructo-oligofructose suitable for use in the composition of the present invention is also easily commercially available, such as RaftilineHP (Orafti).Preferably, the nutritional composition of the present invention comprises at least 25mg FOS / 100ml, more preferably at least 40, even more preferably at least 60mg.Preferably, the composition comprises not more than 250mg FOS / 100ml, more preferably not more than 150mg / 100ml and most preferably not more than 100mg / 100ml.The amount of FOS is preferably 25 to 250g oligofructose / 100ml, preferably 40 to 150g / 100ml, more preferably 60 to 100g / 100ml.Preferably, based on dry weight, the nutritional composition of the present invention comprises at least 0.15 % by weight of FOS, more preferably at least 0.25 % by weight, even more preferably at least 0.4 % by weight.Preferably, based on the dry weight of total composition, the composition comprises not more than 1.5 % by weight of FOS, more preferably not more than 2 % by weight. The presence of FOS showed further improving effects on the microbiota and its SCFA production.

[0109] Preferably, the nutritional composition of the present invention comprises a mixture of galacto-oligosaccharides (including β1,3'-galactosyl lactose) and oligofructose. Preferably, the mixture of galacto-oligosaccharides and oligofructose is present in a weight ratio of 1 / 99 to 99 / 1, more preferably 1 / 19 to 19 / 1, more preferably 1 / 1 to 19 / 1, more preferably 2 / 1 to 15 / 1, more preferably 5 / 1 to 12 / 1, even more preferably 8 / 1 to 10 / 1, even more preferably in a ratio of about 9 / 1. When galacto-oligosaccharides have a low average DP and oligofructose has a relatively high DP, this weight ratio is particularly advantageous. Most preferably, the mixture of oligofructose with an average DP lower than 10, preferably lower than 6, and the oligofructose with an average DP higher than 7, preferably higher than 11, even more preferably higher than 20.

[0110] In a preferred embodiment, the nutritional composition of the present invention comprises a mixture of short-chain (sc) oligofructose and long-chain (lc) oligofructose. Preferably, the mixture of short-chain oligofructose and long-chain oligofructose is present in a weight ratio of 1 / 99 to 99 / 1, more preferably 1 / 19 to 19 / 1, even more preferably 1 / 10 to 19 / 1, more preferably 1 / 5 to 15 / 1, more preferably 1 / 1 to 10 / 1. Preferably, the mixture of short-chain oligofructose with an average DP lower than 10, preferably lower than 6, and the average DP is higher than 7, preferably higher than 11, even more preferably higher than 20.

[0111] In another preferred embodiment, the nutritional composition of the present invention comprises a mixture of short-chain (sc) oligofructose and short-chain (sc) oligogalactose. Preferably, the mixture of short-chain oligofructose and short-chain oligogalactose is present in a weight ratio of 1 / 99 to 99 / 1, more preferably 1 / 19 to 19 / 1, even more preferably 1 / 10 to 19 / 1, more preferably 1 / 5 to 15 / 1, more preferably 1 / 1 to 10 / 1. Preferably, the mixture of short-chain oligofructose and average DP is lower than 10, preferably lower than 6 short-chain oligogalactose.

[0112] The nutritional composition of the present invention preferably comprises 1.75 to 17.5 wt % of total indigestible oligosaccharides, more preferably 2.8 to 10.5 wt %, most preferably 4.2 to 7 wt %, based on the dry weight of the nutritional composition. Based on 100 ml, the nutritional composition of the present invention preferably comprises 0.25 to 2.5 g of total indigestible oligosaccharides, more preferably 0.4 to 1.5 g, most preferably 0.6 to 1 g, based on 100 ml of the nutritional composition. Lower amounts of indigestible oligosaccharides are less effective in improving intestinal barrier function, while too high amounts can cause side effects of bloating and abdominal discomfort. The total amount of indigestible oligosaccharides includes galacto-oligosaccharides (including β3'-GL), fructo-oligosaccharides and any other indigestible oligosaccharides that may further be present in the composition.

[0113] It is also important that the nutritional composition of the present invention does not have an excessive caloric density, yet still provides sufficient calories to feed the subject. Therefore, the liquid food preferably has a caloric density of 0.1 to 2.5 kcal / ml, more preferably a caloric density of 0.5 to 1.5 kcal / ml, even more preferably 0.6 to 0.8 kcal / ml and most preferably 0.65 to 0.7 kcal / ml.

[0114] application

[0115] Nutritional composition of the present invention is preferably infant formula, follow-up formula or toddler formula.The example of toddler formula is toddler milk (toddler milk), toddler formula and growing-up milk.More preferably, nutritional composition is infant formula or follow-up formula.Nutritional composition of the present invention can be advantageously used as complete nutrition for infant.Infant formula is defined as the formula for infant, and can be for example the starting formula that is intended for 0 to 6 months of age or 0 to 4 months of age infant.The follow-up formula is intended for infants from 4 or 6 months of age to 12 months of age.At this age, infants begin to wean and eat other foods.Toddler formula or toddler or growing milk or formula are intended for children from 12 to 36 months of age.Preferably, nutritional composition of the present invention is infant formula.

[0116] Infant formula, follow-on formula or toddler formula can be in liquid form, preferably ready-to-drink liquid, or in powder form. In one embodiment, infant formula, follow-on formula or toddler formula is in powder form, suitable for reconstitution with water to provide ready-to-drink infant formula, follow-on formula or toddler formula. It should be understood that when infant formula, follow-on formula or toddler formula of the present invention is in powder form, the amount of all ingredients including indigestible oligosaccharides, 2'-FL and 3'-GL in the formula is defined as the amount present after reconstitution of the powder with water, i.e., the amount is defined as mg / 100 ml ready-to-drink formula.

[0117] The nutritional composition of the present invention is for providing nutrition to an infant or a young child, preferably an infant, preferably an infant up to 12 months of age.

[0118] The infant formula, follow-on formula or toddler formula according to the invention is used for providing nutrition to an infant or a toddler, preferably an infant, preferably an infant up to 12 months of age.

[0119] The preferred embodiments described above for the infant formula, follow-on formula and toddler formula according to the invention also apply to the infant formula for use, the follow-on formula for use and the toddler formula for use according to the invention.

[0120] The invention also relates to a composition comprising 2-'FL, dietary butyrate and optionally 3-'GL or the composition of the invention for use as a medicament. Preferably, the composition is for improving intestinal health, in particular intestinal barrier function and intestinal maturation, for improving intestinal physiology, for improving intestinal barrier function, for improving intestinal microbiota, in particular for reducing intestinal pathogens, for treating or preventing infections, in particular intestinal infections and / or for treating and / or preventing allergies, and / or for inducing oral tolerance to allergens in infants.

[0121] Preferably, the composition is for improving the immune system, preferably for reducing Th2 responses.

[0122] Since the nutritional composition of the present invention has an improved effect on the intestinal barrier function, it will reduce the translocation of allergens, toxins and / or pathogens, thereby preventing and / or treating allergies and / or preventing or treating infections. Since it has also been found that it has an improved effect on intestinal alkaline phosphatase activity, the nutritional composition reduces intestinal pathogens, thereby preventing and / or treating infections, especially intestinal infections. Improved intestinal barrier maturation is further indicated by improvements in lactase maturation and intestinal cell proliferation. Improved microbiota, increased bifidobacteria, enhanced fermentation acidification and reduced pathogens were observed. In addition, improvements in the intestinal microbiota and / or immune system are beneficial for preventing and / or treating allergies and infections, especially intestinal infections. The effect on the immune system will have an effect on inducing oral tolerance to allergens.

[0123] The effects on both IL-10 and CCL20 levels indicate that human PBMC responses are unexpectedly improved in the presence of a combination of 2'-FL and butyrate, and even further improved when 3'-GL is present.

[0124] Since the nutritional composition of the present invention has an improved effect on reducing Th2 response, it can prevent and / or treat allergies.

[0125] The nutritional composition of the present invention is preferably used for providing nutrition to infants or young children, preferably infants, suffering from allergies or having an increased risk of suffering from allergies.

[0126] The present invention also relates to the use of the nutritional composition according to the present invention for providing nutrition to an infant or a young child, preferably for providing nutrition to an infant. BRIEF DESCRIPTION OF THE DRAWINGS

[0128] Figure 1:

[0129] Effects of different galactosyl lactose (GL) on DON-induced impairment of Caco-2 cell monolayer integrity. Figure 1A and 1B The transepithelial electrical resistance (TEER) of different GLs is shown. Figure 1C and 1D The translocation of Lucifer Yellow (LYF) to the basolateral compartment is shown. TEER is expressed as a percentage of the initial value and LYF is expressed in ng / cm 2xh is expressed in ng / ml. α3'-GL is Gal(α1-3)-Gal(β 1-4)-Glc; β3'-GL is Gal(β 1-3)-Gal(β 1-4)-Glc; β4'-GL is Gal(β 1-4)-Gal(β 1-4)-Glc'; β6'-GL is Gal(β 1-6)-Gal(β1-4)-Glc. Data are mean ± SE. *: p < 0.05 compared with the control; **: p < 0.01 compared with the control; ***: p < 0.001 compared with the control; ^: p < 0.05 compared with the DON control; ^^: p < 0.01 compared with the DON control; ^^^: p < 0.001 compared with the DON control.

[0130] Figure 2 :

[0131] Differential effects of GL on DON-induced increase in IL8 release from Caco-2 cells. IL-8 secretion is expressed as pg / ml and is the mean ± SE. α3'-GL is Gal(α1-3)-Gal(β 1-4)-Glc; β3'-GL is Gal(β 1-3)-Gal(β 1-4)-Glc; β4'-GL is Gal(β 1-4)-Gal(β 1-4)-Glc; β6'-GL is Gal(β 1-6)-Gal(β 1-4)-Glc. Data are the mean ± SE. *: p<0.05 compared with the control; **: p<0.01 compared with the control; ***: p<0.001 compared with the control; ^: p<0.05 compared with the DON control; ^^: p<0.01 compared with the DON control; ^^^: p<0.001 compared with the DON control.

[0132] Example

[0133] Example 1: Infant formula with 2'-FL and dietary butyrate improves intestinal alkaline phosphatase expression

[0134] The two infant formulas were subjected to an in vitro digestion step and after the in vitro digestion step, the effects on intestinal barrier maturation, in particular alkaline phosphatase (AP) maturation, were examined. AP is an intestinal enzyme expressed and secreted by intestinal epithelial cells and used as a differentiation marker. AP plays a key role in intestinal homeostasis and innate immune defense by dephosphorylating harmful substances such as the microbial ligand lipopolysaccharide (endotoxin).

[0135] The control infant formula is a non-fermented infant formula supplemented with non-digestible oligosaccharides (scGOS / lcFOS) in an amount of 0.8 mmg / 100 ml when in ready-to-drink form. scGOS is derived from Vivinal GOS and lcFOS is derived from Raftiline HP. The fat components are mainly vegetable oils, fish oils and microbial oils (source of arachidonic acid). The amount of butyric acid is less than 0.05% by weight, based on total fat.

[0136] The active infant formula was a partially fermented infant formula similar to that of Example 8, i.e., it additionally contained 0.1 g of 2'FL, the lipid component comprised about 50 wt% of bovine milk fat, and had about 1.5 wt% butyric acid based on total fatty acids, about 3.4 g fat / 100 ml, about 0.28 wt% lactic acid based on dry weight, and when in ready-to-drink form about 25 mg 3'-GL / 100 ml.

[0137] In vitro digestion:

[0138] Infant formula was prepared at 13.7% (w / v) in MiliQ water and 35 ml was transferred to a bioreactor of a computer-controlled semi-dynamic gastrointestinal model simulating infant conditions. Each reactor was equipped with a pH electrode and four dosing lines. Each dosing line was connected to a pump that added the following substances: a) hydrochloric acid, 0.25 M; and b) sodium bicarbonate, 0.5 M, for pH control; or c) simulated gastric fluid (SGF); d) simulated intestinal fluid (SIF). The pH was controlled by standardizing the pH to 6.8 at the beginning of digestion and then gradually reducing the pH to 4.3 during the 2-hour gastric phase. During the intestinal digestion phase, the pH was gradually raised from 6.5 to 7.2 over 2 hours. At t = 0 (beginning of digestion), 5.8 ml of simulated saliva (100 mM NaCl, 30 mM KCl, 1.4 mM CaCl2, 14 mM NaHCO3, 0.6 mg / ml α-amylase from Aspergillus oryzae (SIGMA, A9857)) was added as a bolus. From t = 0, 12.25 ml of SGF (100 mM NaCl, 30 mM KCl, 1.4 mM CaCl2, 50 mM sodium acetate, 0.125 mg / ml pepsin from porcine gastric mucosa (SIGMA, P7012) and 0.05 mg / ml lipase from Rhizopus oryzae (Amano)) were gradually added until t = 120 (end of the gastric phase). The pH was increased to 6.5, the continuous enteric phase was started, and 31.5 ml SIF (100 mM NaCl, 10 mM KCl, 1.7 mM CaCl2, 0.17 mg / ml trypsin from bovine pancreas (SIGMA, T9201), 0.18 mg / ml chymotrypsin from bovine pancreas (SIGMA, C4129), 0.09 mg / ml pancreatic lipase from porcine pancreas (SIGMA, L0382), 1.42 mg / ml taurocholate (SIGMA, 86339) and 0.6 mg / ml tauroursodeoxycholate (SIGMA, T0266)) was gradually added. At the end of the simulated gastrointestinal digestion, 5 ml of sample was taken, mixed with 5 ml of enzyme inhibitor buffer (0.1 M sodium phosphate, pH 5.5, 0.58 mg / ml trypsin-chymotrypsin inhibitor from soybean (Glycine max) (SIGMA, T9777), 34.5 μg / ml of Orlistat (SIGMA, O4139)), quick-frozen, and stored at -20°C until further use.

[0139] Cell differentiation

[0140] The human intestinal cell line C2BBe1 ( CRL-2102T M ) were seeded at 5000 cells / well in a 96-well Nunc TM Edge plates and grown to confluence in Dulbecco's modified Eagle's medium (Catalog No. 30-2002) with 10% fetal bovine serum, 1% penicillin / streptomycin and 0.01 mg / ml human transferrin. After reaching confluence, the medium was replaced with predigested infant formula diluted in medium without fetal bovine serum at final concentrations of 0.34%, 0.17% and 0.085% (w / v) in quadruplicate and incubated at 37°C, 5% CO2 for 96 hours, and recovered with diluted predigested infant formula after 48 hours. At the end of the incubation period, 50 μl of medium was collected from each well, the four aliquots were combined and stored at -20°C until AP activity was measured. All wells were then washed with ice-cold phosphate buffered saline and 100 μl of 50 mM Tris-HCL, 150 mM NaCl, 0.5% triton-100, pH 7.0, was added to each well. After incubation on ice for 30 minutes, cell lysates were collected and the protein content was determined using the ThermoFischer, Pierce BCA protein assay kit according to the manufacturer's instructions. AP activity was determined by the Biovision Alkaline Phosphatase Activity Colorimetric Assay Kit according to the manufacturer's instructions. AP activity was expressed as units / mg protein.

[0141] result

[0142] The AP activity in the intestinal epithelial cells treated with the predigested infant formula of the present invention was statistically significantly increased (p<0.05, t-test) when compared to the intestinal epithelial cells treated with the predigested control formula. The effect was dose-dependent and significantly different at all concentrations tested. At infant formula concentrations of 0.34%, 0.17% and 0.085% (w / v), the extracellular AP activity increased by 43%, 36% and 32%, respectively, compared to the control formula, see Table 1. This increase in extracellular AP activity indicates improved maturation of the intestinal barrier function and improved defense against enteric pathogens.

[0143] Table 1: AP activity in intestinal enterocytes exposed to pre-digested control or experimental formulations in mU / mg protein.

[0144] Dilution(x) IF concentration (g / 100ml) Control formula Test formulations average SEM average SEM P＊ 40 0.34 0.84485 0.08992 1.20802 4.601E-02 0.023 80 0.17 1.16073 0.05346 1.57569 6.284E-02 0.007 160 0.085 1.49291 0.11494 1.96274 5736E-02 0.022

[0145] *p-values ​​were determined by t-test, 2-tailed, two-sample, equal variance.

[0146] Example 2: Infant formula with 2'-FL and dietary butyrate improves intestinal lactase expression and cell proliferation

[0147] The nutritional composition of Example 1 was tested in an experiment similar to Example 1. A 13.6% (w / v) formula was used instead of a 13.7 formula. In the gastric stage, 16.6 mg / ml of rabbit lipase (Germ, REG.340) was used instead of the lipase from Rhizopus oryzae. In the intestinal stage, 0.06 mg / ml of pancreatic lipase from porcine pancreas (SIGMA, L0382) and 3.5 mg / ml of porcine pancreatic lipase (SIGMA L0126) were used instead of 0.09 mg / ml of pancreatic lipase from porcine pancreas (SIGMA, L0382).

[0148] Lactase activity was measured by mixing 30 μl of cell lysate with 30 μl of assay buffer (maleic acid 0.625 M, lactose 0.12 M, pH 6.0) and incubating for 4 hours at 37° C. The resulting glucose was quantified. Lactase activity was expressed as μmol glucose / min / mg.

[0149] It was found that lactase activity was significantly increased when cells were treated with the pre-digested experimental test infant formula compared to the pre-digested control formula, see Table 2.

[0150] Table 2: Lactase activity in intestinal enterocytes exposed to pre-digested control or experimental formulations in mU / mg protein.

[0151]

[0152] *p-values ​​were determined by t-test, 2-tailed, two-sample, equal variance.

[0153] In differentiated enterocytes, lactase activity increased, followed by an increase in sucrase activity, after which brush border lactase activity began to decline. Since cells showed no lactase activity when measured (data not shown), increased lactase activity indicates a more differentiated cell state.

[0154] Cell proliferation assay

[0155] Crypt-like human colon cancer HT-29 cells were cultured at 5.10 4 96-well Nunc plated in DMEM TMEdge plates were grown in DMEM with 10% FCS, 1% penicillin / streptomycin and 1 g / L galactose. Cells were allowed to attach for 30 hours, after which the medium was replaced with digested IF diluted in medium without fetal bovine serum at final concentrations of 0.23%, 0.17% and 0.085% (w / v) in triplicate. After 72 hours of incubation, different cell proliferation rates resulted in different cellular protein contents, which were measured by lysing the cells and subsequently measuring protein content using the Thermo Fischer, Pierce BCA protein assay kit according to the manufacturer's instructions.

[0156] As shown by the increased cellular protein content in cells treated with the predigested experimental test infant formula, cell proliferation was significantly increased compared to the predigested control formula (Table 3).

[0157] Table 3: Proliferation of intestinal enterocytes (ug of cell protein / well) exposed to pre-digested control or experimental formulations, expressed in mU / mg protein.

[0158]

[0159] *p-values ​​were determined by t-test, 2-tailed, two-sample, equal variance.

[0160] To fulfill its function as a barrier to the external environment, enterocytes must be constantly renewed. The growth and renewal of enterocytes depends on cells that proliferate in the intestinal crypts. Therefore, it is expected that stimulating the rate of cell proliferation will support intestinal barrier function.

[0161] Example 3: 3'-GL and 2'-FL prevent intestinal barrier damage and prevent increased permeability

[0162] β1,3'-galactosyl lactose (β3'-GL), β1,4'-galactosyl lactose (β4'-GL) and β1,6'-galactosyl lactose (β6'-GL) were obtained from Carbosynth (Berkshire, UK). α1,3'-galactosyl lactose (α3'-GL) was obtained from Elicityl (Crolles, France). Purified deoxydivalenol (DON) (D0156; Sigma Aldrich, St Luis, MO, USA) was dissolved in pure ethanol and stored at -20°C. Human epithelial colorectal adenocarcinoma (Caco-2) cells were obtained from the American Type Tissue Collection (code HTB-37) (Manasse, VA, USA, passages 90-102).

[0163] Caco-2 cells were used according to established methods. Briefly, cells were cultured in Dulbecco's modified Eagle's medium (DMEM) and cultured at 0.3 × 10 5 Cells were seeded at a density of 1.5 × 10 cells / mL into 0.3 cm 24-well plates with polyethylene terephthalate membranes (BD Biosciences, Franklin Lakes, NJ, USA). 2 The Caco-2 cells were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37°C. After 17-19 days in culture, confluent monolayers were obtained with an average transepithelial electrical resistance (TEER) exceeding 400 Ω cm measured by a Millicell-Electrical Resistance System voltmeter (Millipore, Temecula, CA, USA). 2 .

[0164] Caco-2 cell monolayers were therefore grown in a transwell system, which is a model of intestinal barrier function. Before exposure to the fungal toxin deoxynivalenol (DON), which is a trigger and model compound that damages the intestinal barrier, the monolayer was pretreated with different GLs (including β3'-GL, α3'-GL, β4'-GL and β6'-GL) at a concentration of GL of 0.75% by weight for 24 hours. DON was diluted to a final concentration of 4.2 μM in complete cell culture medium and added to the top and basolateral sides of the transwell insert. This DON concentration did not affect the viability of Caco-2 cells. Cultivate with DON for 24 hours.

[0165] Transepithelial resistance (TEER) and fluorescein (LY) permeability measurements were performed to study barrier integrity. For TEER measurements, a Millicel-ERS voltmeter connected to a pair of chopstick electrodes was used to measure TEER values. The results are expressed as a percentage of the initial value. For intercellular tracer flux determination, membrane-impermeable fluorescein (LY) (Sigma, St Luis, MO, USA) was added to the apical compartment in the transwell plate at a concentration of 16 μg / ml, kept for 4 hours, and the intercellular flux was determined by measuring the fluorescence intensity in the basolateral compartment using a spectrophotometric fluorometer (FLUOstar Optima, BMG Labtech, Offenburg, Germany) with excitation wavelength and emission wavelength set to 410nm and 520nm, respectively. Interleukin-8 (IL-8 or CXCL8)--it is a typical marker of inflammation--in response to the release of treatment is quantified in the culture medium on the apical and basolateral sides of the Caco-2 transwell insert. CXCL8 concentration was measured using the human L-8 ELISA assay (BD Biosciences, Pharmingem, San Diego, CA, USA) according to the manufacturer's instructions. For more details on materials and methods, see Akbari et al., 2016, Eur J Nutr. 56(5): 1919-1930.

[0166] The results are shown in Figure 1A , B, C and D and Figure 2 Figure 1 shows the effects of different galactosyl lactose (GL) on DON-induced damage to the integrity of Caco-2 cell monolayer. Figure 1A and 1B The transepithelial electrical resistance (TEER) of different GLs is shown. Figure 1C and 1D The translocation of Lucifer Yellow (LYF) to the basolateral compartment is shown. TEER is expressed as a percentage of the initial value and LYF is expressed in ng / cm 2 xh indicates. α3'-GL is Gal(α1-3)-Gal(β 1-4)-Glc; B3'-GL is Gal(β 1-3)-Gal(β 1-4)-Glc; β4'-GL is Gal(β 1-4)-Gal(β 1-4)-Glc'; β6'-GL is Gal(β 1-6)-Gal(β 1-4)-Glc. Data are mean ± se. *: p < 0.05 compared with the control; **: p < 0.01 compared with the control; ***: p < 0.001 compared with the control; ^: p < 0.05 compared with the DON control; ^^: p < 0.01 compared with the DON control; ^^^: p < 0.001 compared with the DON control.

[0167] from Figure 1A -D, the presence of DON disrupted the barrier function, as shown by the decreased TEER values ​​and increased LY flux in the DON control sample. In addition, the presence of DON increased CXCL8 (IL-8) release, as shown in Figure 2 as shown in . Figure 1A -D further showed that the presence of β3'-GL prevented DON-induced loss of epithelial barrier integrity, as measured by increased TEER values ​​and reduced DON-affected LY flux across the intestinal epithelial cell monolayer. β4'-GL and β6'-GL did not show significant effects on intestinal epithelial cell barrier function. Interestingly, β3'-GL (i.e., galactosyl lactose with β1-3 glycosidic bonds) can effectively protect intestinal barrier function, while α3'-GL (i.e., galactosyl lactose with α1-3 glycosidic bonds) cannot prevent DON-induced intestinal barrier destruction. In contrast, all galactosyl lactose were able to reduce DON-induced IL-8 release, as Figure 2 as shown in .

[0168] These results indicate that β3'-GL (also referred to herein as β1,3'-galactosyl lactose or Gal(β1-3)-Gal(β1-4)-Glc) has a specific effect on protecting the barrier function of intestinal epithelial cells, particularly under challenging conditions, and the effect is beyond and / or independent of the effect of preventing the inflammatory response, and / or the effect on or through the effect of the microbiota. Therefore, these results indicate that β3'-GL has the effect of increasing intestinal barrier function and / or preventing and / or treating intestinal barrier damage. In addition, these results also indicate the effect of β3'-GL on treating, preventing and / or alleviating conditions associated with toxin exposure in subjects, particularly when the toxin is a tricothecene toxin, and more particularly when the toxin is deoxynivalenol.

[0169] In a separate experiment, the effect of 2'fucosyllactose on TEER and LYF flux was determined in the same model. 2'FL was tested at a concentration of 1 mg / ml and was found to statistically significantly prevent DON-induced decreases in TEER and increases in LYF, see Table 4. This suggests that 2'-FL has a favorable effect on intestinal barrier function. Thus, this example shows that when 2'-FL and β3'-GL are combined, the effect on intestinal barrier function in the composition is further improved.

[0170] Table 4: Effects of 2'-FL on DON-induced impairment of Caco-2 cell monolayer integrity

[0171]

[0172] ***p<0.001 compared with the control without DON.

[0173] ^^p<0.01 compared with control with DON; ^p<0.05 compared with control with DON.

[0174] Example 4: Butyric acid improves intestinal barrier function

[0175] To examine the effect of butyrate on intestinal barrier function.

[0176] method

[0177] T84 human intestinal epithelial cells are commonly used to study intestinal barrier integrity in vitro. T84 cells (ATCC, USA) are cultured on 12mm transwell inserts (0.4 μm, Corning Costrar, USA) in DMEM-F12 glutamax with penicillin-streptomycin (100 IU / ml), supplemented with 5% FBS-HI. T84 cells are used after reaching fusion for 14 days. The T84 monolayer cultured on the transwell filter is pre-cultivated for 48 hours with or without butyrate. These samples are then cultivated for another 48 hours in the presence of IL-4 (25 ng / ml). IL-4 is added to the basolateral compartment; the culture medium and additives are replaced every 24 hours.

[0178] Transepithelial electrical resistance (TEER; Ω x cm) was measured by using an epithelial volt-ohmmeter (EVOM; World Precision Instruments, Germany). 2 ) to assess epithelial barrier integrity.

[0179] The results are shown in Table 5, where relative TEER values ​​are given. Columns 48h and 96h are the TEER increase relative to the t=0 value. IL-4 treatment disrupted the intestinal barrier function; however, this disruption was ameliorated in the presence of butyrate.

[0180] Table 5: Effect of butyrate on intestinal barrier function.

[0181] Butyrate concentration (mM) %TEER at 48 hours %TEER(IL-4) at 96 hours - 17(12) 0(4) 4 79(25) 26(16)

[0182] Thus, this example demonstrates that when 2'-FL is combined with dietary butyrate and optionally β3'-GL, the effect on the intestinal barrier function in the composition is further improved.

[0183] Example 5: 2'-FL and (3'-GL and / or butyrate) have different effects on the immune system

[0184] Immune cell activation and responses were determined by culturing human peripheral blood mononuclear cells (PBMCs) in the presence or absence of 2'-FL, 3'-GL and butyrate, with or without T cell-specific stimulation.

[0185] Materials and methods

[0186] Isolation of PBMC from healthy donors: Human peripheral blood mononuclear cells (PBMC) from healthy donors were isolated from buffy coats (Sanquin, Amsterdam, the Netherlands). PBMC were obtained by centrifugation using Leucosep tubes (Greiner Bio-One). PBMC were collected and washed in PBS (Gibco, Thermo Fisher Technologies) + 2% heat-inactivated FCS (Invitrogen), followed by hypotonic lysis of erythrocytes with sterile lysis buffer (0.15M NH4Cl, 0.01M KHCO3 and 0.1mM EDTA, pH 7.4 at 4°C, all from Merck, Darmstadt, Germany). After lysis, PBMCs were resuspended in freezing medium (70% RPMI 1640 medium (Gibco, Thermo Fisher Technologies) supplemented with 10% heat-inactivated FCS and 100 U / ml penicillin-streptomycin, 20% heat-inactivated FCS and 10% DMSO (Sigma)) and stored frozen.

[0187] PBMC activation model: PBMC (0.2·10 6 Cells were cultured in 96-well flat-bottom plates (Corning) with 2'-FL (Jennewein), 3'-GL (0-0.3% w / v; Carbosynth) or sodium butyrate (0.2 mM; Sigma Aldrich) and combinations thereof for 24 h. Subsequently, the cells were additionally subjected to CD3 / CD28 activation (Pelicluster CD3 and Pelicluster CD28, Sanquin) for 24 h. After incubation, IFNy was measured in the supernatant by ELISA (see below). To determine cell activity after stimulation, PBMCs were incubated with the cell proliferation reagent WST-1 (10 μl; Roche) and / or 10% Triton (5 μl; negative control). After 3 h, the absorbance was measured at OD450 nm and OD650 nm, and the cell activity was calculated according to the manufacturer's instructions.

[0188] IFNy production by PBMCs: PBMCs were incubated with the indicated reagents, supernatants were collected after incubation, and mediator levels were measured using a human IFNy ELISA kit (R&D Systems Europe Ltd.) according to the manufacturer's instructions.

[0189] PBMC produces cytokines: PBMC is cultivated with specified reagents. After cultivation, supernatant is collected and IL2, IL6, IL10, IL13, IL21, TNFα, IFNγ, MIF, CCL1, CCL13, CCL17, CCL20, CCL22 and CXCL8-11 levels are measured by performing a validated multiple immunoassay based on Luminex technology (xMAP, Luminex Austin TX USA). Collected with Biorad FlexMAP3D (Biorad laboratories, Hercules USA) in combination with xPONENT software version 4.2 (Luminex). Data were analyzed by 5 parameter curve fitting using Bio-Plex Manager software version 6.1.1 (Biorad).

[0190] After PBMC stimulation, cell culture supernatants were collected and cytokine responses were then measured to test the immune response of the cells. The cytokine levels measured under stimulation conditions were corrected to (low) cytokine levels measured under non-stimulation conditions. In addition, since each donor responds to T cell stimulation with its own efficiency, the individual index of cytokine responses is calculated by dividing the response induced by intervention by the stimulation response of basal.

[0191] IL2, IL6, TNF-α, CCL1, CCL17, and CCL20 are generally believed to be associated with inflammation and / or proliferation. IFN-γ, CXCL9, CXCL10, and CXCL11 are believed to be associated with Th1 responses. IL13, CCL13, and CCL22 are believed to be associated with Th2 responses. IL10 and Galectin-9 are believed to be associated with Treg effects, and IL21 is believed to be associated with B cell effects.

[0192] Statistical analysis: Paired one-tailed (Wilcoxon) t-test was used to compare the CD3 / CD28 stimulation group with the control group, and p < 0.05 was considered to be a significant difference.

[0193] The relative mean ± SEM of the measured and calculated values ​​under stimulation conditions were statistically tested using paired two-tailed (Wilcoxon) t-tests, with p < 0.05 considered significant. The calculated values ​​of the combined effects of the individual components were based on the measured values ​​of each donor.

[0194] Immune cell activity measured by WST increased significantly 24 hours after addition of 2'-FL, whereas decreased activation was detected after addition of 3'-GL. Addition of butyrate did not affect immune cell metabolic activity either under unstimulated conditions or under T cell stimulation conditions (CD3 / CD28).

[0195] The addition of 2'-FL altered the cytokine response, while the addition of 3'-GL did not result in the same changes. Interestingly, the addition of 3'-GL and 2'-FL seemed to significantly improve the performance of 2'-FL. Furthermore, differences were found between the responses produced by 3'-GL and 2'-FL and the metabolic activity of butate on the cells relative to IFN-γ production, suggesting the presence of other immune responses.

[0196] Overall, it was concluded that the total pool of isolated human PBMCs is a diverse pool of immune cells that respond directly and differently to the HMOs provided. Although the cells became more metabolically active, cytokine production in the presence of 2'-FL was not equal to that in the presence of 3'-GL, indicating a differential immune response. The same results were observed for the interaction of 2'-FL and butyrate.

[0197] Results for 2'-FL and butyrate

[0198] Interleukin 10 (IL10) is not a cell type specific cytokine, but is widely expressed by many immune cells. The induction of IL10 usually occurs with other proinflammatory cytokines, although the pathways that induce IL10 may negatively regulate these proinflammatory cytokines. IL10 plays a central role in infection by limiting / regulating the immune response to pathogens, thereby preventing damage to the host. Therefore, IL10 is generally considered to be a regulatory cytokine. IL 10 levels were measured in peripheral blood mononuclear cell (PBMC) cultures from 10 human donors.

[0199] Table 6: IL10 levels in PBMCs under non-stimulated conditions, expressed as relative (normalized) values ​​compared to blank values ​​(blanc), thereby correcting for donor variations.

[0200]

[0201] *: Paired two-tailed (Wilcoxon) t-test, p<0.05 when compared with calculated values.

[0202] In human PBMCs co-cultured with 0.2% 2'-FL, a significant increase in IL10 levels was detected compared to the blank control (p < 0.05), while the addition of butyrate had no effect on IL10 levels. The combination of 0.2% 2'-FL and 0.2 mM butyrate significantly increased IL10 levels compared to the blank control and 0.2 mM butyrate. Interestingly, the combination of 2'-FL and butyrate increased IL10 to a level higher than theoretically expected based on the individual components, see Table 6. This suggests that the regulatory capacity of human PBMCs is unexpectedly and beneficially increased in the presence of the combination of 2'-FL and butyrate compared to the single components.

[0203] Typically, CCL20 and CCR6 play a role in recruiting immature DCs and their precursors to potential antigen entry sites. Depending on the microenvironment of the tissue (e.g., the local presence of TGF-β, IL10, or IL15), immune cells can acquire functional CCR6 and migrate to sites where CCL20 is produced. CCL20 was shown to rapidly induce newly isolated T lymphocyte subsets to adhere firmly to intercellular adhesion molecule-1. Therefore, regulation can be obtained by regulating CCL20 under non-stimulatory conditions.

[0204] Table 7: CCL-20 levels under non-stimulated conditions, expressed as relative values ​​to correct for donor variation

[0205]

[0206] Paired two-tailed (Wilcoxon) t-test: *p<0.05, **p<0.01 when compared with calculated values.

[0207] Compared to the blank control, elevated levels of CCL20 were detected in human PBMCs exposed to 2'-FL, while the addition of butyrate or 3'-GL alone had no statistically significant effect on CCL20 levels. Cultivation of human PBMCs with a combination of 2'-FL and butyrate induced significantly higher CCL20 levels compared to the blank and butyrate alone. In this combination of 2'-FL and butyrate, the additional presence of 3'-GL further increased CCL20 levels. Unexpectedly, the observed levels of the combination of 2'-FL and butyrate were significantly higher than the levels that could be calculated based on the single components. This was also the case when the observed values ​​for the combination of 2'-FL, butyrate and 3'-GL were compared to the theoretically calculated values ​​based on the single components, see Table 7.

[0208] These data indicate that the addition of 2'-FL and butyrate affects the immune response of human PBMCs. The additional presence of 3'-GL further improves the immune response. The total pool of isolated human PBMCs is a diverse pool of immune cells that respond directly and differently to the HMOs provided. The changes in IL-10 and CCL20 levels detected both indicate that the human PBMC response is unexpectedly improved in the presence of a combination of 2'FL and butyrate, and even further improved in the presence of 3'GL.

[0209] Results of 2-'FL, 3'-GL and their combination

[0210] The effects of co-culture with 2'FL and 3'GL and their combination on PBMC cultures from 10 human donors were investigated. First the effect of atopic stimulation of T cells via CD3 / CD28 was determined.

[0211] After stimulating human PBMC, cell culture supernatants were collected and cytokine responses were measured to test the immune response of the cells. After T cell-specific stimulation with CD3 / CD28, several cytokines were detected in the cell supernatants using Luminex technology. Th2 type cytokines IL-4 and IL-13, chemokine CCL17 increased significantly, showing strong T cell stimulation (Table 8).

[0212] Table 8: Cytokine IL-4, IL-13 and chemokine CCL17 levels measured in cell culture supernatants of PBMCs after stimulation with CD3 / CD28 compared to no stimulation conditions (pg / ml)

[0213]

[0214] Paired one-tailed (Wilcoxon) t-test: stimulation vs. no stimulation, *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001

[0215] Subsequently, to test the direct effect of specific compounds on PBMC activity, cells were activated with CD3 / CD28 for 24 hours after pre-incubation with either 2'-FL, 3'-GL and their combination for 24 hours. In addition, since each donor responds to T cell stimulation with its own efficiency, the individual index of cytokine response was calculated by dividing the intervention-induced response by the basal stimulation response (blank set to 1). In this way, interventions were studied in 10 different donors.

[0216] IL-4 and IL-13 are closely related cytokines that are known to regulate many aspects of allergic inflammation. They play an important role in regulating the responses of lymphocytes, myeloid cells, and non-hematopoietic cells. For example, in T cells, IL-4 induces the initial CD4 T cells differentiate into Th2-type T cells; in B cells, IL-4 drives immunoglobulin (Ig) class switching to IgG1 and IgE; in macrophages, IL-4 and IL-13 induce alternative macrophage activation.

[0217] Table 9: Relative levels of IL-4, IL-13 and CCL17 under CD3 / CD28 stimulation conditions using 2'-FL and 3'-GL or in combination.

[0218]

[0219]

[0220] * Paired one-tailed (Wilcoxon) t-test: p<0.05 when compared to the effect calculated for 2'-FL+3'-GL.

[0221] T cell stimulation of human PBMCs resulted in a significant increase in IL-4 and IL-13. Pre-incubation of cells with 2'-FL had no effect on the levels of IL-4 and IL-13 compared to the control. However, a decrease was detected in the presence of 3'-GL compared to the control. In addition, the combination of 2'-FL and 3'-GL induced significantly lower levels of IL-4 and IL-13 compared to the control and 2'-FL. Interestingly, these reduced IL-4 and IL-13 levels were significantly lower than the levels that could be expected based on the calculations of the respective effects of 2'-FL and 3'-GL, see Table 9.

[0222] These data show an unexpected reduction in Th2-type responses to T cell stimulation within the total PBMC population in the presence of a combination of 2'-FL and 3'-GL compared to 3'-GL or 2'FL alone.

[0223] Cytokines regulate cellular responses at the transcriptional level, while chemokines play a role in recruiting inflammatory cells to the site of inflammation. Chemokine CCL17 (thymus and activation-regulated chemokine) is an effective chemoattractant for Th2 lymphocytes and is considered to play an important role in inflammatory diseases such as allergies. For example, serum CCL17 levels clearly reflect the disease activity of atopic dermatitis, which is considered to be an inflammatory skin disease dominated by Th2, especially in the acute phase.

[0224] Human T cell stimulation leads to a significant increase in CCL17, see Table 9. Although pre-cultivation with 2'-FL or 3'-GL has no significant effect on the CCL17 levels in T cell stimulated PBMCs, a significant reduction in CCL17 levels was detected when 2'-FL and 3'-GL were used to pre-cultivate activated PBMCs compared to 2'-FL and 3'-GL alone. Based on the changes induced by each component compared to the control level, the expected changes when the intervention is combined can be calculated. Interestingly, when T cell stimulated PBMCs were cultured in the presence of a combination of 2'-FL and 3'-GL, lower CCL17 levels were induced than expected. The changes in CCL17 levels indicate that the Th2 type response to T cell stimulation in the total PBMC population is unexpectedly further reduced in the presence of a combination of 2'-FL and 3'-GL. These CCL17 data are consistent with IL-4 and IL-13 data.

[0225] The total pool of isolated human PBMCs is a diverse pool of immune cells that can respond directly and differently to the HMOs provided. Although the cells became more metabolically active, 2'-FL exposure alone did not significantly affect the levels of Th2 mediators IL-4, IL-13, and CCL17, while 3'-GL exposure alone resulted in reduced levels of these mediators. Interestingly, simultaneous exposure to 2'-FL and 3'-GL statistically significantly reduced IL-4, IL-13, and CCL17 levels, thereby reducing Th2-type responses. These data suggest that the addition of 3'-GL and 2'-FL has the potential to reduce the occurrence of allergies.

[0226] Example 6: 2'-FL increases butyrate formation by the microbiota, special Especially if GOS still exists

[0227] Fecal samples from healthy 3-month-old infants born by caesarean section, exclusively breast-fed, with no history of antibiotic use, were used as inoculum to simulate quad- Infant gut microbiota in the colonic compartment of a dynamic model of the human gastrointestinal tract consisting of four parallel runs units, and each unit consists of 3 reactors simulating the stomach and small intestine, proximal colon and distal colon.

[0228] The SCFA profiles showed that acetate was the most abundant in the distal colon, followed by propionate (Table 10). In the presence of scGOS / lcFOS and scGOS / lcFOS / 2'-FL, the concentrations of acetate and propionate were higher than in the control group and the unit supplemented with 2'-FL. Similar observations were seen in the proximal colon (data not shown). Interestingly, butyrate was produced earlier in the distal colon and at higher concentrations in the presence of 2'-FL and scGOS / lcFOS / 2'-FL relative to the control and scGOS / lcFOS groups. In the presence of scGOS / lcFOS / 2'-FL and scGOS / lcFOS, the levels of isobutyrate, a branched-chain SCFA produced by proteolytic fermentation, were reduced in the distal colon.

[0229] Table 10: The expression of 1% β-catenin in unsupplemented (control) and supplemented (control) 1% β-catenin in unsupplemented (control) and supplemented (D1-D3), D4-D11 and D12-D15. Short-chain fatty acids produced in the distal colon of the cell.

[0230]

[0231]

[0232] Glycogen profile data indicated that 2'-FL was not metabolized when supplemented alone but was utilized only in the presence of scGOS / lcFOS, where it was metabolized slowly in both the proximal and distal colon. All other carbohydrates, including scGOS, were consumed in the proximal colon within the first hour.

[0233] 2'-FL was shown to ferment only in the presence of other GOS, particularly GOS / lcFOS, thereby forming a microbiota ecosystem that is believed to have health benefits.

[0234] scGOS / lcFOS / 2'-FL promoted the production of butyrate, an important SCFA for intestinal barrier function. scGOS / lcFOS / 2'-FL resulted in unexpectedly low levels of isobutyrate, suggesting lower proteolytic activity in the colon.

[0235] Example 7: Inhibition of pathogens in microbiota by 2'-FL, 3'-GL and butyrate

[0236] Anaerobic fermentation in fecal slurry samples was tested in a BioLector Pro microfluidic micro multi-fermentation tank. Fecal slurry samples were collected from breast-fed infants and formula-fed infants. These fecal slurry samples were processed by adding 0.6 grams of feces to 40 ml Baby Reichardt V.6 medium + mucus + ammonium sulfate + lactic acid and acetic acid. The resulting solution was inserted into a BioLector Pro microfluidic micro multi-fermentation tank. The test leg was supplemented with 3-GL, 2'-FL, 3-GL+2'-FL and GOS / FOS. The control leg was supplemented with sterile water.

[0237] In addition, the test line was supplemented with Clostridium difficile C153 (clostridium difficile agar), Salmonella enteriditis S29 (XLD agar), Cronobactersakasakii E71 (chromogenic agar) or Klebsiella pneumonia K2 (Simons citrate inositol agar). For each DNO and control, pathogen-free cultures were also prepared.

[0238] After fermentation, the fermentation solution was tested for SCFA content (especially acetic acid, propionic acid, butyric acid and isobutyric acid), ammonia content, lactic acid content and pathogen concentration. DNA isolation + identification and 16s sequencing were also performed to determine the composition of the microbiota.

[0239] A 32-well plate that can handle low pH was used. The wells of the plate were filled with fecal solution. According to the template, 2.5% (w / v) of different sterile carbohydrate solutions (3'-GL, 2'-FL, 2'-FL / 3'-GL (2.0+0.5%)) and glucose were added to the fecal slurry.

[0240] The experiment was started with a pH set point of 5.5 (facial inoculum from infant 1, infant 1 vaginally born, breastfed, 5 months old) or pH 6.0 (inoculum from infant 2, infant 2 vaginally born, breastfed, 5 months old), constant pH adjustment, temperature 37°C, humidity 85%, OD control. At 4, 8 and 24 hours, sampling was performed and CFU determinations were performed on TOS-propionic acid MUP agar (total bifidobacteria), XLD agar (for Salmonella S29) and Simons citrate inositol agar (for Klebsiella pneumoniae K2 and SCFA), as well as D- and L-lactic acid and ammonia analysis. 16s DNA sequencing was performed using fecal pellets.

[0241] For both inocula, the extent of fermentation (measured by NaOH consumption, i.e., acid production) was highest with the combination of 3'-GL / 2'-FL compared to either 3'-GL or 2'-FL alone. The initial acidification rates for 3'-GL and 3'-GL / 2'-FL were high. In general, 2'-FL alone resulted in slower and lower acidification. Since the amount of fermentable carbohydrates in the reaction vessel was the same, the higher overall acidification of the combination indicated that the combination of 2'-FL and 3'-GL had an unexpected synergistic effect. The majority of the SCFA produced was acetic acid. L-lactic acid was also produced.

[0242] Bifidobacterium growth was observed using 3'-GL, 2'-FL and the mixture 2'-FL / 3'-GL, and in general, growth stimulation was very similar. However, for infant 1, the highest level was observed at 24 hours using the 3'-GL / 2'-FL mixture. Growth of Enterobacteriaceae was also observed and was very similar under the conditions tested, but was lowest at 8 hours for the combination of 2'-FL / 3'-GL for the inoculum of infant 1. The 16s microbiota sequencing data at this time point showed a relative reduction in Proteobacteria (the main donor was Escherichia). At the end of fermentation, when carbohydrates were exhausted, the 2'-FL / 3'-GL-fed microbiota was able to retain more positive microbiota composition than the control group (glucose and blank). For the inoculum of infant 2, the effect on bifidobacteria was highest in the presence of 3'-GL, and the growth inhibition effect on Enterobacteriaceae was the best when the combination of 3'-GL / 2'-FL was used.

[0243] In general, a slight decrease in acidification was observed under conditions where the pathogen mixture was spiked in the container compared to conditions where no pathogens were spiked. However, the effects of 2'-FL, 3'-GL, and 2'-FL / 3'-GL on acidification, as determined by NaOH consumption, were not affected, and again, were highest with 3'-GL / 2'-FL for both inocula. For the inoculum of Infant 1, Salmonella growth was most restricted by 2'-FL, while Klebsiella growth was most inhibited by the combination of 3'-GL / 2-'FL. For the inoculum of Infant 2, Salmonella growth was most restricted by 2'-FL, while Klebsiella growth was most inhibited by the combination of 3'-GL or 3'-GL / 2-'FL. For both inocula, outward growth of C difficile was restricted under all conditions.

[0244] These results indicate that the combination of 2'-FL and 3'-GL has an improving effect on gut microbiota function and composition beyond the effects of 2'-FL alone or 3'-GL alone. This suggests that a combination of dietary butyrate and 2'FL has a further improving effect on the microbiota when 3'GL is also present.

[0245] Example 8: Infant Formula Square objects

[0246] After reconstitution of 13.7 g of powder to a final volume of 100 ml, an infant formula intended for infants from 0 to 6 months of age comprises, per 100 ml:

[0247] -66kcal,

[0248] -1.3g protein (whey protein / casein weight ratio 1 / 1),

[0249] -7.3g digestible carbohydrates (mainly lactose),

[0250] -3.4 g fat (of which about 50% by weight is milk fat, the rest is vegetable oil, fish oil and microbial oil). Based on the total fatty acids, the amount of butyric acid is 1.48% by weight,

[0251] The amount of arachidonic acid was 0.52% by weight, the amount of eicosapentaenoic acid was 0.11% by weight, the amount of docosahexaenoic acid was 0.52% by weight,

[0252] - 0.9 g indigestible oligosaccharides, including 0.1 g 2'-FL (from Jennewein), 0.08 g long-chain oligofructose (from Raftiline HP), 0.72 g galacto-oligosaccharides (of which about 25 mg 3' galactosyl lactose obtained by fermentation, the rest galacto-oligosaccharides from Vivinal GOS),

[0253] - minerals, vitamins, trace elements and other micronutrients according to the instructions for infant formula,

[0254] - A portion of the formula, about 26% by weight based on dry weight, derived from a Lactofidus product fermented with Streptococcus thermophilus and Bifidobacterium breve (B. breve) strains, producing about 28% by weight of lactic acid based on the dry weight of the composition, of which more than 95% by weight is in the L form.

[0255] Example 9: Subsequent preparation Square objects

[0256] After reconstitution of 14.55 g of powder to a final volume of 100 ml, the subsequent formula intended for infants older than 6 months comprises per 100 ml:

[0257] -68kcal,

[0258] -1.36g protein (whey protein / casein weight ratio 4 / 6),

[0259] -8.1g digestible carbohydrates (mainly lactose),

[0260] -3.2 g fat (of which about 50% by weight is milk fat, the rest is vegetable oil, fish oil and microbial oil). Based on the total fatty acids, the amount of butyric acid is 1.47% by weight,

[0261] The amount of arachidonic acid was 0.29% by weight, the amount of eicosapentaenoic acid was 0.12% by weight, the amount of docosahexaenoic acid was 0.56% by weight,

[0262] -0.85g indigestible oligosaccharides, including 0.05g 2'-FL (from Jennewein), 0.08g long-chain oligofructose (from Raftiline HP), 0.72g Galacto-oligosaccharides (of which about 25 mg is 3'-galactosyl lactose obtained by fermentation and the rest is galacto-oligosaccharides from Vivinal GOS),

[0263] - minerals, vitamins, trace elements and other micronutrients according to the instructions for infant formula,

[0264] - A portion of the formula, about 26% by weight on a dry weight basis, derived from a Lactofidus product fermented by Streptococcus thermophilus and Bifidobacterium breve strains, yielding about 0.28% by weight of lactic acid based on the dry weight of the composition, of which more than 95% by weight is in the L form.

[0265] Example 10: Infant Formula

[0266] The subsequent formula intended for infants from 12 months up to 36 months of age comprises per 100 ml after reconstitution of 15.07 g of powder to a final volume of 100 ml:

[0267] -65kcal,

[0268] -1.3g protein (whey protein / casein weight ratio 4 / 6),

[0269] -8.7g digestible carbohydrates (mainly lactose),

[0270] -2.6 g fat (about 10% by weight of which is milk fat, the rest is vegetable oil and fish oil). Based on the total fatty acids, the amount of butyric acid is about 0.35% by weight, the amount of eicosapentaenoic acid is 0.42% by weight, the amount of docosahexaenoic acid is 0.63% by weight,

[0271] - 1.22 g of indigestible oligosaccharides, including 0.02 g of 2'-FL (from Jennewein), 0.12 g of long-chain oligofructose (from Raftiline HP), 1.08 g of galacto-oligosaccharides (of which about 17 mg of 3'-galactosyl lactose obtained by fermentation and the rest of galacto-oligosaccharides from Vivinal GOS),

[0272] - minerals, vitamins, trace elements and other micronutrients according to the instructions for infant formula,

[0273] - A portion of the formula, about 18% by weight on a dry weight basis, derived from a Lactofidus product fermented by Streptococcus thermophilus and Bifidobacterium breve strains, yielding about 0.2% by weight of lactic acid, based on the dry weight of the composition, of which more than 95% by weight is in the L form.

Claims

1. A nutritional composition for an infant or young child, which is not human milk, comprising: a. 2'-fucosyllactose (2'-FL) in an amount of (i) 0.02 to 1 g per 100 ml of the nutritional composition; (ii) 0.15 to 7.5 wt %, based on dry weight; and / or (iii) 0.03 to 1.5 g per 100 kcal, and b. Dietary butyric acid, which is (i) 0.01 to 0.175 g per 100 ml of the nutritional composition; (ii) 0.075 to 1.3 wt % based on dry weight; and / or (iii) 0.015 to 0.25 g per 100 kcal. 2 . The nutritional composition according to claim 1 , further comprising β3′-galactosyl lactose (β3′-GL).

3. The nutritional composition according to claim 1, wherein the composition is at least partially fermented by lactic acid producing bacteria and comprises 0.1 to 1.5 wt. % of the total lactic acid and lactate, based on the dry weight of the nutritional composition, and wherein at least 90 wt. % of the total lactic acid and lactate is L-lactic acid and L-lactate.

4. The nutritional composition of claim 1, wherein the composition further comprises LC-PUFA selected from the group consisting of DHA, ARA and EPA.

5. The nutritional composition according to claim 4, wherein the nutritional composition comprises at least 1 wt% of the sum of DHA, ARA and EPA, based on total fatty acids. The nutritional composition according to claim 1 , wherein the composition further comprises galacto-oligosaccharides and / or fructo-oligosaccharides.

7. The nutritional composition according to claim 1, wherein the nutritional composition is selected from an infant formula, a follow-on formula or a toddler formula.

8. The nutritional composition of claim 1, wherein the nutritional composition is an infant formula.

9. The nutritional composition according to claim 1, wherein the nutritional composition comprises (i) 0.010 to 0.250 g β3'-GL per 100 ml; (ii) 0.075 to 2 wt% on a dry weight basis; and / or (iii) 0.015 to 0.4 g per 100 kcal.

10. The nutritional composition according to claim 1, wherein the nutritional composition comprises (i) 0.2 to 5 g of the total of galacto-oligosaccharides and fructo-oligosaccharides per 100 ml; and / or (ii) 0.3 to 7.5 g of the total of galacto-oligosaccharides and fructo-oligosaccharides per 100 kcal; and / or 1.5 to 35 wt% of the total of galacto-oligosaccharides and fructo-oligosaccharides, based on dry weight.

11. Use of the nutritional composition according to claim 1 in the manufacture of a medicament for improving intestinal barrier function and / or for improving the immune system and / or for improving intestinal microflora and / or for treating or preventing infection.

12. Use of the nutritional composition according to claim 1 in the manufacture of a medicament for treating or preventing allergies.

13. Use of the nutritional composition according to claim 1 in the manufacture of a medicament for inducing oral tolerance to allergens.

14. Use according to claim 11 or 12, wherein the nutritional composition is administered to an infant or a young child.

15. Use according to claim 11 or 12, wherein the nutritional composition is administered to an infant.

16. Use of a nutritional composition according to any one of claims 1 to 10 for providing nutrition to an infant or young child.

17. Use according to claim 16 for providing nutrition to an infant.

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