BETA-1,3'-GALACTOSYLLACTOSE FOR THE TREATMENT OF GUT BARRIER FUNCTION DISEASES

Abstract

The invention relates to the use of beta1,3′-galactosyllactose for improving the gut barrier function. This can in particular be administered to subjects at risk for increased gut barrier permeability, preferably infants and young children.

Claims

1. A method for increasing the intestinal barrier function and/or for prevention and/or treatment of intestinal barrier disruption in infants or young children that suffer from food allergy and/or atopic dermatitis, the method comprising administering the trisaccharide Gal (beta 1-3)-Gal (beta 1-4)-Glc to the infants or young children.

2. The method according to claim 1, wherein the trisaccharide is present in a nutritional composition.

3. A method for the treatment, prevention and/or alleviation of a toxin exposure associated condition in infants or young children that suffer from food allergy and/or atopic dermatitis, the method comprising administering a nutritional composition comprising the trisaccharide Gal (beta 1-3)-Gal (beta 1-4)-Glc to the infants or young children that suffer from food allergy and/or atopic dermatitis, wherein the toxin exposure associated condition is a toxin mediated intestinal barrier disruption.

4. The method according to claim 1, wherein said trisaccharide or said nutritional composition is administered to infants or young children of 0 to 36 month of age.

5. (canceled)

6. The method according to claim 1, wherein the nutritional composition is an infant formula, follow on formula or young child formula.

7. The method according to claim 1, wherein the nutritional composition comprises 0.07 to 3.75 wt % Gal (beta 1-3)-Gal (beta 1-4)-Glc, based on dry weight of the nutritional composition, and/or wherein the daily dose administered is 0.10 to 6 g Gal (beta 1-3)-Gal (beta 1-4)-Glc.

8. The method according to claim 1, wherein the nutritional composition comprises 10 to 500 mg Gal (beta 1-3)-Gal (beta 1-4)-Glc per 100 ml of the composition.

9. The method according to claim 1, wherein the nutritional composition comprises 1.6 to 4 g protein, 5 to 20 g digestible carbohydrates and 0.35 to 3.7 g galacto-oligosaccharides per 100 kcal of the composition, wherein the amount of Gal (beta 1-3)-Gal (beta 1-4)-Glc is more than 20 wt % based on total galacto-oligosaccharides and/or wherein the amount of Gal (beta 1-3)-Gal (beta 1-4)-Glc is between 150 mg and 250 mg per 100 ml.

10. The method according to claim 1, wherein the nutritional composition comprises 1.6 to 4 g protein, 5 to 20 g digestible carbohydrates and 0.35 to 3.7 g galacto-oligosaccharides per 100 kcal of the composition, wherein the amount of Gal (beta 1-3)-Gal (beta 1-4)-Glc is between 10 mg and 50 mg per 100 ml.

11. The method according to claim 1, wherein the nutritional composition further comprises digestible carbohydrates, protein and lipids, and wherein the lipids comprise LC-PUFA selected from the group consisting of DHA, EPA and ARA.

12. Infant formula, follow on formula or young child formula, comprising: a. 1.6 to 4 g protein based on 100 kcal, b. 5 to 20 g digestible carbohydrates based on 100 kcal, c. 3 to 7 g lipid based on 100 kcal, wherein the lipid comprises: i. LC-PUFA selected from the group consisting of DHA, EPA and ARA, wherein the sum of DHA, ARA and EPA is at least 1 wt % based on total fatty acids, and/or ii. at least 0.1 wt % EPA based on total fatty acids and at least 0.5 wt % DHA based on total fatty acids, and optionally further at least 0.25 wt % ARA based on total fatty acids, d. 0.25 to 2.5 g non-digestible oligosaccharides per 100 ml of ready to drink formula, wherein the non-digestible oligosaccharides comprise Gal (beta 1-3)-Gal (beta 1-4)—Glc in an amount of 10 to 500 mg per 100 ml ready to drink formula.

13. The infant formula, follow on formula or young child formula according to claim 12 wherein the formula comprises 0.25 to 2.5 g galacto-oligosaccharides per 100 ml.

14. The infant formula, follow on formula or young child formula according to claim 12, wherein the formula comprises 0.025 to 0.25 g fructo-oligosaccharides per 100 ml.

15. The infant formula, follow on formula or young child formula according to claim 12, wherein the infant formula, follow on formula or young child formula is in the form of a powder, suitable to reconstitute with water to provide a ready to drink infant formula, follow on formula or young child formula.

16.-25. (canceled)

26. The method according to claim 3, wherein said trisaccharide or said nutritional composition is administered to infants or young children of 0 to 36 month of age.

27. The method according to claim 3, wherein the nutritional composition is an infant formula, follow on formula or young child formula.

28. The method according claim 3, wherein the nutritional composition comprises 0.07 to 3.75 wt % Gal (beta 1-3)-Gal (beta 1-4)-Glc, based on dry weight of the nutritional composition, and/or wherein the daily dose administered is 0.10 to 6 g Gal (beta 1-3)-Gal (beta 1-4)-Glc.

29. The method according to claim 3, wherein the nutritional composition comprises 10 to 500 mg Gal (beta 1-3)-Gal (beta 1-4)-Glc per 100 ml of the composition.

30. The method according to claim 3, wherein the nutritional composition comprises 1.6 to 4 g protein, 5 to 20 g digestible carbohydrates and 0.35 to 3.7 g galacto-oligosaccharides per 100 kcal of the composition, wherein the amount of Gal (beta 1-3)-Gal (beta 1-4)-Glc is more than 20 wt % based on total galacto-oligosaccharides and/or wherein the amount of Gal (beta 1-3)-Gal (beta 1-4)-Glc is between 150 mg and 250 mg per 100 ml.

31. The method according to claim 3, wherein the nutritional composition comprises 1.6 to 4 g protein, 5 to 20 g digestible carbohydrates and 0.35 to 3.7 g galacto-oligosaccharides per 100 kcal of the composition, wherein the amount of Gal (beta 1-3)-Gal (beta 1-4)-Glc is between 10 mg and 50 mg per 100 ml.

32. The method according to claim 3, wherein the nutritional composition further comprises digestible carbohydrates, protein and lipids, and wherein the lipids comprise LC-PUFA selected from the group consisting of DHA, EPA and ARA.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0144] FIG. 1:

[0145] Effects of different galactosyllactoses (GLs) on the DON-induced impairment of the Caco-2 cell monolayer integrity. FIGS. 1A and 1B shows the transepithelial electrical resistance (TEER) for different GLs. FIGS. 1C and 1D show the translocation of lucifer yellow (LYF) to the basolateral compartment. TEER was expressed as a percentage of the initial value and LYF was expressed in ng/cm.sup.2×h, i.e. in ng/ml. alpha3′-GL is Gal (alpha 1-3)-Gal (beta 1-4)-Glc; beta3′-GL is Gal (beta 1-3)-Gal (beta 1-4)-Glc; beta4′-GL is Gal (beta 1-4)-Gal (beta 1-4)-Glc′; beta6′-GL is Gal (beta 1-6)-Gal (beta 1-4)-Glc. Data are the mean±s.e. *: p<0.05 compared to control, **: p<0.01 compared to control, ***: p<0.001 compared to control, {circumflex over ( )}: p<0.05 compared to DON control , {circumflex over ( )}{circumflex over ( )} p<0.01 compared to DON Control, {circumflex over ( )}{circumflex over ( )}{circumflex over ( )} p<0.001 compared to DON Control.

[0146] FIG. 2:

[0147] Different effects of GLs on the DON-induced increase in IL8 release by Caco-2 cells. IL-8 secretion is expressed as pg/ml as mean±s.e. alpha3′-GL is Gal (alpha 1-3)-Gal (beta 1-4)-Glc; beta3′-GL is Gal (beta 1-3)-Gal (beta 1-4)-Glc, beta4′-GL is Gal (beta 1-4)-Gal (beta 1-4)-Glc , beta6′-GL is Gal (beta 1-6)-Gal (beta 1-4)-Glc. Data are the mean±s.e. *: p<0.05 compared to control, **: p<0.01 compared to control, ***: p<0.001 compared to control, {circumflex over ( )}: p<0.05 compared to DON control, {circumflex over ( )}{circumflex over ( )} p<0.01 compared to DON Control, {circumflex over ( )}{circumflex over ( )}{circumflex over ( )} p<0.001 compared to DON Control.

EXAMPLES

Example 1: Label Free Targeted LC-ESI-MS.SUP.2 .Analysis of Beta3′-[and Beta6′]-Galactosyllactose in Human Milk with Enhanced Structural Selectivity

[0148] Fresh human milk volunteer samples from different stages of lactation were spiked with internal standards including e.g. α-Arabinopentaose. Then, 3 anonymized spiked milk specimens (2 healthy donors, 3 different stages of lactation), anonymized spiked milk specimens were diluted 1:10 with H.sub.2O and subjected to ultrafiltration (UF) with 3 kDa cut off. UF permeates were analysed by liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS.sup.2). LC-separation of galactosyllactoses (GLs) and other human milk compounds was facilitated by 2.1×30 mm+2.1×10 mm porous graphitized carbon HPLC columns connected in line with a linear ion trap mass spectrometer.

[0149] Gradient elution of GLs started with 0.3% NH.sub.4OH in H.sub.2O at 0 min and ended with 0.3% NH.sub.4OH in 95% methanol at 27 min. Constant flow rate was at 0.4 ml/min and columns were kept at 45 degrees Celsius. Eluting GLs were characterized by negative ion multiple reaction monitoring (MRM) LC-ESI-MS.sup.2.

[0150] Employed MRM transitions had been determined before by MS/MS experiments with commercially available pure GL-standards using collision induced dissociation (CID) fragmentation. Further details on the methods can be found in Mank M, Welsch P, Heck A J R, Stahl B, Anal Bioanal Chem 2019, 411 (1):231-250.

[0151] beta3′-GL and beta6′-GL were detected directly in human milk samples from various stages of lactation (4, 125 and 159 days post-partum). The results are shown in Table 1. Interestingly, the abundance of beta3′-GL appeared to be relatively stable between colostral and mature milks, whereas the amount of beta6′-GL strongly declined over time.

TABLE-US-00001 TABLE 1 beta3′-GL and beta6′-GL in human milk samples from various stages of lactation. Postpartum day beta3′-GL (mg/100 ml) beta6′-GL 4 10 95 125 3 14 159 4 10

Example 2: Beta1,3′-Galactosyllactose Specifically Protects against Intestinal Barrier Disruption and Prevents Permeability Increase

[0152] beta1,3′-galactosyl-lactose (beta3′-GL), beta1,4′-galactosyllactose (beta4′-GL) and beta1,6′-galactosyl-lactose (beta6′-GL) were obtained from Carbosynth (Berkshire, UK). alpha1,3′-galactosyl-lactose (alpha3′-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 American Type Tissue Collection (Code HTB-37) (Manasse, Va., USA, passage 90-102).

[0153] Caco-2 cells were used according to established methods. In brief: cells were cultured in Dulbecco's modified Eagle medium (DMEM) and seeded at a density of 0.3×10.sup.5 cells into 0.3 cm.sup.2 high pore density (0.4 μm) inserts with a polyethylene terephthalate membrane (BD Biosciences, Franklin Lakes, N.J., USA) placed in a 24-well plate. The Caco-2 cells were maintained in a humidified atmosphere of 95 air and 5% CO.sub.2 at 37° C. After 17-19 days of culturing, a confluent monolayer was obtained with a mean Transepithelial electrical resistance (TEER) exceeding 400 Ωcm.sup.2 measured by a Millicell-Electrical Resistance System voltohm-meter (Millipore, Temecula, Calif., USA).

[0154] Caco-2 cell monolayers were thus grown in a transwell system, which is a model for intestinal barrier function. The monolayers were pretreated for 24 h with different GLs, including beta3′-GL, alpha3′-GL, beta4′-GL and beta6′-GL in a concentration of 0.75 wt % of the GL, before being exposed to the fungal toxin deoxynivalenol (DON), which is a trigger and model compound to impair intestinal barrier. DON was diluted to a final concentration of 4.2 M in complete cell medium and added to the apical side as well as to the basolateral side of the transwell inserts. This DON concentration did not affect the viability of the Caco-2 cells. Incubation with DON was 24 h.

[0155] Measurements of the transepithelial electrical resistance (TEER) and lucifer yellow (LY) permeability were conducted to investigate barrier integrity. For TEER measurements a Millicel-ERS voltohmmeter connected to a pair of chopsticks electrodes was used to measure the TEER values. Results are expressed as a percentage of the initial value. For paracellular tracer flux assay the membrane impermeable lucifer yellow (LY) (Sigma, St Luis, Mo., USA) was added in a concentration of 16 μg/ml to the apical compartment in the transwell plate for 4 h, and the paracellular flux was determined by measuring the fluorescence intensity in the basolateral compartment with a spectrophotofluorimeter (FLUOstar Optima, BMG Labtech, Offenburg, Germany) set at excitation and emission wavelengths of 410 and 520 nm, respectively. Release of interleukin-8 (IL-8 or CXCL8), which is a typical marker for inflammation, was quantified in the medium of the apical side and the basolateral side of the Caco-2 transwell inserts in response to the treatments. CXCL8 concentrations were measured by using the human IL-8 ELISA assay (BD Biosciences, Pharmingem, San Diego, Calif., USA) according to manufacturer's instructions. For more details on materials and methods see Akbari et al, 2016, Eur J Nutr. 56(5):1919-1930.

[0156] The results are shown in FIG. 1 A, B, C and D and in FIG. 2. FIG. 1 shows the effects of different galactosyllactoses (GLs) on the DON-induced impairment of the Caco-2 cell monolayer integrity. FIGS. 1A and 1B show the transepithelial electrical resistance (TEER) for different GLs. FIGS. 1C and 1D show the translocation of lucifer yellow (LYF) to the basolateral compartment. TEER was expressed as a percentage of the initial value and LYF was expressed in ng/cm.sup.2×h. alpha3′-GL is Gal (alpha 1-3)-Gal (beta 1-4)-Glc; beta3′-GL is Gal (beta 1-3)-Gal (beta 1-4)-Glc; beta4′-GL is Gal (beta 1-4)-Gal (beta 1-4)-Glc; beta6′-GL is Gal (beta 1-6)-Gal (beta 1-4)-Glc. Data are the mean±s.e. *: p<0.05 compared to control, **: p<0.01 compared to control, ***: p<0.001 compared to control, {circumflex over ( )}: p<0.05 compared to DON control , {circumflex over ( )}{circumflex over ( )} p<0.01 compared to DON Control, {circumflex over ( )}{circumflex over ( )}{circumflex over ( )} p<0.001 compared to DON Control.

[0157] As can be seen from FIGS. 1A-D, the presence of DON disrupted the barrier function as shown by a decreased TEER value and an increased LY flux for the DON-control samples. Additionally, the presence of DON increased CXCL8 (IL-8) release, as shown in FIG. 2. FIGS. 1A-D further show that the presence of beta3′-GL prevented the DON-induced loss of epithelial barrier integrity as measured by increased TEER values and a reduction in the DON-affected LY flux across the intestinal epithelial monolayer.beta4′-GL and beta6′-GL did not show a significant effect on the intestinal epithelial barrier function. Interestingly, beta3′-GL, i.e. the galactosyl-lactose with a β1-3 glycosidic linkage, was effective in protecting the intestinal barrier function, whereas alpha3′-GL, i.e. the galactosyl-lactose with an α1-3 glycosidic linkage, did not prevent the DON-induced disrupted intestinal barrier. In contrast, all galactosyl-lactoses were able to decrease the DON-induced IL-8 release, as is shown in FIG. 2.

[0158] These results are indicative for the specific effect of beta3′-GL (herein also referred to as beta1,3′-galactosyllactose or Gal (beta 1-3)-Gal (beta 1-4)-Glc) on protecting the intestinal epithelial barrier function, in particular under conditions of challenges, which goes beyond and/or is independent of an effect on preventing an inflammatory response, and/or of an effect on or via the microbiota. These results are thus indicative of an effect that beta3′-GL has on increasing the intestinal barrier function and/or on the prevention and/or treatment of intestinal barrier disruption. In addition, these results are indicative of an effect of beta3′-GL on the treatment, prevention and/or alleviation of a toxin exposure associated condition in a subject, in particular when the toxin is a tricothecene toxin, and more in particular when the toxin is deoxynivalenol.

Example 3: Also in Mixtures of Galacto-Oligosaccharides Beta3′-GL Specifically Protects against Intestinal Barrier Disruption and Prevents Permeability Increase

[0159] Several GOS preparations, comprising varying amounts of beta3′-GL, were tested in this experiment. As a source of beta4′-GL and beta6′-GL VivinalGOS (VGOS) was used. VivinalGOS is available from FrieslandCampina Domo (Amersfoort, The Netherlands) and contains about 59% GOS on dry matter. The DP3 of this GOS is predominantly 4′GL and 6′G.

[0160] Another GOS preparation, herein referred to as TOS, was produced in house by incubating a lactose mixture with Streptococcus thermophilus strain ST065 (this strain is also referred to as strain S. thermophilus CNCM I-1620), as described in more detail in WO 96/06924, FR2723960 and EP0778885, and in LeForestier et. al., 2009 Eur J Nutr, 48:457-464, with the additional step that S. thermophilus cell debris material was removed by centrifugation. The obtained TOS was used as a source of beta4′-GL, beta6′-GL and beta3′-GL. The amount of galacto-oligosaccharides (excluding lactose, galactose and glucose) was 10.2 wt % per 100 ml, of which about 60-65 wt % (62 wt %) was beta3′-GL, the remainder being 6′-GL and non-digestible oligosaccharides with DP 2, 4 and 5 (see also WO 96/06924, FR2723960 and EP0778885 and LeForestier et. al., 2009 Eur J Nutr, 48:457-464).

[0161] Barrier function experiments were performed in a similar way as in example 2 using the following concentrations of Vivinal GOS (VGOS): 0 (Control), 0.25 wt % (VGOS 0.25), 0.5 wt % (VGOS 0.5), 0.75 wt % (VGOS 0.75), 1 wt % (VGOS 1), 1.5 wt % (VGOS 1.5) and 2 wt % (VGOS 2). For TOS concentrations of 0.25 wt % (TOS 0.25) and 0.5 wt % (TOS 0.5) were tested. The results are shown in Tables 2 and 3.

[0162] The effects of different GOS mixtures on the DON-induced impairment of the Caco-2 cell monolayer integrity were measured. Different GLs were tested for transepithelial electrical resistance (TEER), as shown in Table 2, and for the translocation of lucifer yellow (LYF) to the basolateral compartment, as shown in Table 3. TEER was expressed as a percentage of the initial value and LYF was expressed in ng/cm.sup.2×h. GOS is VivinalGOS. TOS is the galacto-oligosaccharide mixture produced by S. thermophilus ST065 beta galactosidase and containing about 65 wt % beta3′-GL based on total galacto-oligosaccharides. Data in Tables 2 and 3 are the mean±s.e.

[0163] *: p<0.05 compared to control,

[0164] **: p<0.01 compared to control;

[0165] ***: p<0.001 compared to control;

[0166] {circumflex over ( )}: p<0.05 compared to DON control;

[0167] {circumflex over ( )} p<0.01 compared to DON Control;

[0168] {circumflex over ( )}{circumflex over ( )}{circumflex over ( )} p<0.001 compared to DON Control.

TABLE-US-00002 TABLE 2 Transepithelial electrical resistance (TEER) for different GOS mixtures, wherein TEER is expressed as a percentage of the initial value. TEER Control DON VGOS 0.25 VGOS 0.5 VGOS 0.75 VGOS 1 VGOS 1.5 VGOS 2 TOS 0.25 TOS 0.5 Mean 99.95 29.93 *** 33.04 *** 34.75 *** 34.83 *** 36.36 *** 47.5 *** 90.66 {circumflex over ( )}{circumflex over ( )}  88.9 {circumflex over ( )}{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}  85.84 {circumflex over ( )}{circumflex over ( )}  Std. 2.488 0.0353  1.632   0.4837  2.387   3.595    0.3964 2.144 7.025 4.165 Error

TABLE-US-00003 TABLE 3 Translocation of lucifer yellow (LYF) to the basolateral compartment for different GOS mixtures, wherein LYF is expressed in ng/cm.sup.2 × h. LYF Control DON VGOS 0.25 VGOS 0.5 VGOS 0.75 VGOS 1 VGOS 1.5 VGOS 2 TOS 0.25 TOS 0.5 Mean 377.8 622.8 *** 618.9 {circumflex over ( )} *** 571.9 *** 574.8 *** 451.1 ** {circumflex over ( )}{circumflex over ( )} 396.3 {circumflex over ( )}{circumflex over ( )}{circumflex over ( )} 384.9 {circumflex over ( )}{circumflex over ( )}{circumflex over ( )} 406.8 {circumflex over ( )}{circumflex over ( )}{circumflex over ( )} 436 {circumflex over ( )}{circumflex over ( )}{circumflex over ( )} Std. 1.208 25.31   13.05 .sup.   9.299  6.053 1.598 10.31  4.166  4.456    1.975 Error

[0169] VGOS only improved the barrier function as determined by preventing TEER decrease at relatively high concentration of 2 wt %. Also the LY flux was only prevented by relatively high concentrations of VGOS of 1 wt % or above. TOS, on the other hand, was already effective at a concentration of 0.25 wt %. Since the main difference between VGOS and TOS is the presence of GOS with a beta1.3′ link between the galactose moieties in TOS, and more in particular the presence of a high amount of beta3′-GL in TOS, this is indicative again for an effect of beta3′-GL on the gut barrier function, and this effect is maintained in the presence of other GOS structures. Also this effect is observed with beta3′-GL concentrations lower than about 0.17 wt %.

Example 4: LC-PUFA with Short Chain Fatty Acid Fermentation Products Improve Gut Barrier Function

[0170] The effect of a long chain polyunsaturated fatty acids (LC-PUFA) and a mixture of short chain fatty acids (SCFA mix) having a concentration profile that is typical for fermentation of a mixture of galacto-oligosaccharides (TOS) derived from VivinalGOS and long chain fructo-oligosaccharides (RaftilineHP) in a ratio of 9/1 (w/w) on barrier permeability was investigated, as disclosed in Example 3 of EP2100520 (incorporated by reference herein) The SCFA mix had a fatty acid profile as shown in FIG. 3A of EP2100520 (incorporated by reference herein) (see second bar in FIG. 3A), i.e. about 75% being acetic acid.

Methods

[0171] T84 human intestinal epithelial cells are commonly used to study intestinal barrier integrity in vitro. T84 cells (ATCC, USA) were cultured on 12 mm 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 were used 14 days after reaching confluence. Monolayers of T84 cultured on transwell filters were pre-incubated with LC-PUFA, SCFA mix or a combination thereof. The EPA-containing samples were incubated for an additional 48 h in the presence of IL-4 (25 ng/ml). IL-4 was added to the basolateral compartment; medium and additives were changed every 24 h.

[0172] Epithelial barrier integrity was assessed by measuring transepithelial resistance (TEER; Ω×cm.sup.2) with the epithelial volt-ohm meter (EVOM; World Precision Instruments, Germany). TEER measurements were performed prior to medium refreshment at 0, 24, 48, 72, 96 h of incubation.

[0173] Results are shown in Table 4.

TABLE-US-00004 TABLE 4 Effect of LC-PUFA on the gut barrier function. Conc SCFA mix % TEER LC-PUFA (mM) (48 h) LC-PUFA (μM) (C2-C4 fatty acids) Mean (s. d.) — — 4  6 (10) ARA/EPA/DHA 100 — 40 (7) ARA/EPA/DHA 100 4 71 (6)

[0174] This example shows an improved effect of LC-PUFA on the gut barrier function, and in particular in combination with the fermentation products. Therefore this example is indicative of a further improved effect on the intestinal barrier function in a composition, when combining beta3′-GL, enhanced levels of LC-PUFA (EPA, DHA and ARA) and GOS fermentation products. Beta3′-GL and the LC-PUFA will have a direct effect on the intestinal barrier function, whereas additional non-digestible oligosaccharides, in particular GOS other than beta3′GL, will beneficially affect the intestinal barrier function via the SCFA formation.

Example 5: Infant Formula

[0175] An infant formula, provided as a powder in a pack with instructions to reconstitute with water to a ready to drink milk. When reconstituted the formula contains per 100 ml: [0176] 68 kcal [0177] about 1.4 g Protein (mainly whey protein and casein from bovine) [0178] about 3.2 g Lipid, wherein the amount of DHA is 0.52 wt %, EPA is 0.11 wt % and ARA is 0.52 wt % based on total fatty acids [0179] about 8.1 g digestible carbohydrates (mainly lactose) [0180] about 815 to 830 mg non-digestible oligosaccharides, comprising of 735 to 750 mg galacto-oligosaccharides, of which 15 to 30 mg beta3′-GL (sources Vivinal GOS and GOS produced by the betagalactosidase from S. thermophilus CNCM-I-1620) and about 80 mg IcFOS (source RaftilinHP) minerals, trace elements, vitamins and other micronutrients as known in the art and according to international directives for infant formula.

Example 6: Follow on Formula

[0181] A follow on formula, provided as a powder in a pack with instructions to reconstitute with water to a ready to drink milk. When reconstituted the formula contains per 100 ml: [0182] 68 kcal [0183] about 1.4 g Protein (mainly whey protein and casein from bovine) [0184] about 3.2 g Lipid, wherein the amount of DHA is 0.52 wt %, EPA is 0.11 wt % and ARA is 0.52 wt % based on total fatty acids [0185] about 8.1 g digestible carbohydrates (mainly lactose) [0186] about 720 mg galacto-oligosaccharides, of which about 140 mg beta3′-GL, (500 mg from Vivinal GOS and 220 mg from GOS produced by the betagalactosidase from S. thermophilus CNCM-I-1620, respectively) and about 80 mg IcFOS (source RaftilinHP) [0187] minerals, trace elements, vitamins and other micronutrients as known in the art and according to international directives for infant formula.