Mixtures of non-digestible oligosaccharides

Abstract

The present invention relates to a nutritional composition comprising a mixture of non-digestible oligosaccharides consisting of a combination of five specific human milk oligosaccharides, beta-galactooligosaccharides and long chain fructooligosaccharides (lcFOS). These nutritional compositions are beneficial for improving intestinal microecology.

Description

Technical Field

[0001] The present invention is in the field of nutritional compositions for infants and young children comprising non-digestible oligosaccharides to improve the intestinal microbiota. Background Art

[0002] Human milk contains a large amount of non-digestible carbohydrates, called human milk oligosaccharides (HMOs). Mature human milk contains 5 to 15 g / l of HMOs. It is estimated that there are over 200 structurally distinct oligosaccharides. The building blocks of HMOs are the monosaccharides D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GlcNAc), L-fucose (Fuc), and sialic acid (N-acetylneuraminic acid (Neu5Ac)). Lactose (Galβ1-4Glc) forms the reducing end and can be extended by repeating N-acetyllactosamine units (Galβ1-3 / 4GlcNAc). The lactose or polylactosamine backbone can be sialylated using α2-3 and / or α2-6 linkages and / or fucosylated using α1-2, α1-3, and / or α1-4 linkages. The structural complexity and abundance of these non-digestible oligosaccharides are unique to human milk, as they are found at much lower levels in the milk of other mammalian species. The role of HMOs in human milk is to improve the intestinal microbiome by stimulating bifidobacteria and other beneficial lactic acid-producing bacteria, thereby inhibiting the growth of potential pathogens. Furthermore, HMOs inhibit the binding of pathogenic microorganisms to the surface of infant epithelial cells, and HMOs have also been reported to have direct beneficial effects on the gastrointestinal epithelium, which serves as the immune system.

[0003] Breastfeeding is the preferred method of feeding infants. However, in some cases, breastfeeding is not possible or less than ideal. In these cases, infant formula is a good alternative. Modern infant formula ingredients have been tailored to meet the many specific nutritional needs of rapidly growing infants.

[0004] In the past, infant formula did not contain non-digestible oligosaccharides. Subsequent development of infant formula contains prebiotics, non-digestible oligosaccharides, to functionally mimic the effects of HMOs. One of the most thoroughly studied prebiotic mixtures is a mixture of galacto-oligosaccharides (GOS) plus long-chain fructo-oligosaccharides (lcFOS) in a weight ratio of 9:1. After administering this specific prebiotic mixture to infants, bifidobacteria in the intestinal microecology increased and pathogens decreased, making the microecology more similar to that in human milk-fed infants (Knol et al., Acta Acta Pediatrics, 2005; 94(Suppl 449); Knol et al., 31–33. Pediatr Gastroenterol Nutr, Vol. 40, No. 1, January 2005; WO 2005 / 039319).

[0005] Recently, milk oligosaccharides with the same structure as HMOs have become available through fermentation by genetically modified microorganisms, and infant formula containing HMOs or HMO mixtures has become available.

[0006] WO 2019 / 110800 discloses a spray-dried human milk oligosaccharide mixture and a nutritional composition comprising the same. The nutritional composition may include prebiotics such as galacto-oligosaccharides (GOS), fructo-oligosaccharides (FOS), inulin or a combination thereof.

[0007] WO 2021 / 116236 discloses age-specific nutritional compositions comprising an HMO mixture. Optionally, at least one of the nutritional compositions comprises a prebiotic, preferably, the prebiotic comprises polydextrose, galacto-oligosaccharide or a combination thereof.

[0008] WO 2020 / 239996 discloses that a combination of galacto-oligosaccharides (GOS) and long-chain fructo-oligosaccharides (lcFOS) with 2'-fucosyllactose (2'-FL) reduces or prevents proteolytic fermentation in the gastrointestinal tract of a subject. The document shows that adding 2'-FL to the combination of GOS and lcFOS does not increase bifidobacteria compared to when the GOS / lcFOS combination is used alone.

[0009] WO 2020 / 245313 discloses that the combination of 2'-fucosyllactose (2'-FL) and 3'-galactosyllactose has a beneficial effect on intestinal barrier function.

[0010] WO 2019 / 055718 discloses compositions for increasing the production of specific metabolites in the intestinal tract of lactating infant mammals (including humans). These compositions typically comprise one or more bacterial strains selected for their growth on mammalian milk oligosaccharides, sources of mammalian milk oligosaccharides, and optionally nutritional components required for the growth of the infant mammal.

[0011] CN 113907144 discloses that certain HMOs can prevent Staphylococcus aureus infection. A series of HMO combinations are proposed, including 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lactose-N-tetraose (LNT), 3'-sialyllactose (3'-SL), and 6'-sialyllactose (6'-SL). The document also discloses that dietary fibers, such as fructooligosaccharides, galacto-oligosaccharides, and polyfructose, can be included in infant formula.

[0012] US2014 / 248415 discloses including an HMO combination comprising 2'-FL, LNT, 3'-SL and 6'-SL in an infant formula, and optionally including 90% GOS, 10% inulin or FOS as prebiotics. The HMO combination is believed to enhance the beneficial effects and efficiency of probiotics.

[0013] US2020 / 354760 relates to a process for purifying L-fucose from fermentation to provide food grade quality L-fucose. In general, a premix for infant or toddler formula is described, which, in addition to L-fucose, also includes a carbohydrate selected from the group consisting of: human milk oligosaccharides (HMOs), galacto-oligosaccharides (GOS), inulin, oligofructose (FOS), lactose, isomaltose, sialic acid, and combinations thereof. As an example, the following infant formula is described: in addition to L-fucose, the infant formula also includes HMOs containing 2'-FL, 3-FL, LNT, 3'-SL, and 6'-SL.

[0014] US2023 / 013644 relates to nutritional compositions containing HMOs for providing nutrition to infants of different ages. Infant formulas containing combinations of 2'-FL, 3-FL, LNT, 3'-SL, and 6'-SL are described. The document suggests further inclusion of prebiotics and provides examples of formulas containing GOS and polydextrose (PDX).

[0015] Parschat et al., Nutrients 2021 13:2871, reported a clinical study evaluating the safety, tolerability, and growth effects of an infant formula containing 5.75 g / L of a combination of five HMOs: 52% 2'-FL, 13% 3-FL, 26% LNT, 4% 3'-SL, and 5% 6'-SL. The formula tested did not contain additional prebiotics. The results showed that the infant formula containing the five HMOs was safe and well-tolerated, while supporting normal growth.

[0016] Conze et al., Food and Chemical Toxicology 2022, 163:112377, performed a weighted analysis of HMO measurements in human milk reported in the literature and calculated a weighted average of 54.2% 2'-FL, 12.0% 3-FL, 19.7% LNT, 5.9% 3'-SL, and 8.2% 6'-SL. These authors, considering that HMOs are thought to play a key role in balancing the intestinal microbiome, imparting antimicrobial effects, developing the intestinal barrier, and regulating immune responses, and given the wide variation in HMO levels found, they recommend appropriate supplementation above the average levels found in human milk.

[0017] There remains a need for infant and toddler formulas having a mixture of non-digestible oligosaccharides that further improve the infant's intestinal microbiome. Summary of the Invention

[0018] By employing an in vitro fermentation model using infant fecal samples, the inventors surprisingly discovered that a mixture of non-digestible oligosaccharides consisting of a combination of 5 specific human milk oligosaccharides (5 HMOs), bGOS, and lcFOS synergistically increased the amount of bifidobacteria and synergistically reduced the number of Gram-negative Enterobacteriaceae when compared to a mixture of β-galacto-oligosaccharides (bGOS) and long-chain fructo-oligosaccharides (lcFOS), or when compared to a combination of 5 specific human milk oligosaccharides (5 HMOs) alone. The combination of the 5 specific HMOs is a combination of 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3'-sialyllactose (3'-SL), and 6'-sialyllactose (6'-SL). When fecal samples from several infants were tested, a synergistic effect was consistently found. Furthermore, when the fermentation supernatant was tested in an intestinal barrier model, it was found that the fermentation supernatant of the mixture of non-digestible oligosaccharides of the present invention synergistically increased barrier resistance. Therefore, the nutritional composition comprising a mixture of five specific HMOs, bGOS, and lcFOS will have a further improving effect on the intestinal microecology by increasing bifidobacteria or reducing pathogens, and will have an improving effect on intestinal barrier function. The risk of gastrointestinal disorders (such as microbial flora imbalance, infection, and inflammation) will be further reduced. DETAILED DESCRIPTION

[0019] The present invention relates to a nutritional composition for infants or young children comprising a mixture of non-digestible oligosaccharides consisting of:

[0020] Component a): beta-galacto-oligosaccharides (bGOS) and long-chain fructo-oligosaccharides (lcFOS), and

[0021] Component b): 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3'-sialyllactose (3'-SL), and 6'-sialyllactose (6'-SL). Component b) can also be referred to as a combination of five HMOs.

[0022] The present invention also relates to the nutritional composition of the present invention for use in therapy.

[0023] The present invention also relates to the use of the nutritional composition according to the invention for improving the intestinal microecology, preferably in infants or young children.

[0024] The present invention further relates to the use of the nutritional composition according to the invention for improving the intestinal barrier function, preferably in infants or young children.

[0025] The present invention also relates to the use of the nutritional composition of the present invention for preventing and / or treating intestinal disorders.

[0026] Component a): β-galacto-oligosaccharides and long-chain fructo-oligosaccharides

[0027] The nutritional composition comprises non-digestible oligosaccharides beta-galacto-oligosaccharides (bGOS) and long-chain oligofructose (lcFOS). Non-digestible oligosaccharides are oligosaccharides with an average degree of polymerization ranging from 2 to 100. Non-digestible oligosaccharides are oligosaccharides that are not digested in the stomach or small intestine and reach the colon intact. Maltodextrin, lactose and monomers such as galactose, fucose and sialic acid are not considered non-digestible oligosaccharides, i.e. they are considered digestible carbohydrates.

[0028] The nutritional composition of the present invention comprises beta-galacto-oligosaccharides (bGOS). bGOS are non-digestible oligosaccharides preferably having the formula ([galactose] n-glucose; wherein n is an integer ranging from 2 to 10, i.e. 2, 3, 4, 5, 6, ..., 10), wherein the galactose units are mostly linked together via beta bonds. bGOS are available, for example, under the trademark Vivinal TM GOS (Borculo DomoIngredients, The Netherlands). Other suitable sources are Oligomate TM (Yakult, Japan). Preferably, the average degree of polymerization (DP) of the bGOS of the present invention is in the range of 3 to 7, more preferably in the range of 3 to 5. Preferably, bGOS comprises mainly β-1,4 and / or β-1,6 linkages between galactose units, more preferably β-1,4 linkages predominate. In a preferred embodiment, bGOS comprises at least 80%, more preferably at least 90%, of β-1,4 and β-1,6 linkages based on the total linkages. bGOS is more capable of stimulating bifidobacteria. Preferably, bGOS comprises less than 10% of β-1,3 linkages based on the total linkages.

[0029] The nutritional composition of the present invention comprises long-chain fructo-oligosaccharides (lcFOS). lcFOS is a non-digestible oligosaccharide comprising a chain of β-linked fructose units with an average degree of polymerization (DP) ranging from 6 to 1000, more preferably from 10 to 100, even more preferably from 20 to 40. lcFOS includes inulin, fructan and / or mixed types of polyfructose. A particularly preferred lcFOS is inulin. Inulin has a structure of chain-terminating glucosyl moieties and repeating fructosyl moieties linked by β-2,1 bonds. lcFOS suitable for use in the nutritional composition of the present invention is also commercially available, for example HP (Orafti company).

[0030] For the purposes of the present invention, when an amount or range is expressed per volume, unless otherwise stated, this refers to the nutritional composition in a ready-to-use form.When an amount is expressed as wt%, unless otherwise stated, this refers to wt% on a dry weight basis.

[0031] Preferably, the nutritional composition according to the present invention comprises 100 mg to 2 g of bGOS plus lcFOS per 100 ml, more preferably 250 mg to 1.5 g, even more preferably 500 mg to 1 g per 100 ml. Based on dry weight, the nutritional composition according to the present invention preferably comprises 0.7 to 14.3 wt%, more preferably 1.8 to 10.7 wt%, even more preferably 3.6 to 7.1 wt% of bGOS plus lcFOS. Based on 100 kcal, the nutritional composition according to the present invention preferably comprises 150 mg to 3 g of bGOS plus lcFOS, more preferably 375 mg to 2.25 g, even more preferably 750 mg to 1.5 g. Preferably, the nutritional composition according to the present invention comprises less than 10 mg of β1,3'-galactosyl lactose (3'-GL) per 100 ml. Based on dry weight, the nutritional composition according to the present invention preferably comprises less than 0.07 wt% 3'-GL. Based on 100 kcal, the nutritional composition according to the present invention preferably comprises less than 15 mg of 3'-GL.

[0032] Preferably, the amount of bGOS ranges from 70% to 95% and the amount of lcFOS ranges from 5% to 30% based on the total weight of bGOS plus lcFOS, with the sum of bGOS and lcFOS being 100%. More preferably, the amount of bGOS ranges from 85% to 95% and the amount of lcFOS ranges from 5% to 15% based on the total weight of bGOS plus lcFOS, with the sum of bGOS and lcFOS being 100%. It is preferred to combine GOS with lcFOS rather than with other longer oligosaccharides such as polydextrose. When compared with either bGOS or lcFOS alone, mixtures of bGOS and lcFOS have been found to have a synergistic effect on the amount of intestinal bifidobacteria and lactobacilli, as well as the formation of short-chain fatty acids. Furthermore, mixtures of GOS and lcFOS have been shown to produce higher amounts of short-chain fatty acids and lactic acid than when polydextrose is used (Vester-Boler et al., Nutr Res [Nutrition Research] 2009, 29:631-639).

[0033] Component b): Combination of 5 HMOs

[0034] The nutritional composition according to the present invention comprises a specific combination of human milk oligosaccharides consisting of 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3'-sialyllactose (3'-SL), and 6'-sialyllactose (6'-SL). This specific mixture was found to be superior when combined with component a). The combination of four HMOs showed a lesser effect than the combination of five HMOs. Because HMO fermentation by the intestinal microbiome leads to interactions (such as cross-feeding and quorum sensing), the results of one specific combination of HMOs cannot be extrapolated to a different combination of HMOs.

[0035] These HMOs can be isolated from natural sources (e.g., animal milk) by chromatography or filtration techniques. Alternatively, they can be produced biotechnologically using specific enzymes (e.g., fucosyltransferases and / or fucosidases for the production of 2'-FL and 3-FL, sialidases and glycosyltransferases for the production of SL and LNT) using enzyme-based fermentation techniques (recombinant or natural enzymes) or microbial fermentation techniques known in the art. In the latter case, the microorganisms can express their native enzymes and substrates, or can be engineered to produce the corresponding substrates and enzymes. Single microbial cultures and / or mixed cultures can be used. Alternatively, these HMOs can be produced by chemical synthesis, for example, from lactose and free monomers (e.g., fucose, sialic acid, N-acetylglucosamine, galactose). These HMOs are commercially available from, for example, Kyowa Hakko, FrieslandCampina, Glycom / DSM, Chr. Hansen, and Sigma-Aldrich.

[0036] The amount of the combination of the above five HMOs is preferably 20 to 400 mg / 100 ml, more preferably 30 to 300 mg / 100 ml, even more preferably 40 to 250 mg / 100 ml. The amount of human milk oligosaccharides is preferably 0.14 to 2.86 wt%, more preferably 0.21 to 2.14 wt%, even more preferably 0.28 to 1.79 wt%, based on the dry weight of the composition. Preferably, the amount of human milk oligosaccharides per 100 kcal is 30 to 600 mg, more preferably 45 to 450 mg, even more preferably 60 to 375 mg.

[0037] Based on the total weight of the HMOs, the amount of each specific HMO is preferably 42 to 62 wt% 2'-FL, 10 to 16 wt% 3-FL, 21 to 31 wt% LNT, 3 to 5 wt% 3'-SL, and 4 to 6 wt% 6'-SL, with the sum of 2'-FL, 3-FL, LNT, 3'-SL, and 6'-SL being 100%, and more preferably 47 to 57 wt% 2'-FL, 11 to 15 wt% 3-FL, 23 to 28 wt% LNT, 3.5 to 4.5 wt% 3'-SL, and 4.5 to 5.5 wt% 6'-SL. Such ratios will ensure an even better effect on the microbiome.

[0038] Combination of multiple HMOs with a mixture of bGOS and lcFOS

[0039] The nutritional composition of the present invention comprises a mixture of non-digestible oligosaccharides consisting of bGOS, lcFOS, 2'-FL, 3-FL, LNT, 3'-SL, and 6'-SL. This mixture was found to synergistically improve microecology and intestinal barrier resistance. Because the effect of improving microecology is complex and considered specific, all seven non-digestible oligosaccharides must be present; omitting or adding additional non-digestible oligosaccharides may yield different results.

[0040] Preferably, the nutritional composition of the present invention comprises a mixture of non-digestible oligosaccharides consisting of:

[0041] Component a) beta-galacto-oligosaccharides (bGOS) having an average degree of polymerization (DP) ranging from 3 to 7 and long chain fructo-oligosaccharides (lcFOS) having an average degree of polymerization (DP) ranging from 20 to 40, and

[0042] Component b) (combination of 5 HMOs) 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL),

[0043] wherein component a) consists of 70 to 95 wt% of bGOS based on the total weight of component a) and 5 to 30 wt% of lcFOS based on the total weight of component a), the sum of bGOS and lcFOS being 100% of the total weight of component a), and

[0044] wherein component b) is composed of 42 to 62 wt% 2'-FL, 10 to 16 wt% 3-FL, 21 to 31 wt% LNT, 3 to 5 wt% 3'-SL and 4 to 6 wt% 6'-SL, and the sum of 2'-FL, 3-FL, LNT, 3'-SL and 6'-SL is 100% of the total weight of component b).

[0045] The amount of non-digestible oligosaccharides is preferably 120 mg to 2.40 g / 100 ml, more preferably 280 mg to 1.80 g / 100 ml, even more preferably 540 mg to 1.25 g / 100 ml. Based on the dry weight of the composition, the amount of non-digestible oligosaccharides is preferably 0.86 to 17.14 wt%, more preferably 2.00 to 12.86 wt%, even more preferably 3.86 to 8.93 wt%. Preferably, the amount of non-digestible oligosaccharides per 100 kcal is 180 mg to 3.60 g, more preferably 420 mg to 2.70 g, even more preferably 810 mg to 1.88 g.

[0046] In a preferred embodiment, in the nutritional composition of the present invention, the amount of bGOS plus lcFOS is 100 mg to 2 g / 100 ml, more preferably 250 mg to 1.5 g, even more preferably 500 mg to 1 g / 100 ml; the amount of bGOS plus lcFOS is 0.7 to 14.3 wt%, more preferably 1.8 to 10.7 wt%, even more preferably 3.6 to 7.1 wt% based on the dry weight of the composition; and / or the amount of bGOS plus lcFOS per 100 kcal is 150 mg to 3 g, more preferably 375 mg to 2.25 g, even more preferably 750 mg to 1.5 g; and

[0047] The amount of the combination of the five HMOs is 20 to 400 mg / 100 ml, more preferably 30 to 300 mg / 100 ml, even more preferably 40 to 250 mg / 100 ml; based on the dry weight of the composition, the amount of the combination of the five HMOs is 0.14 to 2.86 wt%, more preferably 0.21 to 2.14 wt%, even more preferably 0.28 to 1.79 wt%; and / or the amount of the combination of the five HMOs per 100 kcal is 30 to 600 mg, more preferably 45 to 450 mg, even more preferably 60 to 375 mg.

[0048] Preferably, the nutritional composition according to the present invention comprises 50 to 97.5 wt% of component a) based on the total non-digestible oligosaccharides and 2.5 to 50 wt% of component b) based on the total non-digestible oligosaccharides, the sum of component a) and component b) being 100% of the total non-digestible oligosaccharides. More preferably, the nutritional composition according to the present invention comprises 60 to 95 wt% of component a) based on the total non-digestible oligosaccharides and 5 to 40 wt% of component b) based on the total non-digestible oligosaccharides, the sum of component a) and component b) being 100%. Most preferably, the nutritional composition according to the present invention comprises 75 to 90 wt% of component a) based on the total non-digestible oligosaccharides and 10 to 25 wt% of component b) based on the total non-digestible oligosaccharides, the sum of component a) and component b being 100%. Preferably, the weight ratio of component a / b is in the range of 39 to 1, more preferably in the range of 19 to 1.5, even more preferably in the range of 9 to 3. Such a ratio will produce a further improvement effect on the microecology and intestinal barrier function.

[0049] Preferably, based on the total weight, the mixture of non-digestible oligosaccharides consists of 60 to 90 wt% bGOS, 7.5 to 10 wt% lcFOS, 1.5 to 15 wt% 2'-FL, 0.4 to 4 wt% 3-FL, 0.75 to 7.5 wt% LNT, 0.10 to 1.2 wt% 3'-SL, and 0.10 to 1.5 wt% 6'-SL, the total being 100%. More preferably, based on the total weight, the mixture of non-digestible oligosaccharides consists of 65 to 85 wt% bGOS, 7.5 to 9.5 wt% lcFOS, 5 to 12 wt% 2'-FL, 1 to 3 wt% 3-FL, 2 to 6 wt% LNT, 0.2 to 1.0 wt% 3'-SL, and 0.20 to 1.2 wt% 6'-SL. Such a ratio will produce an effect of further improving microecology and intestinal barrier function.

[0050] Nutritional composition

[0051] The nutritional composition according to the present invention is not natural cow's milk or natural milk from another mammal. The nutritional composition of the present invention preferably comprises digestible carbohydrates, protein and lipids, wherein the lipids preferably provide 30% to 60%, preferably 35% to 55% of the total calories, the protein provides 5% to 15%, more preferably 6% to 12%, even more preferably 7% to 9% of the total calories, and the digestible carbohydrates provide 25% to 75%, more preferably 40% to 60% of the total calories. The caloric density of the non-digestible oligosaccharides is 2 kcal / g and preferably accounts for 0.4% to 7% of the total calories. The nutritional composition preferably comprises 3 to 7 g lipids / 100 kcal, preferably 4 to 6 g lipids / 100 kcal, more preferably 4.5 to 5.5 g lipids / 100 kcal; it preferably comprises 1.25 to 4 g protein / 100 kcal, more preferably 1.5 to 3.0 g protein / 100 kcal, more preferably 1.8 to 2.2 g protein / 100 kcal, and it preferably comprises 6 to 20 g digestible carbohydrates / 100 kcal, more preferably 10 to 15 g digestible carbohydrates / 100 kcal.

[0052] Preferably, when the nutritional composition is in ready-to-use form, its energy density is 45 to 75 kcal / 100 ml, more preferably 60 to 70 kcal / 100 ml, even more preferably 65 to 70 kcal / 100 ml. Such a density ensures an optimal balance between hydration and caloric intake.

[0053] The nutritional composition is preferably a solid product, preferably a powder. Suitably, the nutritional composition is in powder form, which can be reconstituted with water to form a ready-to-use liquid. Alternatively, the nutritional composition can be a ready-to-use liquid or in the form of a liquid concentrate that should be diluted with water to form a ready-to-use liquid.

[0054] The nutritional composition preferably includes digestible carbohydrates. Based on heat, the nutritional composition preferably includes 6g to 20g digestible carbohydrates / 100kcal, more preferably 10g to 15g / 100kcal. When in liquid form (e.g., as a ready-to-use liquid), the nutritional composition preferably includes 4g to 15g digestible carbohydrates / 100ml, more preferably 7g to 10g / 100ml. Based on dry weight, the nutritional composition preferably includes 30 to 85wt%, more preferably 40 to 65wt% digestible carbohydrates. Alternatively, when the nutritional composition is in powder form, digestible carbohydrates are preferably present in an amount of 40g to 85g / 100g dry weight, more preferably 40g to 65g / 100g dry weight. Preferred digestible carbohydrate sources are one or more of lactose, glucose, sucrose, fructose, galactose, maltose, starch and maltodextrin. Lactose is the main digestible carbohydrate present in human milk. Lactose advantageously has a low glycemic index. The nutritional composition preferably comprises lactose.The nutritional composition preferably comprises digestible carbohydrates, wherein at least 35 wt%, more preferably at least 50 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.

[0055] The nutritional composition preferably comprises protein. The protein concentration in the nutritional composition is determined by the sum of protein, peptides and free amino acids. Preferably, the nutritional composition comprises 1.25g to 4g protein / 100kcal, even more preferably 1.5g to 3.0g protein / 100kcal, even more preferably 1.8 to 2.2g / 100kcal. A low protein concentration is advantageously closer to human milk because human milk contains a lower amount of protein based on total calories compared to cow's milk. Based on the ready-to-use liquid product, the nutritional composition preferably comprises 0.8 to 2.5g / 100ml, more preferably 1.0g to 2.0g / 100ml, even more preferably 1.2 to 1.5g / 100ml. Based on dry weight, the nutritional composition preferably comprises 6 to 18wt%, more preferably 7 to 15wt%, even more preferably 8 to 11wt% protein. Alternatively, when the nutritional composition is in powder form, the protein is preferably present in an amount of 6 to 18 g / 100 g dry weight, more preferably 7 to 15 g, even more preferably 8 to 11 g / 100 g dry weight. The source of the protein is preferably selected in such a way that the minimum requirements for the essential amino acid content are met and that satisfactory growth is ensured. Thus, protein sources based on bovine milk proteins (such as whey proteins, caseins, and mixtures thereof) and proteins based on soy, potato or pea are preferred. In the case of using whey proteins, the protein source is preferably based on acid whey or sweet whey, modified sweet whey, whey protein isolate or mixtures thereof.

[0056] The nutritional composition of the present invention preferably includes lipid.Lipid is preferably present in an amount of 3g to 7g / 100kcal, more preferably in an amount of 4g to 6g lipid / 100kcal and most preferably in an amount of 4.5g to 5.5g lipid / 100kcal. When in liquid form (e.g., as a ready-to-use liquid), the nutritional composition preferably includes 2.2g to 4.5g lipid / 100ml, more preferably 2.5g to 4.0g, even more preferably 3.0 to 3.75g / 100ml. Based on dry weight, the nutritional composition preferably includes 16 to 32wt%, more preferably 18 to 30wt%, even more preferably 20 to 28wt% lipid. Alternatively, when the nutritional composition is in powder form, lipid is preferably present in an amount of 16g ​​to 32g / 100g dry weight, more preferably 18 to 30g, even more preferably 20 to 28g / 100g dry weight.

[0057] Lipid preferably comprises vegetable lipid.The existence of vegetable lipid advantageously realizes the best fatty acid profile of high polyunsaturated fatty acids (such as essential linolenic acid and α-linolenic acid), and is more similar to human milk fat.Only lipid from non-human mammal milk (such as cow's milk) can not provide best fatty acid profile.The amount of essential fatty acids in non-human mammal milk is too low.Preferably, the nutritional composition comprises at least one, preferably at least two vegetable lipid sources selected from the group consisting of: linseed oil (linseed oil or flaxseed oil), rapeseed oil (rape seed oil) (such as rapeseed oil (colza oil), low erucic acid rapeseed oil and canola oil), sunflower oil, high oleic sunflower oil, safflower oil, high oleic safflower oil, olive oil, coconut oil, soybean oil, palm oil and palm kernel oil.

[0058] In addition, animal fat (such as cow's milk fat) is preferably present in the nutritional composition. Such a lipid source can provide additional desirable components such as butyric acid (BA) and caproic acid (CA) as well as beta-palmitic acid (sn2-PA). Components such as butyric acid are known to have synergistic effects with human milk oligosaccharides on the intestinal barrier, the immune system, and anti-pathogenic effects. Preferably, the nutritional composition comprises at least 0.5 wt% butyric acid, more preferably at least 0.7 to 2 wt%, based on total fatty acids.

[0059] In addition, egg oil and / or fish oil and / or microbial oil (such as oil from fungi and algae) may be present. Such oils are suitable sources of long-chain polyunsaturated fatty acids (such as docosahexaenoic acid (DHA), arachidonic acid (ARA) and / or eicosapentaenoic acid (EPA)). Preferably, the nutritional composition comprises n3 LC-PUFA, such as EPA and / or DHA, more preferably DHA. Preferably, the nutritional composition comprises at least 0.05wt%, preferably at least 0.1wt%, more preferably at least 0.2wt% DHA based on total fatty acids. Preferably, the nutritional composition comprises no more than 2.0wt%, preferably no more than 1.0wt% DHA based on total fatty acids. The nutritional composition preferably comprises ARA. Preferably, the nutritional composition comprises at least 0.05wt%, preferably at least 0.1wt%, more preferably at least 0.2wt% ARA based on total fatty acids.

[0060] Preferably, additional ingredients are present in the nutritional composition, like vitamins, minerals, trace elements, nucleotides and other micronutrients known in the art.

[0061] Preferably, the nutritional composition comprises lactic acid bacteria, particularly Bifidobacterium, selected from the group consisting of Bifidobacterium and / or Lactobacillus. The content of Bifidobacterium is high in the intestinal microecology of infants receiving breastfeeding. Adding one or more strains belonging to the genus Bifidobacterium to the nutritional composition will further improve the intestinal microecology and its activity. More preferably, the nutritional composition comprises Bifidobacterium breve. Such strains of Bifidobacterium breve species are commercially available or can be isolated from the microecology of the infant. An example of a commercially available strain is Bifidobacterium breve M-16V from Morinaga. The amount of Bifidobacterium and / or Lactobacillus is preferably 10 4 to 10 11 cfu / gram dry weight of the nutritional composition.

[0062] The nutritional composition according to the present invention is preferably in the form of infant formula, larger infant formula or toddler formula. This means that the composition to be administered is not human milk. Infant formula or larger infant formula or toddler formula means that it relates to an artificially manufactured composition, or in other words, it is a synthetic composition. In the context of the present invention, toddler formula may also be referred to as growing-up milk. The nutritional composition of the present invention is preferably intended to or used to provide nutrition to an infant or toddler.

[0063] Infant formula is intended to be used for the baby of about 4 to 6 months from birth to age, and is intended as a substitute for human milk. Usually, infant formula is suitable for being used as sole source of nutrition. Such infant formula is also referred to as a section of formula. In the context of the present invention, this is referred to as nutritional composition or infant formula, for the first 6 months of life.

[0064] Follow-on formula is intended for infants from the age of 4 to 6 months up to the age of 12 months and is intended as a complementary feeding for infants who are starting to eat other foods for weaning. In the context of the present invention, this is referred to as a nutritional composition or follow-on formula for infants from the age of 6 to 12 months.

[0065] Infant formula refers to an artificially manufactured nutritional composition intended for children between 12 and 36 months of age, in other words, for children between 1 and 3 years of age, intended as a complementary feeding. In the context of the present invention, this is referred to as a nutritional composition or infant formula for children between 12 and 36 months of age (for children between 1 and 3 years of age).

[0066] Infant formula and follow-on formula are strictly regulated, for example EU Regulations No. 609 / 2013 and 2016 / 127 and the Codex Alimentarius for Infant Formula CODEXSTAN 72-1981. Toddler formula preferably follows the directive provisions for follow-on formula.

[0067] The nutritional composition is preferably an infant formula or a follow-on formula.

[0068] When the nutritional composition is in the form of an infant formula, the nutritional composition preferably comprises 0.25 g to 1.50 g / 100 ml, more preferably 0.50 to 1.00 g / 100 ml of component a), and preferably 0.03 to 0.40 g, more preferably 0.04 to 0.30 g / 100 ml of component b). When the nutritional composition is in the form of an infant formula, the weight ratio a / b is preferably in the range of 2 to 9, more preferably in the range of 3 to 6.

[0069] When the nutritional composition is in the form of a follow-on formula, the nutritional composition preferably comprises 0.25 to 1.50 g / 100 ml, more preferably 0.50 to 1.00 g / 100 ml, of component a) per 100 ml, and preferably 0.03 to 0.30 g, more preferably 0.04 to 0.25 g / 100 ml of component b). When the nutritional composition is in the form of a follow-on formula, the weight ratio a / b is preferably in the range of 5 to 10, more preferably in the range of 7 to 9.

[0070] When the nutritional composition is in the form of a toddler formula, the nutritional composition preferably comprises 0.50 to 2.00 g, more preferably 0.50 to 1.50 g / 100 ml of component a), and preferably 0.02 to 0.30 g, more preferably 0.03 to 0.25 g / 100 ml of component b). When the nutritional composition is in the form of a toddler formula, the weight ratio a / b is preferably in the range of 15 to 39, more preferably in the range of 20 to 30.

[0071] Young children require higher amounts of non-digestible oligosaccharides because formula plays a lower role in their total daily diet than, for example, infant formula, which can be the sole form of nutrition, making it more difficult to correct the microbiome. Similarly, in human milk, the amount of HMOs decreases during lactation as the infant ages.

[0072] application

[0073] The nutritional composition of the present invention is preferably used to provide nutrition to infants or toddlers, preferably to infants. Infants are human children under the age of 12 months. Toddlers are human children between the ages of one and three years, also known as toddlers.

[0074] Preferably, the nutritional composition of the present invention is provided to a human subject during the first 3 years of life. Preferably, the nutritional composition is used in a method of providing nutrition to a human subject in the first 12 months of life, optionally in the first 3 years of life, the method comprising providing a first infant formula for the first 6 months of life, wherein the formula comprises the mixture of non-digestible oligosaccharides of the present invention, wherein component a) provides 0.25 to 1.50 g / 100 ml, more preferably 0.50 to 1.00 g, and component b) provides 0.03 to 0.40 g, preferably 0.04 to 0.30 g / 100 ml, providing a second follow-on formula for infants aged 6 to 12 months, wherein the formula comprises the mixture of non-digestible oligosaccharides of the present invention, wherein component a) provides 0.25 to 1.2 g / 100 ml, more preferably 0.50 to 1.00 g and component b) provides 0.03 to 0.30 g, preferably 0.04 to 0.25 g / 100 ml, and optionally a third infant formula for an age range of 12 to 36 months (i.e. 1 to 3 years) is provided, the third formula comprising a mixture of non-digestible oligosaccharides of the present invention, wherein component a) provides 0.50 to 2.00 g / 100 ml, more preferably 0.50 to 1.50 g, and component b) provides 0.02 to 0.30 g, preferably 0.03 to 0.25 g / 100 ml, provided that the weight ratio a / b of the second formula is higher than the weight ratio a / b of the first formula, and optionally the weight ratio a / b of the third formula is higher than the weight ratio a / b of the second infant formula. Preferably, the weight ratio a / b of the first formula is 2 to 9, the weight ratio a / b of the second formula is 5 to 10, and the weight ratio a / b of the third formula is 15 to 39. More preferably, the weight ratio a / b of the first formula is 3 to 6, the weight ratio a / b of the second formula is 7 to 9, and the weight ratio a / b of the third formula is 20 to 30.

[0075] The inventors discovered that a specific combination of non-digestible oligosaccharides consisting of bGOS, lcFOS, 2'-FL, 3-FL, LNT, 3'-SL, and 6'-SL, as described above, improves the microbiome by increasing the amount of bifidobacteria and reducing the amount of Enterobacteriaceae. Metagenomic analysis showed that KEGG orthologs related to carbohydrate metabolism and KEGG orthologs of enzymes specific for bifidobacteria also increased synergistically. This again indicates that a higher percentage of bacteria (i.e., bifidobacteria) have such genes. Combinations of the four HMOs with or without GOS and lcFOS were less effective in increasing bifidobacteria or reducing Enterobacteriaceae. Therefore, the nutritional composition of the present invention is used to improve the intestinal microbiome in a subject, preferably an infant or toddler, more preferably an infant. Preferably, the improvement in the intestinal microbiome is selected from at least one of the following: increasing bifidobacteria and reducing pathogens in the intestinal microbiome of a subject, preferably an infant or toddler, more preferably an infant, wherein the pathogens are preferably Enterobacteriaceae.

[0076] The inventors have found that fermentation supernatants of a specific combination of non-digestible oligosaccharides consisting of bGOS, lcFOS, 2'-FL, 3-FL, LNT, 3'-SL, and 6'-SL as described above improve intestinal barrier resistance when compared to bGOS plus lcFOS, or when compared to a combination of the five HMOs. Increased intestinal barrier resistance is beneficial because it reduces the translocation of toxins and pathogens.

[0077] Due to this improved microecology and improved intestinal barrier function, the nutritional composition of the present invention will prevent and / or treat gastrointestinal disorders in a subject, preferably an infant or young child, more preferably an infant. Preferably, the gastrointestinal disorder is selected from the group consisting of intestinal microbial dysbiosis in or resulting from intestinal and intestinal inflammation.

[0078] Therefore, the present invention also relates to a method for improving intestinal microecology in an infant or young child, the method comprising administering to the infant or young child a nutritional composition according to the present invention. This method can be regarded as a non-therapeutic method.

[0079] For some jurisdictions, this aspect of the invention may be formulated as the use of a non-digestible oligosaccharide for the manufacture of a nutritional composition according to the invention for improving the intestinal microecology in an infant or young child.

[0080] Alternatively, this may be formulated as a nutritional composition according to the invention for use in improving the intestinal microecology in infants or young children.

[0081] In one embodiment, improving the intestinal microecology is increasing bifidobacteria in the intestine and / or reducing pathogens in the intestine, preferably reducing Enterobacteriaceae in the intestine. In one embodiment, improving the intestinal microecology is improving the intestinal barrier function.

[0082] In one aspect, the present invention also relates to a method for improving intestinal barrier function in an infant or young child, the method comprising administering to the infant or young child a nutritional composition according to the present invention. This method can be regarded as a non-therapeutic method.

[0083] For some jurisdictions, this aspect of the invention may be formulated as the use of a non-digestible oligosaccharide for the manufacture of a nutritional composition according to the invention for improving the intestinal barrier function in an infant or young child.

[0084] Alternatively, this may be formulated as a nutritional composition according to the invention for use in improving the intestinal barrier function in infants or young children.

[0085] The present invention also relates to a method for preventing and / or treating intestinal disorders, which method comprises administering a nutritional composition according to the invention to an infant or young child.

[0086] For some jurisdictions, this aspect of the invention may be formulated as the use of a non-digestible oligosaccharide for the manufacture of a nutritional composition according to the invention for the prevention and / or treatment of intestinal disorders.

[0087] Alternatively, this may be formulated as a nutritional composition according to the invention for use in the prevention and / or treatment of intestinal disorders.

[0088] In one embodiment, the intestinal disorder is selected from the group consisting of dysbiosis in or resulting from the intestinal and intestinal inflammation, infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 : Mean relative TEER% (sd) of confluent caco-2 monolayers after 48 h of treatment with supernatant of fermented non-digestible oligosaccharides, normalized to the mean relative TEER of bGOS / lcFOS.

[0090] Examples

[0091] Example 1: A combination of five HMOs and a mixture of bGOS / lcFOS synergistically improves infant microecology

[0092] Materials and Methods

[0093] Stool samples were selected from:

[0094] - Infants 1, 3 months old breastfed male infants,

[0095] - Infants 2, 3 months old breastfeeding female infants,

[0096] - Infant 3, a 4.5-month-old female infant who is partly breastfed and partly formula-fed, and

[0097] - Infant 4, 4 months old breastfeeding female infant

[0098] The following mixtures were tested:

[0099] - Component a) bGOS and lcFOS, weight ratio 90:10

[0100] - Component b) a combination of five HMOs: 2'-FL, 3-FL, LNT, 3'-SL and 6'-SL, with wt% of 52 wt%, 13 wt%, 26 wt%, 4 wt% and 5 wt% respectively

[0101] - A mixture according to the invention: bGOS, lcFOS, 2'-FL, 3-FL, LNT, 3'-SL and 6'-SL in wt% ratios of 65.9wt%, 8.7wt%, 13.2wt%, 3.3wt%, 6.6wt%, 1.0wt% and 1.3wt%, respectively.

[0102] In addition, stool samples from infants 2 and 3 were tested for the following combination:

[0103] - A combination of 4 HMOs (without LNT): 2'-FL, 3-FL, 3'-SL and 6'-SL, with wt% of 39 wt%, 23 wt%, 10 wt% and 28 wt% respectively

[0104] - Comparative mixture: bGOS, lcFOS, 2'-FL, 3'FL, 3'-SL and 6'-SL with wt ratios of 64.8 wt% bGOS wt%, 8.6 wt% lcFOS wt%, 10.3% 2'-FL wt%, 6.2% 3-FL wt%, 2.4% 3'-SL wt% and 7.5 wt% 6'-SL, respectively.

[0105] Colon microecological medium: yeast extract 1g / l, ammonium sulfate 2g / l, K2HPO4 2g / l, NaHCO33.2g / l, NaCl 4.5g / l, MgSO4.7H2O 0.5g / l, cysteine ​​HCl 0.5g / l, CaCl2.2H2O 0.4g / l, bile salts 25mg / l, menadione, metal and vitamin solution, hemin (10mg / l), mucus 2.5g / l.

[0106] Under anaerobic conditions, the fecal samples were thawed and a 4% (w / v) suspension of each fecal sample was prepared in a carbon-free infant-adapted colonic microecological medium adjusted to the pH of breast-fed infant feces (pH 5.5), containing 25 mM acetate and 12 mM lactate, 25 mg / L bile acid (Sigma), 2.5 g / L porcine gastric mucin, 15 mmol / L ammonium sulfate, 1 g / L tryptone. The diluted fecal samples were homogenized, allowed to settle for 5 minutes, then filtered to remove large particles and subsequently filtered on a Millex 100 μm vacuum filter. After the initial fecal community stabilized (the first 4 hours of fermentation), a refeed / renewal medium without selective acetate and lactate was used, as the microecology itself will be sufficient to produce the selective acetate and lactate.

[0107] A 32-well Biolector Pro plate with a pH optode (BOH2 round well, M2P-labs) was used. All fermentation wells of the plate were filled with 1.52 ml of fecal solution, and one feed row of the plate was filled with sterile 3 M NaOH. For the continuous night feeding mode, the wells of the other feed row were filled with the following: water (blank), 10 wt% bGOS: lcFOS 9: 1, 10 wt% combination of 4 HMOs, 10% combination of 5 HMOs, 10% bGOS: lcFOS: 4 HMOs, and bGOS: lcFOS: 5 HMOs. All conditions were performed four times (per column). The plate was sealed with a ventilated silicone foil with a slit. The plate was incubated in a BioLector Pro (85% moisture, 37°C, 600 rpm, anaerobic). Eighty microliters of 10% (w / v) sterile carbohydrate solution (as described above) were added, or as a control, 80 ul of sterile water (blank) was added. The experiment started with a pH setting of 5.8 and performed continuous pH measurements. The pH was controlled at pH 5.4-5.8. After 4 hours, the experiment was suspended, the fecal slurry of each well was harvested, and centrifuged for a short time under sterile anaerobic conditions. The supernatant was frozen for further analysis, and the fecal sediment was resuspended in fresh culture medium with carbohydrates and pipetted back into the original wells of the BOH2 plate. The procedure was repeated again for 3 days, with manual feeding twice a day (performed every 4 hours), followed by automatic continuous slow feeding (80 μl overnight) via a microfluidic system. After 3 days, the final precipitate was harvested and subjected to metagenomic sequencing.

[0108] Metagenomic sequencing: The genomic DNA was sequenced using a previously described protocol ( Total DNA was extracted from the pellet (Ramírez-Bosca et al., 2018) Scientific Reports 8(1):3812. A total of 50 ng of DNA was amplified using the 16S metagenomic sequencing library Illumina 15044223B protocol (Illumina). The Nextera XT library kit (Illumina) was used according to the manufacturer's instructions. The DNA was simultaneously fragmented and tagged with dual-index sequencing adapters. Next, the NovaSeq 6000 sequencing platform was used in a 150 paired-end read configuration (NovaSeq Control Software (NCS) version 1.6). The Bcl2fastq 2.20 program was used to translate the sequencing reads and remove the sequencing adapters. Optical duplicates were removed using the Clumpify tool from the BBTools suite (Bushnell B, BBMap (2015). sourceforge.net / projects / bbmap / ), and reads with a Phred quality score below Q20 and length <50 nucleotides were filtered using BBMap v38.36 (Bushnell B, 2015). Human genome sequences were filtered using NGLess v1.0.0-Linux64 (Coelho, Alves et al., 2019) Microbiome [Microbiome] 7(1):84, and these reads were assigned to classification using Metaphlan (v.4) (Blanco-Míguez, Beghini et al., 2022 bioRxiv preprint doi: https: / / doi.org / 10.1101 / 2022.08.22.504593). Reads from each sample were aligned to single-copy genetic markers present in almost all bacteria. From these alignments, we calculated the estimated number of reads contributing to a given clade for each identified taxon. For each sample, we divided the counts of reads identified as Bifidobacteriaceae and Enterobacteriaceae by the number of reads that could be assigned to any bacterial taxon to calculate the relative abundance of these (Bifidobacteriaceae and Enterobacteriaceae) bacterial groups.

[0109] result

[0110] The results are shown in Tables 1 and 2. Consistently, when the mixture of 5 HMOs with bGOS and lcFOS was used, the % of Bifidobacteria increased, and the % of Enterobacteriaceae decreased. This increase in Bifidobacteriaceae and decrease in Enterobacteriaceae were greater than would be expected from the combination of 5 HMOs or the combination of bGOS plus lcFOS alone, thus demonstrating a synergistic effect. The bGOS / lcFOS / 5 HMO mixture also outperformed the combination of 4 HMOs and the bGOS / lcFOS / 4 HMO mixture. For infants 2 and 3, the % of total Bifidobacteriaceae was higher, and a synergistic improvement in Bifidobacteriaceae was also observed with the bGOS / lcFOS / 5 HMO mixture. Similarly, the % of Enterobacteriaceae was lower with the bGOS / lcFOS / 5 HMO mixture.

[0111] Table 1: % Bifidobacteria in total bacteria

[0112]

[0113] Table 2: % Enterobacteriaceae in total bacteria

[0114]

[0115] These results demonstrate that a specific combination of five HMOs with a mixture of bGOS and lcFOS has an improved and unexpected synergistic effect. Bifidobacteria increased, while potential pathogens, such as Gram-negative Enterobacteriaceae, decreased. This demonstrates the specificity of the mixture consisting of five specific HMOs in combination with bGOS and lcFOS in improving the microbiome.

[0116] Metagenomic analysis revealed a coordinated increase in KEGG orthologs related to carbohydrate metabolism and enzymes specific to Bifidobacteria. This included, for example, (ko: K22397yjhH, yagE; 2-dehydro-3-deoxy-D-pentose aldolase [EC: 4.1.2.28]), which is involved in the breakdown of fucose derived from fucosyllactose. This suggests that a higher percentage of bacteria (i.e., Bifidobacteria) harbor these genes.

[0117] Example 2: Effect of 3-fucosyllactose on Bifidobacterium Growth and Enterobacteriaceae Inhibition in Combinations of Five HMOs Contribution

[0118] Materials and Methods

[0119] Stool samples were selected from:

[0120] -Baby 1, 6 months mixed feeding male baby,

[0121] - Infants 2, 3 months old breastfeeding female infants,

[0122] The following mixtures were tested:

[0123] - A combination of five HMOs: 2'-FL, 3-FL, LNT, 3'-SL, 6'-SL, with wt% of 52wt%, 13wt%, 26wt%, 4wt% and 5wt% respectively

[0124] - A combination of 4 HMOs (without 3-FL): 2'-FL, LNT, 3'-SL, 6'-SL, with wt% of 59.8 wt%, 29.9 wt%, 4.6 wt% and 5.7 wt% respectively

[0125] The samples were subjected to the same protocol and screened as in Example 1 to obtain the relative abundance of Bifidobacterium and Enterobacteriaceae bacterial groups.

[0126] result

[0127] Selective bifidobacterial plating showed that for both donors, more CFU bifidobacteria were present with the combination of 5 HMOs compared to the combination of HMOs without 3-FL, confirming that 3-FL contributes to the pro-bifidobacterial growth effect of the combination of 5 HMOs.

[0128] The results are further shown in Tables 3 and 4.

[0129] Table 3: % Bifidobacteria in total bacteria

[0130] baby 4 HMOs (minus 3-FL) 5 types of HMOs 1 45.4 61.7 2 47.9 54.8

[0131] Table 4: % Enterobacteriaceae in total bacteria

[0132] baby 4 HMOs (minus 3-FL) 5 types of HMOs 1 53.1 38.2 2 24.0 17.2

[0133] Compared with the combination of 4 HMOs without 3-FL, the combination of 5 HMOs increased the % of Bifidobacteria and decreased the % of Enterobacteriaceae. It can be concluded that 3-FL contributes significantly to the growth-promoting effect on Bifidobacteria and the suppression of opportunistic pathogens in the Enterobacteriaceae family.

[0134] For infant 1, the stool samples were also tested for the presence of lcFOS. Enterobacteriaceae inhibition The impact was tested.

[0135] The following combinations were tested:

[0136] - Comparative mixture bGOS / 5 HMOs (without 1cFOS): bGOS, 2'-FL, 3-FL, LNT, 3'-SL and 6'-SL, with wt ratios of 72.2 wt%, 14.4 wt%, 3.6 wt%, 7.2 wt%, 1.1 wt% and 1.4 wt%, respectively

[0137] - A mixture according to the invention: bGOS, lcFOS, 2'-FL, 3-FL, LNT, 3'-SL and 6'-SL in wt% ratios of 65.9wt%, 8.7wt%, 13.2wt%, 3.3wt%, 6.6wt%, 1.0wt% and 1.3wt%, respectively.

[0138] Table 5: % Enterobacteriaceae in total bacteria

[0139]

[0140] This suggests that lcFOS helps to suppress opportunistic pathogens in the Enterobacteriaceae family.

[0141] Example 3: A combination of five HMOs and fecal fermentation supernatant of GOS / lcFOS synergistically improves intestinal barrier function

[0142] Materials and Methods

[0143] Human colon cancer Caco-2 (ATCC) cells were cultured at 1 x 10 5 Cells were cultured in transwells (Corning 3460) at a density of 10 cells / well. To investigate the effect on barrier development, cell monolayers were treated 6 days after inoculation at confluence with supernatants extracted from fermented bGOS:lcFOS 9:1, combinations of the 5 HMOs fermented, and mixtures thereof. Supernatants from 3 days of fermentation using fecal slurries from infants 1, 3, and 4 of Example 1 (supernatant from infant 2 was not tested) were tested. Supernatants were filtered with a 2 μm pore size and diluted 1 / 10 in DMEM without FCS (Gibco) before addition to the cell monolayers. The medium was refreshed after 24 h of treatment.

[0144] After 48 h of treatment, the permeability of the monolayers was assessed by measuring transepithelial electrical resistance (TEER) using a Millicell ERS-2 volt-ohmmeter (Millipore).

[0145] result

[0146] The results are as follows Figure 1 These values ​​represent the ratio of the TEER value of the treatment group to its baseline value, and were then normalized to the bGOS / lcFOS group of the corresponding donor to 100%. Each donor was treated twice, and the average of three donors was then calculated.

[0147] Surprisingly, the fermentation supernatant of the combination of the five HMOs with a specific mixture of bGOS and lcFOS consistently showed higher TEER values ​​when compared to either the combination of the five HMOs alone or the combination of bGOS plus lcFOS alone, and a synergistic increase was observed. This effect was also observed in the supernatants obtained after fermentation with microbiota from individual infants (data not shown).

[0148] These results suggest that the combination of five HMOs and a specific mixture of bGOS and lcFOS has an ameliorative and synergistic effect on intestinal barrier function after fermentation by the intestinal microecology.

[0149] Example 4: Infant formula

[0150] Infant formula intended for infants aged 0-6 months, each 100 ml (obtained by reconstitution of 13.7 g of powder with water) containing:

[0151] -67kcal

[0152] -Digestible carbohydrates (primarily lactose): 7.3g

[0153] -Protein (whey protein, casein): 1.3g

[0154] -Lipids: 3.4g

[0155] - 0.9g of non-digestible oligosaccharides consisting of:

[0156] o 0.7 g GOS / lcFOS, wt / wt ratio 7.6:1

[0157] 0.2 g of a mixture of five HMOs consisting of 52 wt% 2'-FL, 13 wt% 3-FL, 26 wt% LNT, 4 wt% 3'-SL, and 5 wt% 6'-SL

[0158] - Micronutrients as per the Infant Formula Directive

[0159] Example 5: Infant formula

[0160] Infant formula intended for infants aged 0-6 months, each 100 ml (obtained by reconstitution of 13.7 g of powder with water) containing:

[0161] -66kcal

[0162] -Digestible carbohydrates (primarily lactose): 7.2g

[0163] -Protein (whey protein, casein): 1.3g

[0164] - Lipids: 3.4g

[0165] - Non-digestible oligosaccharides 0.872g consisting of:

[0166] o 0.7 g GOS / lcFOS, wt / wt ratio 9:1

[0167] o 0.172 g of a combination of five HMOs consisting of 52 wt% 2'-FL, 13 wt% 3-FL, 26 wt% LNT, 4 wt% 3'-SL, and 5 wt% 6'-SL

[0168] - Micronutrients as per the Infant Formula Directive

[0169] Example 6: Follow-on Formula

[0170] Follow-on formula is intended for infants aged 6-12 months. Each 100 ml (obtained by reconstituting 14.4 g of powder with water) contains:

[0171] -68kcal

[0172] -Digestible carbohydrates (mainly lactose): 8.2g

[0173] -Protein (whey protein, casein): 1.4g

[0174] - Lipids: 3.2g

[0175] - Non-digestible oligosaccharides 0.786g consisting of:

[0176] o 0.7 g GOS / lcFOS, wt / wt ratio 9:1

[0177] o85 mg of a combination of 5 HMOs consisting of 52 wt% 2'-FL, 13 wt% 3-FL, 26 wt% LNT, 4 wt% 3'-SL, and 5 wt% 6'-SL

[0178] - Micronutrients as per the Follow-up Formula Directive

[0179] Example 7: Infant formula

[0180] Toddler formula is intended for infants aged 12 to 36 months. Each 100 ml (after reconstitution of 14.4 g of powder with water) contains:

[0181] -65kcal

[0182] -Digestible carbohydrates (primarily lactose): 8.3g

[0183] -Protein (whey protein, casein): 1.3g

[0184] - Lipids: 2.7g

[0185] - Non-digestible oligosaccharides 1.243g consisting of:

[0186] o 1.2 g GOS / lcFOS, wt / wt ratio 9:1

[0187] o43 mg of a combination of 5 HMOs consisting of 52 wt% 2'-FL, 13 wt% 3-FL, 26 wt% LNT, 4 wt% 3'-SL, and 5 wt% 6'-SL

[0188] - Micronutrients as per the Follow-up Formula Directive

Claims

1. A nutritional composition for an infant or young child comprising a mixture of non-digestible oligosaccharides consisting of: Component a) beta-galacto-oligosaccharides (bGOS) having an average degree of polymerization (DP) ranging from 3 to 7 and long chain fructo-oligosaccharides (lcFOS) having an average degree of polymerization (DP) ranging from 20 to 40, and Component b) (combination of 5 HMOs) 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL), wherein component a) consists of 70 to 95 wt% of bGOS based on the total weight of component a) and 5 to 30 wt% of lcFOS based on the total weight of component a), the sum of bGOS and lcFOS being 100% of the total weight of component a), and wherein component b) is composed of 42 to 62 wt% 2'-FL, 10 to 16 wt% 3-FL, 21 to 31 wt% LNT, 3 to 5 wt% 3'-SL and 4 to 6 wt% 6'-SL, and the sum of 2'-FL, 3-FL, LNT, 3'-SL and 6'-SL is 100% of the total weight of component b).

2. The nutritional composition for infants or young children according to claim 1, comprising 50 to 97.5 wt% of component a) based on the total weight of non-digestible oligosaccharides and 2.5 to 50 wt% of component b) based on the total weight of non-digestible oligosaccharides, the sum of component a) and component b) being 100% of the total weight of non-digestible oligosaccharides.

3. A nutritional composition for infants or young children according to any one of the preceding claims, wherein component a) consists of 85 to 95 wt% of bGOS, based on the total weight of component a), and 5 to 15 wt% of lcFOS, based on the total weight of component a), the sum of bGOS and lcFOS being 100% of the total weight of component a).

4. A nutritional composition for infants or young children according to any one of the preceding claims, wherein the mixture of non-digestible oligosaccharides consists of 60 to 90 wt% GOS, 7.5 to 10 wt% lcFOS, 1.5-15 wt% 2'-FL, 0.4-4 wt% 3-FL, 0.75 to 7.5 wt% LNT, 0.10 to 1.2 wt% 3'-SL and 0.10-1.5 wt% 6'-SL, the total being 100%.

5. The nutritional composition for infants or young children according to any one of the preceding claims, comprising 120 mg to 2.40 g of non-digestible oligosaccharides consisting of component a) and component b) per 100 ml, more preferably 280 mg to 1.80 g per 100 ml, when in ready-to-use form, or 0.86 to 17.14 wt%, more preferably 2.00 to 12.86 wt% of non-digestible oligosaccharides consisting of component a) and component b) based on dry weight, or 180 mg to 3.60 g, more preferably 420 mg to 2.70 g of non-digestible oligosaccharides consisting of component a) and component b) per 100 kcal, based on energy, when in powder form.

6. A nutritional composition for infants or young children according to any one of the preceding claims, wherein the amount of bGOS plus lcFOS is from 100 mg to 2 g / 100 ml, more preferably from 250 mg to 1.5 g, even more preferably from 500 mg to 1 g / 100 ml; the amount of bGOS plus lcFOS is from 0.7 to 14.3 wt%, more preferably from 1.8 to 10.7 wt%, even more preferably from 3.6 to 7.1 wt% bGOS plus lcFOS based on the dry weight of the composition; and / or the amount of bGOS plus lcFOS per 100 kcal is from 150 mg to 3 g, more preferably from 375 mg to 2.25 g, even more preferably from 750 mg to 1.5 g; and wherein the amount of the combination of the five HMOs is 20 to 400 mg / 100 ml, more preferably 30 to 300 mg / 100 ml, even more preferably 40 to 250 mg / 100 ml; based on the dry weight of the composition, the amount of the combination of the five HMOs is 0.14 to 2.86 wt%, more preferably 0.21 to 2.14 wt%, even more preferably 0.28 to 1.79 wt%; and / or the amount of the combination of the five HMOs per 100 kcal is 30 to 600 mg, more preferably 45 to 450 mg, even more preferably 60 to 375 mg.

7. A nutritional composition for infants or young children according to any one of the preceding claims, comprising 3 to 7 g lipids / 100 kcal, 1.25 to 4 g protein / 100 kcal and 6 to 20 g digestible carbohydrates / 100 kcal.

8. A nutritional composition for infants or young children according to any one of the preceding claims, which is an infant formula, a follow-on formula or a toddler formula.

9. A method for providing nutrition to a human subject during the first 12 months of life, optionally the first 3 years of life, the method comprising: a) providing a first nutritional composition for the first 6 months of life, the first nutritional composition comprising a mixture of non-digestible oligosaccharides according to claim 1 , wherein component a) provides 0.25 to 1.50 g / 100 ml, preferably 0.5 to 1.00 g / 100 ml, and component b) provides 0.03 to 0.40 g / 100 ml, preferably 0.04 to 0.30 g / 100 ml, b) providing a second nutritional composition for an age range of 6 to 12 months, the second nutritional composition comprising a mixture of non-digestible oligosaccharides according to claim 1 , wherein component a) provides 0.25 to 1.50 g / 100 ml, preferably 0.50 to 1.00 g / 100 ml, and component b) provides 0.03 to 0.30 g / 100 ml, preferably 0.04 to 0.25 g / 100 ml, and c) optionally providing a third nutritional composition for an age range of 12 to 36 months, i.e. 1 to 3 years, comprising a mixture of non-digestible oligosaccharides according to claim 1 , wherein component a) provides 0.50 to 2.00 g / 100 ml, preferably 0.50 to 1.50 g / 100 ml, and component b) provides 0.02 to 0.30 g / 100 ml, preferably 0.03 to 0.250 g / 100 ml, Provided that the ratio a / b of the second formula is higher than the ratio a / b of the first formula, and the ratio a / b of the optional third formula is higher than the ratio a / b of the second infant formula.

10. A method for improving intestinal microecology in an infant or young child, the method comprising administering to the infant or young child the nutritional composition according to any one of claims 1 to 8.

11. The method according to claim 10, wherein improving the intestinal microecology is increasing bifidobacteria in the intestine and / or reducing pathogenic bacteria in the intestine, preferably reducing Enterobacteriaceae in the intestine.

12. A method for improving intestinal barrier function in an infant or young child, the method comprising administering to the infant or young child the nutritional composition according to any one of claims 1 to 8.

13. The nutritional composition according to any one of claims 1 to 8, for use in preventing and / or treating intestinal disorders.

14. Nutritional composition for use according to claim 13, wherein the intestinal disorder is selected from the group consisting of microbial dysbiosis in the intestine and intestinal inflammation, infection or microbial dysbiosis resulting from the intestine and intestinal inflammation, infection.

15. Use of non-digestible oligosaccharides for the manufacture of a nutritional composition according to any one of claims 1 to 8, for improving intestinal microecology in infants or young children.

16. The use according to claim 15, wherein improving intestinal microecology is increasing bifidobacteria in the intestine and / or reducing pathogenic bacteria in the intestine, preferably reducing Enterobacteriaceae in the intestine.

17. Use of non-digestible oligosaccharides for the manufacture of a nutritional composition according to any one of claims 1 to 8 for improving intestinal barrier function in infants or young children.

18. Use of non-digestible oligosaccharides for the manufacture of a nutritional composition according to any one of claims 1 to 8, for use in preventing and / or treating intestinal disorders.

19. The use according to claim 18, wherein the intestinal disorder is selected from the group consisting of microbial dysbiosis in the intestine and intestinal inflammation, infection or microbial dysbiosis resulting from the intestine and intestinal inflammation, infection.

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