CN111698993A

CN111698993A - Fermentation formulations containing indigestible oligosaccharides

Info

Publication number: CN111698993A
Application number: CN201980012647.7A
Authority: CN
Inventors: S·蒂姆森; A·R·奥泽尔; J·科诺尔
Original assignee: Nutricia NV
Current assignee: Nutricia NV
Priority date: 2018-02-09
Filing date: 2019-02-11
Publication date: 2020-09-22
Also published as: EP3749328A1; WO2019155043A1

Abstract

Administering to the infant an infant formula comprising non-digestible oligosaccharides, preferably also partially fermented, results in an intestinal metabolomics profile and microbiota function that is more similar to breast-fed infants compared to infants fed conventional infant formulas.

Description

Fermentation formulations containing indigestible oligosaccharides

Technical Field

The present invention relates to the field of infant nutrition for improving the intestinal microbiota.

Background

It is widely accepted that the best nutrition for newborn infants is human milk. Infant Formula (IF) developed based on mature human milk composition is considered the best alternative when the mother is unable to breastfeed his infant or chooses not to breastfeed. Studies to improve the quality of infant formula do not necessarily aim to mimic the exact composition of human milk, but to achieve functional effects beyond those observed only in nutrition in breast-fed infants.

The human gut has a complex microbial ecosystem, the gut microbiota, which is considered to be an important part of human physiology. In adults, the gut microbiota is considered a stable ecosystem, and therefore, the process of microbial colonization early in life, which is closely related to the maturation of the gastrointestinal tract itself, can be considered an essential step in healthy development. Several environmental factors that can exist early in life have been shown to have a long-term impact on the gut microbiota and its activity, increasing the risk of disease later in life. Nutrition early in life is a major factor affecting the development of the intestinal microflora. Bifidobacteria species are usually predominant in the gut microbiota of breast-fed infants, whereas a gut microbiota rich in members of the Firmicutes (Firmicutes) is usually observed in infants fed with conventional formulas. However, what is relevant is not just the composition of the gut microbiota. Furthermore, the function of the microbiota and/or the presence of metabolites will to a large extent influence the intestinal physiology and biology and have an impact on the health of the present and future lives. Differences between breast-fed infants and conventional formula-fed infants were observed in these areas, which may explain why breast-fed infants have improved health outcomes in many areas regarding gut, immune system, brain and metabolic health.

Formulations supplemented with a prebiotic scGOS/lcFOS mixture have been shown to modulate the composition and function of the gut microbiota, i.e. to increase metabolic activity and fermentation properties to levels found in human milk-fed infants (Knol et al, 2005, JPGN 40: 36-42). It has previously been shown that when this scGOS/lcFOS mixture is added to infant milk formula partially fermented with Bifidobacterium breve (Bifidobacterium breve) and Streptococcus thermophilus (Streptococcus thermophilus), its effect on metabolic activity and fermentation properties can be largely preserved (Huet, f. et al, 2016, JPGN 63: e 43-53).

The above prior art documents disclose interventions to modulate specific metabolic parameters, on which targeted analyses are performed, such as the amount and relative contribution of specific short chain fatty acids, and pH values, but without concern for the entire metabolome.

WO 2017/021476 relates to a nutritional composition comprising fucosylated and N-acetylated oligosaccharides for promoting or inducing the overall intestinal microbiota such that it functions closer to a pure breast-fed infant than an infant fed with a conventional nutritional composition.

However, there is a need for a nutritional composition for further improving the overall intestinal microbiota of an infant or young child, even more similar to that of breast-fed infants.

Disclosure of Invention

The inventors applied metabolomics, an omics tool in which hundreds of metabolites (typically small molecules, typically < 1,000- & lt 1,500Da) were measured simultaneously to study the functional profile of the infant intestinal microbiota and the effect of feeding experimental, partially fermented experimental or control formulations containing indigestible oligosaccharides. Metabolites exhibit a more terminal phenotype, which is the result of all gene-encoded functions in the ecosystem, as they represent the final sum of all activated genes, epigenetic expression modifications and other transcriptional regulation, post-translational protein modifications, and environmental factors (both biotic and abiotic). The inventors found that metabolomics data show an increasing extreme difference between the study groups at almost every time point compared to the data for microbiota composition (each comparison of 180-. At baseline, the experimental and control groups were still relatively close, but the pattern changed over time, with the experimental group being close to that observed in breast-fed infants, while the difference between the control and breast-fed reference groups became more pronounced. This set of non-targeted data suggests that the function of the infant gut ecosystem is highly dependent on diet and responsive to diet. Distinctly different metabolites are derived from many functional classes. One example is the different properties of secondary bile salts, where the pattern of the experimental group is more similar or less deviating from the pattern of the breastfeeding reference group.

In this study, the characteristics of the stool samples reflect that infants fed the experimental formula containing indigestible oligosaccharides have intestinal physiological conditions more similar to those of breast-feeding than the control group. The effect was even more pronounced in the group receiving the additional partially fermented formula.

Detailed description of the preferred embodiments

Accordingly, the present invention relates to a method of promoting the development of gut microbiota in a human subject of below 36 months of age, the gut microbiota function being closer to the gut microbiota function of a human subject of the same age that is human breast fed, compared to the gut microbiota function of a human subject of the same age that is fed a nutritional composition not comprising non-digestible oligosaccharides, the method comprising administering a nutritional composition comprising 2.5 to 15 wt.% non-digestible oligosaccharides based on dry weight.

In one embodiment, the method of the invention may be viewed as a non-medical method of promoting the development of gut microbiota function.

The invention can also be expressed as the use of non-digestible oligosaccharides for the manufacture of a nutritional composition for promoting the development of gut microbiota in a human subject of below 36 months of age, the nutritional composition comprising 2.5 to 15 wt.% of non-digestible oligosaccharides on a dry weight basis, the gut microbiota function being closer to the gut microbiota function of a human subject of the same age as human breast fed compared to the gut microbiota function of a human subject of the same age as fed a nutritional composition not comprising non-digestible oligosaccharides.

The present invention can also be expressed as a nutritional composition comprising 2.5 to 15 wt.% on a dry weight basis of non-digestible oligosaccharides for use in promoting the development of gut microbiota in a human subject of below 36 months of age, which gut microbiota function is closer to the gut microbiota function of a human subject of the same age that is human milk fed, compared to the gut microbiota function of a human subject of the same age that is fed a nutritional composition not comprising non-digestible oligosaccharides.

Preferably, in the method or use of the invention, the nutritional composition is at least partially fermented by lactic acid producing bacteria and comprises a total of 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis. Furthermore, it is preferred that in the method or use of the invention the development of gut microbiota function is promoted compared to the gut microbiota function of an elderly human subject fed a nutritional composition which is not at least partially fermented by lactic acid producing bacteria and which does not comprise total 0.02 to 1.5 wt% lactic acid and lactate on a dry weight basis.

Thus, the present invention also relates to a method of promoting the development of gut microbiota in a human subject of below 36 months of age, which gut microbiota functions closer to the gut microbiota function of a human subject of the same age that is human milk fed, compared to the gut microbiota function of a human subject of the same age fed with a nutritional composition that is not at least partially fermented by lactic acid producing bacteria and does not comprise total 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis, not comprising non-digestible oligosaccharides, the method comprising administering the nutritional composition that is at least partially fermented by lactic acid producing bacteria, wherein the nutritional composition comprises total 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis, and wherein the nutritional composition comprises 2.5 to 15 wt.% non-digestible oligosaccharides on a dry weight basis.

The invention can also be expressed as the use of a fermented ingredient and non-digestible oligosaccharides for the preparation of a nutritional composition at least partially fermented by lactic acid producing bacteria for promoting the development of an intestinal microbiota in a human subject below 36 months of age, which intestinal microbiota functions closer to the intestinal microbiota function of a human subject of the same age that is human fed with the nutritional composition at least not partially fermented by lactic acid producing bacteria and not comprising 0.02 to 1.5 wt.% total lactic acid and lactate on a dry weight basis, and not comprising non-digestible oligosaccharides, wherein the nutritional composition comprises 0.02 to 1.5 wt.% total lactic acid and lactate on a dry weight basis, and wherein the nutritional composition comprises 2.5 to 15 wt.% non-digestible oligosaccharides on a dry weight basis.

The present invention may also be expressed as a nutritional composition at least partially fermented by lactic acid producing bacteria, wherein the nutritional composition comprises a total of 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis, and wherein the nutritional composition comprises 2.5 to 15 wt.% non-digestible oligosaccharides on a dry weight basis for promoting the development of gut microbiota in human subjects below 36 months of age, which gut microbiota function is closer to the gut microbiota function of human subjects of the same age who are human milk fed compared to the gut microbiota function of human subjects of the same age fed a nutritional composition at least not partially fermented by lactic acid producing bacteria and not comprising a total of 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis, which nutritional composition does not comprise non-digestible oligosaccharides.

Fermentation ingredient

The nutritional composition in the method or use of the invention (hereinafter also referred to as the present nutritional composition, or the nutritional composition or final nutritional composition of the invention) is preferably at least partially fermented. The partially fermented nutritional composition comprises a composition that is at least partially fermented by lactic acid producing bacteria. It has been shown that the presence of the fermented ingredients in the final nutritional composition after administration makes the intestinal microbiota function more similar to that of breast-fed infants.

Preferably, the fermentation is performed during the preparation of the nutritional composition. Preferably, the nutritional composition does not contain a significant amount of viable bacteria in the final product due to heat inactivation or inactivation by other means after fermentation. Preferably, the fermented ingredient is a milk-derived product which is a milk substrate fermented by lactic acid producing bacteria, wherein the milk substrate comprises at least one substance selected from the group consisting of: milk, whey protein hydrolysate, casein hydrolysate, or a mixture thereof. Suitably, nutritional compositions comprising a fermented ingredient and non-digestible oligosaccharides and methods for their preparation are described in WO 2009/151330, WO 2009/151331 and WO 2013/187764.

The fermentation component preferably comprises bacterial cell debris such as glycoproteins, glycolipids, peptidoglycans, lipoteichoic acids (LTA), lipoproteins, nucleotides and/or capsular polysaccharides. It is advantageous to use the fermented ingredients comprising inactivated bacteria and/or cell debris directly as part of the final nutritional product, as this will result in a higher concentration of bacterial cell debris. When commercial preparations of lactic acid producing bacteria are used, these preparations are typically washed and the material is separated from the aqueous growth medium containing the bacterial cell debris, thereby reducing or eliminating the presence of bacterial cell debris. Furthermore, upon fermentation and/or other interaction of the lactic acid producing bacteria with the milk substrate, additional bioactive compounds may be formed, such as short chain fatty acids, bioactive peptides and/or oligosaccharides and other metabolites, which also result in a gut microbiota function more similar to that of breast-fed infants. Such bioactive compounds produced by lactic acid-producing bacteria or other food grade bacteria during fermentation may also be referred to as post-biotin (post-biological). Compositions comprising such metabiotics are believed to be advantageously closer to breast milk, as breast milk is not a pure synthetic formulation, but contains metabolites, bacterial cells, cell debris, and the like. Thus, the fermented ingredients, in particular the fermented milk-derived products, are believed to have an improved effect on the gut microbiota function compared to unfermented milk-derived products containing no or only lactic acid producing bacteria.

Preferably, 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% fermented ingredients on a dry weight basis. As a means of indicating that the final nutritional composition comprises an at least partially fermented composition and that the degree of fermentation, the total level of lactate in the final nutritional composition may be used, as this is the metabolic end product produced by the lactic acid producing bacteria upon fermentation. The final nutritional composition of the invention comprises a total of 0.02 to 1.5 wt.%, more preferably 0.05 to 1.0 wt.%, even more preferably 0.1 to 0.5 wt.% lactic acid and lactate based on dry weight of the composition. Preferably, at least 50% by weight, even more preferably at least 90% by weight of the total of lactic acid and lactate is in the form of the L (+) -isomer. Thus, 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 lactic acid and lactate. L (+) -lactate and L (+) -lactic acid are also referred to herein as L-lactate and L-lactic acid.

Lactic acid producing bacteria for preparing fermented ingredients

The lactic acid producing bacteria for preparing the fermented ingredients, in particular the fermented milk substrate, are preferably provided in the form of a single culture or a mixed culture. Lactic acid producing bacteria consist of the following genera: bifidobacterium, Lactobacillus, Carnobacterium, Enterococcus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella. Preferably, the lactic acid producing bacteria used for fermentation comprise bacteria of the genus bifidobacterium and/or streptococcus.

Preferably, the streptococcus is a streptococcus thermophilus (s. thermophilus) strain. The selection of suitable strains of Streptococcus thermophilus is described in example 2 of EP 778885 and in example 1 of FR 2723960. In another preferred embodiment of the present invention, the nutritional composition comprises 10²-10⁵cfu viable Streptococcus thermophilus bacteria/g dry weight of the final nutritional composition, preferably the final nutritional composition comprises 10³-10⁴Viable bacteria of Streptococcus thermophilus per g dry weight.

For the purposes of the present invention, the preferred strains of S.thermophilus for preparing the fermentation components have been deposited by Compuginie Gervais Danone at Collection national de Cultures de Microorgansims (CNCM) operated by Institut Pasteur, 25 rue du Docteur Roux, Paris, France, with accession number I-1620 deposited at 23.8.1995 and accession number I-1470 deposited at 25.8.1994. Other strains of Streptococcus thermophilus are commercially available.

Streptococcus thermophilus is not considered a probiotic, since it cannot survive in the stomach.

The bifidobacterium is a gram-positive anaerobic rod-shaped bacterium. For the purposes of the present invention, preferred bifidobacterium species for use in preparing fermentation compositions preferably have at least 95% identity, more preferably at least 97% identity, in the 16S rRNA sequence when compared to a model strain of the corresponding bifidobacterium species, as defined in manuals on this subject, for example, Sambrook, j., Fritsch, e.f. and manitis, T. (1989), Molecular Cloning, a Laboratory Manual, 2 nd edition, Cold Spring Harbor (n.y.) Laboratory Press. The bifidobacteria preferably used are further described by Scardovi, v. in Genus bifidobacterium, p.1418-1434: bergey's management of systematic bacteriology, volume 2. Sneath, p.h.a., n.s.mair, m.e.sharp, and j.g.holt (ed). Williams & Wilkins.1986, page 635. Preferably, the lactic acid producing bacteria used for fermentation comprise or are at least one bifidobacterium selected from the group consisting of: bifidobacterium breve (b.breve), bifidobacterium infantis (b.infarnata), bifidobacterium bifidum (b.bifidum), bifidobacterium catenulatum (b.catenulatum), bifidobacterium adolescentis (b.adolescentis), bifidobacterium thermophilum, bifidobacterium gallnut (b.gallicum), bifidobacterium animalis (b.animalis) or bifidobacterium lactis (b.lactis), bifidobacterium angulus (b.angulus), bifidobacterium pseudocatenulatum (b.pseudocatenulatum), bifidobacterium acidophilum (b.thermaldophilum) and bifidobacterium longum (b.longum), more preferably bifidobacterium breve, bifidobacterium infantis, bifidobacterium bifidum, bifidobacterium catenulatum, bifidobacterium longum, more preferably bifidobacterium longum and bifidobacterium breve, even more preferably bifidobacterium breve, more preferably bifidobacterium breve selected from the following bifidobacterium breve: bifidobacterium breve Bb-03(Rhodia/Danisco), Bifidobacterium breve M-16V (Morinaga), Bifidobacterium breve R0070(Institute Rosell, Lallemand), Bifidobacterium breve BR03(Probiotical), Bifidobacterium breve BR92(CellBiotech) DSM 20091, LMG 11613 and Bifidobacterium breve I-2219 deposited in Paris CNCM, France. Most preferably, the Bifidobacterium breve is Bifidobacterium breve M-16V (Morinaga) or Bifidobacterium breve I-2219, even more preferably Bifidobacterium breve I-2219.

Most preferably, the nutritional composition of the invention comprises a fermented ingredient fermented by lactic acid producing bacteria including both bifidobacterium breve and streptococcus thermophilus. In one embodiment, the lactic acid producing bacterial fermentation is by streptococcus thermophilus and bifidobacterium breve. In one embodiment, the final nutritional composition comprises a fermented ingredient, wherein the lactic acid producing bacteria are inactivated after fermentation.

Preferably, the fermented ingredients are not fermented by Lactobacillus bulgaricus (Lactobacillus bulgaricus). The products fermented by Lactobacillus bulgaricus are considered unsuitable for infants, since the activity of the specific dehydrogenase converting D-lactate to pyruvate is much lower in young infants than the dehydrogenase converting L-lactate.

Preferably, the nutritional composition of the invention comprises inactivated lactic acid producing bacteria and/or bacterial debris derived from lactic acid producing bacteria obtained from more than 1x10 based on dry weight per g of final composition⁴cfu lactic acid producing bacteria, more preferably 1X10⁵cfu, even more preferably 1x10⁶Preferably, the inactivated bacteria or bacterial debris is obtained from less than 1 × 10 based on dry weight per g of final composition¹²cfu of lactic acid producing bacteria, more preferably 1X10¹⁰cfu, even more preferably 1x10⁹cfu. The correlation between the inactivated lactic acid bacteria and cfu can be determined by molecular techniques or by examining the production process known in the art.

Fermentation process

Preferably, the fermented ingredient is a milk-derived product which is a milk substrate fermented by lactic acid producing bacteria and which milk substrate comprises at least one component selected from: milk, whey protein hydrolysate, casein hydrolysate, or a mixture thereof. The milk-derived product or milk substrate to be fermented is suitably present in an aqueous medium. The milk substrate to be fermented comprises at least one component selected from the group consisting of: milk, whey protein hydrolysate, casein hydrolysate, or a mixture thereof. The milk may be whole milk, semi-skimmed milk and/or skimmed milk. Preferably, the milk substrate to be fermented comprises skim milk. The whey may be sweet whey and/or acid whey. Preferably, whey is present at a concentration of 3 to 80g dry weight per l aqueous medium containing milk substrate, more preferably 40 to 60 g/l. Preferably, the whey protein hydrolysate is present in an aqueous medium containing a milk substrate at 2 to 80g dry weight/l, more preferably 5 to 15 g/l. Preferably, lactose is present in 5 to 50g dry weight/l aqueous substrate, more preferably 1 to 30 g/l. Preferably, the aqueous medium containing the milk substrate comprises buffer salts to maintain the pH within the desired range. Sodium dihydrogen phosphate or potassium dihydrogen phosphate is preferably used as a buffer salt, preferably at 0.5 to 5g/l, more preferably 1.5 to 3 g/l. Preferably, the aqueous medium containing the milk substrate comprises cysteine in an amount of 0.1 to 0.5g/l of aqueous substrate, more preferably 0.2 to 0.4 g/l. The presence of cysteine results in a substrate with a low redox potential, which is advantageous for the activity of lactic acid producing bacteria, in particular bifidobacteria. Preferably, the aqueous medium containing a milk substrate comprises yeast extract in an amount of 0.5 to 5g/l of aqueous medium containing a milk substrate, more preferably 1.5 to 3 g/l. Yeast extracts are a rich source of enzyme cofactors and growth factors for lactic acid producing bacteria. The presence of yeast extract will enhance the fermentation of lactic acid producing bacteria.

Suitably, prior to the fermentation step, the milk substrate, in particular the aqueous medium containing the milk substrate, is pasteurised to eliminate the presence of unwanted live bacteria. Suitably, the product is pasteurised after fermentation to inactivate the enzymes. Suitably, the enzyme inactivation is carried out at 75 ℃ for 3 minutes. Suitably, the aqueous medium containing the milk substrate is homogenised prior to fermentation and/or the milk-derived product is homogenised after fermentation. Homogenization makes the substrate and/or fermentation product more stable, especially in the presence of fat.

The seeding density is preferably 1x10²To 5x 10¹⁰Preferably 1X10⁴To 5x 10⁹cfu lactic acid producing bacteria per ml aqueous medium containing a milk substrate, more preferably 1X10⁷To 1x10⁹cfu lactic acid producing bacteria per ml of aqueous medium containing a milk substrate. The final bacterial density after fermentation is preferably 1x10³To 1x10¹⁰More preferably 1x10⁴To 1x10⁹cfu/ml aqueous medium containing milk substrate.

The fermentation is preferably carried out at a temperature of about 20 ℃ to 50 ℃, more preferably 30 ℃ to 45 ℃, even more preferably about 37 ℃ to 42 ℃. The optimal temperature for the growth and/or activity of the lactic acid producing bacteria, more particularly lactobacillus (lactobacillus) and/or bifidobacteria is from 37 ℃ to 42 ℃.

The incubation is preferably carried out at a pH of 4 to 8, more preferably 6 to 7.5. The pH does not cause protein precipitation and/or off-taste, while lactic acid producing bacteria such as lactobacilli and/or bifidobacteria are capable of fermenting the milk substrate.

The incubation time is preferably 10 minutes to 48 hours, preferably 2 hours to 24 hours, more preferably 4 hours to 12 hours. A sufficiently long time enables fermentation to be carried out to a sufficient or large extent and at the same time immunogenic cell fragments such as glycoproteins, glycolipids, peptidoglycans, lipoteichoic acids (LTA), flagella, lipoproteins, DNA and/or capsular polysaccharides and metabolites (metabiol) are produced, however for economic reasons the incubation time does not have to be too long.

Preferably, the milk-derived product or milk substrate, preferably skim milk, is pasteurized, cooled, and fermented with one or more lactic acid producing strains, preferably streptococcus thermophilus strains, to an acidity at which the fermented product is cooled and stored. Preferably, the second milk-derived product is prepared in a similar manner using one or more species of bifidobacterium for fermentation. Subsequently, the two fermentation products are preferably mixed together and with the other components, besides the fat component, which constitute the infant formula. Preferably, the mixture is preheated, then fat is added on-line, homogenized, pasteurized and dried. Or fermenting in a fermenter with Bifidobacterium, preferably Bifidobacterium breve and Streptococcus thermophilus.

Methods for preparing fermentation components suitable for the purposes of the present invention are known per se. EP 778885, incorporated herein by reference, discloses in particular a suitable process for preparing a fermentation component in example 7. FR2723960, incorporated herein by reference, discloses in particular a suitable process for preparing a fermented ingredient in example 6. Briefly, a milk substrate, preferably pasteurized, comprising lactose and optionally other macronutrients (such as fat (preferably vegetable fat), casein, whey protein, vitamins and/or minerals etc.) is concentrated to e.g. 15 to 50% dry matter and then inoculated with streptococcus thermophilus, e.g. with a milk product containing 10⁶To 10¹⁰Individual bacteria per ml of 5% culture were inoculated. Preferably, the milk substrate comprises milk protein peptides. The temperature and duration of the fermentation are as described above. Suitably, after fermentation, the fermented ingredients may be pasteurised or sterilised and, for example, spray dried or freeze dried to provide a form suitable for formulation in the final product.

A preferred method for preparing the fermentation ingredients for the nutritional composition of the present invention is disclosed in WO 01/01785, more specifically in examples 1 and 2. A preferred method for preparing the fermentation ingredients for the nutritional composition of the invention is described in WO 2004/093899, more specifically in example 1.

The viable cells of the lactic acid producing bacteria in the fermented composition are preferably removed after fermentation, e.g. by inactivation and/or physical removal. Preferably, the cells are inactivated. Preferably, the lactic acid producing bacteria are heat inactivated after fermentation of the milk substrate. Preferred heat inactivation methods are (flash) pasteurization, sterilization, ultra high temperature treatment, high/short heat treatment and/or spray drying at temperatures at which bacteria cannot survive. The cell debris is preferably obtained by heat treatment. By said heat treatment preferably at least 90%, more preferably at least 95%, even more preferably at least 99% of the viable microorganisms are inactivated. Preferably, the fermented nutritional composition comprises less than 1x10⁵Viable lactic acid bacteria in colony forming units (cfu) per g dry weight. The heat treatment is preferably carried out at a temperature of from 70 to 180 ℃, preferably from 80 to 150 ℃Preferably at a temperature of from about 3 minutes to 2 hours, preferably at a temperature of from 80 to 140 ℃ for from 5 minutes to 40 minutes. Inactivation of lactic acid bacteria advantageously results in less post-acidification and a safer product. This is particularly advantageous when the nutritional composition is administered to an infant or young child. Suitably, after fermentation, the fermented ingredients may be pasteurised or sterilised and, for example, spray dried or freeze dried to provide a form suitable for formulation in the final product.

Indigestible oligosaccharides

The nutritional composition of the invention comprises non-digestible oligosaccharides, and preferably comprises at least two different non-digestible oligosaccharides, in particular two different sources of non-digestible oligosaccharides. It has been shown that the presence of non-digestible oligosaccharides improves the function of the gut microbiota, making it more similar to that of breast-fed infants. Thus, the simultaneous presence of non-digestible oligosaccharides and fermented ingredients, in particular milk-derived products obtained by fermentation with lactic acid producing bacteria, synergistically and advantageously results in a gut microbiota that functions more like the gut microbiota of mainly or fully breast-fed infants.

The term "oligosaccharide" as used herein refers to a saccharide having a Degree of Polymerization (DP) of 2 to 250, preferably a DP of 2 to 100, more preferably 2 to 60, even more preferably 2 to 10. If the nutritional composition of the invention comprises oligosaccharides with a DP between 2 and 100, this results in a composition that may contain oligosaccharides with a DP between 2 and 5, a DP between 50 and 70 and a DP between 7 and 60. The term "non-digestible oligosaccharides" as used in the present invention refers to oligosaccharides that are not digested in the gut by the action of acids or digestive enzymes present in the human upper gut (such as the small intestine and stomach), but are preferably fermented by the human gut microbiota. For example, sucrose, lactose, maltose and maltodextrin are considered digestible.

Preferably, the indigestible oligosaccharide of the invention is soluble. The term "soluble" as used herein when referring to a polysaccharide, fibre or oligosaccharide means that the substance is at least soluble according to the method described in l.prosky et al, j.assoc.off.anal.chem.71, 1017-.

In the method or use of the present invention, the non-digestible oligosaccharides comprised in the nutritional composition of the present invention preferably comprise a mixture of non-digestible oligosaccharides. The non-digestible oligosaccharides are preferably selected from fructooligosaccharides, such as inulin; (ii) non-digestible dextrin; galactooligosaccharides, such as transgalactooligosaccharides; xylo-oligosaccharides, arabino-oligosaccharides (arabino-oligosaccharides), gluco-oligosaccharides (gluco-oligosaccharides), gentiooligosaccharides (gentio-oligosaccharides), gluco-manno-oligosaccharides (gluco-oligosaccharides), galacto-manno-oligosaccharides (galacto-oligosaccharides), manno-oligosaccharides (manno-oligosaccharides), isomalto-oligosaccharides (isomalto-oligosaccharides), nigero-oligosaccharides (nigero-oligosaccharides), gluco-manno-oligosaccharides (gluco-oligosaccharides), chito-oligosaccharides (chito-oligosaccharides), soy oligosaccharides (soy oligosaccharides), uronic acids (urocanic-oligosaccharides), and mixtures thereof. This oligosaccharide has many biochemical properties and has similar functional benefits, including improved gut microbiota function. It will also be appreciated that some non-digestible oligosaccharides and preferably some mixtures have a further improving effect. Thus the indigestible oligosaccharide is more preferably selected from fructooligosaccharides, such as inulin; galacto-oligosaccharides, such as beta galacto-oligosaccharides and mixtures thereof, even more preferably beta galacto-oligosaccharides and/or inulin, most preferably beta galacto-oligosaccharides. In one embodiment of the nutritional composition of the invention, the indigestible oligosaccharide is selected from the group consisting of galactooligosaccharides, fructooligosaccharides and mixtures thereof, more preferably beta galactooligosaccharides, fructooligosaccharides and mixtures thereof.

The indigestible oligosaccharide is preferably selected from the group consisting of β -galacto-oligosaccharides, α -galacto-oligosaccharides and galactans (galactan). according to a more preferred embodiment, the indigestible oligosaccharide is β -galacto-oligosaccharides preferably the indigestible oligosaccharide comprises galacto-oligosaccharides having β (1, 4), β (1, 3) and/or β (1, 6) glycosidic linkages and terminal glucose

GOS (Domo FrieslandCampinea Ingredients), Bi2muno (Clasado), Cup-oligo (Nissin Sugar), and Oligomate55 (Yakult). These oligosaccharides are described inImprove the function of intestinal microbiota to a great extent.

In other cases, the fructooligosaccharides may have the names of, for example, fructans (fructans), oligofructose (oligofructans), polyfructose (polyfructose), polyfructan (polyfructan), inulin, fructans (levan), and fructans (fructans), and may refer to oligosaccharides comprising β -linked fructose units, which are preferably linked by β (2, 1) and/or β (2, 6) glycosidic bonds and preferably have a DP of 2 to 200. preferably, the fructooligosaccharides comprise terminal β (2, 1) glycosidic bonds to glucose

HP (Orafti) is commercially available. Other suitable sources are raftilose (orafti), fibrilose and fibriline (cosecra) and Frutafit and frutalose (sensus).

Preferably, the nutritional composition of the invention comprises a mixture of galactooligosaccharides and fructooligosaccharides. Preferably, the mixture of galactooligosaccharides and fructooligosaccharides 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 about 9/1. This weight ratio is particularly advantageous when the galacto-oligosaccharide has a low average DP and the fructo-oligosaccharide has a relatively high DP. Most preferred is a mixture of galacto-oligosaccharides with an average DP below 10, preferably below 6 and fructo-oligosaccharides with an average DP above 7, preferably above 11, even more preferably above 20. This mixture synergistically improves the functionality of the infant's gut microbiota to make it more similar to that of breast-fed infants.

Preferably, the nutritional composition of the invention comprises a mixture of short chain fructooligosaccharides and long chain fructooligosaccharides. Preferably, the mixture of short-chain and long-chain fructooligosaccharides 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. Preferred are mixtures of short chain fructooligosaccharides with an average DP below 10, preferably below 6 and fructooligosaccharides with an average DP above 7, preferably above 11, even more preferably above 20.

Preferably, the nutritional composition of the invention comprises a mixture of short chain fructooligosaccharides and short chain galactooligosaccharides. Preferably, the mixture of short chain fructooligosaccharides and short chain galactooligosaccharides is present in a weight ratio of from 1/99 to 99/1, more preferably from 1/19 to 19/1, even more preferably from 1/10 to 19/1, more preferably from 1/5 to 15/1, more preferably from 1/1 to 10/1. Preferred are mixtures of short chain fructooligosaccharides and galactooligosaccharides with an average DP below 10, preferably below 6.

The nutritional composition of the invention comprises a total of 2.5 to 20 wt.%, more preferably 2.5 to 15 wt.%, even more preferably 3.0 to 10 wt.%, most preferably 5.0 to 7.5 wt.% of non-digestible oligosaccharides, based on dry weight of the nutritional composition. The nutritional composition of the invention preferably comprises a total of 0.35 to 2.5 wt.%, more preferably 0.35 to 2.0 wt.%, even more preferably 0.4 to 1.5 wt.% indigestible oligosaccharides based on 100ml nutritional composition. Lower amounts of indigestible oligosaccharides are less effective in improving the function of the intestinal microbiota, while too high amounts may lead to side effects of abdominal distension and abdominal discomfort.

Nutritional composition

The nutritional composition for use according to the invention may also be considered as a pharmaceutical composition, preferably suitable for administration to an infant. The nutritional composition of the invention is preferably enterally administered, more preferably orally administered.

Preferably, the nutritional composition used according to the invention is not a probiotic composition or a composition comprising probiotics. The lactic acid producing bacteria preferably do not replicate or inactivate during production and/or do not survive the conditions of the upper gastrointestinal tract of a human.

The nutritional composition of the invention is preferably an infant formula, follow-on formula, baby milk or baby formula or growing-up milk for infants (growing-up milk). The nutritional composition of the invention can advantageously be used as a complete nutrition for infants. Preferably, the nutritional composition of the invention is an infant formula. Infant formula is defined as a formula for infants and may for example be a starting formula for infants from 0 to 6 or 0 to 4 months of age. The follow-on formula is for infants from 4 or 6 months of age to 12 months of age. At this month of age, the infant begins weaning to eat other food. Toddlers or growing-up milks or formulas are used for children of 12 to 36 months of age. The composition of the present invention preferably comprises a lipid component, a protein component and a carbohydrate component and is preferably administered in liquid form. The nutritional composition of the invention may also be in the form of a dry food product, preferably in the form of a powder, accompanied by instructions to mix the dry food product (preferably powder) with a suitable liquid (preferably water). The nutritional composition used according to the invention preferably comprises other ingredients, such as vitamins, minerals, trace elements and other micronutrients, so that it is a complete nutritional composition. According to international directives, it is preferred that infant formulas contain vitamins, minerals, trace elements and other micronutrients.

The nutritional composition of the present invention preferably comprises lipid, protein and digestible carbohydrate, wherein lipid provides 5 to 50% of the total calories, protein provides 5 to 50% of the total calories, and digestible carbohydrate provides 15 to 90% of the total calories. Preferably, in the nutritional composition of the present invention, the lipid provides 35 to 50% of the total calories, the protein provides 7.5 to 12.5% of the total calories, and the digestible carbohydrate provides 40 to 55% of the total calories. To calculate the percentage of total calories of protein, the total energy provided by protein, peptide and amino acids needs to be considered. Preferably, 3 to 7g lipid per 100kcal nutritional composition, preferably 4 to 6g per 100kcal nutritional composition is provided; providing 1.6 to 4g protein per 100kcal nutritional composition, preferably 1.7 to 2.5g per 100kcal nutritional composition, and providing 5 to 20g digestible carbohydrate per 100kcal nutritional composition, preferably 8 to 15g per 100kcal nutritional composition. Preferably, the nutritional composition of the invention comprises 4 to 6g lipid per 100kcal, 1.6 to 2.0g protein per 100kcal, more preferably 1.7 to 1.9g protein per 100kcal, and 8 to 15g digestible carbohydrate per 100kcal of the nutritional composition. In one embodiment, 3 to 7g lipid per 100kcal nutritional composition, preferably 4 to 6g lipid per 100kcal nutritional composition; 1.6 to 2.1g protein per 100kcal nutritional composition, preferably 1.6 to 2.0g protein per 100kcal nutritional composition; and providing 5 to 20g digestible carbohydrate per 100kcal of the nutritional composition, preferably 8 to 15g digestible carbohydrate per 100kcal of the nutritional composition, and wherein preferably the digestible carbohydrate component comprises at least 60 wt% lactose, based on total digestible carbohydrate, more preferably at least 75 wt%, even more preferably at least 90 wt% lactose, based on total digestible carbohydrate. The total amount of calories is determined by the sum of calories from protein, lipids, digestible carbohydrates and non-digestible oligosaccharides.

The nutritional composition of the present invention preferably comprises a digestible carbohydrate component. Preferred digestible carbohydrate components 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 invention preferably comprises lactose. Since the nutritional composition of the invention comprises fermented ingredients obtained by fermentation of lactic acid producing bacteria, the amount of lactose is reduced relative to its source by fermentation, the lactose being converted to lactate and/or lactic acid by fermentation. Therefore, in the preparation of the nutritional composition of the present invention, lactose is preferably added. Preferably, the nutritional composition of the invention does not comprise a substantial amount of carbohydrates other than lactose. Lactose has a lower glycemic index than digestible carbohydrates such as maltodextrin, sucrose, glucose, maltose and other digestible carbohydrates with a high glycemic index and is therefore preferred. The nutritional composition of the invention preferably comprises digestible carbohydrate, 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 carbohydrate is lactose. The nutritional composition of the invention preferably comprises at least 25 wt.% lactose, preferably at least 40 wt.%, more preferably at least 50 wt.% lactose, based on dry weight.

The nutritional composition of the present invention preferably comprises at least one lipid selected from the group consisting of animal lipids (excluding human lipids) and vegetable lipids. Preferably, the composition of the invention comprises a combination of vegetable lipids and at least one oil selected from the group consisting of fish oil, animal oil, algal oil, fungal oil and bacterial oil. The nutritional composition of the present invention preferably provides 3 to 7g lipid per 100kcal of nutritional composition, preferably 4 to 6g lipid per 100kcal of nutritional composition. When in liquid form, e.g. as a ready-to-eat liquid, the nutritional composition preferably comprises 2.1 to 6.5g lipid per 100ml, more preferably 3.0 to 4.0g per 100 ml. The nutritional composition of the invention preferably comprises 12.5 to 40 wt.%, more preferably 19 to 30 wt.% lipid based on dry weight. Preferably, the lipid comprises the essential fatty acids alpha-linolenic acid (ALA), Linoleic Acid (LA) and/or long chain polyunsaturated fatty acids (LC-PUFA). The LC-PUFA, LA and/or ALA may be provided as free fatty acids, in triglyceride form, diglyceride form, monoglyceride form, phospholipid form or as a mixture of one or more of the foregoing. Preferably, the nutritional composition of the invention comprises at least one, preferably at least two lipid sources selected from the group consisting of: rapeseed oils (e.g., colza oil, canola oil, and canola oil), high oleic sunflower oil, high oleic safflower oil, olive oil, marine oils (marine oil), microbial oils, coconut oil, palm kernel oil. The nutritional composition of the present invention is not human milk.

The nutritional composition of the 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 protein and/or rice protein; and mixtures thereof. The nutritional composition of the invention preferably contains casein and/or whey protein, more preferably bovine whey protein and/or bovine casein. Thus, in one embodiment, the protein in the nutritional composition of the invention comprises a protein selected from whey protein and casein, preferably whey protein and/or casein is derived from bovine milk. Preferably, the protein comprises less than 5 wt.% free amino acids, dipeptides, tripeptides or hydrolysed protein, based on total protein. The nutritional composition of the invention preferably comprises casein and whey protein in a weight ratio of casein to whey protein of from 10: 90 to 90: 10, more preferably from 20: 80 to 80: 20, even more preferably from 35: 65 to 55: 45.

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

The nutritional composition of the invention preferably comprises 1.6 to 4.0g protein per 100kcal nutritional composition, preferably 1.6 to 3.5g protein per 100kcal nutritional composition, even more preferably 1.75 to 2.5g protein per 100kcal nutritional composition. In one embodiment, the nutritional composition of the invention comprises 1.6 to 2.1g protein per 100kcal nutritional composition, preferably 1.6 to 2.0g protein per 100kcal nutritional composition, more preferably 1.75 to 2.1g protein per 100kcal nutritional composition, even more preferably 1.75 to 2.0g protein per 100kcal nutritional composition. In one embodiment, the nutritional composition of the invention comprises protein in an amount of less than 2.0g/100kcal, preferably from 1.6 to 1.9g, even more preferably from 1.75 to 1.85g/100kcal of the nutritional composition. Too low a protein content based on total calories will result in inadequate growth and development in infants and young children. Too high amounts may cause metabolic stress on e.g. the kidneys of infants and young children. When in liquid form, e.g. as a ready-to-feed liquid, the nutritional composition preferably comprises 0.5 to 6.0g protein per 100ml, more preferably 1.0 to 3.0g protein per 100ml, even more preferably 1.0 to 1.5g protein per 100ml, most preferably 1.0 to 1.3g protein per 100 ml. On a dry weight basis, the nutritional composition of the invention preferably comprises 5 to 20 wt.% protein, preferably at least 8 wt.% protein, based on dry weight of the total nutritional composition, more preferably 8 to 14 wt.% protein, even more preferably 8 to 9.5 wt.% protein, based on dry weight of the total nutritional composition.

To meet the caloric requirements of an infant or young child, the nutritional composition preferably comprises 45 to 200kcal per 100ml of liquid. For infants, the nutritional composition more preferably has 60 to 90kcal per 100ml of liquid, even more preferably 65 to 75kcal per 100ml of liquid. This calorie density ensures an optimal ratio between water and calorie consumption. For young children, human subjects 12 to 36 months of age, the nutritional composition more preferably has a caloric density of 45 to 65, even more preferably 50 to 60kcal/100 ml. The osmolality of the composition of the invention is preferably from 150 to 420mOsmol/L, more preferably from 260 to 320 mOsmol/L. The low osmolarity aims to further reduce the gastrointestinal pressure.

When the nutritional composition is in a ready-to-eat, liquid form, the preferred volume administered daily is about 80 to 2500ml per day, more preferably about 200 to 1200ml per day. Preferably, the number of feeds per day is from 1 to 10, preferably from 3 to 8. In one embodiment, the nutritional composition is administered daily in liquid form for at least 2 days, preferably at least 4 weeks, preferably at least 8 weeks, more preferably at least 12 weeks, wherein the total volume administered daily is from 200ml to 1200ml, and wherein the number of feeds per day is from 1 to 10.

When in liquid form, the nutritional composition of the invention preferably has a viscosity of from 1 to 60mpa.s, preferably from 1 to 20mpa.s, more preferably from 1 to 10mpa.s, most preferably from 1 to 6 mpa.s. The low viscosity ensures proper liquid administration, e.g., fitting through the entire nipple. The viscosity is also very similar to that of human milk. Furthermore, the low viscosity results in normal gastric emptying and better energy intake, which is necessary for infants that require energy for optimal growth and development. The nutritional compositions of the present invention are optionally in powder form, suitable for reconstitution with water to form a ready-to-drink liquid. The nutritional composition of the present invention is preferably prepared by mixing the powdered composition with water. Typically, infant formulas are prepared in this manner. The invention therefore also relates to a packaged powder composition, wherein the package is provided with instructions for mixing the powder with an amount of liquid such that a liquid composition having a viscosity of 1 to 60mpa.s is obtained. The Physica Rheometer MCR 300(Physica Messt) was usedechnik GmbH, Ostfilden, Germany) at 20 ℃ for 95s^-1The shear rate of (c) determines the viscosity of the liquid.

Use of

In the context of the present invention, "prevention" of a disease or a certain condition also means "reduction of the risk" of suffering from a disease or a certain condition, and also means treatment of a person who is "at risk" of suffering from said disease or said certain condition.

The methods of the present invention, which include administering the nutritional compositions of the present invention, also refer to administering an effective amount of the nutritional compositions to an individual in need of such treatment.

In this era based on omics technology, increasingly sophisticated tools have made it possible to study the intestinal microbiota at different molecular levels. In the past decade, the most widely used tools to study gut microbiota have been based on sequencing (parts of) bacterial 16S rRNA gene sequences and identifying which bacterial lineages are present. However, analysis of gut microbiota by DNA-based methods does not provide a direct view of the functional level of the gut ecosystem.

Metabolomics is a systematic study of the unique chemical fingerprints left by specific cellular processes, and is a study of their small molecule metabolite profiles. Metabolome represents the collection of all metabolites in a cell, tissue, organ or organism of an organism, which is the final product of a cellular process. Metabolome refers to the entire group of small molecule chemicals found in a biological sample. Small molecule chemicals found in a particular metabolome can include endogenous metabolites (e.g., amino acids, organic acids, nucleic acids, fatty acids, amines, sugars, vitamins, cofactors, pigments, antibiotics, etc.) and exogenous chemicals that are naturally produced by an organism. The molecular weight of small molecules must typically be < 1500Da in order to be identified as metabolites, or to be considered part of the metabolome. Metabolomics can contribute greatly to the functional map of the gut microbiota. After all, metabolites delineate a more final phenotype, which is the result of all gene-encoded functions in the ecosystem, as they represent the final sum of all activating genes, epigenetic expression modifications and other transcriptional regulation, post-translational protein modifications and environmental factors (biological and non-biological).

Metabolites with significant differences represent many functional classes. This non-targeted data set indicates that the function of the infant's intestinal ecosystem is highly dependent on diet and responsive to diet.

The characteristics of the stool samples in this study reflect that infants fed a formula containing indigestible oligosaccharides, preferably a partially fermented formula containing indigestible oligosaccharides, have a composition of gut microbiota more similar to breast feeding and intestinal physiological conditions more similar to breast feeding compared to the control group. Furthermore, more extensive non-targeted analysis showed that the intestinal microbiota of infants fed the preferred partially fermented formula of the present invention functioned less differently from the breast-fed reference group than infants fed the control formula, indicating that the non-digestible oligosaccharide containing formula, preferably the non-digestible oligosaccharide containing partially fermented formula, pushed the intestinal ecosystem towards a more breast-fed-like situation. It has been found that the hyper-pathways associated with amino acids, lipids, xenobiotics, carbohydrates, nucleotides, cofactors and vitamins, energy and peptides are affected. The greatest effect of changes in metabolite numbers is shown in the amino acid, lipid-related pathways, and when relative changes are observed (number of altered pathway metabolites/number of total pathway metabolites tested), very high differences are also observed in pathways related to energy, nucleotides and cofactors, as well as vitamins.

In a study of the metabolite profile, the inventors found that the gut metabolome of an infant is more similar to that of a breast-fed infant when fed with the nutritional composition of the present invention. The gut metabolome is more similar to that of breast-fed infants compared to that of infants fed formulas that do not contain indigestible oligosaccharides, and compared to that of infants fed formulas that do not contain unfermented formulas that do not contain indigestible oligosaccharides.

Thus, in a preferred embodiment of the method or use of the invention, promoting development of gut microbiota function means that the gut metabolome is closer to that of human subjects of the same age that are human breast fed compared to the gut metabolome of human subjects of the same age that are fed a nutritional composition that does not comprise non-digestible oligosaccharides.

Furthermore, in a preferred embodiment of the method or use of the present invention, promoting development of gut microbiota function means that the gut metabolome is more similar to that of a human subject of the same age as human milk fed compared to the gut metabolome fed with a nutritional composition which is not at least partially fermented by lactic acid producing bacteria and which does not comprise total 0.02 to 1.5 wt.% lactic acid and lactate, on a dry weight basis, and which does not comprise non-digestible oligosaccharides.

In one aspect, the invention relates to a method of establishing a metabolome which is more similar to the intestinal metabolome of a human subject of the same age as human milk fed, compared to the intestinal metabolome of a human subject of the same age fed with a nutritional composition which does not comprise non-digestible oligosaccharides, in a human subject below 36 months of age, which method comprises administering a nutritional composition which comprises from 2.5 to 15 wt.% non-digestible oligosaccharides based on dry weight.

In one embodiment, the method of the invention according to the above aspects may be regarded as a non-medical method of establishing a metabolome.

The present invention may also be expressed as the use of non-digestible oligosaccharides for the manufacture of a nutritional composition for establishing a metabolome in a human subject of below 36 months of age, which nutritional composition comprises 2.5 to 15 wt.% non-digestible oligosaccharides on a dry weight basis, which metabolome is more similar to the intestinal metabolome of a human subject of the same age that has been breast-fed, compared to the intestinal metabolome of a human subject of the same age that has been fed a nutritional composition that does not comprise non-digestible oligosaccharides.

The present invention may also be expressed as a nutritional composition comprising 2.5 to 15 wt.% non-digestible oligosaccharides based on dry weight for use in establishing a metabolome in a human subject of below 36 months of age that is more similar to the enterometabolome of a human subject of the same age that has been breast-fed compared to the enterometabolome of a human subject of the same age that has been fed a nutritional composition that does not comprise non-digestible oligosaccharides.

In one embodiment the invention relates to a method of establishing a metabolome which is more similar to the intestinal metabolome of a human subject of the same age fed with human milk, compared to the intestinal metabolome of a human subject of the same age which is fed with a nutritional composition which is at least not partially fermented by lactic acid producing bacteria and which does not comprise total 0.02 to 1.5 wt.% lactic acid and lactate, on a dry weight basis, and which does not comprise non-digestible oligosaccharides, which method comprises administering a nutritional composition which is at least partially fermented by lactic acid producing bacteria, wherein the nutritional composition comprises total 0.02 to 1.5 wt.% lactic acid and lactate, on a dry weight basis, and wherein the nutritional composition comprises 2.5 to 15 wt.% non-digestible oligosaccharides, on a dry weight basis, in a human subject below 36 months of age.

The invention may also be expressed as the use of a fermented ingredient and non-digestible oligosaccharides for the preparation of a nutritional composition at least partially fermented by lactic acid producing bacteria for establishing a metabolome in a human subject below 36 months of age, wherein the nutritional composition comprises a total of 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis, and wherein the nutritional composition comprises 2.5 to 15 wt.% non-digestible oligosaccharides on a dry weight basis, which metabolome is more similar to the intestinal metabolome of a human subject of the same age that has been human milk fed, compared to the intestinal metabolome of a human subject of the same age that has been fed a nutritional composition that is not at least partially fermented by lactic acid producing bacteria and that does not comprise a total of 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis, which non-digestible oligosaccharides.

The present invention may also be expressed as a nutritional composition at least partially fermented by lactic acid producing bacteria, wherein the nutritional composition comprises a total of 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis, and wherein the nutritional composition comprises 2.5 to 15 wt.% non-digestible oligosaccharides on a dry weight basis for establishing a metabolome which is more similar to the intestinal metabolome of human subjects of the same age who are human milk fed, compared to the intestinal metabolome of human subjects of the same age which are fed a nutritional composition which is at least not partially fermented by lactic acid producing bacteria and which does not comprise a total of 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis, which does not comprise non-digestible oligosaccharides.

Preferably, less than 45%, more preferably less than 40%, even more preferably less than 35% of the metabolites with significant differences in the intestinal metabolic profile or metabolome observed in infants fed the nutritional composition of the present invention are at significantly different levels compared to those observed in breast-fed infants. Preferably, more than 10%, more preferably more than 15%, even more preferably more than 20% of the metabolites observed in the gut metabolism profile or metabolome of significantly different levels in infants fed the nutritional composition of the invention are at significantly different levels compared to infants fed the control formula.

In a preferred embodiment of the method or use of the invention, less than 45%, more preferably less than 40%, even more preferably less than 35% of the metabolites with significant differences in their levels in the gut metabolome are at significantly different levels compared to the gut metabolome of human subjects of the same age that are breast-fed, and more than 10%, more preferably more than 15%, even more preferably more than 20% of the metabolites with significant differences in their levels compared to the gut metabolome of human subjects of the same age that are fed a nutritional composition without non-digestible oligosaccharides.

In a preferred embodiment of the method or use of the invention, the metabolites with a significant difference in the intestinal metabolic profile or metabolome (associated with the intestinal metabolome of human subjects of the same age that have been breast-fed) observed in infants fed with the nutritional composition of the invention are reduced by at least 5% compared to the metabolites with a significant difference in the levels in human subjects of the same age that have been fed with a nutritional composition that does not comprise non-digestible oligosaccharides (associated with the intestinal metabolome of human subjects of the same age that have been breast-fed), more preferably the metabolites with a significant difference in the levels in the intestinal metabolome are reduced by at least 10%.

In one aspect, the present invention relates to a method of establishing a metabolome in a human subject of less than 36 months of age, which method comprises administering a nutritional composition comprising 2.5 to 15 wt.% on a dry weight basis of non-digestible oligosaccharides, which metabolome has a significantly different level of metabolites (associated with the intestinal metabolome of human subjects of the same age that have been breast fed) which is reduced by at least 5%, more preferably by at least 10% compared to the level of metabolites (associated with the intestinal metabolome of human subjects of the same age that have been breast fed) which have a nutritional composition which does not comprise non-digestible oligosaccharides.

The present invention may also be expressed as the use of non-digestible oligosaccharides in the manufacture of a nutritional composition for establishing a metabolome in a human subject of under 36 months of age, which nutritional composition comprises 2.5 to 15 wt.% on a dry weight basis of non-digestible oligosaccharides, of which metabolome the levels of metabolites significantly different (related to the intestinal metabolome of a human subject of the same age that is breast fed) are reduced by at least 5%, more preferably of which the levels of metabolites significantly different (related to the intestinal metabolome of a human subject of the same age that is breast fed) are reduced by at least 10% compared to the levels of metabolites significantly different (related to the intestinal metabolome of a human subject of the same age that is breast fed) in a nutritional composition that does not comprise non-digestible oligosaccharides.

In one aspect, the invention may also be expressed as a nutritional composition comprising 2.5 to 15 wt.% on a dry weight basis of non-digestible oligosaccharides for use in establishing a metabolome in a human subject of below 36 months of age, in which metabolites with a significant difference in level (associated with the gut metabolome of a human subject of the same age that has been breast fed) are reduced by at least 5%, more preferably by at least 10%, compared to metabolites with a significant difference in level (associated with the gut metabolome of a human subject of the same age that has been breast fed) in a human subject of the same age that has been fed a nutritional composition not comprising non-digestible oligosaccharides.

In one aspect, the present invention relates to a method for establishing a metabolome in a human subject of less than 36 months of age, wherein less than 45%, more preferably less than 40%, even more preferably less than 35% of the metabolites with significantly different levels in the metabolome are at significantly different levels compared to the intestinal metabolome of a human subject of the same age that is breast-fed, which method comprises administering a nutritional composition comprising 2.5 to 15 wt.% non-digestible oligosaccharides based on dry weight.

The present invention may also be expressed as the use of non-digestible oligosaccharides for the manufacture of a nutritional composition for establishing a metabolome in a human subject of below 36 months of age, which nutritional composition comprises 2.5 to 15 wt.% on a dry weight basis of non-digestible oligosaccharides, of which less than 45%, more preferably less than 40%, even more preferably less than 35% of metabolites with significantly different levels are at significantly different levels compared to the intestinal metabolome of human subjects of the same age that are breast fed.

The present invention may also be expressed as a nutritional composition comprising 2.5 to 15 wt.% on dry weight basis of non-digestible oligosaccharides for use in establishing a metabolome in human subjects below 36 months of age in which less than 45%, more preferably less than 40%, even more preferably less than 35% of the metabolites with significantly different levels are at significantly different levels compared to the intestinal metabolome of human subjects of the same age that are breast fed.

In one aspect, the invention relates to a method for establishing a metabolome in a human subject of less than 36 months of age in which more than 10%, more preferably more than 15%, even more preferably more than 20% of the metabolites with significantly different levels are at significantly different levels compared to the intestinal metabolome of a human subject of the same age fed a nutritional composition which does not comprise non-digestible oligosaccharides.

The present invention may also be expressed as the use of non-digestible oligosaccharides for the manufacture of a nutritional composition for establishing a metabolome in a human subject under 36 months of age comprising, based on dry weight, 2.5 to 15 wt.% of non-digestible oligosaccharides, wherein more than 10%, more preferably more than 15%, even more preferably more than 20% of metabolites with significantly different levels are at significantly different levels compared to the intestinal metabolome of a human subject of the same age fed a nutritional composition which does not comprise non-digestible oligosaccharides.

The present invention may also be expressed as a nutritional composition comprising 2.5 to 15 wt.% on dry weight basis of non-digestible oligosaccharides for use in establishing a metabolome in a human subject of less than 36 months of age in which more than 10%, more preferably more than 15%, even more preferably more than 20% of metabolites with significantly different levels are at significantly different levels compared to the intestinal metabolome of a human subject of the same age fed a nutritional composition which does not comprise non-digestible oligosaccharides.

In a preferred embodiment of the present invention of the established metabolome less than 45%, more preferably less than 40%, even more preferably less than 35% of the metabolites with significantly different levels in the established metabolome are at significantly different levels compared to the intestinal metabolome of human subjects of the same age that are breast-fed, and more than 10%, more preferably more than 15%, even more preferably more than 20% of the metabolites with significantly different levels in the established metabolome are at significantly different levels compared to the intestinal metabolome of human subjects of the same age that are fed a nutritional composition without non-digestible oligosaccharides.

In a preferred embodiment of the method or use of the invention, less than 45%, more preferably less than 40%, even more preferably less than 35% of the metabolites with significant differences in the levels in the gut metabolome are at significantly different levels compared to the gut metabolome of human subjects of the same age that are breast-fed, and more than 10%, more preferably more than 15%, even more preferably more than 20% of the metabolites with significant differences in the levels in the gut metabolome-te are at significantly different levels compared to the gut metabolome of human subjects of the same age that are fed a nutritional composition that is at least not partially fermented by lactic acid producing bacteria and that does not comprise a total of 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis, and that does not comprise non-digestible oligosaccharides.

In a preferred embodiment of the method or use of the present invention, the metabolite with a significantly different level in the gut metabolism profile or metabolome (associated with the gut metabolome of a human subject of the same age that has been breast fed) observed in infants fed with the nutritional composition of the present invention is at least 5% less, more preferably at least 10% less, than the metabolite with a significantly different level in the gut metabolome (associated with the gut metabolome of a human subject of the same age that has been breast fed) observed in infants fed with the nutritional composition of the present invention when fed with a nutritional composition of the present invention which has been fed with a significantly different level of lactate which does not comprise a total of 0.02 to 1.5% by weight on a dry weight basis of lactate.

In one aspect, the present invention relates to a method for establishing a metabolome in a human subject of less than 36 months of age, which method comprises administering a nutritional composition which is at least partially fermented by lactic acid producing bacteria, wherein the nutritional composition comprises a total of 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis, and wherein the nutritional composition comprises 2.5 to 15 wt.% on a dry weight basis of non-digestible oligosaccharides which are reduced by at least 5% compared to metabolites with a significant difference in levels in a human subject of the same age which is fed a nutritional composition which is at least not partially fermented by lactic acid producing bacteria and which does not comprise a total of 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis of non-digestible oligosaccharides (associated with the metabolome of the gut of a human subject of the same age which is human fed with human milk), more preferably, there is at least a 10% reduction in metabolites with significantly different levels in the gut metabolome.

The invention can also be expressed as the use of a fermented ingredient and non-digestible oligosaccharides for the preparation of a nutritional composition at least partially fermented by lactic acid producing bacteria for establishing a metabolome in a human subject below 36 months of age, wherein the nutritional composition comprises a total of 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis, and wherein the nutritional composition comprises 2.5 to 15 wt.% on a dry weight basis of non-digestible oligosaccharides, which are reduced by at least 5% compared to metabolites significantly differing in level in a human subject of the same age fed a nutritional composition which is at least not partially fermented by lactic acid producing bacteria and which does not comprise a total of 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis, which is not comprising non-digestible oligosaccharides (associated with the gut metabolome of a human subject of the same age as human fed with human milk), more preferably, there is at least a 10% reduction in metabolites with significantly different levels in the gut metabolome.

In one aspect, the invention may also be expressed as a nutritional composition at least partially fermented by lactic acid producing bacteria, wherein the nutritional composition comprises a total of 0.02 to 1.5 wt% lactic acid and lactate on a dry weight basis, and wherein the nutritional composition comprises 2.5 to 15 wt% non-digestible oligosaccharides on a dry weight basis for establishing a metabolome in human subjects below 36 months of age in which metabolites with significantly different levels (related to the gut metabolome of human subjects of the same age as human breast feeding) are reduced by at least 5% compared to metabolites with significant differences in levels (related to the gut metabolome of human subjects of the same age as human breast feeding) fed a nutritional composition which is not at least partially fermented by lactic acid producing bacteria and which does not comprise a total of 0.02 to 1.5 wt% lactic acid and lactate on a dry weight basis, which does not comprise non-digestible oligosaccharides, more preferably, there is at least a 10% reduction in metabolites with significantly different levels in the gut metabolome.

In one aspect, the present invention relates to a method for establishing a metabolome in a human subject of less than 36 months of age, in which less than 45%, more preferably less than 40%, even more preferably less than 35% of the metabolites with significantly different levels are at significantly different levels compared to the intestinal metabolome of a human subject of the same age that is breast-fed, the method comprising administering a nutritional composition that is at least partially fermented by lactic acid producing bacteria, wherein said nutritional composition comprises a total of 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis, and wherein said nutritional composition comprises 2.5 to 15 wt.% non-digestible oligosaccharides on a dry weight basis.

The invention may also be expressed as the use of a fermented ingredient and non-digestible oligosaccharides for the preparation of a nutritional composition at least partially fermented by lactic acid producing bacteria for establishing a metabolome in a human subject below 36 months of age, wherein the nutritional composition comprises a total of 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis, and wherein the nutritional composition comprises 2.5 to 15 wt.% non-digestible oligosaccharides on a dry weight basis, of which less than 45%, more preferably less than 40%, even more preferably less than 35% of metabolites with significant differences in levels are at significantly different levels compared to the intestinal metabolome of human subjects of the same age that are breast fed.

The present invention may also be expressed as a nutritional composition at least partially fermented by lactic acid producing bacteria, wherein the nutritional composition comprises a total of 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis, and wherein the nutritional composition comprises 2.5 to 15 wt.% non-digestible oligosaccharides on a dry weight basis for establishing a metabolome in human subjects having an age below 36 months, wherein less than 45%, more preferably less than 40%, even more preferably less than 35% of the metabolites with significantly different levels are at significantly different levels compared to the intestinal metabolome of human subjects of the same age that are breast-fed.

In one aspect, the present invention relates to a method for establishing a metabolome in a human subject of less than 36 months of age, in which more than 10%, more preferably more than 15%, even more preferably more than 20% of metabolites with significantly different levels are at significantly different levels compared to the intestinal metabolome of a human subject of the same age fed a nutritional composition which is not at least partially fermented by lactic acid producing bacteria and which does not comprise total 0.02 to 1.5 wt.% of lactic acid and lactate, based on dry weight, and which does not comprise non-digestible oligosaccharides.

The invention can also be expressed as the use of a fermented ingredient and non-digestible oligosaccharides for the preparation of a nutritional composition at least partially fermented by lactic acid producing bacteria for establishing the metabolome in a human subject under 36 months of age, wherein the nutritional composition comprises a total of 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis, and wherein the nutritional composition comprises 2.5 to 15 wt.% non-digestible oligosaccharides based on dry weight, compared to the gut metabolome of a human subject of the same age fed a nutritional composition which is at least not partially fermented by lactic acid producing bacteria and which does not comprise total 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis, and which does not comprise non-digestible oligosaccharides, greater than 10%, more preferably greater than 15%, even more preferably greater than 20% of the metabolites with significantly different levels in the metabolome are at significantly different levels.

The present invention may also be expressed as a nutritional composition at least partially fermented by lactic acid producing bacteria, wherein the nutritional composition comprises a total of 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis, and wherein the nutritional composition comprises 2.5 to 15 wt.% indigestible oligosaccharides on a dry weight basis for establishing a metabolome in human subjects below 36 months of age in which more than 10%, more preferably more than 15%, even more preferably more than 20% of metabolites with significantly different levels are at significantly different levels compared to the intestinal metabolome of human subjects of the same age fed a nutritional composition which is at least not partially fermented by lactic acid producing bacteria and which does not comprise a total of 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis, and which does not comprise indigestible oligosaccharides.

In a preferred embodiment of the present invention of the established metabolome less than 45%, more preferably less than 40%, even more preferably less than 35% of the metabolites with significant differences in levels in the established metabolome are at significantly different levels compared to the intestinal metabolome of human subjects of the same age that are breast-fed, and more than 10%, more preferably more than 15%, even more preferably more than 20% of the metabolites with significant differences in levels in the established metabolome-te are at significantly different levels compared to the intestinal metabolome of human subjects of the same age that are fed a nutritional composition that is at least not partially fermented by lactic acid producing bacteria and that does not comprise a total of 0.02 to 1.5% by weight on a dry weight basis of lactic acid and lactate, and that does not comprise non-digestible oligosaccharides.

The percentage of metabolites that differ in level is expressed in terms of the total metabolites tested. The nutritional composition of the invention is a nutritional composition comprising 2.5 to 15 wt.% non-digestible oligosaccharides based on dry weight, preferably the nutritional composition of the invention is a nutritional composition at least partially fermented by lactic acid producing bacteria, wherein the nutritional composition comprises 0.02 to 1.5 wt.% in total of lactic acid and lactate based on dry weight, and wherein the nutritional composition comprises 2.5 to 15 wt.% non-digestible oligosaccharides based on dry weight.

The control formulation was a formulation that did not contain indigestible oligosaccharides. Preferably, the control formulation is a formulation that does not contain a fermentation component and does not contain non-digestible oligosaccharides.

In a preferred embodiment, less than 350, more preferably less than 300 metabolites with significantly different levels are at significantly different levels in the gut metabolism profile or metabolome observed in infants fed the nutritional composition of the present invention compared to that observed in human milk-fed infants. In a preferred embodiment, more than 150, more preferably more than 200 metabolites with significantly different levels are present in the gut metabolism profile or metabolome observed in infants fed the nutritional composition of the invention compared to infants fed the control formula.

In a preferred embodiment, the metabolite profile or metabolome of the gut that is not statistically different (in relation to human milk fed infants) observed in infants fed the nutritional composition of the present invention is at least 75 more, preferably at least 100, more preferably at least 125 metabolites observed in infants fed the nutritional composition of the present invention compared to the amount of metabolites that are not statistically different in control formula fed infants (in relation to human milk fed infants).

In a further preferred embodiment of the method and use of the present invention, the present invention is further used for promoting the development of gut microbiota of a human subject of below 36 months of age having a composition closer to the gut microbiota of a human subject of the same age that is human breast fed compared to the gut microbiota of a human subject of the same age that is fed a nutritional composition not comprising non-digestible oligosaccharides. Preferably, promoting the development of gut microbiota means that the gut microbiota has a lower alpha-diversity, preferably determined by the Chao-1 index, compared to the gut microbiota of a human subject fed a nutritional composition not comprising non-digestible oligosaccharides. Furthermore, preferably promoting the development of gut microbiota refers to the gut microbiota having a lower abundance of Blautia and/or erysipelothrichales (Erysipelotrichales), and/or to an increased abundance of lactobacillus, preferably to the gut microbiota having a lower abundance of Blautia and/or erysipelothrichales, more preferably to the gut microbiota having a lower abundance of Blautia, compared to the gut microbiota of a human subject fed a nutritional composition not comprising non-digestible oligosaccharides.

In a further preferred embodiment of the method and use of the present invention, the present invention is further used for promoting the development of gut microbiota of human subjects below 36 months of age having a composition closer to the gut microbiota of human subjects of the same age that are human milk fed compared to the gut microbiota fed to human subjects of the same age that are not at least partially fermented by lactic acid producing bacteria and that do not comprise a total of 0.02 to 1.5 wt.% lactic acid and lactate, based on dry weight, of a nutritional composition that does not comprise non-digestible oligosaccharides. Preferably, promoting development of gut microbiota means that the gut microbiota has a lower alpha-diversity, preferably determined by the Chao-1 index, compared to the gut microbiota of a human subject fed a nutritional composition that is at least not partially fermented by lactic acid producing bacteria and that does not comprise total 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis, not comprising non-digestible oligosaccharides. Furthermore, preferably promoting the development of gut microbiota means that the gut microbiota has a lower abundance of Blautia and/or erysipelothrix, and/or an increased abundance of lactobacillus, preferably Blautia and/or erysipelothrix, more preferably the gut microbiota has a lower abundance of Blautia, compared to the gut microbiota of a human subject fed a nutritional composition which is at least not partially fermented by lactic acid producing bacteria and which does not comprise total 0.02 to 1.5 wt.% lactic acid salts, based on dry weight, and which does not comprise non-digestible oligosaccharides.

The present inventors have discovered an effect on the fecal metabolome and believe that the results obtained from fecal samples are representative of intestinal conditions, particularly the large intestine or colon. In the context of the present invention, "intestinal metabolome" or "intestinal microbiome function" likewise means "large intestinal metabolome" or "large intestinal microbiome function" or "colon metabolome" or "colon microbiome function" or "stool metabolome" or "stool microbiome function".

For bile acids, it has been found that differences in secondary bile acids are observed in the intestinal tract. Secondary bile acids are produced by enzymes of the primary bile acids by intestinal bacteria, thus reflecting the function of the intestinal microbiota. After the intervention period, the pattern of secondary bile acids was found to be more similar in the experimental group than in the control group to that of the breastfeeding reference group. In general, the secondary bile acids in the breast-fed group and experimental group were lower compared to the control group. Of the 23 secondary bile acids measured, 14 were the cases, including 5 of the 6 more abundant secondary bile acids, 1, 2-dehydrocholate, 3-hydrocholate, 7-ketodeoxycholate, 7-ketolithocholate and hyocholate. Of the 6 more abundant secondary bile acids, only taurocholate sulfate (taurocholate sulfate) was higher in the breast-fed group than in the control and experimental groups. For the remaining 9 secondary bile acids present in small or very small amounts, no or very low differences were observed, except for glycocholate sulfate and taurocholate 3, which were all the lowest in the experimental group. These effects are observed in particular in 3 b-hydroxy-5-cholenic acid, 6-oxocholate, 7-ketocholate, 7-ketodeoxycholate, glycocholate sulfate and ursodeoxycholate. Furthermore, it was found that the effect on primary bile acids, which are not a direct result of microbial biotransformation, was generally lower in the experimental group and closer to the breast-fed reference group than the control group, in particular glycochenodeoxycholate and glycocholate.

In a preferred embodiment of the methods and uses of the present invention, promoting development of gut microbiota function refers to a bile acid profile, preferably a secondary bile acid profile.

In a preferred embodiment of the methods and uses of the present invention, promoting development of gut microbiota function refers to a reduction in the level of bile acids, preferably the level of secondary bile acids, compared to the level of bile acids in a human subject fed a nutritional composition not comprising non-digestible oligosaccharides.

In a preferred embodiment of the methods and uses of the present invention, promoting development of gut microbiota function refers to a reduction in the level of bile acids, preferably the level of secondary bile acids, compared to the level of bile acids in a human subject fed a nutritional composition which is at least not partially fermented by lactic acid producing bacteria and which does not comprise total 0.02 to 1.5 wt.% of lactic acid and lactate, based on dry weight, and which does not comprise non-digestible oligosaccharides.

Interestingly, it has been found that the level of intestinal gamma-aminobutyric acid (GABA) is also affected. GABA levels were significantly increased at week 8 and week 17 in the experimental group compared to the control group. Similarly, the level in the breast-fed group was significantly higher than that in the control group. On the other hand, the differences between the experimental group and the breast-fed group were not statistically significant. Intestinal GABA is thought to be beneficial and has beneficial effects on visceral sensitivity and pain perception. GABA is thought to beneficially affect the gut and central nervous system.

In a preferred embodiment of the methods and uses of the present invention, promoting the development of gut microbiota function refers to the level of gamma-aminobutyric acid (GABA).

In a preferred embodiment of the methods and uses of the present invention, promoting development of gut microbiota function refers to an increased level of gamma-aminobutyric acid (GABA) compared to the GABA level of a human subject fed a nutritional composition not comprising indigestible oligosaccharides.

In a preferred embodiment of the methods and uses of the present invention, promoting development of gut microbiota function refers to an increased level of gamma-aminobutyric acid (GABA) compared to the level of GABA in a human subject fed a nutritional composition which is at least not partially fermented by lactic acid producing bacteria and which does not comprise 0.02 to 1.5 wt.% total of lactic acid and lactate, based on dry weight, and which does not comprise indigestible oligosaccharides.

In the context of the present invention, a synonym for promoting the development of gut microbiota function is to develop, improve, induce, maintain, support or drive gut microbiota function.

Compared to the gut microbiota function of an infant administered a nutritional composition that does not comprise a combination of fermented ingredients and non-digestible oligosaccharides, preferably does not comprise a combination of fermented ingredients and non-digestible oligosaccharides, the effect described herein, i.e. promoting, developing, improving, inducing, maintaining or driving gut microbiota function, is observed to be more similar to the gut microbiota function found in breast-fed infants.

In one embodiment, the nutritional composition of the invention is for improving the function of the intestinal microbiota of a human subject of 0 to 36 months of age. In one embodiment the present nutritional composition is for use in improving gut microbiota function in a human subject at an age of 0 to 18 months, even more preferably an infant below 12 months of age, even more preferably an infant at an age of 0 to 6 months, most preferably an infant at an age of 0 to 4 months. In one embodiment the nutritional composition of the invention is for use in improving gut microbiota function in young children of 12 to 36 months of age, most preferably young children of 18 to 30 or to 24 months of age. Preferably, the nutritional composition of the invention is further used to provide nutrition to said human subject. Preferably, the administration of the nutritional composition lasts for at least 1 week, more preferably at least 4 weeks, more preferably at least 8 weeks, even more preferably at least 4 months.

In a preferred embodiment, the method or use of the invention is for infants delivered vaginally. In a preferred embodiment, the method or use of the invention is for full term infants, preferably for healthy full term infants. In a preferred embodiment, the method or use of the invention is for healthy vaginally delivered infants. In a preferred embodiment, the method or use of the invention is for healthy infants born by caesarean section.

In one embodiment, the method or use of the invention is for a human subject below 36 months of age with a fragile or unbalanced intestinal microbiota or a dysregulated intestinal microbiota, or below 36 months of age at risk of a fragile or unbalanced intestinal microbiota or a dysregulated intestinal microbiota, preferably selected from the group consisting of below 36 months of age: premature infants, infants born at a small gestational age, infants with a low birth weight, infants or young children who have received or have received antibiotic treatment, infants born by caesarean section or infants or young children who have suffered from or have suffered from intestinal inflammation or intestinal infection or infants born by mothers who have received prenatal antibiotic treatment. Microbial disorders include, and are preferably, dysbacteriosis. Preferably the microbial or flora dysregulation is a dysregulation in the colon.

In one embodiment, the nutritional composition of the invention is for use in providing healthy gut function and/or for use in preventing and/or treating gut microbiota dysbiosis in a human subject below 36 months of age.

In one embodiment of the method or use of the invention, the final nutritional composition comprises 2.5 to 15 wt.% indigestible oligosaccharides based on dry weight, optionally comprising fermentation ingredients as defined herein.

In this document and in the claims hereof, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, the recitation of 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 elements be present. Thus, the indefinite article "a" or "an" usually means "at least one". Wt% means weight percent.

Drawings

Figure 1 shows numerical information for metabolites with significantly different levels at each visit and comparison of different diet groups.

FIG. 2 shows the distribution of significantly altered metabolites per group per metabolic hyperpathway, per visit; a: the number of metabolites; b: percent metabolites based on total number of metabolites analyzed; and (3) upper plate: breast feeding group and control group; middle plate: breast feeding group and experimental group; a lower plate: experimental group and control group.

Examples

Example 1: effect of partially fermented infant or control formula containing non-digestible oligosaccharides on gut microbiota function compared to breast-fed reference group

In an exploratory clinical study, the growth and safety of the experimental formula (formula 1) and the control formula (formula 2) were studied with 3-4 month intervention on healthy term infants. In a randomized, controlled, multicenter, double-blind, prospective clinical trial, infants were enrolled before 28 days of age and were assigned to receive one of the two formulas up to 17 weeks of age.

Experimental infant formula 1 was a 9: 1 scGOS (source) containing 0.8g/100ml weight ratio

GOS) and lcFOS (Source)

) Infant formula of indigestible oligosaccharides. In this infant formula, 30% was derived from lactofibers on a dry weight basis^TMA commercially available infant formula sold under the trade name galia. Lactofidus^TMIs a fermented milk-derived composition prepared by fermentation with Streptococcus thermophilus and comprising Bifidobacterium breve. A mild heat treatment is used. Infant formula 1 comprises on a dry weight basis about 0.33 wt% (lactate + lactate), of which at least 95% is L-lactic acid + L-lactate. In infant formula 1, the level of colony forming units of the lactic acid producing bacterium Streptococcus thermophilus was about 2X 10⁴cfu/g dry weight and derived from the fermented ingredient Lactofidus^TM。

Control infant formula 2 was a commercially available unfermented infant formula without scGOS/lcFOS. The compositions of the two formulas were similar in energy and macronutrient composition (66 kcal per 100ml, 1.2g protein (bovine whey protein/casein in a weight ratio of 1/1), 7.7g digestible carbohydrates (of which 7.6g lactose), 3.4g fat (mainly vegetable fat). the two infant formulas also contained vitamins, minerals, trace elements and other micronutrients according to the international directive 2006/141/EC for infant formulas.

For reference, a group of infants was listed as being fully breastfed by 17 weeks of age. The intent-to-treat (ITT) population consisted of all subjects who had been randomly assigned infant formula (n-199), and in addition, 100 subjects were listed in the breastfeeding reference group. The ITT population consisted of 94 subjects in the experimental group and 105 subjects in the control group.

Fecal samples were collected on the first day (baseline), 8 weeks of age, and 16-17 weeks of age, not later than the first day after the last study product intake, at random groups or later. Analysis of stool parameters was performed for a small group of infants who chose: natural birth (vaginal delivery), no use of probiotics, thickeners, antibiotics or other drugs that may affect the microbiota from birth to the end of study participation, and no purgative within three days or less prior to stool sampling. This panel consisted of 30 subjects from each of the three study groups-a total of 90 subjects, yielding a total of 270 stool samples. As subjects may have consumed their respective study products for one day, or-even more relevant-may have consumed other commercially available infant formulas containing fermented formulas or prebiotics or probiotics, an impact has been produced compared to the breast-fed reference group.

DNA was extracted from Stool samples using the QIAmp DNA pool Mini Kit (Qiagen) according to the manufacturer's protocol, except that a two-step bead milling step was added. A0.2-0.3 g fecal sample, 300mg 0.1mm glass beads and 1.4mL ASL (lysis) buffer were taken and on the suspension a first step of bead milling step 3X 30 seconds was performed (FastPrep-24 instrmentprogram 5.5). After the inhibitor Ex tablet was added, a second bead milling step was performed for 3X 30 seconds (FastPrep-24instrument program 5.5) to homogenize the sample. After each bead milling step, the samples were cooled on ice for 5 minutes. The extracted DNA was purified using NanoDrop^TMThe quality and concentration of DNA was checked using a spectrophotometer (Thermo Fisher Scientific Inc.) using Quant-iTTM 193 dsDNA BR Assay kit (Invitrogen)^TM) The measurement is performed. The DNA alleles were stored at-80 ℃ until use.

From the purified fecal DNA extract, the V3-V5 region of the bacterial 16S rRNA gene was amplified using primers 357F and 926 Rb. The obtained 16S rRNA gene amplicons were subjected to pyrosequencing using 454 FLX sequencer (454 Life Sciences, Branford, CT, USA) as described above.

Sequence data was analyzed using Quantitative Instruments Intra Microbiological Ecology (QIIME), version 1.8.0. The quality control filter is configured to discard sequences that are less than 200 bases in length, sequences that are more than 1000 bases in length, wherein the average sequence mass fraction is less than 25, have any ambiguous bases, or comprise homopolymer segments of greater than 6 bases. The chimeric sequences were filtered using the Chimeraslayer, owned by QIIME. De novo operation sorting unit (OUT) screening of the filtered sequences was performed using the USEARCH algorithm, which groups sequences with greater than 97% identity. QIIME sparsely processed the OTUs to ensure that the number of reads per sample was the same for alpha-diversity calculations using the following metric: chao1 and observatory species. Representative sequences (i.e., the most abundant sequences) of each OTU were then classified using a ribosomal database project classifier (RDP) by alignment with the SILVA ribosomal RNA database (release 1.0.8).

Counting: for targeted physiological and microbial parameters, the Wilcoxon Rank Sum test was used to calculate the differential p-value between experiments and controls at each time point. If a percentage of the values of a given parameter is detected in more than 70% of the samples, values below the limit of quantitation are replaced by (limit of detection + limit of quantitation)/2, and values below the limit of detection are replaced by limit of detection/root number 2. If the measured percentage of any one group is below the quantitation limit, the parameter is converted to binary (1 for presence, 0 for absence or below the detection limit). For all binary parameters, a Chi-square test (fisher Exact, if expected cell count < 5) was used for reasoning.

For the 16S rRNA gene amplicon sequencing results, the relative abundance of each taxon was mainly subjected to a two-part statistical test (Wagner et al, 2011). In this test, the proportion of zeros in the two sets is compared first, and then the median of the non-zero data in the two sets is compared. The two parts are combined and a p-value is obtained for each taxon at each visit. When the minimum set has at least 10 observations (i.e., non-zero values), the two-part statistical test is unreliable (Wagner et al, 2011), so the data is analyzed as follows: if two groups have 10 non-zero values, two-part statistics are carried out;if any group has < 10 non-zero values, the data is considered binary and a Chi-square test is performed, unless the expected count of 50% cells is < 5, in which case the Barnard test is performed. On the resulting p-values, the multiplex assay was corrected by evaluating the positive false discovery rate (pFDR) (Benjamini)&Hochberg, 1995). The method of using a solution made by Storey, Taylor,&guiding method described in Siegmund (2004) estimates pi₀And then calculate the q-value of the significance measure for each feature. For metabolomics results, the normalized and rescaled signals were only subjected to a two-part statistical test (Wagner et al, 2011) and then q-values were also calculated. The sequencing results were considered statistically significant when p-values < 0.05 and q-values < 0.05. Metabolomics results are considered statistically significant when p values < 0.05 and q values < 0.1.

Frozen stool samples were shipped to a commercial laboratory (Metabolon, Durham, NC) under dry ice for metabolite analysis. The procedure for metabolic profiling has been previously described (Chow, J. et al, Journal of protein Research, 2014.13 (5): p.2534-2542), three platforms for the combined use of analysis including GC/MS and two LC/MS systems, one optimized for positive ionization and one optimized for negative ionization. Data was collected on multiple platform weekdays and scaled to the median of each group balance weekday block for each individual compound. This minimizes any daytime instrument gain or drift, but does not interfere with the variability of the daytime samples. No other adjustments or normalization of the data were made.

As a result:

targeted microbiota quantification studies with qPCR showed that samples from experimental groups at 8 and 17 weeks of age showed statistically significant increases in the amount of bifidobacteria and decreases in the amounts of Clostridium difficile (Clostridium difficile) and Clostridium perfringens (Clostridium perfringens) groups compared to controls, while at baseline these measurements showed no significant differences between the treatment groups. No significant effect on the total amount of bacteria was observed.

Non-targeted 16S rRNA gene amplicon sequencing showed that the various bacterial taxa (4-11 genera, depending on time point) did change consistently with the experimental formulation when compared to the control formulation 4 months after intervention. At 2 months of intervention, an intermediate effect was observed. At the end of the intervention, the levels of these differential bacterial populations in the experimental group appear to more closely match the levels detected in the breastfeeding reference group. See table 1.

Particularly at week 17, the relative abundance of members of the experimental group of Clostridium (Clostridium), Blautia and erysipelothrix was significantly reduced, more comparable to that of the breast-fed reference group. The Bacillus bifidus is increased. It was also found to have an effect on lactobacilli (control decline) and statistically significant differences were observed at week 8. Furthermore, the control group was found to have higher clostridia (Clostridiales), Blautia and erysipelothrix and lower bifidobacteria, more deviating from the experimental and breast-fed reference groups.

The overall microbiota profile diversity can be summarized as an index of diversity for each sample. This diversity index, better known as α -diversity, can be calculated in various ways. The Chao-1 index (estimated based on species abundance of abundance data) for the breastfeeding reference group was consistently low (median Chao1 index 91.37 at 4 months, Q1-Q3 at 66.71-119.2; mean 90.17, 95% Confidence Interval (CI) range 79.48-100.9), while the Chao-1 index for the control group increased over time (median 117.9 at 4 months, Q1-Q3 at 96.46-128.1; mean 114.3, 95% CI range 104.7-124). The experimental group, the Chao-1 index, was still lower and more similar to the breastfeeding reference group (median 96.5 at 4 months, Q1-Q3 of 86.17-114.3; mean 105.2, 95% CI range 93.78-116.6). Consistent with this observation, the median of observed species expressed as OTU (operating classification unit) was lower at 4 months in the experimental group, more similar to the breast-fed reference group, while the median was higher in the control group.

These results indicate that the gut microbiota of infants fed partially fermented formula containing non-digestible oligosaccharides more closely resembles the composition of breast-fed gut microbiota compared to the control group.

Second, non-targeted metabolomics studies were performed on fecal samples to identify a total of 786 unique metabolites (470 and 625 unique metabolites per sample). Metabolomics data were analyzed by Principal Component Analysis (PCA) and a separation of the two formula-fed and breast-fed infants at baseline was observed. During and after the experiment, the separation of the experimental group remained unchanged, but the separation of the control group increased with time (see fig. 1). The separation observed in the metabolomic PCA plots was greater than the PCA plots generated on 16S rRNA gene amplicon sequencing data (data not shown), and in addition, the first two major components account for more variation in metabolomic PCA. These results indicate that metabolomics provides higher resolution biological data, and that the metabolite profiles are more sensitive in showing the differential function of fecal microbiota, and therefore that dietary intervention is also more sensitive to the impact of intestinal metabolomics.

The metabolite numbers between the breast-fed group and the control group and between the experimental group and the control group were significantly different, but at the same time there was no significant difference between the breast-fed group and the experimental group, 58 at week 8 and 91 at week 17.

The detected metabolites showed significant differences at baseline between breast-fed infants and either the control or experimental groups, 268 (34.7%) or 250 (32.6%), respectively, whereas there were no significant differences between the two treatment groups (i.e. only 16 (2.1%) metabolites; fig. 1). This may be explained by the fact that the reference group of breastfeeding already at the time of listing is fully breastfed, whereas infants included in the two formula feeding group are already (partially) formula fed. Interestingly, the metabolite numbers with significant differences between the breastfed group and the control group increased to 404 (51.9%) metabolites over time in the control group, whereas the differential metabolite numbers between the experimental group and the breastfed reference group remained more or less constant, i.e. 261 (34.3%) metabolites at the end of the study, which effect was already largely observed at the intermediate time point of week 8. The levels of 87 metabolites at week 8 and 116 metabolites at week 17 were significantly different (p < 0.05, q value < 0.1) on one hand when the breastfed reference group was compared to the control group and when the control group was compared to the experimental group, and on the other hand, there was no significant difference between the breastfed reference group and the experimental group.

This indicates that the microbiota function gradually deviates from the breastfeeding reference group over time during feeding of the control formula. For the function of fecal microbiota in infants fed experimental formula, baseline differences did not increase over the period of 3-4 months measured herein. When the two treatment groups were compared to the 16S rRNA gene amplicon sequence, the differences in the shown more detailed microbiota composition indicated that the levels of 17-week-old difficile bacteria detected predominantly in the experimental group were the same as those observed in the breastfeeding reference group. These changes appear to follow at a slower rate compared to the targeted physiological parameters measured metabolomically, with significant differences already at week 8 (figure 1). Many of the genera reduced in the experiment were known adult commensal bacteria and were absent/reduced in the breast-fed reference group of infants.

Metabolites with significant differences represent many functional classes. This non-targeted data set indicates that the function of the infant's intestinal ecosystem is highly dependent on diet and response to diet.

The characteristics of the stool samples in this study reflect that infants fed the partially fermented formula had a gut microbiota composition more similar to breast feeding and gut physiological conditions more similar to breast feeding than the control group. Furthermore, more in-depth non-targeted analysis showed that infants fed the partially fermented formula had a smaller deviation in gut microbiota function from the breast-fed reference group than infants fed the control formula, indicating that the partially fermented formula promoted a gut ecosystem more similar to that of breast-fed. The superpathways for amino acids, lipids, xenobiotics, carbohydrates, nucleotides, cofactors and vitamins, energy and peptides are involved (see figure 2). The greatest effect of changes in the number of metabolites is manifested in the amino acid and lipid-related pathways, and when relative changes are observed, very high differences are also observed in the energy, nucleotide, cofactor and vitamin-related pathways.

The bile acids were amplified and differences in secondary bile acids were found to be observed in the intestine. Secondary bile acids are produced by enzymes of the primary bile acids by intestinal bacteria, thus reflecting the function of the intestinal microbiota. After the intervention period, the pattern of secondary bile acids was found to be more similar in the experimental group than in the control group to that of the breastfeeding reference group. In general, the secondary bile acids were lower in the breast-fed group and the experimental group compared to the control group. This is the case for 14 of the 23 secondary bile acids measured, including 5 of the 6 more abundant secondary bile acids, 1, 2-dehydrocholate, 3-hydrocholate, 7-ketodeoxycholate, 7-ketolithocholate and hyocholate, especially 7-ketolithocholate. The taurocholate sulfate in the breast-fed group was higher than that in the control group and the experimental group. There was no or very little difference in the remaining 9 secondary bile acids present in small or very small amounts, except for glycocholate sulfate and taurocholate 3, which were all the lowest in the experimental group. The change is obvious for 3 b-hydroxy-5-cholenic acid, 6-oxocholate, 7-ketocholate, 7-ketodeoxycholate, glycocholenatesulfate and ursodeoxycholate. Furthermore, it was found that the effect on primary bile acids, which are not a direct result of microbial biotransformation, was generally lower in the experimental group and closer to the breast-fed reference group than the control group, in particular glycochenodeoxycholate and glycocholate.

Interestingly, intestinal gamma-aminobutyric acid (GABA) levels were also found to be affected. The GABA level in the experimental group was significantly higher at week 8 and week 17 than in the control group. Similarly, the level in the breast-fed group was significantly higher than that in the control group. On the other hand, the difference between the experimental group and the breast-fed group was not statistically significant. Intestinal GABA is thought to be beneficial and has beneficial effects on visceral sensitivity and pain perception. GABA is thought to beneficially affect the intestinal and central nervous systems.

These results indicate that when a nutritional formula that is partially fermented and comprises non-digestible oligosaccharides is administered, the development of gut microbiota is promoted, making its function closer to that of human milk feeding, compared to a control formula.

Example 2: improving intestinal microbiota by feeding a formulation containing indigestible oligosaccharides

In another randomized, multicenter, double-blind, prospective clinical trial, infants were enrolled before 28 days of age and assigned to receive one of three formulas up to 17 weeks of age:

test group 1: infant formula 1 comprises per 100 ml: 66kcal, 1.35g protein (bovine whey protein/casein in a weight ratio of 1/1), 8.2g digestible carbohydrate (of which 5.6g lactose and 2.1g maltodextrin), 3.0g fat (mainly vegetable fat), 0.8g scGOS (source) comprising a weight ratio of 9: 1

GOS) and lcFOS (Source)

) Is a non-digestible oligosaccharide. On a dry weight basis, about 50% in this infant formula is derived from lactofibers^TM. The infant formula comprises on a dry weight basis about 0.55 wt% lactate, of which at least 95% is L (+) -lactate. The composition further comprises vitamins, minerals, trace elements and other micronutrients according to the international instructions 2006/141/EC of infant formula.

Test group 2: infant formula 2, similar to formula 1, wherein about 15% on a dry weight basis is derived from lactofisus^TM. The infant formula comprises on a dry weight basis about 0.17 wt% lactate, of which at least 95% is L (+) -lactate.

Test group 3: infant formula 3, similar to infant formula 1, but without the indigestible oligosaccharides scGOS and lcFOS.

Test group 4: infant formula 4, an unfermented infant formula comprising 0.8g of indigestible oligosaccharides comprising scGOS (source) in a weight ratio of 9: 1

GOS) and lcFOS (Source)

) But does not contain lactofibers^TMAnd the remainder is similar in composition to infant formula 1.

At baseline and after 17 weeks of intervention, stool samples were collected for microbiological analysis in a manner similar to that described in example 1, except QIIME version (1.6.0), statistical analysis of the 16S rRNA gene amplicon sequencing results using Wilcoxon rank sum test, p-values of differences between groups at various time points were calculated, and combined with pFDR estimation (q-value calculation) to control false findings due to multiple tests. Only a small group of vaginally born subjects with one complete stool sample set (two visits) was analyzed for samples (30 subjects per group, resulting in 240 stool samples), with stool volume sufficient for all analyses. Furthermore, samples from the following infants were excluded: infants who used any systemic antibiotics at any time after birth or used thickening agents added to the formula during the study.

In a selected group of stool samples, the effect of the infant formula used on microbiota was assessed. Following the dry prognosis (at week 17), the measured fecal microbiology parameters from the infants of test group 3 (without fermented ingredients, but with GOS/FOS) showed an increase in bifidobacteria population with a lower incidence of pathogens measured at clostridium difficile levels. Differences were observed in at least 5 taxa relating to lactobacillus, Blautia, clostridium, digestive streptococcaceae and order erysipelothrix, with the results shown in table 2.

Table 2: at 17 weeks, the taxon abundance with a significant q value < 0.1

The lactobacillus content of group 1 was increased compared to groups 3 and 4. Groups 1 and 2 had reduced levels of Blautia and erysipelothrix as compared to groups 3 and 4. In all cases, the difference between group 1 and group 3 was statistically significant, with p < 0.05.

The overall microbiota profile diversity can be summarized as an index of diversity for each sample. For each sample, the Chao-1 index was calculated at the depth of 1496 sequences per sample (possible depth measurement without missing values). At 4 months, the Chao-1 estimate was lower in group 1, thus indicating a lower diversity (median 70.68, Q1-Q3 from 52.5 to 145.9; mean 73.11, 95% CI interval 60.34-85.89) than group 3 (median 104.2, Q1-Q3 from 77.69 to 139.8; mean 104.8, 95% CI interval 88.97 to 120.7) which did not contain indigestible oligosaccharides and than group 4 (median 97.32, Q1-Q3 from 68.99 to 120.5; mean 96.04, 95% CI from 82.71 to 109.4) which did not contain fermented ingredients. The difference between group 1 and group 3 was statistically significant (p ═ 0.005).

Metabonomics:

differences in metabolite numbers between groups fed formulas 1, 3 and 4 were determined in a manner similar to example 1 (group 2 not analyzed). At week 17, the differences in the number and percentage of metabolites were most affected by the presence of NDO. The metabolite difference between the groups fed formulas 4 and 3 was 41.4% (393 metabolites) and the difference between the groups fed formulas 3 and 1 was 34% (323 metabolites).

It was observed that amino acid and peptide hyperpathways were affected by the presence of NDO, and that fermentation formulations present alongside NDO had additional effects on other hyperpathways. This is true of lipid-related hyper-pathways when the number of metabolites is observed, and lipid, nucleotide, vitamin and cofactor, energy, xenobiotic and carbohydrate-related hyper-pathways when the percentage of metabolites is observed.

Furthermore, the difference between formulas 4 and 1 was less than the difference between formulas 3 and 1 for the primary and secondary bile acids, but of the 9 primary bile acids tested, formula 1 had the lowest glycochenodeoxycholate 3 and cholate sulfate. For the 25 secondary bile acids identified, the levels of formula 1 and 4 were lower compared to formula 3, with 3-hydrocholate, 6-oxocholate, dehydrocholate, taurocholate sulfate, and 3 b-hydroxy-5-choleenoic acid being the lowest in the group fed formula 1.

The highest level of 4 hydroxyphenyl pyruvate was observed in group 1, which was also higher in the breast-fed group of example 1, amplifying specific metabolites. The levels of tryptophan, cysteine, homocitrulline (homocitrulline), stearylethanolamine, sphingosine, uridine, 5-methyluridine, beta-alanine, oxalic acid and D-urobilin were lowest in group 1, and also lowest in the breastfeeding reference group of example 1.

When combined with the results of clinical trial examples 1 and 2, a control group and a breastfeeding reference group were present. It can therefore be concluded that an improved effect on gut microbiota function is observed in infants fed a formula containing non-digestible oligosaccharides compared to a formula without non-digestible oligosaccharides, which is more similar to the breast-fed reference group. Further improvement effects are observed when the nutritional composition is partially fermented. An improved effect was observed compared to formulations without fermented ingredients and without indigestible oligosaccharides.

These results show that the indigestible oligosaccharides, in particular GOS and/or FOS, more particularly GOS and FOS, have an effect on the gut metabolome or function of the gut microbiome that is closer to that of the gut microbiome of breast-fed infants compared to formula-fed infants that do not contain indigestible oligosaccharides. This effect is further improved when the infant consumes a formula which is additionally at least partially fermented by lactic acid producing bacteria.

Claims

1. A nutritional composition comprising, on a dry weight basis, 2.5 to 15 wt.% of non-digestible oligosaccharides selected from fructooligosaccharides, non-digestible dextrins, galactooligosaccharides, xylooligosaccharides, arabinooligosaccharides, arabinogalactooligosaccharides, glucooligosaccharides, gentiooligosaccharides, glucomannooligosaccharides, galactomannooligosaccharides, mannooligosaccharides, isomaltooligosaccharides, aspergillus niger oligosaccharides, glucomannooligosaccharides, chitooligosaccharides, soy oligosaccharides, uronic acid oligosaccharides, sialyloligosaccharides and fucooligosaccharides and mixtures thereof, for use in promoting the development of gut microbiota in human subjects below 36 months of age, which function closer to the gut microbiota function of human subjects of human breast fed age than to a gut microbiota function of human subjects of the same age fed nutritional composition not comprising non-digestible oligosaccharides.

2. The nutritional composition for use according to claim 1, wherein promoting development of gut microbiota function means that the gut metabolome is more similar to that of a human subject of the same age that has been human fed, compared to the gut metabolome of a human subject of the same age that has been fed a nutritional composition that does not comprise non-digestible oligosaccharides.

3. A nutritional composition comprising, on a dry weight basis, 2.5 to 15 wt.% of non-digestible oligosaccharides selected from fructooligosaccharides, non-digestible dextrins, galactooligosaccharides, xylooligosaccharides, arabinooligosaccharides, arabinogalactooligosaccharides, glucooligosaccharides, gentiooligosaccharides, glucomannooligosaccharides, galactomannooligosaccharides, mannooligosaccharides, isomaltooligosaccharides, aspergillus niger oligosaccharides, glucomannooligosaccharides, chitooligosaccharides, soy oligosaccharides, uronic acid oligosaccharides, sialyloligosaccharides and fucooligosaccharides and mixtures thereof, for use in establishing a metabolic panel in a human subject under 36 months of age that is more similar to the intestinal metabolic panel of a human subject of the same age as human milk fed, compared to the intestinal metabolic panel of a human subject of the same age fed a nutritional composition that does not comprise non-digestible oligosaccharides.

4. The nutritional composition for use according to claim 2 or 3, wherein the metabolites with significant differences in the gut metabolome at levels of less than 45% compared to the gut metabolome of a human subject of the same age that has been breast-fed and the metabolites with significant differences in the gut metabolome at levels of more than 10% compared to the gut metabolome of a human subject of the same age that has been fed a nutritional composition that does not comprise non-digestible oligosaccharides.

5. The nutritional composition for use according to any one of claims 2 to 4, wherein the metabolites with significantly different levels in the gut metabolome, which is associated with the gut metabolome of a human subject of the same age that has been human milk fed, are reduced by at least 5%, more preferably by at least 10% compared to the metabolites with significantly different levels in a human subject of the same age that has been fed a nutritional composition that does not comprise non-digestible oligosaccharides, which is associated with the gut metabolome of a human subject of the same age that has been human milk fed.

6. The nutritional composition for use according to claim 2, wherein the number of metabolites without statistical difference in the gut metabolome, which is associated with human breast fed human subjects of the same age, is at least 75 more, preferably at least 100, more preferably at least 125, compared to the number of metabolites without statistical difference in human subjects of the same age fed a nutritional composition which does not comprise non-digestible oligosaccharides.

7. Nutritional composition for use according to any of the preceding claims, further for promoting the development of gut microbiota in a human subject below 36 months of age, the composition of gut microbiota being closer to the gut microbiota of a human subject of the same age that is human breast fed, compared to the gut microbiota of a human subject of the same age that is fed a nutritional composition that does not comprise non-digestible oligosaccharides.

8. Nutritional composition for use according to any of the preceding claims, wherein promoting development of gut microbiota function refers to a bile acid profile, preferably a secondary bile acid profile.

9. Nutritional composition for use according to any of the preceding claims, wherein promoting development of gut microbiota function means that the level of bile acids, preferably the level of secondary bile acids, is reduced compared to the level of bile acids of a human subject fed a nutritional composition not comprising non-digestible oligosaccharides.

10. The nutritional composition for use according to any one of claims 1-7 and 9, wherein promoting development of gut microbiota function refers to an increased level of gamma-aminobutyric acid (GABA) compared to the GABA level of a human subject fed a nutritional composition not comprising non-digestible oligosaccharides.

11. Nutritional composition for use according to any one of the preceding claims, which is at least partially fermented by lactic acid producing bacteria, wherein the nutritional composition comprises a total of 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis.

12. Nutritional composition for use according to any one of the preceding claims, wherein the promotion of development of gut microbiota function is compared to the gut microbiota function of an elderly human subject fed a nutritional composition that is not at least partially fermented by lactic acid producing bacteria and that does not comprise a total of 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis.

13. Nutritional composition for use according to any one of the preceding claims, wherein the human subject is an infant of less than 12 months, more preferably less than 6 months.

14. Nutritional composition for use according to any one of the preceding claims, wherein the human subject has or is at risk of having a fragile or unbalanced gut microbiota or gut microbiota dysbiosis.

15. Nutritional composition for use according to any one of claims 11-14, wherein at least 90 wt.% of the total lactate is L (+) -lactic acid and/or L (+) -lactate.

16. Nutritional composition for use according to any one of claims 11-15, wherein the fermented ingredients are fermented by bifidobacteria and/or streptococci.

17. Nutritional composition for use according to any one of claims 11-16, wherein the lactic acid producing bacteria are inactivated to less than 10⁶Level of cfu/g dry weight of the nutritional composition.

18. Nutritional composition for use according to any one of claims 11-17, wherein the amount of fermentation ingredients is 10 to 90 wt% based on the nutritional composition.

19. Nutritional composition for use according to any of the preceding claims, wherein the indigestible oligosaccharide comprises galacto-oligosaccharides and/or fructo-oligosaccharides.

20. Nutritional composition for use according to any of the preceding claims, which is an infant formula, follow-on formula, toddler's milk or toddler's formula, or a growing-up milk for toddlers, preferably an infant formula.