CN112384227A

CN112384227A - Fermentation formulations containing indigestible oligosaccharides

Info

Publication number: CN112384227A
Application number: CN201980012669.3A
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: 2021-02-19
Also published as: WO2019155044A1; EP3749329A1

Abstract

Administration of partially fermented infant formula comprising non-digestible oligosaccharides results in a gut flora composition that is more similar to the gut flora composition of breast-fed infants.

Description

Fermentation formulations containing indigestible oligosaccharides

Technical Field

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

Background

It is widely accepted that the best nutrition for newborn infants is human milk. Infant Formula (IF) based on a mature human milk composition is considered to be 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 in breast-fed infants only in terms of nutrition.

The human intestinal tract possesses a complex microbial ecosystem, and the intestinal flora is considered to be an essential part of our human physiology. In adults, the intestinal flora is considered to be a stable ecosystem, and therefore, in the young, the process of microbial colonization (closely related to the maturation of the gastrointestinal tract itself) can be considered an essential step in the development of health. Several environmental factors that may arise in young age have been shown to have a long-term effect on the intestinal flora and its activity, increasing the risk of illness in later years. Nutrition of young adults is a major factor affecting the development of the intestinal flora. Bifidobacteria species (bifidobacteria species) are usually predominant in the gut flora of breast-fed infants, whereas gut flora rich in Firmicutes members is usually observed in infants fed with conventional formulas.

Formulations supplemented with the prebiotic scGOS/lcFOS mixture have been shown to modulate gut flora composition to a bifidobacteria-rich population (Knol et al, 2005, JPGN 40: 36-42). When this scGOS/lcFOS mixture is added to infant formula partially fermented with Bifidobacterium breve and Streptococcus thermophilus, its effect on intestinal flora has been shown to remain unchanged (Huet, F. et al, 2016, JPGN 63: e 43-53).

WO 2009/151330 discloses a method of feeding an infant delivered by caesarean section and the use of a composition, in particular a product obtained from fermented milk, whey protein hydrolysate, casein hydrolysate and/or lactose, administered to an infant delivered by caesarean section. Thus stimulating a rapid colonization of the intestinal flora of said infant.

The above prior art documents disclose interventions to modulate specific flora taxa, which were analyzed for specificity. For example, increased bifidobacteria or decreased clostridia are produced, but the overall flora is not of concern.

WO 2015/033304 discloses that administration of the probiotic lactobacillus paracasei DG in healthy adults reduces the number of Blautia (Blautia) in the flora.

WO 2017/021476 relates to a nutritional composition comprising fucosylated and N-acetylated oligosaccharides, which promotes or induces a global intestinal flora that is closer to that of infants fed exclusively with human breast milk than infants fed with conventional nutritional compositions.

However, there is a need for nutritional compositions for further improving the overall intestinal flora of infants and young children, even more similar to the intestinal flora of breast-fed infants.

Disclosure of Invention

The inventors have used several independent clinical trials to analyze the flora of infants fed with various test and control formulas by molecular techniques including non-targeted 16S rRNA gene amplicon sequencing. This technique examined the effect on the overall microbiota. It was found that the intestinal flora of infants fed the test formula (i.e. partially fermented and comprising non-digestible oligosaccharides) was more similar to the flora of the breast-fed reference group of infants. The intestinal flora is more similar to that of breast-fed infants compared to the flora of infants fed with a control formula comprising non-digestible oligosaccharides but not fermented, compared to the flora of infants fed with a control formula partially fermented but not comprising non-digestible oligosaccharides, and the difference observed is greatest compared to the flora of infants fed with a conventional non-fermented formula without non-digestible oligosaccharides. This indicates that the effect of the combination of fermented formula and indigestible oligosaccharides on the intestinal flora of infants is further improved. More specifically, the flora of infants fed partially fermented and formula comprising non-digestible oligosaccharides was found to be more similar to that of breast-fed infants, in particular it was found that the alpha-diversity was lower, with a lower abundance of blautia and/or erysipelothrichales (Erysipelotrichales) and/or with an increased abundance of lactic acid bacteria.

Detailed Description

Accordingly, the present invention relates to a method for promoting the development of gut flora in a human subject with an age below 36 months, which gut flora has a composition closer to the gut flora of a human subject of the same age as fed with human milk than the gut flora of a human subject of the same age 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 and/or a nutritional composition not comprising non-digestible oligosaccharides, the method comprising administering a nutritional composition that 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.% non-digestible oligosaccharides on a dry weight basis.

In one embodiment, the method of the invention may be considered a non-medical method for promoting the development of gut flora.

The invention may also be described as the use of a fermented composition and non-digestible oligosaccharides for the preparation of 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 an intestinal flora of a human subject of age below 36 months, which intestinal flora has a composition closer to the intestinal flora of a human subject of the same age that is human milk fed compared to the intestinal flora of a human subject of the same age that is not at least partially fermented by lactic acid producing bacteria and that is not fed a total of 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis and/or a nutritional composition that does not comprise non-digestible oligosaccharides.

The present invention may also be described 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 an intestinal flora of a human subject with an age below 36 months, which intestinal flora constitutes closer to the intestinal flora of a human subject of the same age as human fed human milk compared to the intestinal flora of a human subject of the same age that is not at least partially fermented by lactic acid producing bacteria and does not comprise a total of 0.02 to 1.5 wt.% lactic acid and lactate and/or does not comprise non-digestible oligosaccharides on a dry weight basis.

The present invention also relates to a method for preventing and/or treating a disturbance of the intestinal flora in a human subject with an age below 36 months, said method comprising administering 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 weight of lactic acid and lactate based on dry weight and wherein the nutritional composition comprises 2.5 to 15 weight% non-digestible oligosaccharides based on dry weight.

The invention may also be described as the use of a fermented composition and non-digestible oligosaccharides for the preparation of 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 the prevention and/or treatment of gut flora disturbance in a human subject with an age below 36 months.

The present invention may also be described as a nutritional composition at least partially fermented by lactic acid producing bacteria for use in the prevention and/or treatment of a disturbance of the intestinal flora in a human subject with an age below 36 months, 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.

Fermentation composition

The nutritional composition in the method or use of the invention (hereinafter also referred to as the nutritional composition of the invention, or the nutritional composition or final nutritional composition of the invention) is at least partially fermented. The partially fermented nutritional composition comprises at least in part a composition fermented by a lactic acid producing bacterium. The results show that the presence of the fermented composition in the final nutritional composition results in a more similar intestinal flora as the flora of breast-fed infants upon administration.

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 composition is a milk-derived product, which is a milk substrate fermented by lactic acid producing bacteria, wherein the milk substrate comprises at least one milk substrate selected from the group consisting of: milk, whey protein hydrolysate, casein hydrolysate, or a mixture thereof. Suitably, nutritional compositions comprising a fermented composition and non-digestible oligosaccharides and methods for their preparation are described in WO 2009/151330, WO 2009/151331 and WO 2013/187764.

The fermentation composition preferably comprises bacterial cell debris such as glycoproteins, glycolipids, peptidoglycans, lipoteichoic acids (LTA), lipoproteins, nucleotides and/or integral membrane polysaccharides. It is advantageous to use the fermentation composition 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 make the intestinal flora 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 prebiotics (post-biolics). It is believed that compositions comprising such prebiotics advantageously more closely resemble breast milk, as breast milk is not a fully synthetic formula, but rather comprises metabolites, bacterial cells, cell debris, and the like. Thus, the fermented composition, in particular the fermented milk-derived product, is believed to have an improved effect compared to a non-fermented milk-derived product having no or only lactic acid producing bacteria on the intestinal flora.

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.% of the fermented composition on a dry weight basis. As a way of indicating that the final nutritional composition comprises at least partly the 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.

In the era of omics-based technology, more and more sophisticated tools are available for studying intestinal flora at different molecular levels. In the last decade, the most widely used tool for studying intestinal flora has been based on (partial) sequencing of bacterial 16S rRNA gene sequences and determining which bacterial lineages exist. However, the functional level of the intestinal ecosystem cannot be directly understood by performing an analysis of the intestinal flora by a DNA-based method.

Lactic acid producing bacteria for producing fermented ingredients

It is hypothesized that the beneficial effect of the fermentation composition to be obtained is not dependent on the exact species of lactic acid producing bacteria used for the fermentation. The lactic acid producing bacteria for the preparation of the fermentation composition, in particular for the fermentation of the 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 (Bifidobacterium), Lactobacillus (Lactobacillus), Carnobacterium (Carnobacterium), Enterococcus (Enterococcus), Lactococcus (Lactococcus), Leuconostoc (Leuconostoc), Oenococcus (Oenococcus), Pediococcus (Pediococcus), Streptococcus (Streptococcus), Tetragenococcus (Tetragenococcus), Rogococcus (Vagococcus) and Weissella (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. Selection of a suitable strain 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 as 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, such as Sambrook, j., Fritsch, e.f., and manitis, T. (1989), Molecular Cloning, a Laboratory Manual, 2 nd edition, Cold Spring Harbor (n.y.) Laboratory Press. Preferred bifidobacteria for use are also described by Scardovi, v. in Genus Bifidobacterium, page 1418-1434, in Bergey's manual of systematic bacteriology, volume 2, Sneath, p.h.a., n.s.mair, m.e.sharp and j.g.holt (eds.). Baltimore: 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 (b.thermophilum), bifidobacterium galloides (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, still more preferably bifidobacterium longum and bifidobacterium breve, even more preferably bifidobacterium breve, and more preferably bifidobacterium breve selected from the group consisting of: bifidobacterium breve Bb-03(Rhodia/Danisco), Bifidobacterium breve M-16V (Morinaga), Bifidobacterium breve R0070(Institute Rosell, Lallemand), Bifidobacterium breve BR03(Probiotical), Bifidobacterium breve BR92(Cell Biotech) 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 composition fermented by lactic acid producing bacteria including both bifidobacterium breve and streptococcus thermophilus (s. In one embodiment, the lactic acid producing bacterial fermentation is by fermentation of streptococcus thermophilus and bifidobacterium breve. In one embodiment, the final nutritional composition comprises a fermented composition, wherein the lactic acid producing bacteria are inactivated after fermentation.

Preferably, the fermented composition is 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 1 x10 based on dry weight per g of final composition⁴cfu, more preferably 1X 10⁵cfu, even more preferably 1X 10⁶cfu of lactic acid producing bacteria. Preferably, the inactivated bacteria or bacterial debris is obtained from less than 1 x10 based on dry weight per g of final composition¹²cfu, more preferably 1X 10¹⁰cfu, even more preferably 1X 10⁹cfu of lactic acid producing bacteria. The correlation of the inactivated lactic acid bacteria and cfu can be determined by molecular techniques known in the art or by examining the production process.

Fermentation process

Preferably, the fermented composition is a milk-derived product, which is a milk substrate fermented by lactic acid producing bacteria, said milk substrate comprising at least one milk substrate selected from the group consisting of: 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 milk substrate 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/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 at 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 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, the milk substrate, in particular the aqueous medium containing the milk substrate, is pasteurised prior to the fermentation step 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 results in a more stable substrate and/or fermentation product, especially in the presence of fat.

The seeding density is preferably 1X 10²To 5X 10¹⁰Preferably 1X 10⁴To 5X 10⁹cfu lactic acid producing bacteria per ml aqueous medium containing a milk substrate, more preferably 1X 10⁷To 1X 10⁹cfu lactic acid producing bacteria per ml of aqueous medium containing a milk substrate. The final bacterial density after fermentation is preferably 1X 10³To 1X 10¹⁰More preferably 1X 10⁴To 1X 10⁹cfu/ml aqueous medium containing a 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 lactic acid bacteria 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 lactic acid bacteria 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 proceed to a sufficient or large extent and at the same time produces immunogenic cell fragments such as glycoproteins, glycolipids, peptidoglycans, lipoteichoic acids (LTA), flagella, lipoproteins, DNA and/or entamoesin and metabolites (prebiotics), however for economic reasons the incubation time need not 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 bacterial 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. Alternatively, the fermentation is carried out in a fermentor which has both bifidobacteria (preferably Bifidobacterium breve) and Streptococcus thermophilus.

Methods for preparing fermentation compositions 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. FR 2723960, incorporated herein by reference, discloses in particular in example 6 a suitable process for preparing a fermented ingredient. 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 (s. thermophilus), e.g. with a milk 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 fermentation ingredients may be subjected toPasteurized or sterilized and, for example, spray dried or lyophilized to provide a form suitable for formulation in the final product.

A preferred method of preparing a fermentation composition to be used in the nutritional composition of the present invention is disclosed in WO 01/01785, more specifically in examples 1 and 2. A preferred method of preparing a fermentation composition to be used in the nutritional composition of the present 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. Preferably at least 90%, more preferably at least 95%, even more preferably at least 99% of the viable microorganisms are inactivated by said heat treatment. Preferably, the fermented nutritional composition comprises less than 1 x10⁵Colony forming units (cfu) of live lactic acid producing bacteria per g dry weight. The heat treatment is preferably carried out at a temperature of 70 to 180 c, preferably 80 to 150 c, preferably for about 3 minutes to 2 hours, preferably at a temperature of 80 to 140 c, for 5 minutes to 40 minutes. Inactivation of the lactic acid producing 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. The presence of indigestible oligosaccharides has been shown to improve the flora, making it more similar to that of breast-fed infants. Thus, the presence of both non-digestible oligosaccharides and a fermented composition, in particular by a synergistic effect with a milk-derived product obtained by fermentation with lactic acid producing bacteria, advantageously makes the overall flora more similar to that of infants fed mainly or completely by breast milk.

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 intestinal tract by the action of acids or digestive enzymes present in the human upper digestive tract (such as the small intestine and stomach), but are preferably fermented by the human intestinal flora. 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, 1017-1023 (1988).

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 (uronic acids oligosaccharides), and mixtures thereof. Such oligosaccharides have many biochemical properties and have similar functional advantages, including improved flora. It will also be appreciated that some non-digestible oligosaccharides and preferably some mixtures have an even further improved effect. Thus, more preferably, the indigestible oligosaccharide is selected from fructooligosaccharides, such as inulin; galactooligosaccharides, such as beta-galactooligosaccharides; and mixtures thereof, even more preferably beta-galactooligosaccharides and/or inulin, most preferably beta-galactooligosaccharides. In one embodiment of the nutritional composition of the invention, the indigestible oligosaccharide is selected from the group consisting of galacto-oligosaccharides, fructo-oligosaccharides and mixtures thereof, more preferably beta-galacto-oligosaccharides, fructo-oligosaccharides and mixtures thereof.

The indigestible oligosaccharide is preferably selected from the group consisting of beta-galactooligosaccharides, alpha-galactooligosaccharides and galactans (galactan). According to a more preferred embodiment, the indigestible oligosaccharide is a β -galactooligosaccharide. Preferably, the indigestible oligosaccharide comprises a galactooligosaccharide having beta (1, 4), beta (1, 3) and/or beta (1, 6) glycosidic linkages and a terminal glucose. Trans-galacto-oligosaccharides can be sold, for example, by the tradename

GOS (Domo FrieslandCampinea Ingredients), Bi2muno (Clasado), Cup-oligo (Nissin Sugar), and Oligomate55 (Yakult). These oligosaccharides improve the flora to a greater extent.

The indigestible oligosaccharide preferably comprises fructooligosaccharides. In other cases, the fructooligosaccharide may have the names of, for example, fructan (fructan), fructooligosaccharide (oligofructose), polyfructose (polyfructose), polyfructan (polyfructan), inulin, fructan (levan), and fructan (fructan), and may refer to oligosaccharides comprising β -linked fructose units, which are preferably linked by β (2, 1) and/or β (2, 6) glycosidic linkages and preferably have a DP of 2 to 200. Preferably, the fructooligosaccharides contain glucose with a terminal β (2, 1) glycosidic linkage. Preferably, the fructooligosaccharides contain at least 7 β -linked fructose units. In another preferred embodiment, inulin is used. Inulin is a fructooligosaccharide in which at least 75% of the glycosidic linkages are beta (2, 1) linkages. Typically, inulin has an average chain length of 8 to 60 monosaccharide units. Fructooligosaccharides suitable for use in the compositions of the invention are available under the trade name fructooligosaccharides

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 intestinal flora of infants 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 flora, 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 and is 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. Preferably, the lactic acid producing bacteria are not replicated or inactivated during production and/or do not survive the conditions present in the human upper gastrointestinal tract.

The nutritional composition of the invention is preferably an infant formula, follow-on formula, toddler milk (toddler milk) or toddler formula or growing-up milk for young children (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 age, the infant begins weaning and eats other food. Baby 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 7.0 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, the lipids provide 3 to 7g/100kcal of the nutritional composition, preferably 4 to 6g/100kcal of the nutritional composition; the protein provides 1.6 to 4g/100kcal of the nutritional composition, preferably 1.7 to 2.5g/100kcal of the nutritional composition, and the digestible carbohydrate provides 5 to 20g/100kcal of the nutritional composition, preferably 8 to 15g/100kcal of the nutritional composition. Preferably, the nutritional composition of the invention comprises lipids providing 4 to 6g/100kcal of the nutritional composition, proteins providing 1.6 to 2.0g/100kcal of the nutritional composition, more preferably 1.7 to 1.9g/100kcal of the nutritional composition, and digestible carbohydrates providing 8 to 15g/100kcal of the nutritional composition. In one embodiment, the lipids provide 3 to 7g of lipids per 100kcal of nutritional composition, preferably 4 to 6g per 100kcal of nutritional composition; the protein provides 1.6 to 2.1g/100kcal of the nutritional composition, preferably 1.6 to 2.0g/100kcal of the nutritional composition; and digestible carbohydrates provide 5 to 20g/100kcal of the nutritional composition, preferably 8 to 15g/100kcal of the nutritional composition, and wherein preferably the digestible carbohydrate component comprises at least 60 wt% lactose, based on total digestible carbohydrates, more preferably at least 75 wt%, even more preferably at least 90 wt% lactose, based on total digestible carbohydrates. 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 a fermented composition obtained by fermentation of lactic acid producing bacteria, the amount of lactose is reduced relative to its source due to the fermentation, the conversion to lactate and/or lactic acid by fermenting lactose. 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 high content 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 (fungal oil) and bacterial oil (bacterial oil). The lipids of the nutritional composition of the present invention preferably provide 3 to 7g/100kcal of the nutritional composition, preferably the lipids provide 4 to 6g/100kcal of the 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.% lipid, more preferably 19 to 30 wt.%, 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% protein based on dry weight of the nutritional composition of the invention was calculated according to kjeldahl method by measuring total nitrogen and using a conversion factor of 6.38 in case of casein or 6.25 for other proteins 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 protein to provide 1.6 to 4.0g/100kcal of the nutritional composition, preferably 1.6 to 3.5g, even more preferably 1.75 to 2.5g/100kcal of the nutritional composition. In one embodiment, the nutritional composition of the invention comprises protein to provide 1.6 to 2.1g/100kcal of the nutritional composition, preferably 1.6 to 2.0g, more preferably 1.75 to 2.1g, even more preferably 1.75 to 2.0g/100kcal of the 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 lead to inadequate growth and development of the infant. Too high a content may cause metabolic stress (bude) on e.g. the kidneys of infants. 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. 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 the 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 heat density ensures an optimal ratio between water and heat consumption. For young children, human subjects 12 to 36 months of age, the nutritional composition more preferably has a caloric density of 45 to 65kcal/100ml, 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 the form of a ready-to-eat liquid, the preferred volume administered per day 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. Alternatively, the nutritional compositions of the present invention are in the form of a powder suitable for reconstitution with water as a ready-to-drink liquid. The composition of the invention is preferably prepared by mixing a 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. Using a Physica Rheometer MCR 300(Physica Messtechnik GmbH, Ostfilden, Germany) at 20 ℃ for 95s^-1The shear rate of (c) determines the viscosity of the liquid.

Applications 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 present method comprising administering the present nutritional composition also refers to administering an effective amount of the nutritional composition to an individual in need of such treatment.

The inventors found that using non-targeted 16S rRA gene amplicon sequencing, the intestinal flora of the infant is more similar to that of breast-fed infants when consuming the nutritional composition of the present invention. This technique examines the effect on the overall intestinal microbiota and not only obtains results known to target bacteria. It is known that the results obtained in stool samples by this technique are representative for the flora in the intestine, in particular the large intestine. When referring to the intestinal flora, preferably the flora in the large intestine is meant. The intestinal flora is more similar to that of breast-fed infants compared to the intestinal flora of infants fed with a formula comprising non-digestible oligosaccharides but not fermented, and when compared to the intestinal flora of infants fed with a formula partially fermented but not comprising non-digestible oligosaccharides. The differences observed were greatest when compared to the intestinal flora of infants fed conventional non-fermented formula without indigestible oligosaccharides. This indicates that the fermentation formulation and the indigestible oligosaccharides have a synergistic effect. Compared to feeding only or even mainly nutritional compositions not according to the invention, in particular nutritional compositions not at least partially fermented by lactic acid producing bacteria and/or not comprising non-digestible oligosaccharides, it was found that the flora of infants fed with a partially fermented and non-digestible oligosaccharide comprising formula is more similar to the intestinal flora of breast-fed infants in the following respects: lactic acid bacteria having low alpha-diversity (determined using the Chao-1 index), having a lower abundance of blautia and/or order erysipelothrix, and/or having an increased abundance.

Thus, in a preferred embodiment of the method or use of the invention, promoting development of the intestinal flora means that the intestinal flora has a lower a-diversity, preferably as determined by the Chao-1 index, compared to the intestinal flora of a human subject 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.% of lactic acid and lactate on a dry weight basis and/or does not comprise non-digestible oligosaccharides.

In one aspect, the invention relates to a method for reducing the alpha-diversity of the intestinal flora of a human subject with an age below 36 months comprising administering 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 based on dry weight, and wherein the nutritional composition comprises 2.5 to 15 wt.% indigestible oligosaccharides based on dry weight, preferably the alpha-diversity of the intestinal flora is reduced compared to the alpha-diversity of the intestinal flora of a human subject of the same age 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 based on dry weight and/or a nutritional composition that does not comprise indigestible oligosaccharides.

The method of the invention for reducing the alpha-diversity of the intestinal flora may also be regarded as a non-medical method for reducing the alpha-diversity of the intestinal flora.

The method of the invention may also be expressed as the use of a fermented composition and non-digestible oligosaccharides for the preparation of 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 reducing the alpha-diversity of the intestinal flora of a human subject with age below 36 months, preferably for reducing the alpha-diversity of the intestinal flora, compared to the alpha-diversity of the intestinal flora of a human subject of the same age that is not at least partially fermented by lactic acid producing bacteria and that is not fed with a total of 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis and/or a nutritional composition that does not comprise 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 use in reducing the alpha-diversity of the intestinal flora of a human subject of the same age, preferably for use in reducing the alpha-diversity of the intestinal flora, compared to the alpha-diversity of the intestinal flora of a human subject of the same age that is not at least partially fermented by lactic acid producing bacteria and that is fed a nutritional composition that does not comprise a total of 0.02 to 1.5 wt.% lactic acid and lactate and/or does not comprise non-digestible oligosaccharides on a dry weight basis.

The order Erysipelamiales is an order belonging to the class Erysipelamiales and the phylum firmicutes. This population is more abundant in adults on a high fat diet, and is generally lower in breast-fed infants.

Blautia (a part recently reclassified as Ruminococcus (mainly related to Ruminococcus obeum)) is present in adult intestinal flora and is one of the major members of normal gastrointestinal flora. The abundance is reported to be between 2.5% and 16%. IBS patients are associated with increased blautia. A common feature of bransted species is the formation of acetate salts by hydrogen and carbon dioxide. The species of blautia are hydrogen-producing organisms that have been shown to participate in the digestion of plant fibers. Blautia globosa (Blautia coccoides) can be considered as an adult type symbiotic bacteria because they are almost absent during the development of the juvenile flora. Healthy infants have little gas formation from the gut flora and it is therefore believed that the presence of blautia in relation to the micro flora of the infant indicates an increased amount of gut gas formation and therefore reflects disturbed or at least different microbial fermentation characteristics compared to breast-fed infants.

The genus lactobacillus is a lactic acid producing bacterium that is found in higher amounts in the intestinal flora of breast-fed infants than in the flora of infants fed with conventional formula.

In one aspect, the present invention relates to a method for reducing the abundance of blautia and/or erysipelothrix bacteria and/or increasing the abundance of lactic acid bacteria, preferably for reducing the abundance of blautia and/or erysipelothrix bacteria, more preferably for reducing the abundance of blautia, in the gut flora of a human subject with an age below 36 months, the method comprising administering 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.% on a dry weight basis of non-digestible oligosaccharides, and no total of 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis and/or no non-digestible oligosaccharides in the gut flora of a human subject of the same age fed a nutritional composition that is at least partially fermented by lactic acid producing bacteria and does not comprise a total of 0.02 to 1.5 wt.% lactic acid and lactate on a dry weight basis and/or does not comprise non digestible oligosaccharides The abundance of the order toxellales and/or lactic acid bacteria is preferably used for reducing the abundance of the order blautia and/or erysipelothrix in the gut flora and/or for increasing the abundance of lactic acid bacteria, preferably for reducing the abundance of the order blautia and/or erysipelothrix, more preferably for reducing the abundance of the order lawsonia.

The method of the invention for reducing the abundance of the target blautia and/or erysipelothrix bacteria and/or increasing the abundance of lactic acid bacteria in the gut flora may also be seen as a non-medical method for reducing the abundance of the target blautia and/or erysipelothrix bacteria and/or increasing the abundance of lactic acid bacteria in the gut flora.

The method of the invention can also be expressed as the use of a fermented composition and non-digestible oligosaccharides for the preparation of 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 based on dry weight, and wherein the nutritional composition comprises 2.5 to 15 wt.% non-digestible oligosaccharides based on dry weight, for reducing the abundance of blautia and/or erysipelothrix spp and/or increasing the abundance of lactic acid bacteria in the intestinal flora of a human subject with the same age as compared to the abundance of blautia and/or lactic acid bacteria in the intestinal flora of a human subject not at least partially fermented by lactic acid producing bacteria and not fed a total of 0.02 to 1.5 wt.% lactic acid and/or a nutritional composition not comprising non-digestible oligosaccharides based on dry weight, preferably for reducing the abundance of blautia and/or erysipelothrix target and/or increasing the abundance of lactic acid bacteria in the gut flora, preferably for reducing the abundance of blautia and/or erysipelothrix target, more preferably for reducing the abundance of blautia.

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 based on dry weight, and wherein the nutritional composition comprises 2.5 to 15 wt.% indigestible oligosaccharides based on dry weight, for reducing the abundance of blautia and/or erysipelothrix purpose and/or increasing the abundance of lactic acid bacteria in the intestinal flora of a human subject of the same age as compared to the abundance of blautia and/or erysipelothrix purpose and/or lactic acid bacteria in the intestinal flora of a human subject of the age below 36 months of age of the same age as a nutritional composition which is not at least partially fermented by lactic acid producing bacteria and does not comprise a total of 0.02 to 1.5 wt.% lactic acid and/or does not comprise indigestible oligosaccharides based on dry weight, preferably for reducing the abundance of blautia and/or erysipelothrix bacteria, more preferably for reducing the abundance of blautia, preferably for reducing the abundance of blautia and/or erysipelothrix bacteria in the gut flora and/or for increasing the abundance of lactic acid bacteria, preferably for reducing the abundance of blautia and/or erysipelothrix bacteria, more preferably for reducing the abundance of blautia.

The Chao1 score indicates the abundance of intestinal flora. It estimates the number of different species present in the sample. For adults, an increased or high abundance of the intestinal flora is desirable, which is not the case for infants. Azad et al, 2013, CMAJ, 185(5)385- "394 observed an increase in the Chao1 index in healthy infants not breastfed but formula-fed at 4 months of age compared to the flora of breastfed infants. Preferably the alpha-diversity, preferably the Chao-1 index, of the intestinal flora of infants fed with the nutritional composition according to the invention is at least 5% lower, more preferably at least 10% lower than the intestinal flora of infants fed with conventional formulas without fermented composition and/or indigestible oligosaccharides. Preferably the alpha-diversity, preferably the Chao-1 index, of the intestinal flora of infants fed with the nutritional composition according to the invention is at least 5%, more preferably at least 10% lower than the intestinal flora of infants fed with a conventional formula without the fermented composition and non-digestible oligosaccharides. Preferably the intestinal flora is alpha-diverse, preferably the Chao-1 index is not lower than the intestinal flora of human milk fed infants.

In the context of the present invention, synonyms for promoting the development of the intestinal flora are developing, improving, inducing, maintaining, supporting or driving the intestinal flora.

Compared to the intestinal flora of an infant administered a nutritional composition not comprising the fermented composition and/or the non-digestible oligosaccharides, preferably not comprising the combination of the fermented composition and the non-digestible oligosaccharides, the effect described herein is observed, i.e. promoting, developing, improving, inducing, maintaining or driving the intestinal flora to be more similar to the intestinal flora found in breast-fed infants.

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

In a preferred embodiment, the method or use of the invention is for infants delivered via the birth canal. In a preferred embodiment, the method or use of the invention is for term infants, preferably for healthy term infants. In a preferred embodiment, the method or use of the invention is for a healthy infant delivered via the birth canal. 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 with a fragile or unbalanced intestinal flora or a dysregulated intestinal flora at an age below 36 months, or a human subject with an age below 36 months at risk of a fragile or unbalanced intestinal flora or a dysregulated intestinal flora, preferably a human subject with an age below 36 months is selected from the group consisting of a preterm infant, a small for gestational age infant, a low birth weight infant, an infant or toddler that has received or is receiving antibiotic treatment, an infant or toddler that has received or suffers from intestinal inflammation or intestinal infection, or an infant or toddler that has received perinatal antibiotic treatment, or an infant or toddler that has suffered from or suffers from intestinal inflammation or intestinal infection, or a mother that has undergone perinatal. 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 providing healthy gut function and/or for preventing and/or treating gut dysbiosis in a human subject with an age below 36 months.

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, an element introduced 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" generally means "at least one". Wt% means weight percent.

Examples

Example 1: effect of partially fermented infant formula or control formula with indigestible oligosaccharides on the intestinal flora composition compared to a breast-fed reference group

In an exploratory clinical study, 3-4 months of intervention was performed on full-term healthy infants to study the growth and safety of the experimental formula (formula 1) versus the control formula (formula 2). 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 until 17 weeks of age.

Experimental infant formula 1 is an infant formula comprising 0.8g/100ml scGOS (source) in a weight ratio of 9: 1

GOS) and lcFOS (Source)

) Is a non-digestible oligosaccharide. In this infant formula, 30% on a dry weight basis is from lactofibers^TMIt is a commercially available infant formula marketed under the trade name Gallia. Lactofidus^TMIs a fermented milk-derived composition produced by fermentation with streptococcus thermophilus and comprising bifidobacterium breve. A mild heat treatment is used. Infant formula 1 comprises about 0.33 wt% (lactate + lactate) on a dry weight basis, with at least 95% being L-lactate + L-lactate. In the infantIn infant formula 1, the level of colony forming units of lactic acid producing bacteria (Streptococcus thermophilus) was about 2X10⁴cfu/g dry weight, and is derived from the fermentation composition Lactofidus^TM。

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

As a reference, a group of infants who were breast-fed only to the age of 17 weeks was included. The intent-to-treat (ITT) population consisted of all subjects who had been randomly assigned infant formula (n-199), with an additional 100 subjects included 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 at 8 weeks of age and 16-17 weeks of age were collected at random or on subsequent (baseline) days, no later than the day of the last study product intake. Analysis of stool parameters in a subgroup of infants, selected as follows: spontaneous labor (birth canal), without the use of probiotics, thickening agents, antibiotics or other drugs that may affect the flora from birth to the end of the study, and without the use of laxatives three days or less prior to stool sampling. This subgroup consisted of 30 subjects per each of the three study groups-90 subjects in total, yielding 270 stool samples in total.

As subjects potentially may have consumed a day of each study product, or-even more relevant-may have consumed other commercial infant formulas containing fermented formulas, or prebiotics or probiotics, the impact may have been produced compared to the breastfed reference group.

Stool samples were subjected to D using the QIAmp DNA Stool Mini kit (Qiagen) according to the manufacturer's protocol, except that a double beating (bcad-waiting) step was addedAnd (5) NA extraction. To a stool sample of 0.2-0.3g 300mg of 0.1mm glass beads and 1.4mL of ASL (lysis) buffer were added and a first beating step of 3x 30 seconds was performed on the suspension (FastPrep-24 instrument procedure 5.5). After the addition of the InhibitEx tablets, the second beating step was carried out for 3x 30 seconds (FastPrep-24 instrument program 5.5) to homogenize the samples. After each beating step, the samples were cooled on ice for 5 minutes. Using NanoDrop^TMThe purity of the extracted DNA was checked with a spectrophotometer (Thermo Fisher Scientific Inc.) using Quant-iTTM 193 dsDNA BR Assay kit (Invitrogen)^TM) The mass and concentration of the DNA was determined. DNA aliquots were stored at-80 ℃ until use.

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

Sequence data was analyzed using the pipeline of Quantitative instruments intro microbiological Ecology, version 1.8.0 (QIIME). Quality control filters (Quality control filters) are set to discard sequences less than 200 bases in length, greater than 1000 bases in length, an average sequence Quality score of less than 25, with any ambiguous bases, or homopolymer strands containing greater than 6 bases. The chimeric sequences were filtered using QIIME's own Chimeraslayer. On the filtered sequences, Operational Taxonomic Units (OTUs) screening was performed ab initio using the USEARCH algorithm, which groups sequences with more than 97% identity. OTUs were diluted by QIIME to ensure the same number of reads per sample in order to perform alpha diversity calculations using the following metrics: chao1 and the species observed. Subsequently, classification criteria were assigned to representative sequences (i.e., the most abundant sequences) of each OTU by alignment with the SILVA Ribosomal RNA Database (release 1.0.8) using a Ribosomal Database entry Classifier (RDP).

Counting: the Wilcoxon Rank Sum test was used to calculate the p-value of the difference between the experiment and the control at each time point for physiological and microbial target parameters. If a percentage of a given parameter value is detected in 70% or more 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 the square root of the limit of detection/(2). In the case where the measured percentage in either group is below the quantitation limit, then the parameter is converted to binary (1 for presence, 0 for absence or below the detection limit). For all binary parameters, reasoning was performed using the chi-square test (fisher exact test if expected cell count < 5).

For the 16S rRNA gene amplicon sequencing results, the relative abundance of each taxon was mainly subjected to two-part statistical tests (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 fractions are combined and each classification unit obtains a p-value at each visit. When there are at least 10 observations in the smallest group (i.e., non-zero values), the two-part statistical test is unreliable (Wagner et al, 2011), and therefore, the data is analyzed as follows: if both groups have 10 non-zero values, then two part statistics are performed; if any group has < 10 non-zero values, the data is processed as binary data and a chi-squared test is performed unless the desired frequency of 50% of the cells is < 5, in which case a Barnard test will be performed. For the resulting p-values, a correction of multiple tests was performed by evaluating the positive false discovery rate (pFDR) (Benjamini)&Hochberg, 1995). The results of the tests by Storey, Taylor,&the bootstrap method described by Siegmund (2004) was used to estimate π₀Then, a q-value is calculated, which is a measure of the significance of each feature. The sequencing results were considered statistically significant when p-value < 0.05 and q-value < 0.05.

As a result:

quantitative analysis of target flora by qPCR showed that samples from experimental groups at 8 and 17 weeks of age showed statistically significant increased amounts of bifidobacteria and lower amounts of Clostridium difficile (Clostridium) and Clostridium perfringens (Clostridium perfringens groups) compared to control groups, whereas at baseline these measurements were not significantly different between treatment groups. No significant effect on the total amount of bacteria was observed.

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

Particularly at week 17, the relative abundance of members of the orders clostridia, blautia and erysipelothrix in the experimental group was significantly reduced, comparable to the breast-fed reference group. The number of bifidobacteria increases. Effects on lactic acid bacteria were also found (control group decreased) and statistical differences were observed at week 8. It was also found that the control group had a higher content of clostridia, blautia and erysipelothrix targets and a lower content of bifidobacteria, which is more different from the experimental group and the breast-fed reference group.

The overall population distribution diversity can be summarized as one diversity index per sample. This diversity index can be calculated in a number of ways, more broadly referred to as a-diversity. The Chao-1 index (based on an estimate of abundance of the species based on abundance data) of the breast-fed reference group remained consistently and at a low level (median Chao1 index at 4 months 91.37, Q1-Q3 66.71-119.2; mean 90.17, 95% Confidence Interval (CI) range 79.48-100.9), while the control group was timely increased (median at 4 months 117.9, Q1-Q3 at 96.46-128.1; mean 114.3, 95% CI range 104.7-124). For the experimental group, the Chao-1 index remained low, more similar to the breast-fed control group (median at 4 months 96.5, Q1-Q3 of 86.17-114.3; mean 105.2, 95% CI ranging from 93.78-116.6). Consistent with this observation, the median number of species (in OTU (operating taxonomic unit)) observed in the experimental group was lower at 4 months, more similar to the breast-fed control group, and higher in the control group.

These results indicate that the intestinal flora of infants fed the partially fermented formula containing non-digestible oligosaccharides has a flora composition more like that in the intestine of breast-fed infants than the control group.

Example 2: consumption of partially fermented formula containing indigestible oligosaccharide for improving intestinal flora composition

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

test group 1: infant formula 1 contained 66kcal per 100ml, 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 of origin) in 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 infant formula 1, wherein about 15% is derived from lactofibers on a dry weight basis^TM. The infant formula comprises about 0.17 dry weight basisWeight% lactic acid + lactate, at least 95% of which is L (+) -lactic acid/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, with 0.8g scGOS (source) comprising a weight ratio of 9: 1

GOS) and lcFOS (Source)

) Non-digestible oligosaccharides of (4), but does not comprise lactofisus^TMAnd the balance is similar to the composition of infant formula 1.

In a similar manner as described in example 1, except for QIIME version (1.6.0), stool samples were collected for microbiological analysis at baseline and 17 weeks post-intervention, and a Wilcoxon Rank Sum test was performed on statistical analysis of 16S rRNA gene amplicon sequencing results to calculate p-values for differences between groups at each time point, combined with pFDR estimation (q-value calculation) to control false findings due to multiple tests. Only a subset of the production subjects from the birth canal (30 subjects in each group, 240 stool samples in total) were analyzed, these samples had a complete set of stool samples (two visits) and the stool volume was sufficient for all analyses. Furthermore, samples of infants using any systemic antibiotics at any time after birth or using thickeners added to the formula during the study were excluded.

In a selected set of stool samples, the effect of the infant formula used on the flora was evaluated. After the dry prognosis (week 17), the fecal microbiology parameters (no fermented components but containing GOS/FOS) determined from group 3 infants showed an increase in bifidobacteria population with a low incidence of pathogenic bacteria, as determined by clostridium difficile levels. Differences were observed in at least 5 taxa involving lactic acid bacteria, blautia, clostridiales, peptostridia and erysipelothrix, and the results are listed in table 2.

Table 2: significance q value & lt 0.1 abundance of taxa-17 weeks

The number of lactic acid bacteria was found to increase in group 1 compared to groups 3 and 4. The numbers of blautia and erysipelothrix in groups 1 and 2 were found to be reduced 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 population distribution diversity can be summarized as one diversity index per sample. For each sample, the Chao-1 index (the deepest measurement possible, no missing values) was calculated at the depth of 1496 sequences per sample. At month 4, the Chao-1 estimate was lower for group 1, thus indicating a lower diversity (median 70.68, Q1-Q3 of 52.5-145.9; mean 73.11, 95% Cl interval of 60.34-85.89) than group 3 without indigestible oligosaccharides (median 104.2, Q1-Q3 of 77.69-139.8; mean 104.8, 95% Cl interval of 88.97-120.7) and also lower than group 4 without fermentation composition (median 97.32, Q1-Q3 of 68.99-120.5; mean 96.04, 95% Cl of 82.71-109.4). The difference between group 1 and group 3 was statistically significant (p ═ 0.005).

When combining the results of clinical trial examples 1 and 2, there were all control and breastfeeding reference groups. It can therefore be deduced that an improved effect on the intestinal flora was observed in infants fed with a partially fermented formula in combination with non-digestible oligosaccharides compared to a formula without fermented composition and/or without non-digestible oligosaccharides, which is more similar to the breast-fed control group. These effects were observed for diversity (lower), blautia (lower), erysipelothrix (lower), lactic acid bacteria (higher), bifidobacteria (higher). Effects have been observed at intermediate time points. For products with 15 wt% of the fermentation composition, an effect has been observed, but a higher effect is observed for compositions with 30 or 50 wt% of the fermentation composition.

Example 3: effect of fermented infant formula and indigestible oligosaccharides on the flora composition of infants

In another randomized, double-blind, control, parallel, prospective, multicenter, multinational, intervention study, subjects were equally randomized into four treatment groups. Furthermore, infants who were purely breastfed since birth (never received any infant formula) and whose mother had an intention to continue purely breastfeeding until the infant was at least 4 months of age were included in the breastfeeding reference group. A total of 350 subjects were enrolled, 280 of which were randomly given any of the four test products, and 70 subjects were included in the breastfeeding reference group.

Test group 1: infant formula 1 was the experimental test formula: a modified cow's milk-based infant formula for use with bottle-fed 0-6 month old infants. The formula contains about 0.9g/100ml of non-digestible oligosaccharides (a mixture of galactooligosaccharides with an average degree of polymerisation below 6 and fructooligosaccharides with an average degree of polymerisation above 20 (raftilin hp from Orafti)) and comprises fermented infant formula

(sold by Gallia, France) comprising Bifidobacterium breve and Streptococcus thermophilus (heat inactivated after the fermentation process), bacterial fermentation metabolites (such as L- (+) lactate). The amount of L-lactate is greater than 0.05 wt.% based on the dry weight of the composition. No added indigestible oligosaccharides are present.

Test group 2: infant formula 2 is a modified cow's milk-based infant formula for bottle-fed infants of 0-6 months of age (Nutrilon 1, sold by Nutricia, the netherlands). The formulation contains non-digestible oligosaccharides (NDO) -galactooligosaccharides (available from Frieslandcampina Domo)

GOS, average degree of polymerization less than 6) and fructooligosaccharides (raftilin hp available from Orafti, average degree of polymerization greater than 20) in a w/w ratio of about 9: 1 in an amount of about 0.8g/100 ml. The formulation does not contain a fermentation composition.

Test group 3: infant formula 2 is a cow's milk based modification for bottle-fed 0-6 month old infantsInfant formula and is a fermented infant formula comprising Bifidobacterium breve and Streptococcus thermophilus (heat inactivated after fermentation process), a bacterial fermentation metabolite (such as L- (+) lactic acid)

(sold by Gallia in France). The amount of L-lactate is higher than 0.05 wt.%, based on the dry weight of the composition. No added indigestible oligosaccharides are present.

Control group: infant formula 4 was a control formula: a modified cow's milk-based infant formula for use in bottle-fed 0-6 month old infants, free of non-digestible oligosaccharides and free of fermented components.

All four test formulations contained nucleotides and a fat blend containing long chain fatty acids. The formulas are similar in calorie content, protein content, fat blend, and have similar amounts of digestible carbohydrates. The number of viable bacteria in test products 2 and 3 was less than 10³cfu/g. The formula also comprises vitamins, minerals, trace elements and other micronutrients according to international instructions 2006/141/EC for infant formulas.

At the end of the study, 198 randomized subjects completed the study, while 82 randomized subjects prematurely exited the study. There was no statistically significant difference in the number and nature of early withdrawers compared to the control group for the effective test product group. Subjects were well balanced among the study groups in terms of demographic and baseline characteristics.

At each visit, parents collected a stool sample into a stool container. After parental collection, the samples were directly frozen at-20 ℃ and kept at this temperature until the samples were handed to the investigator. In the field, samples were stored at-80 ℃ for subsequent analysis. At each collection, 2 tubes must be half filled. Samples were analyzed by Fluorescence In Situ Hybridization (FISH), which is a cytogenetic technique for bacterial identification and enumeration by fluorescent bacterial-specific DNA probes. FISH probes are used in a manner known in the art. More specifically, for the Burkita globularis group, probe Erec48(Franks, 1998, AEM 64: 3336-45) was used.

As a result:

at month 4, bifidobacteria increased in experimental group 1, while several clostridial species decreased (data not shown). For blautia globosa, improved effects were observed in the experimental formulation. At baseline, there was no difference in the percentage of B.globularis in any of the formulations (p value > 0.05; Kruskal-Wallis). However, at month 4, statistically significant differences between the formulations were detected (p-value < 0.001; Kruskal-Wallis test). The overall difference at month 4 was mainly influenced by Calismax (p-value < 0.001 relative to control; p-value < 0.001 relative to Calisma; test Wilcoxon Sum Rank) and Nutrilon (p-value < 0.001 relative to control; p-value 0.015 relative to Calisma; test Wilcoxon Sum Rank) formulations, see table 3.

Table 3: percentage of Braudneria spheroidis, determined by FISH.

a: p < 0.05 group 1 vs group 2, b: p < 0.05 group 1 vs group 3, c: p < 0.05 group 1 vs group 4, d: p < 0.05 group 2 vs group 3, e: p < 0.05 group 2 vs group 4, f: p < 0.05 group 3 vs group 4. Determined by pairwise comparison using the Wilcoxon Sum Rank test.

The observed results again show that an improved effect on the intestinal flora was observed in formula-fed infants with a combination of partially fermented formula and non-digestible oligosaccharides compared to formula without fermented composition and/or without non-digestible oligosaccharides. These effects were observed for alpha diversity (lower) and blautia (lower).

Claims

1. 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.% on a dry weight basis of non-digestible oligosaccharides selected from fructooligosaccharides, non-digestible dextrins, galactooligosaccharides, xylooligosaccharides, arabinooligosaccharides, arabinogalactooligosaccharides, oligoglucosides, gentiooligosaccharides, glucomannooligosaccharides, galactomannooligosaccharides, mannooligosaccharides, isomaltooligosaccharides, aspergillus niger oligosaccharides, glucomannooligosaccharides, chitooligosaccharides, soy oligosaccharides, uronic acid oligosaccharides, sialyloligosaccharides and fucooligosaccharides and mixtures thereof for promoting the development of an intestinal flora in a human subject with an age below 36 months that is at least partially fermented with lactic acid producing bacteria and that does not comprise a total of 0.02 to 1.5 wt.% lactic acid and lactic acid based on a dry weight basis The composition of the gut flora of an elderly human subject fed with salt and/or a nutritional composition not comprising non-digestible oligosaccharides is closer to the gut flora of an elderly human subject fed with human milk than to the gut flora of an elderly human subject fed with salt and/or a nutritional composition not comprising non-digestible oligosaccharides.

2. Nutritional composition for use according to claim 1, wherein promoting development of the intestinal flora means that the intestinal flora has a lower a-diversity, preferably as determined by the Chao-1 index, compared to the intestinal flora of a human subject not being fed by a nutritional composition being at least partially fermented by lactic acid producing bacteria and not comprising a total of 0.02 to 1.5 wt.% lactic acid and lactate based on dry weight and/or not comprising non-digestible oligosaccharides.

3. Nutritional composition for use according to claim 1, wherein the promotion of gut flora development is a gut flora with a lower abundance of blautia and/or lactobacillus of the order erysipelothrix and/or with an increased abundance of lactic acid bacteria, preferably of the species blautia with a lower abundance of gut flora, compared to gut flora of a human subject not being fed by the lactic acid producing bacteria and not comprising a total of 0.02 to 1.5 wt.% lactic acid and lactate based on dry weight and/or a nutritional composition not comprising non-digestible oligosaccharides.

4. 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.% on a dry weight basis of non-digestible oligosaccharides selected from the group consisting of fructooligosaccharides, non-digestible dextrins, galactooligosaccharides, xylooligosaccharides, arabinooligosaccharides, arabinogalactooligosaccharides, oligoglucosides, gentiooligosaccharides, glucomannanoligosaccharides, galactooligosaccharides, mannooligosaccharides, isomaltooligosaccharides, aspergillus niger oligosaccharides, glucomannanoligosaccharides, chitooligosaccharides, soy oligosaccharides, uronic acid oligosaccharides, sialyloligosaccharides and fucooligosaccharides and mixtures thereof, for use in the prevention and/or treatment of gut flora imbalance in a human subject aged below 36 months.

5. 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.

6. 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 intestinal flora or intestinal dysbiosis.

7. Nutritional composition for use according to any one of the preceding claims, wherein the nutritional composition comprises a total of 0.1 to 1.5 wt.% of lactic acid and lactate based on dry weight of the nutritional composition.

8. Nutritional composition for use according to any one of the preceding claims, wherein at least 90 wt.% of the lactic acid and lactate in total are L (+) -lactic acid and/or L (+) -lactate.

9. Nutritional composition for use according to any one of the preceding claims, wherein the fermented composition is fermented by Bifidobacterium and/or Streptococcus.

10. Nutritional composition for use according to any one of the preceding claims, wherein the nutritional composition comprises bifidobacterium breve and/or streptococcus thermophilus.

11. Nutritional composition for use according to any of the preceding claims, wherein the lactic acid producing bacteria are inactivated to below 10⁶Level of cfu/g dry weight of the nutritional composition.

12. Nutritional composition for use according to any one of the preceding claims, wherein the amount of the fermentation composition is from 10 to 90 wt. -%, based on the total nutritional composition.

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

14. Nutritional composition for use according to any one of the preceding claims, wherein the nutritional composition comprises protein, digestible carbohydrate and lipid and wherein protein provides 1.6 to 2.5g/100kcal of nutritional composition, digestible carbohydrate provides 5 to 20g/100kcal of nutritional composition and lipid provides 3 to 7g/100kcal of nutritional composition.

15. Nutritional composition for use according to any one of the preceding claims, which is an infant formula, follow-on formula, toddler milk or toddler formula or growing-up milk for young children, preferably an infant formula.